in vitro method for reprogramming human or other mammalian somatic cells to induced pluripotent stem

专利摘要:
RNA preparations comprising purified modified RNA to reprogram cells. the present invention provides compositions and methods for reprogramming somatic cells using purified RNA preparations comprising single-stranded mRNA encoding an ips cell induction factor. the purified RNA preparations are preferably substantially free of contaminating RNA molecules that: i) would activate an immune response in somatic cells, ii) decrease the expression of single-stranded mRNA in somatic cells, and / or iii) activate RNA sensors in somatic cells. in certain embodiments, the purified RNA preparations are substantially free of partial mRNAs, double-stranded mRNAs, uncapped RNA molecules and / or single-stranded mRNAs.
公开号:BR112012013875B1
申请号:R112012013875
申请日:2010-12-07
公开日:2020-04-14
发明作者:Person Anthony；Weissman Drew；Dahl Gary；Jendrisak Jerome；Meis Judith；Kariko Katalin
申请人:Person Anthony；Weissman Drew；Dahl Gary；Jendrisak Jerome；Meis Judith；Kariko Katalin；
IPC主号:

专利说明:

IN VITRO METHOD FOR REPROGRAMMING HUMAN SOMATIC CELLS OR OTHER MAMMALS FOR INDUCED PLURIPOTENT STEM CELLS (IPS OR IPSCS CELLS) AND COMPOSITION
This application claims priority for provisional application US 61 / 267,312, filed on December 7, 2009, which is hereby incorporated by reference in its entirety.
This application also claims priority for provisional application US 11 / 990,646, filed on March 27, 2009, which is an entry into the national phase in the USA of PCT / US06 / 32372, filed on August 21, 2006, which claims priority for application US 60 / 710,164, filed on August 23, 2005, all of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
The present invention relates to compositions and methods for altering or reprogramming the differentiating state of eukaryotic cells, including human cells or other animal cells, by contacting cells with purified RNA preparations that comprise or consist of one or more strand mRNA molecules different codes that each encode a reprogramming factor (for example, an iPS cell induction factor). Single-stranded mRNA molecules preferably comprise at least one modified nucleoside (for example, selected from the group consisting of a pseudouridine (abbreviated by the Greek letter “psi” or “Ψ”), 5-methylcytosine (m ⁵ C), 5 -methyluridine (m ⁵ U), 2'-O-methyluridine (Um or m ² '° U), 2-thiouridine (s ² U), and N ⁶ -methyladenosine (m ⁶ A)) in place of at least one part of the corresponding unmodified canonical nucleoside (for example, in place of substantially all unmodified canonical nucleosides A, C, G, or T). In addition, single-stranded mRNA molecules are preferably purified to be substantially free of contaminating RNA molecules that could activate an unwanted response and decrease single-stranded mRNA expression and / or activate RNA sensors in cells. In certain embodiments, purified RNA preparations are preferably substantially free of contaminating RNA molecules which are: smaller or larger than single-stranded mRNA molecules, full-length, double-stranded and / or uncapped RNA.
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FUNDAMENTALS
In 2006, it was reported (Takahashi and Yamanaka 2006) that the introduction of genes encoding four protein factors (OCT4 (Octamer-4; POU class 5 homeobox 1), SOX2 (SRY (sex-determining region Y) -box 2) , KLF4 (Krueppel type factor 4), and cMYC) in differentiated somatic cells from mice induced those cells to become pluripotent stem cells, (referred to here as “induced pluripotent stem cells,” “iPS cells,” or “iPSCs”) . After this original report, pluripotent stem cells were further induced by transforming human somatic cells with genes encoding similar human protein factors (OCT4, SOX2, KLF4, and c-MYC) (Takahashi et al. 2007), or transforming somatic human cells with genes encoding human factors OCT4 and SOX2 plus genes encoding two other human factors, NANOG and LIN28 (Lin-28 homologue A) (Yu et al. 2007). All of these methods used retroviruses or lentiviruses to integrate the genes encoding the reprogramming factors into transformed cell genomes, and somatic cells were reprogrammed into iPS cells only for a long period of time (for example, a week).
The generation of iPS cells from differentiated somatic cells offers great promise as a possible means of treating diseases through cell transplantation. The possibility of generating iPS cells from somatic cells from individual patients may also allow the development of patient-specific therapies with less risk due to immune rejection. In addition, the generation of iPS cells from disease-specific somatic cells offers promise as a means to study and develop drugs to treat specific disease states (Ebert et al. 2009, Lee et al. 2009, Maehr et al. 2009) .
Viral release of genes encoding protein reprogramming factors (or “iPSC factors”) provides a highly efficient way to create iPS cells from somatic cells, but the integration of exogenous DNA into the genome, whether random or non-random, creates unpredictable results and can ultimately lead to cancer (Nakagawa et al. 2008). New reports show that iPS cells can be created (at lower efficiency) using other methods that do not require genome integration. For example, repeated transfections of
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3/141 expression plasmids containing genes for OCT4, SOX2, KLF4 and c-MYC in mouse embryonic fibroblasts to generate iPS cells have been demonstrated (Okita et al. 2008). The induced pluripotent stem cells were also generated from human somatic cells by the introduction of a plasmid that expressed genes encoding human OCT4, SOX2, c-MYC, KLF4, NANOG and LIN28 (Yu et al. 2009). Other successful approaches to generating iPS cells include treating somatic cells with: recombinant protein reprogramming factors (Zhou et al. 2009); non-integrating adenoviruses (Stadtfeld et al. 2008); or piggyBac transposons (Woltjen et al. 2009) to release reprogramming factors. Currently, the generation of iPS cells using these nonviral release techniques to release reprogramming factors is extremely inefficient. Future methods for generating iPS cells for potential clinical applications need to increase the speed and efficiency of iPS cell formation, while maintaining the integrity of the genome.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods for reprogramming the differentiating state of eukaryotic cells, including human cells or other animal cells, by contacting the cells with purified RNA preparations that comprise or consist of one or more different single-stranded mRNA molecules that each encodes a reprogramming factor (for example, an iPS cell induction factor). The purified single-stranded mRNA molecules preferably comprise at least one modified nucleoside (for example, selected from the group consisting of a pseudouridine (Ψ), ^5- methylcytosine (m ⁵ C), 5-methyluridine (m ⁵ U), 2'- O-methyluridine (Um or m ² ' ^-O U), 2-thiouridine (s ² U), and N6-methyladenosine (m ⁶ A)) in place of at least a part (for example, including substantially all) of the nucleosides corresponding unmodified canonicals of the corresponding unmodified canonical nucleosides A, C, G or T. In addition, single-stranded mRNA molecules are preferably purified to be substantially free of contaminating RNA molecules that could activate an unwanted response, decrease the expression of single-stranded mRNA and / or activate RNA sensors in cells (for example, double-stranded RNA-dependent enzymes) in cells. In certain embodiments, purified RNA preparations are preferably substantially free of contaminating RNA molecules which are: smaller or larger than single-stranded mRNA molecules, full-length, double-stranded or
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4/141 and / or uncapped RNA. In some preferred embodiments, the invention provides compositions and methods for reprogramming differentiated eukaryotic cells, including human or other somatic cells, by contacting the cells with purified RNA preparations comprising or consisting of one or more different single-stranded mRNA molecules each encodes an iPS cell induction factor.
In some embodiments, the present invention provides methods for changing the differentiating state of a somatic cell comprising: introducing an mRNA that encodes an iPS cell induction factor into a somatic cell to generate a reprogrammed differentiated cell, wherein the mRNA comprises at least one 5-methylcytidine (or others modified based on the description here).
In certain embodiments, the present invention provides methods for reprogramming a human cell that has a first differentiated state or phenotype into a cell that has a second differentiated state or phenotype comprising: introducing into the cell that exhibits a first differentiated state an RNA preparation comprising molecules of purified mRNA that encode at least one reprogramming factor and cell culture under conditions where the cell exhibits a second differentiated state. In certain embodiments, the modified mRNA molecules contain at least one modified nucleoside selected from the group consisting of pseudouridine or 5-methylcytidine. In certain modalities, the cell is a human or animal. In other embodiments, the purified RNA preparation: i) comprises first single-stranded mRNAs that encode a first iPS cell induction factor, wherein substantially all of the first complete single-stranded mRNAs comprise at least one pseudouridine residue and / or at least one 5-methylcytidine residue, and ii) is substantially free of contaminating RNA molecules that are capable of activating RNA sensors in said somatic cell. In certain embodiments, the contaminating RNA molecules are selected from the group consisting of: partial mRNAs encoding only a part of said iPS cell induction factor, RNA molecules that are smaller than full-length mRNA, RNA molecules which are larger than full-length mRNA, double-stranded mRNA molecules and molecules of one-capped mRNA molecules.
In some embodiments, the present invention provides methods for reprogramming a somatic cell (for example, de-differentiate or trans-differentiate) comprising: contacting
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5/141 a somatic cell with a purified RNA preparation to generate a reprogrammed cell, in which the purified RNA preparation: i) comprises first single-stranded mRNAs that encode a first iPS cell induction factor, in which substantially all first complete single-stranded mRNAs comprise at least one pseudouridine residue and / or at least one 5-methylcytidine residue, and ii) is substantially free of contaminating molecules (eg, contaminating RNA molecules) that are capable of activating the sensors of RNA in the somatic cell. In particular embodiments, the contaminating RNA molecules comprise: partial mRNAs that encode only a part of the iPS cell induction factor, single run-on strand mRNAs that encode the iPS cell induction factor and encode at least an additional part of iPS cell induction factor, double-stranded mRNA molecules, and un-capped mRNA molecules. In certain embodiments, the first single-stranded mRNAs do not yet encode an additional part of the first iPS cell induction factor.
In some embodiments, the reprogrammed cell is a de-differentiated cell (for example, stem cell or stem cell type cell). In other embodiments, the reprogrammed cell is a transdifferentiated cell (for example, a skin cell is reprogrammed into a neuronal cell, or other type of change). In other embodiments, the first single-stranded mRNAs encode the first complete iPS induction factors (for example, the mRNA encodes the entire coding sequence for a particular iPS induction factor). In other modalities, the other contact comprises contacting the somatic cell with a growth factor and / or cytokine (for example, after a period of time). In other embodiments, the other contact comprises contacting the somatic cell with an immune response inhibitor.
In certain embodiments, all or almost all of the uridine nucleosides in the first single stranded mRNA are replaced by pseudouridine nucleosides. In other embodiments, all or almost all of the cytidine nucleosides in the first single-stranded mRNA are replaced by 5-methylcytidine nucleosides or another base mentioned here.
In particular embodiments, the present invention provides methods for generating a reprogrammed cell comprising: contacting a somatic cell with a purified RNA preparation to generate a reprogrammed cell that is capable of surviving in culture for at least 10 days (for example, at least 10 days, at least 13 days, at least 16
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6/141 days, at least 20 days, at least 40 days or is capable of forming a cell line), where the purified RNA preparation comprises single stranded first mRNAs that encode an iPS cell induction factor, and where most early single-stranded mRNAs comprise at least one pseudouridine residue and / or at least one 5-methylcystidine residue.
In certain embodiments, the purified RNA preparation is free of a quantity of contaminating RNA molecules that could activate an immune response in the somatic cell sufficient to prevent a reprogrammed cell from surviving at least 10 days in culture (for example, at least 10 days , at least 15 days, at least 20 days, at least 40 days, or more). In other embodiments, contaminating RNA molecules include: partial mRNAs that encode only a part of the iPS cell induction factor, single run-on strand mRNAs that encode the iPS cell induction factor and encode at least an additional part of iPS cell induction factor, double-stranded mRNA molecules, and un-capped mRNA molecules and mixtures thereof. In certain embodiments, the reprogrammed cell that is generated is able to form a reprogrammed cell line. In other embodiments, the preparation of purified RNA and free of an amount of contaminating RNA molecules that could activate an immune response in the somatic cell sufficient to prevent the generation of the reprogrammed cell line.
In particular embodiments, the contaminating RNA molecules are selected from the group consisting of: partial mRNAs that encode only a part of the iPS cell induction factor, single-stranded run-on mRNAs that encode the iPS cell induction factor and encode at least an additional part of the iPS cell induction factor, double-stranded mRNA molecules, and un-capped mRNA molecules and mixtures thereof.
In some embodiments, the present invention provides methods for generating reprogrammed cell line comprising: a) contacting a somatic cell with a purified RNA preparation to generate a reprogrammed cell, wherein a purified RNA preparation comprises mRNAs encoding an induction factor of iPS cell, and where most mRNAs comprise at least one pseudouridine residue and / or at least one 5-methylcystidine residue and, b) culturing the de-differentiated cell to generate a reprogrammed cell line. In other embodiments, the preparation of purified RNA is free of an amount of contaminating molecules that could activate an immune response in the
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7/141 enough somatic cell to prevent generation of the reprogrammed cell line. In certain embodiments, the immune response involves activation of RNA sensors in the somatic cell.
In some embodiments, the present invention provides methods for reprogramming a somatic cell comprising: contacting a somatic cell with a purified RNA preparation to generate a reprogrammed cell, wherein the purified RNA preparation: i) comprises first single-stranded encoding mRNAs a first iPS cell induction factor, in which substantially all of the first single-stranded mRNAs comprise at least one pseudouridine residue and / or at least one 5-methylcystidine residue, and ii) is substantially free of: a) partial mRNAs that they encode only a portion of the first iPS cell induction factor and b) double-stranded mRNA molecules. In other embodiments, the first single-stranded mRNA does not yet encode an additional part of the first iPS cell induction factor. In particular embodiments, the first single-stranded mRNA completely encodes the first iPS cell induction factor. In other embodiments, the purified RNA preparation is still substantially free (or essentially free or virtually free or free) of single-stranded run-on mRNAs encoding the first iPS cell induction factor and encoding at least an additional portion of the first iPS cell induction factor. In other embodiments, substantially all of the first complete single stranded mRNAs are 5 'capped. In other embodiments, the purified RNA preparation is still substantially free of uncapped mRNA molecules. In some embodiments, substantially all of the first single-stranded mRNAs comprise at least one pseudouridine residue. In additional embodiments, substantially all of the first single stranded mRNAs comprise at least one 5-methylcystidine residue. In other embodiments, substantially all of the first single-stranded mRNAs comprise at least one pseudouridine residue and at least one 5-methylcytidine residue.
In certain embodiments, the purified RNA preparation comprises a transfection reagent. In other embodiments, the preparation of purified RNA is obtained by HPLC purification of an RNA sample that contains a substantial amount of the partial mRNAs and the double-stranded mRNAs. In other embodiments, the preparation of purified RNA is essentially free of partial mRNAs and single-stranded run-on mRNAs. In some
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8/141 embodiments, the purified RNA preparation is essentially free or virtually free or free of double-stranded mRNA molecules. In other embodiments, the purified RNA preparation is essentially free or virtually free of uncapped mRNA molecules. In some embodiments, substantially all of the first single stranded mRNAs are polyadenylated. In other embodiments, single-stranded complete mRNAs are capped with 7-methylguanosine.
In some embodiments, the first iPS cell induction factor is selected from the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2. In other embodiments, the purified RNA preparation: i) further comprises single-stranded mRNAs that encode a second iPS cell induction factor, wherein the second single-stranded mRNAs comprise at least one pseudouridine residue and / or at least one 5-methylcystidine residue, and ii) is still substantially free of: a) partial mRNAs that encode only a part of the second iPS cell induction factor, and b) double-stranded mRNAs. In other embodiments, the purified RNA preparation is still substantially free of single-stranded run-on mRNAs that encode a second iPS cell induction factor and encode at least an additional part of the second iPS cell induction factor. In some embodiments, the second iPS cell induction factor is selected from the group consisting of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2. In certain embodiments, the somatic cell is a fibroblast. In other modalities, the reprogrammed cell is a pluripotent trunk cell. In other modalities, the differentiated cell expresses NANOG and TRA-160. In some embodiments, the cell is in vitro. In other modalities, the cell resides in culture. In particular modalities, the cell resides in MEF conditioned medium.
In some embodiments, the present invention provides compositions comprising a purified RNA preparation, wherein the purified RNA preparation: i) comprises first single stranded mRNAs that encode a first iPS cell induction factor, wherein the first stranded mRNAs simple comprise at least one pseudouridine residue and / or at least one 5-methylcystidine residue, and ii) it is substantially free of contaminating RNA molecules, which are capable of activating RNA sensors in a somatic cell. In certain embodiments, the present invention provides compositions comprising a purified RNA preparation, wherein the purified RNA preparation: i) comprises first single-stranded mRNAs that encode a first induction factor
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9/141 of iPS cell, wherein single stranded full-length mRNAs comprise at least one pseudouridine residue and / or at least one 5-methylcystidine residue, and ii) is substantially free of: a) partial mRNAs that encode only one part of the first iPS cell induction factor, and b) double-stranded RNA.
In certain embodiments, the purified RNA preparation is still substantially free of single-stranded run-on mRNAs encoding the first iPS cell induction factor and encoding at least an additional part of the first iPS cell induction factor. In some embodiments, the purified RNA preparation: i) further comprises second single stranded mRNAs encoding a second iPS cell induction factor, wherein the second complete single stranded mRNAs comprise at least one pseudouridine residue and / or at least minus a 5-methylcystidine residue, and ii) is substantially free of: a) partial mRNAs that encode only a portion of the second iPS cell induction factor, and b) single-stranded run-on mRNAs that encode second first induction factor iPS cell and encodes at least an additional part of the second iPS cell induction factor.
In some embodiments, the present invention provides compositions comprising an in vitro synthesized mRNA that encodes the MYC gene, wherein the in vitro synthesized mRNA comprises at least one pseudouridine residue and / or at least one 5-methylcystidine residue. In certain embodiments, the compositions are substantially free of contaminating RNA molecules that are capable of activating RNA sensors in a somatic cell.
In particular embodiments, the present invention provides methods for inducing a mammalian cell to produce the MYC protein comprising: contacting a mammalian cell with an in vitro synthesized mRNA encoding the MYC gene, wherein the in vitro synthesized mRNA comprises at least one residue of pseudouridine and / or at least one 5-methylcystidine residue, thus inducing the mammalian cell to produce the MYC protein. In other embodiments, the mammalian cell is a dendritic cell. In other embodiments, the mammalian cell is an alveolar cell, an astrocyte, a microglia cell, or a neuron.
In some embodiments, the present invention provides methods of treating a subject comprising contacting a subject with the MYC protein that produces the mammalian cell described above and here.
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In additional embodiments, the present invention provides methods of synthesizing an in vitro transcribed RNA molecule encoding the MYC gene comprising: combining an isolated RNA polymerase, a template nucleic acid sequence encoding the MYG gene, unmodified nucleotides, and pseudouridine nucleotides or 5-methylcystidine modified under conditions such that an in vitro RNA molecule encoding the MYC gene is generated that comprises at least one residue and pseudouridine or 5-methylcystidine.
Experiments conducted during the development of modalities of the present invention demonstrated that mRNA molecules can be administered to cells and induce a process of de-differentiation to generate de-differentiated cells - including pluripotent stem cells. Thus, the present invention provides compositions and methods for generating iPS cells. Surprisingly, mRNA administration can provide highly efficient generations of iPS cells.
The present invention further provides RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside, gene therapy vectors comprising them, methods of synthesizing them, and methods for gene replacement, gene therapy, gene silencing transcription, and the release of therapeutic proteins into tissue in vivo, comprising the molecules. The present invention further provides methods of reducing the immunogenicity of RNA, oligoribonucleotide, and polyribonucleotide molecules.
In some embodiments, the present invention provides methods for de-differentiating a somatic cell comprising: introducing mRNA that encodes one or more iPSC-inducing factors into a somatic cell to generate a de-differentiated cell.
In some embodiments, the present invention provides methods for de-differentiating a somatic cell comprising: introducing mRNA that encodes one or more iPSC inducing factors into a somatic cell and maintaining the cell under conditions where the cell is viable and the mRNA being introduced in the cell it is translated in sufficient quantity and for sufficient time to generate a differentiated cell. In some preferred embodiments, the de-differentiated cell is an induced pluripotent stem cell (iPSC).
In some embodiments, the present invention provides methods for altering the state of de-differentiation (or de-differentiated state) of a eukaryotic cell comprising: introducing mRNA that encodes one or more reprogramming factors into a cell and maintaining
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11/141 the cell in conditions where the cell is viable and the mRNA that is introduced into the cell is translated in sufficient quantity and for sufficient time to generate a cell that presents an altered state of differentiation compared to the cell in which the mRNA was introduced .
In some embodiments, the present invention provides methods for altering the undifferentiated state of a eukaryotic cell comprising: introducing mRNA that encodes one or more reprogramming factors into a cell and maintaining the cell in conditions where the cell is viable and the mRNA that is introduced into the cell is translated in sufficient quantity and for sufficient time to generate a cell that presents an altered state of differentiation compared to the cell in which the mRNA was introduced. In some embodiments, the altered state of differentiation is a differentiated state of differentiation compared to the cell into which the mRNA was introduced. For example, in some embodiments, the cell that has the altered state of differentiation is a pluripotent stem cell that is differentiated compared to a somatic cell into which the mRNA was introduced (for example, a somatic cell that is differentiated into a fibroblast, cardiomyocyte, or other differentiated cell type). In some embodiments, the cell into which the mRNA is introduced is a somatic cell of a lineage, phenotype, or function, and the cell that has the altered state of differentiation is a somatic cell that has a different lineage, phenotype, or function than the cell in which the mRNA was introduced; thus, in these modalities, the method results in transdifferentiation (Graf and Enver 2009).
The methods of the invention are not limited with respect to a particular cell in which the mRNA is introduced. In some embodiments of any of the above methods, the cell into which the mRNA is introduced is derived from any multicellular eukaryote. In some embodiments of any of the above methods, the cell into which the mRNA is introduced is selected from a human cell and an animal cell. Although the work presented here was carried out using cells from humans or other animals, applicants still claim that the methods of the present invention comprising reprogramming human and animal cells by contacting the cells with a purified RNA preparation consisting of one or more mRNA molecules purified single-stranded cells, each of which encodes a protein reprogramming factor (for example, a transcription factor) still belongs to the reprogramming of other eukaryotic cells (for example, plant cells and fungal cells). In some modalities of any of the above methods, the cell in
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12/141 that the mRNA is introduced is a normal cell that is from an organism that is free of a known disease. In some embodiments of any of the above methods, the cell into which the mRNA is introduced is a cell in an organism that has a known disease. In some embodiments of any of the above methods, the cell into which the mRNA is introduced is a cell that is free of a known pathology. In some embodiments of any of the above methods, the cell into which the mRNA is introduced is a cell that has a known disease state or pathology (for example, a cancer cell, or a pancreatic beta cell that has characteristic metabolic properties diabetic cell).
The invention is not limited to the use of a specific cell type (for example, a specific somatic cell type) in modalities of methods comprising introducing mRNA that encodes one or more iPSC cell inducing factors to generate a de-differentiated cell (for example, example an iPS cell). Any cell that is subject to de-differentiation using iPS cell induction factors is contemplated. Said cells include, among others, fibroblasts, keratinocytes, adipocytes, lymphocytes, T cells, B cells, mononuclear cells in cord blood, oral mucosa cells, liver cells, HeLa, MCF-7 or other cancer cells. In some embodiments, the cells reside in vitro (for example, in culture) or in vivo. In some embodiments, when generated in culture, a cell-free conditioned medium (for example, MEF conditioned medium) is used. As shown below, this means providing improved generation of iPS cells. The invention is not limited, however, to the culture conditions used. Any culture condition or medium now known or subsequently identified as useful for the methods of the invention (for example, for generating iPS cells from somatic cells and maintaining said cells) is contemplated for use with the invention. For example, although not preferred, in some embodiments of the method, a layer of feeder cell is used instead of conditioned medium to grow the cells that are treated using the method.
In some embodiments of any of these methods, the step of introducing mRNA comprises releasing the mRNA into the cell (for example, a human cell or other animal somatic cell) with a transfection reagent (for example, TRANSIT ™ mRNA transfection reagent, MirusBio, Madison, WI).
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However, the invention is not limited by the nature of the transfection method used. In fact, any known or identified future transfection process that is capable of releasing mRNA molecules in cells in vitro or in vivo, is contemplated, including methods that release mRNA in cells in culture or in a life support medium. , whether said cells comprise isolated cells or cells comprising a eukaryotic tissue or organ, or methods that release mRNA in vivo into cells in an organism, such as a human, animal, plant or fungus. In some embodiments, the transfection reagent comprises a lipid (for example, liposomes, micelles, etc.). In some embodiments, the transfection reagent comprises a nanoparticle or nanotube. In some embodiments, the transfection reagent comprises a cationic compound (for example, polyethylene imine or PEI). In some embodiments, the transfection method uses an electric current to release mRNA into the cell (for example, by electroporation). In some embodiments, the transfection method uses a ballistic method to release mRNA into the cell (for example, a “gene gun” or biolistic particle delivery system.)
The data presented here show that, with respect to the mRNA introduced into the cell, certain amounts of mRNAs used in the EXAMPLES described here resulted in greater efficiency and faster induction of pluripotent stem cells from particular somatic cells used than other amounts of mRNA . However, the methods of the present invention are not limited to using a specific amount of mRNA to introduce it into the cell. For example, in some embodiments, a total of three doses, each dose comprising 18 micrograms of each of the six different mRNAs, each encoding a different reprogramming human factor, was used to introduce the mRNA into approximately 3 x 10 ⁵ cells of human fibroblasts in a 10 cm plate (for example, released using a transfection reagent containing lipid), although in other embodiments, greater or lesser amounts of mRNAs were used to introduce into cells.
The invention is not limited to a particular chemical form of the mRNA used, although certain forms of mRNA can produce more efficient results. However, in some preferred embodiments, the mRNA comprises at least one modified nucleoside (for example, selected from the group consisting of a pseudouridine (Ψ), 5-methylcytosine (m ⁵ C), 5-methyluridine (m ⁵ U) , 2'-O-methyluridine (Um or m ² '° U), 2-thiouridine (s ² U), and N ⁶ Petition 870180166239, of 12/21/2018, page 32/167
14/141 methyladenosine (m ⁶ A)) in place of at least part of the corresponding unmodified canonical nucleoside (for example, in some preferred embodiments, at least one modified nucleoside in place of substantially all of the corresponding unmodified canonical nucleosides A, C, G, or T). In some embodiments, the mRNA is polyadenylated. In some preferred embodiments, mRNA is prepared by polyadenylation of an in vitro transcribed RNA (IVT), the method comprising contacting the IVT RNA using a poly (A) polymerase (for example, yeast RNA polymerase or poly (A) polymerase) E. coli). In some embodiments, the mRNA is polyadenylated during IVT using a DNA template that encodes the poly (A) tail. Regardless of whether the RNA is polyadenylated using a poly (A) polymerase or during IVT of a DNA template, in some preferred embodiments, the mRNA comprises a poly-A tail (for example, a poly-A tail containing 50-200 nucleotides , for example, preferably 100-200, 150-200 nucleotides, or more than 150 nucleotides), although in some embodiments, a longer or shorter poly-A tail is used. In some embodiments, the mRNA used in the methods is capped. To maximize the efficiency of expression in cells, it is preferable that most mRNA molecules contain a skin. In some preferred embodiments, the mRNA molecules used in the methods are synthesized in vitro by incubating the uncapped primary RNA in the presence of capping enzyme. In some preferred embodiments, the primary RNA used in the capping enzyme reaction is synthesized by in vitro transcription (IVT) of a DNA molecule that encodes the RNA to be synthesized. The DNA encoding the RNA to be synthesized contains an RNA polymerase promoter, to which an RNA polymerase binds and initiates its transcription. It is also known in the art that mRNA molecules generally contain regions of different sequence located before the translation start codon and after the translation stop codon that are not translated. These regions, called five untranslated prime regions (5 'UTR) and three prime untranslated regions (3' UTR), respectively, can affect the stability of the mRNA, the location of the mRNA, and the efficiency of the translation of the mRNA to which are united. Certain 5 'and 3' RTUs, such as those for alpha and beta globins are known to improve mRNA stability and mRNA expression. Thus, in some preferred modalities, the mRNAs that encode the reprogramming factors (for example, iPSC induction factors) present 5 'RTU and / or 3' RTU which results in greater
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15/141 mRNA stability and increased mRNA expression in cells (for example, an alpha globin or a beta globin 5 'UTR and / or 3' UTR; for example, a Xenopus or human alpha globin or a beta globin 5 'UTR and / or 3 'UTR, or, for example, tobacco etch virus (TEV) 5' UTR).
IVT can be performed using any RNA polymerase as long as the synthesis of mRNA from the DNA template encoding the RNA is specifically and sufficiently initiated from a respective cognate RNA polymerase promoter and full length mRNA is obtained. In some preferred embodiments, RNA polymerase is selected from T7 RNA polymerase, SP6 RNA polymerase and T3 RNA polymerase. In some other modalities, capped RNA is synthesized co-transcribed using a kappa dinucleotide analog in the IVT reaction (for example, using an AMPLICAP ™ T7 kit or a MESSAGEMAX ™ T7 ARCACAPPED MESSAGE transcription kit; EPICENTRE or CellScript, Madison, WI, USA) . If capping is performed co-transcribed, preferably the kappa dinucleotide analogue is an anti-reverse kappa analogue (ARCA). However, the use of a separate IVT reaction, followed by capping with a capping enzyme system, which results in approximately 100% of the RNA being capped, super cotranscriptional capping is preferred, which typically results in only about 80% of RNA being capped. Thus, in some preferred embodiments, a high percentage of mRNA molecules used in a method of the present invention are capped (for example, more than 80%, more than 90%, more than 95%, more than 98% , more than 99%, more than 99.5%, or more than 99.9% of the population of mRNA molecules are capped). In some preferred embodiments, the mRNA used in the methods of the present invention has a cap with a cap1 structure, meaning that 2 'hydroxyl of the ribose in the penultimate nucleotide with respect to the cap nucleotide is methylated. However, in some embodiments, mRNA used in the methods has a cap with the cap0 structure, meaning that the 2 'hydroxyl of ribose in the penultimate nucleotide with respect to the cap nucleotide is not methylated. With some, but not all transcripts, transfection of eukaryotic cells with mRNA containing a cap with a cap1 structure results in a higher level or longer duration of protein expression in the transfected cells compared to transfection of the same cells with the same mRNA but with a cover containing a cap0 structure. In some modalities, the mRNA used in
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16/141 methods of the present invention have a modified kappa nucleotide. In some experiments carried out before the experiments presented in the EXAMPLES here, the present applicants demonstrated that when 1079 or IMR90 human fibroblast cells were transfected with OCT4 mRNA that contained uridine, or pseudouridine in place of uridine, the mRNA containing pseudouridine was translated into a higher level or for a longer duration than the mRNA containing uridine. Therefore, in some preferred embodiments, one or more of all uridines contained in the mRNAs used in the methods of the present invention are replaced by pseudouridine (for example, replacing pseudouridine-5'-triphosphate in the IVT reaction to synthesize RNA in place of uridine- 5'triphosphate). However, in some embodiments, the mRNA used in the methods of the invention contains uridine and does not contain pseudouridine. In some preferred embodiments, the mRNA comprises at least one modified nucleoside (for example, selected from the group consisting of a pseudouridine (Ψ), 5-methylcytosine (m ⁵ C), 5-methyluridine (m ⁵ U), 2'- Omethyluridine (Um or m ² '° U), 2-thiouridine (s ² U), and N ⁶ -methyladenosine (m ⁶ A)) in place of at least a part of the corresponding unmodified canonical nucleoside (for example, in place of substantially all the corresponding unmodified canonical nucleosides A, C, G or T). In some preferred embodiments, the mRNA comprises at least one modified nucleoside selected from the group consisting of a pseudouridine (Ψ) and ⁵ methylcytosine (m ⁵ C). In some preferred embodiments, the mRNA comprises both pseudouridine (Ψ) and 5-methylcytosine (m ⁵ C). In addition, to achieve specific objectives, a nucleic acid base, sugar fraction, or internucleotide bond in one or more of the mRNA nucleotides that is introduced into a eukaryotic cell in any of the methods of the invention can comprise a nucleic acid base modified sugar fraction, or internucleotide binding.
The invention is also not limited with respect to the source of the mRNA that is released in the eukaryotic cell in any of the methods of the invention. In some embodiments, such as those described in EXAMPLES, mRNA is synthesized by an in vitro transcription of a DNA model comprising a gene cloned into a linearized plasmid vector or by an in vitro transcription of a DNA template that is synthesized by PCR or RT-PCR (ie, by IVT of a PCR amplification product), capping using a capping enzyme system, or by co-transcription capping by incorporating a capping analog
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17/141 dinucleotide (for example, an ARCA) during IVT, and polyadenylation using a poly (A) polymerase. In some preferred embodiments, the mRNA is synthesized by IVT from a DNA template comprising a gene cloned into a linearized plasmid vector or a PCR or RT-PCR amplification product, where the DNA template encodes a 3'poli tail (THE). In some other embodiments, the mRNA that is released into the eukaryotic cell in either method of the invention is derived directly from a cell or a biological sample. For example, in some embodiments, mRNA derived from a cell or biological sample is obtained by amplifying the cell's mRNA or biological sample using an RNA amplification reaction, and capping the amplified mRNA using a capping enzyme system or by capping co-transcriptional by incorporating a dinucleotide cover analogue (for example, an ARCA) during IVT, and, if the amplified mRNA does not yet contain a template-coded poly (A) tail, the RNA amplification reaction, polyadenylating the mRNA amplified using a poly (A) polymerase.
With respect to methods comprising introducing mRNA that encodes one or more iPS cell induction factors to generate a differentiated cell (e.g., an iPS cell), the invention is not limited by the nature of the iPS cell induction factors used. Any mRNA encoding one or more protein induction factors now known, or later discovered, which find use in de-differentiation, are contemplated for use in the present invention. In some embodiments, one or more mRNAs that code for KLF4, LIN28, c-MYC, NANOG, OCT4, or SOX2 are employed. Oct3 / 4 and certain elements of the Sox gene family (Sox1, Sox2, Sox3, and Sox15) have been identified as transcriptional regulators involved in the induction process. Additional genes, however, including certain elements of the Klf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (C-myc, L-myc, and N-myc), Nanog, and LIN28, were identified for increasing induction efficiency. Any or more of such factors can be used as desired.
While the compositions and methods of the invention can be used to generate iPS cells, the invention is not limited to the generation of these cells. For example, in some embodiments, mRNA that encodes one or more reprogramming factors is introduced into the cell to generate a cell with an altered state of differentiation compared to the cell in
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18/141 that the mRNA was introduced. For example, in some embodiments, mRNA that encodes one or more iPS cell induction factors is used to generate a differentiated cell that is not an iPS cell. These cells find use in research, drug screening, and other applications.
In some embodiments, the present invention further provides methods for employing the de-differentiated cells generated by the above methods. For example, said cells find use in research, drug screening, and therapeutic applications in humans or other animals. For example, in some modalities, the generated cells find use in the identification and characterization of iPS cell induction factors as well as other factors associated with differentiation or de-differentiation. In some embodiments, the generated differentiated cells are transplanted into an organism or tissue that resides in vitro or in vivo. In some embodiments, an organism, tissue or culture system that houses the generated cells is exposed to a test compound and the effect of the test compound on the cells or on the organism, tissue or culture system is observed or measured.
In some other embodiments, a de-differentiated cell generated using the above methods (for example, an iPS cell) is further treated to generate a de-differentiated cell that has the same state of differentiation or cell type compared to a somatic cell from which the de-differentiated cell was generated. In some other embodiments, a de-differentiated cell generated using the above methods (for example, an iPS cell) is further treated to generate a de-differentiated cell that has the same state of differentiation or cell type compared to a somatic cell from which the de-differentiated cell was generated. In some embodiments, the differentiated cell is generated from the de-differentiated cell (for example, the generated iPS cell) by introducing mRNA that encodes one or more reprogramming factors in the iPS cell generated during one or more treatments and keeping the cell in which the mRNA is introduced under conditions where the cell is viable and is differentiated into the cell that has the altered state of differentiation or cell type compared to the generated de-differentiated cell (for example, the generated iPS cell) in which the mRNA encoding one or more reprogramming factors is introduced. In some of these modalities, the generated differentiated cell that has the altered state of differentiation is used for research, drug screening, or therapeutic applications (for example, in humans or other animals). For example, the differentiated cells generated find use in the identification and characterization of
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19/141 reprogramming factors associated with differentiation. In some embodiments, the differentiated cells generated are transplanted into an organism or tissue that resides in vitro or in vivo. In some embodiments, an organism, tissue or culture system that houses the generated cells is exposed to a test compound and the effect of the test compound on the cells or on the organism, tissue or culture system is observed or measured. In some preferred embodiments of the method comprising introducing mRNA that encodes one or more iPSC-inducing factors into a somatic cell and maintaining the cell in conditions where the cell is viable and the mRNA that is introduced into the cell is expressed in sufficient quantity and by enough time to generate a de-differentiated cell (for example, where the de-differentiated cell is an induced pluripotent stem cell), enough time to generate a de-differentiated cell is less than a week. In some preferred embodiments of this method, the reprogramming efficiency for generating de-differentiated cells is more than or equal to 50 de-differentiated cells (e.g., iPSCs) per 3 x 10 ⁵ input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating de-differentiated cells is more than or equal to 100 de-differentiated cells (e.g., iPSCs) per 3 x 10 ⁵ input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating de-differentiated cells is more than or equal to 150 de-differentiated cells (e.g., iPSCs) per 3 x 10 ⁵ input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating de-differentiated cells is more than or equal to 200 de-differentiated cells (e.g., iPSCs) per 3 x 10 ⁵ input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating de-differentiated cells is more than or equal to 300 de-differentiated cells (e.g., iPSCs) per 3 x 10 ⁵ input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating de-differentiated cells is more than or equal to 400 de-differentiated cells (e.g., iPSCs) per 3 x 10 ⁵ input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating de-differentiated cells is more than or equal to 500 de-differentiated cells (for example, iPSCs)
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20/141 by 3 x 10 ⁵ input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating de-differentiated cells is more than or equal to 600 de-differentiated cells (eg, iPSCs) per 3 x 10 ⁵ input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating de-differentiated cells is more than or equal to 700 de-differentiated cells (e.g., iPSCs) per 3 x 10 ⁵ input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating de-differentiated cells is more than or equal to 800 de-differentiated cells (e.g., iPSCs) per 3 x 10 ⁵ input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating de-differentiated cells is more than or equal to 900 de-differentiated cells (e.g., iPSCs) per 3 x 10 ⁵ input cells into which the mRNA is introduced. In some preferred embodiments of this method, the reprogramming efficiency for generating de-differentiated cells is more than or equal to 1000 de-differentiated cells (e.g., iPSCs) per 3 x 10 ⁵ input cells into which the mRNA is introduced. Thus, in some preferred modalities, this method was more than 2 times more efficient than the published protocol comprising the release of reprogramming factors with a viral vector (for example, a lentivirus vector). In some preferred modalities, this method was more than 5 times more efficient than the published protocol comprising releasing factors from reprogramming with a viral vector (for example, a lentivirus vector). In some preferred embodiments, this method was more than 10 times more efficient than the published protocol comprising releasing factors from reprogramming with a viral vector (eg, a lentivirus vector). In some preferred embodiments, this method was more than 20 times more efficient than the published protocol comprising releasing reprogramming factors with a viral vector (eg, a lentivirus vector). In some preferred embodiments, this method was more than 25 times more efficient than the published protocol comprising releasing factors from reprogramming with a viral vector (eg, a lentivirus vector). In some preferred embodiments, this method was more than 30 times more efficient than the published protocol comprising releasing factors from reprogramming with a viral vector (eg, a lentivirus vector).
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In some preferred embodiments, this method was more than 35 times more efficient than the published protocol comprising releasing factors from reprogramming with a viral vector (eg, a lentivirus vector). In some preferred embodiments, this method was more than 40 times more efficient than the published protocol comprising releasing factors from reprogramming with a viral vector (eg, a lentivirus vector).
The present invention further provides compositions (systems, kits, reaction mixtures, cells, mRNA) used or useful in the methods and / or generated by the methods described here. For example, in some embodiments, the present invention provides an mRNA that encodes an iPS cell inducing factor, the pseudouridine-containing mRNA in place of uridine.
The present invention further provides compositions comprising a transfection reagent and an mRNA that encodes an iPS cell induction factor (for example, a mixture of transfection reagent and mRNA). In some embodiments, the compositions comprise mRNA that encodes a plurality (for example, 2 or more, 3 or more, 4 or more, 5 or more, or 6) of iPS cell inducing factors, including but not limited to KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.
The compositions may further comprise any other reagent or component sufficient, necessary or useful for the practice of any of the methods described herein. Said reagents or components include, but are not limited to, transfection reagents, culture medium (for example, MEF condition medium), cells (for example, somatic cells, iPS cells), containers, boxes, buffers, inhibitors (for example, inhibitors RNase), markers (e.g. fluorescent, luminescent, radioactive, etc.), positive and / or negative control molecules, reagents to generate capped mRNA, dry ice or other refrigerants, instructions for use, cell culture equipment, detection / analysis equipment, and the like.
This invention provides RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside, gene therapy vectors comprising the same, methods of gene therapy and methods of silencing transcription genes comprising the same, methods of reducing their immunogenicity, and methods of synthesizing them.
In one embodiment, the present invention provides a messenger RNA comprising a pseudouridine residue. In another embodiment, the present invention provides an RNA molecule that encodes a protein of interest, said RNA molecule
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22/141 comprising a pseudouridine residue. In another embodiment, the present invention provides an RNA molecule transcribed in vitro, comprising pseudouridine or a modified nucleoside. In another embodiment, the present invention provides an oligoribonucleotide synthesized in vitro, comprising a pseudouridine or a modified nucleoside, wherein the modified nucleoside is m ⁵ C, m ⁵ U, m ⁶ A, s ² U, Ψ, or 2'- Omethyl-U. In another embodiment, the present invention provides a gene therapy vector, comprising a polynucleotide molecule synthesized in vitro, wherein the polyribonucleotide molecule comprises a pseudouridine or a modified nucleoside.
In another embodiment, the present invention provides a double-stranded RNA (dsRNA) molecule containing, as part of its sequence, a pseudouridine or a modified nucleoside and another comprising a siRNA or shRNA. In another embodiment, the dsRNA molecule is more than 50 nucleotides in length. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the present invention provides a method for inducing a mammalian cell to produce a recombinant protein, comprising contacting a mammalian cell with an in vitro synthesized RNA molecule encoding the recombinant protein, the in vitro synthesized RNA molecule comprising a pseudouridine or a modified nucleoside, thereby inducing a mammalian cell to produce a recombinant protein.
In another embodiment, the present invention provides a method for treating anemia in a subject, comprising contacting a subject's cell with an in vitro synthesized RNA molecule, the in vitro synthesized RNA molecule encoding erythropoietin, thereby treating anemia in a subject.
In another embodiment, the present invention provides a method for treating a vasospasm in a subject, comprising contacting a subject's cell with an in vitro synthesized RNA molecule, the in vitro synthesized RNA molecule encoding inducible nitric oxide synthase (iNOS), thus treating a vasospasm in a subject.
In another embodiment, the present invention provides a method for improving the survival rate of a cell in a subject, comprising contacting the cell with an RNA molecule synthesized in vitro, the RNA molecule synthesized in vitro that encodes
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23/141 a heat shock protein, thereby improving a cell's survival rate in a subject.
In another embodiment, the present invention provides a method for decreasing an incidence of a blood vessel restenosis following a blood vessel enlarging procedure, comprising contacting a blood vessel cell with an in vitro synthesized RNA molecule, the RNA molecule synthesized in vitro that encodes a heat shock protein, thereby reducing an incidence of restenosis in a subject.
In another embodiment, the present invention provides a method for increasing hair growth from a hair follicle on a subject's scalp, comprising contacting a scalp cell with an in vitro synthesized RNA molecule, the RNA molecule synthesized in vitro that encodes a telomerase or an immunosuppressive protein, thereby increasing hair growth from a hair follicle.
In another embodiment, the present invention provides a method of inducing the expression of an enzyme with antioxidant activity in a cell, comprising contacting the cell with an RNA molecule synthesized in vitro, the RNA molecule synthesized in vitro that encodes the enzyme, as well inducing the expression of an enzyme with antioxidant activity in a cell.
In another embodiment, the present invention provides a method for treating cystic fibrosis in a subject, comprising contacting a cell of the subject with an in vitro synthesized RNA molecule, the in vitro synthesized RNA molecule encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), thus treating cystic fibrosis in a subject.
In another embodiment, the present invention provides a method for treating an X-linked agammaglobulinemia in a subject, comprising contacting a subject's cell with an in vitro synthesized RNA molecule, the in vitro synthesized RNA molecule encoding a Bruton tyrosine kinase , thus treating an X-linked agammaglobulinemia.
In another embodiment, the present invention provides a method for treating severe adenosine deaminase combined with immunodeficiency (ADA SCID) in a subject, comprising contacting a subject's cell with an in vitro synthesized RNA molecule,
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24/141 the RNA molecule synthesized in vitro that encodes an ADA, thus treating an ADA SCID.
In another embodiment, the present invention provides a method for producing a recombinant protein, comprising contacting an in vitro translation apparatus with an in vitro synthesized polyrubonucleotide, the in vitro synthesized polyrubonucleotide comprising a modified pseudouridine or nucleoside, thereby producing a recombinant protein.
In another embodiment, the present invention provides a method of synthesizing an in vitro transcribed RNA molecule comprising a modified nucleotide with a modified pseudouridine nucleoside, comprising contacting an isolated polymerase with a mixture of unmodified nucleotides and the modified nucleotide.
In another embodiment, the present invention provides an in vitro transcription apparatus, comprising: an unmodified nucleotide, a nucleotide containing a pseudouridine or a modified nucleoside, and a polymerase. In another embodiment, the present invention provides an in vitro transcription kit, comprising: an unmodified nucleotide, a nucleotide containing a modified pseudouridine or nucleoside, and a polymerase. Each possibility represents a separate embodiment of the present invention.
DESCRIPTION OF THE FIGURES
The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention can be better understood by reference to one or more of these figures in combination with the detailed description of specific modalities presented here.
Figure 1 shows that mRNAs that encode each of the six human reprogramming factors, prepared as described in EXAMPLES, are translated and located at the predicted subcellular locations after transfection in 1079 human newborn fibroblasts. 1079 untreated human fibroblasts: Photos A, E, I, M, Q, and U show phase contrast images of 1079 untreated human fibroblasts that have not been transfected with an mRNA encoding a reprogramming factor and photos B, F, J, N, R, and V show fluorescent images of the same fields after the cells have been stained with an antibody specific to each reprogramming factor; these results show that
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25/141 there was little or none of these endogenous reprogramming factor proteins in 1079 untreated human fibroblasts. 1079 treated human fibroblasts: Photos C, G, K, O, S, and W show phase contrast images of 1079 human fibroblasts that were transfected with an mRNA that encodes the indicated reprogramming factor, and photos D, H, L, P, T, and X show fluorescent images of the same fields after the cells have been stained with a specific antibody for each reprogramming factor 24 hours after transfection. These results show that each of the reprogramming factor proteins was expressed in 1079 human fibroblast cells 24 hours after transfection with the respective mRNAs encoding the reprogramming factor and that the reprogramming factor proteins were located in the predicted subcellular sites. A-T are at 20x magnification. U-X are at 10x magnification.
Figure 2 shows that mRNA encoding human reprogramming factors (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) produce iPS cells in human somatic cells. Figure 2 shows images of bright field (A, C) and immunofluorescent (B, D) from an iPS cell colony in 12 days after the final transfection with mRNA encoding reprogramming factors. NANOG staining is observed in colony # 1 (B, D). Images A and B are at 10x magnification. C and D are at 20x magnification.
Figure 3 shows that iPS colonies derived from human somatic cells 1079 and IMR90 are positive for NANOG and TRA-1-60. Figure 3 shows phase contrast (A, D, G) and immunofluorescent (B, C, E, F, H, I) images of iPS colonies derived from 1079 cells (A, D) and IMR90 (G) cells. The same iPS colony shown in (A) is positive for both NANOG (B) and TRA-1-60 (C). The iPS colony shown in (D) is positive for NANOG (E) and positive for TRA-1-60 (F). The iPS colony generated from IMR90 (G) fibroblasts is still positive for both NANOG (H) and TRA-1-60 (I). All images are at 20x magnification.
Figure 4 shows that the rapid formation of the improved efficiency iPSC colony is achieved by transfecting the cells with mRNA that encodes reprogramming factors in MEF conditioned medium. Over 200 colonies were detected 3 days after the final transfection; in the 10 cm dish, IMR90 cells were transfected three times with 36 pg of each reprogramming mRNA (ie, encoding KLF4, LIN28, c-MYC, NANOG, OCT4, and
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SOX2). Representative iPSC colonies are shown at 4x (A, B), 10x (CE) and 20x (F) magnification. Eight days after the final mRNA transfection with mRNAs encoding the six reprogramming factors, more than 1000 iPSC colonies were counted in IMR90 cells transfected with 18 pg (G, I) or 36 pg (H) from each of the six mRNAs . Representative colonies are shown at 4x magnification (G-H) and at 10x magnification (I).
Figure 5 shows that the iPSC colonies derived from 1079 and IMR90 are positive for both NANOG and TRA-1-60. Eight days after the final mRNA transfection with 36 pg of mRNA for each of the six reprogramming factors, the 1079-derived iPSC colonies (shown in A, D, and G) are positive for NANOG (B, E, and H) and TRA-1-60 (C, F, and I). Eight days after the final mRNA transfection with 18 pg (JL) or 36 pg (MO) of mRNA for each of the six reprogramming factors, iPS colonies derived from IMR90 are still positive for NANOG (K, N) and TRA-1-60 (L, O).
Figure 6. Production of TNF-α by MDDCs transfected with natural RNA, demonstrating that unmodified RNA synthesized in vitro and bacterial RNA and mammalian mitochondrial RNA is highly immunogenic, while other mammalian RNAs are weakly immunogenic. Human MDDCs were incubated with Lipofectin® alone, or complexed with R-848 (1 pg / ml), or RNA (5 pg / ml) from 293 cells (total, nuclear and cytoplasmic RNAs), mouse heart (poliA + mRNA ), Human platelet mitochondrial RNA, bovine tRNA, bacterial tRNA and total RNA (E. coli) with or without RNase digestion. After 8 h, TNF-α was measured in the supernatants by ELISA. Mean values ± SEM are shown. The results are representative of 3 independent experiments.
Figure 7. RNA-dependent activation of TLR demonstrates that modification of m6A and s2U blocks TLR3 signaling, while all modifications block TLR7 and TLR8 signaling, and that the least modified bacterial RNA and unmodified RNA transcript in vitro activates all three TLRs. (A) Aliquots (1 pg) of RNA 1571 transcribed in vitro without (none) or with nucleoside modifications m ⁵ C, m ⁶ A, Ψ, m ⁵ U or S ² U were analyzed on denatured agarose gel followed by staining in ethidium bromide and UV lighting. (B) 293 cells expressing human TLR3, TLR7, TLR8 and control vectors were treated with Lipofectin® alone, Lipofectin®-R-848 (1 pg / ml) or RNA (5 pg / ml). The modified nucleosides present in RNA-730 and RNA-1571 are noticed. (Ç)
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27/141 CpG ODN-2006 (5 pg / ml), LPS (1.0 pg / ml) and isolated RNA were obtained from rat liver, mouse cell line (TUBE) and human spleen (total), Mitochondrial RNA from human platelets, or from two different sources of E. coli. 293hTLR9 cells served as controls. After 8 h, IL-8 was measured in the supernatants by ELISA. Mean values ± SEM are shown. Cell lines containing hTLR3-labeled siRNA are indicated with an asterisk. The results are representative of four independent experiments.
Figure 8. Cytokine production by RNA-transfected DC demonstrates that all modifications block cytokine activation generated DC, while only uridine modifications blocked the activation of blood-derived DC. MDDC generated with GMCSF / IL-4 (A, C) or GM-CSF / IFN-α MDDCs (B), and primary DC1 and DC2 (D) were treated for 8 to 16 h with Lipofectin® alone, Lipofectin®-R -848 (1 pg / ml) or RNA (5 pg / ml). The modified nucleosides present in RNA-1571 are noticed. TNF-α, IL-12 (p70) and IFN-α were measured in the supernatant by ELISA. Mean values ± SEM are shown. The results are representative of 10 (A and C), 4 (B), and 6 (D) independent experiments. E. Activation of DC by RNA. MDDC were treated for 20 h with Lipofectin® alone or complexed with 1 pg / ml poly (I) :( C) or R-848 as positive controls (upper frame) or Lipofectin® complexed with the indicated RNA (5 pg / ml; bottom frame). The modified nucleosides present in RNA-1886 are noticed. The expression of CD83, CD80, and HLA-DR was determined by flow cytometry.
Figure 9. Activation of DC by RNA demonstrates that all nucleoside modifications inhibit RNA-mediated activation. MDDC were treated for 20 h with Lipofectin® alone, Lipofectin®-R-848 (1 pg / ml) or RNA-1571, modified as indicated (5 pg / ml). (A) CD83 and HLA-DR staining. (B) the levels of TNF-α in the supernatants and the average fluorescence of CD80 and CD86 in response to incubation with RNA. The volume of medium was increased 30 times for flow cytometry, as indicated by the asterisk. The data are representative of four independent experiments.
Figure 10. Capped RNA-1571 containing different amounts (0, 1, 10, 50, 90, 99 and 100% modified nucleoside, compared to the corresponding unmodified NTP) were transcribed, and it was shown that the modification of only a few nucleosides resulted in an inhibition of DC activation. A. All transcripts were digested with monophosphates and
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28/141 analyzed by reverse phase HPLC to determine the relative amount of modified nucleoside incorporation. The representative absorbance profiles obtained in the indicated proportions (Ψ: ϋ) are shown. Elution times are noted for pseudouridine 3'-monophosphates (Ψ), cytidine (C), guanosine (G), uridine (ϋ), 7-methylguanosine (m7G) and adenosine (A). (B) The modified nucleoside content of RNA-1571. The expected percentage of m ⁶ A, Ψ (pseudouridine), or m ⁵ C in RNA-1571 was calculated based on the relative amount of modified NTP in the transcription reaction in the nucleoside composition of RNA-1571 (A: 505, U: 451 , C: 273, G: 342). The values for the modified nucleoside content were determined based on the quantification of the HPLC chromatograms. Notes: A: values (%) for m ⁶ ATP, ΨΤΡ in ⁵ CTP in relation to ATP, UTP and CTP, respectively. B: values for m ⁶ A, Ψ in ⁵ C monophosphates in relation to all NMPs. (C) MDDC were transfected with Lipofectin® complexed RNA -1571 capped (5 pg / ml) containing the indicated amount of m ⁶ A, Ψ or m ⁵ C. After 8 h, TNF-α was measured in the supernatants. The data are expressed as relative inhibition of TNF-α. Mean ± SEM values obtained from 3 independent experiments are shown.
Figure 11. Expression of TNF-α by DCs transfected with oligoribonucleotide demonstrates that as little as a modified nucleoside reduces the activation of DC. (A) Sequences of chemically synthesized oligoribonucleotides (ORN) (ORN1-4) (SEQ ID NOs: 6-9) or transcripts in vitro (ORN5-6) (SEQ ID NOs: 10-11) are shown. The modified nucleoside positions Um (2'-O-methyluridine), m ⁵ C and Ψ are highlighted. Human MDDCs were transfected with Lipofectin® alone (medium), R-848 (1 pg / ml) or
Lipofectin® complexed with RNA (5 pg / ml). Where noted, the cells were treated with 2.5 pg / ml cycloheximide (CHX). (B). After 8 h of incubation, TNF-α was measured in the supernatant. (C) RNA from the cells was analyzed by Northern blot. Representative mean values ± SEM from 3 independent experiments are shown.
Figure 12. A. Modified mRNA Ψ do not stimulate the production of proinflammatory cytokine in vivo. Serum samples (6 h after injection) were analyzed by ELISA and revealed that 3 pg of unmodified mRNA induced a higher level of IFN-α than did 3 pg of Ψ-modified mRNA (P <0.001). Levels of IFN-α induced by 3 pg of Ψmodified mRNA were similar to those obtained when animals were injected with
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29/141 non-complexed lipofectin. Values are expressed as the mean ± s.e.m. (n = 3 or 5 animals / group). B. Similar results were seen with TNF-α.
Figure 13. mRNA containing pseudouridine (Ψ) does not activate PKR. Ψ: pseudouridine. Control: Unmodified RNA. m5C: mRNA with m ⁵ C modification.
Figure 14. Increased expression of luciferase from mRNA containing pseudouridine in rabbit reticulocyte lysate. Luc-Y: mRNA with pseudouridine modification; luc-C: unmodified RNA. Data are expressed by normalizing luciferase activity to unmodified luciferase RNA.
Figure 15. Increased expression of renilla from mRNA containing pseudouridine in cultured cells. A. 293 cells. B. Primary murine, mouse dendritic cells derived from bone marrow. renilla-Y: mRNA with pseudouridine modification; renillaC: Unmodified RNA. RNA was modified with m ⁵ C, m ⁶ A, and m ⁵ U as noted.
Figure 16. A. Additive effect of 3 'and 5' elements on the translation efficiency of modified mRNA Ψ. 293 cells were transfected with conventional and Ψ-modified firefly luciferase mRNAs that had 5 'cap (capLuc), 50 nt-long 3' polyA-tail (TEVlucA50), both or none of these elements (capTEVlucA50 and Luc, respectively) . The cells were lysed 4 h afterwards and luciferase activities were measured in aliquots (1 / 20th) of the total lysates. B. Ψ-modified mRNA is more stable than unmodified mRNA. CapTEVlucAn transfected 293 cells containing unmodified or Ψmodified nucleosides were lysed at the indicated times after transfection. Aliquots (1 / 20th) of the lysates were evaluated for luciferase. Standard errors are too small to be displayed with error bars. C. β-galactosidase expression is increased using Ψ-modified mRNA compared to conventional mRNA. 293 cells seeded in 96-well plates were transfected with mRNAs complexed with lipofectin (0.25 pg / well) encoding bacterial β-galactosidase (lacZ). The transcripts had a cap and 3 'polyA tail that was 30 nt long (caplacZ) or ~ 200 nt long (caplacZ-An). Constructs made using conventional U or Ψ nucleosides were tested. The cells were fixed and stained with X-gal, 24 h after transfection. The images were taken by inverted microscopy (40 and 100X magnification) from the representative wells.
Figure 17. A. Renilla expression after intracerebral injection of modified or unmodified coding mRNA. Rat brain cortex was injected at 8 sites / animals. a
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30/141 hemisphere was injected with RNA that encodes renilla capped with pseudouridine modification (capRenilla-Y), while the corresponding hemisphere with capped RNA with no nucleoside modification (capRenilla-C). Data for 2 animals (6 injection sites) are shown. BG; lower level of detection of the assay. B. Ψ-modified mRNA released intravenously is expressed in the spleen. i // mRNA complexed with lipofectin (0.3 pg capTEVlucAn / mouse) was administered by injection into the tail vein. The animals were sacrificed at 2 and 4 h after the injection and the luciferase activities were measured in aliquots (1 / 10th) of the homogenized organs in lysis buffer. The values represent the luciferase activities in the total organs. C. Ψ-modified mRNA shows greater stability and translation in vivo. capTEVlucAn complexed with lipofectin (0.3 pg / 60 μΐ / animal) with or without Ψ modifications was released i.v. to mice. The animals were sacrificed at 1, 4 and 24 h after injection, and 1/2 of their spleens were processed for measurements of luciferase enzyme (left frame) and the other half for RNA analysis (right frame). Luciferase activities were measured in aliquots (1/5) of the homogenate made from half of the spleens. The plotted values represent luciferase activities in the total spleen and are expressed as the mean ± s.e.m. (n = 3 or 4 / point). D. Expression of firefly luciferase after intratracheal injection of mRNA. capTEVluc-Y: modified RNA capped with pseudouridine that encodes firefly luciferase. CapTEVluc-C: RNA capped with no nucleoside modifications.
Figure 18. Protein production is independent of the amount of mRNA released intravenously in mice. The indicated amounts of nucleic acids complexed with lipofectin, capTEVlucAn mRNA with or without Ψ constituents and plasmid DNA pCMVluc in a volume of 60 pl / animal were released by i.v. injection into mice. Animals injected with mRNA or plasmid DNA were sacrificed at 6 h or 24 h after injection, respectively, and luciferase activities were measured in aliquots (1 / 10th) of their spleens homogenized in lysis buffer. The value of each animal is shown, and the horizontal lines indicate the average; N.D., not detectable.
Figure 19. Expression of firefly luciferase after intratracheal release of coding mRNA. mRNA were complexed to lipofectin (or PEI, as noted) and animals were injected with 0.3 µg of mRNA encoding firefly luciferase with or
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31/141 without modification, then sacrificed 3 hours later. The lungs were harvested and homogenized and the luciferase activity was measured in aliquots of the lysed organs.
Figure 20. Ψ-modified mRNA does not include inflammatory mediators after lung release. The induction of TNF-α and IFN-α in serum after intratracheal release of mRNA encoding unmodified luciferase or Ψ-modified mRNA. Serum levels of TNF-α and IFN-α were determined by ELISA 24 hours after mRNA release.
Figure 21 shows the results of Example 35: mRNA encoding Luciferase Firefly or Renilla with the indicated modifications were complexed to lipofectin and released to murine dendritic cells (A) and HEK293T (B). Human DCs were transfected with mRNA complexed with TransIT that encodes luciferase firefly or renilla with the indicated modifications (C). The data are expressed as changes at times compared to unmodified mRNA.
Figure 22 shows the results of Example 36: The T7 polymerase transcription reactions used for the generation of mRNA results in larger amounts of RNA of the correct size, but still contains contaminants. This is visualized by applying RNA to a reverse phase HPLC column that separates size-based RNA under denaturing conditions. TEV-luciferase-A51 Ψ-modified RNA was applied to the HPLC column in 38% Buffer B and subjected to a linear gradient of buffer B increasing to 55%. The profile demonstrated both lower than expected and higher than expected contaminants.
Figure 23 shows the results of Example 37: (A) mRNA encoding EPO with the indicated modifications and with or without HPLC purification were released to levels of murine DCs and EPO in the supernatants were measured 24 hours later. While m5C / 'l'-inodiicate mRNA had the highest level of translation prior to HPLC purification, Ψ-modified mRNA has the highest translation after HPLC purification. (B) Human DCs were transfected with mRNA that encodes renilla with the modifications indicated with or without HPLC purification.
Figure 24 shows the results of Example 38: (B) human DCs were transfected with RNA complexed to TransIT with the indicated modifications with or without HPLC purification. IFN-α levels were measured after 24 hours. HPLC purification increased the immunogenicity of unmodified RNA, which is sequence dependent,
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32/141 according to other unmodified RNAs had similar levels of IFN-α or reduced levels after purification by HPLC. Ψ-modified RNA had unmeasured levels of IFN-α, similar to DCs treated with control. (B) Ψ-modified RNA before (-) and after HPLC purification (P1 and P2) was analyzed for dsRNA using dot blotting with a monoclonal antibody specific for dsRNA (J2). Purification of RNA removed dsRNA contamination. (C) Ψ-modified RNA encoding iPS factors are immunogenic, which is removed by HPLC purification of the RNA.
Figure 25 provides an mRNA coding sequence for KLF4 (SEQ ID NO: 12) and LIN28 (SEQ ID NO: 13).
Figure 26 provides an mRNA coding sequence for cMYC (SEQ ID NO: 14) and NANOG (SEQ ID NO: 15).
Figure 27 provides an mRNA coding sequence for OCT4 (SEQ ID NO: 16) and SOX2 (SEQ ID NO: 17).
Figure 28 shows that mRNA encoding human reprogramming factors (KLF4, c-MYC, OCT4, and SOX2) produce iPS cells in primary human keratinocyte cells. Figure 28 shows phase contrast images of HEKn cells at 2 days (A) and iPS colony formation at 11 days (B) and 20 days (C) after the final transfection with mRNA encoding 4 reprogramming factors. The images are at 10x magnification.
Figure 29 shows that mRNA encoding human reprogramming factors (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) produce iPS cells in human keratinocytes that are positive for known iPS cell markers. Figure 29 shows phase contrast images of colonies derived from HEKn cells (A, D, and G). The same iPS colony shown in (A) is positive for both KLF4 (B) and LIN28 (C). The iPS colony shown in (D) is positive for SSEA4 (E) and positive for TRA-1-60 (F). The iPS colony shown in (G) is positive for NANOG (H). All images are at 20x magnification.
Figure 30 shows increases in the expression of 3 iPS-associated messages in HEKn cells transfected with 4 reprogramming mRNAs (KLF4, c-MYC, OCT4, and SOX2) that do not include the NANOG reprogramming factor. The increased expression of the messages was detected by qPCR and is normalized to the GAPDH expression. The level of
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33/141 expression of each message is described in relation to the level found in the original cell line.
DEFINITIONS
The present invention will be understood and interpreted based on the terms defined below.
As used here substantially all, in reference to complete single stranded mRNAs comprising a pseudouridine or 5-methylcytidine residue, means that of all the complete single stranded mRNAs present in a sample, at least 95% have a pseudouridine residue or 5 - methylcytidine or both.
As used here essentially all, in reference to complete single-stranded mRNAs that comprise a pseudouridine or 5-methylcytidine residue, means that of all the complete single-stranded mRNAs present in a sample, at least 99% have either a pseudouridine or 5 residue -methylcitidine.
As used here, contaminating RNA molecules are molecules that comprise RNA residues and that can, at least partially, activate an immune response when transfected into a cell (for example, by activating RNA as RNA protein kinase dependent (PKR) sensors, retinoic acid-inducible gene-I (RIG-I), the Toll-like receptor (TLR) 3, TLR7, TLR8, and oligoadenylate synthase (OAS), or RNA molecules that can at least partially activate an interfering RNA response ( RNAi) (for example, including a response to large double-stranded RNA molecules or small double-stranded RNA molecules (siRNAs) in cells. Examples of contaminating RNA molecules include, but are not limited to, full or partial full-length mRNAs encoding only a part of a reprogramming factor (for example, a non-full length iPS cell induction factor); single-stranded mRNAs that are larger than the length mRNA complete code encoding a reprogramming factor (for example, an iPS cell induction factor), for example, without being bound by a theory, “IVT run-on or other mechanisms; large or small double-stranded mRNA molecules, and uncapped mRNA molecules.
As used here, a purified RNA preparation is substantially free of contaminating RNA molecules (or a given contaminating RNA), when less than 0.5% of the total RNA in the purified RNA preparation is made up of
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34/141 contaminating RNA molecules (or a particularly recited contaminating RNA). The amounts and relative amounts of non-contaminating mRNA molecules and contaminating RNA molecules (or one contaminating RNA in particular) can be determined by HPLC or other methods used in the art to separate and quantify RNA molecules.
As used here, a purified RNA preparation is essentially free of contaminating RNA molecules (or a particular contaminated RNA mentioned), when less than 1.0% of the total RNA in the purified RNA preparation consists of contaminating RNA molecules ( or a particular contaminating RNA). The amounts and relative amounts of non-contaminating mRNA molecules and contaminating RNA molecules ((or a particular contaminating RNA) can be determined by HPLC or other methods used in the art to separate and quantify RNA molecules.
As used here, a purified RNA preparation is virtually free of contaminating RNA molecules (or a particular contaminated RNA mentioned), when less than 0.1% of the total RNA in the purified RNA preparation consists of contaminating RNA molecules (or a particular contaminating RNA cited). The amounts and relative amounts of non-contaminating mRNA molecules and contaminating RNA molecules (or a particular contaminating RNA) can be determined by HPLC or other methods used in the art to separate and quantify RNA molecules.
As used here, a purified RNA preparation is free of contaminating RNA molecules (or a particular said contaminating RNA), when less than 0.01% of the total RNA in the purified RNA preparation consists of contaminating RNA molecules (or a particularly cited contaminating RNA). The amounts and relative amounts of non-contaminating mRNA molecules and contaminating RNA molecules (or one RNA contaminant in particular) can be determined by HPLC or other methods used in the art to separate and quantify RNA molecules.
The terms comprising, containing, having, including and even should be interpreted as among others unless otherwise indicated. The terms one, one and / or similar and referring in the context of the description of the invention and, specifically, in the context of the appended claims, should be interpreted to cover both the singular and the plural, unless otherwise stated. The use of any and all examples or exemplary language (for example, how) is intended only to illustrate aspects or modalities of
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35/141 invention, and should not be construed as limiting the scope of the invention, unless claimed otherwise
Regarding the use of the word derivative, as for an RNA (including mRNA) or a polypeptide that is “derived” from a sample, biological sample, tumor cell, or similar, it means that the RNA or polypeptide, or was present in the sample, biological sample, tumor cell, or similar, or was made using the RNA in the sample, biological sample, tumor cell, or similar, by a process such as an in vitro transcription reaction, or an RNA amplification reaction, where the RNA or polypeptide is either encoded by either a copy of all or part of the RNA or polypeptide molecules in the original sample, biological sample, tumor cell, or the like. By way of example, said RNA may be an in vitro transcription or an RNA amplification reaction, with or without cDNA cloning, rather than being obtained directly from the sample, biological sample, tumor cell, or the like as long as the Original RNA used for in vitro transcription or an RNA amplification reaction has been from the sample, biological sample, tumor cell, or the like. The terms sample and biological sample are used in their broadest sense and cover samples or specimens obtained from any source that contains or may contain eukaryotic cells, including biological and environmental sources. As used here, the term sample when used to refer to biological samples obtained from organisms, includes body fluids (eg blood, or saliva), feces, biopsies, smears (eg, oral smears), isolated cells , exudates, and the like. Organisms include fungi, plants, animals and humans. However, these examples are not to be interpreted as limiting the types of samples or organisms that find use with the present invention. In addition, in order to carry out the research or study the results related to the use of a method or composition of the invention, in some embodiments, a sample or biological sample comprises fixed cells, treated cells, cell lysates, and the like. In some modalities such as method modalities in which mRNA is released into a cell from an organism that has a known disease or into a cell that has a known disease state or pathology, the biological sample or sample also comprises bacteria or virus.
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36/141
As used here, the term incubating and its variants means contacting one or more components of a reaction with another component or components, under conditions and for sufficient time such that a desired reaction product is formed.
As used here, a nucleoside consists of a nucleic acid base (for example, the canonical nucleic acid bases: guanine (G), adenine (A), thymine (T), uracil (U), and cytosine (C)) ; or a modified nucleic acid base (eg, 5-methylcytosine (m ⁵ C)), which is covalently linked to a pentose sugar (eg, ribose or 2'deoxyribose), while a nucleoside's nucleotide or mononucleotide which is phosphorylated in one of the hydroxyl groups of the pentose sugar. Linear nucleic acid molecules are said to have a "5 'terminal"(5' end) and a '3' terminal "(3 'end), because, except with respect to capping or adenylation (for example, adenylation by a ligase) mononucleotides are joined in one direction via a phosphodiester bond to produce oligonucleotides or polynucleotides, so that a phosphate at the 5 'carbon end of a sugar portion of the mononucleotide is attached to an oxygen over the 3' carbon portion sugar from its neighboring mononucleotide. Therefore, one end of a single stranded linear or polynucleotide oligonucleotide or one end of a strand of a double stranded linear nucleic acid (RNA or DNA) is referred to as a 5 'end if its 5' phosphate is not joined or bound to the oxygen from the 3 'carbon of a mononucleotide sugar molecule, and like the 3'-end and its 3' oxygen is not associated with a 5 'phosphate, which is attached to a sugar from another mononucleotide. A terminal nucleotide, as used herein, is the nucleotide at the final position of the 3 'or 5' terminal.
In order to achieve the specific objectives, a nucleic acid base, sugar portion, or internucleoside (or internucleotide) linkage in one or more of the mRNA nucleotides that is introduced into a eukaryotic cell, in any of the methods of the invention may comprise a modified base, sugar portion, or internucleoside bond. For example, in addition to the other modified nucleotides discussed herein for carrying out the methods of the present invention, one or more of the mRNA nucleotides may also have a modified nucleic acid base comprising or consisting of: xanthine; aliaminouracil; aliamino-thymidine; hypoxanthine, 2-aminoadenine; 5-propynyl uracil; 5-propynyl cytosine; 4-thiouracil; 6-thioguanine; a uracil aza or desaza; a thymidine aza or desaza; a cytosine aza or deaza; an adenine aza or desaza; or a guanine aza or desaza, or
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37/141 a nucleic acid base that is derivatized with a part of biotin, a part of digoxigenin, a fluorescent or chemiluminescent part, an extinction part or some other portion in order to accomplish one or more other specific purposes; and / or one or more of the mRNA nucleotides may have a sugar part, such as, among others: 2 'fluoro - 2'deoxyribose or 2'-O-methyl-ribose, which provide resistance to some nucleases, or 2' -amino2'-deoxyribose or 2'-azido-2'-deoxyribose, which can be marked by reacting them with visible, fluorescent, fluorescent infrared or other detectable dyes or chemicals that have an electrophilic, photoreactive, alkaline, or other chemical part reactive portion.
In some embodiments of the invention, one or more of the mRNA nucleotides comprise a modified internucleoside bond, such as a phosphorothioate, phosphorodithioate, phosphoroselenate, or a phosphorodiselenate bond, which are resistant to some nucleases, including an analogous cover dinucleotide (Grudzien -Nogalska et al. 2007) which is used in an IVT reaction for co-transcriptional RNA capping, or in the poly (A) tail (for example, by incorporating a nucleotide that has the phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkages modified during IVT of the RNA or, for example, by incorporating ATP which contains the phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkages modified into a poly (A) tail in the RNA by polyadenylation using a poly (A) polymerase). The invention is not limited to the modified nucleic acid bases, sugar moieties, or listed internucleoside bonds, which are presented to show examples that can be used for a particular purpose in a method.
As used herein, a nucleic acid or a "polynucleotide" or an oligonucleotide is a sequence of covalently linked nucleotides in which the 3 'position of the sugar portion of a nucleotide is linked by a phosphodiester bond to the 5' position of the sugar portion of the next nucleotide (i.e., a 3 'to 5' phosphodiester bond), and where the nucleotides are linked in a specific sequence, i.e., a linear order of nucleotides. In some embodiments, the nucleic acid or polynucleotide or oligonucleotide consists of or includes 2'-deoxyribonucleotides (DNA). In some embodiments, the oligonucleotide consists of or includes ribonucleotides (RNA).
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38/141
The terms isolated or purified when used in relation to a nucleic acid or polynucleotide, as in isolated RNA or purified RNA refer to a nucleic acid that is identified and separated from at least one contaminant with which it is ordinarily associated in its origin. Thus, an isolated or purified nucleic acid (for example, DNA and RNA) is present in a form or configuration different from that found in nature, or a form or configuration different from that which existed before being subjected to a treatment or method of purification. For example, a given DNA sequence (for example, a gene) is found on the host cell's chromosome along with other genes, as well as structural and functional proteins, and a specific RNA (for example, a specific mRNA that encodes a specific protein ), is found in the cell as a mixture with numerous other RNAs and other cellular components. The isolated or purified polypeptide or nucleic acid can be present in the form of single strand or double strand.
A cap or cap of nucleotides means a 5'-triphosphate nucleoside which, under suitable reaction conditions, is used as a substrate by a capping enzyme system and which is thus joined to the 5 'end of an uncapped RNA comprising primary RNA or RNA transcripts having a 5'-diphosphate. The nucleotide that joined the RNA is also referred to as a nucleotide cap in this document. A nucleotide cap is a guanine nucleotide that joins through its 5 'end to the 5' end of a primary RNA transcript. The RNA that has the nucleotide cap attached to its 5 'end is referred to as capped RNA or capped RNA transcription or capped transcription ”. A common capped nucleoside is 7-methylguanosine or N ⁷ methylguanosine (sometimes referred to as the standard cap), which has a structure designated as m ⁷ G, in which case the capped RNA or m ⁷ G-capped RNA has a structure designated as m ⁷ G (5 ') ppp (5') N1 (pN) x-OH (3 '), or more simply, as m ⁷ GpppN1 (pN) x or m ⁷ G [5'] ppp [5 '] N, where m ⁷ G represents nucleoside 7-methylguanosine layer, ppp represents the triphosphate bridge between the 5 'carbons of the nucleoside layer and the first nucleotide of the primary RNA transcript, N1 (pN) x-OH (3') represents the primary RNA transcript, of which N1 is the nucleotide plus 5 ', p represents a phosphate group, G represents a guanosine nucleoside, m ⁷ represents the methyl group at position 7 of guanine, and [5'] indicates the position at which op joins the ribose of the nucleotide layer and the first
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39/141 nucleoside from mRNA (N) transcription. In addition to this standard cover, a variety of naturally occurring cover and synthetic analogues are known in the art. RNA that has some nucleotide cap is referred to as capped RNA. Capped RNA can be naturally occurring from a biological sample, or can be obtained by capping RNA in vitro that has a 5 'triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (for example, example vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system). Alternatively, the capped RNA can be obtained by in vitro transcription (IVT) of a DNA template containing an RNA polymerase promoter, in which, in addition to the GTP, the IVT reaction also contains an analogous dinucleotide cap (for example, a cap analogue m ⁷ GpppG or an N7-methyl, 2'-O-methylGpppG ARCA analogue or an N7-methyl 3'-O-methyl-GpppG ARCA analogue) using methods known in the art (for example, using an AMPLICAP ™ T7 capping kit or a MESSAGEMAX ™ T7 ARCA-CAPPED MESSAGE - Transcription Kit, Epicenter or CellScript).
Capping of a primary 5'-triphosphorylated mRNA transcript in vivo (or using an in vitro capping enzyme system) occurs through several enzymatic steps (Higman et al. 1992, Martin et al. 1975, Myette and Niles, 1996) .
The following enzymatic reactions are involved in capping eukaryotic mRNA:
(1) RNA triphosphatase cleaves mRNA 5'-triphosphate to a diphosphate, pppN1 (p) Nx-
OH (3 ') ppN1 (pN) x-OH (3') + Pi; and then (2) RNA guanyltransferase catalyzes GTP binding for diphosphate 5'-o plus 5 'of the mRNA nucleotides (N1), ppN1 (pN) x-OH (3') + GTP G (5 ') ppp (5 ') N1 (pN) x-OH (3') + PPi and, finally, (3) guanine-7-methyltransferase, using S-adenosyl-methionine (AdoMet) as a cofactor, catalyzes the methylation of 7 - guanine nitrogen in the nucleotide cap, G (5 ') ppp (5') N1 (pN) x-OH (3 ') + AdoMet M7G (5') ppp (5 ') N1 (pN) x-OH (3 ') + AdoHyc. RNA that results from the action of RNA triphosphatase and enzymatic activities of RNA guanyltransferase, as well as RNA that is additionally methylated by the enzymatic activity of guanine-7methyltransferase, is referred to here as 5 'capped RNA or capped RNA and a capping or enzyme system , more simply, a capping enzyme here
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40/141 means any combination of one or more polypeptides with the enzymatic activities that result in capped RNA. Capping enzyme systems, including cloned forms of such enzymes, have been identified and purified from many sources and are well known in the art (Banerjee 1980, Higman et al. 1992, Higman et al. 1994, Myette and Niles 1996 , 1995 Shuman, Shuman 2001, Shuman et al. 1980, Wang et al. 1997). Any capping enzyme system, which can convert uncapped RNA that has a 5 'polyphosphate to capped RNA can be used to provide capped RNA for any of the embodiments of the present invention. In some embodiments, the capping enzyme system is a poxvirus capping enzyme system. In some preferred embodiments, the enzyme capping system is a vaccinia virus capping enzyme. In some embodiments, the capping enzyme system is the capping enzyme of Saccharomyces cerevisiae. Furthermore, taking into account the fact that the genes encoding RNA triphosphatase, RNA guanyltransferase and guanine-7-methyltransferase from one source can complement deletions of one or all of these genes from another source, the capping enzyme system can be from one source, or one or more of the RNA triphosphatase, RNA guanyltransferase, and / or guanine-7-methyltransferase activities can comprise a polypeptide from a different source.
A modified nucleotide layer of the present invention means a nucleotide layer where the sugar, the nucleic acid base, or the internucleoside bond is chemically modified compared to the corresponding canonical 7-methylguanosine layer of nucleotides. Examples of a modified nucleotide layer include a nucleotide layer comprising: (i) a modified 2'-or 3'-deoxyguanosine-5'-triphosphate (or 2'guanine or 3'-deoxyribonucleic acid-5'-triphosphate) in whereas the 2'-, or 3'-deoxy position of the sugar deoxyribose moiety is replaced by a group comprising an amino group, an azido group, a fluorine group, a methoxy group, a thiol (or mercapto) group or a methylthio group ( or methylmercapto), or (ii) a modified guanosine-5'-triphosphate, wherein the O ⁶ oxygen of the guanine base is methylated. or (iii) 3'-deoxyguanosine For the sake of clarity, it should be understood in this document that an alkoxy-substituted deoxyguanosine-5'-triphosphate may also be referred to as an O-alkyl-substituted guanosine-5'-triphosphate; by way of example, but without limitation, (2'-methoxy-2'-deoxyguanosine-5'-triphosphate (2'-methoxy-2'dGTP) and 3'-methoxy-3'-deoxyguanosine-5'-triphosphate 3 '-methoxy- 3'-dGTP) can also be here
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41/141 referred to as 2'-O-methylguanosine-5'-triphosphate (2'-OMe-GTP) and 3'-O-methylguanosine-5'triphosphate (3'-OMe-GTP), respectively. After adhesion of the modified nucleotide cap to the 5'-end of the uncapped RNA comprising primary RNA transcripts (or RNA having a 5'- diphosphate), the portion of the modified nucleotide cap which is joined to the uncapped RNA comprising primary transcripts of RNA (or RNA that has a 5'- diphosphate) can be referred to here as a modified nucleoside layer (that is, without referring to the phosphate groups to which it is attached), but is sometimes referred to as a “modified nucleotide layer. .
A modified nucleotide capped RNA is a capped RNA molecule that is synthesized using a capping enzyme system and a modified nucleotide cap, where the nucleotide cap at its 5 'end comprises a modified cap nucleus, or capped RNA that is synthesized co-transcriptionally in an in vitro transcription reaction that contains a modified dinocleotide layer analog where the modified dinocleotide layer contains the chemical modification in the nucleotide layer. In some embodiments, a modified analogous dinocleotide layer is an anti-reverse analogous layer or ARCA (Grudzien et al. 2004 Jemielity, et al. 2003, Grudzien-Nogalska et al. 2007, Peng et al. 2002, Stepinski et al. 2001 ).
A primary RNA or primary RNA transcript means an RNA molecule that is synthesized by an RNA polymerase in vivo or in vitro and that has a 5'-carbon triphosphate RNA molecule from its majority of the 5 'nucleotides
An RNA amplification reaction or an RNA amplification method means a method for increasing the amount of RNA corresponding to one or more desired RNA sequences in a sample. For example, in some embodiments, the RNA amplification method comprises: (a) synthesizing the first complementary cDNA strand to one or more desirable RNA molecules by extending one or more primers by RNA-dependent DNA polymerase that ring the molecules of Desired RNA; (b) synthesizing double-stranded cDNA from the first cDNA strand using a process where a functional RNA polymerase promoter is joined to it, and (c) contacting the double-stranded cDNA with an RNA polymerase that binds to said promoter under transcriptional conditions, through which the RNA corresponding to one or more desired RNA molecules is obtained. Unless otherwise indicated in relation to the specific modality
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42/141 of the invention, an RNA amplification reaction according to the present invention means a sense RNA amplification reaction, which means an RNA amplification reaction that synthesizes sense RNA (for example, RNA having the same sequence of mRNA or other primary RNA transcript, rather than the complement of that sequence). The sense RNA amplification reactions known in the art, which fall within this definition include, among others, the methods that synthesize sense RNA described in Ozawa et al. (Ozawa et al 2006.) and in US Patent Applications 20090053775; 20050153333; 20030186237; 20040197802; and 20040171041. The RNA amplification method described in US Patent Application 20090053775 is a preferred method for obtaining amplified RNAs derived from one or more cells, whose amplified RNAs are then used to make mRNA for use in the methods of the present invention.
Poly-A polymerase (PAP) means an independent model of RNA polymerase found in most eukaryotes, prokaryotes, eukaryotes and viruses that selectively use ATP to incorporate AMP residues into 3'-hydroxylated ends of RNA. Since PAP enzymes that have been studied from plants, animals, bacteria and viruses all catalyze the same global reaction (Edmonds 1990) are highly structurally conserved (Gershon 2000) and have no intrinsic specificity for particular sequences or sizes of molecules. If PAP RNA is separated from proteins that recognize AAUAAA polyadenylation signals (Wilusz and Shenk, 1988), purified wild-type and recombinant PAP enzymes from any of a variety of sources can be used for the present invention. In some embodiments, a Saccharomyces PAP enzyme (for example, from S. cerevisiae) is used for polyadenylation to make purified RNA preparations comprising or consisting of one or more modified mRNAs, each of which encodes a reprogramming factor ( for example, an iPS cell induction factor). In some embodiments, an E. coli PAP enzyme is used for polyadenylation to make purified RNA preparations comprising or consisting of one or more modified mRNAs, each of which encodes a reprogramming factor (for example, a cell-inducing factor iPS).
A reprogramming factor means a protein, polypeptide, or other biomolecule that, when used alone or in combination with other factors or conditions,
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43/141 causes a change in the differentiation state of a cell in which the reprogramming factor is introduced or expressed. In some preferred embodiments of the methods of the present invention, the reprogramming factor is a protein or polypeptide that is encoded by an mRNA that is introduced into a cell, thereby generating a cell that has an altered state of differentiation compared to the cell in which the mRNA was introduced. In some preferred embodiments of the methods of the present invention, the reprogramming factor is a transcription factor. One embodiment of a reprogramming factor used in a method of the present invention is an iPS cell induction factor.
An iPS cell induction factor or iPSC induction factor is a protein, polypeptide, or other biomolecule that, when used alone or in combination with other reprogramming factors, causes the generation of iPS cells from somatic cells. Examples of iPS cell induction factors include OCT4, SOX2, c-MYC, KLF4, NANOG and LIN28. Factors of iPS cell induction include full-length polypeptide sequences or their biologically active fragments. Likewise, an mRNA encoding an iPS cell induction factor can encode a full-length polypeptide or biologically active fragments. The mRNA that codes for the sequence of iPS induction specimens are shown in Figures 25 (KLF4 and LIN28), 26 (c-myc and NANOG), and 27 (OCT4 and SOX2). In certain embodiments, the present invention employs the sequences or similar sequences shown in these figures, including mRNA molecules that further comprise, attached to these mRNA sequences, oligoribonucleotides that exhibit any of the 5'and 3 'UTR sequences, Kozak sequences, the IRES sequences, nucleotide cap, and / or poly (A) sequences used in the experiments described herein, or which are generally known in the art and which can be used in place of those used herein, by linking them to these protein sequences encoding mRNA in order to optimize the translation of the respective mRNA molecules in the cells and improve their stability in the cell, in order to carry out the methods described here.
Cell differentiation or differentiation means the naturally occurring biological process by which a cell that has a less specialized state of differentiation or cell type (for example, a fertilized egg cell, a cell in a
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44/141 embryo, or a cell of a eukaryotic organism) becomes a cell that has a more specialized state of differentiation or cell type. Scientists, including biologists, cell biologists, immunologists, and embryologists, use a variety of methods and criteria to define, describe or categorize different cells according to their cell type, “differentiated state, or differentiated state. In general, a cell is defined, described, or categorized in relation to its cell type, “differentiated state, or differentiated state. based on one or more phenotypes displayed by that cell, such phenotypes may include shape, a metabolic or biochemical activity or function, the presence of determined biomolecules in the cell (for example, based on staining that react with specific biomolecules), or on the cell (for example, based on the binding of one or more antibodies that react with specific biomolecules on the cell surface). For example, in some embodiments, different types of cells are identified and classified using a cell sorter or fluorescence activated cell sorter equipment (FACS). Cell differentiation or differentiation can also occur in cultured cells.
The term reprogramming, as used herein, means cell differentiation or differentiation that occurs in response to the delivery of one or more reprogramming factors into the cell, directly (for example, by the delivery of reprogramming factors proteins or polypeptides into the cell). cell) or indirectly (for example, by distributing the purified RNA preparation of the present invention comprising one or more mRNA molecules, each of which encodes a reprogramming factor) and maintaining the cells in conditions (for example, medium, temperature , oxygen and CO2 levels, matrix and other environmental conditions) that are favorable for differentiation. The term reprogramming when used here is not intended to mean or refer to a specific differentiation direction or route (for example, from a less specialized cell type to a more specialized cell type) and does not exclude processes that proceed in a differentiation direction or route than what is normally observed in nature. Thus, in different embodiments of the present invention, reprogramming means and includes any and all of the following:
(1) De-differentiation means a process of a cell that has a more specialized state of differentiation or type of cell (for example, a fibroblast of
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45/141 mammal, a keratinocyte, a muscle cell, or a neural cell) going to a cell that has a less specialized state of differentiation or cell type (for example, an iPS cell);
(2) Transdifferentiation, means a process of a cell that has a more specialized state of differentiation or type of cells (for example, a mammalian fibroblast, a keratinocyte, or a neural cell) going to another more specialized state of differentiation or cell type (for example, from a fibroblast or keratinocytes to a muscle cell) and (3) Redifferentiation or expected differentiation or natural differentiation, means, a process of a cell that exhibits any particular state of differentiation or type of cell going to another state of differentiation or cell type, as would be expected in nature, if the cell was present in its natural place and environment (for example, in an embryo or an organism), if that process occurs in vivo in a organism or in culture (for example, in response to one or more reprogramming factors).
DESCRIPTION OF THE INVENTION
The present invention provides compositions and methods for reprogramming the differentiating state of eukaryotic cells, including human or other animal cells, by contacting the cells with purified RNA preparations comprising or consisting of one or more different single stranded mRNA molecules that each one encodes a reprogramming factor (for example, an iPS cell induction factor). The purified single-stranded mRNA molecules preferably comprise at least one modified nucleoside selected from the group consisting of a pseudouridine (Ψ), 5-methylcytosine (m ⁵ C), 5-methyluridine (m ⁵ U), 2'-O-methyluridine (One or m ² ' ^- ° U), 2-thiouridine (s ² U), and N ⁶ methyladenosine (m ⁶ A) in place of at least part (for example, including substantially all) of the corresponding unmodified canon nucleoside of unmodified canonical nucleosides A, C, G, or T. In addition, single-stranded mRNA molecules are preferably purified to be substantially free of contaminating RNA molecules that could activate an unwanted response, decrease mRNA expression in simple ribbon, and / or activate RNA sensors in the cells. In certain embodiments, purified RNA preparations are substantially free of contaminating RNA molecules that are: short or longer than mRNA length molecules
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46/141 total single strand, double stranded, and / or uncapped RNA. In some preferred embodiments, the invention provides compositions and methods for differentiated reprogramming of eukaryotic cells, including human or other animal somatic cells, by contacting the cells with purified RNA preparations comprising or consisting of one or more different single-stranded mRNA molecules encoding each an iPS cell induction factor.
In certain embodiments, the mRNA used in the purified RNA preparations is purified to remove substantially, essentially, or virtually all contaminants, including substantially, essentially, or virtually all RNA contaminants. The present invention is not limited with respect to the purification methods used to purify the mRNA, and the invention includes the use of any method that is known in the art or to be developed in the future, in order to purify the mRNA and remove contaminants, including RNA contaminants, that interfere with the intended use of mRNA. For example, in preferred embodiments, mRNA purification removes contaminants that are toxic to cells (for example, by inducing an innate immune response in cells, or, in the case of double-stranded RNA contaminants comprising RNA, through induction of interfering RNA (RNAi), for example, through siRNA or long RNAi molecules) and contaminants that directly or indirectly decrease mRNA translation in cells). In some embodiments, the mRNA is purified by HPLC using a method described herein, included in the Examples. In certain embodiments, the mRNA is purified using a polymeric resin substrate comprising a C18 derivatized styrene-divinylbenzene copolymer and a triethylamine acetate (TEAA) ion-pairing agent used in the column buffer, along with the use of a gradient of acetonitrile to elute mRNA and separate it from contaminating RNAs depending on size, in some embodiments, mRNA purification is performed using HPLC, but in some other embodiments a gravity flow column is used for purification. In some embodiments, mRNA is purified using a method described in the book entitled RNA Purification and Analysis ”by Douglas T. Gjerde, Lee Hoang, and David Hornby, published by Wiley-VCH, 2009, incorporated herein by reference. In some embodiments, mRNA purification is carried out in a non-denaturing manner (for example, at a temperature below about 50 degrees C,
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47/141 for example, at room temperature). In some embodiments, the purification of mRNA is carried out in a partially denaturing manner (for example, at a temperature below about 50 degrees C and 72 degrees C). In some embodiments, mRNA purification is carried out in denaturation mode (for example, at a temperature greater than about 72 degrees C). Of course, those skilled in the art will know that the denaturation temperature depends on the melting temperature (Tm) of the mRNA to be purified, as well as on the melting temperatures of RNA, DNA, or RNA / DNA hybrids that contaminate the mRNA. In other embodiments, the mRNA is purified as described by Mellits KH et al. (Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNA1 from a T7 vector. Nucleic Acids Research 18: 5401-5406, 1990, Nucleic Acids Research 18: 5401-5406, 1990, incorporated herein by reference in its totality). These authors used a three-step purification to remove contaminants that can be used in embodiments of the present invention. Step 1 was 8% polyacrylamide gel electrophoresis in 7M urea (denaturation conditions). The main band was excised from RNA from the gel slice and subjected to 8% polyacrylamide gel electrophoresis under non-denaturing conditions (without urea) and the main band recovered from the gel slice. Further purification was performed on a CF-11 cellulose column using a mobile ethanol-salt buffer phase that separates double-stranded RNA from single-stranded RNA (RM Franklin 1966 Proc Natl Acad Sei USA 55: 1504-1511; Barber. R. 1966 Biochem Biophys Acta 114: 422; and Zelcer A et al 1982 J. Gen. Virol 59: 139-148, all of which are incorporated herein by reference) and the final purification step was cellulose chromatography. In some other embodiments the mRNA is purified using a hydroxylapatite (PAH) column, either under non-denaturing conditions or at higher temperatures (for example, as described by Pays E. 1977 Biochem J. 165: 237-245; Lewandowski LJ et al 1971 J. Virol 8: 809-812; Clawson GA and EA Smuckler 1982 Cancer Research 42: 3228-3231, and / or Andrews-Pfannkoch C et al 2010 Applied and Environmental Microbiology 76: 5039 - .. 5045, all which are hereby incorporated by reference). In some other embodiments, mRNA is purified by weak anion exchange liquid chromatography under non-denaturing conditions (for example, as described by Easton LE et al. 2010. RNA 16: 647-653 to clean up in vitro transcription reactions, incorporated herein as reference). In other embodiments, the mRNA is purified using a combination of any of the methods
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48/141 above or another method known in the art or to be developed in the future. In yet another embodiment the mRNA used in the compositions and methods of the present invention is purified using a process that comprises treating the mRNA with an enzyme that specifically acts (for example, digests) one or more contaminating RNA or contaminating nucleic acids (for example, including DNA), but does not act on (for example, does not digest) the desired mRNA. For example, in some embodiments, the mRNA used in the compositions and methods of the present invention is purified using a process that comprises treating the mRNA with a ribonuclease III (RNase III) enzyme (for example, E. coli RNase III) and the mRNA is then purified away from RNase III digestion products. The ribonuclease III (RNase III) enzyme here means an enzyme that digests double stranded RNA greater than about twelve base pairs to the margin of double stranded RNA fragments. In some embodiments, the mRNA used in the compositions and methods of the present invention is purified using a process that comprises treating the mRNA with one or more other enzymes that specifically digest one or more contaminating RNAs or contaminating nucleic acids (for example, including DNA) .
This invention provides RNA, oligoribonucleotide, and polyribonucleotide molecules, which comprise pseudouridine or a modified nucleoside, gene therapy vectors comprising the same, gene therapy methods and gene transcription silencing methods comprising the same, methods of reducing genes immunogenicity, and the methods for their synthesis. These modified sequences are preferably present in the purified RNA preparations described herein.
In one embodiment, the present invention provides a messenger RNA that comprises a pseudouridine residue. In another embodiment, the messenger RNA encodes a protein of interest. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides an RNA molecule that encodes a protein of interest, this RNA molecule comprising a pseudouridine residue. In another embodiment, the present invention provides an RNA molecule transcribed in vitro, comprising a pseudouridine. In another embodiment, the present invention provides an RNA molecule transcribed in vitro, comprising a modified nucleoside.
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As provided herein, the present invention provides methods for synthesizing RNA molecules transcribed in vitro, comprising pseudouridine and / or modified nucleosides. In another embodiment, the present invention provides a messenger RNA molecule that comprises a pseudouridine residue.
In another embodiment, RNA molecules transcribed in vitro from the methods and compositions of the present invention are synthesized by T7 phage RNA polymerase. In another embodiment, synthesized by RNA polymerase from phage SP6. In another embodiment, synthesized by phage T3 RNA polymerase. In another embodiment, the molecule is synthesized by a polymerase selected from the above polymerases. In another embodiment, the RNA molecule transcribed in vitro is an oligoribonucleotide. In another embodiment, the RNA molecule transcribed in vitro is a polyribonucleotide. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides an in vitro-synthesized oligoribonucleotide, comprising a pseudouridine or a modified nucleoside, where the modified nucleoside is m ⁵ C, m ⁵ U, m ⁶ A, s ² U, Ψ, or 2'-Omethyl -U. In another embodiment, the present invention provides a synthesized polyribonucleotide, comprising a pseudouridine or a modified nucleoside, where the modified nucleoside is m ⁵ C, m ⁵ U, m ⁶ A, s ² U, Ψ, or 2'-O-methyl -U.
In another modality, the oligoribonucleotide synthesized in vitro or polyribonucleotide is (sh) short hairspin RNA. In another modality, the oligoribonucleotide synthesized in vitro is a small interference RNA (siRNA). In another embodiment, the oligoribonucleotide synthesized in vitro is any other type of oligoribonucleotide known in the art. Each possibility represents a separate embodiment of the present invention.
In another embodiment, an RNA molecule, oligoribonucleotide, or polyribonucleotide of the methods and compositions of the present invention further comprises an open reading frame encoding a functional protein. In another embodiment, the RNA molecule or oligoribonucleotide molecule functions without encoding a functional protein (for example, in transcriptional silencing), such as an RNzyme, etc. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the RNA molecule, oligoribonucleotide, or polyribonucleotide further comprises a poly-A tail. In another modality, the molecule of
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RNA, oligoribonucleotide, or polyribonucleotide does not include a poly-A tail. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the RNA molecule, oligoribonucleotide, or polyribonucleotide further comprises a m7GpppG layer. In another embodiment, the RNA molecule, oligoribonucleotide, or polyribonucleotide does not include a m7GpppG layer. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the RNA molecule, oligoribonucleotide, or polyribonucleotide further comprises an independent layer that improves translation. In another embodiment, the RNA molecule, oligoribonucleotide, or polyribonucleotide does not include an independent translation enhancer layer. In another modality, the independent translation enhancer layer is an etch tobacco virus (TEV) -cap independent translation enhancer. In another embodiment, the independent translation enhancer layer is any other independent translation enhancer layer known in the art. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the present invention provides a gene therapy vector, comprising a polyrubonucleotide molecule synthesized in vitro, where the polyrubonucleotide molecule comprises a pseudouridine or a modified nucleoside.
In another embodiment, an RNA molecule, oligorubonucleotide, or polyrubonucleotide of the methods and compositions of the present invention comprises a pseudouridine. In another embodiment, the RNA molecule or oligorubonucleotide molecule comprises a modified nucleoside. In another embodiment, the RNA molecule or oligorubonucleotide molecule is an RNA molecule synthesized in vitro or an oligoribonucleotide. Each possibility represents a separate embodiment of the present invention.
Pseudouridine refers, in another embodiment, to am ¹ acp ³ T (1-methyl-3- (3-amino-3carboxypropyl) pseudouridine. In another embodiment, the term refers to am ¹ T (1 methylpseudouridine) In another embodiment, the term refers to Tm (2'-O-methylpseudouridine. In another embodiment, the term refers to am ⁵ D (5-methyldihydrouridine). In another embodiment, the term refers to am ³ T (3-methylpseudouridine) In another embodiment, the term refers to a pseudouridine part that is no longer modified In another embodiment, the term refers to a monophosphate, diphosphate or triphosphate from any of the above pseudouridines.
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51/141 embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.
In another embodiment, an RNA, oligoribonucleotide, or polyribonucleotide molecule of the methods and compositions of the present invention is a therapeutic oligoribonucleotide.
In another embodiment, the present invention provides a method for releasing a recombinant protein to a subject, the method comprising the step of contacting the subject with an RNA, oligoribonucleotide or polyribonucleotide molecule or a gene therapy vector of the present invention, thus providing a recombinant protein to a subject.
In another embodiment, the present invention provides a double-stranded RNA (dsRNA) molecule comprising a pseudouridine or a modified nucleoside and further comprising a siRNA or short hairpin RNA (shRNA). In another embodiment, the dsRNA molecule is greater than 50 nucleotides in length. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the pseudouridine or a modified nucleoside is within the siRNA sequence. In another embodiment, the pseudouridine or a modified nucleoside is outside the siRNA sequence. In another embodiment, one or more pseudouridines and / or a modified nucleoside residue are present both inside and outside the siRNA sequence. Each possibility represents a separate embodiment of the present invention.
In another embodiment, siRNA or shRNA is contained internally in the dsRNA molecule. In another embodiment, siRNA or shRNA is contained at one end of the dsRNA molecule. In another embodiment, one or more siRNA or shRNA are contained at one end of the dsRNA molecule while the other one is contained internally. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the length of an RNA, oligoribonucleotide or polyribonucleotide molecule (e.g., a single-stranded RNA (ssRNA) or dsRNA molecule) of the methods and compositions of the present invention is greater than 30 nucleotides in length. In another embodiment, the RNA or oligoribonucleotide molecule is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another
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52/141 modality, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides. Each possibility represents a separate embodiment of the present invention.
In another embodiment, a dsRNA molecule of the methods and compositions of the present invention is manufactured by in vitro transcription. In another embodiment, the step for in vitro transcription uses T7 phage RNA polymerase. In another modality, transcription in
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53/141 vitro uses RNA polymerase from phage SP6. In another embodiment, in vitro transcription uses T3 phage RNA polymerase. In another embodiment, in vitro transcription uses an RNA polymerase selected from the above polymerases. In another embodiment, in vitro transcription uses any other RNA polymerase known in the art. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the dsRNA molecule is capable of being processed by a cellular enzyme to produce siRNA or shRNA. In another embodiment, the cellular enzyme is an endonuclease. In another embodiment, the cell enzyme is Dicer. Dicer is a nuclease of the RNase III family that initiates interfering RNA (RNAi) and phenomena linked by the generation of small RNAs that determine the specificity of these gene silencing pathways ((Bernstein E, Caudy AA et al, Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001; 409 (6818): 363-6). In another embodiment, the cellular enzyme is any other cellular enzyme known in the art that is capable of cleaving a dsRNA molecule. Each possibility represents a modality separate from the present invention.
In another embodiment, the dsRNA molecule contains two siRNA or shRNA. In another embodiment, the dsRNA molecule contains three siRNA or shRNA. In another embodiment, the dsRNA molecule contains more than three siRNA or shRNA. In another embodiment, siRNA and / or shRNA are released from the dsRNA molecule by a cellular enzyme. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the present invention provides a method for administering a siRNA or shRNA to a cell, comprising administering a dsRNA molecule of the present invention, where the cell processes the dsRNA molecule to obtain the siRNA or shRNA, as well , administering a siRNA or shRNA to a cell.
In another embodiment, the nucleoside that is modified into an RNA molecule, oligoribonucleotide, polyribonucleotide of the methods and compositions of the present invention is uridine (U). In another embodiment, the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenosine (A). In another embodiment, the modified nucleoside is guanosine (G). Each possibility represents a separate embodiment of the present invention.
In another embodiment, the modified nucleoside of the methods and compositions of the present invention is m ⁵ C (5-methylcytidine). In another embodiment, the modified nucleoside is
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54/141 m ⁵ U (5-methyluridine). In another embodiment, the modified nucleoside is m ⁶ A (N ⁶ methyladenosine). In another embodiment, the modified nucleoside is s ² U (2-thiouridine). In another modified nucleoside modality it is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).
In other embodiments, the modified nucleoside is m ¹ A (1-methyladenosine); m ² A ( ² methyladenosine); Am (2'-O-methyladenosine); ms ² m ^G A (2-methylthio-N ^6- methyladenosine); ^G A (~ isopentenyladenosine); ms ^z i6A (2-methylthio-N ⁶ isopentenyladenosine); io ⁶ A (N ⁶ - (cishhydroxyisopentenyl) adenosine); ms ² io ⁶ A (2-methylthio-N ^6- (cis-hydroxyisopentenyl) adenosine); g ⁶ A / KT ⁶ item 1 m rn I po tT> nm / - i lar! on z ci tio) · 4- ⁶ A / NT ⁶ 4 · τ * ο / η · 1 c * q τ4λ qm / xi ln / 4 on rtcmoV mc ² + ⁶ A i m ofi l Ή i ~ x NT ⁶ (n-glyciiiylcaibamoyladeiiosiiia); ta (n-iieonylcaibamoyladenosine); MS rt (2-methylthio-N 1 1 Ιτολιί in τ4λ qm / 1 -μ n / 4 on r ci Noy m ^6/6/6 NT AT 1 ofi m ^ d / 4on / ci in) · tan ⁶ A / ^ NT ⁶ thioneoniiccabamoyladenosine); mta (n -methyl-N-tIeonylcaIbamoyladenosine); iin a (n-hydroxynorvalylcarbamoyladenosine); ms ² hn ⁶ A (2-methylthio-N ⁶ -hydroxynorvalyl carbamoyladenosine); Ar (p) (2'-O-ribosyladenosine (phosphate)); I (inosine); m ¹ I (1-methylinosine); m ^l Im (1,2'-O-dimethylinosine); m ³ C (3-methylcytidine); Cm (2'-O-methylcytidine); S ² C ( ² thiocytidine); ac ⁴ C (N ⁴ -acetylcitidine); f5C (5-formylcitidine); m ⁵ Cm (5,2'-O-dimethylcytidine); ac ⁴ Cm (N ⁴ -acetyl-2'-O-methylcytidine); k ² C (lysidine); m ¹ G (1-methylguanosine); m ² G (N ² methylguanosine); m ⁷ G (7-methylguanosine); Gm (2'-O-methylguanosine); m ² 2G (N ² , N ² dimethylguanosine); m ² Gm (N ² , 2'-O-dimethylguanosine); m ² 2Gm (N ² , N ² , 2'-O-trimethylguanosine); Gr (p) (2'-O-ribosylguanosine (phosphate)); iW (wibutosine); o2iW (peroxiwibutosine); OHiW (hydroxywibutosine); OHiW * (submodified hydroxywibutosine); imG (wiosin); mimG (methylwiosin); Q (queuosin); oQ (epoxyqueuosine); galQ (galactosyl-queuosin); manQ (mannosilqueuosina); price (7-cyano-7-deazaguanosine); preQl (7-aminomethyl-7deazaguanosine); G + (archaeosine); D (dihydrouridine); m ⁵ Um (5,2'-O-dimethyluridine); S ⁴ U ( ⁴ thiouridine); m ⁵ s ² U (5-methyl-2-thiouridine); s ² Um (2-thio-2'-O-methyluridine); acp ³ U (3- (3-amino3-carboxypropyl) uridine); ho ⁵ U (5-hydroxyuridine); mo ⁵ U (5-methoxyuridine); as ⁵ U (uridine 5-oxyacetic acid); mcmo ⁵ U (5-oxyacetic acid uridine methyl ester); chm ⁵ U (5 (carboxyhydroxymethyl) uridine); mchm ⁵ U (5 (carboxyhydroxymethyl) methyl ester uridine); mcm ⁵ U (5-methoxycarbonylmethyluridine); mcm ⁵ Um (5-methoxycarbonylmethyl-2'-O-methyluridine);
mcm ⁵ s ² U (5-methoxycarbonylmethyl-2-thiouridine); nm ⁵ s ² U (5-aminomethyl-2-thiouridine); mnm ⁵ U (5-methylaminomethyluridine); mnm ⁵ s ² U (5-methylaminomethyl-2-thiouridine); mnmse ² U (5methylaminomethyl-2-selenouridine); ncm ⁵ U (5-carbamoylmethyluridine); ncm ⁵ Um (5carbamoylmethyl-2'-O-methyluridine); cmnm ⁵ U (5-carboxymethylaminomethyluridine); cmnm ⁵ um
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55/141 (5-carboxymethylaminomethyl-2 '- methyluridine); cmnm ⁵ s ² U (5-carboxymethylaminomethyl-2thiouridine); m ⁶ 2A (N6, N6-dimethyladenosine); Im (2'-O-methylinosine); m ⁴ C (N ⁴ -methylcytidine); m ⁴ Cm (N ⁴ , 2'-O-dimethylcytidine); hm ⁵ C (5-hydroxymethylcitidine); m ³ U (3-methyluridine); cm ⁵ U (5-carboxymethyluridine); m ⁶ Am (N ⁶ , 2'-Odimethyladenosine); m ⁶ 2Am (N ⁶ , N ⁶ , 2'-Otrimethyladenosine); m ^2.7 G (N ² , 7-dimethylguanosine); m ^2.2.7 G (N ² , N ² , 7-methylmethanesma); m ³ Um (3,2'-O-dimethyluridine); m ⁵ D (5-methyldihydrouridine); f5Cm (5-formyl-2'-O-methylcytidine); m ¹ Gm (1,2'-O-dimethylguanosine); m ¹ Am (1,2'-O-dimethyladenosine); im ⁵ U ( ⁵ taurinomethyluridine); Tm5s2U (5-taurinomethi1-2-thiouridine)); imG-14 (4-demethylwiosin); imG2 (isowiosin); or ac ⁶ A (N ⁶ -acetyladenosine). Each possibility represents a separate embodiment of the present invention.
In another embodiment, an RNA molecule, oligoribonucleotide, or polyribonucleotide of the methods and compositions of the present invention comprises a combination of two or more of the above modifications. In another embodiment, the RNA molecule or oligorubonucleotide molecule comprises a combination of 3 or more of the above modifications. In another embodiment, the RNA molecule or oligorubonucleotide molecule comprises a combination of more than 3 of the above modifications. Each possibility represents a separate embodiment of the present invention.
In another embodiment, between 0.1% and 100% of the residues in the RNA molecule, oligoribonucleotides, polyribonucleotides of the methods and compositions of the present invention are modified (for example, either by the presence of pseudouridine or a modified nucleoside base In another embodiment, 0 , 1% of the residues are modified. In another modality, 0.2%. In another modality, the fraction is 0.3%. In another modality, the fraction is 0.4%. In another modality, the fraction is 0.5%. In another modality, the fraction is 0.6%. In another modality, the fraction is 0.8%. In another modality, the fraction is 1%. In another modality, the fraction is 1.5%. In another modality, the fraction is 2%. In another modality, the fraction is 2.5%. In another modality, the fraction is 3%. In another modality, the fraction is 4 % In another modality, the fraction is 5%. In another modality, the fraction is 6%. In another modality, the fraction is 8%. In another modality, the fraction is d and 10%. In another modality, the fraction is 12%. In another modality, the fraction is 14%. In another modality, the fraction is 16%. In another modality, the fraction is 18%. In another modality, the fraction is 20%. In another modality, the fraction is 25%. In another
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56/141 modality, the fraction is 30%. In another modality, the fraction is 35%. In another modality, the fraction is modality, the fraction is modality, the fraction is
40%. In another modality,
50%. In another modality,
70%. In another modality, the fraction is 45%. In another, the fraction is 60%. In another, the fraction is 80%. In another modality, the fraction is 90%. In another modality, the fraction is 100%.
In another modality, the fraction is less than 5%. In another modality, the fraction is less than 3%. In another modality, the fraction is less than 1%. In another modality, the fraction is less than 2%. In another modality, the fraction is less than 4%. In another modality, the fraction is less than 6%. In another modality, the fraction is less than 8%. In another modality, the fraction is less than 10%. In another modality, the fraction is less than 12%. In another modality, the fraction is less than 15%. In another modality, the fraction is less than 20%. In another modality, the fraction is less than 30%. In another modality, the fraction is less than 40%. In another modality, the fraction is less than 50%. In another modality, the fraction is less than 60%. In another modality, the fraction is less than 70%.
In another embodiment, 0.1% of the residues of a given nucleotide (uridine, cytidine, guanosine, or adenine) are modified. In another embodiment, the nucleotide fraction is 0.2%. In another modality, the fraction is 0.3%. In another modality, the fraction is 0.4%. In another modality, the fraction is 0.5%. In another modality, the fraction is 0.6%. In another modality, the modality fraction, the modality fraction, the fraction is 0.8%. In another, it is 1.5%. In another, it is 2.5%. In another modality, the modality fraction, the modality fraction, the fraction is 1%. In another it is 2%. In another it is 3%. In another modality, the fraction is 4%. In another modality, the fraction is 5%. In another modality, the fraction is 6%. In another modality, the fraction is 8%. In another modality, the fraction is
10%. In another modality, the fraction is 12%. In another modality, the fraction is 14%. In another modality, the fraction is 16%. In another modality, the fraction is 18%. In another modality, the fraction is modality, the fraction is modality, the fraction is modality, the fraction is modality, the fraction is modality
20%. In another modality,
30%. In another modality,
40%. In another modality,
50%. In another modality,
70%. In another modality, the fraction is 25%. In another, the fraction is 35%. In another, the fraction is 45%. In another, the fraction is 60%. In another, the fraction is 80%. In another modality, the fraction is 90%. In another modality, the fraction is 100%.
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In another embodiment, the fraction of a given nucleotide is less than 8%. In another modality, the fraction is less than 10%. In another modality, the fraction is less than 5%. In another modality, the fraction is less than 3%. In another modality, the fraction is less than 1%. In another modality, the fraction is less than 2%. In another modality, the fraction is less than 4%. In another modality, the fraction is less than 6%. In another modality, the fraction is less than 12%. In another modality, the fraction is less than 15%. In another modality, the fraction is less than 20%. In another modality, the fraction is less than 30%. In another modality, the fraction is less than 40%. In another modality, the fraction is less than 50%. In another modality, the fraction is less than 60%. In another modality, the fraction is less than 70%.
In another embodiment, the terms "ribonucleotide," "oligoribonucleotide", and polyribonucleotide refer to a sequence of at least 2 combinations of sugar-phosphate bases. The term includes, in another embodiment, compounds comprising the nucleotides in which the sugar moiety is ribose. In another embodiment, the term includes RNA and RNA derivatives in which the backbone is modified. Nucleotides refer, in another embodiment, to the monomeric units of nucleic acid polymers. RNA can be, in another embodiment, in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, small interfering RNA (siRNA ), micro RNA (miRNA) and ribozymes. The use of siRNA and miRNA has been described (Caudy AA et al, Genes and Devel 16: 2491-96 and references cited here). In addition, these forms of RNA can be single, double, triple, quadruple strand. The term also includes, in another embodiment, artificial nucleic acids that may contain other types of backbones, but the same bases. In another embodiment, the nucleic acid is an artificial PNA (peptide nucleic acid). PNAs contain the backbones of peptides and nucleotide bases and are capable of binding, in another modality, to both DNA and RNA molecules. In another embodiment, the nucleotide is modified oxetane. In another embodiment, the nucleotide is modified by replacing one or more phosphodiester bonds with a phosphorothioate bond. In another embodiment, the artificial nucleic acid contains any other variant of the phosphate structure of native nucleic acids known in the art. The use of nucleic acids and phosphotiorates and PNA are known to those of skill in the art, and are described in, for example, Neilsen PE, Curr Opin Struct Biol 9: 353-57 ~ and Raz NK et al Biochem Biophys Res Commun. 297: 1075-84. Production and
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The use of nucleic acids is known to those skilled in the art and is described, for example, in Molecular Cloning, (2001), Sambrook and Russell, eds. and Methods in Enzymology: Methods for molecular cloning in eukaryotic cells (2003) Purchio and G. C. Fareed. . Each nucleic acid derivative represents a separate embodiment of the present invention
In another embodiment, the term oligoribonucleotide refers to a chain that comprises less than 25 nucleotides (nt). In another embodiment, "oligoribonucleotide" refers to a chain of less than 24 nucleotides. In another embodiment, "oligoribonucleotide" refers to a chain of less than 23 nucleotides. In another embodiment, "oligoribonucleotide" refers to a chain of less than 22 nucleotides. In another embodiment, “oligoribonucleotide” refers to a chain of less than 21 nucleotides. In another embodiment, "oligoribonucleotide" refers to a chain of less than 20 nucleotides. In another embodiment, “oligoribonucleotide” refers to a chain of less than 19 nucleotides. In another embodiment, “oligoribonucleotide” refers to a chain of less than 18 nucleotides. In another embodiment, "oligoribonucleotide" refers to a chain of less than 17 nucleotides. In another embodiment, "oligoribonucleotide" refers to a chain of less than 16 nucleotides. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the term "polyribonucleotide" refers to a chain that comprises more than 25 nucleotides (nt). In another embodiment, "polyribonucleotide" refers to a chain of more than 26 nucleotides. In another embodiment, "polyribonucleotide" refers to a chain of more than 28 nucleotides. In another embodiment, the term refers to a sequence of more than 30 nucleotides. In another modality, the term refers to a
sequence in more in 32 nucleotides. In another modality, O term refers to The an sequence in more in 35 nucleotides. In another modality, O term refers to The an sequence in more in 40 nucleotides. In another modality, O term refers to The an sequence in more in 50 nucleotides. In another modality, O term refers to The an sequence in more in 60 nucleotides. In another modality, O term refers to The an sequence in more in 80 nucleotides. In another modality, O term refers to The an sequence in more in 100 nucleotides. In another modality, O term refers to The an sequence in more in 120 nucleotides. In another modality, O term refers to The an
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59/141 sequence of more than 150 nucleotides. In another embodiment, the term refers to a sequence of more than 200 nucleotides. In sequence of more than 300 nucleotides. In sequence of more than 400 nucleotides. In another embodiment, the term refers to another modality, the term refers to another modality, the term refers to a sequence of more than
500 nucleotides.
In another embodiment, the term refers to a sequence of more than 600 nucleotides. In another embodiment, the term refers to a sequence of more than 800 nucleotides. sequence of more than 1000 nucleotides. sequence of more than 1200 nucleotides. sequence of more than 1400 nucleotides. sequence of more than 1600 nucleotides. sequence of more than 1800 nucleotides.
In another modality, the term refers to an In another modality, the term refers to an
In another modality, the term refers to a
In another modality, the term refers to a
In another modality, the term refers to a
In another embodiment, the term refers to a sequence of more than 2000 nucleotides. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the present invention provides a method for inducing a mammalian cell to produce a protein of interest, comprising contacting the mammalian cell with an in vitro synthesized RNA molecule encoding the recombinant protein, an in vitro synthesized RNA molecule it comprises a pseudouridine or a modified nucleoside, thereby inducing a mammalian cell to produce a protein of interest. In another embodiment, the protein of interest is a recombinant protein. Each possibility represents a separate embodiment of the present invention.
Encoding refers, in another embodiment, to an RNA molecule that encodes the protein of interest. In another embodiment, the RNA molecule comprises an open reading frame that encodes the protein of interest. In another embodiment, one or more proteins are also encoded. In another embodiment, the protein of interest is the only protein encoded. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the present invention provides a method for inducing a mammalian cell to produce a recombinant protein, comprising contacting the mammalian cell with an in vitro transcribed RNA molecule encoding the recombinant protein, the in vitro transcribed RNA molecule further comprises a pseudouridine or a
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60/141 modified nucleoside, inducing a mammalian cell to produce a recombinant protein.
In another embodiment, an RNA molecule, oligoribonucleotide, or polyribonucleotide of the methods and compositions of the present invention is translated into the cell more efficiently than an unmodified RNA molecule with the same sequence. In another embodiment, the RNA molecule, oligoribonucleotide, or polyribobonucleotide, exhibits greater capacity to be translated by a target cell. In another modality, the translation is improved by a factor of 2 compared to its unmodified counterpart. In another modality, the translation is improved by a factor of 3 times. In another modality, the translation is improved by a factor of 5 times. In another modality, the translation is improved by a factor of 7 times. In another modality, the translation is improved by a factor of 10 times. In another modality, the translation is improved by a factor of 15 times. In another modality, the translation is improved by a factor of 20 times. In another modality, the translation is improved by a factor of 50 times. In another modality, the translation is improved by a factor of 100 times. In another modality, the translation is improved by a factor of 200 times. In another modality, the translation is reinforced by a factor of 500 times. In another modality, the translation is improved by a factor of 1000 times. In another modality, the translation is reinforced by a factor of 2000 times. In another embodiment, the factor is 10-1000 times. In another modality, the factor is 10-100- times. In another modality, the factor is 10-200 times. In another mode, the factor is 10-300 times. In another mode, the factor is 10-500 times. In another embodiment, the factor is 20-1000 times. In another embodiment, the factor is 30-1000 times. In another embodiment, the factor is 50-1000 times. In another modality, the factor is 1001000 times. In another embodiment, the factor is 200-1000 times. In another modality, translation is enhanced by any significant amount or range of values. Each possibility represents a separate embodiment of the present invention.
Methods of determining translation efficiency are well known in the art, and include, for example, measuring the activity of an encoded reporter protein (for example, luciferase or renilla or [examples here] or fluorescent green protein [[Wall AA, Phillips AM et al, Effective translation of the second cistron in two Drosophila dicistronic transcripts is determined by the absence of in-frame AUG codons in the first cistron. J Biol Chem 2005; 280 (30): 27670-8]), or by measuring radioactive marking incorporated into the protein
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61/141 translated (Ngosuwan J, Wang NM et al, Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-chaperones moleculars in post-translational translocation of presecretory proteins into the endoplasmic reticulum J Biol Chem 2003; 278 (9): 7034. -42). Each method represents a separate embodiment of the present invention.
In some expression studies provided here, translation was measured from RNA complexed with Lipofectin ® (Gibco BRL, Gaithersburg, MD, USA) and injected into the tail vein of rats. In spleen lysates, modified pseudouridine RNA was translated significantly more efficiently than unmodified RNA (Figure 17B). Under the conditions used here, the transfection efficiency based on methods of the present invention correlates with the transfection reagent's ability to penetrate tissues, providing an explanation for the most pronounced effect on spleen cells. Splenic blood flow is an open system, with blood content directly in contact with elements of red and white pulp, including lymphoid cells.
In another experiment, in vitro phosphorylation assays were performed using recombinant human PKR and its substrate, eIF2a in the presence of coding mRNA for capped renilla (0.5 and 0.05 ng / uL). mRNA containing pseudouridine (Ψ) did not activate PKR, as detected by the lack of both PKR auto-phosphorylation and e! F2u phosphorylation, while RNA without nucleoside modification and m5C-modified mRNA activated PKR. phosphorylated efF2u is known to block the initiation of mRNA translation, therefore, the lack of phosphorylation allows, in another modality to increase translation, the mRNA containing pseudouridine (Ψ).
In another embodiment, the improved translation is in a cell (in relation to the translation in the same cell of an unmodified RNA molecule with the same sequence; Examples 13-14). In another embodiment, translation is improved in vitro (for example, in a mixture of in vitro translation or a reticulocyte lysate. Examples 13-14 In another embodiment, translation is improved in vivo (Example 13) In each case, the improved translation, relates to an unmodified RNA molecule with the same sequence, under the same conditions, each possibility representing a separate embodiment of the present invention.
In another embodiment, the RNA, oligoribonucleotide or polyribonucleotide molecule of the methods and compositions of the present invention is significantly less immunogenic than an unmodified RNA molecule synthesized in vitro with the
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62/141 same sequence. In another embodiment, the modified RNA molecule is 2-times less immunogenic than its unmodified counterpart. In another embodiment, immunogenicity is reduced by a factor of three. In another embodiment, immunogenicity is reduced by a factor of 5. In another modality, immunogenicity is reduced by a factor of 7 times. In another embodiment, immunogenicity is reduced by a factor of 10. In another modality, immunogenicity is reduced by a factor of 15 times. In another modality, immunogenicity is reduced by a factor of 20. In another embodiment, immunogenicity is reduced by a factor of 50. In another embodiment, immunogenicity is reduced by a factor of 100. In another modality, immunogenicity is reduced by a factor of 200 times. In another modality, immunogenicity is reduced by a factor of 500 times. In another embodiment, immunogenicity is reduced by a factor of 1000 times. In another modality, immunogenicity is reduced by a factor of 2000 times. In another embodiment, immunogenicity is reduced by a different factor.
In another embodiment, significantly less immunogenic refers to a detectable decrease in immunogenicity. In another embodiment, the term refers to a decrease in immunogenicity times (for example, one of the dimunition factors listed above). In another embodiment, the term refers to a decrease such that an effective amount of the RNA, oligoribonucleotide, or polyribonucleotide molecule can be administered without triggering a detectable immune response. In another embodiment, the term refers to a decrease in such a way that the RNA, oligoribonucleotide, or polyribonucleotide molecule can be repeatedly administered without inducing an immune response sufficient to detectably reduce the expression of the recombinant protein. In another embodiment, the decrease is such that an RNA, oligoribonucleotide, or polyribonucleotide molecule can be repeatedly administered without inducing an immune response sufficient to eliminate the detectable expression of the recombinant protein.
Effective amount of an RNA, oligoribonucleotide, or polyribonucleotide molecule, in another embodiment, refers to an amount sufficient to exert a therapeutic effect. In another embodiment, the term refers to a sufficient amount
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63/141 to elicit the expression of a detectable amount of the recombinant protein. Each possibility represents a separate embodiment of the present invention.
Reduced immunogenicity of RNA, oligoribonucleotide, and polyribonucleotide molecules of the present invention is demonstrated here (Examples 4-11).
Methods for determining immunogenicity are well known in the art, and include, for example, measurement of cytokine secretion (for example, IL-12, IFN-α, TNF-α, RANTES, MlP-Ια or β, IL-6, IFN -β, or IL-8; Examples here), measuring the expression of DC activation markers (for example, CD83, HLA-DR, CD80 and CD86; Examples here), or measuring the ability to act as an adjunct to a response adaptive immune system. Each method represents a separate embodiment of the present invention.
In another embodiment, the relative immunogenicity of the modified nucleotide and its unmodified counterpart are determined by determining the amount of the modified nucleotide needed to elicit one of the above responses, of the same degree as a given amount of the unmodified nucleotide. For example, if twice as many modified nucleotides are needed to elicit the same response, then the modified nucleotide is twice less immunogenic than the unmodified nucleotide.
In another embodiment, the relative immunogenicity of the modified nucleotide and its unmodified counterpart are determined by determining the amount of cytokine (for example, IL-12, IFN-α, TNF-α, RANTES, MlP-Ια or β, IL-6 , IFN -β, or IL-8) secreted in response to administration of the modified nucleotide, in relation to the same amount of the unmodified nucleotide. For example, if a medium, at most, of cytokine is secreted, then the modified nucleotide is twice less immunogenic than the unmodified nucleotide. In another embodiment, background levels of stimulation are subtracted before calculating immunogenicity in the above methods. Each possibility represents a separate embodiment of the present invention.
In another embodiment, a method of the present invention further comprises mixing RNA, oligoribonucleotide, or polyribonucleotide molecules with a transfection reagent before the contact step. In another embodiment, a method of the present invention further comprises administering RNA, oligoribonucleotide, or polyribonucleotide molecules together with the transfection reagent. In another embodiment, the transfection reagent is a cationic lipid reagent (Example 6).
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In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin® or Lipofectamine®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.
In another embodiment, the transfection reagent forms a liposome. Liposomes, in another modality, increase intracellular stability, increase absorption efficiency and improve biological activity. In another embodiment, liposomes are hollow spherical vesicles composed of lipids in an arrangement similar to those lipids that form the cell membrane. They have, in another embodiment, an aqueous internal space for capturing water-soluble compounds and vary in size from 0.05 to several microns in diameter. In another embodiment, liposomes can deliver RNA to cells in a biologically active form.
Each type of transfection reagent represents a separate embodiment of the present invention.
In another embodiment, the target cell of the methods of the present invention is an antigen presenting cell. In another embodiment, the cell is an animal cell. In another embodiment, the cell is a dendritic cell (Example 14). In another embodiment, the cell is a neural cell. In another embodiment, the cell is a brain cell (Example 16). In another embodiment, the cell is a spleen cell. In another embodiment, the cell is a lymphoid cell. In another embodiment, the cell is a lung cell (Example 16). In another embodiment, the cell is a skin cell. In another embodiment, the cell is a keratinocyte. In another embodiment, the cell is an endothelial cell. In another embodiment, the cell is an astrocyte, a microglial cell, or a neuron (Example 16). In another embodiment, the cell is an alveolar cell (Example 16). In another embodiment, the cell is an alveolar surface cell (Example 16). In another embodiment, the cell is an alveolar macrophage. In another embodiment, the cell is an alveolar pneumocyte. In another embodiment, the cell is a vascular endothelial cell. In another embodiment, the cell is a mesenchymal cell. In another embodiment, the cell is an epithelial cell. In another embodiment, the cell is a cell
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65/141 hematopoietic. In another embodiment, the cell is the cell of the colon epithelium. In another embodiment, the cell is a cell in the lung epithelium. In another embodiment, the cell is a bone marrow cell.
In other embodiments, the target cell is a Claudius cell, Hensen cell, Merkel cell, Muller cell, Paneth cell, Purkinje cell, Schwann cells, Sertoli cell, acidophilic cell, acinar cell, adipoblast, adipocytes, alpha cell white or brown, amacrine cells, beta cell, capsular cell, cementocyte, main cell, chondroblast, chondrocytes, chromaffin cells, chromophobic cell, corticotrophic, delta cells, Langerhans cells, follicular dendritic cells, enterochromaffin cells, ependymocytes, epithelial cells, cells basal cells, squamous cells, endothelial cell, transition cells, erythroblasts, erythrocytes, fibroblasts, fibrocyte, follicular cell, germ cells, gametes, egg, sperm, egg, primary oocyte, secondary oocyte, spermatoids, spermatocytes, primary spermatocyte, secondary spermatocyte germinal epithelium, giant cells, cells glials, astroblasts, astrocytes, oligodendroblastoids, oligodendrocytes, glioblasts, goblet cells, gonadotrophic cells, granulosa cells, hemocytoblasts, hair cells, hepatoblasts, hepatocytes, cells, interstitial hyalocyte, justaglomerular cells, basal cells, keratinocytes, keratinocytes, keratinocytes, ceramics eosinophils, neutrophils, lymphoblasts, B-lymphoblasts, T-lymphoblasts, lymphocytes, B-lymphocytes, T-lymphocytes, induced T-lymphocyte helper, T-lymphocyte Th1 cells, Th2 T-lymphocytes, natural killer cells, thymocytes, macrophages, Kup cell , alveolar macrophage, foam cell, histiocytes, luteal cell, lymphoid stem cells, lymphoid stem cells, lymphoid stem cells, macroglial cells, mamotrophic cells, mast cells, medulloblasts, megakaryoblasts, megakaryocytes, melanoblasts, mesangial melanocyte cells, mesothelial cells, metothelial cells, metothelial cells, metothelial cells, monoblasts, monocytes, neck mucosa cells muscle cells, cardiac muscle cells, skeletal muscle cells, smooth muscle cells, myelocyte, myeloid cell, myeloid stem cells, myoblasts, myoepithelial cells, myofibroblast, neuroblasts, neuroepithelial cells, neuron, odontoblast, osteoblasts, osteoclasts, osteocytes, osteocytes, osteocytes, osteocytes oxyntic cell, parafolicular cells, paraluteal cell, peptic cell, pericyte, peripheral blood mononuclear cells, cell, phalangeal pherochromocyte, pinalocyte, pituocyte, plasma cell, platelets, podocytes, preritroblast, promonocytoe, promyeloblast, promyelocyte, retormulocyte, pronormoblastoma pigment epithelial cells of the retina,
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66/141 retinoblast, small cells, somatotrophic, stem cells, sustaining cells, teloglial cell, or zymogenic cell. Each possibility represents a separate embodiment of the present invention.
A variety of disorders can be treated using the methods of the present invention, including, but not limited to, monogenic disorders, infectious diseases, acquired diseases, cancer, and the like. Examples of monogenic disorders include ADA deficiency, cystic fibrosis, familial hypercholesterolemia, hemophilia, chronic granulomatosis disease, Duchenne muscular dystrophy, Fanconi anemia, sickle cell anemia, Gaucher disease, Hunter syndrome, X-linked SCID, and the like . In another embodiment, the disorder treated involves one of the proteins listed below. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the recombinant protein encoded by an RNA, oligoribonucleotide, or polyribonucleotide molecule of the methods and compositions of the present invention is ecto nucleside-triphosphate diphosphohydrolase.
In another embodiment, the recombinant protein is erythropoietin (EPO). In other embodiments, the recombinant encoded protein is ABCA4; ABCD3; ACADM; AGL; AGT; ALDH4AI; ALPL; AMPD1; APOA2; AVSD1; BRCD2; C1QA; C1QB; C1QG; C8A; C8B; CACNA1S; CCV; CD3Z; CDC2L1; CHML; CHS1; CIAS1; CLCNKB; CMD1A; CMH2; CMM; COL11A1; COL8A2; COL9A2; CPT2; CRB1; CSE; CSF3R; CTPA; CTSK; DBT; DIO1; DISC1; DPYD; EKV; ENO1; ENO1P; EPB41; EPHX1; F13B; F5; FCGR2A;
FCGR2B; FCGR3A; FCHL; FH; FMO3; FMO4; FUCA1; FY; GALLEY; GBA; GFND; GJA8; GJB3; GLC3B; HF1; HMGCL; HPC1; HRD; HRPT2; HSD3B2; HSPG2; KCNQ4; KCS; KIF1B; LAMB3; LAMC2; LGMD1B; LMNA; LOR; MCKD1; MCL1; MPZ; MTHFR; MTR; MUTYH; MYOC; NB; NCF2; NEM1; NPHS2; NPPA; NRAS; NTRK1; OPTA2; PBX1; PCHC; PGD; PHA2A; PHGDH; PKLR; PKP1; PLA2G2A; PLOD; PPOX; PPT1; PRCC; PRG4; PSEN2; PTOS1; REN; RFX5; RHD; RMD1; RPE65; SCCD; SERPINC1; SJS1; SLC19A2; SLC2A1; SPG23; SPTA1; TAL1; TNFSF6; TNNT2; TPM3; TSHB; UMPK; UOX; UROD; USH2A; VMGLOM; VWS; WS2B; ABCB11; ABCG5; ABCG8; ACADL; ACP1; AGXT; AHHR; ALMS1; ALPP; ALS2; APOB; BDE; BDMR; BJS; BMPR2; CHRNA1; CMCWTD; CNGA3; COL3A1; COL4A3; COL4A4; COL6A3; CPSI; CRYGA; CRYGEP1; CYP1B1; CYP27A1; DBI; DES; DYSF; AND GIVE; EFEMPI; EIF2AK3;
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ERCC3; FSHR; GINGF; GLC1B; GPD2; GYPC; HADHA; HADHB; HOXD13; HPE2; IGKC; IHH; IRSI; ITGA6; KHK; KYNU; LCT; LHCGR; LSFC; MSH2; MSH6; NEB; NMTC; NPHP1; PAFAH1P1; PAX3; PAX8; PMS1; PNKD; PPH1; PROC; REG1A; SAG; SFTPB; SLC11A1; SLC3A1; SOS1; SPG4; SRD5A2; TCL4; TGFA; TMD; TPO; UGT1A @; UV24; WSS; XDH; ZAP70; ZFHX1B; ACAA1; AGS1; AGTR1; AHSG; AMT; ARMET; BBS3; BCHE; BCPM; BTD; CASR; CCR2; CCR5; CDL1; CMT2B; COL7A1; CP; CPO; CRV; CTNNB1; DEM; ETM1; FANCD2; FIH; FOXL2; GBE1; GLB1; GLC1C; GNAI2; GNATI; GP9; GPX1; HGD; HRG; ITIH1; KNG; LPP; LRS1; MCCCI; MDS1; MHS4; MITF; MLH1; MYL3; MYMY; OPA1; P2RY12; PBXP1; PCCB; POU1F1; PPARG; PROS1; PTHR1; RCA1; RHO; SCA7; SCLC1; SCN5A; SI; SLC25A20; SLC2A2; TF; TGFBR2; THPO; THRB; TKT; TM4SF1; TRH; UMPS; UQCRC1; USH3A; VHL; WS2A; XPC; ZNF35; ADH1B; ADH1C; AFP; AGA; AIH2; ALB; ASMD; BFHD; CNGA1; CRBM; DCK; DSPP; DTDP2; ELONG; ENAM; ETFDH; AND YOU; F11; FABP2; FGA; FGB; FGFR3; FGG; FSHMD1A; GC; GNPTA; GNRHR; GYPA; HCA; HCL2; HD; HTN3; HVBS6; IDUA; IF; JPD; KIT; KLKB1; LQT4; MANBA; MLLT2; MSX1; MTP; NR3C2; PBT; PDE6B; PEE1; PITX2; PKD2; QDPR; SGCB; SLC25A4; SNCA; SOD3; STATH; TAPVR1; TYS; WBS2; WFS1; WHCR; ADAMTS2; ADRB2; AMCN; AP3BI; APC; ARSB; B4GALT7; BHR1; C6; C7; CCAL2; CKN1; CMDJ; CRHBP; CSF1R; DHFR; DIAPH1; DTR; EOS; EPD; ERVR; F12; FBN2; GDNF; GHR; GLRA1; GM2A; HEXB; HSD17B4; ITGA2; KFS; LGMD1A; LOX; LTC4S; MAN2A1; MCC; MCCC2; MSH3; MSX2; NR3C1; PCSK1; PDE6A; PFBI; RASAI; SCZDI; SDHA; SGCD; SLC22A5; SLC26A2; SLC6A3; SM1; SMA @; SMN1; SMN2; SPINK5; TCOF1; TELAB1; TGFBI; ALDH5A1; ARG1; AT; ASSP2; BCKDHB; BF; C2; C4A; CDKN1A; COL10A1; COL11A2; CYP21A2; DYX2; EJM1; ELOVL4; EPM2A; ESR1; EYA4; F13A1; FANCE; GCLC; GJA1; GLYS1; GMPR; GSE; HCR; HFE; HLA-A; HLA-DPBI; HLA-DRA; HPFH; ICS1; IDDM1; IFNGR1; IGAD1; IGF2R; ISCW; LAMA2; LAP; LCA5; LPA; MCDR1; MOCS1; MUT; MYB; NEU1; NKS1; NYS2; OA3; ODDD; OFC1; PARK2; PBCA; PBCRA1; PDB1; PEX3; PEX6; PEX7; PKHD1; PLA2G7; PLG; POLH; PPAC; PSORS1; PUJO; RCD1; RDS; RHAG; RP14; RUNX2; RWS; SCA1; SCZD3; SIASD; SOD2; ST8; TAPl; TAP2; TFAP2B; TNDM; TNF; TPBG; TPMT; TULP1; WISP3; AASS; ABCB1; ABCB4; ACHE; AQP1; ASL; ASNS; AUTS1; BPGM; BRAF; C7orf2; CACNA2D1; CCM1; CD36; CFTR; CHORDOMA; CLCN1; CMH6; CMT2D; COL1A2;
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CRS; CYMD; DFNA5; D, L [D; DYT11; EEC1; ELN; ETV1; FKBP6; GCK; GHRHR; GHS; GLI3; GPDS1; GUSB; HLXB9; HOXA13; HPFH2; HRX; IAB; IMMP2L; KCNH2; LAMB1; LEP; MET; NCF1; NM; OGDH; OPN1SW; PEX1; PGAM2; PMS2; PON1; PPP1R3A; PRSS1; PTC; PTPN12; RP10; RP9; SERPINE1; SGCE; SHFM1; SHH; SLC26A3; SLC26A4; SLOS; SMAD1; TBXAS1; TWIST; ZWS1; ACHM3; ADRB3; ANK1; CA1; CA2; CCAL1; CLN8; CMT4A; CNGB3; COH1; CPP; CRH; CYP11B1; CYP11B2; DECR1; DPYS; DURS1; EBS1; ECA1; EGI; EXT1; EYA1; FGFR1; GNRH1; GSR; GULOP; HR; KCNQ3; KFM; KWE; LGCR; LPL; MCPH1; MOS; MYC; NAT1; NAT2; NBS1; PLAT; PLEC1; PRKDC; PXMP3; RP1; SCZD6; SFIPC; SGM1; SPG5A; STAR; TG; TRPS1; TTPA; VMD1; WRN; ABCA1; ABL1; ABO; ADAMTS13; AK1; ALAD; ALDH1A1; ALDOB; AMBP; AMCD1; ASS; BDMF; BSCL; C5; CDKN2A; CHAC; CLA1; CMD1B; COL5A1; CRAT; DBH; DNAI1; DYS; DYT1; ENG; FANCC; FBP1; FCMD; FRDA; GALT; GLDC; GNE; GSM1; GSN; HSD17B3; HSN1; IBM2; INVS; JBTS1; LALL; LCCS1; LCCS; LGMD2H; LMX1B; MLLT3; MROS; MSSE; NOTCH1; ORM1; PAPPA; PIP5K1B; PTCH; PTGS1; RLN1; RLN2; RMRP; ROR2; RPD1; SARDH; SPTLC1; STOM; TDFA; TEK; TMC1; TRIM32; TSC1; TYRP1; XPA; CACNB2; COL17Al; CUBN; CXCL12; CYP17; CYP2C19; CYP2C9; EGR2; EMX2; ERCC6; FGFR2; HK1; HPS1; IL2RA; LGI1; LIPA; MAT1A; MBL2; MKI67; MXI1; NODAL; OAT; OATL3; PAX2; PCBD; PEO1; PHYH; PNLIP; PSAP; PTEN; RBP4; RDPA; RET; SFTPA1; SFTPD; SHFM3; SIAL; THC2; TLX1; TNFRSF6; UFS; UROS; AA; ABCC8; ACAT1; ALX4; AMPD3; ANC; APOA1; APOA4; APOC3; ATM; BSCL2; BWS; PANTS; CAT; CCND1; CD3E; CD3G; CD59; CDKN1C; CLN2; CNTF; CPT1A; CTSC; DDB1; DDB2; DHCR7; DLAT; DRD4; ECB2; DI4; EVR1; EXT2; F2; FSHB; FTH1; G6PT1; G6PT2; GIF; HBB; HBBP1; HBD; HBE1; HBG1; HBG2; HMBS; HND; HOMG2; HRAS; HVBS1; IDDM2; IGER; INS; JBS; KCNJ11; KCNJ1; KCNQ1; LDHA; LRP5; MEN1; MLL; MYBPC3; MYO7A; NNO1; OPPG; OPTB1; PAX6; PRAÇA; PDX1; PGL2; PGR; PORC; PTH; PTS; PVRL1; PYGM; RAG1; RAG2; ROM1; RRAS2; SAA1; SCA5; SCZD2; SDHD; SERPING1; SMPD1; TCIRG1; TCL2; TECTA; TH; TREH; TSG101; TYR; USH1C; VMD2; VRNI; WT1; WT2; ZNFl45; A2M; AAAS; ACADS; ACLS; ACVRL1; ALDH2; AMHR2; AOM; AQP2; ATD; ATP2A2; BDC; C1R; CD4; CDK4; CNA1; COL2A1; CYP27B1; DRPLA; ENUR2; FEOM1; FGF23; FPF; GNB3; GNS; HAL; HBP1; HMGA2; HMN2; HPD; IGF1; KCNA1; KERA; KRAS2; KRT1; KRT2A; KRT3;
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KRT4; KRT5; KRT6A; KRT6B; KRTHB6; LDHB; LYZ; MGCT; MPE; MVK; MYL2; OAP; PAH; PPKB; PRB3; PTPN11; PXR1; RLS; RSN; SAS; SAX1; SCA2; SCNN1A; SMAL; SPPM; SPSMA; TBX3; TBX5; TCF1; TPI1; TSC3; ULR; VDR; VWF; ATP7B; BRCA2; BRCD1; CLN5; CPB2; DI2; EDNRB; ENUR1; ERCC5; F10; F7; GJB2; GJB6; IPF1; MBS1; MCOR; NYS4; PCCA; RB1; RHOK; SCZD7; SGCG; SLC10A2; SLC25A15; STARP1; ZNF198; ACHM1; ARVD1; BCH; CTAA1; DAD1; DFNB5; EML1; GALC; GCH1; IBGC1; IG H@; IGHC group; IGHG1; IGHM; IGHR; IV; LTBP2; MCOP; MJD; MNG1; MPD1; MPS3C; MYH6; MYH7; NP; NPC2; PABPN1; PSEN1; PYGL; RPGRIP1; SERPINA1; SERPINA3; SERPINA6; SLC7A7; SPG3A; SPTB; TCL1A; TGMI; TITF1; TMIP; TRA @; TSHR; USH1A; VP; ACCPN; AHO2; ANCR; B2M; BBS4; BLM; CAPN3; CDAN1; CDAN3; CLN6; CMH3; CYP19; CYP1A1; CYP1A2; DYX1; EPB42; ETFA; EYCL3; FAH; FBN1; FES; HCVS; HEXA; IVD; LCS1; LIPC; MY05A; OCA2; OTSC1; PWCR; RLBP1; SLC12A1; SPG6; TPM1; UBE3A; WMS; ABCC6; ALDOA; APRT; ATP2A1; BBS2; CARD15; CATM; CDH1; CETP; CHST6; CLN3; CREBBP; CTH; CTM; CYBA; CYLD; DHS; DNASE1; DPEP1; ERCC4; FANCA; GALNS; GAN; HAGH; HBA1; HBA2; HBHR; HBQ1; HBZ; HBZP; HP; HSD11B2; IL4R; LIPB; MC1R; MEFV; MHC2TA; MLYCD; MMVP1; PHKB; PHKG2; PKD1; PKDTS; PMM2; PXE; SALL1; SCA4; SCNN1B; SCNN1G; SLC12A3; TAT; TSC2; VDI; WT3; ABR; THE HUNT; ACADVL; ACE; ALDH3A2; APOH; ASPA; AXIN2; BCL5; BHD; BLMH; BRCA1; CACD; CCA1; CCZS; CHRNB1; CHRNE; CMT1A; COL1A1; CORD5; CTNS; EPX; ERBB2; G6PC; GAA; GALK1; GCGR; GFAP; GH1; GH2; GP1BA; GPSC; GUCY2D; ITGA2B; ITGB3; ITGB4; KRT10; KRT12; KRT13; KRT14; KRT14L1; KRT14L2; KRT14L3; KRT16; KRT16L1; KRT16L2; KRT17; KRT9; MAPT; MDB; MDCR; MGI; MHS2; MKS1; MPO; MYO15A; NAGLU; NAPB; NF1; NME1; P4HB; PAFAH1B1; PECAM1; PEX12; PHB; PMP22; PRKAR1A; PRKCA; PRKWNK4; PRP8; PRPF8; PTLAH; RARA; RCV1; RMSA1; RP17; RSS; SCN4A; SERPINF2; SGCA; SGSH; SHBG; SLC2A4; SLC4A1; SLC6A4; SMCR; SOST; SOX9; SSTR2; SYM1; SYNSI; TCF2; THRA; TIMP2; TOC; TOP2A; TP53; TRIM37; VBCH; ATP8B1; BCL2; CNSN; CORD1I; CYB5; DCC; F5F8D; FECH; FEO; LAMA3; LCFS2; MADH4; MAFD1; MC2R; MCL; MYP2; NPC1; SPPK; TGFBRE; TGIF; TTR; AD2; AMH; APOC2; APOE; ATHS; BAX; BCKDHA; BCL3; BFIC; C3; CACNA1A; CCO; CEACAM5; COMP; CRX; DBA; DDU; DFNA4; DLL3; DM1; DMWD; E11S; ELA2;
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EPOR; ERCC2; ETFB; EXT3; EYCLI; FTL; FUT1; FUT2; FUT6; GAMT; GCDH; GPI; GUSM; HB1; HCL1; HHC2; HHC3; ICAM3; INSR; JAK3; KLK3; LDLR; LHB; LIG1; LOH19CR1; LYL1; MAN2B1; MCOLN1; MDRV; MLLT1; NOTCH3; NPHS1; OFC3; OPA3; PEPD; PRPF31; PRTN3; PRX; PSG1; PVR; RYR1; SLC5A5; SLC7A9; STK11; TBXA2R; TGFB1; TNNI3; TYROBP; ADA; AHCY; AVP; CDAN2; CDPD1; CHED1; CHED2; CHRNA4; CST3; EDN3; EEGV1; FTLL1; GDF5; GNAS; GSS; HNF4A; JAG1; KCNQ2; MKKS; NBIA1; PCK1; PI3; PPCD; PPGB; PRNP; THBD; TOP1; AIRE; APP; CBS; COL6Al; COL6A2; CSTB; DCR; DSCR1; FPDMM; HLCS; HPE1; ITGB2; KCNE1; KNO; PRSS7; RUNX1; SOD1; TAM; ADSL; ARSA; BCR; CECR; CHEK2; COMT; CRYBB2; CSF2RB; CTHM; CYP2D6; CYP2D7P1; DGCR; DAY 1; EWSR1; GGT1; MGCR; MN1; NAGA; NE2; OGS2; PDGFB; PPARA; PRODH; SCO2; SCZD4; SERPIND1; SLC5AI; SOXI0; TCN2; TIMP3; TST; YOU F; ABCD1; ACTL1; ADFN; AGMX2; AHDS; AIC; AIED; AIH3; ALAS2; AMCD; AMELX; ANOP1; AIR; ARAF1; ARSC2; ASS; ARTS; ARX; ASAT; ASSP5; ATP7A; ATRX; AVPR2; BFLS; BGN; BTK; BZX; C1HR; CACNA1F; CALB3; CBBM; CCT; CDR1; CFNS; CGF1; CHM; CHR39C; CIDX; CLA2; CLCN5; CLS; CMTX2; CMTX3; CND; COD1; COD2; COL4A5; COL4A6; CPX; CVD1; CYBB; DCX; DFN2; DFN4; DFN6; DHOF; DIAPH2; DKC1; DMD; DSS; DYT3; EBM; EBP; DI1; ELK1; IN D; EVR2; F8; F9; FCP1; FDPSL5; FGD1; FGS1; FMR1; FMR2; G6PD; GABRA3; GATA1; GDI1; GDXY; GJB1; GK; GLA; GPC3; GRPR; GTD; GUST; HMS1; HPRT1; HPT; HTC2; HTR2C; HYR; IDS; IHG1; IL2RG; INDX; IP1; IP2; JMS; KAL1; KFSD; LICAM; LAMP2; MAA; MAFD2; MAOA; MAOB; MCF2; MCS; MEAX; MECP2; MF4; MGCI; MIC5; MID1; MLLT7; MLS; MRSD; MRX14; MRX1; MRX20; MRX2; MRX3; MRX40; MRXA; MSD; MTMI; MYCL2; MYPI; NDP; NHS; NPHLI; NROBI; NSX; NYSI; NYX; OAI; OASD; OCRL; ODTI; OFD1; OPA2; OPD1; OPEM; OPN1LW; OPN1MW; OTC; P3; PDHA1; PDR; PFC; PFKFB1; PGK1; PGK1P1; PGS; PHEX; PHKA1; PHKA2; PHP; PIGA; PLP1; POF1; POLA; POU3F4; PPMX; PRD; PRPSI; PRPS2; PRS; RCCP2; RENBP; RENS1; RP2; RP6; RPGR; RPS4X; RPS6KA3; RS1; S11; SDYS; SEDL; SERPINA7; SH2D1A; SHFM2; SLC25A5; SMAX2; SRPX; SRS; STS; SYN1; SYP; TAF1; TAZ; TBX22; TDD; TFE3; THAS; THC; TIMM8A; TIMP1; TKCR; TNFSF5; UBE1; UBE2A; WAS; WSN; WTS; WWS; XIC; XIST; XK; XM; XS; ZFX; ZIC3; ZNF261; ZNF41; ZNF6; AMELY; ASSP6; AZFI; AZF2; DAZ; GCY; RPS4Y; SMCY;
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SRY; ZFY; ABAT; AEZ; AFA; AFD1; ASAH1; ASD1; ASMT; CCAT; CECR9; STRAIN; CLA3; CLN4; CSF2RA; CTSI; DF; DIH1; DWS; DYT2; DYT4; EBR3; ECT; EEF1A1L14; EYCL2; FANCB; GCSH; GCSL; GIP; GTS; HHG; HMI; HOAC; HOKPP2; HRPT1; HSD3B3; HTC1; HV1S; ICHQ; ICR1; ICR5; IL3RA; KAL2; KMS; KRT18; KSS; LCAT; LHON; LIMM; MANBB; MCPH2; MEB; MELAS; MIC2; MPFD; MS; MSS; MTATP6; MTCOI; MTCO3; MTCYB; MTND1; MTND2; MTND4; MTND5; MTND6; MTRNR1; MTRNR2; MTTE; MTTG; MTTI; MTTK; MTTL1; MTTL2; MTTN; MTTP; MTTS1; NAMSD; OCD1; OPD2; PCK2; PCLD; PCOS1; PFKM; PKD3; PRCA1; PRO1; PROP1; RBS; RFXAP; RP; SHOX; SLC25A6; SPG5B; STO; SUOX; THM; or TTD. Each recombinant protein represents a separate embodiment of this invention.
In another embodiment, the present invention provides a method for treating anemia in a subject, comprising contacting a subject's cell with an in vitro synthesized RNA molecule, the in vitro synthesized RNA molecule encoding erythropoietin, thereby treating the anemia in a subject. In another embodiment, the RNA molecule synthesized in vitro further comprises a pseudouridine or a modified nucleoside. Each possibility represents a separate embodiment of the present invention. In another embodiment, the cell is a cell of subcutaneous tissue. In another embodiment, the cell is a lung cell. In another embodiment, the cell is a fibroblast. In another embodiment, the cell is a lymphocyte. In another embodiment, the cell is a smooth muscle cell. In another embodiment, the cell is any other type of cell known in the art. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the present invention provides a method for treating a vasospasm in a subject, comprising contacting a subject's cell with an in vitro synthesized RNA molecule, the in vitro synthesized RNA molecule encoding inducible nitric oxide synthase (iNOS ), thus treating a vasospasm in a subject.
In another embodiment, the present invention provides a method for improving the survival rate of a cell in a subject, comprising contacting the cell with an RNA molecule synthesized in vitro, the RNA molecule synthesized in vitro encoding a heat shock protein , thereby improving a cell's survival rate in a subject.
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In another embodiment, the cell, whose survival rate is improved, is an ischemic cell. In another modality, the cell is not ischemic In another modality, the cell was exposed to an ischemic environment. In another modality, the cell was exposed to environmental stress. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the present invention provides a method of decreasing the incidence of restenosis of a blood vessel by following a procedure that enlarges the blood vessel, comprising contacting a blood vessel cell with an in vitro synthesized RNA molecule, the RNA synthesized in vitro encoding a heat shock protein molecule, thereby decreasing an incidence of restenosis in a subject.
In another modality, the procedure is an angioplasty. In another embodiment, the procedure is any other procedure known in the art that enlarges the blood vessel. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the present invention provides a method for increasing hair growth from a hair follicle that is a subject's scalp, comprising contact of a scalp cell with an in vitro synthesized RNA molecule, a molecule of RNA synthesized in vitro encoding a telomerase protein or an immunosuppressant, thereby increasing hair growth from a hair follicle.
In another modality, the immunosuppressive protein is the melanocyte alpha stimulating hormone (α-MSH). In another modality, the immunosuppressive protein is transforming the growth factor β-1 (TGF-β 1). In another modality, the immunosuppressive protein is an insulin-like growth factor (IGF-I). In another embodiment, the immunosuppressive protein is any other immunosuppressive protein known in the art. Each possibility represents a separate embodiment of the present invention.
In another embodiment the present invention provides a method for inducing the expression of an enzyme with antioxidant activity in a cell, comprising contact of the cell with an RNA molecule synthesized in vitro, the RNA molecule synthesized in vitro encoding the enzyme, thereby inducing the expression of an enzyme with antioxidant activity in a cell.
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In one embodiment, the enzyme is catalase. In another embodiment, the enzyme is glutathione peroxidase. In another embodiment, the enzyme is phospholipid hydroperoxide glutathione peroxidase. In another embodiment, the enzyme is superoxide dismutase-1. In another embodiment, the enzyme is superoxide dismutase-2. In another embodiment, the enzyme is any other enzyme with antioxidant activity that is known in the art. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the present invention provides a method for treating cystic fibrosis in a subject, comprising contacting a subject's cell with an in vitro synthesized RNA molecule, the in vitro synthesized RNA molecule encoding the enzyme, encoding the regulator of transmembrane conductance in cystic fibrosis (CFTR), thus treating cystic fibrosis in a subject.
In another embodiment, the present invention provides a method for treating an X-linked agammaglobulinemia in a subject, comprising contacting a subject's cell with an in vitro synthesized RNA molecule, the in vitro synthesized RNA molecule encoding tyrosine Bruton kinase thus treating an X-linked agammaglobulinemia.
In another embodiment, the present invention provides a method for treating Severe Combined Immunodeficiency due to adenosine deaminase deficiency (ADA SCID) in a subject, comprising contacting a subject's cell with an in vitro synthesized RNA molecule, the molecule of RNA synthesized in vitro encoding adenosine deaminase (ADA) thus, treating a Severe Combined Immunodeficiency due to adenosine deaminase deficiency (ADA SCID).
In another embodiment, the present invention provides a method for reducing the skin's immune response capacity and improving the skin pathology, comprising contact of a subject cell with an in vitro synthesized RNA molecule, the RNA molecule synthesized in vitro. encoding an ecto-nucleoside triphosphate diphosphohydrolase ', thereby reducing the skin's immune response capacity and improving skin pathology.
In another embodiment, an RNA molecule or ribonucleotide molecule of the present invention is encapsulated in nanoparticles. Methods for packaging the nanoparticles are well known in the art, and are described, for example, in Bose S, et al Bose S, et al (Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells.
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J. Virol. 78: 8146. 2004); Dong Y et al. Poli (d, l-lactide-co-glycolide) / montmorillonite nanoparticles for oral delivery of anticancer drugs. Biomaterials 26: 6068. 2005); Lobenberg R. et al (Improved body distribution of 14C-labeled AZT bound to nanoparticles in rats determined by radioluminography. J Drug Target 5: 171. 1998);); Sakuma SR et al (Mucoadhesion of polistyrene nanoparticles having surface hydrophilic polymeric chains in the gastrointestinal tract. Int J Pharm 177: 161. 1999); Virovic L et al. Novel delivery methods for treatment of viral hepatitis: an update. Expert Opin Drug Deliv 2: 707.2005); and Zimmermann E et al., Electrolyte-and pH-stabilities of aqueous solid lipid nanoparticle (SLN) dispersions in artificial gastrointestinal media. Eur J Pharm Biopharm 52: 203.2001). Each method represents a separate embodiment of the present invention.
Various dosage range modalities of the compounds of the present invention can be used in the methods of the present invention. In one embodiment, the dosage is in the range of 1-10 pg / day. In another embodiment, the dosage is 2-10 pg / day. In another embodiment, the dosage is 3-10 pg / day. In another embodiment, the dosage is 5-10 pg / day. In another embodiment, the dosage is 2-20 pg / day. In another embodiment, the dosage is 3-20 pg / day. In another embodiment, the dosage is 5-20 pg / day. In another embodiment, the dosage is 10-20 pg / day. In another embodiment, the dosage is 3-40 pg / day. In another embodiment, the dosage is 5-40 pg / day. In another embodiment, the dosage is 10-40 pg / day. In another embodiment, the dosage is 20-40 pg / day. In another embodiment, the dosage is 5-50 pg / day. In another embodiment, the dosage is 10-50 pg / day. In another embodiment, the dosage is 20-50 pg / day. In another embodiment, the dosage is 1-100 pg / day. In another embodiment, the dosage is 2-100 pg / day. In another embodiment, the dosage is 3-100 pg / day. In another embodiment, the dosage is 5-100 pg / day. In another embodiment, the dose is 10-100 pg / day. In another embodiment, the dose is 20-100 pg / day. In another embodiment, the dose is 40-100 pg / day. In another modality the dosage is 60-100 pg / day.
In another embodiment, the dosage is 0.1 µg / day. In another embodiment, the dosage is 0.2 pg / day. In another modality, the dosage is 0.3 pg / day. In another modality, the dosage is 0.5 pg / day. In another modality, the dosage is 1 pg / day. In another embodiment, the dosage is 2 mg / day. In another modality, the dosage is 3 pg / day. In another modality, the dosage is 5 pg / day. In another modality the dosage is 10 pg / day. In another modality, the dosage is 15 pg / day. In another modality, the dosage is 20
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75/141 pg / day. In another modality, the dosage is 30 pg / day. In another modality, the dosage is 40 pg / day. In another modality, the dosage is 60 pg / day. In another modality, the dosage is 80 ug / day. In another modality, the dosage is 100 pg / day.
In another embodiment, the dosage is 10 pg / dose. In another modality, the dosage is 20 pg / dose. In another modality, the dosage is / dose. In another embodiment, the dosage is 30 pg / dose. In another embodiment, the dosage is 40 pg / dose. In another embodiment, the dosage is 60 pg / dose. In another embodiment, the dosage is 80 pg / dose. In another embodiment, the dosage is 100 pg / dose. In another embodiment, the dosage is 150 pg / dose. In another embodiment, the dosage is 200 pg / dose. In another embodiment, the dosage is 300 pg / dose. In another modality, the dosage is 400 pg / dose In another modality, the dosage is 600 pg / dose. In another embodiment, the dosage is 800 pg / dose. In another embodiment, the dosage is 1000 pg / dose. In another embodiment, the dosage is 1.5 mg / dose. In another embodiment, the dosage is 2 mg / dose. In another embodiment, the dosage is 3 mg / dose. In another embodiment, the dosage is 5 mg / dose. In another embodiment, the dosage is 10 mg / dose. In another embodiment, the dosage is 15 mg / dose. In another embodiment, the dosage is 20 mg / dose. In another embodiment, the dosage is 30 mg / dose. In another embodiment, the dosage is 50 mg / dose. In another embodiment, the dosage is 80 mg / dose. In another embodiment, the dosage is 100 mg / dose.
In another embodiment, the dosage is 10-20 pg / dose. In another embodiment, the dosage is 20-30 pg / dose. In another embodiment, the dosage is 20-40 pg / dose. In another embodiment, the dosage is 30 - 60 pg / dose. In another embodiment, the dosage is 40-80 pg / dose. In another embodiment, the dosage is 50-100 pg / dose. In another mode, the dosage is 50-150 pg / dose. In another embodiment, the dosage is 100-200 pg / dose. In another embodiment, the dosage is 200-300 pg / dose. In another embodiment, the dosage is 300-400 pg / dose. In another embodiment, the dosage is 400-600 pg / dose. In another embodiment, the dosage is 500-800 pg / dose. In another embodiment, the dosage is 800-1000 pg / dose. In another embodiment, the dosage is 1000-1500 pg / dose. In another embodiment, the dosage is 1500-2000 pg / dose. In another embodiment, the dosage is 2-3 mg / dose. In another embodiment, the dosage is 2-5 mg / dose. In another embodiment, the dosage is 2-10 mg / dose. In another embodiment, the dosage is 2-20 mg / dose. In another embodiment, the dosage is 2-30 mg / dose. In another embodiment, the dosage is 2-50 mg / dose. In another modality, the dosage is 2-80
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76/141 mg / dose. In another embodiment, the dosage is 2-100 mg / dose. In another embodiment, the dosage is 3-10 mg / dose. In another embodiment, the dosage is 3-20 mg / dose. In another embodiment, the dosage is 3-30 mg / dose. In another embodiment, the dosage is 3-50 mg / dose. In another embodiment, the dosage is 3-80 mg / dose. In another embodiment, the dosage is 3-100 mg / dose. In another embodiment, the dosage is 5-10 mg / dose. In another embodiment, the dosage is 5-20 mg / dose. In another embodiment, the dosage is 5-30 mg / dose. In another embodiment, the dosage is 5-50 mg / dose. In another embodiment, the dosage is 5-80 mg / dose. In another embodiment, the dosage is 5-100 mg / dose. In another embodiment, the dosage is 1020 mg / dose. In another embodiment, the dosage is 10-30 mg / dose. In another embodiment, the dosage is 10-50 mg / dose. In another embodiment, the dosage is 10-80 mg / dose. In another embodiment, the dosage is 10-100 mg / dose.
In another embodiment, the dosage is a daily dose. In another embodiment, the dosage is a weekly dose. In another embodiment, the dosage is a monthly dose. In another embodiment, the dosage is an annual dose. In another embodiment, the dose is one of a series of a defined number of doses. In another embodiment, the dose is a single dose. As described below, in another embodiment, an advantage of the RNA, oligoribonucleotide, or polyribonucleotide molecules of the present invention is its greater potency, allowing the use of smaller doses.
In another embodiment, the present invention provides a method for producing a recombinant protein, comprising contacting an in vitro translation apparatus with an in vitro-synthesized oligoribonucleotide, the in-vitro synthesized oligoribonucleotide comprising a pseudouridine or a modified nucleoside, thereby producing a protein recombinant.
In another embodiment, the present invention provides a method for producing a recombinant protein, comprising contacting an in vitro translation apparatus with an in vitro transcribed RNA molecule of the present invention, the in vitro transcribed RNA molecule comprising a pseudouridine or a modified nucleoside, thus producing a recombinant protein.
In another embodiment, the present invention provides an in vitro transcription apparatus, comprising: an unmodified nucleotide, a nucleotide containing a modified pseudouridine or nucleoside, and a polymerase. In another modality, the
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The present invention provides an in vitro transcription kit, comprising: an unmodified nucleotide, a nucleotide containing a pseudouridine or a modified nucleoside, and a polymerase. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the in vitro translation apparatus comprises a reticulocyte lysate. In another embodiment, the reticulocyte lysate is a rabbit reticulocyte lysate.
In another embodiment, the present invention provides a method of reducing the immunogenicity of an oligoribonucleotide molecule or RNA molecule, the method comprising the step of replacing a nucleotide of the oligorubonucleotide molecule or RNA molecule with a modified nucleotide containing a modified nucleoside or a pseudouridine, thereby reducing the immunogenicity of an oligoribonucleotide molecule or RNA molecule.
In another embodiment, the present invention provides a method of reducing the immunogenicity of a gene therapy vector that comprises a polyribonucleotide molecule or RNA molecule, the method comprising the step of replacing a nucleotide of the polyrubonucleotide molecule or RNA molecule with a modified nucleotide that contains a modified nucleoside or a pseudouridine, thereby reducing the immunogenicity of a gene therapy vector.
In another embodiment, the present invention provides a method of enhancing in vitro translation from an oligoribonucleotide molecule or RNA molecule, the method comprising the step of replacing a nucleotide of the oligoribonucleotide molecule or RNA molecule with a modified nucleotide that contains a modified nucleoside or a pseudouridine, thereby enhancing in vitro translation from an oligoribonucleotide molecule or RNA molecule.
In another embodiment, the present invention provides a method of enhancing in vivo translation from a gene therapy vector that comprises a polyribonucleotide molecule or RNA molecule, the method comprising the step of replacing a nucleotide from the polyrubonucleotide molecule or molecule of RNA with a modified nucleotide that contains a modified nucleoside or a pseudouridine, thereby increasing in vivo translation from a gene therapy vector.
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In another embodiment, the present invention provides a method of increasing the efficiency of delivery of a recombinant protein by a gene therapy vector comprising a polyribonucleotide molecule or RNA molecule, the method comprising the step of replacing a nucleotide of the polyrubonucleotide molecule or RNA molecule with a modified nucleotide that contains a modified nucleoside or a pseudouridine, thus increasing the efficiency of the delivery of a recombinant protein by a gene therapy vector.
In another embodiment, the present invention provides a method of increasing the in vivo stability of the gene therapy vector that comprises a polyribonucleotide molecule or RNA molecule, the method comprising the step of replacing a nucleotide of the polyrubonucleotide molecule or RNA molecule with a modified nucleotide containing a modified nucleoside or a pseudouridine, thereby increasing the in vivo stability of the gene therapy vector.
In another embodiment, the present invention provides a method of synthesizing an in vitro transcribed RNA molecule comprising a pseudouridine nucleoside, comprising contacting an isolated polymerase with a mixture of unmodified nucleotides and the modified nucleotides (Examples 5 and 10).
In another embodiment, the in vitro transcription methods of the present invention use an extract from an animal cell. In another embodiment, the extract is from reticulocytes or a cell with similar efficiency of in vitro transcription. In another embodiment, the extract is from any other type of cell known in the art. Each possibility represents a separate embodiment of the present invention.
Any of the RNA molecules or oligoribonucleotide molecules of the present invention can be used, in another embodiment, in any of the methods of the present invention.
In another embodiment, the present invention provides a method of enhancing an immune response to an antigen, comprising administration of the antigen, in combination with mitochondrial (mt) RNA (Examples 4 and 8).
In another embodiment, the present invention provides a method of reducing the ability of an RNA molecule to stimulate a dendritic cell (DC), comprising the
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79/141 modification of a nucleoside of the RNA molecule by a method of the present invention (for example, see EXAMPLES).
In another embodiment, the DC is a DC1 cell. In another embodiment, the DC is a DC2 cell. In another embodiment, DC is a subtype of a DC1 or DC2 cell. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the present invention provides a method of reducing the ability of an RNA molecule to stimulate TLR3 signaling, comprising modifying a nucleoside of the RNA molecule by a method of the present invention. In another embodiment, the present invention provides a method of reducing the ability of an RNA molecule to stimulate TLR7 signaling, comprising modifying a nucleoside of the RNA molecule by a method of the present invention. In another embodiment, the present invention provides a method of reducing the ability of an RNA molecule to stimulate TLR8 signaling, comprising modifying a nucleoside of the RNA molecule by a method of the present invention. Each possibility represents a separate embodiment of the present invention.
In another embodiment, all internucleoside or internucleotide bonds in the RNA, oligoribonucleotide, or polyrubonucleotide molecule are phosphodiester. In another embodiment, the inter-nucleotide bonds are predominantly phosphodiester. In another embodiment, the majority of internucleotide bonds are phosphorothioate. In another embodiment, most inter-nucleotide bonds are phosphodiester. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the percentage of inter-nucleotide bonds in the RNA, oligoribonucleotide, or polyrubonucleotide molecule that are phosphodiester is greater than 50%. In another modality, the percentage is higher than 10%. In another modality, the percentage is higher than 15%. In another modality, the percentage is higher than 20%. In another modality, the percentage is higher than 25%. In another modality, the percentage is higher than 30%. In another modality, the percentage is higher than 35%. In another modality, the percentage is higher than 40%. In another modality the percentage is higher than 45%. In another modality, the percentage is greater than 55%. In another modality, the percentage is higher than 60%. In another modality, the percentage is higher than 65%. In another modality, the percentage is higher than 70%. In another modality, the percentage is above 75%. In another
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80/141 modality, the percentage is higher than 80%. In another modality, the percentage is higher than 85%. In another modality, the percentage is greater than 90%. In another modality, the percentage is greater than 95%.
In another embodiment, a method of the present invention comprises increasing the number, percentage, or frequency of modified nucleosides in the RNA molecule to decrease immunogenicity or increase the efficiency of translation. As provided herein (for example, see EXAMPLES), the number of residues modified in an RNA, oligoribonucleotide, or polyrubonucleotide molecule determines, in another embodiment, the magnitude of the effects observed in the present invention.
In another embodiment, the present invention provides a method for introducing a recombinant protein into a subject's cell, comprising contacting the subject with an in vitro transcribed RNA molecule encoding the recombinant protein, the in vitro transcribed RNA molecule which further comprises a pseudouridine or other modified nucleoside, thereby introducing a recombinant protein into a subject's cell.
In another embodiment, the present invention provides a method for decreasing the production of TNF-α in response to a gene therapy vector in a subject, comprising the engineering step of the vector to include a pseudouridine or a modified nucleoside base, thereby decreasing the production of TNF-α in response to a gene therapy vector in a subject.
In another embodiment, the present invention provides a method for decreasing the production of IL-12 in response to a gene therapy vector in a subject, comprising the engineering step of the vector to include a pseudouridine or a modified nucleoside base, thereby decreasing the production of IL-12 in response to a gene therapy vector in a subject.
In another embodiment, the present invention provides a method of reducing the immunogenicity of a gene therapy vector, comprising introducing a modified nucleoside into said gene therapy vector, thereby reducing the immunogenicity of a gene therapy vector
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As provided in this document, the results of the present invention show that the primary DC has an additional RNA signaling entity that recognizes modified m5C and m6A RNA and whose signaling is inhibited by the modification of U residues.
In another embodiment, an advantage of the RNA, oligoribonucleotide, and polyribonucleotide molecules of the present invention is that the RNA is not incorporated into the genome (as opposed to DNA-based vectors). In another embodiment, an advantage is that the translation of RNA, and therefore the appearance of the encoded product, is instantaneous. In another embodiment, an advantage is that the amount of protein generated from the mRNA can be regulated by presenting more or less RNA. In another embodiment, an advantage is that repeated delivery of purified pseudouridine or other modified RNA molecules, oligoribnucleotides, polyribonucleotides does not induce an immune response, while repeated delivery of unmodified RNA could induce signaling pathways between RNA sensors.
In another embodiment, an advantage is the lack of immunogenicity, allowing for repeated delivery without the generation of inflammatory cytokines. In another embodiment, the stability of the RNA is increased by circularization, decreasing the degradation by exonucleases.
In another embodiment, the present invention provides a method of treating a subject with a disease that comprises an immune response against a self-RNA molecule itself, comprising administering to the subject an antagonist of a TLR-3 molecule, thereby treating the subject with a disease that comprises an immune response against an auto-RNA molecule.
In another embodiment, the present invention provides a method of treating a subject with a disease that comprises an immune response against an autoRNA molecule, comprising administering to the subject an antagonist of a TLR-7 molecule, thereby treating the subject with a disease that comprises an immune response against an auto-RNA molecule.
In another embodiment, the present invention provides a method of treating a subject with a disease that comprises an immune response against an autoRNA molecule, comprising administering to the subject an antagonist of a TLR-8 molecule, thereby treating the subject with a disease that comprises an immune response against an auto-RNA molecule.
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In another embodiment, the disease that comprises an immune response against an auto-RNA molecule is an autoimmune disease. In another modality, the disease is systemic lupus erythematosus (SLE). In another embodiment, the disease is another disease known in the art that comprises an immune response against an auto-RNA molecule. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the present invention provides a kit comprising a reagent used in carrying out a method of the present invention. In another embodiment, the present invention provides a kit comprising a composition, tool or instrument of the present invention.
In another embodiment, the present invention provides a kit for measuring or studying signaling by a TLR-3, TLR-7 and TLR-8 receptor, as exemplified in Example 7.
In another embodiment, a treatment protocol of the present invention is therapeutic. In another modality, the protocol is prophylactic. Each possibility represents a separate embodiment of the present invention.
In one embodiment, the phrase contacting a cell or contacting a population refers to a method of exposure, which can be direct or indirect. In one method, such contact comprises direct injection of the cell through any means well known in the art, such as microinjection. In another embodiment, the feeding of the cell is indirect, as through the provision of a culture medium that involves the cell, or administration to a subject, or through any route known in the art. In another embodiment, the term contacting means that the molecule of the present invention is introduced into a subject being treated, and the molecule comes into contact with the cell in vivo. Each possibility represents a separate embodiment of the present invention.
Methods for quantifying the frequency of reticulocytes and for measuring the biological activity of EPO are well known in the art, and are described, for example, in Ramos, AS et al (Biological evaluation of recombinant human erythropoietin in pharmaceutical products. Braz J Med Biol Res 36: 1561). Each method represents a separate embodiment of the present invention.
The compositions of the present invention may, in another embodiment, be administered to a subject by any method known to a person skilled in the art, such as parenteral, paracanceral, transmucosal, transdermal, intramuscular, intravenous, intradermal,
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In another embodiment of the methods and compositions of the present invention, the compositions are administered orally, and are thus formulated in a form suitable for oral administration, that is, as a solid or liquid preparation. Suitable oral solid formulations include tablets, capsules, pills, granules, lozenges and the like. Suitable oral liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In another embodiment of the present invention, the active ingredient is formulated in a capsule. According to this embodiment, the compositions of the present invention comprise, in addition to the active compound and the inert carrier or diluent, a hard gelatin capsule.
In other embodiments, the pharmaceutical composition is administered via intravenous, intra-arterial, or intramuscular injection of a liquid preparation. Suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In another embodiment, the pharmaceutical compositions are administered intravenously and are formulated in a manner suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intraarterially and are formulated in a manner suitable for intraarterial administration. In another embodiment, the pharmaceutical compositions are administered intramuscularly and are formulated in a manner suitable for administration via intramuscular.
In another embodiment, the pharmaceutical compositions are administered topically on the surfaces of the body and are formulated in a manner suitable for topical administration. Suitable formulations include gels, topical ointments, creams, lotions, drops and the like. For topical administration, the compositions or their physiologically tolerated derivatives are prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier.
In another embodiment, the composition is administered as a suppository, for example, a rectal suppository or a urethral suppository. In another embodiment, the pharmaceutical composition is administered by subcutaneous implantation of a pellet. In another embodiment, the pellet provides controlled release of the agent over a period of time.
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In another embodiment, the active compound is distributed in a vesicle, for example, a liposome (see Langer, Science 249: 1527-1533 (1990) Tratar et al, Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).
As used herein, pharmaceutically acceptable carriers or diluents are well known to those skilled in the art. The carrier or diluent can be, in various embodiments, a solid carrier or diluent for solid formulations, liquid carrier or diluent for liquid formulations for, or mixtures thereof.
In another embodiment, solid carriers / diluents include, among others, a gum, a starch (for example, starch with, pre-gelatinized starch), a sugar (for example, lactose, mannitol, sucrose, dextrose), a cellulosic material ( for example, microcrystalline cellulose), an acrylate (for example, polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
In other embodiments, pharmaceutically acceptable carriers for liquid formulations can be aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, and Including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable or of synthetic origin, for example, peanut oil, soy oil, mineral oil, olive oil, sunflower oil, and fish liver.
Parenteral vehicles (for subcutaneous, intravenous, intra-arterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer and fixed oils. Intravenous vehicles include fluid and nutrient stores, electrolyte stores like those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils such as, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related solutions of sugars, and glycols such as propylene glycol or polyethylene glycol are the preferred liquid carriers, particularly for injectable solutions. Examples of oils are Examples of oils are those of petroleum, animal, vegetable or
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In another embodiment, the compositions comprise binders (for example, acacia, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (for example, corn starch, starch potato, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (eg, Tris-HCI., acetate, phosphate) of various pH and ionic forces, additives such as albumin or gelatine, to prevent the absorption of surfaces, detergents (eg Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (eg sodium lauryl sulphate), permeation promoters, solubilizing agents (eg, glycerol, polyethylene) glycerol, antioxidants (eg, ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (eg, hydroxypropyl cellulose, hydroxypro pilmetyl cellulose), agents for increasing viscosity (eg carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (eg aspartame, citric acid), preservatives (eg, thimerosal, benzyl alcohol, parabens) , lubricants (eg stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow aids (eg colloidal silicon dioxide), plasticizers (eg, diethyl phthalate, triethyl citrate), emulsifiers.
In another embodiment, the pharmaceutical compositions provided herein are controlled release compositions, that is, compositions in which the compound is released over a period of time after administration 15. Controlled or sustained release compositions include formulation in lipophilic deposits (for example, fatty acids, waxes, oils). In another embodiment the composition is an immediate release composition, that is, a composition in which the entire compound is released immediately after administration.
In another embodiment, the molecules of the present invention are modified by covalently bonding water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. The modified compounds are known to have substantially longer half-lives in the blood after intravenous injection than the corresponding unmodified compounds (Abuchowski et al, 1981 ;. Newmark et al, 1982; ..
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And Katre et al, 1987). Such modifications also increase, in another embodiment, the solubility of the compound in aqueous solution, eliminate the aggregation, improve the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired biological activity in vivo can be achieved by administering such a compound polymer sequestering less frequently or in lower doses than with the unmodified compound.
An active component is, in another embodiment, formulated in the composition as pharmaceutically acceptable neutralized salt forms. Pharmaceutically acceptable salts include acid addition salts (for example, formed with the free amino groups of a polypeptide or antibody molecule), which are formed with inorganic acids, such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine , and the like.
Each of the above additives, excipients, formulations and methods of administration represents a separate embodiment of the present invention.
EXAMPLES
The following experimental protocols were used in the examples provided below, unless otherwise indicated.
MATERIALS AND METHODS FOR EXAMPLES 1-3
Cell culture 1079 Fibroblast cells from the foreskin of human newborns (Cat # CRL-2097, ATCC, Manassas, VA) and human IMR90 cells (Cat # CCL-186, ATCC) were grown in Advanced MEM Medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT), 2mM Glutamax (Invitrogen), 0.1mM β-mercaptoethanol (Sigma, St. Louis, MO), and Penicillin / Streptomycin (Invitrogen). All cells grew at 37 ° C and 5% CO2. In some experiments, human iPS cells that were induced using methods described here were maintained in irradiated mouse embryonic fibroblasts (MEFs) (R&D Systems, Minneapolis, MN) in 0.1 cm 10 cm pre-coated plates
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87/141 gelatin (Millipore, Phillipsburg, NJ) in DMEM / F12 medium supplemented with 20% serum replacement KnockOut, 0.1mM L-glutamine (all from Invitrogen), 0.1mM βmercaptoethanol (Sigma) and 100ng / ml factor basic fibroblast growth (Invitrogen). In some experiments, human iPS cells that were induced using the methods described here were maintained in conditioned MEF medium that was collected as previously described (Xu et al. 2001).
Construction of Vectors. The cDNAs for the open reading regions (ORFs) of KLF4, LIN28, NANOG, and OCT4 were amplified in PCR from cDNA clones (Open Biosystems, Huntsville, AL), cloned into a downstream plasmid vector from a promoter of T7 RNA polymerase (Mackie 1988, Studier and Moffatt 1986) (e.g., several pBluescript ™, Agilent, La Jolla, CA or pGEM ™, Promega, Madison, WI, vectors) and sequenced. SOX2 ORFs were amplified by PCR from a cDNA clone (Invitrogen) and c-MYC ORF was isolated by HeLa cell total RNA RT-PCR. Both SOX2 and c-MYC ORF were further cloned into a plasmid vector downstream of a T7 RNA polymerase promoter and sequenced.
Alternative plasmid vectors containing human open reading regions of (KLF4, LIN28, c-MYC, NANOG, OCT4 and SOX2) were cloned into pBluescriptII. These pBluescriptII vectors were constructed by linking the reading regions opened above at EcoRV (cMyc) or EcoRV / SpeI (KLF4, LIN28, NANOG, OCT4, and SOX2) sites between 5 'and 3' regions of non-Xenopus laevis beta-globin translated (Krieg and Melton 1984).
MRNA Production Plasmid constructs containing T7 RNA polymerase promoter (pT7-KLF4, pT7-LIN28, pT7-c-MYC, pT7-OCT4, pT7-SOX2, or pT7-XBgKLF4, pT7-XBg-LIN28, pT7-XB- c-MYC, pT7-XBg-OCT4, and pT7-XBg-SOX2) were linearized with BamHI and pT7-NANOG and pT7-XBg-NANOG were linearized with Xba I. The mSCRIPT ™ mRNA production system (EPICENTRE or CellScript, Madison, WI, USA) was used to produce mRNA with a 5 'Cap1 structure and a 3' Poly (A) tail (for example, with approximately 150 residues of A), except that pseudouridine-5'-triphosphate (TRILINK, San Diego, CA) was used in place of uridine-5'-triphosphate in T7 RNA polymerase transcription reactions in vitro.
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Purification and analysis of mRNA. In some experimental modalities, the mRNA was purified by HPLC, column fractions were collected, and the mRNA fractions were analyzed for purity and immunogenicity as described in “Materials and Methods for Examples 35-38” and / or as described and shown for Figures 22-24. In some experimental embodiments, purified RNA preparations comprising or consisting of mRNAs encoding one or more reprogramming factors that exhibited little or no immunogenicity were used for the experiments to reprogram human somatic cells to iPS cells.
Reprogramming Human Somatic Cells in MEFs. 1079 fibroblasts were plated in 1 x 10 ⁵ cells / well of a 6-well plate pre-coated with 0.1% gelatin (Millipore) and grown overnight. The 1079 fibroblasts were transfected with equal amounts of each mRNA reprogramming factor (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) using TransIT transfection reagent mRNA (MirusBio, Madison, WI). A total of three transfections were performed, with one transfection being performed on alternate days, with average changes on the day after the first and second transfections. The day after the third transfection, the cells were trypsinized in 3.3 x 10 ⁵ cells were plated in 1079 medium in 0.1% gelatin 10 cm plate pre-coated with 7.5 x 10 ⁵ MEFs on the previous day. The day after plating the 1079 fibroblasts transfected in MEFs, the medium was changed to iPS cell medium. The iPS cell medium was changed every day. Eight days after plating the transfected cells in MEFs, MEF conditioned medium was used. MEF conditioned medium was collected as previously described (Xu et al. 2001). The plates were screened every day for the presence of colonies with an iPS morphology using an inverted microscope.
Alternative protocols for reprogramming 1079 and IMR90 fibroblasts in MEFs have also been used. MEFs were plated in 1.25 x 10 ⁵ cells / well of a 6-well plate pre-coated with 0.1% gelatin and incubated overnight in complete fibroblast medium. Fibroblasts 1079 or IMR90 were plated in 3 x 10 ⁴ cells / well of a 6-well plate seeded with MEFs the previous day and grown overnight at 37 ^o C / 5% CO2. The mScript kit was then used to generate Cap1 / polyadenylated mRNA from the following vectors (pT7-X3g-KLF4, pT7-X3g-LIN28, pT7-X $ gc-MYC, pT7-X3g
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NANOG, pT7-X3g-OCT4, and pT7-X3g-SOX2) for use in these daily transfections. All of their reprogramming mRNAs were diluted to 100 ng / pl of each mRNA. The equal molarity of each mRNA was added together using the following conversion factors (OCT4 is set to 1 and all other mRNAs are multiplied by these conversion factors to obtain an equal molarity in each mRNA mix). KLF = 1.32, LIN28 = 0.58, c-MYC = 1.26, NANOG = 0.85, OCT4 = 1, and SOX2 = 0.88. To obtain equal molarity of each factor 132 pl of KLF4, 58 pl of LIN28, 126 pl of c-MYC, 85 pl of NANOG, 100 pl of OCT4 and 88 pl of SOX2 mRNA (each in 100ng / pl) could be added together. A total dose of 600pg for transfections could mean that 100ng (using molarity conversions above) of each of the six reprogramming mRNAs was used. The Trans-IT mRNA transfection reagent was used to transfect these mRNA doses. For all transfections, pools of mRNA were added to 250 pl of DMEM / F12 medium without additives or Advanced MEM medium without additives. 5pl of mRNA boosting reagent and 5pl of TransIT transfection reagent was added to each tube at room temperature for two minutes before adding the transfection mix to 2.5ml of Advanced MEM medium with 10% FBS + 100ng / ml of hFGFb or iPS medium containing 100ng / ml hFGFb. The transfections were repeated every day for 10-16 days. The medium was changed every 4 hours after each transfection. In some experiments, cells were trypsinized and replated in new MEF plates between 5-8 days after the initial transfection. 1079 cells were divided 1/6 or 1/12 on new MEF plates while IMR90 cells were divided 1/3 or 1/6 on new MEF plates.
Reprogramming Human Somatic Cells in MEF Conditioning Medium. Fibroblasts 1079 or IMR90 were plated in 3 x 10 ⁵ cells by 10 cm plates pre-coated with 0.1% gelatin (Millipore) and grown overnight. Fibroblasts 1079 or IMR90 were transfected with equal amounts of each mRNA reprogramming factor (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) using TransIT transfection reagent mRNA (MirusBio, Madison, WI). For each transfection, 6 pg, 18 pg, or 36 pg of each reprogramming mRNA (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) was used per 10 cm plate. A total of three transfections were performed, with one transfection being performed every other day with the medium being changed the day after each of the first and
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90/141 second transfections. All transfections were performed in conditioned MEF medium. The day after the third transfection, the cells were trypsinized and 3 x 10 ⁵ cells were plated on new 10 cm plates pre-coated with 0.1% gelatin (Millipore). The cells were grown in MEF medium conditioned by the duration of the experiment.
Similar daily mRNA transfections were also performed as described in the previous section with the only difference that MEFs were not used as feeder layers, only conditioned MEF medium was used.
Immunofluorescence. The 1079 cells or plates of iPS cells derived from 1079 were washed and fixed in 4% paraformaldehyde in PBS for 30 minutes at room temperature. The iPS cells were then washed 3 times for 5 minutes each wash with PBS followed by three washes in PBS + 0.1% Triton X-100. The iPS cells were then blocked in blocking buffer (PBS + 0.1% Triton, 2% FBS, and 1% BSA) for 1 hour at room temperature. The cells were then incubated for 2 hours at room temperature with the primary antibody (anti-mouse human OCT4 Cat # sc-5279, Santa Cruz Biotechnology, Santa Cruz, CA), (anti-human rabbit NANOG Cat # 3580, anti - Cat # 4038 rabbit human KLF4, Cat # 5930 mouse anti-human LIN28, Cat # 5605 rabbit anti-c-MYC human, Cat # 3579 anti-rabbit human SOX2, and CAT anti-TRA-1-60 from mouse all from Cell Signaling Technology, Beverly, MA) in a 1: 500 dilution in blocking buffer. After washing 5 times in PBS + 0.1% Triton X-100, iPS cells were incubated for 2 hours with rabbit anti-Alexa Fluor 488 antibody (Cat # 4412, Cell Signaling Technology), secondary mouse anti-FITC (Cat # F5262, Sigma), or a mouse anti-Alexa Fluor 555 (Cat # 4409, Cell Signaling Technology) in 1: 1000 dilutions in blocking buffer. The images were taken using a Nikon TS100F inverted microscope (Nikon, Tokyo, Japan) with a 2 megapixel mochromatic digital camera (Nikon) using the NIS-elements (Nikon) software.
EXAMPLE 1
This Example describes the tests to determine whether mRNA transfections encoding KLF4, LIN28, c-MYC, NANOG, OCT4 and SOX2 resulted in the appropriate expression and subcellular location of each respective protein product in 1079 fetal foreskin fibroblasts of newborns . The mRNAs used in the experiments were prepared with pseudouridine-5'-triphosphate substituting for uridine-5'-triphosphate (Kariko et al. 2008). The
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91/141 1079 fibroblasts were transfected with 4 pg of each mRNA per well of a 6-well plate and immunofluorescence analysis was performed 24 hours after transfection. Levels of endogenous proteins KLF4, LIN28, NANOG, OCT4 and SOX2 were not detectable by immunofluorescence in untransfected 1079 cells (Fig.1: B, F, N, R, V). Endogenous levels of c-MYC were relatively high in 1079 non-transfected cells (Fig. 1J). Transfections with mRNAs encoding the transcription factors, KLF4, c-MYC, NANOG, OCT4, and SOX2 all resulted in a primarily nuclear localization of each protein 24 hours after mRNA transfections (Figure 1: D, L, P, T , X). The cytoplasmic mRNA binding protein, LIN28, was localized to the cytoplasm (Fig. 1: H).
EXAMPLE 2
Having demonstrated efficient mRNA transfection and appropriate subcellular location of the reprogramming proteins, this Example describes the development of a protocol for the generation of iPS cells from somatic fibroblasts. Equal amounts (by weight) of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2 mRNAs were transfected into 1079 fibroblasts three times (once every other day). The day after the third transfection, the cells were plated on irradiated MED feeder cells and grown in iPS cell medium. Six days after plating the 1079 fibroblasts in irradiated MEFs, two putative iPS cell colonies became apparent on the 10 cm plate transfected with 3 pg of each mRNA reprogramming factor (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2). Colonies were allowed to grow up to 12 days after the last transfection before being fixed for immunofluorescence analysis. The specific NANOG marker for internal cell mass is generally used to assess whether iPS cell colonies are truly iPS colonies (Gonzalez et al. 2009, Huangfu et al. 2008). NANOG expression from mRNAs that were transfected 12 days earlier could be negligible based on previous reports on the duration of mRNA stability and expression (Kariko et al. 2008). Staining for NANOG showed that the two colonies of iPS cells were positive for NANOG (Fig. 2 B, D, and not shown). The surrounding fibroblasts that were not part of the iPS cell colony were negative for NANOG, suggesting that they were not reprogrammed in iPS cells.
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In a subsequent experiment using the same protocol, both 1079 human IMR90 fibroblasts and fibroblasts were transfected with the same reprogramming mRNAs. Several colonies were detected as early as 4 days after plating the transfected cells in irradiated MEFs. When 6 pg of each mRNA (KLF4, LIN28, cMYC, NANOG, OCT4, and SOX2) were used in 6-well plate transfections, 3 putative iPS cell colonies were then detected in both cell lines after plating on MEFs in plates 10 cm (Figure 3). In addition to analyzing these colonies for expression of NANOG, TRA-1-60, a more rigid marker of completely reprogrammed iPS cells (Chan et al. 2009), it was also used for immunofluorescence analysis. The iPS colonies generated from 1079 fibroblasts (Fig. 3 AF) and IMR90 fibroblasts (Fig. 3 GI) were positive for both NANOG and TRA-1-60, indicating that these colonies are completely reprogrammed type III cell colonies . This protocol comprising three mRNA transfections that encode all six reprogramming factors and then plating on MEF feeder cells resulted in similar reprogramming efficiency (3-6 iPS colonies per 1 x 106 cells entered) as previously reported by protocols comprising release of the same reprogramming factors by transfection of an expression plasmid (Aoi et al. 2008).
EXAMPLE 3
This Example describes an attempt to improve the efficiency of differentiated reprogramming cells using mRNA. In one approach, a protocol was used that comprised transfecting 1079 or IMR90 fibroblasts three times (once every other day) with the mRNAs that encode the six reprogramming factors in conditioned MEF medium rather than in fibroblast medium and then the fibroblasts 1079 treated in conditioned MEF medium instead of plating them in an MEF feeder layer after treatments. At the highest transfection dose used (36 pg of each reprogramming factor per 10 cm plate), 208 iPS cell colonies were detected three days after the final transfection (Figure A-F). Interestingly, no iPS cell colony was detected on the 6 or 18 pg plates of each of the reprogramming factors at the time point of day 3, suggesting that a dose above 18 pg was important, under these conditions, for the colony formation of iPS cells occurs within 3 days in MEF medium
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93/141 conditioned. IMR90 cells showed an even greater number of iPS cell colonies, with about 200 colonies 8 days after the last transfection on the transfected plate with three 6 pg doses of each of the six mRNA reprogramming factors and> 1000 colonies in cells IMR90 transfected three times with doses of 18 pg or 36 pg of each of the six reprogramming mRNAs (Figure 4 GI). Colonies were visible 3 days after the final transfection in 1079 cells, while only colonies became visible 6-7 days after the final transfection in IMR90 cells. Therefore, the more mature colonies derived from 1079 cells were larger and denser and were darker in bright field images compared to IMR90 colonies (Figure 4). All colonies on plate 1079 transfected three times with 36 pg of each reprogramming mRNA were positive for both NANOG and TRA-1-60 8 days after the final mRNA transfection (Figure 5 AI). All the more immature iPS IMR90 colonies were still positive for both NANOG and TRA-1-60 (Figure 5 OJ), but showed less robust staining for both markers due to the less dense cellular nature compared to the more mature 1079 colonies (Figure 5 AI ). The present protocol comprising releasing mRNAs in 1079 or IMR90 cells in conditioned MEF medium had a reprogramming efficiency of 200 to> 1000 colonies per 3 x 10 ⁵ cells entered. This protocol for inducing iPS cells was faster and almost 2-3 orders of magnitude more efficient than published protocols comprising transfecting fibroblasts with DNA plasmids that encode the same six reprogramming factors in fibroblast medium (Aoi et al. 2008 ). In addition, this protocol was more than 7-40 times more efficient than the published protocol comprising the release of lentivirus reprogramming factors, based on published data that the release of lentiviral reprogramming factors in 1079 newborn fibroblasts, which resulted in approximately 57 colonies of iPS cells per 6 x 10 ⁵ cells entered (Aoi et al. 2008). This protocol is even faster than the published methods.
EXAMPLE 4
NATURAL OCCURRENCE RNA MOLECULES HAVE DIFFERENTIAL CAPACITIES TO ACTIVATE DENDRITIC CELLS, MATERIALS AND EXPERIMENTAL METHODS
Plasmids and Reagents
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Plasmids pT7T3D-MART-1 and pUNO-hTLR3 were obtained from ATCC (Manassas, VA) and InvivoGen (San Diego, CA), respectively. pTEVluc was obtained from Dr Daniel Gallie (UC Riverside), contains pT7-TEV (the viral ethanol RNA sequence of tobacco etch) -luciferase-A50, and is described in Gallie, DR et al, 1995. The leading tail 5 tobacco etch and poly (A) viruses are functionally synergistic translation regulators. Gene 165: 233) pSVren was generated from p2luc (Grentzmann G, Ingram JA, et al, A dualluciferase reporter system for studying recoding signals. RNA 1998; 4 (4): 479-86) by removing the luciferase encoding sequence from firefly with BamHI and NotI digestion, end padding, and rewiring.
human TLR3 specific siRNA, pTLR3-sh was constructed by inserting synthetic ODN encoding shRNA with 20 nt of homology in length to human TLR3 (nt 703-722, access: NM_003265) in plasmid pSilencer 4.1-CMV-neo (Ambion, Austin, TX). pCMV-hTLR3 was obtained by first cloning a hTLR3-specific PCR product (nt 80-2887; Access NM_003265) in pCRII-TOPO (Invitrogen, Carlsbad, CA), then released with Nhe I-Hind III by cutting and subcloning the sites corresponding to pcDNA3.1 (Invitrogen). LPS (E. coli 055: B5) was obtained from Sigma Chemical Co, St. Louis, MO. CpG ODN2006 and R-848 were obtained from InvivoGen.
Cells and cell culture
Human embryonic reindeer cells 293 (ATCC) were propagated in DMEM supplemented with glutamine (Invitrogen) and 10% FCS (Hyclone, Ogden, UT) (complete medium). In all cases here, 293 cells refer to human embryonic kidney (HEK) 293 cells. The 293-hTLR3 cell line was generated by transforming 293 cells with pUNO-hTLR3. The cell lines 293-hTLR7, 293-hTLR8 and 293-hTLR9 (InvivoGen) were grown in complete medium supplemented with blasticidine (10 pg / ml) (Invivogen). The 293-ELAM-Iuc and TLR7-293 cell lines (M. Lamphier, Eisai Research Institute, Andover MA), and TLR3-293 cells were cultured as written (Kariko et al, 2004, mRNA is an endogenous ligand for the receptor type Toll 3. J Biol Chem 279: 12542-12550). Cell lines 293, 293-hTLR7 and 293-hTLR8 were stably transfected with pTLR3-sh and selected with G-418 (400 pg / ml) (Invitrogen). Neo-resistant colonies were selected and only those that do not express TLR3, determined as lack of secretion of IL-8 loin response to poly (I) :( C),
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95/141 have been used in other studies. Leukopheresis samples were obtained from HIV-infected volunteers using an IRB-approved protocol.
Generation of murine DC
Murine DCs were generated by collecting bone marrow cells from the tibia and femurs of 6-8 week old C57BL / 6 mice and lysing the red cells. The cells were seeded in 6-well plates in 10 ⁶ cells / well in 2 ml DMEM + 10% FCS and 20 ng / ml muGM-CSF (R & D Systems). On day 3, 2 ml of fresh medium with muGM-CSF was added. On day 6, 2 ml medium / well was collected, and the cells were pelleted and resuspended in fresh medium with muGM-CSF. On day 7 of the culture, muDC was harvested, washed.
Natural RNA
The mitochondria was isolated and the platelets obtained from the University of Pennsylvania Blood Bank using a lysis fractionation procedure (Mitochondria isolation kit; Pierce, Rockford, IL). RNA was isolated from purified mitochondria, cytoplasmic and nuclear fractions of 293 cells, unfractionated 293 cells, rat liver, mouse TUBE cell line, and E. coli DH5alpha strain by Master Blaster® (BioRad, Hercules, CA) . bovine tRNA, wheat tRNA, yeast tRNA, E. coli tRNA, poly (A) + mouse heart and poly (I) mRNA :( C) were acquired from Sigma, total human spleen RNA and E. coli RNA were purchased from Ambion. Oligoribonucleotide-5'-rnonophosphates have been chemically synthesized (Dharmacon, Lafayette, CO).
Aliquots of RNA samples were incubated on the Benzonase nuclease (1 U per 5 pl RNA in 1 microgram per microliter (pg / pl) for 1 h) (Novagen, Madison, WI). Aliquots of RNA-730 were digested with alkaline phosphatase (New England Biolabs). RNA samples were analyzed by denaturing agarose or polyacrylamide gel electrophoresis for quality assurance. Assays for LPS in RNA preparations using the Limulus Amebocyte Lysate gel coagulation assay were negative with a sensitivity of 3 picograms per milliliter (pg / ml) (University of Pennsylvania, Core Facility).
HPLC analysis
The monophosphate nucleosides were separated and visualized via HPLC. To release free 3'-monophosphate nucleoside, 5 pg aliquots of RNA were digested with 0.1 U of
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T2 RNase (Invitrogen) in 10 μΐ of 50 mM NaOAc and 2 mM EDTA buffer (pH 4.5) overnight, then the samples were injected into an Agilent 1100 HPLC using a Waters Symmetry C 18 column (Waters, Milford , MA). At a flow rate of 1 mL / min, a gradient of 100% buffer A (30 mM KH2PO4 and 10 mM tetraethylammonium phosphate [PicA reagent, Waters], pH 6.0) to 30% buffer B (acetonitrile) runs in 60 minutes. The nucleotides were detected using an array and photodiode at 254 nm. Identities were checked for retention times and spectacles.
Dendritic cell assays
Dendritic cells in 96-well plates (approximately 1.1 x 10 ⁵ cells / well) were treated with R-848, Lipofectin®, or Lipofectin®-RNA for 1 h, than the medium was changed. At the end of 8 am (unless otherwise stated), the cells were harvested by RNA isolation or flow cytometry, while the culture medium collected was subjected to cytokine ELISA. Levels of IL-12 (p70) (BD Biosciences Pharmingen, San Diego, CA), IFN-α, TNF-α, and IL-8 (Biosource International, Camarillo, CA) were measured in supernatants by sandwich ELISA. Cultures were performed in triplicate or quadruplicate and measured in duplicate.
Northern blot analysis
RNA was isolated from MDDCs after an 8 h incubation after treatment as described above. Where noted, the cells were treated with 2.5 μg / ml cycloheximide (Sigma) 30 min before stimulation and throughout the total duration of the incubation. RNA samples were processed and analyzed in Northern blots as described (Kariko et al, 2004, ibid) using human TNF-α and GAPDH probes derived from plasmids (pE4 and pHcGAP, respectively) obtained from ATCC.
RESULTS
To determine the immune stimulatory potential of different cell RNA subtypes, RNA was isolated from different subcellular compartments, that is, cytoplasm, nucleus and mitochondria. These RNA fractions, as well as total RNA, tRNA and mRNA selected from the polyA tail, all from mammalian sources, were complexed with Lipofectin® and added to MDDC. While the total mammalian, nuclear and cytoplasmic RNA all stimulated MDDC, as evidenced by detectable TNF-α secretion, TNF-α levels were much lower than those induced by in vitro synthesized mRNA
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97/141 (Figure 6). In addition, mammalian tRNA does not induce any detectable level of TNF-α, while mitochondrial (mt) RNA induced much more TNF-α than other types of mammalian RNA. Total bacterial RNA was also a potent activator of MDDC; in contrast, bacterial tRNA induced only a low level of TNF-α. tRNA from other sources (yeast, wheat germ, bovine) were non-stimulatory. Similar results were seen when RNA from other mammalian sources was tested. When RNA samples were digested with Benzonase, which cleaves ssRNA and dsRNA, RNA signaling was abolished in MDDC, verifying that TNF-α secretion was due to RNA in the preparations. The activation potentials of RNA types tested showed an inverse correlation with the extent of the nucleoside modification. Similar results were obtained in the experiments described in this Example for both types of DC generated by cytokine.
These findings demonstrate that RNA immunogenicity is affected by the extent of the nucleoside modification, with a greater degree of modification tending to decrease immunogenicity.
EXAMPLE 5
IN VITRO SYNTHESIS OF RNA MOLECULES WITH MODIFIED NUCLEOSIDE MATERIALS AND EXPERIMENTAL METHODS
RNA transcribed in vitro
Using in vitro transcription assay (MessageMachine and MegaScript kits; Ambion,) the following long RNAs were generated by T7 RNA polymerase (RNAP) as described (Kariko et al, 1998, phosphate-enhanced transfection of cationic lipidcomplexed mRNA and plasmid DNA. Biochim Biophys Acta 1369, 320-334) (Note: model names are indicated in parentheses; the number in the RNA name specifies the length): RNA-1866 (I-linearized Node pTEVluc) encodes firefly luciferase and a tail 50 nt polyA length. RNA-1571 (Ssp I-linearized pSVren) encodes Renilla luciferase. RNA-730 (Hind III-linearized pT7T3D-MART-1) encodes the human melanoma antigen MART-I. RNA-713 (I-linearized EcoR pTIT3D-MART-1) corresponds to the antisense sequence of MART-1, RNA497 (Bgl II-linearized pCMV-hTLR3) encodes a partial 5 'fragment of hTLR3. The sequences of RNA molecules are as follows:
RNA-I866:
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98/141 ggaauucucaacacaacauauacaaaacaaacgaaucucaagcaaucaagcauucuacuucuauugcagcaauuu aaaucauuucuuuuaaagcaaaagcaauuuucugaaaauuuucaccauuuacgaacgauagccauggaagacgccaaaaac auaaagaaaggcccggcgccauucuauccucuagaggauggaaccgcuggagagcaacugcauaaggcuaugaagagauac gcccugguuccuggaacaauugcuuuuacagaugcacauaucgaggugaacaucacguacgcggaauacuucgaaauguc cguucgguuggcagaagcuaugaaacgauaugggcugaauacaaaucacagaaucgucguaugcagugaaaacucucuuc aauucuuuaugccgguguugggcgcguuauuuaucggaguugcaguugcgcccgcgaacgacauuuauaaugaacguga auugcucaacaguaugaacauuucgcagccuaccguaguguuuguuuccaaaaagggguugcaaaaaauuuugaacgugc aaaaaaaauuaccaauaauccagaaaauuauuaucauggauucuaaaacggauuaccagggauuucagucgauguacacgu ucgucacaucucaucuaccucccgguuuuaaugaauacgauuuuguaccagaguccuuugaucgugacaaaacaauugca cugauaaugaauuccucuggaucuacuggguuaccuaaggguguggcccuuccgcauagaacugccugcgucagauucuc gcaugccagagauccuauuuuuggcaaucaaaucauuccggauacugcgauuuuaaguguuguuccauuccaucacgguu uuggaauguuuacuacacucggauauuugauauguggauuucgagucgucuuaauguauagauuugaagaagagcuguu uuuacgaucccuucaggauuacaaa auucaaagugcguugcuaguaccaacccuauuuucauucuucgccaaaagcacucu gauugacaaauacgauuuaucuaauuuacacgaaauugcuucugggggcgcaccucuuucgaaagaagucggggaagcgg uugcaaaacgcuuccaucuuccagggauacgacaaggauaugggcucacugagacuacaucagcuauucugauuacacccg agggggaugauaaaccgggcgcggucgguaaaguuguuccauuuuuugaagcgaagguuguggaucuggauaccgggaa aacgcugggcguuaaucagagaggcgaauuaugugucagaggaccuaugauuauguccgguuauguaaacaauccggaag cgaccaacgccuugauugacaaggauggauggcuacauucuggagacauagcuuacugggacgaagacgaacacuucuuc auaguugaccgcuugaagucuuuaauuaaauacaaaggauaucagguggcccccgcugaauuggaaucgauauuguuaca acaccccaacaucuucgacgcgggcguggcaggucuucccgacgaugacgccggugaacuucccgccgccguuguuguuu uggagcacggaaagacgaugacggaaaaagagaucguggauuacguggccagucaaguaacaaccgcgaaaaaguugcgc ggaggaguuguguuuguggacgaaguaccgaaaggucuuaccggaaaacucgacgcaagaaaaaucagagagauccucau aaaggccaagaagggcggaaaguccaaauuguaaaauguaacucuagaggauccccaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaca (SEQ ID NO: 1).
RNA 1571: ggcuagccaccaugacuucgaaaguuuaugauccagaacaaaggaaacggaugauaacugguccgcaguggugggccaga uguaaacaaaugaauguucuugauucauuuauuaauuauuaugauucagaaaaacaugcagaaaaugcuguuauuuuuuu acaugguaacgcggccucuucuuauuuauggcgacauguugugccacauauugagccaguagcgcgguguauuauaccag accuuauugguaugggcaaaucaggcaaaucugguaaugguucuuauagguuacuugaucauuacaaauaucuuacugca ugguuugaacuucuuaauuuaccaaagaagaucauuuuugucggccaugauuggggugcuuguuuggcauuucauuaua gcuaugagcaucaagauaagaucaaagcaauaguucacgcugaaaguguaguagaugugauugaaucaugggaugaaugg
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99/141 ccugauauugaagaagauauugcguugaucaaaucugaagaaggagaaaaaaugguuuuggagaauaacuucuucgugga aaccauguugccaucaaaaaucaugagaaaguuagaaccagaagaauuugcagcauaucuugaaccauucaaagagaaagg ugaaguucgucguccaacauuaucauggccucgugaaaucccguuaguaaaaggugguaaaccugacguuguacaaauug uuaggaauuauaaugcuuaucuacgugcaagugaugauuuaccaaaaauguuuauugaaucggacccaggauucuuuucc aaugcuauuguugaaggugccaagaaguuuccuaauacugaauuugucaaaguaaaaggucuucauuuuucgcaagaaga ugcaccugaugaaaugggaaaauauaucaaaucguucguugagcgaguucucaaaaaugaacaaaugucgacgggggccc cuaggaauuuuuuagggaagaucuggccuuccuacaagggaaggccagggaauuuucuucagagcagaccagagccaaca gccccaccagaagagagcuucaggucugggguagagacaacaacucccccucagaagcaggagccgauagacaaggaacug uauccuuuaacuucccucagaucacucuuuggcaacgaccccucgucacaauaaagauaggggggcaacuaaagggaucgg ccgcuucgagcagacaugauaagauacauugaugaguuuggacaaaccacaacuagaaugcagugaaaaaaaugcuuuauu ugugaaauuugugaugcuauugcuuuauuuguaaccauuauaagcugcaauaaacaaguuaacaacaacaauugcauuca uuuuauguuucagguucagggggaggugugggagguuuuuuaaagcaaguaaaaccucuacaaaugugguaaaaucgau aaguuuaaacagauccag guggcacuuuucggggaaaugugcgcggaaccccuauuuguuuauuuuucuaaauacauuca aauauguauccgcucaugagacaauaacccugauaaaugcuucaauaau (SEQ ID No: 2).
RNA 730 gggaauuuggcccucgaggccaagaauucggcacgaggcacgcggccagccagcagacagaggacucucauuaaggaagg uguccugugcccugacccuacaagaugccaagagaagaugcucacuucaucuaugguuaccccaagaaggggcacggccac ucuuacaccacggcugaagaggccgcugggaucggcauccugacagugauccugggagucuuacugcucaucggcuguug guauuguagaagacgaaauggauacagagccuugauggauaaaagucuucauguuggcacucaaugugccuuaacaagaa gaugcccacaagaaggguuugaucaucgggacagcaaagugucucuucaagagaaaaacugugaaccugugguucccaau gcuccaccugcuuaugagaaacucucugcagaacagucaccaccaccuuauucaccuuaagagccagcgagacaccugaga caugcugaaauuauuucucucacacuuuugcuugaauuuaauacagacaucuaauguucuccuuuggaaugguguaggaa aaaugcaagccaucucuaauaauaagucaguguuaaaauuuuaguagguccgcuagcaguacuaaucaugugaggaaaug augagaaauauuaaauugggaaaacuccaucaauaaauguugcaaugcaugauaaaaaaaaaaaaaaaaaaaacugcggcc GCA (SEQ ID NO: 3).
RNA-713 gggaauaagcuugcggccgcaguuuuuuuuuuuuuuuuuuuuaucaugcauugcaacauuuauugauggaguuuuccca auuuaauauuucucaucauuuccucacaugauuaguacugcuagcggaccuacuaaaauuuuaacacugacuuauuauua gagauggcuugcauuuuuccuacaccauuccaaaggagaacauuagaugucuguauaaauucaagcaaaagugugagaga aauaauuucagcaugucucaggugucucgcuggcucuuaaggugaauaaggugguggugacuguucugcagagaguuuc ucauaagcagguggagcauugggaaccacagguucacaguuuuucucuugaagagacacuuugcugucccgaugaucaaa
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100/141 cccuucuugugggcaucuucuuguuaaggcacauugagugccaacaugaagacuuuuauccaucaaggcucuguauccau uucgucuucuacaauaccaacagccgaugagcaguaagacucccaggaucacugucaggaugccgaucccagcggccucuu cagccgugguguaagaguggccgugccccuucuugggguaaccauagaugaagugagcaucuucucuuggcaucuugua gggucagggcacaggacaccuuccuuaaugagaguccucugucugcuggcuggccgcgugccucgugccgaauu (SEQ ID NO: 4).
RNA-497:
gggagacccaagcuggcuagcagucauccaacagaaucaugagacagacuuugccuuguaucuacuuuuggggg ggccuuuugcccuuugggaugcugugugcauccuccaccaccaagugcacuguuagccaugaaguugcugacugcagcca ccugaaguugacucagguacccgaugaucuacccacaaacauaacaguguugaaccuuacccauaaucaacucagaagauu accagccgccaacuucacaagguauagccagcuaacuagcuuggauguaggauuuaacaccaucucaaaacuggagccaga auugugccagaaacuucccauguuaaaaguuuugaaccuccagcacaaugagcuaucucaacuuucugauaaaaccuuugc cuucugcacgaauuugacugaacuccaucucauguccaacucaauccagaaaauuaaaaauaaucccuuugucaagcagaa gaauuuaaucacauua (SEQ ID NO: 5).
To obtain modified RNA, the transcription reaction was set up by replacing one (or two) of the basic NTPs with the corresponding triphosphate derivatives of the modified nucleotide nucleotide 5-methylcystidine, 5-methyluridine, 2-thiouridine, N6-methyladenosine or pseudouridine ( TriLink, San Diego, CA). In each transcription reaction, all 4 nucleotides or their derivatives were present in a concentration of 7.5 millimolar (mM). In selected experiments, as indicated, 6 mM of cover analogue m7GpppG (New England BioLabs, Beverly, MA) was further included for on-capped RNA. ORN5 and ORN6 were generated using oligodeoxynucleotide and T7 RNAP DNA templates (Silencer® siRNA construction kit, Ambion).
RESULTS
To further test the effect of nucleoside modifications on immunogenicity, an in vitro system was developed for the production of RNA molecules with pseudouridine or modified nucleosides. In vitro transcription reactions were performed in which 1 or 2 of the 4 nucleotide triphosphate (NTP) were replaced with a corresponding modified nucleoside NTP. Several sets of RNA with different primary sequences ranging in length between 0.7-1.9 kb, and containing or none, 1 or 2 types of modified nucleosides have been transcribed. The modified RNAs were not distinguishable from their unmodified counterparts in their mobility in denaturing gel electrophoresis,
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101/141 showing that they were intact and otherwise unmodified (Figure 7A). This procedure worked efficiently with any of T7, SP6, and T3 phage polymerases, and therefore is generalizable to a wide variety of RNA polymerases.
These findings provide a new in vitro system for the production of RNA molecules with modified nucleosides.
EXAMPLE 6
RNA TRANSCRIPTED IN VITRO STIMULATES HUMAN TLR3 AND NUCLEOSIDE MODIFICATIONS REDUCES RNA IMMUNOGENICITY MATERIALS AND EXPERIMENTAL METHODS
Parental cells-293; 293-hTLR7 and 293-hTLR8, all expressing TLR3-specific siRNA, and 293hTLR9, TLR3-293 were seeded in 96-well plates (5 x 10 ⁴ cells / well) and grown without antibiotics. On the subsequent day, the cells were exposed to R-848 or RNA complexed with Lipofectin® (Invitrogen) as described (Kariko et al, 1998, ibid). RNA was removed after one hour (h), and the cells were further incubated in an incomplete medium for 7 h. Supernatants were collected for measurement of IL-8.
RESULTS
To determine whether the nucleoside modification influences RNA-mediated activation of TLRs, human embryonic kidney cells 293 were stably transformed to express human TLR3. Cell lines were treated with RNA complexed with Lipofectin ® and TLR activation was monitored as indicated 10 by the release of interleukin (IL) -8. Several different RNA molecules were tested. RNA transcribed without modifications, in vitro generated a high level of IL-8 secretion. RNA containing m6A or s2U nucleoside modifications, in contrast, did not induce detectable IL-8 secretion (Figure 7B). The other nucleoside modifications tested (ie, m5C, m5U, Ψ and mSC / Ψ) had a lesser suppressive effect on TLR3 stimulation (Figure 7B). Ψ refers to pseudouridine.
Thus, modifications of nucleosides such as m ⁶ AS ² U, m ⁵ C, m ⁵ U, Ψ, reduce the immunogenicity of RNA as mediated by TLR3 signaling.
EXAMPLE 7
RNA TRANSCRIPTED IN VITRO STIMULATES HUMAN TLR7 AND TLR8, AND NUCLEOSIDE MODIFICATIONS REDUCED RNA IMMUNOGENICITY
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To test the possibility that 293 expresses endogenous TLR3 that interferes with the evaluation of the effects of RNA on specific TLR receptors, the expression of endogenous TLR3 was eliminated from the 293-TLR8 cell line by stably transfecting according to cells with a plasmid expressing RNA type short hairpin (sh) TLR3-specific RNA (also known as siRNA). This cell line was used for further study, since it did not respond to poly (I) :( C), LPS, and oligodeoxynucleotides containing CpG (ODNs), indicating the absence of TLR3, TLR4 and TLR9, but not responds to R-848, the human TLR8 cognate ligand (Figure 7B). When 293-hTLR8 cells expressing TLR3-labeled shRNA (293-hTLR8 cells shRNATLR3) were transfected with RNA transcribed in vitro, they secreted large amounts of IL-8. In contrast, the RNA containing most of the nucleoside modifications (m ⁵ C, m ⁵ U, Ψ, at ⁵ C / T, S ² U) eliminated stimulation (no more IL-8 production than control negative, ie empty vector). The m6A modification had a variable effect, in some cases eliminating and in other cases reducing the release of IL-8 (Figure 7B).
The results of this Example and the previous Example show that (a) RNA with natural inter-nucleotide phosphodiester bonds (for example RNA transcribed in vitro) stimulates human TLR3, TLR7 and TLR8; and (b) modifications of nucleosides such as m6A, m5C, m5U, s2U and Ψ, alone or in combination, reduce RNA immunogenicity as mediated by TLR3, TLR7 and TLR8 signaling. In addition, these results provide a new system for studying signaling by specific TLR receptors.
EXAMPLE 8
NUCLEOSIDE MODIFICATIONS REDUCED RNA IMMUNOGENICITY AS MEDIATED BY TLR7 AND TLR8 SIGNALING
The next set of experiments tested the ability of RNA isolated from natural sources to stimulate TLR3, TLR7 and TLR8. RNA from different mammalian species have been transfected into 293 cell lines that express TLR3, TLR7 and TLR8 as described in the previous example. None of the mammalian RNA samples induced IL-8 secretion above the level of the negative control. On the other hand, total bacterial RNA obtained from two different sources of E. coli induced robust IL-8 secretion in cells transfected with TLR3, TLR7 and TLR8, but not TLR9 (Figure 7C). Neither LPS nor unmethylated DNA (CpG ODN) (the potential contaminants in bacterial RNA isolates)
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103/141 activated the tested TLR3, TLR7 or TLR8. Mitochondrial RNA isolated from human platelets stimulated TLR8, but not TLR3 or TLR7.
These results demonstrate that bacterial and unmodified in vitro transcribed RNA are activators of TLR3, TLR7 and TLR8, and mitochondrial RNA stimulates TLR8. In addition, these results confirm the finding that RNA nucleoside modification decreases its ability to stimulate TLR3, TLR7 and TLR8.
EXAMPLE 9
NUCLEOSIDE MODIFICATIONS REDUCES RNA CAPACITY IN INDUCING CYTOKIN SECRETION AND ACTIVATION OF DC EXPRESSION MARKER
EXPERIMENTAL MATERIALS AND METHODS
DC stimulation tests
After 20 h of incubation with RNA, DCs were stained with CD83-phycoerythrin mAb (Research Diagnostics Inc, Flanders, NJ), HLA-DR-Cy5PE, and CD80 or CD86-fluorescein mAb isothiocyanate and analyzed in a FACScalibur® flow cytometer using CellQuest® software (BD Biosciences). Cell culture supernatants were collected at the end of a 20h incubation and subjected to cytokine ELISA. Levels of IL-12 (p70) (BD Biosciences Pharmingen, San Diego, CA), IFN-α, and TNF-α (Biosource International, Camarillo, CA) were measured in supernatants by ELISA. Cultures were performed in triplicate or quadruplicate and each sample was measured in duplicate.
RESULTS
The following experiments tested the ability of RNA containing modified or unmodified nucleosides to stimulate cytokine-generated MDDC. Reproducible nucleoside modifications decrease the ability of 5 RNAs to induce TNF-α and IL-12 secretion by MDDC generated in GM-CSF / IL-4 and MDCC generated by (GMCSF) / IFNα, in most cases for levels no more than the negative control (Figures 8A and B). The results were similar when other sets of RNA with the same base modifications but different primary sequences and lengths were tested, or when the RNA was further modified by adding a 5 'cap and / or polyA tail at the 3' end or removing the 5 'triphosphate fraction. RNAs of different lengths and sequences induced
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104/141 varying amounts of TNF-α from DC, typically less than a two-fold difference (Figure 8C).
Then the test was performed on DC1 and primary DC2. The primary monocytoid (DCI, BDCA1 ⁺ ) and plasmacytoid (DC2, BDCA4 ⁺ ) DC were purified from peripheral blood. Both cell types produced TNF-α when exposed to R-848, but only DC1 responded to poly (I) :( C), at a very low level, indicating the absence of TLR3 activity in DC2. Transfection of transcripts in vitro induced TNF-α secretion in both DC1 and DC2, while modified m5U, Ψ or s2U-transcripts were not stimulatory (Figure 8D). In contrast to cytokine-generated DC, m5C and m6A modifications of RNA did not decrease its stimulatory capacity in primary DC1 and DC2. The double-modified ιη6Λ / Λ transcripts were non-stimulatory, while a mixture of RNA molecules with a single type of modification (móA + Ψ) was a potent cytokine inducer. Thus, modification of uridine exerted a dominant suppressive effect on an in cis RNA molecule in primary DC. These results were consistent across all donors tested.
These findings show that RNA transcribed in vitro stimulates cytokine production by DC. In addition, since DC2 does not express TLR3 or TLR8, and the m5C and m6A modification of RNA reduced its TLR7 stimulatory capacity, these findings show that primary DC has an additional RNA signaling entity that recognizes the m5C- RNA modification and m6A- and whose signaling is inhibited by modifying U residues.
According to additional indicators of immunogenicity, the cell surface expression of CD80, CD83, CD86 and MHC class II molecules, and TNF-α secretion were measured by FACS analysis of MDDC treated with RNA-1571 and its modified versions. Modification of RNA with pseudouridine and modified nucleosides (m5C, m6A, s2U and mOA / '!') Reduced these markers (Figure 9), confirming the previous findings.
In summary, the ability of RNA to induce DCs to mature and secrete cytokines depends on the DC subtype as well as on the characteristics of the nucleoside modification present in the RNA. An increasing amount of modification decreases the immunogenicity of RNA.
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EXAMPLE 10
RNA-MEDIATED IMMUNE STIMULATION SUPPRESSION IS PROPORTIONAL TO THE NUMBER OF MODIFIED NUCLEOSIDS PRESENT IN RNA
MATERIALS AND EXPERIMENTAL METHODS
Human DC
For cytokine generated by DC, monocytes were purified from PBMC by discontinuous Percoll gradient centrifugation. The low density fraction (enriched in monocyte) was depleted of B, T, and NK cells using magnetic beads (Dynal, Lake Success, NY) specific for CD2, CD16, CD19, and CD56, yielding highly purified monocytes as determined by cytometry flow rate using anti-CD14 (> 95%) or anti-CD11c (> 98%) mAb.
To generate immature DC, purified monocytes were cultured in serum-free AIM V medium (Life Technologies), supplemented with GM-CSF (50 ng / ml) + IL-4 (100 ng / ml) (R & D Systems, Minneapolis, MN) in AIM V medium (Invitrogen) for the generation of DC-derived monocyte (MDDC) as described (Weissman, D et al, 2000. J Immunol 165: 4710-4717). DC were also generated by treatment with GM-CSF (50 ng / ml) + IFN-α (1,000 V / ml) (R & D Systems) to obtain IFN-α MDDC (Santini et al., 2000. Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice. J Exp Med 191: 1777-178).
Primary myeloid and plasmacytoid DCs (DC1 and DC2) were obtained from peripheral blood using BDCA-1 and BDCA-4 cell isolation kits (Miltenyi Biotec Auburn, CA), respectively.
RESULTS
Most of the modified nucleoside RNA used thus contained a type of modification that occurs in approximately 25% of the nucleoside nucleotides in the RNA (for example all uridine bases). To define the minimum frequency of modifications of particular nucleosides that is sufficient to reduce immunogenicity under the conditions used here, RNA molecules with limited numbers of modified nucleosides were generated. In the first set of experiments, RNA was transcribed in vitro in the presence of varying proportions of m6A, Ψ (pseudouridine) or m5C to their NTPs
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106/141 unmodified correspondents. The amount of incorporation of modified phosphate nucleosides into RNA was expected to be proportionate to the proportion contained in the transcription reaction, since the RNA yields obtained with T7 RNAP showed that the enzyme uses mTPA, Ψ or m5C NTPs almost as efficiently as basic NTPs. To confirm this expectation, the RNA transcribed in the presence of UTP ^ in a 50:50 ratio was digested and shown to contain UMP and Ψ in an almost 50:50 ratio (Figure 10A).
RNA molecules with increasing levels of modified nucleosides were transfected into MDDC, and TNF-α secretion was evaluated. Each modification (m6A, Ψ and m5C) inhibited the secretion of TNF-α in proportion to the fraction of the modified bases. Even the smallest amounts of modified bases tested (0.2-0.4%, corresponding to 3-6 nucleosides modified per 1571 nt molecule), was sufficient to measurably inhibit cytokine secretion (Figure 10B). RNA with 1.7-3.2% modified nucleoside levels (14-29 modifications per molecule) showed a 50% reduction in the induction of TNF-α expression. In 293 cells expressing TLR, a higher percentage (2.5%) of modified nucleoside content was required to inhibit RNA-measured signaling events.
Thus, pseudouridine and modified nucleosides reduce the immunogenicity of RNA molecules, even when present as a small fraction of residues.
In additional experiments, 21-mer oligoribonucleotides (ORN) with inter-nucleotide phosphodiester bonds were synthesized in which the modified nucleosides (m5C, Ψ or 2'-O-methyl-U [Um]) were replaced in a particular position (Figure 11A ). While the unmodified ORN induced TNF-α secretion, this effect was abolished in the presence of a single nucleoside modification (Figure 11B). Similar results were obtained with TLR-7 and TLR-8 transformed 293 cells that express TLR3-labeled siRNA.
The above results were confirmed by measuring the levels of TNF-α mRNA in MDDC by Northern blot assay, using both 21-mer ORN (ORN1) and 31-mer above (ORN5 and ORN6) in vitro transcripts. To amplify the signal, cycloheximide, which blocks the degradation of selected mRNAs, was added to some samples, as shown in the Figure. Unmodified ODN increased levels of TNF-α mRNA, while
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ORNs containing a single modified nucleoside were significantly less stimulatory; ORN2-Um show the greatest decrease in TNF-α production (Figure 11C). Similar results were seen in mouse macrophage RAW cells and in human DC.
In summary, each of the tested modifications (m6A, m5C, m5U, s2U, Ψ and 2'-Omethyl) suppressed RNA-mediated immune stimulation, even when present in a small fraction of residues. Another suppression was observed when the modified nucleoside propulsion was increased.
EXAMPLE 11
MODIFICATION OF RNA PSEUDOURIDINE REDUCES ITS IMMUNOGENICITY IN VIVO
To determine the effect of pseudouridine modification on RNA immunogenicity in vivo, 0.25 µg RNA) was complexed to Lipofectin® and injected intratracheally into mice, mice were bled 24 h later, and circulating levels of TNF-α and IFN-α were evaluated from serum samples. capped mRNA modified in pseudouridine induced significantly less TNF-α. and IFN-α mRNA than was stimulated by unmodified mRNA (Figure 12A-B).
These results provide evidence that pseudouridine-modified mRNA is significantly less immunogenic in vivo than unmodified RNA.
EXAMPLE 12
RNA CONTAINING PSEUDOURIDINE HAS CAPACITY
REDUCED IN ACTIVATING PRK
EXPERIMENTAL MATERIALS AND METHODS
PKR phosphorylation assays
Aliquots of PKR active agarose (Upstate) were incubated in the presence of magnesium / ATP cocktail (Upstate), kinase buffer and ATP mix [range ³² p] and RNA molecules for 30 min at 30 ° C. Unmodified RNA and nucleoside modifying RNA (m5C, pseudouridine, m6A, m5U) and dsRNA were tested. Recombinant human eIF2a (BioSource) was added, and samples were further incubated for 5 min, 30 ° C. The reactions were stopped by adding NuPage LDS sample buffer with reducing reagent (Invitrogen), denatured for 10 min, 70 ° C, and analyzed at 10% PAGE. The gels were dried
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RESULTS
To determine whether mRnA containing pseudouridine activates dsRNA-dependent protein kinase (PKR), in vitro phosphorylation assays were performed using recombinant human PKR and its substrate, eIF2a (eukaryotic initiation factor 2 alpha) in the presence of capped mRNA encoding renilla ( 0.5 and 0.05 ng / μ ^. MRNA containing pseudouridine (Ψ) did not activate PKR, as detected by the lack of both PKR auto-phosphorylation and efF2u phosphorylation, while RNA without nucleoside modification and mRNA with m modification ⁵ C activated PKR (Figure 13), thus modifying pseudouridine decreases RNA immunogenicity.
EXAMPLE 13
ENHANCED TRANSLATION OF PROTEINS FROM RNA CONTAINING PSEUDOURIDIN AND m ⁵ C IN VITRO
EXPERIMENTAL MATERIALS AND METHODS
In vitro translation of mRNA in rabbit reticulocyte lysate
In vitro translation was performed on rabbit reticulocyte lysate (Promega, Madison WI). A 9 μl aliquot of the lysate was supplemented with 1 μl (1 μg) mRNA and incubated for 60 min at 30 ° C. A one μl aliquot was removed for analysis using firefly and renilla assay systems (Promega, Madison WI), and a LUMAT LB 950 luminometer (Berthold / EG & G Wallac, Gaithersburg, MD) with a measurement time of 10 seconds.
RESULTS
To determine the effect of pseudouridine modification on RNA translation efficiency in vitro, (0.1 μg / μl) uncapped pseudouridine mRNA encoding firefly luciferase was incubated in rabbit reticulocyte lysate for 1 h 30 ° C, and luciferase activity was determined. mRNA containing pseudouridine was translated more than 2 times more efficiently than RNA without pseudouridine in rabbit reticulocyte lysate, but not in wheat extract or E. coli lysate (Figure 14), showing that modifying pseudouridine increases efficiency of RNA translation. Similar results were obtained with m ⁵ C-modified RNA. When a polyA tail was
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Thus, modifying pseudouridine at ⁵ C increases the efficiency of RNA translation, and the addition of a polyA tail to the mRNA containing pseudouridine further increases the efficiency of translation.
EXAMPLE 14
IMPROVED TRANSLATION OF PROTEINS FROM RNA CONTAINING PSEUDOURIDIN IN CULTIVATED CELLS
EXPERIMENTAL MATERIALS AND METHODS
Translation assays in cells
The 96-well plates were seeded with 5 x 10 ⁴ cells per well 1 day before transfection. Lipofectin®-mRNA complexes were assembled and added directly to the cell monolayers after removal of the culture medium (0.2 pg mRNA-0.8 pg lipofectin in 50 pl per well). The cells were incubated with the transfection mixture for 1 h at 37 ° C, 5% CO2 incubator, then the mixture was replaced with fresh preheated medium containing 10% FCS, then the cells were analyzed as described in the previous Example.
RESULTS
To determine the effect of pseudouridine modification on RNA translation in cultured cells, 293 cells were transfected with in vitro transcribed mRNA capped with modified nucleoside encoding the renilla reporter protein. The cells were lysed 3 h after the start of transfection, and renilla levels were removed by enzymatic assays. In 293 cells, DNA modified by pseudouridine and m5C were translated almost 10 times and 4 times more efficiently, respectively, than unmodified mRNA (Figure 15A).
Then, the experiment was carried out with mouse DC derived from primary bone marrow, in this case lysing the cells 3 h and 8 h after transfection. RNA containing the pseudouridine modification was translated 15-30 times more efficiently than unmodified RNA (Figure 15B).
Similar expression results were obtained using human DC and other primary cells and established cell lines, including CHO and RAW type cells
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110/141 mouse macrophage. In all cell types, modification of pseudouridine produced the best improvement of the tested modifications.
Thus, modification of pseudouridine increased the efficiency of RNA translation in all types of cells tested, including different types of both professional antigen presenting cells and non-professional antigen presenting cells, providing further evidence that pseudouridine modification increases the efficiency of RNA translation.
EXAMPLE 15
5 'AND 3' ELEMENTS STILL IMPROVE TmRNA TRANSLATION IN MAMMALIAN CELLS
To test the effect of additional RNA structural elements in improving translation by modifying pseudouridine, a set of ymRNAs encoding luciferase was synthesized that contained combinations of the following modifications: 1) a single 5 'untranslated sequence (TEV, a potentiate of independent translation of cover), 2) cover and 3) polyA tail. The ability of these modifications to improve the translation of conventional ymRNA or mRNA was assessed (Figure 16A). These structural elements additively improved the translation efficiency of both conventional ymRNA, with ymRNA showing better protein production of all constructs.
The protein's expression capacity from the efficient ymRNA construct of firefly luciferase, capTEVlucA50 (containing TEV, cap, and an extended poly (A) tail) was then evaluated for 24 hours in 293 cells (Figure 16B). ymRNA produced more protein at each time point tested and conferred more persistent luciferase expression than equivalent conventional mRNA constructs, showing that ψmodifications stabilize mRNA.
To test whether ψ-modification of mRNA improved translation efficiency in mammalian cells in situ, caplacZ ^ mRNA constructs with or without extended polyA (An) tails and encoding β-galactosidase (lacZ) were generated and used to transfect 293 cells. 24 h after mRNA release, significant increases in β-galactosidase levels were detected by X-gal visualization, in both caplacZ and caplacZ-An, compared to the corresponding (conventional) transcript control (Figure 16C). This trend was observed
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EXAMPLE 13
IMPROVED TRANSLATION OF PROTEINS FROM RNA CONTAINING PSEUDOURIDIN IN VIVO MATERIALS AND EXPERIMENTAL METHODS
Intracerebral RNA injections
All animal procedures were in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by Institutional Animal Care and Use Committee. Male Wistar rats (Charles River Laboratories, Wilmington, MA) were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg / kg body weight). The heads were placed in a stereotaxic frame, and eight 1.5 mm diameter evenly spaced holes were made bilaterally (coordinates in relation to the bregma: anterior / posterior +3.0, -3, -6 mm; lateral ± 2, 5 mm) leaving the dura intact. Intracerebral injections were made using a 25 pl syringe (Hamilton, Reno, NV) with 30 gauge, sterile 1-inch needle (Beckton 25 Dickinson Labware, Franklin Lakes, NJ) that was attached to a large probe holder and stereotactic arm. To avoid air space in the syringe, the needle hub was filled with 55 pl of complex before the needle was attached, and the rest of the sample was removed through the needle. The injection depth (2 mm) was determined in relation to the dura surface, and 4 µl of complex (32 ng mRNA) was administered in a single rapid bolus infusion. 3 hours (h) later, the rats were sacrificed halothane, and the brains were removed in cooled phosphate buffered saline.
Injection of RNA into the mouse tail vein
The tail veins of female BALB / c mice (Charles River Laboratories) were injected (bolus) with 60 pl RNA complexed with Lipofectin® (0.26 pg). The organs were removed and homogenized in luciferase or Renilla lysis buffer in microcentrifuge tubes using a pestle. The homogenates were centrifuged and the supernatants were analyzed for activity.
Release of RNA into the lung
Female BALB / c mice were anesthetized using ketamine (100 mg / kg) and xilasine (20 mg / kg). Small incisions were made in the skin adjacent to the trachea. When the
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112/141 trachea was exposed, 501-11 RNA complexed with Lipofectin® (0.2 pg) was instilled into the trachea towards the lung. The incisions were closed and the animals were left to recover. 3 hours after RNA release, the mice were sacrificed by cervical dislocation and the lungs were removed, homogenized in luciferase or Renilla lysis buffer (250 μ1), and evaluated for activity. In a different set of animals, blood samples (100 μl / animal) were collected from tail veins, coagulated and centrifuged. Serum fractions were used to determine levels of TNF and IFNa by ELISA as described in the Examples above, using mouse specific antibodies.
RESULTS
To determine the effect of pseudouridine modification on RNA translation in vivo, each hemisphere of the rat brain cortex was injected with capped RNA modified by pseudouridine encoding renilla or unmodified RNA, and the translation of the RNA was measured. Pseudouridine-modified RNA was translated significantly more efficiently than unmodified RNA (Figure 17A). Then expression studies were performed in mice. mRNAs encode firefly luciferase because no endogenous mammalian enzyme interferes with its detection. The transcripts (unmodified and ymRNA) were constructed with cap, TEV (capTEVA50) and extended (200 nt) poly (A) tails. 0.25 μg of Lipofectin®-complexed RNA was injected into mice (intravenous (i.v.) into the tail vein). A range of organs were screened for luciferase activity to determine the optimal measurement site. The administration of 0.3 μg capTEVlucAn ymRNA induced high expression of luciferase in the spleen and moderate expression in bone marrow, but little expression in lung, liver, heart, kidney or brain (Figure 17B). In subsequent studies, the spleens were studied.
The translation efficiencies of conventional and ψ mRNA (0.015 mg / kg; 0.3 pg / animal administered intravenously) were subsequently compared in time course experiments. Luciferase activity was readily detectable in 1 h, peaking in 4 h and declined in 24 h after administration of conventional or ψ mRNA, but each time it was substantially higher in animals that received ψmRNA (Figure 17C, left frame). In 24 h, only animals injected with ψmRNA showed splenic luciferase activity (4 times above the background). A similar relative pattern of expression (between
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In the next experiment, 0.25 pg of mRNA-Lipofectin® was delivered to the mouse's lungs by intratracheal injection. Pseudouridine-modified capped RNA was translated more efficiently than capped RNA without pseudouridine modification (Figure 17D).
Thus, modification of pseudouridine increases the efficiency of RNA translation in vitro, in cultured cells, and models of various animals in vivo and by various routes of administration, showing its wide application as a meiod and increasing the efficiency of RNA translation.
EXAMPLE 17
PSEUDOURIDIN MODIFICATION IMPROVES RNA STABILITY IN VIVO
Northern analysis of splenic RNA at 1 and 4 h after injection in the animals of the previous Example revealed that the administered mRNAs, in their intact and partially degraded forms, were readily detectable (Figure 17C, table at right). In contrast, at 24 h, unmodified capTEVlucAn mRNA was below the detection level, while capTEVlucAn ψmRNA, although partially degraded, was still clearly detectable. Thus, ψmRNA is more stably preserved in vivo than the control mRNA.
To test whether protein production in vivo is quantitatively dependent on the concentration of mRNA released intravenously, mRNAs were administered to mice at 0.015-0.150 mg / kg (0.3-3.0 pg capTEVlucAn per animal) and spleens were analyzed 6 hours after as described above. Luciferase expression was quantitatively correlated with the amount of injected RNA (Figure 18) and at each concentration.
These findings confirm the results of Example 15, which demonstrate that ψmRNA is more stable than unmodified RNA. Still, the immunogenicity of ψ-mRNA was lower than unmodified RNA, as described here above (Figure 12 and Figure 17C, table on the right).
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To summarize Examples 16-17, the 3 advantages of ψ-mRNA compared to conventional mRNA (improved translation, increased stability and reduced immunogenicity) seen in vitro are still seen in vivo.
EXAMPLE 18
TmRNA RELEASED THROUGH THE RESPIRATORY TRACT BEHAVES LIKE THE MRNA ADMINISTERED THROUGH THE INTRAVENOUS VIA
To test the ability of ψmRNA to be released by inhalation, Lipofectin®-or PEI-complexed mRNAs encoding firefly luciferase were released to mice intratracheally, when a needle was placed in the trachea and the mRNA solution dispersed in lungs. Similar to intravenous release, significantly greater expression of luciferase was observed with ψmRNA compared to unmodified mRNA (Figure 19), although significantly less protein was produced with intratracheal as compared to intravenous routes. Unmodified mRNA administered intratracheally was associated with significantly higher concentrations of inflammatory cytokines (IFN-α and TNF-α) compared to vehicle control, whereas ψmRNA was not (Figure 19).
Thus, ψmRNA can be released by inhalation without activating the innate immune response.
EXAMPLE 19
RELEASE OF EPO-TmRNA TO CELLS 293 ψmRNA was generated from the plasmid containing the human EPO cDNA. When 0.25 pg of EPO ^ mRNA was transfected in 10 ⁶ cultured 293 cells, more than 600 mU / ml of EPO protein was produced. Thus, modified RNA molecules of the present invention are effective in delivering recombinant proteins to cells.
EXAMPLE 20
PREPARATION OF TmRNA CONSTRUCTS THAT IMPROVE EPO ENHANCED
MATERIALS AND EXPERIMENTAL METHODS
This EPO coding sequence is cloned using restriction enzyme techniques to generate 2 new plasmids, pTEV-EPO and pT7TS-EPO, which are used as templates for producing EPO ^ mRNA. EPO ^ mRNAs are produced from these templates
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115/141 by in vitro transcription (MessageMachine® and MegaScript® kits; Ambion,) using T7 RNA polymerase (RNAP), incorporating nucleoside in equimolar concentrations (7.5mM). To incorporate the nucleoside modifications, ψ triphosphate (TriLink, San Diego, CA) replaces UTP in the transcription reaction. To ensure eamentomRNA capping, a non-reversible 6 mM 3'-O-Me-m7GpppG analog (New England BioLabs, Beverly, MA) is also included. ΨmRNAs are poly (A) -flow in a reaction of ~ 1.5 pg / pl RNA, 5 mM ATP, and 60 U / p1 poly (A) yeast polymerase (USB, Cleveland, OH) mixed at 30 ° C for 3 to 24 h. The quality of ψmRNAs is assessed by denaturation using agarose gel electrophoresis. The assays for LPS in mRNA preparations using the Limulus Amebocyte Lysate gel clot assay with a sensitivity of 3 pg / ml are further performed.
RESULTS
The 3 'untranslated (3'UTR) proximal region of EPO ^ mRNA preserves a pyrimidine-stabilizing element of ~ 90 nt length from the nascent EPO mRNA, which stabilizes EPO mRNA by specific association with a ubiquitous protein, erythropoietin mRNA (ERBP). To maximize the stability of EPO ^ mRNA, 2 changes are incorporated into an EPO plasmid to improve the stability and translation efficiency of the transcribed mRNA: 1) A 5'UTR sequence of tobacco etch virus (TEV) is incorporated upstream of the coding sequence of EPO to generate pTEV-EPO. 2) A plasmid, pT7TS-EPO, is generated, in which the EPO cDNA is flanked by sequences corresponding to 5 'and 3' Xenopus beta-globin mRNA RTUs.
In addition, the poly (A) tail length during ψmRNA production from these plasmid templates is extended, increasing the incubation period for the poly (A) polymerase reaction. The larger poly (A) tail decreases the rate at which ψmRNA degrades during translation.
These improvements result in increased translation efficiency in vivo, thus minimizing the therapeutic dose of the final product.
EXAMPLE 21
IN VITRO ANALYSIS OF PROTEIN PRODUCTION FROM EPO mRNA CONSTRUCTS
MATERIALS AND EXPERIMENTAL METHODS
Preparation of mammalian cells.
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Human embryonic reindeer cells 293 (ATCC) were propagated in DMEM supplemented with glutamine (Invitrogen) and 10% FCS (Hyclone, Ogden, UT) (complete medium). Leukopheresis samples are obtained from HIV-infected volunteers using an IRB-approved protocol. DCs are produced as described above and grown with GM-CSF (50 ng / ml) + IL-4 (100 ng / ml) (R & D Systems) in AIM V ® medium (Invitrogen).
Mice and DC liver cells are obtained by published procedures. Briefly, the spleens of BALB / c mice are aseptically removed and chopped with forceps in complete medium. Tissue fragments are sedimented by gravity and the single cell suspension washed and lysed with AKC lysis buffer (Sigma). Murine DCs are derived from bone marrow cells collected from femurs and tibia of 6-9-week-old BALB / c mice. The cells are cultured in DMEM containing 10% FCS (Invitrogen) and 50 ng / ml muGM-CSF (R&D) and used on day 7.
Cell transfection and detection of EPO and pro-inflammatory cytokines
Transfections are performed with Lipofectin in the presence of phosphate buffer, an effective release method for splenic cell expression and in vitro. EPO-ymRNA (0.25 pg / well; 100,000 cells) is added to each cell type in triplicate for 1 hour and the supernatant replaced with fresh medium. 24 hours later, the supernatant is collected for measurement by ELISA of EPO, IFN-α or β and TNF-α.
RESULTS
To assess the impact of single RTUs on improving the translation efficiency of ymRNA, EPO-ymRNA containing, or not containing, each improvement (element 5 'TEV, (βglobin 5' and 3 'RTU) with long poly (A) tails are tested for protein production in vitro and immune activation in vitro using EPO mRNA containing conventional nucleosides according to controls.The efficiency of protein production from each mRNA is evaluated in mammalian cell lines, (HEK293, CHO), human DCs and primary murines, and spleen cells for each mRNA.The measurement of total EPO produced in all cell types and immunogenicity (pro-inflammatory cytokines associated with the supernatant) in primary cells is assessed. The mRNA construct that demonstrates the ideal combination high EPO production (in 1 or more cell types) and low cytokine stimulation is used in subsequent studies. Improvements in EPO-wmRNA 5 'and 3'UTRs and larger tails of
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EXAMPLE 22
EPO PRODUCTION CHARACTERIZATION AND BIOLOGICAL RESPONSE FOR EPO-ψ mRNA IN VIVO
MATERIALS AND EXPERIMENTAL METHODS
Administration of EPO-ymRNA to mice.
All animal studies described are performed in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by Institutional Animal Care and Use Committee of the University of Pennsylvania. Female BALB / c mice (n = 5 per experimental condition; 6 weeks, 18-23 g; Charles River Laboratories) are anesthetized using 3.5% halothane in a mixture of N2O and O2 (70:30), then halothane reduced at 1% and anesthesia was maintained using a nasal mask. Animal body temperatures are maintained throughout the procedure using a heating pad heated to 37 ° C. EPO-ymRNA-lipofectin complexes (constructed by mixing varying amounts of nucleic acid with 1 pl of lipofectin in a final volume of 60 pl are injected into a lateral tail vein. Blood samples are collected 3 times a day for 3 days after injection of mRNA during the course of the study, at an ideal time point in dose-response studies, and daily from days 2-6 in the studies for reticulocytosis.
Determination of reticulocytes by flow cytometry.
Whole blood samples are stained using Retic-COUNT reagent (BD Diagnostics) and the event data acquired on a FACScan flow cytometer. Red blood cells (RBCs) are selected for their direct and lateral dispersion properties and analyzed for absorption of Tiazol Orange. Cells stained with Retic-COUNT reagent are detected by fluorescence and reticulocytes expressed as the percentage of total RBC. At least 50,000 events are counted per sample.
RESULTS
To optimize the production of biologically functional human EPO protein (hEPO) in response to mRNA encoding EPO, the following studies are performed:
Time course of EPO production after single injection of EPO-wmRNA. After intravenous administration of 1 pg of EPO-ymRNA, hEPO is measured serially from 1 Petition 870180166239, of 12/21/2018, p. 136/167
118/141 h after administration of EPO-ymRNA by ELISA, to determine the half-life of EPO protein in serum. This half-life is a result of both the EPO protein half-life and the functional half-life of EPO-ymRNA. The resulting ideal time point for measuring the EPO protein after administration of EPO-ymRNA is used in subsequent studies.
Dose-response of EPO production after single injection of EPO-wmRNA. To determine the correlation between the amount of EPO protein produced and the amount of EPOymRNA administered, increasing concentrations of EPO-ymRNA (0.01 to 1 pg / animal) are administered and EPO is measured at the ideal time point.
Relationship between hEPO production and reticulocyte. To measure the effect of EPO-ymRNA on a biological correlate of EPO activity, flow cytometry is used to determine the frequency of reticulocyte in the blood). Flow cytometry has a coefficient of variation of <3%. Mice receive a single dose of EPO-ymRNA, and blood is collected from mice daily from days 2-6. The relationship between the EPO-ymRNA dose and reticulocyte frequency is then assessed at the maximum reticulocyte time point. The dose of EPO-ymRNA that leads to at least a 5% increase in reticulocyte count is used in subsequent studies. Serum hEPO concentrations in mice estimated at 50 mU / ml and / or an estimated 5% increase in reticulocyte frequency are obtained.
EXAMPLE 23
MEASUREMENT OF IMMUNE RESPONSES TO EPO ^ mRNA IN VIVO
MATERIALS AND EXPERIMENTAL METHODS
Detection of cytokines in plasma.
Serum samples obtained from blood collected at different times during and after 7 daily administrations of mRNA complexed with lipofectin are analyzed for IFN-α, TNF-α, and IL-12 from mice using ELISA kits.
Northern blot analysis
Aliquots (2.0 pg) of RNA samples isolated from spleen and separated by 1.4% electrophoresis in denaturing agarose gel, transferred to loaded membranes (Schleicher and Schuell) and hybridized in MiracleHyb® (Stratagene). The membranes are probed for TNF-α, IFN down-stream signaling molecules (for example IRF7, IL12 p35 and p40, and GAPDH) and other immune activation markers. The specificity of all
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119/141 probes are confirmed by sequencing. To probe the membranes, 50 ng of DNA is labeled using Redivue [alpha- ³² P] dCTP® (Amersham) with a random prime targeting kit (Roche). The hybridized membranes are exposed to Kodak BioMax MS film using a screen intensifier MS at -70 ° C.
Histopathology.
The spleens of mice treated with EPO-ymRNAe treated with positive and negative control are harvested, fixed, sectioned, stained with hematoxylin and eosin and evaluated by a veterinary pathologist for signs of immune activation.
RESULTS
To confirm the reduced immunogenicity of RNA molecules of the present invention, mice (n = 5) receive daily doses of EPO-ymRNA for 7 days, then are evaluated for adverse events measured by immune, as indicated by serum cytokine concentrations, at splenic expression of mRNAs encoding inflammatory proteins, and pathological evaluation. The maximum doses administered are 3 pg or 5 x the effective single dose as determined above. unmodified mRNA and Lipofectin® alone are used as positive and negative controls, respectively.
These studies confirm that the reduced immunogenicity of RNA molecules of the present invention.
EXAMPLE 24
ANOTHER IMPROVEMENT OF EPO-chRNA RELEASE METHODS
Nanoparticle Complexation.
Polymer and ymRNA solutions are mixed to form complexes. Several formulation conditions are tested and optimized: (1) sub-22 nm polyethyleneimine (PEI) / mRNA complexes are prepared by adding 25 volumes of mRNA to 1 volume of PEI in water without mixing for 15 minutes. (2) rod-like poly-L-lysine-polyethylene glycol (PLL-PEG) with average dimensions of 12x150 nm is synthesized by slowly adding 9 volumes of mRNA to 1 volume of CK30-PEG10k in acetate contraction buffer while vortexing. (3) for the synthesis of a carrier polymer of a biodegradable gene, polyaspartic anhydride coetylene glycol (PAE) is synthesized by opening a polycondensation ring of N (Benzyloxycarbonyl) L-aspartic anhydride and ethylene glycol. The aspartic acid pendant amine is then unprotected and protonated by acidification with hydrogen chloride and
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Intratracheal release.
The mice are anesthetized with 3% halothane (70% N2O + 30% O2) in an anesthetic chamber and maintained with 1% halothane (70% N2O + 30% O2) during operation with a nasal cone. The trachea is exposed, and 50 pl of mRNA complex is infused with 150 pl of air in the lung through the trachea using 250 pl of Hamilton syringe (Hamilton, Reno, NV) with a 27 G 1/2 needle.
RESULTS
To improve the efficiency of the release and expression of ymRNA administered intratracheally (i.t.), ymRNA is encapsulated in nanoparticles. Nanoparticle packaging involves condensing and encapsulating DNA (for example) into particles that are smaller than the nuclear membrane pore, using chemicals including poly-L-lysine and polyethylene glycol. RNA is packaged in 4 different nanoparticle formulations (PEI, PLL, PAE, and CK30PEGl0k), and ymRNA release efficiency is compared to ymRNA encoding luciferase compared to (Luc-ymRNA). The release and dose-response kinetics are then characterized using EPO-ymRNA.
EXAMPLE 25
PREVENTION OF RESTENOSIS BY RELEASE TO THE CAROTID ARTERY OF MODIFIED MRNA THAT CODES RECOMBINANT THERMAL SHOCK PROTEIN
EXPERIMENTAL MATERIALS AND METHODS
Experimental draw
RNA is administered to the rat carotid artery by intra-arterial injection close to the time of balloon angioplasty, after which blood flow is reintegrated. The rats are sacrificed 3 h after injection, sections of the carotid artery are excised, cells from the vascular endothelium are harvested and homogenized, and the luciferase activity is determined as described in the Examples above.
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RESULTS
Pseudouridine-modified RNA encoding Luciferase is administered to the rat carotid arteries. 3 hours later, RNA luciferase can be detected at a release site but not at adjacent sites.
Then, this protocol is used to prevent restenosis of a blood vessel after balloon angioplasty in an animal restenosis model, by releasing modified RNA that encodes a heat shock protein, for example HSP70; a growth factor (eg platelet-derived growth factor (PDGF), vascular endothelial growth factor (V-EGF), or insulin-like growth factor (IGF); or a protein that down-regulates or antagonizes signaling growth factor.The administration of modified RNA reduces the incidence of restenosis.
EXAMPLE 26
TREATMENT OF CYSTIC FIBROSIS BY RELEASING MRNA MOLECULES THAT CODE RESPIRATORY EPITHELIUM CFTRP
Modified nucleoside RNA or pseudouridin encoding CFTR is released, as described in Example 16, into the lungs of an animal cystic fibrosis model, and its effect on the disease is assessed as described in Scholte BJ, et al (Animal models of cystic fibrosis. J Cyst Fibros 2004; 3 Suppl2: 183-90) or Copreni E, et al, Lentivirus-mediated gene transfer to the respiratory epithelium: a promising approach to gene therapy of cystic fibrosis. GeneTher 2004; 11 Suppl 1: S67-75). Administration of RNA alleviates cystic fibrosis.
In additional experiments, the modified mRNA molecules of the present invention are used to release the lungs, other recombinant proteins of therapeutic value, for example through an inhaler that releases RNA.
EXAMPLE 27
TREATMENT OF XLA BY RELEASE OF MODIFIED MRNA MOLECULES THAT CODES, IN ADDITION TO HEMATOPOIETIC CELLS
Modified nucleoside or pseudouridine RNA encoding ADA is released into the hematopoietic cells of an X-linked agammaglobulinemia in an animal model, and its effect on the disease is assessed as described in Tanaka M, Gunawan F, et al, Inhibition of heart transplant injury and graft coronary artery disease after prolonged organ ischemia by selective protein kinase C regulators. J Thorac Cardiovasc Surg 2005; 129 (5): 1160-7) or
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Zonta S, Lovisetto F, et al, Uretero-neocystostomy in a swine model of kidney transplantation: a new technique. J Surg Res. 2005 Apr; 124 (2): 250-5). Administration of RNA has been shown to increase XLA.
EXAMPLE 28
PREVENTION OF ORGAN REJECTION BY RELEASE OF MODIFIED MRNA MOLECULES THAT CODE IMMUNOMODULATORY PROTEIN IN A TRANSPLANTATION PLACE
Modified nucleoside RNA or pseudouridine encoding a cytokine, a chemokine, or an IS interferon (for example IL-4, IL-13, IL-I0, or TGF-β) is released at a transplant site in a transplant model organ, and its effect on the incidence of rejection is assessed as described in Yu PW, Tabuchi RS et al, Sustained correction of B-cell development and function in a murine model of X-linked agammaglobulinemia (XLA) using retroviral-mediated gene transfer. Blood. 2004 104 (5): 1281-90) or Satoh M, Mizutani A et al, X-linked immunodeficient mice spontaneously produce lupus-related anti20 RNA helicase A autoantibodies, but are resistant to pristane-induced lupus. Int Immunol 2003, 15 (9): 1117-24). Administration of RNA reduces the incidence of transplant rejection.
EXAMPLE 29
THE TREATMENT OF NIEMANN-PICK DISEASE DISEASE, MUCOPOLISACARIDOSIS, AND OTHER INABLE METABOLIC ERRORS BY RELEASE OF MODIFIED MRNA FOR BODY TISSUES
Modified nucleoside RNA or pseudouridine encoding sphingomyelinase is released to the lung, brain, or other Niemann-Pick Type A and B disease tissue in animal models, and its effect on the disease is assessed as described in Passini MA, Macauley SL, et al , AAV vector-mediated correction of brain pathology in a mouse model of Niemann-Pick A disease. Mol Ther 2005; 11 (5): 754-62) or Buccoliero R, Ginzburg L, et al, Elevation of lung surfactant phosphatidylcholine in mouse models of Sandhoff and of Niemann-Pick A disease. J Inherit Metab Dis 2004; 27 (5): 641-8). Administration of RNA has been shown to improve the disease.
Modified nucleoside RNA or pseudouridine encoding alpha-L-iduronidase, iduronate-2-sulfatase, or a related enzyme is released into the body tissues of an animal model of mucopolysaccharidosis, and its effect on the disease is evaluated as described in
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Simonaro CM, D'Angelo M, et al, Joint and bone disease in mucopolisaccharidosis VI and VII: identification of new therapeutic targets and biomarkers using animal models. Pediatr Res 2005; 57 (5 Pt 1): 701-7) or McGlynn R, Dobrenis K, et al, Differential subcellular localization of cholesterol, gangliosides, and glycosaminoglycans in murine models of mucopolisaccharide storage disorders. J Comp Neurol 2004 20; 480 (4): 415-26). Administration of RNA improves the disease.
In additional experiments, the mRNA molecules of the present invention are used to provide clotting factors (for example for hemophiliacs). In additional experiments, modified mRNA molecules of the present invention are used to provide acid-b-glucosidase to treat Gaucher disease. In additional experiments, modified mRNA molecules of the present invention are used to provide alpha-galactosidase A to treat Fabry's disease. In additional experiments, modified mRNA molecules of the present invention are used to provide cytokines for the treatment of infectious diseases.
In additional experiments, modified mRNA molecules of the present invention are used to correct other inborn errors of metabolism, by administering mRNA molecules that encode, for example ABCA4; ABCD3; ACADM; AGL; AGT; ALDH4Al; ALPL; AMPD1; APOA2; AVSD1; BRCD2; C1QA; C1QB; C1QG; C8A; C8B; CACNA1S; CCV; CD3Z; CDC2L1; CHML; CHS1; CIAS1; CLCNKB; CMD1A; CMH2; CMM; COL11AI; COL8A2; COL9A2; CPT2; CRB1; CSE; CSF3R; CTPA; CTSK; DBT; DIO1; DISC1; DPYD; EKV; ENO1; ENO1P; EPB41; EPHX1; F13B; F5; FCGR2A; FCGR2B; FCGR3A; FCHL; FH; FMO3; FMO4; FUCA1; FY; GALLEY; GBA; GFND; GJA8; GJB3; GLC3B; HF1; HMGCL; HPC1; HRD; HRPT2; HSD3B2; HSPG2; KCNQ4; KCS; KIF1B; LAMB3; LAMC2; LGMD1B; LMNA; LOR; MCKD1; MCL1; MPZ; MTHFR; MTR; MUTYH; MYOC; NB; NCF2; NEM1; NPHS2; NPPA; NRAS; NTRK1; OPTA2; PBX1; PCHC; PGD; PHA2A; PHGDH; PKLR; PKP1; PLA2G2A; PLOD; PPOX; PPT1; PRCC; PRG4; PSEN2; PTOS1; REN; RFX5; RHD; RMD1; RPE65; SCCD; SERPINC1; SJS1; SLC19A2; SLC2A1; SPG23; SPTA1; TAL1; TNFSF6; TNNT2; TPM3; TSHB; UMPK; UOX; UROD; USH2A; VMGLOM; VWS; WS2B; ABCB11; ABCG5; ABCG8; ACADL; ACP1; AGXT; AHHR; ALMS1; ALPP; ALS2; APOB; BDE; BDMR; BJS; BMPR2; CHRNA1; CMCWTD; CNGA3; COL3A1; COL4A3; COL4A4; COL6A3; CPS1; CRYGA; CRYGEP1; CYP1B1; CYP27A1; DBI; DES; DYSF; AND GIVE; EFEMP1; EIF2AK3; ERCC3;
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FSHR; GINGF; GLC1B; GPD2; GYPC; HADHA; HADHB; HOXD13; HPE2; IGKC; IHH; IRS1; ITGA6; KHK; KYNU; LCT; LHCGR; LSFC; MSH2; MSH6; NEB; NMTC; NPHP1; PAFAH1P1; PAX3; PAX8; PMS1; PNKD; PPH1; PROC; REGIA; SAG; SFTPB; SLC11A1; SLC3Al; SOS1; SPG4; SRD5A2; TCL4; TGFA; TMD; TPO; UGT1A @; UV24; WSS; XDH; ZAP70; ZFHX1B; ACAA1; AGS1; AGTR1; AHSG; AMT; ARMET; BBS3; BCHE; BCPM; BTD; CASR; CCR2; CCR5; CDL1; CMT2B; COL7A1; CP; CPO; CRY; CTNNB1; DEM; ETM1; FANCD2; F1H; FOXL2; GBE1; GLB1; GLC1C; GNAI2; GNAT1; GP9; GPX1; HGD; HRG; ITIH1; KNG; LPP; LRS1; MCCC1; MDS1; MHS4; MITF; MLH1; MYL3; MYMY; OPA1; P2RY12; PBXPI; PCCB; POU1FI; PPARG; PROS1; PTHR1; RCA1; RHO; SCA7; SCLC1; SCN5A; SI; SLC25A20; SLC2A2; TF; TGFBR2; THPO; THRB; TKT; TM4SF1; TRH; UMPS; UQCRC1; USH3A; VHL; WS2A; XPC; ZNF35; ADH1B; ADH1C; AFP; AGA; AIH2; ALB; ASMD; BFHD; CNGA1; CRBM; DCK; DSPP; DTDP2; ELONG; ENAM; ETFDH; AND YOU; F11; FABP2; FGA; FGB; FGFR3; FGG; FSHMD1A; GC; GNPTA; GNRHR; GYPA; HCA; HCL2; HD; HTN3; HVBS6; IDUA; IF; JPD; KIT; KLKB1; LQT4; MANBA; MLLT2; MSX1; MTP; NR3C2; PBT; PDE6B; PEE1; PITX2; PKD2; QDPR; SGCB; SLC25A4; SNCA; SOD3; STATH; TAPVR1; TYS; WBS2; WFS1; WHCR; ADAMTS2; ADRB2; AMCN; AP3BI; APC; ARSB; B4GALT7; BHR1; C6; C7; CCAL2; CKN1; CMDJ; CRHBP; CSF1R; DHFR; DIAPH1; DTR; EOS; EPD; ERVR; F12; FBN2; GDNF; GHR; GLRA1; GM2A; HEXB; HSD17B4; ITGA2; KFS; LGMD1A; LOX; LTC4S; MAN2A1; MCC; MCCC2; MSH3; MSX2; NR3C1; PCSK1; PDE6A; PFBI; RASA1; SCZD1; SDHA; SGCD; SLC22A5; SLC26A2; SLC6A3; SM1; SMA @; SMN1; SMN2; SPINK5; TCOF1; TELAB1; TGFBI; ALDH5Al; ARG1; AT; ASSP2; BCKDHB; BF; C2; C4A; CDKN1A; COL10A1; COL11A2; CYP21A2; DYX2; EJM1; ELOVL4; EPM2A; ESR1; EYA4; F13A1; FANCE; GCLC; GJA1; GLYS1; GMPR; GSE; HCR; HFE; HLA-A; HLA-DPB1; HLA-DRA; HPFH; ICS1; IDDM1; IFNGR1; IGAD1; IGF2R; ISCW; LAMA2; LAP; LCA5; LPA; MCDR1; MOCS1; MUT; MYB; NEU1; NKS1; NYS2; OA3; OODD; OFC1; PARK2; PBCA; PBCRA1; PDB1; PEX3; PEX6; PEX7; PKHD1; PLA2G7; PLG; POLH; PPAC; PSORS1; PUJO; RCD1; RDS; RHAG; RP14; RUNX2; RWS; SCA1; SCZD3; SIASD; SOD2; ST8; TAP1; TAP2; TFAP2B; TNDM; TNF; TPBG; TPMT; TULP1; WISP3; AASS; ABCB1; ABCB4; ACHE; AQP1; ASL; ASNS; AUTS1; BPGM; BRAF; C7orf2; CACNA2D1; CCM1; CD36; CFTR; CHORDOMA; CLCN1; CMH6; CMT2D; COL1A2;
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CRS; CYMD; DFNA5; D, L [D; DYT11; EEC1; ELN; ETV1; FKBP6; GCK; GHRHR; GHS; GLI3; GPDS1; GUSB; HLXB9; HOXA13; HPFH2; HRX; IAB; IMMP2L; KCNH2; LAMB1; LEP; MET; NCF1; NM; OGDH; OPN1SW; PEX1; PGAM2; PMS2; PON1; PPP1R3A; PRSS1; PTC; PTPN12; RP10; RP9; SERPINE1; SGCE; SHFM1; SHH; SLC26A3; SLC26A4; SLOS; SMAD1; TBXAS1; TWIST; ZWS1; ACHM3; ADRB3; ANKI; CA1; CA2; CCAL1; CLN8; CMT4A; CNGB3; COH1; CPP; CRH; CYP11B1; CYP11B2; DECR1; DPYS; DURS1; EBS1; ECA1; EGI; EXT1; EYA1; FGFR1; GNRH1; GSR; GULOP; HR; KCNQ3; KFM; KWE; LGCR; LPL; MCPH1; MOS; MYC; NAT1; NAT2; NBS1; PLAT; PLEC1; PRKDC; PXMP3; RP1; SCZD6; SFTPC; SGM1; SPG5A; STAR; TG; TRPS1; TTPA; VMD1; WRN; ABCA1; ABL1; ABO; ADAMTS13; AK1; ALAD; ALDH1A1; ALDOB; AMBP; AMCD1; ASS; BDMF; BSCL; C5; CDKN2A; CHAC; CLA1; CMD1B; COL5A1; CRAT; DBH; DNAI1; DYS; DYT1; ENG; FANCC; FBP1; FCMD; FRDA; GALT; GLDC; GNE; GSM1; GSN; HSD17B3; HSN1; IBM2; INVS; JBTS1; LALL; LCCS1; LCCS; LGMD2H; LMX1B; MLLT3; MROS; MSSE; NOTCH1; ORM1; PAPPA; PIP5K1B; PTCH; PTGS1; RLN1; RLN2; RMRP; ROR2; RPD1; SARDH; SPTLC1; STOM; TDFA; TEK; TMC1; TRIM32; TSC1; TYRP1; XPA; CACNB2; COLl7A1; CUBN; CXCL12; CYP17; CYP2C19; CYP2C9; EGR2; EMX2; ERCC6; FGFR2; HK1; HPSI; IL2RA; LGI1; LIPA; MAT1A; MBL2; MKI67; MXI1; NODAL; OAT; OATL3; PAX2; PCBD; PEO1; PHYH; PNL1P; PSAP; PTEN; RBP4; RDPA; RET; SFTPA1; SFTPD; SHFM3; SIAL; THC2; TLX1; TNFRSF6; UFS; UROS; AA; ABCC8; ACAT1; ALX4; AMPD3; ANC; APOA1; APOA4; APOC3; ATM; BSCL2; BWS; PANTS; CAT; CCND1; CD3E; CD3G; CD59; CDKN1C; CLN2; CNTF; CPT1A; CTSC; DDB1; DDB2; DHCR7; DLAT; DRD4; ECB2; DI4; EVR1; EXT2; F2; FSHB; FTH1; G6PT1; G6PT2; GIF; HBB; HBBP1; HBD; HBE1; HBG1; HBG2; HMBS; HND; HOMG2; HRAS; HVBS1; IDDM2; IGER; INS; JBS; KCNJ11; KCNJ1; KCNQ1; LDHA; LRP5; MEN1; MLL; MYBPC3; MYO7A; NNO1; OPPG; OPTB1; PAX6; PRAÇA; PDX1; PGL2; PGR; PORC; PTH; PTS; PVRL1; PYGM; RAG1; RAG2; ROM1; RRAS2; SAA1; SCA5; SCZD2; SDHD; SERPING1; SMPD1; TCIRG1; TCL2; TECTA; TH; TREH; TSG101; TYR; USH1C; VMD2; VRN1; WT1; WT2; ZNF145; A2M; AAAS; ACADS; ACLS; ACVRL1; ALDH2; AMHR2; AOM; AQP2; ATD; ATP2A2; BDC; C1R; CD4; CDK4; CNA1; COL2A1; CYP27B1; DRPLA; ENUR2; FEOM1; FGF23; FPF; GNB3; GNS; HAL; HBP1; HMGA2; HMN2; HPD; IGF1; KCNA1; KERA; KRAS2; KRT1; KRT2A;
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KRT3; KRT4; KRT5; KRT6A; KRT6B; KRTHB6; LDHB; LYZ; MGCT; MPE; MVK; MYL2; OAP; PAH; PPKB; PRB3; PTPN11; PXR1; RLS; RSN; SAS; SAX1; SCA2; SCNN1A; SMAL; SPPM; SPSMA; TBX3; TBX5; TCF1; TPI1; TSC3; ULR; VDR; VWF; ATP7B; BRCA2; BRCD1; CLN5; CPB2; DI2; EDNRB; ENUR1; ERCC5; F10; F7; GJB2; GJB6; IPF1; MBS1; MCOR; NYS4; PCCA; RB1; RHOK; SCZD7; SGCG; SLC10A2; SLC25A15; STARP1; ZNFl98; ACHM1; ARVDI; BCH; CTAA1; DAD1; DFNB5; EML1; GALC; GCH1; IBGC1; IG H@; IGHC group; IGHG1; IGHM; IGHR; IV; LTBP2; MCOP; MJD; MNG1 MPD1; MPS3C; MYH6; MYH7; NP; NPC2; PABN1; PSEN1 PYGL; RPGRIP1; SERPINA1; SERPINA3; SERPINA6; SLC7A7; SPG3A; SPTB; TCL1A; TGMI; TITF1; TMIP; TRA @; TSHR; USH1A; VP; ACCPN; AHO2; ANCR; B2M; BBS4; BLM; CAPN3; CDAN1; CDAN3; CLN6; CMH3; CYP19; CYP1A1; CYP1A2; DYX1; EPB42; ETFA; EYCL3; FAH; FBN1; FES; HCVS; HEXA; IVD; LCS1; LIPC; MYO5A; OCA2; OTSC1; PWCR; RLBP1; SLC12A1; SPG6; TPM1; UBE3A; WMS; ABCC6; ALDOA; APRT; ATP2A1; BBS2; CARD15; CATM; CDH1; CETP; CHST6; CLN3; CREBBP; CTH; CTM; CYBA; CYLD; DHS; DNASE1; DPEP1; ERCC4; FANCA; GALNS; GAN; HAGH; HBA1; HBA2; HBHR; HBQ1; HBZ; HBZP; HP; HSD11B2; IL4R; LIPB; MC2R; MEFV; MHC2TA; MLYCD; MMVP1; PHKB; PHKG2; PKD1; PKDTS; PMM2; PXE; SALL1; SCA4; SCNN1B; SCNN1G; SLC12A3; TAT; TSC2; VDI; WT3; ABR; THE HUNT; ACADVL; ACE; ALDH3A2; APOH; ASPA; AXIN2; BCL5; BHD; BLMH; BRCA1; CACD; CCA1; CCZS; CHRNB1; CHRNE; CMT1A; COL1A1; CORD5; CTNS; EPX; ERBB2; G6PC; GAA; GALK1; GCGR; GFAP; GH1; GH2; GP1BA; GPSC; GUCY2D; ITGA2B; ITGB3; ITGB4; KRT10; KRT12; KRT13; KRT14; KRT14L1; KRT14L2; KRT14L3; KRT16; KRT16L1; KRT16L2; KRT17; KRT9; MAPT; MDB; MDCR; MGI; MHS2; MKS1; MPO; MYO15A; NAGLU; NAPB; NF1; NME1; P4HB; PAFAH1B1; PECAM1; PEX12; PHB; PMP22; PRKAR1A; PRKCA; PRKWNK4; PRP8; PRPF8; PTLAH; RARA; RCV1; RMSA1; RP17; RSS; SCN4A; SERPINF2; SGCA; SGSH; SHBG; SLC2A4; SLC4A1; SLC6A4; SMCR; SOST; SOX9; SSTR2; SYM1; SYNS1; TCF2; THRA; TIMP2; TOC; TOP2A; TP53; TRIM37; VBCH; ATP8B1; BCL2; CNSN; CORD1; CYB5; DCC; F5F8D; FECH; PEO; LAMA3; LCFS2; MADH4; MAFD1; MC2R; MCL; MYP2; NPC1; SPPK; TGFBRE; TGIF; TTR; AD2; AMH; APOC2; APOE; ATHS; BAX; BCKDHA; BCL3; BFIC; C3; CACNA1A; CCO; CEACAM5; COMP; CRX; DBA; DDU; DFNA4; DLL3; DM1; DMWD; E11S; ELA2;
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EPOR; ERCC2; ETFB; EXT3; EYCL1; FTL; FUT1; FUT2; FUT6; GAMT; GCDH; GPI; GUSM; HB1; HCL1; HHC2; HHC3; ICAM3; INSR; JAK3; KLK3; LDLR; LHB; LIG1; LOH19CR1; LYL1; MAN2B1; MCOLN1; MDRV; MLLT1; NOTCH3; NPHS1; OFC3; OPA3; PEPD; PRPF31; PRTN3; PRX; PSG1; PVR; RYR1; SLC5A5; SLC7A9; STK11; TBXA2R; TGFB1; TNNI3; TYROBP; ADA; AHCY; AVP; CDAN2; CDPD1; CHED1; CHED2; CHRNA4; CST3; EDN3; EEGV1; FTLL1; GDF5; GNAS; GSS; HNF4A; JAG1; KCNQ2; MKKS; NBIA1; PCK1; PI3; PPCD; PPGB; PRNP; THBD; TOP1; AIRE; APP; CBS; COL6A1; COL6A2; CSTB; DCR; DSCR1; FPDMM; HLCS; HPE1; ITGB2; KCNE1; KNO; PRSS7; RUNX1; SOD1; TAM; ADSL; ARSA; BCR; CECR; CHEK2; COMT; CRYBB2; CSF2RB; CTHM; CYP2D6; CYP2D7P1; DGCR; DAY 1; EWSR1; GGT1; MGCR; MN1; NAGA; NF2; OGS2; PDGFB; PPARA; PRODH; SCO2; SCZD4; SERPIND1; SLC5A1; SOX10; TCN2; TIMP3; TST; YOU F; ABCD1; ACTL1; ADFN; AGMX2; AHDS; AIC; AIED; AIH3; ALAS2; AMCD; AMELX; ANOP1; AIR; ARAF1; ARSC2; ASS; ARTS; ARX; ASAT; ASSP5; ATP7A; ATRX; AVPR2; BFLS; BGN; BTK; BZX; C1HR; CACNA1F; CALB3; CBBM; CCT; CDR1; CFNS; CGF1; CHM; CHR39C; CIDX; CLA2; CLCN5; CLS; CMTX2; CMTX3; CND; COD1; COD2; COL4A5; COL4A6; CPX; CVD1; CYBB; DCX; DFN2; DFN4; DFN6; DHOF; DIAPH2; DKC1; DMD; DSS; DYT3; EBM; EBP; DI1; ELK1; IN D; EVR2; F8; F9; FCP1; FDPSL5; FGD1; FGS1; FMR1; FMR2; G6PD; GABRA3; GATA1; GDI1; GDXY; GJB1; GK; GLA; GPC3; GRPR; GTD; GUST; HMS1; HPRT1; HPT; HTC2; HTR2C; HYR; IDS; IHG1; IL2RG; INDX; IP1; IP2; JMS; KAL1; KFSD; L1CAM; LAMP2; MAA; MAFD2; MAOA; MAOB; MCF2; MCS; MEAX; MECP2; MF4; MGC1; MIC5; MID1; MLLT7; MLS; MRSD; MRX14; MRX1; MRX20; MRX2; MRX3; MRX40; MRXA; MSD; MTM1; MYCL2; MYP1; NDP; NHS; NPHL1; NR0B1; NSX; NYS1; NYX; OA1; OASD; OCRL; ODT1; OFD1; OPA2; OPD1; OPEM; OPN1LW; OPN1MW; OTC; P3; PDHA1; PDR; PFC; PFKFB1; PGK1; PGK1P1; PGS; PHEX; PHKA1; PHKA2; PHP; PIGA; PLP1; POF1; POLA; POU3F4; PPMX; PRD; PRPS1; PRPS2; PRS; RCCP2; RENBP; RENS1; RP2; RP6; RPGR; RPS4X; RPS6KA3; RS1; S11; SDYS; SEDL; SERPINA7; SH2D1A; SHFM2; SLC25A5; SMAX2; SRPX; SRS; STS; SYN1; SYP; TAF1; TAZ; TBX22; TDD; TFE3; THAS; THC; TIMM8A; TIMP1; TKCR; TNFSF5; UBE1; UBE2A; WAS; WSN; WTS; WWS; XIC; XIST; XK; XM; XS; ZFX; ZIC3; ZNF261; ZNF41; ZNF6; AMELY; ASSP6; AZF1; AZF2; DAZ; GCY; RPS4Y; SMCY; SRY;
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ZFY; ABAT; AEZ; AFA; AFD1; ASAH1; ASD1; ASMT; CCAT; CECR9; STRAIN; CLA3; CLN4; CSF2RA; CTS1; DF; DIH1; DWS; DYT2; DYT4; EBR3; ECT; EEF1A1L14; EYCL2; FANCB; GCSH; GCSL; GIP; GTS; HHG; HMI; HOAC; HOKPP2; HRPT1; HSD3B3; HTC1; HV1S; ICHQ; ICR1; ICR5; IL3RA; KAL2; KMS; KRT18; KSS; LCAT; LHON; LIMM; MANBB; MCPH2; MEB; MELAS; MIC2; MPFD; MS; MSS; MTATP6; MTCO1; MTCO3; MTCYB; MTND1; MTND2; MTND4; MTND5; MTND6; MTRNR1; MTRNR2; MTTE; MTTG; MTTI; MTTK; MTTL1; MTTL2; MTTN; MTTP; MTTS1; NAMSD; OCD1; OPD2; PCK2; PCLD; PCOS1; PFKM; PKD3; PRCA1; PRO1; PROP1; RBS; RFXAP; RP; SHOX; SLC25A6; SPG5B; STO; SUOX; THM; or TTD.
EXAMPLE 30
TREATMENT OF VASOESPASM BY RELEASE OF MODIFIED MRNA MOLECULES THAT CODES INTO BODY TISSUES
Modified nucleoside RNA or pseudouridine encoding inducible nitric oxide synthase (iNOS) is released to the vascular endothelium of animal models of vasospasm (eg subarachnoid hemorrhage), and its effect on the disease is assessed as described in Pradilla G, Wang PP, et al , Prevention of vasospasm by anti-CD11 / CD18 monoclonal antibody therapy following subarachnoid hemorrhage in rabbits. J Neurosurg 2004; 101 (1): 8892) or Park S, Yamaguchi M, et al, Neurovascular protection reduces early brain injury after subarachnoid hemorrhage. Stroke 2004; 35 (10): 2412-7). Administration of RNA improves the disease.
EXAMPLE 31
HAIR GROWTH RESTORATION BY RELEASE OF MODIFIED MRNA THAT CODES AN IMMUNOSUPPRESSIVE PROTEIN
Modified nucleoside RNA or pseudouridine encoding a telomerase or an immunosuppressive protein (eg α-MSH, TGF-β 1, or IGF-I is released into the hair follicles of animals used as models of weight loss or baldness, and their effect in hair growth is evaluated as described in Jiang J, Tsuboi R, et al, Topical application of ketoconazole stimulates hair growth in C3H / HeN mice. J Dermatol 2005; 32 (4): 243-7) or McElwee KJ, Freyschmidt- Paul P, et al, Transfer of CD8 (+) cells induces localized hair loss whereas CD4 (+) / CD25 (-) cells promote systemic alopecia areata and
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CD4 (+) / CD25 (+) cells blockade disease onset in the C3H / HeJ mouse model. J Invest Dermatol 2005; 124 (5): 947-57). Administration of RNA restores hair growth.
EXAMPLE 32
SYNTHESIS OF A RNA MOLECULE TRANSCRIBED IN VITRO WITH ALTERED NUCLEOSIDS CONTAINING A SYRNA
One double-stranded RNA (dsRNA) molecule comprising pseudouridine or a modified nucleoside and another comprising a small interfering RNA (siRNA) or short hairpin RNA (shRNA) is synthesized by the following procedure: RNA strips complementary to the desired sequence containing uridine or 1 or more modified nucleosides are synthesized by in vitro transcription (for example by T7, SP6, or T3 RNA phage polymerase) as described in Example 5. DsRNA molecules show reduced immunogenicity. In other experiments the dsRNA molecules are designed to be processed by an intracellular enzyme to yield the desired siRNA or shRNA. Because dsRNA molecules of several hundred nucleotides are easily synthesized, each dsRNA can further be designed to contain multiple siRNA or shRNA molecules, to facilitate the release of several siRNA or shRNA into a single target cell.
EXAMPLE 33
USE OF AN RNA MOLECULE TRANSCRIBED IN VITRO WITH ALTERED NUCLEOSIDS TO RELEASE SYRNA
The dsRNA molecule of the previous Example is complexed with a transfection reagent (for example a cationic transfection reagent, a lipid-based transfection reagent, a protein-based transfection reagent, a polyethyleneimine-based transfection reagent, or calcium phosphate) and released into a target cell of interest. Enzymes on or on the target cell surface degrade dsRNA into the desired siRNA or shRNA molecules. This method effectively silences the transcription of 1 or more cellular genes that correspond to the siRNA or shRNA sequences.
EXAMPLE 34
TESTING THE EFFECT OF ADDITIONAL NUCLEOSIDE MODIFICATIONS ON RNA IMMUNOGENICITY AND TRANSLATION EFFICIENCY
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Additional nucleoside modifications are introduced into RNA transcribed in vitro, using the methods described above in Examples 5 and 10, and their effects and immunogenicity and translation efficiency are tested as described in Examples 4-11 and 12-18, respectively. Certain additional modifications have been shown to decrease immunogenicity and improve translation. These modifications are additional modalities of methods and compositions of the present invention.
Tested modifications include, for example:
γη ¹ A 'rn ² A' Αιττ π'ΐ ^^ 'ιΆ' i ^ A · triéióA 'r ⁶ A * mc ^ A' tm ⁶ A '
Hl TA; AAA r .; rUU; ms 111 TA; ZY; ms 1UA; To ZA; ms TO ZV; g ZV; ZY; ms L zA; AAA L ZA; AAAA ZA;
mQ ² hn ⁶ A * A'γΓή '^' T * γπ ¹ ^ ιύ ^ Τ ^ π 'tn ³ C · Cnr Q ² ^ * qc ⁴ ^ * rn ⁵ ^^ n íic ⁴ Cnr 1c ² c iyi ¹ ^ · Γπ ² ^ τ m ⁷ É ~ r * ms mi zA; rviip); i; mi; m im; m C; Cm; s C; ac C; The. ç; m Cm; ac Cm; κ c; m G; m G; m G;
Gm; m ² 2G; m ² Gm; m ² 2Gm; Gr (p); yW; o2yW; OHyW; OHyW *; imG; mimG; Q; oQ; galQ; manQ; preQ0; preQ1; G +; D; m ⁵ Um ', m ¹ T; Tm; S ⁴ U; -m ⁵ s ² U ', S ² Um,' acp ³ U ·, ho ⁵ u., mo ⁵ U ·; as ⁵ U ·; mcmo ⁵ U; chm ⁵ U; mchm ⁵ U ·; mcm ⁵ U; mcm ⁵ um; mcm ⁵ s ² U; nm ⁵ s ² U; mnm ⁵ U; mnm ⁵ s ² U; mnm ⁵ if ² U; ncm ⁵ U; ncm ⁵ One; cmnm ⁵ U; cmnm ⁵ um; cmnm ⁵ s ² U; m ⁶ 2A; im; m ⁴ C;
('m hni ⁵ C · τη ³ TT' m ¹ cm ⁵ TT 'm ⁶ A ^ n ^- iri ^ Am · m2 ^{, 7} G · γπ ² , ² , ⁷ ^ τ · τη ³ ΤΤ ^ π ^- τη ⁵ ^ 'τη ³ Ψ' m V / AAA; ΑΑΑΑΑ V /; AAA LJ; AAA dC ^ T; CAAA; AAA ζΑΑΑΑ; AAA 2A1H; m G; m G; AAA UAAA; AAA J_Z; AAA I;
f5Cm; m ^l Gm; m ^l Am; im ⁵ U; rm ⁵ s ² U; imG-14; imG2; and ac ⁶ A.
MATERIALS AND METHODS FOR EXAMPLES 35-38
HPLC Purification of RNA: mRNA produced by T7 polymerase transcription was purified by HPLC using a column matrix of polystyrene-divinylbenzene non-porous alkylated copolymer (PS-DVB) (2.1 pm) (21 mm x 100 mm) column) and a Triethylammonium acetate (TEAA) buffer system with an acetonitrile gradient. Buffer A containing 0.1 M TEAA and buffer B containing 0.1 M TEAA and 25% acetonitrile. The columns were equilibrated with 38% buffer B in buffer A, loaded with RNA, and then run with a linear gradient to 55% buffer B for 30 minutes at 5 ml / minute. The fractions corresponding to the desired peak were collected. The RNA analyzes were performed with the same column matrix and buffer system, but using a 7.8 mm x 50 mm column at 1.0 ml / min and a gradient duration of 25 minutes.
Isolation of RNA from column fractions: The collected fractions were combined and first their RNA contents were concentrated using Amicon Ultra-15 centrifugal filter units with 30K membrane (Milipore). The filter device was filled with 15 ml of sample and spun at 4,000x g for 10 min (4 ° C) in a Thermo Scientific Sorvall ST16R centrifuge using a mobile bucket rotor. Under these conditions, ~ 98% of the solvent volume
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131/141 can be removed. When the collected fractions had a volume of more than 15 ml, the filter unit was reused to fill with additional fractions from the column and centrifuging again until all the RNA was in a tube. To remove the salt and solvent from the concentrated RNA, the water-free nuclease was added (up to 15 ml) and the filter device was rotated again. The “washing” process was repeated until the acetonitrile concentration was <0.001%. The desalted, solvent-free sample was removed from the filtration device and the RNA was recovered by overnight precipitation at -20 ° C in NaOAc (0.3 M, pH 5.5), isopropanol (1 volume) and glycogen (3 pl). The precipitated RNA was collected, washed twice with 75% ethanol cooled on ice and reconstituted in water.
dsRNA dot blot: RNA (25-100 ng) was blotted on a nitrocellulose membrane, allowed to dry, blocked with 5% dry skimmed milk in TBS buffer supplemented with 0.05% Tween-20 (TBS-T), and incubated with specific mAb for dsRNA J2 or K1 (English & Scientific Consulting) for 60 minutes. The membranes were washed 6 times with TBS-T and then reacted with donkey HRP conjugated anti-mouse antibody (Jackson Immunology). After washing 6 vees, dsRNA was detected with the addition of SuperSignal West Pico Chemiluminescent substrate (Pierce) and image capture for 30 seconds to 2 minutes in a Fujifilm LAS1000 digital imaging system.
Generation of dendritic cell: Monocytopheresis samples were obtained from normal volunteers using an IRB approved protocol. Human DCs were produced by monocytes under treatment with GM-CSF (50 ng / ml) + IL-4 (100 ng / ml) (R&D Systems) in AIM V medium (Invitrogen) for 7 days. On days 3 and 6, a 50% volume of new media with cytokines was added.
Murine DC were generated by isolation of bone marrow mononuclear cells from Balb / c mice and cultured in RPMI + 10% FBS medium supplemented with murine GMCSF (20 ng / ml, Peprotech). On days 3 and 6, a 50% volume of new medium with GM-CSF was added. Non-adherent cells were used after 7 days of culture.
RNA Lipofectin Complexation: Phosphate stock buffer was added to serum-free DMEM to generate final concentrations of 20 mM potassium phosphate and 100 ng / ml BSA, pH 6.4. For 3 wells of a 96-well plate, RNA complexed with lipofectin was prepared in the following proportions: 2.4 pl of lipofectin was added to 21.3 pl of serum-free DMEM medium with phosphate buffer and incubated at room temperature
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132/141 for 10 minutes. Then, 0.75 pg of RNA in 9.9 pl of serum-free DMEM was added and the mixture was incubated for an additional 10 minutes at room temperature. Finally, 116.4 ml of serum-free DMEM medium was added to increase the final volume to 150 ml. The mixture was vortexed.
RNA TransIT Complexation: For each well of a 96-well plate, 0.25 pg of RNA was added to 17.3 µl of ice-free DMEM. TransIT mRNA reagent (0.3 µl) was added with vortexing followed by 0.2 µl mRNA boosting reagent and vortexing. Complexed RNA was added within 5 minutes of formation.
Cell transfections: For RNA complexed with lipofectin, 50 pl (0.25 pg RNA / well) was added directly to the cells, 5 x 10 ⁵ per well. The transfected cells were incubated for 1 h at 37 ° C in a 5% CO2 incubator. The lipofectin-RNA mixture was removed and replaced with 200 µl of preheated serum containing medium. For RNA complexed with TransIT, 17 µl of complex was added to the cells, 5 x 10 ⁵ per well, in 200 µl of medium containing serum. The cells were lysed in specific lysis medium, 3 to 24 hr after transfection and the activity of firefly or renilla luciferase was measured with specific substrates in a luminometer.
Immunogenicity analysis of RNA: DCs (murine or human) in 96-well plates (5 x 10 ⁵ cells / well) were treated with medium, or lipofectin or RNA complexed with TransIT. The supernatant was collected after 24 hr and subjected to analysis. Levels of IFN-α (TransIT released RNA) or TNF-α (Lipofectin released RNA) (Biosource International, Camarillo, CA) were measured in supernatants by ELISA. Cultures were performed in triplicate to quadruplicate and measured in duplicate.
EXAMPLE 35
This example assesses the sequence and type dependence of translation cells for Ψ-, m5C, and Ψ / m5C-modified mRNA in relation to unmodified RNA (U). mRNA encoding Luciferase Firefly or Renilla with the indicated modifications were complexed to lipofectin and released to murine dendritic cells (A) and HEK293T (B). Human DCs were transfected with mRNA encoding firefly or renilla luciferase with the indicated modifications complexed with TransIT (C). The data shows that depending on the RNA sequence and the type of cell that translates this, the optimal change varies. It must still be demonstrated that the improvement caused by the incorporation of modified nucleosides is
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133/141 substantially higher for primary cells compared to transformed cell lines. Enzyme activity in lysed cells was measured using specific substrates and measured light produced in a luminometer and expressed as an increase sometimes compared to unmodified RNA (U).
EXAMPLE 36
The transcription reactions of T7 phage polymerase used for the generation of mRNA results in larger amounts of RNA of the correct size, but still contains contaminants. This is visualized by applying RNA to a reverse phase HPLC column that separates size-based RNA under denaturing conditions. Ymodified TEV-luciferase-A51 RNA was applied to the HPLC column in 38% Buffer B and subjected to a linear gradient of buffer B increasing to 55%. The profile demonstrated both lower than expected and higher than expected contaminants. These results are shown in Figure 22.
EXAMPLE 37
HPLC purification increases the translation of all types of modified or unmodified RNA, but Ψ-modified mRNA is better translated. The results are shown in Figure 23. (A) mRNA encoding EPO with the indicated modifications and with or without HPLC purification were released to levels of murine DCs and EPO in the supernatants were measured 24 hours later. While modified m5C / Y mRNA had the highest level of translation prior to HPLC purification, Ψ-modified mRNA had the highest translation after HPLC purification. (B) Human DCs were transfected with mRNA that encodes renilla with the modifications indicated with or without HPLC purification. Similar to murine DCs and EPO mRNAs, after HPLC purification, Y-modified mRNA had the highest level of translation.
EXAMPLE 38 Ψ, m5C, and Ψ / mSC modified mRNA have low levels of immunogenicity which is reduced to control levels with HPLC purification. The results are shown in Figure 24. (B) Human DCs were transfected with RNA complexed with TransIT with the indicated modifications with or without HPLC purification. IFN-α levels were measured after 24 hours. HPLC purification increased the immunogenicity of unmodified RNA, which
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134/141 is sequence dependent, as other unmodified RNA had similar levels of IFN-a levels or reduced levels. Ψ-modified RNA had unmeasured levels of IFNa, similar to DCs treated with control. (B) Ψ-modified RNA before (-) and after HPLC purification (P1 and P2) was analyzed for dsRNA using dot blotting with a monoclonal antibody specific for dsRNA (J2). Purification of RNA removed dsRNA contamination. (C) Ψ-modified RNA encoding iPS factors are immunogenic, which is removed by HPLC purification of the RNA.
MATERIALS AND METHODS FOR EXAMPLES 39-41
Cell culture Neonatal human epidermal keratinocyte (HEKn) cells (Invitrogen) were cultured in EpiLife medium supplemented with Penicillin / Streptomycin keratinocyte growth supplement (Invitrogen). All cells grew at 37 ° C and 5% CO2. The human iPS cells that were induced using the methods described here were transferred to qualified hESC matrix matrix (BD Biosciences) in coated 6-well plates after transfection.
Construction of Vectors. Generally the same as Examples 1-3.
MRNA production. Generally the same as Examples 1-3.
Purification and analysis of mRNA. In some experimental modalities, the mRNA was purified by HPLC, column fractions were collected, and the mRNA fractions were analyzed for purity and immunogenicity as described in “Materials and Methods for Examples 35-38” and / or as described and shown for Figures 22-24. In some experimental embodiments, purified RNA preparations comprising or consisting of mRNAs encoding one or more reprogramming factors that exhibited little or no immunogenicity were used for the experiments to reprogram human somatic cells to iPS cells.
Reprogramming of Primary Keratinocytes. HEKn cells were plated in 1 x 10 ⁵ cells / well of a 6-well plate in EpiLife medium and grown overnight. The cells were transfected with equal amounts of each mRNA reprogramming factor (KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2) or a subset of factors using TransIT ™ transfection reagent mRNA (MirusBio, Madison, WI). Three transfections were performed, on alternate days, with half changes each day. The day after the third
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135/141 transfection, cells were trypsinized and plated in mTeSR1 medium (StemCell Technologies) on plates coated with 6 well matrigel. The mTeSR cell medium was changed daily. The cells were maintained at 37 ^o C with 5% CO2. The plates were screened for changes in morphology using an inverted microscope.
HEKn cells were further reprogrammed by a single transfection by electroporation with equal amounts of each mRNA reprogramming factor. The cells were plated directly onto the matrigel-coated plates at a density of 1 x 10 ⁵ cells per 6-well plate or 7.5 x 10 ⁵ cells per 10 cm plate in a mTeSR1 medium that was changed daily.
Immunofluorescence. Generally the same as Examples 1-3.
Quantitative RT-PCR (qPCR) cellular RNA was reverse transcribed using standard methods and an oligo d (T) 21 primer of equivalent amounts of cellular RNA. Three messages were amplified using gene-specific primers and real-time PCR using SYBR green detection and GAPDH normalization. The expression levels were determined in relation to the expression level in the original HEKn cell line, and described as changes in the cycle threshold level (Ct).
EXAMPLE 39
This example describes the development of a protocol for the generation of iPS cells from somatic keratinocytes. Equal amounts (by weight) of KLF4, c-MYC, OCT4, and SOX2 mRNAs were transfected into HEKn cells three times (once every other day) with TransIT ™ mRNA Reagent. The medium was changed daily. The day after the third transfection, the cells were plated on matrigel-coated plates and grown in mTeSR1 cell medium. For 11 days after the first transfection, the morphology of the reprogrammed cell began to appear (Fig. 28).
EXAMPLE 40
This example describes the characterization of cells that result from the transfection of primary keratinocytes with equal amounts of KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2 mRNAs. One million HEKn cells were electroporated once with 5 micrograms of each mRNA and plated on 10 cm plates coated with matrigel in mTeSR1 cell medium. 15 days after transfection the cells were fixed for analysis of
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136/141 immunofluorescence. The resulting colonies were positive for their iPS markers KLF4, LIN28, SSEA4, TRA-1-60, and NANOG (Fig. 29).
EXAMPLE 41
This example describes the differences in expression between primerous keratinocytes and reprogrammed keratinocytes with equal amounts of KLF4, c-MYC, OCT4, and SOX2 mRNAs. 7.5 x 10 ⁵ HEKn cells were electroporated once with 3 or 5 micrograms of each mRNA and plated on plates coated with 10 cm matrix in mTeSR1 medium. The medium was changed daily. 13 days after transfection, the cells were transferred to freshly coated matrigel plates. 21 days after transfection, the total cell RNA was purified from untransfected HEKn cells and two wells of reprogrammed cells. Equal amounts of each cellular RNA were converted to cDNA and analyzed by qPCR. Increased levels of NANOG, CRYPT and REX1 were detected by qPCR using specific message primers (Fig. 30). These three messages have been shown to be elevated in iPS cells (Aasen T et al. 2008. Nature Biotech 26: 1276). None of these factors were introduced into the cells by transfection; therefore, changes in expression are due to the influence of the reprogramming factors that were introduced.
EXAMPLE 42
Transdifferentiation cells with mRNAs
The cells can be transdifferentiated using the purified mRNA preparations described here, or the modified mRNA as described here, or the purified mRNA preparations containing modified mRNA as described here. In this Example, a purified RNA preparation containing OCT4 mRNA that has at least one pseudouridine or 5-methylcytidine is employed. Said purified and modified OCT4 mRNAs are substituted for the OCT4 coding vectors in the protocol described in Szabo et al. (Nature 468: 521-528, 2010, which is incorporated herein as a reference in its entirety as if completely established here) and in the protocol described in Racila et al. (Gene Therapy, 1-10, 2010, hereby incorporated by reference in its entirety as if completely established here). In an embodiment of each of these methods, the purified RNA preparation comprises or consists of OCT 4 mRNA, in which all uridine nucleosides are replaced by pseudouridine nucleosides. In an embodiment of each of these methods, the preparation of purified RNA comprises or consists of OCT 4
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137/141 mRNA, where all cytidine nucleosides are replaced by 5 methyl cystidine nucleosides. In an embodiment of each of these methods, the purified RNA preparation comprises or consists of OCT 4 mRNA, in which all uridine nucleosides are replaced by pseudouridine nucleosides and all cytidine nucleosides are replaced by 5-methylcystidine nucleosides. In preferred models, the OCT4 mRNA is purified to be free of contaminating RNAs. The reference by Racilla et al. describes a system in which human keratinocytes have been transdifferentiated for being redirected and an alternative differentiation pathway. In particular, transient transfection of human hair keratinocytes with the transcription factor OCT4 was employed. After 2 days, these transfected cells showed expression of endogenous embryonic genes and showed reduced genomic methylation. It has been shown that these cells could be converted into contractile neuronal and mesenchymal cell types.
The reference by Szabo et al. demonstrated the ability to generate progenitors and mature cells of the hematopoietic fate directly from human dermal fibroblasts without establishing pluripotency. In particular, the ectopic expression of OCT4 activated hematopoietic transcription factors, along with treatment with the specific cytokine, allowed the generation of cells that express the pan-leukocyte CD45 marker. These unique fibroblast-derived cells gave rise to granulocytic, monocytic, megakaryocytic and erythroid lineages and demonstrated in vivo graft capacity.
In addition to the use of OCT4, both of these protocols also used cytokines or growth factors, such as growth and transformation factor (TGF), PDGF-BB, cell factor (SCF), and tyrosine kinase ligand type FMS 3 (Flt3L). Other growth factors and cytokines could be used as granulocyte colony stimulating factor (G-CSF), IL3, IL-6, erythropoietin, basic fibroblast growth factor (bFGF), insulin-like growth factor 2 (IGFII), and bone morphogenetic protein 4 (BMP-4). As such, in certain modalities, the protocols by Racilla et al. or Szabo et al. are repeated with the replacement of modified OCT4 mRNA (eg, psuedouridine-modified and / or 5-methylcytidine-modified), along with the use of the growth factors or cytokines mentioned above. In some embodiments, the cells are contacted with the cytokine and / or growth factor proteins that are used. In some other embodiments, cells are contacted with modified mRNAs (for example, modified mRNAs
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138/141 as described in the present application, for example, for example, modified psuedouridine and / or modified 5-methylcytidine) which encodes one or more of the cytokines and / or growth factors that are used in the transdifferentiation protocol. It will be clear from this description that the present invention includes contacting a human or animal cell with a purified RNA preparation comprising or consisting of mRNA that encodes a reprogramming factor to transdifferentiate a cell containing a first state of differentiation or phenotype for a cell containing a second state of differentiation or phenotype.
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权利要求:
Claims (11)
[1]
1. In vitro method for reprogramming human or other mammalian somatic cells, which exhibit a first differentiated state or phenotype, for an induced pluripotent stem cell (iPS or iPSCs cells), characterized by the fact that it comprises:
a) introducing into said cells that exhibit a first state or differentiated phenotype a purified RNA preparation comprising modified and synthesized in vitro mRNA molecules that
- encode a plurality of 4 or more, 5 or more, or 6 iPSC induction factors selected from the group consisting of OCT4, SOX2, KLF4, MYC, NANOG and LIN28,
- comprise a modified nucleoside selected from the group consisting of pseudouridine (Ψ), 5-methyluridine (m ⁵ U) and 2-thiouridine (S ² U) in place of the unmodified uridine nucleoside and 5-methylcytidine (m ⁵ C) in place of the corresponding unmodified cytidine nucleoside, and
- exhibit a 5'-cap and a 3 'poly (A) tail;
wherein said modified mRNA molecules make up said purified RNA preparation which is purified by a purification process comprising:
purifying said modified mRNA molecules using HPLC or gravity flow column purification; and / or treating said modified mRNA molecules with a ribonuclease III (RNase III) enzyme such that small RNase III digestion products are generated, and purifying said small RNase III digestion products away from said molecules of modified mRNA, in which this process removes: contaminating RNA molecules that are immunogenic and toxic to the cell by inducing an innate immune response, as detectable by measuring the decreased secretion of IFN-α or TNF-α cytokine transfected with dendritic cells said modified purified mRNA molecules compared to the secretion of said cytokine from transfected dendritic cells with the unpurified modified mRNA molecules;
such that said purified RNA preparation is free of contaminating RNA molecules that, if present, would activate an immune response in said cells
Petition 870200009792, of 01/21/2020, p. 21/24
[2]
2/4 sufficient to prevent the survival of said cells and the generation of reprogrammed cells; and
b) repeating said introduction of said purified RNA preparation over several days and culturing the cell, where the cell is reprogrammed for an iPSC.
2. Method according to claim 1, characterized in that the modified mRNA molecules, synthesized in vitro, exhibit a 3 'poly-A tail comprising 50 to 200 or more than 150 nucleotides.
[3]
3. Method according to claim 1 or 2, characterized by the fact that said RNA contaminating molecules are selected from the group consisting of dsRNA molecules and mRNA molecules without cap.
[4]
4. Method according to claim 1 or 2, characterized in that said purified RNA preparation comprises synthesized in vitro modified mRNA molecules, in which:
(i) more than 98% of said mRNA molecules are capped;
(ii) said mRNA molecules exhibit a cap with a cap1 structure, in which the 2'hydroxyl of the ribose in the penultimate nucleotide with respect to the cap nucleotide is methylated;
(iii) said poly-A tail comprises 50 to 200 or more than 150 nucleotides;
(iv) said modified mRNA molecules exhibit a particular sequence selected from the group consisting of: a 5 'UTR sequence, a Kozak sequence, an IRES sequence, and a 3' UTR sequence, in which said particular sequence results in greater translation of the modified mRNA molecules compared to the same mRNA molecules that do not exhibit said particular sequence, including wherein said 5 'UTR sequence or 3' UTR sequence is from an Xenopus or human alpha globin or beta globin mRNA , or wherein said 5 'UTR sequence is an etch tobacco virus (TEV) RNA sequence.
(v) the pseudouridine-modified nucleoside (Ψ) is present in place of all corresponding unmodified uridine nucleosides;
(vi) said in vitro modified and synthesized mRNA molecules further comprise the 5-methylcytidine-modified nucleoside (m ⁵ C) in place of a portion of the corresponding unmodified cytidine nucleosides; and / or
Petition 870200009792, of 01/21/2020, p. 22/24
3/4 (vii) said modified mRNA molecules encode said plurality of 5 or 6 iPSC induction factors and / or said MYC is selected from c-MYC, 1MYC and N-MYC.
[5]
5. Method according to claim 4, characterized in that said in vitro modified and synthesized mRNA molecules comprise 5-methylcytidine-modified nucleoside (m ⁵ C) in place of all corresponding unmodified cytidine nucleosides.
[6]
6. Method according to claim 2, characterized by the fact that each said modified nucleoside is 5-methyluridine (m ⁵ U) or pseudouridine which is not further modified (Ψ) and is present in place of all said nucleosides of corresponding unmodified uridine and / or wherein said modified nucleoside is 5-methylcytidine (m ⁵ C) and is present in place of all said unmodified corresponding cytidine nucleosides.
[7]
Method according to claim 2, characterized in that said modified nucleoside is 2-thiouridine (s ² U) and is present in place of only a portion of the corresponding unmodified uridine nucleoside and / or is 5methylcytidine (m ⁵ C) and is present in place of only a portion of the corresponding unmodified cytidine nucleoside.
[8]
8. Method, according to claim 7, characterized by the fact that:
(a) the percentage fraction of uridine residues in said in vitro synthesized mRNA, which are modified by the presence of said modified 2-thiouridine nucleoside (s2U), is selected from the group consisting of less than 20% of said uridine residues, less than 30% of said uridine residues, less than 40% of said uridine residues, less than 50% of said uridine residues, less than 60% of said uridine residues and less than 70% of said uridine residues, particularly in that said percentage fraction of uridine residues in said in vitro synthesized mRNA, which are modified by the presence of said modified 2thiouridine (s2U) nucleoside, is selected from the group consisting of 10%, 12%, 14%, 16% , 18%, 20%, 30%, 35%, 40%, 45%, 50% and 60% of said uridine residues; and / or (b) the percentage fraction of cytidine residues in said in vitro synthesized mRNA, which are modified by the presence of m ⁵ C (5-methylcytidine) in place of cytidine, is selected from the group consisting of less than 20% of said cytidine residues, less than 30% of said cytidine residues, less than 40% of said cytidine residues
Petition 870200009792, of 01/21/2020, p. 23/24
4/4 cytidine, less than 50% of said cytidine residues, less than 60% of said cytidine residues and less than 70% of said cytidine residues, particularly where said percentage fraction of cytidine residues, in said mRNA synthesized in vitro, which are modified by the presence of said modified 5-methylcytidine nucleoside, is selected from the group consisting of 10%, 12%, 14%, 16%, 18%, 20%, 30%, 35%, 40% , 45%, 50% and 60% of said cytidine residues.
[9]
Method according to any one of claims 1 to 8, characterized in that it further comprises, after said introduction, the contact of said cell with a growth factor and / or cytokine.
[10]
Method according to any one of claims 1 to 8, characterized in that it comprises introducing into the said cell the modified mRNA encoding a cytokine or growth factor.
[11]
11. Composition characterized by comprising a mammalian cell containing the purified RNA preparation or the in vitro modified and synthesized mRNA molecules that comprise said purified RNA preparation, as defined in any one of claims 1 to 8.

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法律状态:
2017-12-12| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-05-02| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|

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2018-06-12| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-10-09| B07B| Technical examination (opinion): publication cancelled [chapter 7.2 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 7.4 NA RPI NO 2475 DE 12/06/2018 POR TER SIDO INDEVIDA. |

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2018-10-23| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-04-24| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2019-10-22| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-02-11| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-04-14| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/12/2010, OBSERVADAS AS CONDICOES LEGAIS. |

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2020-06-23| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: REF. RPI 2571 DE 14/04/2020 QUANTO A CAPA DA CARTA PATENTE DUPLICADA. |

优先权:

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申请号 | 申请日 | 专利标题

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US26731209P| true| 2009-12-07|2009-12-07|

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PCT/US2010/059305|WO2011071931A2|2009-12-07|2010-12-07|Rna preparations comprising purified modified rna for reprogramming cells|

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