STABLE CELL LINES FOR RETROVIRAL PRODUCTION

专利摘要:
The present invention relates to a retroviral producer cell comprising retroviral nucleic acid sequences encoding: gag and pol proteins, an envelope protein or a functional substitute thereof; and the genomic RNA of the retroviral vector particle, wherein said nucleic acid sequences are all located at a single locus within the genome of the retroviral producer cell.
公开号:FR3044016A1
申请号:FR1661255
申请日:2016-11-21
公开日:2017-05-26
发明作者:Sabine Johnson；Celeste Pallant；Eirini Vamva；Conrad Vink
申请人:GlaxoSmithKline Intellectual Property Development Ltd；
IPC主号:

专利说明:

FIELD OF THE INVENTION The invention relates to nucleic acid vectors comprising genes necessary for retroviral production and their uses. The invention also relates to methods for producing packaging / retrovirus cell lines comprising the nucleic acid vectors as described herein.
BACKGROUND OF THE INVENTION
In gene therapy, genetic material is administered to endogenous cells in a subject in need of treatment. The genetic material can introduce new genes into the subject or introduce additional copies of preexisting genes, or introduce different alleles or variants of the genes that are present in the subject. Viral vector systems are proposed as a method of efficiently delivering genes for use in gene therapy (Verma and Somia (1997) Nature 389: 239-242).
In particular, these viral vectors are based on members of the retrovirus family because of their ability to integrate their genetic inheritance into the genome of the host. Retroviral vectors are designed to retain the essential proteins necessary for packaging and distribution of the retroviral genome, but any non-essential accessory protein, including that responsible for their pathogenic profile, is eliminated. Examples of retroviral vectors include lentiviral vectors, such as those based on human immunodeficiency virus type 1 (HIV-1), which are widely used because they are capable of integrating into non-proliferative cells.
Currently, the majority of viral vectors are produced by transient co-transfection of viral genes into a host cell line. The viral genes are introduced using bacterial plasmids that exist in the host cell only for a limited period of time because the viral genes remain on the plasmids and are not integrated into the genome. As such, transiently transfected genetic material is not passed on to subsequent generations during cell division.
There are many disadvantages associated with transient transfection, such as batch-to-batch variability, the high cost of transfection reagents, and the difficulty of maintaining quality control (see Segura et al (2013) Expert Opin. Ther 13 (7): 987-1011). The process of transfection itself also requires many operations and its scaling up is a challenge. It is also difficult to remove the plasmid impurities that are carried during the preparation of the vector (see Pichlmair et al (2007) J. Virol 81 (2): 539-47).
In order to solve the problems associated with transient transfection, there is an aspiration to develop retroviral packaging and retroviral-producing cell lines to simplify the production of retroviral vectors.
Packing cell lines were generated by transfection of a cell line capable of packaging retroviral vectors with plasmids, wherein the individual plasmids carry unique retroviral packaging genes and eukaryotic selection markers. Packaging genes are integrated into the genome of packaging cell lines and are described as being stably transfected. In the last 20 years, various attempts have been made to produce stable packaging and production cell lines for retroviral vectors.
Several problems have been reported regarding production and packaging cell lines produced through the integration of retroviral vector components into the host cell genome. In the first place, the sequential introduction of the components of the retroviral vector can be laborious and inflexible. Problems with genetic and / or transcriptional instability of retroviral vector components are also encountered when they are integrated into the genome of host cells because the integration site is impossible to predict (Ni et al (2005) J. Gene Med 1: 818-834.). A significant drop in viral vector productivity has also been reported during suspension adaptation and upscaling of producer cell lines (Farson et al., (2001) Hum.) Gene Ther., 12: 981- 997, Guy et al (2013) Hum Gene Ther., Methods 24 (2): 125-39).
It is therefore an object of the present invention to provide a method of producing stable cell lines for the production and packaging of retroviruses which overcomes one or more of the disadvantages associated with existing methods.
SUMMARY OF THE INVENTION
The present inventors have developed a new mode of production of packaging and production cell lines which involves the use of nucleic acid vectors comprising a non-mammalian origin of replication and have the capacity to contain at least 25 kilobases (kb). of DNA, such as artificial bacterial chromosomes, comprising the retroviral genes essential for the production of a retroviral vector. This allows the expression of the retroviral genes necessary for the production of replication defective retroviral vector particles to solve the problems associated with transient transfection methods. The use of a nucleic acid vector comprising a non-mammalian origin of replication and having the capacity to contain at least 25 kb of DNA (i.e., a large hybrid DNA) has several advantages. First, the vectors can be first manipulated in non-mammalian cells (eg microbial cells, such as bacterial cells) in contrast to mammalian host cells, which makes them easier to use (eg chromosomes artificial bacterial can be first manipulated in E. coli). Once the nucleic acid vector has been prepared, it can be introduced into a host cell and any mammalian cell that has the nucleic acid vector integrated into its endogenous chromosomes can be selected to isolate a stable cell line. The introduction of retroviral nucleic acids into the mammalian host cell is also accomplished in one step, thereby reducing the selection pressure and the silencing time interval. This allows for faster screening of potential packaging cells and reduces the cost of materials since only a single vector is used in contrast to previous methods that utilize multiple plasmid vectors. In particular, the use of the current system reduces the manufacturing cost by saving the cost of plasmids, necessary transfection reagents (eg polyethyleneimine [PEI]) by reducing the amount of benzonase treatment. required (there is a reduced amount of DNA in the lentiviral harvest, therefore, less benzonase is required to eliminate the excess during the downstream treatment), and in terms of test costs (it is not no longer necessary to carry out tests for the residual plasmid in the lentiviral product).
In addition, the retroviral genes essential for retroviral production (with or without the transfer vector) are present within the nucleic acid vector so that, if the vector is introduced into the mammalian host cells, all of the Retroviral genes incorporated into the nucleic acid vector will integrate into a locus within the endogenous genome of the mammalian host cell. This solves problems such as gene silencing that may occur when the retroviral genes are randomly integrated and at different loci within the genome of the host cell. The use of the nucleic acid vectors of the invention therefore provides advantages in terms of generation of retrovirus production and packaging cell lines.
Therefore, according to a first aspect of the invention, there is provided a retroviral producer cell comprising nucleic acid sequences encoding: gag and pol proteins, an env protein or a functional substitute thereof; and the genomic RNA of the retroviral vector particle, wherein said nucleic acid sequences are all located at a single locus within the genome of the retroviral producer cell.
BRIEF DESCRIPTION OF THE FIGURES FIGURE 1: Step by step guide for the construction of BACpack-WTGP-277delU5 and BACpack-SYNGP-277delU5. FIGURE 2: Selection of a stable polyclonal batch. Adherent HEK293T cells were transfected with BACpackWTGagPol-Transfer using calcium phosphate. Stable batches were produced after 2 weeks of selection with Zeocin. To see which stable transfectants were able to generate viruses, stable poly batches were induced with doxycycline for 48 hours. The viral supernatant was harvested 48 hours after induction, filtered through a 0.22 μπι filter and titrated by transduction of HEK293T cells. GFP positive transduced cells were used to calculate the transduction units / ml (TU / ml). FIG. 3: Generation of stable transfection clones in suspension. HEK293 6E cells were transfected with BACpackWTGagPol-Transfer using the reagent 293fectin. Stable batches were produced after 2 weeks of selection with Zeocin. Stable batches were cloned by limiting the dilution in 96-well plates to obtain non-cellular clones, which were then expanded. The GFP detected by fluorescence microscopy the best Clones 1, 14, 15 and 16 obtained with the adherent medium (DMEM + FBS), then a suspension adaptation (FreeStyle medium) was carried out. FIGURE 4; Induction of lentivirus in clones in suspension. To see if stable HEK6E transfectants were able to generate viruses, 20 ml of stable suspension clones were induced with doxycycline (2 μg / ml) for 48 hours. The viral supernatant was harvested 48 hours after induction, filtered through a 0.45 μτα filter and titrated by transduction of HEK293T cells. GFP-positive transduced cells were used to calculate the transduction units / ml (TU / ml). FIG. 5: Vector titers of the clones produced according to Example 4. The results show that the vector titers from clones 1 and 16 increased modestly between passage 5 and passage 21.
DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS
Unless otherwise stated, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.
The term "comprising" includes "including" or "consisting", for example, a composition "comprising" X may consist exclusively of X or may include something more, for example X + Y.
The term "essentially consisting of" limits the extent of the characteristic to the specified materials or steps and those which do not materially affect the basic characteristics of the claimed feature.
The term "constituted of" excludes the presence of any additional components.
The term "about" in relation to a numerical value x means, for example, x ± 10%, 5%, 2% or 1 I.
The term "vector" or "nucleic acid vector" refers to a vehicle that is able to artificially carry foreign (ie exogenous) genetic material into another cell, where it can be replicated and / or expressed. Examples of vectors include non-mammalian nucleic acid vectors, such as artificial bacterial chromosomes (BAC); artificial chromosomes of yeast (YAC), artificial chromosomes derived from PI (PAC), cosmids or fosmids.
Other examples of vectors include viral vectors such as retroviral and lentiviral vectors, which are of particular interest in the present application. Lentiviral vectors, such as those based on human immunodeficiency virus type 1 (HIV-1), are widely used since they are capable of integrating into nonproliferative cells. The viral vectors can be made defective by replication by breaking their viral genome into separate parts, for example by placing it on separate plasmids. For example, what is called the first generation of lentiviral vectors, developed by the Salk Institute for Biological Studies, was constructed as a three-plasmid expression system consisting of an expression cassette. packaging, the envelope expression cassette and the vector expression cassette. The "packaging plasmid" contains all the gag-pol sequences, the regulatory sequences (tat and rev) and accessories (vif, vpr, vpu, nef). The "envelope plasmid" contains the vesicular stomatitis virus glycoprotein (VSVg) in substitution for the native HIV-1 envelope protein, under the control of a cytomegalovirus (CMV) promoter. The third plasmid (the "transfer plasmid") carries the long terminal repeats (LTRs), the encapsulation sequence (ψ), the Rev response element sequence (RRE), and the CMV promoter to express the transgene at the same time. inside the host cell.
The second generation of lentiviral vectors was characterized by the deletion of virulence sequences vpr, vif, vpu and nef. The packaging vector has been reduced to the gag, pol, tat and rev genes, thereby increasing the safety of the system.
To improve the lentiviral system, the third-generation vectors were designed by eliminating the tat gene from the packaging hybrid and inactivating the LTRs from the vector cassette, thereby reducing problems with the effects of mutagenesis. insertional.
The different generations of lentiviruses are described in the following references: First generation: Naldini et al. (1996) Science 272 (5259): 263-7; Second generation: Zufferey et al. (1997) Nat. Biotechnol. (9): 871-5; Third generation: Dull et al. (1998) J. Virol. 72 (11): 8463-7. A summary of the development of lentiviral vectors can be found in Sakuma et al. (2012) Biochem. J. 443 (3): 603-18 and Picanço-Castro et al. (2008) Exp. Opin. Therap. Patents 18 (5): 525-539.
The term "non-mammalian origin of replication" refers to a nucleic acid sequence where the replication is initiated and which is derived from a non-mammalian source. This allows the nucleic acid vectors of the invention to stably replicate and segregate alongside endogenous chromosomes in a suitable host cell (e.g. a microbial cell, such as a bacterial or yeast cell) so that be transmissible to the offspring of the host cell unless the host cell is a mammalian host cell. In mammalian host cells, nucleic acid vectors having non-mammalian origins of replication will either be integrated into the endogenous chromosomes of the mammalian host cell, or lost during replication of the mammalian host cell. For example, nucleic acid vectors having non-mammalian origins of replication such as artificial bacterial chromosomes (BAC), artificial chromosome derived from PI (PAC), cosmids or fosmids, are capable of stable replication and segregate with endogenous chromosomes in bacterial cells (such as E. coli), but if they are introduced into mammalian host cells, the BAC, PAC, cosmid or fosmid will either be integrated or lost during replication of the mammalian host cell. Artificial yeast chromosomes (YAC) are able to stably replicate and segregate alongside endogenous chromosomes in yeast cells, but if introduced into mammalian host cells, the YACs will either be integrated or lost during replication of the mammalian host cell. Therefore, in this context, the nucleic acid vectors of the invention act as DNA reservoirs (i.e., for genes essential for retroviral production) that can be easily transferred into mammals to generate stable cell lines for retroviral production. Examples of non-mammalian origins of replication include the origins of bacterial replications, such as oriC, oriV or oriS; or the origins of yeast replication known as autonomous replication sequences (ARS elements).
The nucleic acid vectors of the present invention comprise a non-mammalian origin of replication and are capable of containing at least 25 kilobases (kb) of DNA. In one embodiment, the nucleic acid vector has the ability to contain at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110 , 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 or 350 kb d DNA. It is understood that the references to the "capacity to contain" have their usual meaning and imply that the upper limit for the size of the insert for the nucleic acid vector is not less than the claimed size (c '). that is, not less than 25 kb of DNA).
The object of the present invention is to include genes essential to retroviral packaging in a single hybrid (i.e., the nucleic acid vector). Therefore, the nucleic acid vectors of the invention must be capable of containing large DNA inserts. For the avoidance of doubt, it is understood that references to "nucleic acid vectors" or "artificial chromosomes" do not relate to natural bacterial plasmids (for example as the plasmids currently used in transient transfection methods) because they are not able to contain at least 25 kb of DNA. The maximum size insert that a plasmid can contain is about 15 kb. These nucleic acid vectors also do not refer to bacteriophages, which generally contain only maximal inserts of 5 to 11 kb. Therefore, in one embodiment, the nucleic acid vector of the invention is not a plasmid, nor a bacteriophage, nor an episome.
The term "endogenous chromosomes" refers to genomic chromosomes in the host cell prior to the generation or introduction of an exogenous nucleic acid vector, such as an artificial bacterial chromosome.
The terms "transfection," "transformation," and "transduction," as used herein, can be used to describe the insertion of the non-mammalian or viral vector into a target cell. Vector insertion is usually referred to as transformation for bacterial cells and transfection for eukaryotic cells, although insertion of a viral vector may also be termed transduction. Those skilled in the art will be familiar with the various commonly used non-viral transfection methods, which include but are not limited to the use of physical methods (e.g., electroporation, cell compression, sonoporation, optical transfection, protoplast fusion, impalfection, magnetofection, gene gun or bombardment of particles), chemical reagents (for example calcium phosphate, highly branched organic compounds or cationic polymers) or cationic lipids (for example lipofection). Several methods of transfection require the contact of plasmid DNA solutions with the cells which are then cultured and selected for expression of a marker gene.
The term "promoter" refers to a sequence that impulses gene expression. In order to drive a high expression level, it may be beneficial to use a highly effective promoter, such as a highly effective non-retroviral promoter. Examples of suitable promoters may include a promoter such as the human cytomegalovirus (CMV) immediate early promoter, the spleen focus-forming virus (SFFV) promoter, the Rous sarcoma virus (RSV) promoter or the factor 1 promoter. -alpha of human elongation (pEF).
A Tet operon (transcriptional activation controlled by tetracycline) can be used in an inducible gene expression method, the transcription being started or stopped reversibly in the presence of the antibiotic tetracycline or a derivative thereof (eg doxycycline) By nature, the Ptet promoter expresses TetR, the repressor, and TetA, the protein that pumps the tetracycline antiobiotic out of the cell. In the present invention, the Tet operon may be present or absent, for example, in one embodiment, the Tet operon may be present in the promoter.
The term "selectable marker" refers to a gene that will assist in the selection of cells actively expressing an inserted gene (eg a transgene). Examples of suitable selection markers include the enzymes coding for antibiotic resistance (i.e., an antibiotic resistance gene), for example, kanamycin, neomycin, puromycin, hygromycin, blasticidin or zeocin. Another example of suitable selection markers are fluorescent proteins, for example Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP) or Blue Fluorescent Protein (BFP).
The term "polyA signal" refers to a polyadenylation signal sequence, for example in place 3 'of a transgene, which allows host factors to add a polyadenosine tail (polyA) at the end of the nascent mRNA when the transcription. The polyA tail is a stretch of up to 300 adenosine ribonucleotides that protects the mRNA from enzymatic degradation and also facilitates translation. Therefore, the nucleic acid vectors of the present invention may include a polyA signal sequence such as polyA signals of human beta globin or rabbit beta globin, early or late polyA signals of simian virus 40 (SV40), the polyA signal of human insulin, or the polyA signal of bovine growth hormone. In one embodiment, the polyA signal sequence is the polyA signal of human beta globin.
The term "intron sequence" refers to a nucleotide sequence that is removed from the final gene product by splicing the RNA. The use of an intron downstream of the activator / promoter region and upstream of the cDNA insert has been found to increase the level of gene expression. The increase in expression depends in particular on the cDNA insert. Therefore, the nucleic acid vector of the present invention may comprise introns such as the intron of human beta globin, intron II of rabbit beta globin, or a chimeric human beta-globulin intron. In one embodiment, the intron is an intron of human beta globin and / or an intron II of rabbit beta globin.
The term "packaging cell line" refers to a cell line comprising gag and pol protein and stably inserted envelope glycoprotein genes. Alternatively, the term "producer cell line" refers to a packaging cell line having a stably inserted transfer vector containing a transgene of interest. One skilled in the art will appreciate that the nucleic acid vectors described herein can be used to generate packaging cell lines (i.e., when at least the gag, pol and env genes are present on the acid vector nucleic and incorporated into a host cell) or producing cell lines (i.e., wherein the nucleic acid vector further comprises the components of the transfer vector to be incorporated into a host cell along with the gag, pol and env genes).
The term "stably transfected" refers to cell lines that are capable of transmitting to their offspring (i.e., daughter cells) the retroviral genes introduced, either because the transfected DNA has been incorporated into the endogenous chromosomes either by stable inheritance of exogenous chromosomes.
NUCLEIC ACID VECTORS
According to one aspect of the invention, there is provided a nucleic acid vector comprising a non-mammalian origin of replication and having the capacity to contain at least 25 kilobases (kb) of DNA, characterized in that said nucleic acid vector comprises retroviral nucleic acid sequences encoding: gag and pol proteins, and an env protein or functional substitute thereof.
In particular, each of the retroviral nucleic acid sequences may be arranged as individual expression hybrids within the nucleic acid vector.
Current methods for retroviral vector generation involve transient transfection of retroviral genes into a host cell. However, several disadvantages are related to this method because it is expensive, requires a lot of operations and it is difficult to scale it up. One solution would be to manipulate a packaging cell line that stably incorporates retroviral packaging genes to avoid problems with transient transfection and to reduce the emission of variable retroviral vectors.
The present inventors have found that the nucleic acid vectors described herein can be used to produce a retroviral packaging cell line that eliminates prior difficulties related to retroviral vector production methods. For example, known methods for producing retroviral packaging cell lines involve multiple rounds of selection after the introduction of each retroviral gene. This operation can take up to six months and also requires a lot of manpower. By including all of the retroviral genes in the nucleic acid vector, the retroviral genes can then be inserted into the endogenous chromosomes of a mammalian host cell in a single step. Therefore, the use of a nucleic acid vector, as proposed herein, will reduce the selection pressure, reduce the time interval of silencing and allow for faster screening of the packaging cells. potential. In addition, the retroviral genes included on the nucleic acid vector will all be integrated into the endogenous chromosomes of the mammalian host cell at a single locus, which will reduce the risk that the individual retroviral genes will become silent and will guarantee that all retroviral genes will be expressed regularly.
In one embodiment, the nucleic acid vector further comprises nucleic acid sequences that encode the genomic RNA of a retroviral vector particle. It is understood that the genomic RNA of the retroviral vector particle is usually included on the "transfer vector" used in the transient transfection methods. The transfer vector plasmid contains a promoter (such as CMV), the 3 'LTR (which may or may not be a self-activating 3' LTR (ie, SIN)), the 5 'LTR (which may or may not contain the U5 region), the encapsidation sequence (ψ) and potentially the promoter-linked transgene.
In one embodiment, multiple copies of the genomic RNA of the retroviral vector particle (i.e., the transfer vector) are included in the nucleic acid vector. Multiple copies of the transfer vector are expected to produce a higher viral vector titer. For example, the nucleic acid vector may comprise two or more copies, such as three, four, five, six, seven, eight, nine or ten or more copies of the genomic RNA of the retroviral vector particle (this is that is, the transfer vector).
In one embodiment, the nucleic acid vector contains a site or a plurality of recombination sites. This allows the target sequences to be integrated into the endogenous chromosomes of the mammalian host cell in a site-specific manner in the presence of a recombinase enzyme. The recombinase enzyme catalyzes the recombination reaction between two recombination sites.
Several types of site specific recombination systems are known in the art and any suitable recombination system can be used in the present invention. For example, in one embodiment, the recombination site (s) are chosen or derived from the lambda phage int / att system, the bacteriophage PI Cre / lox system, the yeast FLP / FRT system, the Gin recombinase system. / gix of Mu phage, Cin recombinase system, E. coli recombinase system Pin and R / RS system of plasmid pSR1, or any combination thereof. In another embodiment, the recombination site is an att site (eg from phage lambda), where the att site allows oriented integration in the presence of lambda integrase. It is understood that the reference to lambda integrase includes references to mutant integrases that are still compatible with the int / att system, for example modified lambda integrases, described in WO 2002/097059.
In one embodiment, the nucleic acid vector is selected from: an artificial bacterial chromosome (BAC), an artificial yeast chromosome (YAC), an artificial chromosome derived from PI (PAC), a fosmid or a cosmid. In another embodiment, the nucleic acid vector is a bacterial artificial chromosome (BAC).
Bacterial artificial chromosomes
The term "artificial bacterial chromosome" or "BAC" refers to a hybrid DNA derived from bacterial plasmids that is capable of containing a large exogenous DNA insert. They can generally contain a maximum DNA insert of about 350 kb. BACs were developed from the well-characterized bacterial functional fertility plasmid (plasmid F) that contains distribution genes that promote regular distribution of plasmids after bacterial cell division. This allows BACs to be stably replicated and segregated alongside endogenous bacterial genomes (such as E. coli). The BAC usually contains at least one copy of an origin of replication (such as the oriS or oriV gene), the repE gene (for plasmid replication and copy number regulation), and distribution genes (such as sopA, sopB , parA, parB and / or parC), which ensures the stable maintenance of the BAC in bacterial cells. BACs are naturally circular and super-coiled, making them easier to recover than linear artificial chromosomes such as YACs. They can also be introduced into bacterial host cells relatively easily, using simple methods such as electroporation.
In one embodiment, the bacterial artificial chromosome comprises an oriS gene. In one embodiment, the bacterial artificial chromosome comprises a repE gene. In one embodiment, the artificial bacterial chromosome comprises distribution genes. In another embodiment, the distribution genes are selected from sopA, sopB, parA, parB and / or parC. In yet another embodiment, the artificial bacterial chromosome comprises a sopA and sopB gene.
The BAC for use in the present invention can be obtained from commercial sources, for example the LUCIGEN ™ pSMART BAC (see Genome number EU101022.1 for the complete backbone sequence). This BAC contains the "copy-plus" system of L-arabinose which also contains the copy replication origin of the oriV medium, which is active only in the presence of the TrfA replication protein. The gene for TrfA can be incorporated into the genome of bacterial host cells under the control of the araC-PsAD L-arabinose inducible promoter (see Wild et al., (2002) Genome Res., 12 (9): 1434-1444). The addition of L-arabinose induces the expression of TrfA, which activates oriV, which causes plasmid replication of up to 50 copies per cell.
Artificial chromosomes of yeast
The term "artificial chromosome of yeast" or "YAC" refers to chromosomes in which the yeast DNA is incorporated into bacterial plasmids. They contain an autonomous replication sequence (ARS) (that is, an origin of replication), a centromere, and telomeres. In contrast to BACs, YAC is linear and therefore contains yeast telomeres at each end of the chromosome to protect its ends from degradation as it is transmitted to the progeny of the host cell. YACs may contain a range of DNA insert sizes, from about 100 to 2000 kb.
Artificial chromosomes derived from PI
The term "PI derived artificial chromosome" or "PAC" refers to hybrid DNAs derived from bacteriophage PI DNA and bacterial F plasmid. They can usually contain a maximum DNA insert of about 100 to 300 kb and are used as cloning vectors in E. coli. PACs have similar benefits to BACs, such as being easily purified and being able to enter bacterial host cells.
Cosmids and fosmids
The term "cosmid" refers to hybrid DNAs derived from bacterial plasmids which additionally contain cos sites derived from bacteriophage lambda. Cosmids generally contain an origin of bacterial replication (such as oriV), a selection marker, a cloning site and at least one cos site. Cosmids can usually accept a maximum DNA insert of 40 to 45 kb.
Cosmids have been shown to be more effective in the infection of E. coli cells than standard bacterial plasmids. The term "fosmids" refers to non-mammalian nucleic acid vectors that are similar to cosmids except that they are based on bacterial F plasmid. In particular, they use the origin of plasmid F replication and distribution mechanisms that allow the cloning of large fragments of DNA. Fosmids can usually accept a maximum DNA insert of 40 kb.
RETROVIRUS
Retroviruses are a family of viruses that contain pseudo-diploid single-stranded genomic RNA. They encode a reverse transcriptase that produces DNA from genomic RNA, which can then be inserted into the DNA of the host cell. The invention described herein can be used to produce replication defective retroviral vector particles. The retroviral vector particle of the present invention may be selected from or derived from any suitable retrovirus.
In one embodiment, the retroviral vector particle is derived from or selected from a lentivirus, an alpha-retrovirus, a gamma retrovirus, or a foamy retrovirus, such as a lentivirus or gamma retrovirus, particularly a lentivirus. In another embodiment, the retroviral vector particle is a lentivirus selected from the group consisting of HIV-1, HIV-2, VIS, VIF, VAIE and Visna. Lentiviruses are capable of infecting non-dividing (i.e., quiescent) cells, making them retroviral vectors of interest for gene therapy. In yet another embodiment, the retroviral vector particle is HIV-1 or is derived from HIV-1. The genome structure of some retroviruses may be in the art. For example, details on HIV-1 can be found at GenBank NCBI (Genome Entry # AF033819). HIV-1 is one of the best understood retroviruses and is therefore often used as a retroviral vector. Retroviral genes
The nucleic acid sequences common to all retroviruses can be further explained as follows: Long terminal repeats (LTRs): The basic structure of a retrovirus genome comprises a 5 'LTR and a 3' LTR between or at within which are located the genes necessary for retroviral production. LTRs are necessary for retroviral integration and transcription. They can also act as promoter sequences to regulate the expression of retroviral genes (which means that they are cis-acting genes). The LTRs are composed of three subregions named U3, R, U5: U3 is derived from the unique sequence at the 3 'end of the RNA, R is derived from a repeat sequence at both ends of the RNA; and U5 is derived from the unique sequence at the 5 'end of the RNA. Therefore, in one embodiment, the nucleic acid vector further comprises a 5 'and 3' LTR. In another embodiment, the U5 region of the 5 'LTR can be deleted and replaced with a non-HIV-1 polyA tail (see Hanawa et al (2002) Mol Ther 5 (3): 242-51). .
In order to address safety concerns regarding the generation of replication-competent viruses, a self-inactivating vector (SIN) has been developed by deleting a section in the U3 region of the 3 'LTR, which includes the TATA box and binding sites for Spl and NF-κΒ transcription factors (see Miyoshi et al (1998) J. Virol 72 (10): 8150-7). The deletion is transferred to the 5 'LTR after reverse transcription and integration into infected cells, resulting in the transcriptional inactivation of the LTR. This is known as a self-inactivating lentivirus-based vector system that may be included in the present invention. L': Encapsidation of retroviral RNAs occurs by virtue of a sequence ψ (psi) located at the 5 'end of the retroviral genome. It is also well known in the art that sequences downstream of the psi sequence and extending into the gag coding region are involved in the efficient production of retroviral vectors (see Cui et al (1999) J. Virol. (7): 6171-6176). In one embodiment, the nucleic acid vector further comprises a sequence ψ (psi).
Primer Binding Site (PBS): The retroviral genome contains a PBS that is present after the U5 region of the 5 'LTR. This site binds to the tRNA primer necessary for initiation of reverse transcription. In one embodiment, the nucleic acid vector further comprises a PBS sequence. PPT: Retroviral genomes contain short stretches of purines, called polypurine tract (PPT) near the 3 'end of the retroviral genome. These PPTs serve as RNA primers for DNA synthesis to more strands during reverse transcription. Complex retroviruses (such as HIV-1) contain a second, more centrally located PPT (ie, a central polypurine tract (cPPT)) that provides a second site for initiation of synthesis of the DNA. Retroviral vectors encoding a cPPT have been shown to have enhanced transduction and transgene expression (see Barry et al (2001) Hum Gene Ther 12 (9): 1103-8). In one embodiment, the nucleic acid vector further comprises a 3 'PPT sequence and / or a cPPT sequence.
The genomic structure of the non-coding regions described above is well known to those skilled in the art. For example, details on the genomic structure of non-coding regions in HIV-1 can be found in the GenBank NCBI database at Genome Entry # AF033819, or for HIV-1 HXB2 (a strain of commonly used HIV-1 reference) at Genome Entry No. K03455. In one embodiment, the non-coding regions are derived from the sequences available under Genome K03455 entry number, e.g. from base pairs 454-1126 (for R-U5-PBS-Gag), 7622- 8479 (for RRE) or 7769-8146 (for RRE), 4781-4898 (for cPPT), 9015-9120 & 9521-9719 (for dNEF-PPT-sinU3-R-U5).
Gag / pol: The expression of the gag and pol genes is based on a translation shift of the reading frame between gag and gagpol. Both are polyproteins that are cleaved during ripening. The major structural proteins of template, capsid and nucleocapsid of the retroviral vector, are encoded by gag. The pol gene codes for retroviral enzymes: i) reverse transcriptase, essential for the reverse transcription of retroviral genomic RNA to a double-stranded DNA, ii) integrase, which allows integration of retroviral genomic DNA into a chromosome of the host cell, and iii) protease which cleaves the synthesized polyprotein to produce the mature and functional retrovirus proteins. In one embodiment, the retroviral nucleic acid sequence encoding the gag and pol proteins is derived from the HXB2 sequence of HIV-1, which is available at the Genome KO3455 entry number, for example from bases 790-5105,
Env - The env gene ("envelope") encodes the transmembrane surface and components of the retroviral envelope (eg glycoproteins gp120 and gp41 of HIV-1) and is involved in membrane fusion of the retroviral cell. In order to broaden tropism of the retroviral vector tissues, the retroviral vectors described herein may be pseudotyped with an envelope protein from another virus. Pseudotyping refers to the method by which the range of host cells of the retroviral vectors, including lentiviral vectors, can be extended or modified by changing the glycoproteins (GPs) on the retroviral vector particles (eg using the obtained or derived GPs). other enveloped viruses or using synthetic / artificial GPs). The glycoprotein most commonly used for pseudotyping retroviral vectors is the vesicular stomatitis virus GP (VSVg), because of its wide tropism and the high stability of its vector particles. However, it is well understood by those skilled in the art that other glycoproteins may be used for pseudotyping (see Cronin et al (2005) Curr, Gene Ther 5 (4): 387-398). The choice of the virus used for the pseudotyping may also depend on the type of cell and / or organ to be targeted because certain pseudotypes have been found to have preferences for a type of tissue.
In one embodiment, the env protein or a functional substitute thereof is obtained or derived from a virus selected from a vesiculovirus (e.g., vesicular stomatitis virus), a lyssavirus (e.g., rabies virus, Mokola), an arenavirus (eg lymphocytic choriomeningitis virus (LCMV)), an alphavirus (eg Ross River virus (RRV), Sindbis virus, Semliki forest virus (SFV), encephalitis virus Venezuelan equine), a filovirus (eg Reston Ebola virus, Zaire Ebola virus, Lassa virus), an alpharetrovirus (eg avian leukosis virus (ALV)), a betarovirus (eg retrovirus Jaagsiekte sheep (JSRV)), a gammaretrovirus (eg Moloney murine leukemia virus (MLV), Gibbon monkey leukemia virus (GALV), feline endogenous retrovirus (RD114)), a deltartrovirus ( for example a human T-lymphotropic virus 1 (HTLV -1)), a spumavirus (for example human foam virus), a lentivirus (for example Maedi-visna virus (MW)), a coronavirus (for example SARS-coV), a respirovirus (for example Sendai virus, virus respiratory syncytial syndrome (RSV)), a hepacivirus (eg, hepatitis C virus (HCV)), an influenza virus (eg, influenza A virus), and a nucleopolyhedrovirus (eg, multiple nucleopolyhedrovirus Autographa californica (AcMNPV)). In another embodiment, the env protein or a functional substitute thereof is obtained or derived from the vesicular stomatitis virus.
In this embodiment, vesicular stomatitis virus glycoprotein (VSVg) protein can be used which allows the retroviral particles to infect a wider range of host cells and eliminate the chances of recombination to produce wild-type envelope proteins. In another embodiment, the nucleic acid sequence encoding the env protein or a functional substitute thereof is derived from the sequence available in Genome No. J02428.1, for example from base pairs 3071 to 4720.
The structural genes described herein are common to all retroviruses. Other helper genes can be found in different types of retroviruses. For example, lentiviruses, like HIV-1, contain six other auxiliary genes known as rev, vif, vpu, vpr, nef, and tat. Other retroviruses may have helper genes that are analogous to the genes described herein, but may not always be indicated under the same name as in the literature. References such as Tomonaga and Mikami (1996) J. Gen. Virol. 77 (Pt 8): 1611-1621 describe various retrovirus helper genes.
Rev: The rev auxiliary gene ("virion regulator") encodes an accessory protein that binds to the Rev response element (RRE) and facilitates the export of retroviral transcripts. The protein product of the gene allows retroviral mRNA fragments containing the Rev response element (RRE) to be exported from the nucleus into the cytoplasm. The RRE sequence is predicted to form a complex folded structure. This particular role of rev reflects a tight coupling of the splicing and nuclear export stages. In one embodiment, the nucleic acid vector comprises an RRE sequence. In another embodiment, the RRE sequence is derived from the HXB2 sequence of HIV-1 which is available at Genome Accession No. K03455, e.g. from base pairs 7622 to 8479 or 7769 to 8146. , especially base pairs 7622 to 8479.
Rev binds to RRE and facilitates the export of single-spliced (env, alive, vpr and vpu) or unspliced viral transcripts (qag, pol and genomic RNA), thus causing downstream events such as translation and packaging genes (see Suhasini and Reddy (2009) Curr HIV Res.7 (1): 91-100). In one embodiment, the nucleic acid vector further comprises the rev helper gene or a gene analogous thereto (i.e. from other retroviruses or a functionally similar system). The inclusion of the rev gene ensures the efficient export of the retroviral vector genome RNA transcripts from the nucleus to the cytoplasm, particularly if an RRE element is also included in the transcript to be transported. In another embodiment, the rev gene comprises a sequence identity of at least 60%, as at least 70%, with the base pairs 970 to 1320 of the Genome M11840 entry number (c '). i.e. HIV-1 clone 12 cDNA, locus HIVPCV12). In an alternative embodiment, the rev gene comprises a sequence identity of at least 60%, as at least 70%, 80%, 90% or 100% with the base pairs 5970 to 6040 and 8379 to 8653 Genome K03455.1 (ie Human Immunodeficiency Virus type 1, HXB2).
Auxiliary genes are thought to play a role in retroviral replication and pathogenesis, therefore, several current viral vector production systems do not include some of these genes. The exception is rev which is usually present or a system similar to the rev / RRE system is potentially used. Therefore, in one embodiment, the nucleic acid sequences encoding one or more vpr, vif, vpu, tat, and nef auxiliary genes or helper genes are terminated such that said helper genes are removed from the RNA genomics of the retroviral vector particle or are unable to encode functional helper proteins. In another embodiment, at least two or more auxiliary genes, three or more, four or more, or all of the vpr, vif, vpu, tat, and nef auxiliary genes, or like helper genes are terminated so that said genes auxiliaries are removed from the genomic RNA of the retroviral vector particle or are unable to encode functional helper proteins. Removal of the functional helper gene may not require removal of the entire gene, removal of a portion of the gene or interruption of the gene will be sufficient.
It is understood that the nucleic acid sequences encoding the replication defective retroviral vector particle may be the same as, or derived from, the retrovirus wild-type genes on which the retroviral vector particle is based, or that the sequences may be versions of genetically modified or otherwise altered sequences present in the wild-type virus. Therefore, retroviral genes incorporated into nucleic acid vectors or genomes of the host cell can also be referred to as codon-optimized versions of the wild type genes.
ADDITIONAL COMPONENTS
The nucleic acid vectors of the invention may comprise other additional components. These additional features can be used to help, for example, stabilize transcripts for translation, increase the level of gene expression, and turn on / off gene transcription.
The retroviral vector particles produced by the invention can be used in gene therapy methods. Therefore, in one embodiment, the nucleic acid vector comprises one or more transgenes. This transgene may be a therapeutically active gene that encodes a gene product that can be used to treat or ameliorate a target disease. The transgene may encode, for example, antisense RNA, a ribozyme, a protein (for example a tumor suppressor protein), a toxin, an antigen (which may be used to induce antibodies or helper T cells or cytotoxic T lymphocytes) or an antibody (such as a single-stranded antibody). In one embodiment the transgene encodes beta globin.
Multiple copies of the transgene-containing transfer vector are expected to increase the titer of the retroviral vector, therefore, in one embodiment, the nucleic acid vector comprises multiple copies of the transgene, such as two copies. transgene or more, especially three or more. In some cases, more than one gene product is required to treat a disease, therefore, in another embodiment, the nucleic acid vector further comprises two or more different transgenes, such as three or more, or four or more.
References to a "transgene" refers to heterologous or foreign DNA that is not present or not sufficiently expressed in the host cell of a mammal into which it is introduced. This may include, for example, the case where a target gene is not correctly expressed in the mammalian host cell, therefore a corrected version of the target gene is introduced as a transgene. Therefore, the transgene may be a gene of potential therapeutic interest. The transgene may have been obtained from another cell type, or from another species, or synthetically prepared. Alternatively, the transgene may have been obtained from a host cell, but operably linked to regulatory regions that are different from those present in the native gene. Alternatively, the transgene may have a different allele or variant different from a gene present in the host cell.
The goal of gene therapy is to modify the genetic material of living cells for therapeutic purposes, and this involves the insertion of a functional gene into a cell to achieve a therapeutic effect. The retroviral vector produced using the nucleic acid vectors and the host cells described herein can be used to transfect target cells and induce the expression of the gene of potential therapeutic interest. The retroviral vector can therefore be used for the treatment of a mammalian subject, such as a human subject, afflicted with a disease including, but not limited to, hereditary pathologies, cancer, and certain viral infections.
In one embodiment, the nucleic acid vector further comprises a transcription regulatory element. For example, any of the elements described herein may be operably linked to a promoter so that expression can be controlled. The promoters referred to herein may include promoters known, in whole or in part, which may be constitutively active or inducible, for example in the presence of a regulatory protein. In one embodiment, the nucleic acid vector further comprises a high efficiency promoter such as a CMV promoter. This promoter has the advantage of promoting a high level of expression of the elements encoded on the non-mammalian nucleic acid vector. In another embodiment, the CMV promoter comprises a sequence derived from the human cytomegalovirus strain AD169. This sequence is available under accession number Genome X17403, for example from base pairs 173731 to 174404.
In one embodiment, the promoter (such as the CMV promoter) further comprises at least one Tet operon. The Tet operon system can be used to control the expression of the retroviral sequences contained within the nucleic acid vector. In short, the Tet repressor protein blocks expression by site-binding of the Tet operon, there is no gene expression. Upon addition of tetracycline or doxycycline, the Tet repressor is sequestered, thereby allowing promoter activity, and gene expression is then initiated. Tet operon systems are widely available, such as the Tet operon used in the pcDNA ™ 4 / T0 mammalian expression vector available from Invitrogen.
In one embodiment, the nucleic acid vector further comprises a repressor protein of the tetracycline resistance operon ("Tet repressor" or "TetR"). In another embodiment, the Tet repressor is optimized at the codon level.
In one embodiment, the nucleic acid vector further comprises an insulator, such as a chromatin insulator. The term "insulator" refers to a genetic sequence that blocks the interaction between promoters and activators. In another embodiment, the insulator (as a chromatin insulator) is present between each of the retroviral nucleic acid sequences. This helps prevent promoter interference (i.e., when the promoter of a transcription unit influences the expression of an adjacent transcription unit) between adjacent retroviral nucleic acid sequences. It will be understood that if the insulators are present in the nucleic acid vector between each of the retroviral nucleic acid sequences, then these can be arranged as individual expression hybrids within the vector of the nucleic acid. nucleic acid. For example, each sequence encoding retroviral nucleic acid sequences has its own promoter and / or intron and / or polyA signal. In one embodiment, the chromatin insulator has a sequence identity of at least 90%, for example at least 95%, with the sequence of chicken HS4 isolate (Gallus gallus) (e.g. Genome U78775.2, base pairs 1 to 1205).
In one embodiment, the nucleic acid vector further comprises a selectable marker. This allows cells that have been incorporated into the nucleic acid sequences encoding a replication defective retroviral vector particle to be selected. In another embodiment, the selectable marker is an antibiotic resistance gene, such as a zeocin, kanamycin, or puromycin resistance gene, particularly a zeocin resistance gene (ZeoR). In yet another embodiment, the zeocin resistance gene is derived from the Hindustans blep Streptoalloteichus gene, see for example the Gen X52869.1 entry number from base pairs 3 through 377.
In one embodiment, the nucleic acid vector further comprises a polyA signal. The use of a polyA signal has the advantage of protecting the mRNA from enzymatic degradation and of facilitating translation. In one embodiment, the polyA signal is obtained or derived from SV40, bovine growth hormone and / or human beta globin. In one embodiment, the polyA signal is derived from the early SV40 polyA signal (e.g., see Genome Entry No. EF579804.1, base pairs 2668 to 2538 from the minus strand). In one embodiment, the polyA signal is derived from the polyA signal of human beta globin (e.g., see Genome GU324922.1 entry, base pairs 3394 to 4162).
In one embodiment, the nucleic acid vector further comprises an intron sequence. The use of an intron downstream of the activator / promoter region and upstream of the cDNA insert (i.e., transgene) is known to increase the expression rate of the insert. In another embodiment, the intron sequence is an intron sequence of human beta globin or intron II of rabbit beta globin. In one embodiment, the intron of human beta globin is derived from the sequence available under Genome No. KM504957.1 (eg, from base pairs 476 to 1393). In one embodiment, intron II of the rabbit beta globin is derived from the sequence available under Genome V00882.1 entry # (eg from base pairs 718 to 1290).
In one embodiment, the nucleic acid vector further comprises a posttranscription regulatory element of the groundhog virus (WPRE). The presence of the WPRE has been shown to enhance expression and, as such, is likely to be beneficial in achieving high levels of expression. In another embodiment, the WPRE is derived from the sequence available at Genomic Entry No. J04514.1 (e.g., from base pairs 1093 to 1684).
In one embodiment, the nucleic acid vector further comprises an internal ribosome entry site (IRES). An IRES is a structured RNA element that is usually found in the 5 'untranslated region upstream of the 5' cap (which is required for assembly of the initiation complex). IRES is recognized by translation initiation factors and allows independent translation of the cap. In another embodiment, IRES is derived from the encephalomyocarditis virus (EMCV) genome (eg see Genome Accession No. KF836387.1, base pairs 151 to 724).
In one embodiment, the nucleic acid vector further comprises a multiple cloning site (MC S). An MC S is a short segment of DNA within the nucleic acid vector that contains multiple restriction sites (eg 10, 15 or 20 sites). These sites are usually present only once within the nucleic acid vector to ensure that the endonuclease cleaves only at a site. This allows the retroviral genes to be easily inserted using the appropriate endonucleases (i.e., restriction enzymes). It will be understood by those skilled in the art that the hybrids may be arranged in any order within the nucleic acid vector. In one exemplary embodiment, the nucleic acid vector comprises the following insert: a retroviral nucleic acid sequence encoding the gag and pol proteins, a retroviral nucleic acid sequence encoding env protein or a substitute function thereof (such as VSVg), a retroviral nucleic acid sequence encoding the rev helper gene (such as an optimized codon rev sequence) or a gene analogous thereto or a functionally analogous system, a repressor protein of the tetracycline resistance operon (TetR), an internal ribosome entry site, and a selectable marker (such as a zeocin resistance selection marker) (i.e., a BAC sequence); Retaining GagPol-Env-Rev-RepressorTet-IRES-Antibiotic Resistance Marker ("BAC Skeleton"); such as: GagPol- (wild-type) VSVg- (codon optimized) Rev-RepressorTet-IRES-Zeocin Resistance -pSMARTBAC). In another embodiment, an insulator (such as a chromatin insulator) is present between each gagpol, env, and rev. In another embodiment, a promoter is present between each of the gagpol, env, and rev sequences. In yet another embodiment, at least one copy of the transfer vector sequence (i.e. comprising the nucleic acid sequences that encode the genomic RNA of a retroviral vector particle) is present before the gagpol sequence.
In one embodiment, the nucleic acid vector comprises the following insert: an insulator (such as a chromatin insulator), a promoter (such as a CMV promoter, optionally comprising a Tet operon sequence), an intron (such as a intron human beta globulin), a retroviral nucleic acid sequence encoding gag and pol proteins, a retroviral nucleic acid encoding RRE, a polyA signal (as a polyA signal of human beta globulin), an insulator (such as a chromatin insulator ), a promoter (such as a CMV promoter, optionally comprising a Tet operon sequence), an intron (such as a human beta globulin intron), a retroviral nucleic acid sequence encoding the env protein, or a functional substitute for the env protein; (as VSVg), a polyA signal (such as a polyA signal of human beta globulin), an insulator (such as a chromatin insulator), a promoter (such as a CMV promoter, optionally comprising an operon sequence Te t), a retroviral nucleic acid sequence encoding the rev helper gene or a gene analogous thereto or a functionally analogous system, a polyA signal (such as a polyA signal of human beta globulin), an insulator (such as an insulator chromatin), a promoter (such as a CMV promoter), an intron (such as a rabbit beta globulin intron), a repressor protein of the tetracycline resistance operon (TetR), a ribosome entry site internally, a selectable marker (such as a zeocin resistance selection marker), a polyA signal, and a multiple cloning site.
The nucleic acid sequences can be introduced into the nucleic acid vector sequentially. This allows selection after each integration to ensure that all the necessary nucleic acid sequences are successfully integrated into the nucleic acid vector. Alternatively, at least two or more nucleic acid sequences are introduced into the nucleic acid vector simultaneously.
It will be understood that the additional genes described herein can be introduced into the nucleic acid vector by standard molecular cloning techniques known in the art, for example using restriction endonucleases and ligation techniques. In addition, the nucleic acid vector, in particular the BACs, PACs, fosmides and / or cosmids, can be introduced into bacterial host cells (such as E. coli cells, in particular E. coli strain DH10B). by standard techniques, such as electroporation.
USES
According to another aspect of the invention, the nucleic acid vector as defined herein is provided for use in the production of retroviral packaging or production cell lines.
The nucleic acid vectors described herein can be used to create a retroviral packaging cell line that would greatly simplify the production of retroviral vectors. It is understood that if a transgene is included on the nucleic acid vector, it would then be used to create a producer cell line.
As indicated herein, it would be useful to develop a stable retroviral (or retrovirus producing) cell line to overcome the difficulties associated with transient transfection. The nucleic acid vectors described in this specification can be used to prepare said packaging cell lines as they are capable of containing large DNA inserts containing the essential genes required for retroviral packaging, which can then be integrated. in the endogenous genome of mammalian host cells in one step.
HOST CELLS
According to another aspect of the invention there is provided a retroviral packaging cell comprising nucleic acid sequences encoding: gag and pol proteins, and an env protein or functional substitute thereof; wherein said nucleic acid sequences are all located at a single locus within the genome of the retroviral packaging cell. The advantage of including all of the retroviral genes on a large nucleic acid vector is that they can be prepared in microbial cells (such as bacterial or yeast cells) first, which are easier to use and manipulate, before introducing them into mammalian cells in a single step. This allows the selection pressure to be relaxed and the time interval of silencing to be reduced once the retroviral genes have been integrated into a mammalian host cell. The hallmark of this method is that all of the retroviral genes required to create a packaging cell line are present in a single locus in the endogenous genome, rather than being randomly distributed throughout the endogenous genome. This provides the advantage of producing a retroviral packaging cell that expresses all of the retroviral genes at the same rate because they are located on the same locus, as compared to prior methods where the retroviral genes are randomly integrated throughout the endogenous genome which can lead to irregular expression rates.
In one embodiment, the retroviral packaging cell further comprises nucleic acid sequences that encode the genomic RNA of the retroviral vector particle. This may also be located on the same locus as the nucleic acid sequences encoding the gag and pol proteins and the env protein or a functional substitute thereof.
Therefore, according to another aspect of the invention, there is provided a retroviral producer cell comprising nucleic acid sequences encoding: gag and pol proteins, an env protein or a functional substitute thereof; and the genomic RNA of the retroviral vector particle, wherein said nucleic acid sequences are all located at a single locus within the genome of the retroviral producer cell.
In one embodiment, the retroviral packaging cell is a mammalian cell. In another embodiment, the mammalian cell is selected from HEK 293, CHO, Jurkat, KS62, PerC6, HeLa, or a derivative or functional equivalent thereof . In yet another embodiment, the mammalian host cell is a HEK 293 cell or derived from a HEK 293 cell. These cell lines could be adherent cell lines (i.e., they grow into a monolayer attached to a surface) or adapted / non-adherent cell lines in suspension (i.e., they grow in suspension in a culture medium). In yet another embodiment, the HEK 293 cell is a HEK 293T cell. The term "HEK 293 cell" refers to the human embryonic kidney cell line 293 which is commonly used in biotechnology. In particular, HEK 293T cells are commonly used for the production of various retroviral vectors. Other examples of suitable commercially available cell lines include T REX ™ cell lines (Life Technologies).
It will be understood that all the embodiments described above for the nucleic acid vector may also be applied to the retroviral production / packaging cells of the invention.
PROCESSES
According to another aspect of the invention there is provided a method for producing a stable retroviral packaging cell line, comprising: (a) introducing the nucleic acid vector as defined herein into a culture mammalian host cells; and (b) selecting, within the culture, a mammalian host cell that has the nucleic acid sequences encoded on the integrated vector in an endogenous chromosome of the mammalian host cell.
In one embodiment, the mammalian host cell is selected from a HEK 293 cell, a HEK 6E cell, a CHO cell, a Jurkat cell, a KS62 cell, a PerC6 cell, a HeLa cell, or a derivative or equivalent functional of these. In another embodiment, the mammalian host cell is a HEK 293 cell or derived from a HEK 293 cell. These cell lines could be adherent cell lines (i.e. they grow into a fixed monolayer at one surface) or adapted / non-adherent cell lines in suspension (i.e. they grow in suspension in a culture medium). In yet another embodiment, the HEK 293 cell is a HEK 293T cell or a HEK 6E cell. Other examples of suitable commercially available cell lines include T REX ™ cell lines (Life Technologies). Those skilled in the art will appreciate that introduction of the nucleic acid vector into the host cell can be accomplished using appropriate methods known in the art, for example, lipid-mediated transfection, microinjection, cell fusion (as microcellular), electroporation, or microprojectile bombardment. In one embodiment, the nucleic acid vector is introduced into the host cell by electroporation. It will be understood that the choice of the method to be used for the introduction of the nucleic acid vector may depend on the type of mammalian host cell used.
Once within the mammalian host cell, the nucleic acid vector will randomly integrate the endogenous genome of the mammalian host cell. Therefore, the method further comprises selecting the mammalian host cell that has integrated the nucleic acids encoded on the nucleic acid vector (e.g., using an antibiotic resistance selection marker, such as a zeocin resistance). Those skilled in the art will know the methods for promoting the integration of the nucleic acid vector, for example the linearization of the nucleic acid vector if it is naturally circular (for example BAC, PAC, cosmid or fosmid). The nucleic acid vector may further include areas of homology shared with the endogenous chromosomes of the mammalian host cell to direct integration into a selected site within the endogenous genome. In addition, if recombination sites are present on the nucleic acid vector, then these can be used for targeted recombination. For example, the nucleic acid vector may contain a loxP site that allows for targeted integration when combined with Cre recombinase (i.e. using the Cre / lox system derived from bacteriophage PI). Alternatively (or in addition), the recombination site is an att site (eg from lambda phage), where the att site allows oriented integration in the presence of a lambda integrase. This would allow the retroviral genes to be targeted at a locus within the endogenous genome, allowing for high and / or stable expression. Other targeted integration methods are well known in the art. For example, methods for inducing targeted cleavage of genomic RNA can be used to promote targeted recombination at a selected chromosomal locus. These methods frequently involve the use of cleavage systems manipulated to induce a double-strand break (DSB) or gap in the endogenous genome to induce breakage repair by natural processes such as non-terminal junction. counterparts (NHEJ) or repair using a repair matrix (ie, homology-oriented or HDR-oriented repair).
Cleavage can occur through the use of specific nucleases, such as manipulated zinc finger nucleases (ZFNs), transcriptional activator-like nucleases (TALENs), using the CRISPR / Cas9 system with tracRNA / RNA manipulated ('single guide RNA') to guide specific cleavage and / or using Argonaute-based nucleases (eg from T. thermophilus, known as 'TtAgo', see Swarts et al. 2014) Nature 507 (7491): 258-261). Targeted cleavage using one of these nuclease systems can be exploited to insert a nucleic acid into a specific target location using either HDR or NHEJ induced processes. Therefore, in one embodiment, the method further comprises integrating the nucleic acid sequences encoded on the nucleic acid vector into the genome (i.e., an endogenous chromosome) of the host cell. mammal using at least one nuclease, wherein the at least one nuclease cleaves the genome of the mammalian host cell such that the nucleic acid sequences are integrated into the genome of the cell. In another embodiment, the at least one nuclease is selected from the group consisting of zinc finger nuclease (ZFN), TALE nuclease (TALEN), CRISPR / Cas nuclease system, and combinations thereof.
According to another aspect of the invention, there is provided a retroviral packaging cell obtained by the process as defined in this specification.
The cell line obtained using the methods as defined herein can be used to produce a high titer of retroviral vector.
References herein to the term "high titer" refers to an effective amount of a retroviral vector or particle that is capable of transducing a target cell, such as a patient cell. In one embodiment, a high titer is a titer exceeding 106 TU / ml without concentration (TU = transduction units).
According to another aspect of the invention there is provided a method for producing a replication defective retroviral vector particle, comprising: (a) introducing the nucleic acid vector as defined herein into a culture mammalian host cells; (b) selecting, within the culture, a mammalian host cell that has the nucleic acid sequences encoded on the vector integrated into an endogenous chromosome of the mammalian host cell; and (c) further culturing the mammalian host cell under conditions to produce the defective retroviral vector particle in replication.
As described above, in one embodiment, the mammalian host cell is selected from a HEK 293 cell, a CHO cell, a Jurkat cell, a KS62 cell, a PerC6 cell, a HeLa cell, or a derivative or functional equivalent thereof. In another embodiment, the mammalian host cell is a HEK 293 cell or derived from a HEK 293 cell. These cell lines could be adherent cell lines (i.e. they grow into a fixed monolayer at one surface) or adapted / non-adherent cell lines in suspension (i.e. they grow in suspension in a culture medium). In yet another embodiment, the HEK 293 cell is a HEK 293T cell. Other examples of suitable commercially available cell lines include T REX ™ cell lines (Life Technologies). Those skilled in the art will understand that the conditions used in the process described herein are dependent on the host cell used. Typical conditions, for example the culture medium or the temperature to be used, are well known in the art. In one embodiment, the culture is performed by incubating the mammalian host cell under humid conditions. In another embodiment, the humidified conditions include incubating transfected cells at 37 ° C to 5% CO 2. In one embodiment, the culture is carried out using a culture medium selected from: Dubelcco Modified Eagle's Medium (DMEM) containing 10% (vol / vol) fetal bovine serum (FBS), an UltraCULTURE medium ™ serum-free (Lonza, Cat # 12-725F) or FreeStyle ™ expression medium (Thermo fisher Cat # 12338-018).
In one embodiment, the method further comprises isolating the defective retroviral vector particle in replication. For example, in one embodiment, isolation is achieved using a filter. In another embodiment, the filter is a membrane of low protein binding (e.g., a low protein binding membrane of 0.22 μm or a membrane of low protein binding of 0.45 μm), such as membranes. Artificial polyvinylidene fluoride (PVDF) or polyethersulfone (PES).
Once within the mammalian host cell, the retroviral nucleic acids present on the nucleic acid vector will integrate into a single random locus within the endogenous genome. The integration steps can be promoted as described in the foregoing, for example using linearization and / or shared homology areas. Recombination sites can be used for targeted recombination.
If the target genes are integrated into endogenous chromosomes with a selective marker, such as an antibiotic resistance gene, then the method may further include selecting mammalian host cells that have successfully integrated the retroviral nucleic acids.
Once isolated, the retroviral vector particles can be concentrated for in vivo applications. Concentration methods include, for example, ultracentrifugation, precipitation or anion exchange chromatography. Ultracentrifugation is useful as a fast method for small-scale retroviral vector concentration. Alternatively, anion exchange chromatography (using, for example, Mustang Q anion exchange membrane cartridges) or precipitation (e.g. using PEG 6000) is particularly useful for processing large volumes of lentiviral vector supernatants.
According to another aspect of the invention, there is provided a defective retroviral vector particle in replication obtained by the method as defined in this specification. The invention will now be described in more detail with the aid of the following non-limiting examples.
EXAMPLES EXAMPLE 1 Hybrid Construction Guide
Figure 1 shows a step-by-step guide for building BACpack-WTGP-277delU5 and BACpack-SYNGP-277delU5. With the compatible ends of an Xbal and Nhel digest, the lentiviral packaging genes were progressively loaded into the pSmart BAC vector. At the time of GagPol addition, 2 hybrids were made which contained either wild type GagPol (WTGP) or optimized codon GagPol, SYNGP. The nomenclature of BACpack-WTGP and BACpack-SYNGP was given to them respectively. The transfer cassette was then loaded on these two hybrids, generating BACpackWTGP-277delU5 and BACpackSYNGP-277delU5. EXAMPLE 2 Selection of a stable polyclonal batch
Polyclonal stable transfectant batches were generated by transfection of the adherent cell line, HEK293T, with BACpackSYNGP-277delU5 and BACpackWTGP-277delU5. Successful integration events were then selected with Zeocin.
To evaluate the ability of these polyclonal lots to generate a lentiviral vector, the cells were induced with doxycycline (I) or left uninduced (IU) and compared to untransfected HEK293T cells.
The results show the transduction unit (TU) titre / ml of the lentiviral vector supernatant harvested from each transfection condition. It can be seen from the titration results in Figure 2 that stable polyclonal batches, produced either with BACpackSYNGP-277delU5 or with BACpackWTGP-277delU5, are capable of producing the lentiviral vector at concentrations in the region of 107 TU / ml. , which is comparable to the current transient transient system.
These results confirm that the single BAC vector containing all of the packaging genes required for lentiviral production can produce cell lines capable of producing the lentiviral vector in a suitable way. EXAMPLE 3 Generation of Stable Transfection Clones in Suspension
The primary goal of generating lentiviral vector-producing cell lines using BAC technology is to rapidly apply new advances to the platform. These advances are likely to include a modification of the specialized cell lines. For example, increasing the yield by producing organic products in suspended cells is an industry standard since they grow at higher densities than adherent cells. However, the current lentiviral vector production system relies on high transfection rates, which are more difficult to obtain in suspended cells than in adherent HEK293T cells. Since the efficiency of transfection is less important in the generation of a stable cell line due to the selection of successful integrants, the BAC hybrid is an ideal solution for generating cell lines in suspension producing a lentiviral vector.
As previously demonstrated, the BAC hybrid is capable of generating lentiviral vector-producing cell lines from adherent HEK293T cells. In order to prove the flexibility of the BAC hybrids, stable transfectant cell lines were generated from the HEK293 6E suspension cell line. The HEK293 6E cells were transfected with the BACpackWTGP-277delU5 BAC hybrid and then selected with Zeocin. This operation was followed by cloning to generate clonal cell lines. The results in Figure 3 show the GFP signal generated by stable cell lines. This indicates both the presence of Zeocin resistance and a functional GFP expression cassette in the transfer vector segment.
This result suggests that the BAC hybrid is capable of generating stable clones from multiple cell lines. EXAMPLE 4 Induction of Lentiviruses in Suspension Clones
In order to confirm the ability of stable suspension clones to produce the lentiviral vector, clones 1, 14, 15 and 16 were induced with 2 μg / ml of doxycycline and the viral titer of the supernatant was measured by transduction of HEK293T cells.
The results in Figure 4 show the transduction unit (TU) titre / ml of the lentiviral vector supernatant harvested from each clone. The results clearly show that cell lines generated by stable transfection of the suspended HEK293 6E cell line with BACpackWTGP-277delU5 are capable of producing the lentiviral vector in yields comparable to those of the current transient transfection system. EXAMPLE 5 Titres of the Vector of Clones
Clones 1 and 16 as described in Figure 4 were cultured and induced and titrated at later times to determine whether vector production was stable from these heavily producing clones. As shown in Figures 5A and 5B, the vector titers from these clones actually increased modestly between passage 5 and passage 21, possibly due to an increase in the concentration of sodium butyrate introduced into the induction process.
It will be understood that the embodiments described herein can be applied to all aspects of the invention.

权利要求:
Claims (15)
[1" id="c-fr-0001]
A retroviral producer cell comprising nucleic acid sequences encoding: gag and pol proteins; an env protein; and the genomic RNA of the retroviral vector particle, wherein said nucleic acid sequences are all located at a single locus within the genome of the retroviral producer cell.
[2" id="c-fr-0002]
The retroviral producer cell according to claim 1, which further comprises the rev auxiliary gene,
[3" id="c-fr-0003]
3. A retroviral producer cell according to claim 1 or claim 2, wherein the retroviral nucleic acid sequences are derived from a retrovirus selected from lentiviruses, alpha-retroviruses, gamma-retroviruses or foamy retroviruses.
[4" id="c-fr-0004]
The retroviral producer cell of claim 3, wherein the retroviral nucleic acid sequences are derived from a lentivirus selected from the group consisting of HIV-1, HIV-2, VIS, VIF, VAIE and Visna.
[5" id="c-fr-0005]
The retroviral producer cell of claim 4, wherein the retroviral nucleic acid sequences are derived from HIV-1.
[6" id="c-fr-0006]
The retroviral producer cell according to any of claims 1 to 5, wherein the env is derived from the vesicular stomatitis virus.
[7" id="c-fr-0007]
The retroviral producer cell of any one of claims 1 to 6, which further comprises a transcriptional regulatory element.
[8" id="c-fr-0008]
The retroviral producer cell of claim 7, wherein the transcriptional regulatory element is a CMV promoter,
[9" id="c-fr-0009]
The retroviral producer cell of claim 8, wherein the CMV promoter further comprises at least one Tet operon.
[10" id="c-fr-0010]
The retroviral producer cell of any one of claims 1 to 9 which further comprises the tetracycline resistance operon repressor protein (TetR).
[11" id="c-fr-0011]
The retroviral producer cell of any one of claims 1 to 10, which further comprises an insulator.
[12" id="c-fr-0012]
12. Retroviral producer cell; according to claim 11, wherein an insulator is present between each of the retroviral nucleic acid sequences.
[13" id="c-fr-0013]
13. A retroviral producer cell according to any one of claims 1 to 12, which further comprises a selectable marker.
[14" id="c-fr-0014]
The retroviral producer cell according to any one of claims 1 to 13, which further comprises one or more transgenes.
[15" id="c-fr-0015]
The retroviral producer cell of any one of claims 1 to 14, wherein the cell is a mammalian cell.

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优先权:

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申请号 | 申请日 | 专利标题

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GBGB1520761.6A|GB201520761D0|2015-11-24|2015-11-24|Stable cell lines for retroviral production|

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GBGB1609303.1A|GB201609303D0|2016-05-26|2016-05-26|Stable cell lines for retroviral production|

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