METHOD TO INCREASE THE EFFICIENCY OF LENTIVIRAL TRANSDUCTION OF PROGENITOR CELLS OR CD34+ HEMATO

专利摘要:
compounds for improved viral transduction. these are methods and compositions for improving the efficiency of viral transduction of cells. more particularly, the present invention provides methods and materials useful for safely and reliably improving and efficiently transducing methods for transducing cells, such as human hematopoietic stem cells (hsc), with viruses and/or viral vectors. the compositions and methods are useful for therapeutic indications that favor treatment with hematopoietic stem cell gene therapies.
公开号:BR112014007782B1
申请号:R112014007782-7
申请日:2012-09-28
公开日:2021-06-01
发明作者:Garrett Collins Heffner；Abraham Isaac Bassan
申请人:Bluebird Bio, Inc.；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDER
[001] This order claims benefit under United States Code 35 section 119(e) of provisional order no. US 61/541,736, filed September 30, 2011, which is incorporated herein by reference in its entirety. STATEMENT RELATED TO SEQUENCE LISTING
[002] The sequence listing associated with this application is provided in text format rather than a hard copy, and is incorporated herein by reference in the specification. The name of the text file containing the string listing is BLBD_006_01WO_ST25.txt. The text file is 30 KB, was created on September 27, 2012, and is being submitted electronically via EFS-Web. FUNDAMENTALS FIELD OF TECHNIQUE
[003] The present invention generally relates to improving the efficiency of viral cell transduction methods. More particularly, the present invention provides methods and materials useful for improving the transduction efficiency of cells, such as human hematopoietic stem cells (HSC), with viruses and/or viral vectors that may be useful for therapeutic indications. DESCRIPTION OF RELATED TECHNIQUE
[004] The Food and Drug Administration (FDA) has not yet approved any human gene therapy product for sale. Current human gene therapy is experimental and has not been shown to be very successful in clinical trials. Little progress has been made since the first clinical trial of gene therapy began in 1990. In 1999, gene therapy suffered a major setback with the death of 18-year-old Jesse Gelsinger. Jesse was participating in a trial of gene therapy for ornithine transcarboxylase (OTCD) deficiency. He died of multiple organ failure 4 days after starting treatment. Its death is believed to have been triggered by a severe immune response to the adenovirus vehicle.
[005] Another hard blow came in January 2003, when the FDA placed a temporary break in all gene therapy trials that use retroviral vectors in blood stem cells. The FDA employed this action after it learned that a second child treated in a French gene therapy trial had developed a condition similar to leukemia. Both this child and another child who had developed a similar condition in August 2002 had been successfully treated by gene therapy for X-linked severe combined immunodeficiency disease (X-SCID), also known as “bubble boy syndrome” . The FDA's Biological Response Modifiers Advisory Committee (BRMAC) met in late February 2003 to discuss possible measures that could allow a series of retroviral gene therapy trials to treat cancer. life-threatening illnesses to proceed with adequate precautions. In April 2003, the FDA released its embargo on gene therapy tests that use retroviral vectors in blood stem cells.
[006] Recently, however, several groups have conducted moderately successful gene therapy trials in combating various diseases. In 2008, UK researchers at the UCL Institute of Ophthalmology and Moorfields Eye Hospital NIHR Biomedical Research Center announced a successful clinical trial of gene therapy for the treatment of Leber's congenital amaurosis, a type of inherited blindness. The results showed that the experimental treatment is safe and can improve vision (Maguire et al., N Engl J Med. 358(21):2240 (2008)).
[007] In 2011, Neurologix, Inc. announced positive results in a phase 2 trial of its investigative gene therapy for advanced Parkinson's disease (PD), NLX-P101. Study participants who received NLX-P101 experienced statistically and clinically significant improvements in unmeasured motor scores compared to control subjects who received sham surgery. In the trial, this benefit was seen at one month and remained virtually unchanged throughout the six-month blind study period. The results also demonstrated a positive safety profile for NLX-P101, with no serious adverse effects related to gene therapy or surgical procedure reported. Patients enrolled in the trial had moderate to advanced PD and were not adequately responsive to current therapies.
[008] In 2009, a French group of scientists reported the use of hematopoietic stem cell-mediated gene therapy to successfully treat X-linked adrenoleukodystrophy (ALD). Autologous stem cells were removed from the patients, genetically corrected ex vivo, and then reinfused into the patients after they had received myeloablative treatment. During a 24- to 30-month follow-up period, polyclonal reconstitution, with 9 to 14% of granulocytes, monocytes, and T and B lymphocytes expressing the ALD protein was detected. These results strongly suggest that hematopoietic stem cells were transduced in patients. Starting 14 to 16 months after the infusion of the genetically corrected cells, progressive cerebral demyelination in the two patients stopped.
[009] Recent progress in the field of gene therapy has brought hope that patients afflicted with hemoglobinopathies such as β-thalassemia and sickle cell anemia will benefit from new therapeutic approaches. Transplantation of hematopoietic cells (HCs) modified with lentiviral vectors carrying the β-globin gene has resulted in long-term correction of several mouse models of hemoglobin disorders, Imren et al., Proc Natl Acad Sci US A. 2002; 99(22):14380-14385; Malik et al., Ann NY Acad Sci. 2005;1054:238-249; May et al., Nature. 2000;406(6791):82-86; Pawliuk et al., Science. 2001;294(5550): 2368-2371), but in contrast, has led to transfusion independence in only one β-thalassemia patient (Cavazzana-Calvo et al., Nature. 2010;467(7313):318-322 ).
[0010] Although the main advantages of genetically modified autologous cell infusion are avoiding the risks of GVHD and immunosuppressive pre-transplant conditioning, as well as addressing the lack of compatible donors, current therapy faces at least three substantive caveats: the requirement for myeloablation toxic (Dunbar et al, Hum Gene Ther. 1998;9(17):2629-2640); current gene transfer methods are unable to transduce more than a fraction of hematopoietic stem cells (HSCs) (Santoni de Sio and Naldini, Methods Mol Biol. 2009;506:59-70); and several available in vivo selection strategies suffer from suboptimal efficacy and safety (Beard et al., J Clin Invest. 2010;120(7):2345-2354; Cornetta et al., Cancer Gene Ther. 2006;13(9) :886-895; Milsom et al., Cancer Res. 2008;68(15):6171-6180). For example, in disorders responsive to hematopoietic stem cell therapy, e.g., sickle cell disease, β-thalassemia, adrenoleukodystrophy, and adrenomyeloneuropathy, limitations include, but are not limited to, ineffective transduction of hematopoietic stem or stem cells, a requirement for myeloablative or toxic myelosuppressive therapy and a lack of optimal methods for in vivo selection of transduced cells.
[0011] Consequently, there is a need in the art for improved methods of gene therapy and, in particular, for the treatment or prevention of hematopoietic disorders. The present invention offers solutions to these and other problems that plague the art. BRIEF SUMMARY
[0012] The present invention generally provides methods and compositions comprising a compound that enhances EP prostaglandin receptor signaling for the improvement of viral transduction efficiency. The inventive compositions and methods further provide safer and more reliable methods for transducing cells, such as human hematopoietic stem cells (HSC), with viruses and/or viral vectors. The compositions and methods are useful for therapeutic indications sensitive to treatment with hematopoietic stem cell gene therapies.
[0013] In various embodiments, the present invention contemplates, in part, a method for increasing the transduction efficiency of cells cultured with a retrovirus that comprises culturing the cells and the retrovirus in a culture medium that comprises one or more compounds that enhance the EP prostaglandin receptor signaling. In one embodiment, the compound is a small molecule.
[0014] In one embodiment, the cells consist of progenitor or stem cells.
[0015] In a particular modality, the progenitor or stem cells are selected from the group consisting of: embryonic stem cells and induced pluripotent stem cells.
[0016] In an additional modality, the stem cell or progenitor is selected from the group consisting of: mesenchymal stem cells, hematopoietic stem cells, neuronal stem cells, retinal stem cells, cardiac muscle stem cells , skeletal muscle stem cells, adipose-derived stem cells, chondrogenic stem cells, liver stem cells, kidney stem cells, and pancreatic stem cells.
[0017] In a given modality, the progenitor or stem cells consist of progenitor or hematopoietic stem cells.
[0018] In an additional modality, cells are selected from the group consisting of: osteoblasts, chondrocytes, adipocytes, skeletal muscle, cardiac muscle, neurons, astrocytes, oligodendrocytes, Schwann cells, retinal cells, corneal cells, cells of skin, monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, dendritic cells, T lymphocytes, B lymphocytes, NK cells, gastric cells, intestinal cells, smooth muscle cells, vascular cells, bladder cells, pancreatic alpha cells , pancreatic beta cells, pancreatic delta cells, hepatocytes, renal cells, adrenal cells and lung cells.
[0019] In a further particular embodiment, the cells consist of hematopoietic stem cells or hematopoietic progenitor cells.
[0020] In one embodiment, at least about 50% of the progenitor or hematopoietic stem cells undergo transduction.
[0021] In another modality, at least about 75% of the progenitor or hematopoietic stem cells are subjected to transduction.
[0022] In yet another modality, at least about 90% of the progenitor cells or hematopoietic stem are subjected to transduction.
[0023] In particular embodiments, any one of the compositions or methods disclosed herein comprises one or more compounds that enhance EP prostaglandin receptor signaling selected from the group consisting of: PGA2; PGB2; PGD2; PGE1; PGE2; PGF2; PGI2; PGH2; PGJ2; and precursors, metabolites, derivatives and analogues thereof.
[0024] In certain embodiments, any one of the compositions or methods disclosed herein comprises one or more compounds that enhance EP prostaglandin receptor signaling selected from the group consisting of: 15d-PGJ2; delta12-PGJ2; 2-hydroxyheptadecatrienoic acid (HHT); Thromboxane A2; Thromboxane B2; Iloprost; Treprostinil; Travoprost; Carboprost tromethamine; Tafluprost; Latanoprost; Bimatoprost; isopropyl unoprostone; Cloprostenol; Oestrophan; Superphan; Misoprostol; Butaprost; Linoleic acid; 13(s)-HODE; LY171883; Mead acid; Eicosatrienoic acid; Epoxy eicosatrienoic acid; ONO-259; Cay1039; a PGE2 receptor agonist; 16,16-dimethyl PGE2; 19(R)-hydroxy PGE2; 16,16-dimethyl PGE2 p-(p-acetamidobenzamido)phenyl ester; 11-deoxy-16,16-dimethyl PGE2; 9-deoxy-9-methylene-16,16-dimethyl PGE2; 9-deoxy-9-methylene PGE2; Sulprostone; PGE2 serinol amide; PGE2 methyl ester; 16-phenyl tetranor PGE2; 15(S)-15-methyl PGE2; 15(R)-15-methyl PGE2; Corey-A alcohol; Corey-B alcohol; Corey diol; BIO; 8-bromo-cAMP; Forskolin; Batta-AM; Fendyline; Nicardipine; Nifedipine; Pimozide; Strophantidine; Lanatoside; L-Arg; Sodium nitroprusside; Sodium vanadate; Bradykinin; Mebeverine; Flurandrenolid; Atenolol; Pindolol; Gaboxadol; kynurenic acid; Hydralazine; Thiabendazole; Bicuclin; Vesamicol; Peruvoside; Imipramine; Chlorpropamide; 1,5- Pentamethylene tetrazole; 4-Aminopyridine; Diazoxide; Benfotiamine; 12-Methoxydodecenoic acid; N-Formyl-Met-Leu-Phe; Galamine; IAA 94; and Chlorotrianisene.
[0025] In some embodiments, any one of the compositions or methods presented herein comprises one or more compounds that enhance prostaglandin EP signaling selected from the group consisting of: prostaglandin E2(PGE2) or 16,16 - dimethyl PGE2.
[0026] In further embodiments, any of the methods presented herein further comprises culturing the cells and retroviruses in the presence of a histone deacetylase (HDAC) inhibitor.
[0027] In one embodiment, the HDAC inhibitor is selected from the group consisting of: Trichostatin A (TSA), valproic acid (VPA), sodium butyrate, suberoylanilide hydroxamic acid (SAHA), sodium phenylbutyrate, depsipeptide, trapoxin (TPX), cyclic hydroxamic acid containing peptide 1 (CHAPl), MS-275, LBH589 and PXD-101.
[0028] In various embodiments, any of the compositions or methods disclosed herein comprises a retrovirus consisting of a lentivirus.
[0029] In particular embodiments, any one of the compositions or methods disclosed herein comprises a retrovirus that consists of a human immunodeficiency virus (HIV).
In certain embodiments, any of the compositions or methods disclosed herein comprise a pseudotype retrovirus with a vesicular stomatitis virus G protein envelope protein (VSV-G).
In further embodiments, any of the methods presented herein comprises culturing the cells in the presence of the compound that enhances EP prostaglandin receptor signaling prior to transduction.
In particular embodiments, cells are cultured with the compound that enhances EP prostaglandin receptor signaling for at least about 2 hours.
In additional embodiments, cells are cultured with the compound that enhances EP prostaglandin receptor signaling for at least about 4 hours.
In certain embodiments, cells are cultured in the presence of the compound that increases EP prostaglandin receptor signaling during transduction.
In additional embodiments, cells are cultured in the presence of the compound that enhances EP prostaglandin receptor signaling for at least about twenty-four hours.
In additional embodiments, cells are cultured in the presence of the compound that enhances EP prostaglandin receptor signaling during the first twenty-four hours of transduction.
In some embodiments, cells are cultured in the presence of the compound that increases EP prostaglandin receptor signaling during the first forty-eight hours of transduction.
In particular embodiments, any one of the compositions or methods disclosed herein comprises a retrovirus comprising a vector comprising: a left (5') retroviral LTR; an expression control sequence operably linked to a gene of interest; and a right (3') retroviral LTR.
In certain embodiments, any one of the compositions or methods disclosed herein comprises a retrovirus comprising a vector comprising: a left (5') HIV-1 LTR; a Psi packing sequence (Φ+); a central tract of HIV-1 polypurine/DNA flap (cPPT/FLAP); a rev response element (RRE); a β-globin promoter and a β-globin site control region (LCR) operably linked to a gene of interest; and a right retroviral (3') LTR comprising: one or more isolating elements, or a rabbit β-globin polyA (rβgpA) sequence. In several modalities, hematopoietic stem or progenitor cells are administered to a patient suffering from a haemoglobinopathy.
[0040] In several particular modalities, hemoglobinopathy consists of β-thalassemia or sickle cell disease.
In certain embodiments, any one of the compositions or methods disclosed herein comprises a vector comprising: a left (5') HIV-1 LTR; a Psi packing signal (^); a cPPT/FLAP; an RRE; an MND promoter operably linked to a polynucleotide encoding a human ABCD1 polypeptide; a right HIV-1 (3') LTR; and a rabbit β-globin polyadenylation sequence. In a variety of modalities, the hematopoietic stem or progenitor cells are administered to a patient suffering from an adrenoleukodystrophy or an adrenomyeloneuropathy.
[0042] In several modalities, the retrovirus has imperfect replication. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0043] Figure 1 shows the results of a screening for compounds that promote viral transduction of CD34+ cells. CD34+ cells were thawed and prestimulated with SCF, TPO, FltL and IL3, then transduced with GFP+ lentivirus. Cells were additionally exposed to soluble factors at high, medium or low concentrations (see Table 1) during the prestimulation period (0 to 24 hours) or during the transduction period (24 to 48 hours). Cells were then washed and analyzed by flow cytometry after approximately 1 week in culture. The percentage of cells that were GFP+ was determined and illustrated as a heat map. Gray represents approximately 45% of transduced cells and the dynamic range was from 0% (black) to ~92% (white). BRIEF DESCRIPTION OF SEQUENCE IDENTIFIERS
[0044] SEQ ID NO: 1 shows a polynucleotide sequence of a human alpha globin cDNA.
SEQ ID NO: 2 shows an amino acid sequence of a human alpha globin polypeptide.
[0046] SEQ ID NO: 3 shows an amino acid sequence of a mouse alpha globin polypeptide.
SEQ ID NO: 4 shows an amino acid sequence of a rat alpha globin polypeptide.
[0048] SEQ ID NO: 5 shows a polynucleotide sequence of a human beta globin cDNA.
[0049] SEQ ID NO: 6 shows an amino acid sequence of a human beta globin polypeptide.
SEQ ID NO: 7 shows an amino acid sequence of a mutant human beta globin polypeptide.
[0051] SEQ ID NO: 8 shows an amino acid sequence of a mouse beta globin polypeptide.
SEQ ID NO: 9 shows an amino acid sequence of a rat beta globin polypeptide.
SEQ ID NO: 10 shows a polynucleotide sequence of a human gamma globin cDNA.
[0054] SEQ ID NO: 11 shows an amino acid sequence of a human gamma globin polypeptide.
SEQ ID NO: 12 shows an amino acid sequence of a mouse gamma globin polypeptide.
SEQ ID NO: 13 shows an amino acid sequence of a rat gamma globin polypeptide.
SEQ ID NO: 14 shows a polynucleotide sequence of a human delta globin cDNA.
[0058] SEQ ID NO: 15 shows an amino acid sequence of a human delta globin polypeptide.
[0059] SEQ ID NO: 16 shows a cDNA sequence encoding an ACBD1 polynucleotide.
[0060] SEQ ID NO: 17 shows a cDNA sequence encoding an ACBD1 polynucleotide.
[0061] SEQ ID NO: 18 shows an amino acid sequence of an ACBD1 polypeptide. DETAILED DESCRIPTION A. OVERVIEW
The present invention generally relates to improved gene therapy compositions and methods of using them to treat, prevent or alleviate genetic disorders. A significant challenge for gene therapy is to increase the transduction efficiency of a cell comprising the therapeutic gene that will be delivered to an individual, where corrected cells do not have an intrinsic selective advantage over non-transduced cells.
[0063] The present invention is based, in part, on the unexpected discovery that the novel cell transduction methods of the invention can be used to expand or increase the numbers of therapeutic cells, i.e., corrected cells, in vitro, ex vivo or in vivo to further increase the effectiveness of gene therapy. Without being bound by any particular theory, the present invention considers, in part, that by increasing the transduction efficiency of cells, more corrected cells are generated by transduction, and thus the gene therapy methods of the present invention require the administering fewer numbers of cells to provide therapeutic, preventative, or ameliorative criteria for individuals receiving the gene therapy. Furthermore, due to the fact that a greater number of transduced cells are released to the patient, myelosuppressive or myeloablative therapy is not necessarily required to meet therapeutic, preventive, or ameliorative criteria.
[0064] Consequently, the present invention addresses an unmet clinical need for improving the efficiency of gene therapy in the treatment of genetic diseases, so that a greater number of therapeutic cells within a transduced cell population can be administered to an individual to provide a therapeutic, preventative or ameliorative effect. The invention specifically relates to surprisingly effective cell transduction methods, vectors and genetically engineered cells to facilitate the desired clinical outcomes for gene therapy.
[0065] The practice of the present invention will employ, except where specifically stated otherwise, conventional methods of molecular biology and recombinant DNA techniques within technical skill, many of which are described below for the purpose of illustration. Such techniques are fully explained in the literature. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); A Practical Guide to Molecular Cloning (B. Perbal, ed., 1984).
[0066] All publications, patents and patent applications mentioned herein are hereby incorporated by reference in their entirety. B. DEFINITIONS
[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. For purposes of the present invention, the following terms are defined below.
[0068] For use in the present invention, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Retroviruses are a common tool for gene delivery (Miller, 2000, Nature. 357: 455-460). Once the virus is integrated into the host genome, it is referred to as a “provirus”. The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules that encode the structural proteins and enzymes needed to produce new viral particles.
Illustrative retroviruses include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mammary tumor virus murine (MuMTV), gibbon monkey leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV) and Rous sarcoma virus (RSV) ) and lentiviruses.
[0070] For use in the present invention, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV virus (human immunodeficiency virus; which includes HIV type 1 and HIV type 2); visna-maedi virus (VMV); the caprine arthritis-encephalitis virus (CAEV); Equine Infectious Anemia Virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector structures (i.e., cis-acting sequence elements of HIV) are preferred.
[0071] Retroviral vectors, and more particularly lentiviral vectors, can be used in the practice of the present invention. Accordingly, the term "retrovirus" or "retroviral vector" for use in the present invention is intended to include "lentiviruses" and "lentiviral vectors", respectively.
The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked, for example, inserted into, to the vector nucleic acid molecule. A vector can include sequences that direct autonomous replication in a cell, or it can include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (for example, DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-deficient retroviruses and lentiviruses.
[0073] As will be apparent to a person skilled in the art, the term "viral vector" is widely used to refer to a nucleic acid molecule (eg, a transfer plasmid) that includes nucleic acid elements derived from viruses that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes host cell components in addition to the nucleic acid(s).
The term viral vector can refer to a virus or viral particle capable of transferring a nucleic acid to a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and/or functional genetic elements that are primarily derived from a virus. The term "retroviral vector" refers to a viral vector or plasmid that contains structural or functional genetic elements, or parts thereof, that are primarily derived from a retrovirus. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural or functional genetic elements, or parts thereof, which include LTRs that are primarily derived from a lentivirus. The term "hybrid" refers to a vector, LTR or other nucleic acid that contains both retroviral, eg, lentiviral, and non-lentiviral viral sequences. In one embodiment, a hybrid vector refers to a transfer vector or plasmid that comprises retroviral sequences, e.g., lentivirals, for reverse transcription, replication, integration and/or packaging.
In particular embodiments, the terms "lentiviral vector", "lentiviral expression vector" may be used to refer to lentiviral transfer plasmids and/or infectious lentiviral particles. Where reference is made herein to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is to be understood that the sequences of these elements are present in RNA form in lentiviral particles of the invention and are present in the form of DNA in the DNA plasmids of the invention.
[0076] At each end of the provirus are structures called "long terminal repeats" or "LTRs". The term "long terminal repeat (LTR)" refers to base pair domains located at the ends of retroviral DNAs that, in their natural sequence context, are direct repeats and contain U3, R, and U5 regions. LTRs generally provide fundamental functions for the expression of retroviral genes (eg, promotion, initiation and polyadenylation of gene transcripts) and for viral replication. The LTR contains numerous regulatory signals that include transcriptional control elements, polyadenylation signals, and sequences necessary for viral genome replication and integration. Viral LTR is divided into three regions called U3, R and U5. The U3 region contains the promoter and enhancer elements. The U5 region consists of the sequence between the primer binding site and the R region and contains the polyadenylation sequence. Region R (repeat) is flanked by regions U3 and U5. The LTR is composed of the U3, R and U5 regions and appears at both the 5' and 3' ends of the viral genome. Adjacent to the 5' LTR are sequences necessary for the reverse transcription of the genome (the tRNA primer binding site) and for the efficient packaging of viral RNA into particles (the Psi site).
[0077] For use in the present invention, the term "packaging signal" or "packaging sequence" refers to sequences located within the retroviral genome, which are required for insertion of the viral RNA into the viral particle or capsid, see, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pages 2101-2109. Several retroviral vectors utilize the minimal packaging signal (also referred to as the psi sequence [^] or [Φ+]) necessary for encapsidation of the viral genome. Thus, for use in the present invention, the terms "packaging sequence", "packaging signal", "psi" and the symbol "Φ" are used in reference to the non-coding sequence required for encapsidation of retroviral RNA strands during viral particle formation.
[0078] In several embodiments, the vectors comprise 5' LTR and/or modified 3' LTRs. Modifications to the 3' LTR are often made to improve the security of lentiviral or retroviral systems by rendering the virus replication-deficient. For use in the present invention, the term "replication deficient" refers to a virus that is not capable of fully effective replication, such that infectious virions are not produced (e.g., replication deficient lentiviral progeny). The term "replication-competent" refers to wild-type virus or mutant virus that is capable of replication, such that viral replication of the virus is capable of producing infectious virions (eg, replication-competent lentiviral progeny).
[0079] "Self-inactivating" (SIN) vectors refer to replication-deficient vectors, for example, retroviral or lentiviral vectors, in which the direct LTR enhancer-promoter (3') region, known as the U3 region , has been modified (eg, by deletion and/or substitution) to prevent viral transcription beyond the first cycle of viral replication. This is due to the fact that the direct LTR (3') U3 region is used as a template for the left (5') LTR U3 region during viral replication and thus viral transcription cannot be done without the U3 enhancer-promoter. In a further embodiment of the invention, the 3' LTR is modified in such a way that the U5 region is replaced, for example, by a heterologous or synthetic poly(A) sequence, one or more insulating elements and/or an inducible promoter. It should be noted that modifications to the LTRs, such as modifications to the 3' LTR, the 5' LTR, or both the 3' and 5' LTRs, are also included in the invention.
[0080] An additional security optimization is provided by replacing the U3 region of the 5' LTR by a heterologous promoter to drive transcription of the viral genome during the production of viral particles. Examples of heterologous promoters that can be used include, for example, simian viral 40 (SV40) promoters (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), murine leukemia virus Moloney (MoMLV), Rous sarcoma virus (RSV) and herpes simplex virus (HSV) (thymidine kinase). Typical promoters are capable of driving high levels of transcription in a Tat-independent manner. This substitution reduces the possibility of recombination to generate replication competent virus, due to the fact that there is no complete U3 sequence in the virus production system. In certain embodiments, the heterologous promoter may be inducible such that transcription of all or part of the viral genome will only occur when one or more induction factors are present. Inducing factors include, but are not limited to, one or more chemical compounds or physiological conditions, for example, temperature or pH, at which host cells are cultured.
[0081] In some embodiments, viral vectors comprise a TAR element. The term “TAR” refers to the genetic “transactivation response” element located in the R region of lentiviral LTRs (eg, HIV). This element interacts with the lentiviral trans-activating genetic element (tat) to optimize viral replication. However, this element is not required in modalities where the U3 region of the 5' LTR is replaced by a heterologous promoter.
The "R region" refers to the region within retroviral LTRs that begins at the beginning of the termination group (ie, the beginning of transcription) and ends immediately before the beginning of the poly A tract. The R region is also defined as being flanked by the U3 and U5 regions. The R region plays a role during reverse transcription by allowing the transfer of nascent DNA from one end of the genome to the other.
[0083] For use in the present invention, the term "FLAP element" refers to a nucleic acid whose sequence induces the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in patent no. U.S. 6,682,907 and in Zennou, et al., 2000, Cell, 101:173. During HIV-1 reverse transcription, the central initiation of positive-stranded DNA in the central polypurine tract (cPPT) and central termination in the central termination sequence (CTS) lead to the formation of a three-stranded DNA structure: the DNA HIV-1 central flap. Without being bound by any theory, flap DNA may act as a cis-active determinant of lentiviral genome nuclear import and/or may increase virus titer. In particular embodiments, the retroviral or lentiviral vector structures comprise one or more FLAP elements upstream or downstream of the heterologous genes of interest in the vectors. For example, in particular embodiments, a transfer plasmid includes a FLAP element. In one embodiment, a vector of the invention comprises a FLAP element isolated from HIV-1.
[0084] In one embodiment, the retroviral or lentiviral transfer vectors comprise one or more export elements. The term “export element” refers to a post-transcriptional regulatory element that acts in cis, which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the rev response element (RRE) of human immunodeficiency virus (HIV) (see, for example, Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), and the hepatitis B virus post-transcriptional regulatory element (HPRE). Generally, the RNA export element is placed within the 3' UTR of a gene, and can be inserted as one or multiple copies.
[0085] In particular embodiments, expression of heterologous sequence in viral vectors is increased by incorporating post-transcriptional regulatory elements, effective polyadenylation sites and, optionally, transcription termination signals in the vectors. A variety of post-transcriptional regulatory elements can increase the expression of a heterologous nucleic acid in the protein, for example, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73 :2886); the post-transcriptional regulatory element present in the hepatitis B virus (HPRE) (Huang and Yen, 1995, Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766). In particular embodiments, the vectors of the invention are devoid of or do not comprise a post-transcriptional regulatory element, such as a WPRE or HPRE, due to the fact that, in some cases, these elements increase the risk of cell transformation and/or not substantially or significantly increase the amount of mRNA transcription or increase mRNA stability. Therefore, in some embodiments, the vectors of the invention are devoid of or do not comprise a WPRE or HPRE as an added security measure.
Elements leading to effective termination and polyadenylation of heterologous nucleic acid transcripts increase expression of the heterologous gene. Transcription termination signals are generally found downstream of the polyadenylation signal. The term "polyA site" or "polyA sequence", for use in the present invention, denotes a DNA sequence that directs both the termination and polyadenylation of nascent RNA transcription by RNA polymerase II. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and rapidly degraded. Illustrative examples of polyA signals can be used in a vector of the invention, include an ideal polyA sequence (eg AATAAA, ATTAAA AGTAAA), a bovine growth hormone polyA sequence (BGHpA), a polyA sequence of rabbit β-globin (rβgpA), or other suitable heterologous or endogenous polyA sequence known in the art.
[0087] In certain embodiments, a retroviral or lentiviral vector additionally comprises one or more isolating elements. Insulating elements can contribute to the protection of sequences expressed by lentiviruses, for example, therapeutic polypeptides, against integration side effects, which can be mediated by elements that act on cis present in genomic DNA and lead to the unregulated expression of transferred sequences ( that is, position effect, see, for example, Burgess-Beusse et al., 2002, Proc. Natl. Acad. Sci., USA, 99:16433; and Zhan et al., 2001, Hum. Genet., 109 :471). In some embodiments, the transfer vectors comprise one or more isolating element at the 3' LTR and upon integration of the provirus into the host genome, the provirus comprises the one or more isolators at both the 5' LTR and 3' LTR, by virtue of the doubling of the 3' LTR. Insulators suitable for use in the invention include, but are not limited to, chicken β-globin insulator (see Chung et al., 1993. Cell 74:505; Chung et al., 1997. PNAS 94:575; and Bell et al., 1999. Cell 98:387, incorporated herein by reference). Examples of insulating elements include, but are not limited to, an insulator of a β-globin site, such as chicken HS4.
According to certain specific embodiments of the invention, most or all of the viral vector framework sequences are derived from a lentivirus, e.g. HIV-1. However, it should be understood that many different sources of lentiviral sequences can be used, and numerous substitutions and changes in certain lentiviral sequences can be accommodated without impairing the ability of a transfer vector to perform the functions described herein. In addition, a variety of lentiviral vectors are known in the art, see Naldini et al., (1996a, 1996b and 1998); Zufferey et al., (1997); Dull et al., 1998, patent nos. U.S. 6,013,516; and 5,994,136, many of which can be adapted to produce a viral vector or transfer plasmid of the present invention.
[0089] For use in the present invention, the term "compound" encompasses small organic molecule, prostaglandins, cAMP enhancers, Wnt pathway agonists, cAMP/PI3K/AKT pathway agonists, Ca2+ second messenger pathway agonists, nitric oxide (NO)/angiotensin signaling agonists and inorganic chemicals, which include, without limitation, all analogues and derivatives thereof.
[0090] A "small molecule", "small organic molecule" or "small molecule compound" refers to a low molecular weight compound that has a molecular weight of less than about 5 kD, less than about 4 kD, less than about 3 kD, less than about 2 kD, less than about 1 kD, or less than about 0.5kD. In particular embodiments, small molecules can include nucleic acids, peptides, peptidomimetics, peptoids, other drugs or small organic compounds, and the like. Libraries of chemical and/or biological mixtures, such as fungal, bacterial or algal extracts, are known in the art and can be screened with any of the assays of the invention. Examples of methods for synthesizing molecular libraries can be found in: (Carell et al., 1994a; Carell et al., 1994b; Cho et al., 1993; DeWitt et al., 1993; Gallop et al., 1994 ; Zuckermann et al., 1994).
[0091] Compound libraries can be presented in solution (Houghten et al., 1992) or in beads (Lam et al., 1991), in flakes (Fodor et al., 1993), bacteria, spores (Ladner et al. ., US Patent No. 5,223,409, 1993), plasmids (Cull et al., 1992) or in phage (Cwirla et al., 1990; Devlin et al., 1990; Felici et al., 1991; Ladner et al., ., US Patent No. 5,223,409, 1993; Scott and Smith, 1990). The invention presented in this document encompasses the use of different libraries for the identification of small molecules that increase prostaglandin EP signaling at any point in the cell signaling pathway. Libraries useful for the purposes of the invention include, but are not limited to, (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries that comprise peptides, oligonucleotides, and/or random organic molecules.
[0092] The chemical libraries consist of structural analogs and derivatives of known compounds or compounds that are identified as "linkers (hits)" or "leaders (leads)" through natural product screening. Natural product libraries are derived from collections of microorganisms, animals, plants or marine organisms that are used to create mixtures by screening by: (1) fermentation and extraction of broth from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variables thereof. For an analysis, see, Cane, D.E., et al., (1998) Science 282:63-68. Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds as a mixture. They are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are combinatorial peptide and oligonucleotide libraries.
[0093] More specifically, a combinatorial chemical library consists of a collection of diverse chemical compounds generated by chemical synthesis or biological synthesis, by combining a series of chemical "building blocks", such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide library, is formed by combining a set of chemical building blocks (amino acids) in every possible shape for a given length of compound (ie, the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
[0094] For an analysis of combinatorial chemistry and libraries created from it, see Huc, I. and Nguyen, R. (2001) Comb. Chem. High Throughput Screen 4:53-74; Lepre, C A. (2001) Drug Discov. Today 6:133-140; Peng, S.X. (2000) Biomed. Chromatography 14:430-441; Bohm, H.J. and Stahl, M. (2000) Curr. Opinion Chem. Biol. 4:283-286; Barnes, C and Balasubramanian, S. (2000) Curr. Opinion Chem. Biol. 4:346-350; Lepre, Enjalbal,C, et al., (2000) Mass Septrom Rev. 19:139-161; Hall, D.G., (2000) Nat. Biotechnol. 18:262-262; Lazo, J.S., and Wipf, P. (2000) J. Pharmacol. Exp. Ther. 293:705-709; Houghten, R.A., (2000) Ann. Rev. Pharmacol. Toxicol. 40:273-282; Kobayashi, S. (2000) Curr. Opinion Chem. Biol. (2000) 4:338-345; Kopylov, A.M. and Spiridonova, V.A. (2000) Mol. Biol. (Mosk) 34:1097-1113; Weber, L. (2000) Curr. Opinion Chem. Biol. 4:295-302; Dolle, R.E. (2000) J. Comb. Chem. 2:383433; Floyd,CD., et al., (1999) Prog. Med. Chem. 36:91-168; Kundu, B., et al., (1999) Prog. Drug Res. 53:89-156; Cabilly, S. (1999) Mol. Biotechnol. 12:143-148; Lowe, G. (1999) Nat. Prod. Rep. 16:641-651; Dolle, R.E. and Nelson, K.H. (1999) J. Comb. Chem. 1:235-282; Czarnick, A.W. and Keene, J.D. (1998) Curr. Biol. 8:R705-R707; Dolle, R.E. (1998) Mol. Divers. 4:233-256; Myers, P.L., (1997) Curr. Opinion Biotechnol. 8:701-707; and Pluckthun, A. and Cortese, R. (1997) Biol. Chem. 378:443.
Devices for preparing combinatorial libraries are commercially available (see, for example, 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, for example, ComGenex, Princeton, NJ, Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd., Moscow, UK , 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).
[0096] For use in the present invention, the term "metabolic precursor" refers to a form of a compound that metabolizes into a desired compound.
[0097] For use in the present invention, the term "metabolite" refers to a resultant form of a compound that has been metabolized.
[0098] In reference to chemicals such as organic chemicals, "analog" or "derivative" refers to a chemical molecule that is similar to another chemical in structure and function, often structurally differs by a single element or group, but may differ by modifying more than one group (eg 2, 3 or 4 groups) if it maintains the same function as the parent chemical. Such modifications are routine to those of skill in the art, and include, for example, additional or substituted chemical moieties such as esters or amides of an acid, protecting groups such as a benzyl group for an alcohol or thiol, and tertiary groups. butoxylcarbonyl for an amine. Also included are modifications to alkyl side chains, such as alkyl substitutions (e.g., methyl, dimethyl, ethyl, etc.), modifications to the level of saturation or unsaturation of side chains, and the addition of modified groups, such as such as phenyl and substituted phenoxy. Derivatives can also include conjugates, such as biotin or avidin moieties, enzymes, such as horseradish peroxidase, and the like, and which include radiolabeled, bioluminescent, chemiluminescent, or fluorescent moieties. In addition, moieties can be added to the agents described herein to alter their pharmacokinetic properties, such as to increase their half-life in vivo or ex vivo, or to increase their cell penetration properties, among other desirable properties. Also included are pro-drugs, which are known to optimize numerous desirable qualities of pharmaceutical products (eg solubility, bioavailability, manufacturing, etc.) (see, for example, document under no. WO/2006/047476 for exemplary EP agonist prodrugs, which is incorporated herein by reference for its description of such agonists).
[0099] For use in the present invention, the terms "polynucleotide" or "nucleic acid" refer to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive strand RNA (RNA(+)), strand RNA negative (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA) or DNA. Polynucleotides include double-stranded or single-stranded polynucleotides. It is preferred that polynucleotides of the invention include polynucleotides or variables that are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99% or 100% sequence identity with any of the reference sequences described herein (see, for example, sequence listing), typically, where the variable maintains at least one biological activity of the reference string. In various illustrative embodiments, the present invention considers, in part, transfer plasmid and vector polynucleotide sequences and compositions comprising them. In particular embodiments, the invention provides polynucleotides that encode one or more therapeutic polypeptides and/or other genes of interest. In particular embodiments, the present invention provides polynucleotides that encode a globin polypeptide or an ATP binding cassette, member 1 polypeptide (ABCD1) of the D subfamily (ALD), as discussed elsewhere herein.
[00100] For use in the present invention, the terms "polynucleotide variable" and "variable", and the like, refer to polynucleotides that exhibit substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize to a reference sequence under strict conditions that are defined later in this document. These terms include polynucleotides in which one or more nucleotides have been added or deleted, or replaced by different nucleotides compared to a reference polynucleotide. In this regard, it is well understood in the art that certain alterations including mutations, additions, deletions and substitutions can be made to a reference polynucleotide such that the altered polynucleotide retains the biological activity or function of the reference polynucleotide.
[00101] For use herein, the term "isolated" refers to material, for example, a polynucleotide, a polypeptide, a cell, that is substantially or essentially free of components that normally accompany it in its native state. In particular embodiments, the term "obtained" or "derived" is used synonymously with isolated. For example, an "isolated polynucleotide", for use in the present invention, refers to a polynucleotide that has been purified from sequences flanking it in a naturally occurring state, e.g., a DNA fragment that has been removed. of the sequences that are normally adjacent to the fragment.
Terms that describe the orientation of polynucleotides include: 5' (usually the end of the polynucleotide that has a free phosphate group) and 3' (usually the end of the polynucleotide that has a free hydroxyl group (OH)). Polynucleotide sequences can be annotated in the 5' to 3' orientation or in the 3' to 5' orientation.
[00103] The terms "complementary" and "complementarity" refer to polynucleotides (ie a sequence of nucleotides) related by base pairing rules. For example, the complementary strand of the DNA sequence 5' A G T C A T G 3' is 3' T C A G T A C 5'. The last sequence is often written as the inverse complement with the 5' end on the left and the 3' end on the right, 5' C A T G A C T 3'. A sequence that is equal to its inverse complement is referred to as a palindromic sequence. Complementarity can be "partial", in which only some of the nucleic acid bases are combined according to base pairing rules. Or, there may be "complete" or "full" complementarity between nucleic acids.
The term "nucleic acid cassette", for use in the present invention, refers to genetic sequences within the vector that can express an RNA, and subsequently a polypeptide. In one embodiment, the nucleic acid cassette contains a gene(s)-of-interest, e.g., a polynucleotide(s)-of-interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences and a gene(s) of interest, e.g., a polynucleotide(s) of interest. Vectors can comprise one, two, three, four, five or more nucleic acid cassettes. The nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or polypeptide, subjected to appropriate post-translational modifications required for activity in the transformed cell, and be transferred to the appropriate compartment for biological activity by targeting to appropriate intracellular compartments or secretion into extracellular compartments. It is preferred that the cassette have its 3' and 5' ends adapted for insertion available in a vector, e.g. it has restriction endonuclease sites at each end. In a preferred embodiment of the invention, the nucleic acid cassette contains the sequence of a therapeutic gene used to treat, prevent or ameliorate a genetic disorder, such as a hematopoietic disorder. The cassette can be removed and inserted into a plasmid or viral vector as a single unit.
[00105] Polynucleotides include a polynucleotide(s)-of-interest. For use herein, the term "polynucleotide(s)-of-interest" refers to one or more polynucleotides, e.g., a polynucleotide encoding a polypeptide (i.e., a polypeptide-of-interest), inserted into a expression vector that is desired to be expressed. In preferred embodiments, the vectors and/or plasmids of the present invention comprise one or more polynucleotides of interest, for example, a globin gene or ABCD1 gene. In certain embodiments, a polynucleotide-of-interest encodes a polypeptide that provides a therapeutic effect in the treatment, prevention or amelioration of a hematopoietic disorder or disease, which may be referred to as a "therapeutic polypeptide", e.g., a globin gene . See, for example, patents nos. US 6,051,402 and 7,901,671, the full description and claims of which are specifically incorporated herein by reference. See, for example, SEQ ID NOs: 1, 5, 10 and 14.
[00106] In certain other embodiments, a polynucleotide-of-interest encodes a polypeptide that provides a therapeutic effect in the treatment, prevention or amelioration of an adrenoleukodystrophy or adrenomyeloneuropathy, which may be referred to as a "therapeutic polypeptide", for example, a ABCD1 gene. See, for example, SEQ ID NOs: 16-17. See, for example, patents nos. US 5,869,039; and 6,013,769, the full description and claims of which are specifically incorporated herein by reference.
[00107] The term "globin", for use in the present invention, refers to all proteins or protein subunits that are capable of binding covalently or non-covalently to a heme moiety, and can therefore transport or store oxygen . Vertebrate and invertebrate hemoglobin subunits, vertebrate and invertebrate myoglobins or mutants thereof are encompassed by the term globin. Examples of globins include α-globin or a variable thereof, β-globin or a variable thereof, a Y—globin or a variable thereof, and δ-globin or a variable thereof.
[00108] In one embodiment, the polynucleotide-of-interest consists of a gene that encodes a polypeptide that provides a therapeutic function for the treatment of a hemoglobinopathy, for example, α-globin, β-globin or β-globin A-T87Q . Polynucleotides-of-interest, and polypeptides encoded therefrom, include both polynucleotides encoding wild-type polypeptides, as well as functional variables and fragments thereof. In particular embodiments, a functional variable has at least 80%, at least 90%, at least 95%, or at least 99% identity to a corresponding wild-type reference polypeptide or polynucleotide sequence. In certain modalities, a functional variable or fragment has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or at least 110% or more of an activity of a corresponding wild-type polypeptide. Representative polynucleotide sequences suitable for use in the present invention include, but are not limited to, polynucleotides encoding α-globin, β-globin and β-globin A-T87Q.
[00109] The polynucleotides of the present invention, regardless of the length of the coding sequence itself, can be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, sites of additional restriction enzymes, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (eg, LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, and polynucleotides that encode self-cleaving polypeptides, epitope tags, as set forth elsewhere herein or as known in the art, such that their overall length can vary considerably. Therefore, it is observed that a polynucleotide fragment of almost any length can be employed, with the total length being preferably limited by ease of preparation and use in the intended recombinant DNA protocol.
[00110] The term "expression control sequence" refers to a polynucleotide sequence that comprises one or more promoters, enhancers or other transcriptional control elements or combinations thereof that are capable of directing, increasing, regulating or controlling transcription or expression of an operably linked polynucleotide. In particular embodiments, vectors of the invention comprise one or more expression control sequences that are specific to particular cells, cell types or cell lines, e.g., target cells; that is, expression of polynucleotides operably linked to an expression control sequence specific to particular cells, cell types or cell lines is expressed in target cells and not in other non-target cells. Each of one or more expression control sequences in a vector that is cell specific can express in the same or different types of cells depending on the desired therapy. In preferred embodiments, the vectors comprise one or more expression control sequences specific to hematopoietic cells, for example, hematopoietic stem or progenitor cells. In other preferred embodiments, the vectors comprise one or more erythroid cell-specific expression control sequences.
[00111] The term "promoter", for use in the present invention, refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The term “enhancer” refers to a segment of DNA that contains sequences capable of providing optimized transcription and, in some cases, may function independently of its orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and/or other enhancer elements. The term "promoter/enhancer" refers to a segment of DNA that contains sequences capable of providing both promoter and enhancer functions.
[00112] In particular embodiments, a vector of the invention comprises exogenous, endogenous or heterologous control sequences, such as promoters and/or enhancers. An “endogenous” control sequence is one that is naturally linked to a particular gene in the genome. An “exogenous” control sequence is one that is placed in juxtaposition to a gene through genetic manipulation (ie, molecular biology techniques) such that transcription of that gene is directed by the linked enhancer/promoter. A “heterologous” control sequence is an exogenous sequence that is from a different species of cell that is genetically manipulated. A "synthetic" control sequence may comprise elements from one or more endogenous and/or exogenous sequences and/or in vitro or in silico determined sequences that provide optimal promoter and/or enhancer activity for the particular gene therapy.
[00113] The term "operably linked" refers to a juxtaposition in which the described components are in a relationship that allows them to function in their intended manner. In one embodiment, the term refers to a functional link between a nucleic acid expression control sequence (such as a promoter, and/or enhancer or other expression control sequence) and a second polynucleotide sequence, for example , a polynucleotide-of-interest, in which the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
[00114] For use in the present invention, the term "constitutive expression control sequence" refers to a promoter, enhancer or promoter/enhancer that continuously or continuously allows the transcription of an operably linked sequence. A constitutive expression control sequence can be a "ubiquitous" promoter, enhancer or promoter/enhancer that allows expression in a wide variety of cell and tissue types, or a "cell-specific" promoter, enhancer or promoter/enhancer, " cell type specific”, “cell lineage specific” or “tissue specific” which allows expression in a restricted range of cell and tissue types, respectively. Illustrative ubiquitous expression control sequences include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a simian viral 40 (SV40) (e.g., early or late) promoter, a swine virus LTR promoter. Moloney murine leukemia (MoMLV), a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) promoter (thymidine kinase), H5, P7.5 and P11 promoters from smallpox viruses , a promoter of elongation factor 1-alpha (EF1a), early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), 70kDa heat shock protein 5 (HSPA5), 90kDa beta heat shock protein, member 1 (HSP90B1), 70kDa heat shock protein (HSP70), β-kinesin (β-KIN), ROSA 26 site (Irions et al., (2007) Nature Biotechnology 25, 1477 - 1482), a ubiquitin C (UBC) promoter, a phosphoglycerate promoter kinase-1 (PGK), a cytomegalovirus enhancer/chicken β-actin (CAG) promoter, and a β-actin promoter.
[00115] In a particular embodiment, it may be desirable to use a cell, cell type, cell lineage or tissue-specific expression control sequence to achieve cell-type, lineage-specific, or tissue-specific expression of a desired polynucleotide sequence (for example, to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lines or tissues or during specific developmental stages).
[00116] Illustrative examples of tissue-specific promoters include, but are not limited to: a B29 promoter (B cell expression), a runt transcription factor (CBFa2) promoter (stem cell specific expression), a promoter of CD14 (monocytic cell expression), a CD43 promoter (leukocyte and platelet expression), a CD45 promoter (hematopoietic cell expression), a CD68 promoter (macrophage expression), a CYP450 3A4 promoter (expression of hepatocyte), a desmin promoter (muscle expression), an elastase 1 promoter (pancreatic acinar cell expression), an endoglin promoter (endothelial cell expression), a fibroblast-specific protein 1 (FSP1) promoter (expression of fibroblast cell), a fibronectin promoter (fibroblast cell expression), an fms-related tyrosine kinase 1 (FLT1) promoter (endothelial cell expression), a glial fibrillary acidic protein (GFAP) promoter (expression of star cyto), an insulin promoter (pancreatic beta cell expression), an alpha 2b integrin promoter (ITGA2B) (megakaryocytes), an intracellular adhesion molecule 2 (ICAM-2) promoter (endothelial cells), an interferon promoter beta (IFN-β) (hematopoietic cells), a keratin 5 promoter (keratinocyte expression), a myoglobin (MB) promoter (muscle expression), a myogenic differentiation promoter 1 (MYOD1) (muscle expression), a nephrin promoter (podocyte expression), a bone gamma-carboxyglutamate protein 2 (OG-2) promoter (osteoblast expression), a 3-oxoacid CoA transferase 2B (Oxct2B) promoter, (haploid-spermatid expression) ), a surfactant protein B (SP-B) promoter (lung expression), a synapsin promoter (neuron expression), a Wiskott-Aldrich syndrome protein (WASP) promoter (hematopoietic cell expression).
[00117] In one embodiment, a vector of the present invention comprises one or more tissue or hematopoietic cell specific enhancers and/or promoters selected from the group consisting of: a human β-globin promoter; a human β-globin CRL; and an HS40 human α-globin enhancer and an ankyrin-1 promoter operably linked to a polynucleotide encoding a globin polypeptide.
[00118] In another embodiment, a vector of the present invention comprises a promoter active in a microglial cell, operably linked to a polynucleotide encoding an ATP-binding cassette polypeptide, subfamily D, member 1 (ABCD1). In certain embodiments, the promoter comprises a myeloproliferative sarcoma virus enhancer, deleted negative control region, substituted dl587rev primer binding site (MND) promoter, or transcriptionally active fragments thereof.
[00119] For use herein, "conditional expression" may refer to any type of conditional expression that includes, but is not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular disease state, physiological or biological, etc. This definition is not intended to exclude cell or tissue type specific expression. Certain embodiments of the invention provide conditional expression of a polynucleotide-of-interest, e.g., expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or to causes an increase or decrease in expression of the polynucleotide encoded by the polynucleotide-of-interest.
[00120] Illustrative examples of inducible promoters/systems include, but are not limited to, steroid-inducible promoters, such as promoters for genes encoding glucocorticoids or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter ( inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the mifepristone-regulated system “GeneSwitch” (Sirin et al., (2003) Gene, 323:67), the coumate-inducible switch gene (WO 2002/088346), tetracycline dependent regulatory systems, etc.
[00121] Conditional expression can also be achieved with the use of a site-specific DNA recombinase. According to certain embodiments of the invention, the vector comprises at least one (typically two) site(s) for recombination mediated by a site-specific recombinase. For use herein, the terms "recombinase" or "site-specific recombinase" include integrative or scavenger proteins, enzymes, cofactors, or associated proteins that are involved in recombination reactions that involve one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, forty, etc.), which may be wild-type proteins (see Landy, (1993) Current Opinion in Biotechnology 3:699-707 ), or mutants, derivatives (for example, fusion proteins which contain the recombination protein sequences or fragments thereof), fragments and variables thereof. Illustrative examples of recombinases suitable for use in particular embodiments of the present invention include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1 and ParA.
[00122] Vectors may comprise one or more recombination sites for any one of a wide variety of site-specific recombinases. It should be understood that the target site for a site-specific recombinase is in addition to any site(s) required for vector integration, for example, a retroviral vector or lentiviral vector. For use in the present invention, the terms "recombination sequence", "recombination site" or "site-specific recombination site" refer to a particular nucleic acid sequence to which a recombinase recognizes and binds.
[00123] For example, a recombination site for Cre recombinase consists of loxP which is a 34 base pair sequence comprising two 13 base pair inverted repeats (which serve as the recombinase binding sites) that flank a sequence of 8 base pair core (see Figure 1 of Sauer, B., (1994) Current Opinion in Biotechnology 5:521-527). Other exemplary loxP sites include, but are not limited to: lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).
Suitable recognition sites for FLP recombinase include, but are not limited to: FRT (McLeod, et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), FRT(RE) (Senecoff et al., 1988).
[00125] Other examples of recognition sequences are the sequences of attB, attP, attL and attR, which are recognized by the recombinase enzyme À Integrase, for example, pi-c31. The ΦC31 SSR mediates recombination only between the heterotypic sites attB (34 bp in length) and attP (39 bp in length) (Groth et al., 2000). attB and attP, named after the attachment sites for phage integrase in the phage and bacterial genomes, respectively, both contain imperfect inverted repeats that are likely linked by ΦC31 homodimers (Groth et al., 2000). The product sites, attL and attR, are effectively inert to additional ΦC31-mediated recombination (Belteki et al., 2003), making the reaction irreversible. To catalyze insertions, DNA containing attB has been found to insert into a genomic attP locus more readily than an attP locus into a genomic attB locus (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, typical strategies position by recombination a “coupling site” containing attP at a defined location, which is then associated with an input sequence containing attB for insertion.
[00126] For use in the present invention, an "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein coding region), thus leading to termination independent translation of the gene. See, for example, Jackson et al., (1990) Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. (1995) RNA 1(10):985-1000. In particular embodiments, vectors contemplated by the invention include one or more polynucleotides of interest that encode one or more polypeptides. In particular embodiments, to achieve efficient translation of each of the plurality of polypeptides, the polynucleotide sequences can be separated by one or more IRES sequences or polynucleotide sequences that encode self-cleaving polypeptides.
[00127] For use in the present invention, the term "Kozak sequence" refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the small subunit of the ribosome and increases translation. The consensus Kozak sequence is (GCC)RCCATGG, where R is a purine (A or G) (Kozak, (1986) Cell. 44(2):283-92, and Kozak, (1987) Nucleic Acids Res. 15(20) ):8125-48). In particular embodiments, vectors contemplated by the invention comprise polynucleotides which have a consensus Kozak sequence and which encode a desired polypeptide.
[00128] In certain embodiments, vectors comprise a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, hygromycin, methotrexate, Zeocin, Blastocidin or tetracycline, (b) complement auxotrophic deficiencies or (c) supply non-critical nutrients available from complex media, for example, the gene encoding D-alanine racemase for Bacilli. A number of different sorting systems can be used to retrieve transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase genes ( Wigler et al., (1977) Cell 11:223-232) and adenine phosphoribosyltransferase ( Lowy et al., (1990) Cell 22: 817-823) which can be used in tk or aprt cells, respectively.
[00129] In various embodiments, the vectors of the invention are used to increase, establish and/or maintain the expression of one or more polypeptides, for example, globins. The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and synthetic variables and analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues consist of synthetic non-naturally occurring amino acids, such as a chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. . Illustrative examples of globin polypeptides suitable for use in the compositions and methods of particular embodiments of the invention, for example, SEQ ID NOs: 2 to 4, 6 to 9, 11 to 13 and 15. In addition, see, for example, patents nos. US 6,051,402 and 7,901,671, the full description and claims of which are specifically incorporated herein by reference.
[00130] Illustrative examples of ABCD1 polypeptides suitable for use in the compositions and methods of particular embodiments of the invention, e.g., SEQ ID NO: 18. In addition, see, e.g., patent nos. 5,869,039; and 6,013,769, the full description and claims of which are specifically incorporated herein by reference.
Particular embodiments of the invention also include polypeptide "variables". The term polypeptide "variable" refers to polypeptides that are distinguished from a reference polypeptide by the addition, deletion, truncations and/or substitution of at least one amino acid residue, and that retain a biological activity. In certain embodiments, a polypeptide variable is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative as known in the art.
[00132] In certain embodiments, a variant polypeptide includes an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence similarity or identity to a corresponding sequence of a reference polypeptide. In certain embodiments, amino acid additions or deletions occur at the C-terminal end and/or the N-terminal end of the reference polypeptide.
[00133] As noted above, the polypeptides of the invention can be altered in various ways that include amino acid substitutions, deletions, truncations and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variables of a reference polypeptide can be made by mutations in DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Academic Sci. USA 82: 488-492, Kunkel et al., (1987) Methods in Enzymol, 154: 367-382, patent no. U.S. 4,873,192, Watson, J.D. et al., (1987) Molecular Biology of the Gene, fourth edition, Benjamin/Cummings, Menlo Park, Calif., and references mentioned therein. Guidance regarding suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model by Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found. , Washington, DC).
A "host cell" includes cells transfected, infected or transduced in vivo, ex vivo or in vitro with a recombinant vector or a polynucleotide of the invention. Host cells can include packaging cells, producer cells and cells infected with viral vectors. In particular embodiments, host cells infected with the vital vector of the invention are administered to an individual in need of therapy. In certain embodiments, the term "target cell" is used interchangeably with the host cell and refers to transfected, infected, or transduced cells of a desired cell type. In preferred embodiments, the target cell is a stem cell or progenitor cell. In certain preferred embodiments, the target cell is a somatic cell, e.g., adult stem cell, progenitor cell, or differentiated cell. In particular preferred embodiments, the target cell is a hematopoietic cell, for example, a hematopoietic stem or progenitor cell. Additional therapeutic target cells are discussed, infra.
[00135] The term "stem cell" refers to a cell that consists of an undifferentiated cell capable of (1) long-term self-renewal, or the ability to generate at least one identical copy of the original cell, (2) differentiation at the level of single cell into multiple, and in some case only one specialized cell type and (3) functional in vivo tissue regeneration. Stem cells are subclassified according to their developmental potential as totipotent, pluripotent, multipotent, and oligo/unipotent. “Self-renewal” refers to a cell with a unique ability to produce unaltered daughter cells and to generate specialized cell types (potency). Self-renewal can be achieved in two ways. Asymmetric cell division produces a daughter cell that is identical to the parent cell and a daughter cell that is different from the parent cell and is a progenitor or differentiated cell. Asymmetric cell division does not increase the number of cells. Symmetrical cell division produces two identical daughter cells. "Proliferation" or "expansion" of cells refers to symmetrically dividing cells.
[00136] For use in the present invention, the term "totipotent" refers to the ability of a cell to form cell lines of an organism. For example, in mammals, only the zygote and early cleavage stage blastomeres are totipotent. For use in the present invention, the term "pluripotent" refers to the ability of a cell to form all lineages of the body or soma (i.e., the proper embryo). For example, embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three layers of the dermis, the ectoderm, the mesoderm and the endoderm. For use herein, the term "multipotent" refers to the ability of an adult stem cell to form multiple cell types from one lineage. For example, hematopoietic stem cells are capable of forming all cells of the blood cell lineage, eg lymphoid and myeloid cells. For use herein, the term "oligopotent" refers to the ability of an adult stem cell to differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells are capable of forming cells of the lymphoid or myeloid lineages, respectively. For use herein, the term "unipotent" refers to the ability of a cell to form a single cell type. For example, spermatogonial stem cells are only able to form sperm cells.
[00137] For use in the present invention, the term "progenitor" or "progenitor cells" refers to cells that have the ability to self-renew and differentiate into more mature cells. Many progenitor cells differentiate along a single lineage, but they may have completely extensive proliferative capacity.
[00138] Hematopoietic stem cells (HSCs) give rise to committed hematopoietic progenitor cells (HPCs) that are capable of generating the entire repertoire of mature blood cells over the entire lifetime of an organism. The term “hematopoietic stem cell” or “HSC” refers to multipotent stem cells that give rise to all types of blood cells in an organism, which include myeloid lineages (eg, monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoids (e.g., T cells, B cells, NK cells), and others known in the art (see Fei, R., et al., US Patent No. 5,635,387; McGlave, et al., US Patent No. 5,460,964; Simmons, P., et al., US Patent No. 5,677,136; Tsukamoto, et al., US Patent No. 5,750,397; Schwartz, et al. , US Patent No. 5,759,793; DiGuisto, et al., US Patent No. 5,681,599; Tsukamoto, et al., US Patent No. 5,716,827). When transplanted into lethally irradiated animals or humans, hematopoietic stem and progenitor cells can repopulate the pool of hematopoietic erythroid, neutrophil-macrophage, megakaryocyte, and lymphoid cells.
[00139] Large-scale viral particle production is often necessary to achieve a reasonable viral titer. Viral particles are produced by transfecting a transfer vector into a packaging cell line comprising viral accessory and/or structural genes, e.g. gag, pol, env, tat, rev, vif, vpr, vpu, vpx genes or nef or other retroviral genes.
[00140] For use herein, the term "packaging vector" refers to an expression vector or viral vector that is devoid of a packaging signal and comprises a polynucleotide encoding one, two, three, four or more genes viral accessories and/or structural. Typically, packaging vectors are included in a packaging cell, and are introduced into the cell via transfection, transduction, or infection. Methods for transfection, transduction or infection are well known to those skilled in the art. A retroviral/lentiviral transfer vector of the present invention can be introduced into a packaging cell line, via transfection, transduction or infection, to generate a producer cell or cell line. The packaging vectors of the present invention can be introduced into human cells or cell lines by standard methods which include, for example, calcium phosphate transfection, lipofection or electroporation. In some embodiments, packaging vectors are introduced into cells along with a dominant selectable marker, such as neomycin, hygromycin, puromycin, blastocidin, zeocin, thymidine kinase, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. A selectable marker gene can be physically linked to genes that encode by the packaging vector, for example, by IRES or self-cleaving viral peptides.
[00141] Viral envelope proteins (env) determine the range of host cells that can ultimately be infected and transformed by the recombinant retrovirus generated from the cell lines. In the case of lentiviruses such as HIV-1, HIV-2, SIV, FIV and EIV, the env proteins include gp41 and gp120. It is preferred that the viral env proteins expressed by packaging cells of the invention are encoded in a vector separate from the viral genes gag and pol, as has been described above.
Illustrative examples of retrovirus-derived env genes that can be employed in the invention include, but are not limited to: MLV envelopes, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (Avian Flu ), and influenza virus envelopes. Similarly, genes encoding envelopes from RNA viruses (e.g., Picornaviridae RNA virus families, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Re, Arenaviridae, Bunyaviridae , Retroviridae), as well as from the DNA virus (families of Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae and Iridoviridae) can be used. Representative examples include, FeLV, VEE, HFVW, WDSV, SFV, Rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10 and EIAV .
In other embodiments, envelope proteins for creating a pseudotype of a virus of the present invention include, but are not limited to, any of the following viruses: Influenza A viruses, such as H1N1, H1N2, H3N2 and H5N1 (group avian), Influenza B, Influenza C, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rotavirus, any virus of the Norwalk virus group, enteric adenovirus, parvovirus, virus of Dengue Fever, Monkey Syphilis, Mononegavirales, Lissaviruses such as Rabies Virus, Lagos Bat Virus, Mokola Virus, Duvenhage Virus, European Bat Virus 1 & 2 and Australian Bat Virus, Ephemerovirus, Vesiculovirus, Virus vesicular stomatitis (VSV), herpes virus such as herpes simplex virus type 1 and 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpes virus (HHV), human herpes virus type 6 and 8, human immunodeficiency virus (HIV), papilloma virus , murine gamma herpes virus, Arenavirus, such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, sabiá associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, virus Lymphocytic choriomeningitis (LCMV), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, renal syndrome causing virus with hemorrhagic fever, Rift Valley fever virus, Filoviridae (filovirus) which includes Ebola hemorrhagic fever and hemorrhagic fever of Marburg, Flaviviridae which includes Kaysanur forest disease virus, Omsk hemorrhagic fever virus, tick-borne encephalitis virus and Paramyxoviridae such as Hendra virus and Nipah virus, major smallpox and minor smallpox (smallpox), alphavirus, such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western encephalitis virus, SARS-associated coronavirus (SARS-CoV), Nile virus or accidental, any virus causing encephalitis.
[00144] In one embodiment, the invention provides packaging cells that produce recombinant retroviruses, for example, lentiviruses, pseudotyped with the glycoprotein VSV-G.
[00145] The terms "pseudotype" or "pseudotyping", for use in the present invention, refer to a virus whose viral envelope proteins have been replaced by those of another virus having preferred characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus G protein (VSV-G) envelope proteins, which allows HIV to infect a wider range of cells due to the fact that HIV envelope proteins (encoded by env gene) normally target the virus to cells that have CD4+. In a preferred embodiment of the invention, the lentiviral envelope proteins are pseudotyped with VSV-G. In one embodiment, the invention provides packaging cells that produce recombinant retroviruses, e.g., lentiviruses, pseudotyped with the envelope glycoprotein of VSV-G.
[00146] For use in the present invention, the term "packaging cell lines" is used in reference to cell lines that do not contain a packaging signal, but stably or provisionally express viral structural proteins and replication enzymes ( eg gag, pol and env) which are necessary for the correct packaging of viral particles. Any suitable cell line can be employed to prepare packaging cells of the invention. Cells are usually mammalian cells. In a particular embodiment, the cells used to produce the packaging cell line are human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos- cells 2, Huh7 cells, HeLa cells, W163 cells, 211 cells and 211A cells. In preferred embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells. In another preferred embodiment, the cells are A549 cells.
[00147] For use in the present invention, the term "producer cell line" refers to a cell line that is capable of producing recombinant retroviral particles, comprising a packaging cell line and a transfer vector construct comprising a packaging sign. The production of infectious viral particles and viral stock solutions can be carried out using conventional techniques. Methods of preparing viral stock solutions are known in the art and are illustrated, for example, by Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N.R. Landau et al. (1992) J. Virol. 66:5110-5113. Infectious virus particles can be collected from the packaging cells using conventional techniques. For example, infectious particles can be collected by cell lysis, or cell culture supernatant collection, as is known in the art. Optionally, the collected virus particles can be purified if desired. Suitable purification techniques are well known to those skilled in the art.
[00148] The term "optimize", "promote", "increase" or "expand" generally refers to the ability of the compositions and/or methods of the invention to obtain, cause or produce greater numbers of transduced cells compared to the number of cells transduced by vehicle or a control molecule/composition. In one embodiment, a hematopoietic stem cell transduced with compositions and methods of the present invention comprises an increase in the number of cells transduced compared to existing transduction compositions and methods. The increase in cell transduction can be confirmed using methods known in the art, such as reporter assays, RT-PCR, and cell surface protein expression, among others. An "increased" or "optimized" amount of transduction is typically a "statistically significant" amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 30 or more times (eg 500, 1000 times) (which includes all whole numbers and decimal points between and above 1, eg 1.5, 1.6 , 1.7, 1.8, etc.) the number of cells transduced per vehicle, a control composition or other transduction method.
[00149] The term "lower", "lower", "attenuate", "reduce" or "decline" generally refers to compositions or methods that result in comparably fewer transduced cells compared to cells transduced with compositions and/or methods of according to the present invention. A "decreased" or "reduced" amount of transduced cells is typically a "statistically significant" amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (eg 500, 1000 times) (which includes all whole numbers and decimal points between and above 1, eg 1.5, 1, 6, 1.7, 1.8, etc.) the number of transduced cells (reference response) produced by compositions and/or methods according to the present invention.
[00150] The term "maintain", "conserve", "maintenance", "no change", "no substantial change" or "no substantial decrease" generally refers to a physiological response that is comparable to a response caused by the vehicle, a control molecule/composition, or the response in a particular cell line. A comparable response is one that is not significantly different or measurably different from the reference response.
[00151] The articles “a” and “o” are used in this document to refer to one or more than one (ie, at least one) of the grammatical object of the article. By way of example, “an element” refers to one element or more than one element.
[00152] The use of the alternative (eg, “or”) should be understood as one, both or any combination of the same of the alternatives. For use herein, the terms "include" and "comprise" are used synonymously.
[00153] For use in the present invention, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, content, weight or length that varies by a maximum of 15 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of a quantity, level, value, number, frequency, percentage, dimension, size, content , weight or reference length. In one embodiment, the term "about" or "approximately" refers to a range of quantity, level, value, number, frequency, percentage, dimension, size, content, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1% about a quantity, level, value, number, frequency, percentage, dimension , size, content, weight or reference length.
[00154] Throughout this descriptive report, except where the context requires otherwise, it should be understood that the words "understand", "understand" and "understanding" imply the inclusion of a step, element or group of steps or elements mentioned , but not the deletion of any other step, element or group of steps or elements. The term "consists of" is intended to include, and is not limited to, anything that follows the phrase "consists of". Thus, the phrase “consists of” indicates that the elements listed are required or mandatory, and that no other elements can be present. The term “consists essentially of” is intended to include any elements listed after the sentence, and is not limited to other elements that do not interfere with or contribute to the activity or action specified in the description for the listed elements. Thus, the phrase “consist essentially of” indicates that the listed elements are required or mandatory, but that no element is optional and may or may not be present depending on whether or not they affect the activity or action of the listed elements.
[00155] Throughout this specification, reference to "one (1) embodiment" or "an embodiment" means that a particular feature, structure or resource described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one (1) modality” or “in a modality” in various places throughout this descriptive report are not necessarily referring to the same modality. Additionally, particular features, structures or features may be combined in any suitable way in one or more modalities.
[00156] In the following description, certain specific details are presented in order to provide a complete understanding of various embodiments of the invention. However, one skilled in the art will understand that the invention can be practiced without these details. In addition, it should be understood that individual vectors, or groups of vectors, derived from the various combinations of structures and substituents described herein, are presented by this application to the same extent as if each vector or group of vectors were presented individually. . Thus, the selection of particular vector structures or particular substituents is within the scope of the present description. C. VIRAL VECTORS
[00157] Retroviral and lentiviral vectors have been tested and revealed as suitable delivery vehicles for the stable introduction of genes of interest, for example, encoding therapeutic polypeptides, into the genome of a wide range of target cells. The present invention contemplates, in part, improved delivery of gene therapy vectors to a population of cells that are administered to an individual to provide gene therapy.
[00158] The present invention additionally provides transfer vectors, which can be used to practice the methods of the present invention. Although a person skilled in the art will note that such transfer vectors can be produced using a variety of different viral vectors, in particular embodiments, the transfer vector consists of a retroviral vector or a lentiviral vector, in part, as lentiviral vectors are able to provide effective long-term release, integration and expression of transgenes in non-dividing cells both in vitro and in vivo. A variety of lentiviral vectors are known in the art, see Naldini et al. (1996a, 1996b and 1998); Zufferey et al., (1997); Dull et al., 1998, patent nos. U.S. 6,013,516; and 5,994,136, any of which can be adapted to produce a transfer vector of the present invention.
In general, these vectors are plasmid-based or virus-based and are configured to load the essential sequences for transferring a nucleic acid encoding a therapeutic polypeptide into a host cell.
[00160] In illustrative embodiments, the retroviral vector consists of a lentiviral vector. Thus, the vectors can be derived from human immunodeficiency-1 (HIV-1), human immunodeficiency-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), virus bovine immunodeficiency (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), and the like. HIV-based vector structures (i.e., cis-acting sequence elements of HIV and HIV gag, pol, and rev genes) are generally preferred in connection with most aspects of the present invention, in that the HIV-based constructs are the most effective in transducing human cells.
Although the particular illustrative embodiments include the more detailed description of vectors, compositions and methods used to correct hematopoietic disorders, e.g., hemoglobinopathies, the invention should not be considered limited by this description. One skilled in the art would readily observe that the principles illustrated herein can be applied to gene therapy in other systems, for example, the nervous system, which includes the eyes, central nervous system, and peripheral nervous system; the circulatory system; the muscular system; the skeletal system; organs, which include the skin, heart, lungs, pancreas, liver, kidney, intestine, and the like.
[00162] In one embodiment, the present invention provides vectors, for example, lentiviral vectors, which comprise an expression control sequence that directs the expression of polynucleotide-of-interest, for example, a globin gene, in a type of particular cell or cell lineage. The use of a cell type or cell lineage expression control sequence offers safety advantages by restricting polynucleotide expression to a desired stage of cell differentiation into a single lineage; and, thus, vectors of the invention alleviate concerns that address ectopic expression of polypeptides in unwanted cell types.
[00163] In a non-limiting example, the expression control sequence may be a ubiquitous expression control sequence, as presented elsewhere in this document.
[00164] In another non-limiting example, the expression control sequence may be a stem cell-specific expression control sequence that directs stem cell-specific expression of the polynucleotide-of-interest in an embryonic stem cell, a neural stem cell, a mesenchymal stem cell, a liver stem cell, a pancreatic stem cell, a cardiac stem cell, a kidney stem cell, or a hematopoietic stem cell.
[00165] In yet another non-limiting example, the expression control sequence may consist of a cell type or cell lineage-specific expression control sequence that directs the expression of the polynucleotide-of-interest in a hematopoietic stem cell , a hematopoietic progenitor cell, a myeloid cell, a lymphoid cell, a thrombopoietic lineage, a mast cell, an erythropoietic lineage cell, a granulopoietic lineage cell, and a monocytopoietic lineage cell.
[00166] In particular embodiments, a vector of the invention can be used to express a polynucleotide, e.g., gene-of-interest in one or more or all hematopoietic cells including, but not limited to, hematopoietic stem cells, hematopoietic progenitors, myeloid progenitors, lymphoid progenitors, thrombopoietic progenitors, erythroid progenitors, granulopoietic progenitors, monocytopoietic progenitors, megakaryoblasts, promegakaryocytes, megakaryocytes, thrombocytes/platelets, proerythroblasts, erythroblasts, erythroblastic erythroblasts, erythroblasts, erythroblasts, erythroblasts, erythroblasts, erythroblasts, erythroblasts , basophilic promyelocytes, basophilic myelocytes, basophilic metamyelocytes, basophilic, neutrophilic promyelocytes, neutrophilic myelocytes, neutrophilic metamyelocytes, neutrophils, eosinophilic promyelocytes, eosinophilic myelocytes, macrophages, dendritic cells natural killers (NK), small lymphocytes, T lymphocytes, B lymphocytes, plasma cells and lymphoid dendritic cells.
In preferred embodiments, a vector of the invention can be used to express a polynucleotide, e.g., gene-of-interest in one or more erythroid cells, e.g., proerythroblast, basophilic erythroblast, polychromatic erythroblast, orthochromatic erythroblast, polychromatic erythrocyte and erythrocyte (RBC).
In one embodiment, the vector comprises a hematopoietic cell promoter, enhancer or promoter/enhancer operably linked to a gene of interest, e.g. globin.
Cell type or cell lineage specific expression control sequences include, but are not limited to, hematopoietic cell expression control sequences, such as, for example, a hematopoietic stem cell promoter, and a hematopoietic progenitor cell promoter. In embodiments where expression of the gene of interest is desired in one or more erythroid cells, a suitable hematopoietic cell expression control sequence may include, but is not limited to, an erythroid cell-specific promoter and, optionally, a specific enhancer of erythroid cell, a human β-globin promoter, a human β-globin CRL, or a human HS40 α-globin enhancer and an ankyrin-1 promoter.
[00170] In one embodiment, suitable cell type or cell lineage expression control sequences include, but are not limited to, a promoter active in a microglial cell. In certain embodiments, the promoter comprises an MND promoter or transcriptionally active fragment thereof operably linked to a gene of interest, e.g., ABCD1.
[00171] The use of a cell type or cell lineage expression control sequence offers safety advantages in restricting polynucleotide expression to this desired stage of cell differentiation in a single lineage; and, thus, the vectors of the invention alleviate concerns dealing with ectopic expression of polypeptides in unwanted cell types. In one embodiment, the invention provides, a vector that comprises one or more LTRs, and an expression control sequence operably linked to a gene of interest. In a related embodiment, the expression control sequence consists of an erythroid cell-specific expression control sequence that is selected from the group consisting of: a human β-globin promoter; a human β-globin CRL; and a human HS40 α-globin enhancer and an ankyrin-1 promoter.
[00172] In several modalities, vector design will be done with the aim of treating, preventing or ameliorating a particular hematopoietic disease, disorder or condition. For example, the present invention contemplates vectors for gene therapy of hemoglobinopathies comprising a gene of interest selected from the group consisting of: human α-globin, human β-globin, human δ-globin and Y-human globin, or biologically active variables or fragments thereof. In one embodiment, the globin gene is selected from the group consisting of a wild-type human β-globin gene, a deleted human β-globin gene that comprises one or more intron sequence deletions, and a gene of mutant human β-globin that encodes one less antisickling amino acid residue.
In a particular embodiment, where the condition being treated consists of a sickle cell hemoglobinopathy, the gene of interest may consist of an anti-sickling protein. For use in the present invention, the "antisickling protein" refers to a polypeptide that prevents or reverses the pathological events that lead to erythrocyte sickling under sickle cell conditions. In one embodiment of the invention, the transduced cells of the invention are used to deliver anti-sickling proteins to an individual with a condition of hemoglobinopathy. Antisickling proteins also include mutant β-globin genes that comprise antisickling amino acid residues.
In a preferred embodiment, such a globin variable consists of the human βA-globin gene encoding a threonine to glutamine mutation at codon 87 (βA-T87Q) or a human βA-globin gene (the mature form of the polypeptide of globin has been processed by N-terminal methionine cleavage, codon 87 of the mature globin polypeptide is threonine; codon 88 of the full-length uncleaved globin polypeptide consists of threonine). Other antisickling amino acid residues are known in the art and may be useful in the present invention. For example, see Patent no. U.S. 6,051,402; Patent no. U.S. 5,861,488; Patent no. U.S. 6.670,323; Patent no. U.S. 5,864,029; Patent no. U.S. 5,877,288; and Levasseur et al., Blood 102:4312-4319 (2003), which are incorporated herein by reference.
In certain embodiments, a vector comprising an erythroid-specific expression control sequence is used to treat, prevent, or ameliorate a vast number of disorders that extend beyond hemoglobinopathies. Red blood cell precursors consist of a useful cell population in which they express polypeptides that can be secreted into the circulation and thus released systematically. An example of such protein release in vivo is human factor IX, a clotting factor that is lost in hemophilia B patients, see, for example, AH Chang, et al., Molecular Therapy (2008), which is here incorporated by way of reference.
In one embodiment, cells transduced with vectors of the invention can be used as "factories" for protein secretion, in vitro, ex vivo or in vivo. For example, a vector comprising an erythroid cell-specific expression control sequence can be used for large-scale in vitro protein production from erythroid cells differentiated from HSCs or from embryonic stem cells.
[00177] Polynucleotides-of-interest that could be expressed in this way include, but are not limited to: adenosine deaminase, enzymes affected in lysosomal storage diseases, apolipoprotein E, brain-derived neurotrophic factor (BDNF), morphogenetic protein bone 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), cardiotrophin 1 (CT-1), CD22, CD40, ciliary neurotrophic factor (CNTF), CCL1 - CCL28, CXCL1-CXCL17, CXCL1, CXCL2, CX3CL1, vascular endothelial cell growth factor (VEGF), dopamine, erythropoietin, Factor IX, Factor VIII, epidermal growth factor (EGF), estrogen, FAS ligand, factor fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2), fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5), fibroblast growth factor fibroblast 6 (FGF-6), fibroblast growth factor 1 (FGF-7), fibroblast growth factor 1 ( FGF-10), Flt-3, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage stimulating factor (GM-CSF), growth hormone, hepatocyte growth factor (HGF), interferon alpha (IFN-a), interferon beta (IFN-b), interferon gamma (IFNg), insulin, glucagon, insulin-like growth factor 1(IGF-1), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 13 (IL -13), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 19 (IL-19), macrophage colony stimulating factor (M-CSF), monocyte chemotactic protein 1 (MCP-1 ), macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), growth factor nerve (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), parathyroid hormone, platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB (PDGF-AB) , platelet-derived growth factor BB (PDGF-BB), platelet-derived growth factor CC (PDGF-CC), platelet-derived growth factor DD (PDGF-DD), RANTES, stem cell factor (SCF) , stromal cell-derived factor 1 (SDF-1), testosterone, transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a) , Wnt1, Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14 hedge, Wnt15 Desert hedgehog and Indian hedgehog.
[00178] In one embodiment, a vector of the invention comprises at least one modified or unmodified retroviral LTR, for example, lentiviral LTR, a β-globin promoter and a β-globin site control region (LCR) linked to operable mode to a polynucleotide of interest, for example, which encodes a globin polypeptide. Suitable modifications of the LTRs include, but are not limited to: replacement of the 5' LTR is with a heterologous promoter, e.g., cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, a thymidine promoter kinase, or a simian virus 40 (SV40) promoter; and one or more modifications, additions and/or deletions of a 3' LTR, as discussed elsewhere herein.
[00179] In a particular embodiment, erythroid-specific expression of a polynucleotide is achieved with the use of a human β-globin promoter, a β-globin LCR that comprises one or more of the 2, 3 hypersensitive DNAase I sites 2, 3 and 4 from human β-globin LCR and/or a human 3' β-globin enhancer element.
[00180] In various embodiments, a vector of the invention comprises one or more elements selected from the group consisting of: a Psi packing sequence (^+), a central polypurine/DNA flap (cPPT/FLAP) tract, a retroviral export element, a post-transcriptional regulatory element, one or more isolating elements, a polyadenylation sequence, a selectable marker, and a cell suicide gene, as discussed elsewhere herein.
[00181] In various embodiments, the vectors of the invention comprise a hematopoietic cell operable-mode promoter operably linked to a gene encoding a polypeptide that provides therapy for hemoglobinopathies. Vectors can have one or more LTRs, where any LTR comprises one or more modifications, such as one or more nucleotide substitutions, additions or deletions. Vectors may further comprise one or more of the additional elements to increase transduction efficiency (e.g., a cPPT/FLAP), viral packaging (e.g., a Psi (^), RRE packaging signal, and/or other elements which increase therapeutic gene expression (e.g., poly(A) sequences).
In one embodiment, a vector comprises a left (5') retroviral LTR, a Psi (Φ+) packaging sequence, central polypurine/DNA flap tract (cPPT/FLAP), a retroviral export element, a promoter of β-globin, a β-globin site control region (LCR) and, optionally, a 3' β-globin enhancer operably linked to a polynucleotide of interest, and a right retroviral (3') LTR that comprises one or more insulating elements, or a polyadenylation sequence.
In particular embodiment, a vector of the invention consists of a lentiviral vector comprising a left (5') HIV-1 LTR, a Psi (Φ+) packaging sequence, a central polypurine/HIV flap DNA tract -1 (cPPT/FLAP), a rev response element (RRE), a β-globin promoter, a β-globin site control region (LCR) and, optionally, a 3'β-globin enhancer operably linked to a polynucleotide of interest, and a straight (3') retroviral LTR comprising one or more insulating elements, and a rabbit β-globin polyA (rβgpA) sequence.
In various embodiments, vectors of the invention comprise a promoter operably in a microglial cell operably linked to a gene encoding a polypeptide that provides therapy for adrenoleukodystrophies and/or adrenomyeloneuropathies. Vectors can have one or more LTRs, where each LTR comprises one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. Vectors may additionally comprise one or more of the additional elements to increase transduction efficiency (e.g., a cPPT/FLAP), viral packaging (e.g., a Psi (^), RRE packaging signal, and/or others elements that enhance therapeutic gene expression (eg, poly(A) sequences).
[00185] In a particular embodiment, the transfer vector of the invention comprises a left (5') retroviral LTR; a central polypurine/DNA flap (cPPT/FLAP) tract; a retroviral export element; a promoter active in a microglial cell, operably linked to a polynucleotide encoding an ATP-binding cassette polypeptide, subfamily D, member 1 (ABCD1); and a right (3') retroviral LTR.
[00186] In a particular embodiment, the invention provides a lentiviral vector comprising: a left HIV-1 (5') LTR; a Psi packing signal (^); a cPPT/FLAP; an RRE; an MND promoter operably linked to a polynucleotide encoding a human ABCD1 polypeptide; a right (3') self-inactivating HIV-1 LTR (SIN); and a rabbit β-globin polyadenylation sequence.
[00187] The person skilled in the art would note that many other different modalities can be created from existing embodiments of the invention, such that the transgene or gene of therapeutic interest is expressed in a cell type or target cell lineage in addition of the hematopoietic lineage, for example, the neuronal lineage. D. TRANSDUCTION METHODS
[00188] The present invention considers, in part, methods and compositions that significantly increase the transduction efficiency of target cells. Without being bound by any particular theory, it is considered that the compositions and methods of the present invention can be used to transduce significantly more cells with significantly less virus, thus minimizing the risk of genomic alteration and/or activation of proto-insertion. oncogenes in the genome of the therapeutic cell. Minimizing the risk of activation of protooncogene insertions and other genomic changes in the therapeutic cell is an important consideration in developing an appropriate gene therapy protocol because it minimizes the chance that transduced cells that comprise cancer-like characteristics will be clonally expanded in vivo and give rise to cancers, tumors or other diseases that involve abnormal cell proliferation. Furthermore, the technique has observed that transduction with large amounts of virus can generally be cytotoxic to the transduced cell. Thus, the compositions and methods of the present invention further optimize the survivability of transduced cells. Consequently, the present invention provides a safer and more effective gene therapy.
[00189] The release of a gene(s) or other polynucleotide sequences using a retroviral or lentiviral vector through viral infection rather than transfection is referred to as "transduction". In one embodiment, retroviral vectors are transduced into a cell through infection and provirus integration. In certain embodiments, a cell, eg, a target cell, is "transduced" if it comprises a gene or other polynucleotide sequence released into the cell through infection using a viral or retroviral vector. In particular embodiments, a transduced cell comprises one or more genes or other polynucleotide sequences released by a retroviral or lentiviral vector into its cell genome.
In particular embodiments, host cells or target cells transduced with a viral vector of the invention express a therapeutic polypeptide and are administered to an individual to treat and/or prevent a disease, disorder or condition.
[00191] The production of infectious viral particles and viral stock solutions can be performed using conventional techniques. Methods of preparing viral stock solutions are known in the art and are illustrated, for example, by Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N.R. Landau et al. (1992) J. Virol. 66:5110-5113.
[00192] In particular embodiments, viral particles based on HIV type 1 (HIV-1) can be generated through the co-expression of the virion packaging elements and the transfer vector in a producer cell. These cells can be provisionally transfected with a series of plasmids. Typically, from three to four plasmids are employed, but the number can be higher depending on the degree to which the lentiviral components are broken down into separate units. For example, a plasmid can encode the core and enzymatic components of virion, derived from HIV-1. This plasmid is called a packaging plasmid. Another plasmid typically encodes the envelope protein(s), but commonly the vesicular stomatitis virus (VSV G) protein, due to its high stability and broad tropism. This plasmid can be called an envelope expression plasmid. Yet another plasmid encodes the genome to be transferred to the target cell, that is, the vector itself, and is called the transfer vector. Packaging plasmids can be introduced into human cell lines by known techniques, which include calcium phosphate transfection, lipofection or electroporation. Recombinant virus with titers of several million transduction units per milliliter (TU/ml) can be generated using this technique and its variables. After ultracentrifugation, concentrated stocks of about 108 TU/ml, 109 TU/ml, 1010 TU/ml, 1011 TU/ml, 1012 TU/ml or about 1013 TU/ml can be obtained.
[00193] Infectious virus particles can be collected from the packaging cells using conventional techniques. For example, infectious particles can be collected by cell lysis, or cell culture supernatant collection, as is known in the art. Optionally, the collected virus particles can be purified if desired. Suitable purification techniques are well known to those skilled in the art.
[00194] The virus can be used to infect cells in vivo, ex vivo or in vitro using well known techniques. For example, when cells, eg, CD34+ cells, dendritic cells, peripheral blood cells or stem cells, are subjected to ex vivo transduction, the vector particles can be incubated with the cells using a dose usually in the order between 1 to 50 multiplicities of infection (MOI) which also corresponds to 1x105 to 50x105 viral vector transduction units per 105cells. This, of course, includes the vector quantity that corresponds to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 and 50 MOI.
[00195] The virus can also be released to an individual in vivo, through direct injection into the cell, tissue or organ in need of therapy. Direct injection requires the order of 1 to 50 multiplicities of infection (MOI) which also corresponds to 1x105 to 50x105 viral vector transduction units per 105 cells.
[00196] The virus can also be shed according to viral titer (TU/mL), which can be measured, for example, using a commercially available p24 titration assay, which is an ELISA against the protein. of p24 viral coat. The following formula can be used to calculate the pg/ml of p24: there are approximately 2000 molecules of p24 per physical particle (PP) of lentiviruses: (2 x 103) x (24 x 103 Da of p24 per PP), 48 x 106 / Avogadro = (48 x 106) / (6 x 1023) = 8 x 10-17 g p24 per PP, approximately 1 PP per 1 x 10-16 g p24, 1 x 104 PP per pg p24. A reasonably well packaged lentiviral vector with VSV-G pseudotype will have an infectivity index in the range of 1 TU per 1000 physical particles (PP) to 1 TU per 100 PP (or less). Thus, the range is approximately 10 to 100 TU/pg of p24. It is through this conversion that TU/mL is obtained.
[00197] Based on previous experience, the amount of lentivirus directly injected is determined by the total TU and may vary based on both the volume that could be feasibly injected at the site and the type of tissue to be injected. For example, a brain injection site may only allow a very small volume of virus to be injected, so a high titration preparation would be preferred, a TU of about 1 x 106 to 1 x 107, about 1 x 106 to 1 x 108, 1 x 106 to 1 x 109, about 1 x 107 to 1 x 1010, 1 x 108 to 1 x 1011, about 1 x 108 to 1 x 1012, or about 1 x 1010 to 1 x 1012 or more per injection could be used. However, a systematic release could accommodate a much larger TU, a load of 1 x 108, 1 x 109, 1 x 1010, 1 x 1011, 1 x 1012, 1 x 1013, 1 x 1014 or 1 x 1015 could be released.
[00198] The present invention considers compositions and methods that provide high-efficiency transduction of cells in vitro, ex vivo and in vivo, with the use of lower viral titers than those presented above to achieve comparable transduction efficiencies in the absence of the compositions and methods provided in this document.
[00199] Certain aspects of the present invention arise from the unexpected discovery that transduction efficiency is significantly increased by placing cells, in vitro, ex vivo or in vivo, in contact with a retrovirus and one or more compounds that stimulate EP prostaglandin receptor signaling pathway, such as, for example, a small molecule, or those compounds disclosed in the document under no. WO 2007/112084 and WO2010/108028, each of which are incorporated herein by reference in their entirety. For use in the present invention, the terms "stimulate prostaglandin EP receptor signaling", "activate prostaglandin EP receptor signaling" or "increase prostaglandin EP receptor signaling" generally refer to the ability of a compound to increase the signaling activity of the cell downstream of a prostaglandin EP receptor in the cell placed in contact with the one or more compounds, compared to the signaling activity of the cell downstream of the prostaglandin EP receptor in the absence of the one or more compounds. Assays that can be used to measure activation or stimulation of the EP prostaglandin receptor signaling pathway are known in the art and are described, for example, in the document under no. WO2010/108028, which is incorporated herein by reference in its entirety.
Illustrative examples of compounds that stimulate the EP prostaglandin receptor signaling pathway include, but are not limited to, small molecules, e.g., small organic molecules, prostaglandins, Wnt pathway agonists, cAMP pathway agonists /PI3K/AKT, Ca2+ second messenger pathway agonists, nitric oxide (NO)/angiotensin signaling agonists, and other compounds known to stimulate the prostaglandin signaling pathway selected from the group consisting of: Mebeverine, Flurandrenolid , Atenolol, Pindolol, Gaboxadol, Quinurenic Acid, Hydralazine, Thiabendazole, Bicuclin, Vesamicol, Peruvoside, Imipramine, Chlorpropamide, 1,5-Pentamethylenetetrazol, 4-Aminopyridine, Diazoxide, Benfothiamine, 12-Methoxydodecenoic Acid, N-Methoxydecenoic Acid - Phe, Galamine, IAA 94, Chlorotrianisene, and derivatives of these compounds.
[00201] In a preferred embodiment, the compound that stimulates the prostaglandin pathway consists of a synthetic or naturally occurring chemical polypeptide or molecule that binds to and/or interacts with an EP receptor, typically to activate or augment one or more of the pathways signaling pathways associated with an EP prostaglandin receptor as described herein and known in the art.
[00202] In one embodiment, the compound that stimulates the prostaglandin pathway is selected from the group consisting of: PGA2; PGB2; PGD2; PGE1 (Alprostadil (CaverjectTM; EdexTM; MuseTM; Prostin VRTM); PGE2; PGF2; PGI2 (Epoprostenol (FlolanTM; ProstacyclinTM)); PGH2; PGJ2; and precursors, metabolites, derivatives and analogues thereof.
Additional illustrative compounds that stimulate the prostaglandin thr pathway include, but are not limited to, 15d-PGJ2; delta12-PGJ2; 2-hydroxyheptadecatrienoic acid (HHT); Thromboxane (TXA2 and TXB2); PGI2 analogues, for example, Iloprost (VentavisTM) and Treprostinil (RemodulinTM); PGF2 analogues, for example, Travoprost (TravatanTM), Carboprost tromethamine (HemabateTM), Tafluprost (ZioptanITM), Latanoprost (XalatanTM), Bimatoprost (LumiganTM; LatisseTM), Isopropyl Unoprostone (ResculaTM), Cloprostenol (CycixateTM, EstrusinTM), ™, Lutaprost™, Onsett™, Planate™), Oestrophan, and Superphan; PGE1 analogues, for example Misoprostol (CytotecTM) and Butaprost; and Corey A alcohol [[3aα,4α,5β,6aα]-(-)-[Hexahydro-4-(hydroxymethyl)-2-oxo-2H-cyclopenta/b/furan-5-yl][1,1'- biphenyl]-4-carboxylate]; Corey B alcohol [2H-Cyclopenta[b]furan-2-on,5-(benzoyloxy)hexahydro-4-(hydroxymethyl)[3aR-(3aα,4α,5β,6aα)]]; and Corey diol ((3aR,4S,5R,6aS)-hexahydro-5-hydroxy-4-(hydroxymethyl)-2H-cyclopenta[b]furan-2-one).
In one embodiment, the compound consists of a prostaglandin EP receptor ligand that includes, but is not limited to, prostaglandin E2(PGE2), as well as "analogs" or "derivatives" thereof. Prostaglandins generally refer to hormone-like molecules that are derived from fatty acids that contain 20 carbon atoms, which include a 5-carbon ring, as described herein and known in the art.
Illustrative examples of "analogs" or "derivatives" of PGE2 include, but are not limited to, 16,16-dimethyl PGE2, 16-16 dimethyl PGE2 p-(p-acetamidobenzamido) phenyl ester, 11-deoxy- 16,16-dimethyl PGE2, 9-deoxy-9-methylene-16,16-dimethyl PGE2, 9-deoxy-9-methylene PGE2, 9-keto Fluprostenol, 5-trans PGE2, 17-phenyl-omega-trinor PGE2, PGE2 serinol amide, PGE2 methyl ester, 16-phenyl tetranor PGE2, 15(S)-15-methyl PGE2, 15(R)-15-methyl PGE2, 8-iso-15-keto PGE2, 8-iso PGE2 isopropyl ester, 20-hydroxy PGE 2 , 11-deoxy PGE 1 , nocloprost, sulprostone, butaprost, 15-keto PGE 2 , and 19 (R) hydroxy PGE 2 .
Also included are prostaglandin analogues or derivatives which have a structure similar to PGE2 which are substituted by halogen at position 9 (see, for example, document under No. WO 2001/12596, incorporated herein by reference , in its entirety), as well as 2-decarboxy-2-phosphinic prostaglandin derivatives such as those described in publication no. U.S. 2006/0247214, incorporated herein by reference in its entirety).
[00207] In some embodiments, the compound is a binder with a base other than PGE2. In certain embodiments, the non-PGE2 based ligand is selected from the group consisting of an EP1 agonist, an EP2 agonist, an EP3 agonist, and an EP4 agonist.
In particular embodiments, the EP prostaglandin receptor is selected from EP1, EP2, EP3 and EP4.
Illustrative examples of EP1 agonists based other than PGE2 include, but are not limited to, ONO-DI-004 and ONO-8713. Illustrative examples of EP2 agonists based other than PGE2 include, but are not limited to, CAY10399, ONO_8815Ly, ONO-AE1-259, and CP-533,536. Additional examples of EP2 agonists based other than PGE2 include the carbazoles and fluorenes disclosed in the document under no. WO 2007/071456 , incorporated herein by reference for its description of such agents. Illustrative examples of EP3 agonist based other than PGE2 include, but are not limited to, AE5-599, MB28767, GR 63799X, ONO-NT012 and ONO-AE-248. Illustrative examples of EP4 agonist based other than PGE2 include, but are not limited to, ONO-4819, APS-999 Na, AH23848, and ONO-AE 1-329. Additional examples of EP4 agonists based other than PGE2 can be found in the document under no. WO/2000/038663; patent no. U.S. 6,747,037; and patent no. U.S. 6,610,719, each of which is incorporated herein by reference, for their description of such agonists.
In one embodiment, the compound that stimulates the prostaglandin EP PE receptor signaling pathway is a Wnt agonist. Illustrative examples of Wnt agonists include, but are not limited to, Wnt polypeptides and glycogen synthase kinase 3 (GSK3) inhibitors. Illustrative examples of wnt polypeptides suitable for use as compounds that stimulate the EP prostaglandin receptor signaling pathway include, but are not limited to, Wnt1, Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14, Wnt15 or Wnt15a and biologically active fragments thereof.
GSK3 inhibitors suitable for use as compounds that stimulate the EP prostaglandin receptor signaling pathway bind to and decrease the activity of GSK3α or GSK3β. Illustrative examples of GSK3 inhibitors include, but are not limited to, BIO (6-bromoindirubin-3'-oxime), LiCl or other GSK-3 inhibitors as exemplified in patent nos. U.S. 6,057,117 and 6.608,063; and ordering us. U.S. 2004/0092535 and 2004/0209878; selective and competitive GSK-3 inhibitors with ATP CHIR-911 and CHlR-837 (also referred to as CT-99021 and CT-98023, respectively). Chiron Corporation (Emeryville, CA).
[00212] In another embodiment, the compound that stimulates the EP prostaglandin receptor signaling pathway enhances signaling through the cAMP/P13K/AKT second messenger pathway and is selected from the group consisting of dibutyryl cAMP (DBcAMP) , phorbol ester, forskolin, sclareline, 8-bromo-cAMP, cholera toxin (CTx), aminophylline, 2,4 dinitrophenol (DNP), norepinephrine, epinephrine, isoproterenol, isobutylmethylxanthine (IBMX), caffeine, theophylline (dimethylxanthine), dopamine , rolipram, iloprost, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) and derivatives of these agents.
In yet another modality, the compound that stimulates the EP prostaglandin receptor signaling pathway increases signaling through the Ca2+ second messenger pathway and is selected from the group consisting of Bapta-AM, Fendiline, Nicardipine and derivatives of these compounds.
[00214] In another embodiment, the compound that stimulates the EP prostaglandin receptor signaling pathway increases signaling through the NO/Angiotensin signaling pathway and is selected from the group consisting of L-Arg, sodium nitroprusside, Sodium vanadate, Bradykinin, and derivatives thereof.
[00215] In one embodiment, the present invention provides a method of improving transduction efficiency comprising culturing a population of cells with a retrovirus and one or more compounds that enhance EP prostaglandin receptor signaling selected from the group consisting of in: a prostaglandin, PGE2; PGD2; PGI2; Linoleic acid; 13(s)-HODE; LY171883; Mead acid; Eicosatrienoic acid; Epoxy eicosatrienoic acid; ONO-259; Cay1039; a PGE2 receptor agonist; 16,16-dimethyl PGE2; 19(R)-hydroxy PGE2; 16,16-dimethyl PGE2 p-(p-acetamidobenzamido)phenyl ester; 11-deoxy-16,16-dimethyl PGE2; 9-deoxy-9-methylene-16,16-dimethyl PGE2; 9-deoxy-9-methylene PGE2; Butaprost; Sulprostone; PGE2 serinol amide; PGE2 methyl ester; 16-phenyl tetranor PGE2; 15(S)-15-methyl PGE2; 15(R)-15-methyl PGE2; BIO; 8-bromo-cAMP; Forskolin; Bata-AM; Fendyline; Nicardipine; Nifedipine; Pimozide; Strophantidine; Lanatoside; L-Arg; Sodium nitroprusside; Sodium vanadate; Bradykinin; Mebeverine; Flurandrenolid; Atenolol; Pindolol; Gaboxadol; kynurenic acid; Hydralazine; Thiabendazole; Bicuclin; Vesamicol; Peruvoside; Imipramine; Chlorpropamide; 1,5- Pentamethylene tetrazole; 4-Aminopyridine; Diazoxide; Benfotiamine; 12-Methoxydodecenoic acid; N-Formyl-Met-Leu-Phe; Galamine; IAA 94; and Chlorotrianisene.
[00216] In a particular embodiment, the present invention provides a method of improving transduction efficiency comprising culturing a population of cells with a retrovirus and one or more compounds that are ligands for an EP prostaglandin receptor selected from the group that consists of: 16,16-dimethyl PGE2, 16-16 dimethyl PGE2 p-(p-acetamidobenzamido) phenyl ester, 11-deoxy-16,16-dimethyl PGE2, 9-deoxy-9-methylene-16,16-dimethyl PGE2 , 9-deoxy-9-methylene PGE2, 9-keto Fluprostenol, 5-trans PGE2, 17-phenyl-omega-trinor PGE2, PGE2 serinol amide, PGE2 methyl ester, 16-phenyl tetranor PGE2, 15(S)-15- methyl PGE2, 15(R)-15-methyl PGE2, 8-iso-15-keto PGE2, 8-iso PGE2 isopropyl ester, 20-hydroxy PGE2, 11-deoxy PGE1 , nocloprost, sulprostone, butaprost, 15-keto PGE2, and 19 (R) hydroxy PGE2 .
[00217] The present invention also considers that cell transduction efficiency can be increased by culturing cells in the presence of a retrovirus, a compound that stimulates an EP prostaglandin receptor pathway, e.g., PGE2, and one or more histone deacetylase (HDAC) inhibitors.
[00218] Illustrative examples of HDAC inhibitors suitable for use in the compositions and methods of the present invention include, but are not limited to: HDAC inhibitors include, but are not limited to, TSA (Tricostatin A) (see, for example, , Adcock, (2007) British Journal of Pharmacology 150:829-831), VPA (valproic acid) (see, for example, Munster, et al, (2007) Journal of Clinical Oncology 25:18S:1065), sodium butyrate (NaBu) (see, for example, Han, et al., (2007) Immunology Letters 108: 143-150), SAHA (suberoylanilide hydroxamic acid or vorinostat) (see, for example, Kelly, et al., (2005) Nature Clinical Practice Oncology 2: 150-157), sodium phenylbutyrate (see, for example, Gore, et al., (2006) Cancer Research 66:6361-6369), depsipeptide (FR901228, FK228) (see, for example, Zhu, et al., (2003) Current Medicinal Chemistry 3(3):187-199), trapoxin (TPX) (see, for example, Furumai, et al., (2001) PNAS 98(1):87-92 ), peptide containing cyclic hydroxamic acid 1 (C HAP1) (see, Furumai supra), MS-275 (see, e.g., Carninci, et al., WO2008/126932, incorporated herein by reference)), LBH589 (see, e.g., Goh, et al., WO2008/108741 incorporated herein by reference) and PXD-101 (see, Goh, supra).
[00219] The present invention considers that cells that can be cultured in the presence of a retrovirus can be exposed to (contacted with) a compound that stimulates the EP prostaglandin receptor signaling pathway and/or an HDAC inhibitor by a duration of about 10 minutes to about 72 hours, about 30 minutes to about 72 hours, about 30 minutes to about 48 hours, about 30 minutes to about 24 hours, about 30 minutes to about 12 hours, about 30 minutes to about 8 hours, about 30 minutes to about 6 hours, about 30 minutes to about 4 hours, about 30 minutes to about 2 hours, about 1 hour to about 2 hours, or any intermediate time period.
[00220] In one embodiment, cells cultured with a retrovirus are exposed to (contacted with) a compound that stimulates the EP prostaglandin receptor signaling pathway and/or an HDAC inhibitor for about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 48 hours, or about 72 hours, or any length of time in between.
The present invention contemplates that cells can be cultured with one or more compounds that stimulate the EP prostaglandin receptor signaling pathway and/or one or more HDAC inhibitors prior to culture with a retrovirus, during culture with a retrovirus, or after culturing with a retrovirus, or any combination thereof for any of the aforementioned time periods set forth herein.
The present invention further contemplates that cells can be cultured with one or more compounds that stimulate the EP prostaglandin receptor signaling pathway and a retrovirus prior to culture with one or more HDAC inhibitors, during culture with one or more plus HDAC inhibitors or after culturing with one or more HDAC inhibitors, or any combination thereof, for any of the aforementioned time periods set forth herein.
[00223] The present invention also contemplates that cells can be cultured with a retrovirus prior to culture with one or more compounds that stimulate the EP prostaglandin receptor signaling pathway and/or one or more HDAC inhibitors, during culture with one or more compounds that stimulate the EP prostaglandin receptor signaling pathway and/or one or more HDAC inhibitors, or after culturing with one or more compounds that stimulate the EP prostaglandin receptor signaling pathway and/or one or plus HDAC inhibitors, or any combination thereof for any of the aforementioned time periods, set forth herein.
[00224] Additionally, one skilled in the art would note that the present inventive methods for enhancing transduction include culturing cells with retroviruses, one or more compounds that stimulate the EP prostaglandin receptor signaling pathway, and/or one or more HDAC inhibitors , during the first 6 hours of transduction, the first 12 hours of transduction, the first 24 hours of transduction, the first 48 hours of transduction or the first 72 hours of transduction, or any intermediate duration of transduction.
[00225] Furthermore, the present invention contemplates that cells can be transduced 1, 2, 3 or more times in the presence of a retrovirus and one or more compounds that stimulate the EP prostaglandin receptor signaling pathway and/or one or more HDAC inhibitors. In another embodiment, the present invention contemplates that cells can be transduced 1, 2, 3 or more times in the presence of a retrovirus and exposed to (contacted with) one or more compounds that stimulate the prostaglandin receptor signaling pathway EP and/or one or more HDAC inhibitors only once or twice.
[00226] In a particular embodiment, the invention contemplates that cells can be cultured in the retrovirus, one or more compounds that stimulate the EP prostaglandin receptor signaling pathway and/or one or more HDAC inhibitors, in which the cells are exposed to or brought into contact with those mentioned above during the same or different period of time as set forth elsewhere in this document.
[00227] The present invention also contemplates that the compositions and methods of the invention can increase the transduction of virtually any cell type to at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98 %, at least about 99% or at least about 100%.
[00228] In particular modalities, the increase in transduction efficiency represents at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times or at least 100 times, or more times the enrichment of cells transduced compared to cells transduced with vector alone.
[00229] Before, during and/or after transduction, cells can be cultured in media suitable for the maintenance, growth or proliferation of cells. Suitable culture conditions and media are well known in the art. Such media include, but are not limited to, Dulbecco ® Modified Eagle Medium (DMEM), DMEM F12 ® Medium, Eagle Minimal Essential ® Medium, F-12K ® Medium, Iscove ® Modified Dulbecco Medium, RPMI-1640 ® and serum-free medium for the culture and expansion of SFEM® hematopoietic cells. Many media are also available as low-glucose formulations, with or without sodium pyruvate.
[00230] Additional supplements can also be used to advantage to supply cells with the residual elements necessary for optimal growth and expansion. Such supplements include insulin, transferrin, sodium selenium and combinations thereof. These components may be included in a saline solution, such as, but not limited to, Hanks ® Balanced Salt Solution (HBSS), Earle ® Saline Solution, Antioxidant Supplements, MCDB-201® Supplements, Phosphate Buffered Saline ( PBS), ascorbic acid and ascorbic acid-2-phosphate, as well as additional amino acids. Many cell culture media already contain amino acids, however some require supplementation before culturing cells. Such amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L- histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. It is convenient for one skilled in the art to determine the proper concentrations of these supplements.
Hormones can also be advantageously used in the cell cultures of the present invention and include, but are not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, β-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin /human growth hormone (HGH), thyrotropin, thyroxine and L-thyronine.
[00232] Lipid and lipid vehicles can also be used to supplement cell culture media, depending on the cell type and fate of the differentiated cell. Such lipids and vehicles may include, but are not limited to, cyclodextrin (α, β, y), cholesterol, albumin-conjugated linoleic acid, linoleic acid and albumin-conjugated oleic acid, unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugated to albumin and unconjugated oleic acid and conjugated to albumin, among others.
Cells can also be cultured in serum-free or low-serum culture medium. Serum-free medium used to culture cells is described, for example, in patent no. U.S. 7,015,037. Many cells have been grown in serum-free or low-serum medium.
[00234] After transduction, transduced cells can be cultured under conditions suitable for their maintenance, growth or proliferation. In particular embodiments, transduced cells are cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days prior to transplantation.
[00235] Before, during and/or after transduction, cells can be cultured under conditions that promote the expansion of stem cells or progenitor cells. Any method known in the art can be used. In certain embodiments, before, during or after transduction, cells are cultured in the presence of one or more growth factors that promote the expansion of stem cells or progenitor cells. Examples of growth factors that promote the expansion of stem cells or progenitor cells include, but are not limited to, fetal liver tyrosine kinase ligand (Flt3), stem cell factor, and interleukins 6 and 11, which have demonstrated to promote self-renewal of murine hematopoietic stem cells. Others include Sonic hedgehog, which induces the proliferation of primitive hematopoietic progenitors through activation of bone morphogenetic protein 4, Wnt3a, which stimulates self-renewal of HSCs, brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF). ), fibroblast growth factor (FGF), ciliary neurotrophic factor (CNF), transforming growth factor β (TGF-β), a fibroblast growth factor (FGF, eg, basic FGF, acidic FGF, FGF- 17, FGF-4, FGF-5, FGF-6, FGF-8b, FGF-8c, FGF-9), granulocyte colony stimulating factor (GCSF), a platelet-derived growth factor (PDGF, for example , PDGFAA, PDGFAB, PDGFBB), granulocyte macrophage colony stimulating factor (GMCSF), stem cell factor (SCF), stromal cell-derived factor (SCDF), insulin-like growth factor (IGF), thrombopoietin ( TPO) or interleukin-3 (IL-3). In particular embodiments, before, during or after transduction, cells are cultured in the presence of one or more growth factors that promote the expansion of stem cells or progenitor cells.
[00236] Although the description and examples provided in this document focus on the transduction and selection of multipotent cells, which include hematopoietic stem cells, in particular, the methods and compositions of the present invention can also be used to transduce and select other types cells, which include other types of pluripotent or multipotent stem cells and fragile cells previously not sensitive to selection of transduced cells for therapeutic uses.
[00237] The cell used in accordance with the methods of the present invention may be obtained from any animal, preferably a mammal, for example a human or non-human primate, and more preferably a human, and it can be transplanted into any animal, preferably a mammal, and most preferably a human.
Cells suitable for transduction and administration in the gene therapy methods of the invention include, but are not limited to, stem cells, progenitor cells and differentiated cells.
[00239] Illustrative examples of stem cells suitable for transduction with the compositions and methods of the present invention include, but are not limited to, embryonic stem cells, induced pluripotent stem cells, mesodermal stem cells, endodermal stem cells and ectodermal stem cells.
[00240] In particular embodiments, the population or source of cells transduced using the compositions and methods contemplated herein comprises progenitor cells and/or mesenchymal stem, progenitor cells and/or mesodermal stem, progenitor cells and/or endodermal stem or ectodermal stem and/or progenitor cells. In certain embodiments, the population or source of cells used in the methods contemplated herein comprises bone marrow stem cells, stem cells and/or umbilical cord blood stem, stem cells and/or bone stem cells, stem cells and/or or muscle stem, stem cells and/or hematopoietic stem, stem cells and/or fat stem, stem cells and/or cartilage stem, stem cells and/or neural stem, stem cells and/or skin stem, stem cells and/or liver stem, pancreatic stem and/or stem cells, kidney stem and/or stem cells, gastric stem and/or stem cells, and intestinal stem and/or stem cells.
[00241] In certain embodiments, the population or source of cells transduced using the composition and methods of the present invention include, but are not limited to, osteoblasts, chondrocytes, adipocytes, skeletal muscle, cardiac muscle, neurons, glial cells (astrocytes , oligodendrocytes, Schwann cells), retinal cells (stem cells, cone cells), corneal cells, skin cells, monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells, T cells, B cells, NK cells, gastric cells, intestinal cells, smooth muscle cells, vascular cells, bladder cells, pancreatic islet cells (pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells), hepatocytes, kidney cells, adrenal cells and lung cells.
[00242] In several modalities, the use of stem cells is preferred due to the fact that they have the ability to differentiate into the appropriate cell types when administered to a particular biological niche, in vivo.
In preferred embodiments, the compositions and methods of the present invention are used to enhance transduction of hematopoietic stem or progenitor cells.
[00244] The present invention also contemplates the isolation and transduction of a population of cells. For use herein, the term "population of cells" refers to a plurality of cells that can be composed of any number and/or combination of homogeneous or heterogeneous cell types, as described elsewhere herein. For example, for the transduction of hematopoietic stem or progenitor cells, a population of cells can be isolated or obtained from umbilical cord blood, placental blood, bone marrow, or peripheral blood. A cell population may comprise about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of the target cell type to be transduced. In certain embodiments, hematopoietic stem or progenitor cells can be isolated or purified from a heterogeneous cell population using methods known in the art. In particular embodiments, hematopoietic stem or progenitor cells are purified after transduction of a population of cells, and in other embodiments, hematopoietic stem or progenitor cells are isolated prior to transduction.
The cells of the invention can also be cryopreserved before transduction or after transduction using methods known in the art. Once established in culture, cells can be used fresh or frozen and stored as frozen stocks, using, for example, DMEM with 40% FCS and 10% DMSO. Other methods for preparing frozen stocks for cultured cells are also available for those skilled in the art.
[00246] In particular embodiments, a population of cells comprising progenitor or stem cells is contacted with a retrovirus, eg, lentiviruses, and one or more compounds that enhance prostaglandin signaling, eg, a receptor ligand for prostaglandin EP, such as PGE2 or an analogue or derivative thereof. In certain embodiments, the cell population is further contacted with one or more HDAC inhibitors. In several modalities, the cell population is brought into contact ex vivo or in vivo.
[00247] In certain preferred embodiments, the progenitor or stem cells consist of hematopoietic stem or progenitor cells. E. CELL CULTURE COMPOSITIONS
The present invention further contemplates cell-based compositions comprising a cell culture in a culture medium that comprises a retrovirus and one or more compounds that enhance prostaglandin signaling. As discussed throughout this document, in particular embodiments, the present compositions and methods are useful for ex vivo and in vivo cell-based gene therapies. In some embodiments, the cell culture medium is a pharmaceutically acceptable cell culture medium.
[00249] A therapeutic culture, cell culture, culture system or cell culture compositions comprising a cell-based composition of the present invention may be administered separately via enteral or parenteral administration methods or in combination with other compounds suitable for effecting the desired treatment goals, for example, one or more growth factors.
[00250] In an illustrative embodiment, a therapeutic culture, cell culture, culture system or cell culture composition comprising a transduced cell of the present invention is systematically administered by means of direct injection into a tissue. F. COMPOSITIONS AND FORMULATIONS
[00251] The formulations and compositions of the invention may comprise a combination of any number of transduced or non-transduced cells or a combination thereof, viral vectors, polypeptides, polynucleotides and one or more compounds, e.g., compounds that enhance prostaglandin signaling and/or HDAC inhibitors, as described herein, formulated in pharmaceutically acceptable or physiologically acceptable solutions (e.g., culture medium) for administration to a cell, tissue, organ or animal, alone, or in combination with one or plus other therapy modalities.
The particular ex vivo and in vitro formulations and compositions of the invention may comprise a combination of transduced or non-transduced cells or a combination thereof, viral vectors, and one or more compounds, e.g., compounds that enhance prostaglandin signaling and/or HDAC inhibitors, as described herein, formulated in pharmaceutically acceptable or physiologically acceptable solutions (e.g., culture medium) for administration to a cell, tissue, organ or animal, alone, or in combination with one or plus other therapy modalities.
[00253] Particular in vitro formulations and compositions of the invention may comprise a combination of viral vectors, and one or more compounds, e.g., compounds that enhance prostaglandin signaling and/or HDAC inhibitors, as described herein, formulated in pharmaceutically acceptable or physiologically acceptable solutions (eg, culture medium) for the administration and transduction of a cell or tissue in an animal, alone, or in combination with one or more other modalities of therapy.
In certain embodiments, the present invention provides compositions comprising a therapeutically effective amount of transduced cells, as described herein, formulated together with one or more pharmaceutically acceptable diluents and/or carriers (additives) (e.g., medium of pharmaceutically acceptable cell culture).
In certain other embodiments, the present invention provides compositions comprising a retroviral vector and one or more compounds that enhance EP prostaglandin receptor signaling, as described herein, formulated together with one or more diluents and/or pharmaceutically acceptable carriers (additives) (e.g., pharmaceutically acceptable cell culture medium).
In particular embodiments, the present invention provides compositions comprising a population of cells comprising progenitor or stem cells, a retroviral vector and one or more compounds that enhance EP prostaglandin receptor signaling, as described herein, formulated together with one or more pharmaceutically acceptable diluents and/or carriers (additives) (e.g., pharmaceutically acceptable cell culture medium). In a related embodiment, the cell population comprises stem cells and hematopoietic progenitors.
[00257] The present invention further includes pharmaceutical compositions comprising transduced cells produced according to the methods described herein and a pharmaceutically acceptable carrier. In other embodiments, the present invention provides pharmaceutical compositions comprising a retroviral vector and one or more compounds, e.g., compounds that enhance prostaglandin signaling and/or HDAC inhibitors, as described herein.
[00258] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce a similar allergic or unpleasant reaction when administered to a human being. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, as liquid suspensions or solutions; solid forms suitable for solution or suspension in liquid prior to injection may also be prepared. The preparation can also be emulsified.
[00259] For use in the present invention, "vehicle" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, vehicle solutions, suspensions, colloids and the like. The use of such media and agents for active pharmaceutical substances is well known in the art. Except where any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
[00260] For use in the present invention, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible, which include pharmaceutically acceptable cell culture medium. In one embodiment, a composition comprising a vehicle is suitable for parenteral administration, for example, intravascular (intravenous or intra-arterial), intraperitoneal or intramuscular administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except where any conventional media or agent is incompatible with the transduced cells, their use in the pharmaceutical compositions of the invention is contemplated.
The compositions of the invention may comprise one or more polypeptides, polynucleotides, vectors comprising the same, compounds that enhance EP prostaglandin receptor signaling, HDAC inhibitors, and transduced cells, etc., as described herein, formulated in pharmaceutically acceptable or physiologically acceptable solutions for administration to a cell or an animal, alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions of the invention can be administered in combination with other agents as well, such as, for example, cytokines, growth factors, hormones, small molecules or various pharmaceutically active agents. There is virtually no limit to other components that can also be included in the compositions, as long as the additional agents do not adversely affect the composition's ability to deliver the intended gene therapy.
[00262] In the pharmaceutical compositions of the invention, the formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of treatment and dosage regimens suitable for the use of the particular compositions described herein in a variety of treatment regimens, which include, for example, oral, parenteral, intravenous, intranasal and intramuscular administration and formulation.
In certain circumstances, it will be desirable to release the compositions disclosed herein parenterally, intravenously, intramuscularly or even intraperitoneally, as described, for example, in patent no. U.S. 5,543,158; patent no. U.S. 5,641,515 and patent no. U.S. 5,399,363 (each specifically incorporated herein by reference in their entirety). Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under usual conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (US Patent No. 5,466,468, specifically incorporated herein by reference in its entirety ). In all cases, the form should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The vehicle can be a solvent or dispersion medium that contains, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity can be maintained, for example, through the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferred to include isotonic agents, for example sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about through the use in the compositions of agents which delay absorption, for example, aluminum monostearate and gelatin.
[00265] For parenteral administration in an aqueous solution, for example, the solution should be adequately buffered if necessary and the liquid diluent first made isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those skilled in the art in light of the present description. For example, a dosage can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypodermoclysis fluid or injected into the proposed infusion site (see, for example, Remington: The Science and Practice of Pharmacy, 20th edition. Baltimore, MD: Lippincott Williams & Wilkins, 2005). Some variation in dosage will necessarily occur depending on the condition of the individual being treated. The individual responsible for administration will, in any event, determine the appropriate dose for the particular individual. In addition, for human administration, preparations should meet the standards of sterility, pyrogenicity and general safety and purity as required by FDA Office of Biologicals Office standards.
[00266] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients mentioned above as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those mentioned above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques which produce a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution the same.
[00267] The compositions presented herein may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric , mandelic and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Under formulation, the solutions will be administered in a manner compatible with the dosage formulation and in an amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like.
[00268] In certain embodiments, the compositions can be released through intranasal sprays, inhalation and/or other aerosol delivery vehicles. Methods for delivering gene, polynucleotide and peptide compositions directly to the lungs through nasal aerosol sprays have been described, for example, in patent no. U.S. 5,756,353 and patent no. U.S. 5,804,212 (each specifically incorporated herein by reference in its entirety). Similarly, drug delivery using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (US Patent No. 5,725,871, specifically incorporated herein by reference in its entirety) also are well known in the pharmaceutical art. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is described in patent no. U.S. 5,780,045 (specifically incorporated herein by reference in its entirety).
[00269] In certain modalities, release may occur through the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, optionally mixing with CPP polypeptides, and the like, for the introduction of the compositions of the present invention into host cells appropriate. In particular, the compositions of the present invention can be formulated for release encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticle, or the like. The formulation and use of such delivery vehicles can be carried out using conventional and known techniques. The formulations and compositions of the invention may comprise one or more repressors and/or activators comprising a combination of any number of polypeptides, polynucleotides and small molecules as described herein, formulated in pharmaceutically acceptable or physiologically acceptable solutions (e.g., medium of culture) for administration to a cell or an animal, alone, or in combination with one or more other therapy modalities. It will also be understood that, if desired, the compositions of the invention may be administered in combination with other agents as well, such as, for example, cells, other proteins or polypeptides, or various pharmaceutically active agents.
In certain embodiments, the present invention provides formulations or compositions suitable for the delivery of viral vector systems (i.e., viral-mediated transduction) that include, but are not limited to, retroviral vectors (e.g., lentivirals).
[00271] Exemplary formulations for ex vivo delivery may also include the use of various transfection agents known in the art, such as calcium phosphate, electroporation, heat shock, and various liposome formulations (ie, lipid-mediated transfection). Liposomes, as described in more detail below, are lipid bilayers that capture a fraction of aqueous fluid. DNA spontaneously associates with the outer surface of cationic liposomes (by virtue of their charge) and these liposomes will interact with the cell membrane.
[00272] In certain aspects, the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more polynucleotides or polypeptides, as described herein, formulated together with one or more pharmaceutically acceptable diluents and/or carriers (additives) acceptable (e.g., pharmaceutically acceptable cell culture medium).
Particular embodiments of the invention may comprise other formulations, such as those which are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy, 20th edition. Baltimore, MD: Lippincott Williams & Wilkins, 2005.
In certain embodiments, compositions of the present invention comprise an effective amount of a composition and optionally comprise one or more adjunctive therapies. In certain embodiments of the present invention, compositions which comprise a cell-based composition and which optionally comprise one or more adjunctive therapies may comprise sterile saline, Ringer's solution, Hanks' balanced saline solution (HBSS), or Isolyte S , pH 7.4, serum-free cell medium, or other pharmaceutically acceptable medium (eg, cell culture medium), as discussed elsewhere herein.
In particular embodiments, a composition comprises a population of cells that is treated (e.g., contacted) with one or more compounds that enhance EP prostaglandin receptor signaling and/or one or more HDAC inhibitors, each one independently at a final concentration of about 1 µM to about 100 µM. In certain embodiments, a population of cells is treated with one or more compounds that enhance EP prostaglandin receptor signaling and/or one or more HDAC inhibitors, each independently at a final concentration of about 1 x 10-14 M to about 1 x 10-3 M, about 1 x 10-13 M to about 1 x 10-4 M, about 1 x 10-12 M to about 1 x 10-5 M, about 1 x 10-11 M to about 1 x 104 M, about 1 x 10-11 M to about 1 x 10-5 M, about 1 x 10-10 M to about 1 x 10-4 M, about 1 x 10-10 M to about 1 x 10-5 M, about 1 x 10-9 M to about 1 x 10-4 M, about 1 x 10-9 M to about 1 x 10-5 M, about 1 x 10-8 M to about 1 x 10-4 M, about 1 x 10-7 M to about 1 x 10-4 M, about 1 x 10-6 M to about 1 x 10-4 M, or any intermediate ranges of final concentrations.
[00276] In another particular embodiment, a population of cells is contacted with one or more compounds that enhance EP prostaglandin receptor signaling and/or one or more HDAC inhibitors, each independently at a final concentration of about 1 x 10-14 M, about 1 x 10-13 M, about 1 x 10-12 M, about 1 x 10-10 M, about 1 x 10-9 M, about 1 x 10-8 M, about 1 x 10-7 M to about 1 x 10-6 M, about 1 x 10-5 M, about 1 x 10-4 M, about 1 x 10-3 M, or any concentration intermediate end. In compositions comprising one or more one or more compounds that enhance EP prostaglandin receptor signaling and/or one or more HDAC inhibitors, the compounds may be at different concentrations from one another or at the same concentration.
[00277] One of ordinary skill in the art would be able to use routine methods to determine the proper route of administration and the correct dosage of an effective amount of a composition comprising transduced cells and/or one or more compounds that enhance receptor signaling. prostaglandin EP and/or one or more HDAC inhibitors for methods of the present invention. It would also be well known to those skilled in the art to recognize that, in certain therapies, multiple administrations of pharmaceutical compositions of the invention will be required to effect the therapy.
[00278] For example, a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more times over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more.
In addition, multiple administrations of the same or different compositions of the present invention can be administered multiple times for extended periods of time, as noted above.
[00280] Additionally, administration of the transduced cells and/or one or more compounds that enhance prostaglandin EP signaling and/or one or more HDAC inhibitors can be by the same or different routes, as discussed elsewhere in this document. Administration of the transduced cells and/or one or more compounds that enhance prostaglandin EP signaling and/or one or more HDAC inhibitors can also be performed at different sites using the same or different route of administration. Additionally, administration of the transduced cells and/or one or more compounds that increase prostaglandin EP signaling and/or one or more HDAC inhibitors can be done at the same site by the same route, at the same time or at different times. G. GENE THERAPY METHODS
[00281] Transduced cells and corresponding retroviral vectors provide improved methods of gene therapy. For use in the present invention, the term "gene therapy" refers to introducing a gene into a cell's genome. In various embodiments, a viral vector of the invention comprises a hematopoietic expression control sequence that expresses a therapeutic transgene that encodes a polypeptide that provides curative, preventative, or ameliorative benefits to an individual diagnosed with or suspected of having a condition, disorder or disease monogenic or a disease, disorder or condition that is responsive to hematopoietic stem cell therapy.
[00282] In a preferred embodiment, the invention provides transduced cells with the potential to develop into brain microglial cells. In particular embodiments, hematopoietic stem cells are transduced with a vector of the invention and administered to an individual in need of therapy for an adrenoleukodystrophy or adrenomyeloneuropathy. Hematopoietic stem cells are the origin of brain microglial cells and, therefore, are preferred.
[00283] In particular embodiments, transduced hematopoietic stem or progenitor cells comprise viral vectors that have a hematopoietic expression control sequence that expresses a therapeutic transgene that encodes a polypeptide that provides curative, preventative, or ameliorative benefits to an individual diagnosed with or who is suspected of having a monogenic condition, disorder, or disease or a disease, disorder, or condition of the hematopoietic system.
A composition comprising a virus, e.g. lentivirus, and/or one or more compounds that enhance prostaglandin EP receptor signaling and/or one or more HDAC inhibitors can infect or transduce cells at increased efficiencies in vivo , ex vivo, or in vitro, compared to cells transduced with vector alone. In ex vivo and in vitro modalities, the transduced cells can then be administered to an individual in need of therapy. The present invention contemplates that the vector, viral particles and transduced cells of the invention are to be used to treat, prevent and/or ameliorate a monogenic condition, disorder or disease or a disease, disorder or condition of the hematopoietic system in an individual, for example, a hemoglobinopathy.
[00285] For use in the present invention, "hematopoiesis" refers to the formation and development of blood cells from progenitor cells, as well as the formation of progenitor cells from stem cells. Blood cells include, but are not limited to, erythrocytes or red blood cells (RBCs), reticulocytes, monocytes, neutrophils, megakaryocytes, eosinophils, basophils, B cells, macrophages, granulocytes, mast cells, thrombocytes, and leukocytes.
[00286] For use herein, the term "hemoglobinopathy" or "hemoglobinopathic condition" includes any disorder that involves the presence of an abnormal hemoglobin molecule in the blood. Examples of hemoglobinopathies include, but are not limited to, hemoglobin C disease, hemoglobin sickle cell disease (SCD), sickle cell anemia, and thalassemias. Also included are hemoglobinopathies in which a combination of abnormal hemoglobins is present in the blood (eg, sickle cell/Hb-C disease).
[00287] The term “sickle cell anemia” or “sickle cell disease” is defined herein to include any symptomatic anemic condition that results from sickling of red blood cells. Manifestations of sickle cell disease include: anemia; ache; and/or organ dysfunction such as renal failure, retinopathy, acute chest syndrome, ischemia, priapism and stroke. For use herein, the term "sickle cell disease" refers to a variety of clinical problems that accompany sickle cell anemia, especially in those individuals who are homozygous for sickle cell replacement in HbS. Among the constitutional manifestations mentioned herein through the use of the term sickle cell disease are growth and developmental delay, an increased tendency to develop serious infections, particularly due to pneumococcus, marked impairment of splenic function, prevention of effective clearance of circulating bacteria, with recurrent infarctions and eventual destruction of splenic tissue. Also included in the term “sickle cell disease” are acute episodes of musculoskeletal pain, which primarily affects the lumbar spine, abdomen and femoral axis, and which are similar in mechanism and severity to the curves. In adults, such attacks commonly manifest as mild or moderate attacks of short duration with intervals of a few weeks or months interspersed with agonizing attacks lasting 5 to 7 days that affect an average of about once a year. Among the events known to trigger such crises are acidosis, hypoxia, and dehydration, all of which potentiate the intracellular polymerization of HbS (JH Jandl, Blood: Textbook of Hematology, 2nd edition, Little, Brown and Company, Boston, 1996, pages 544- 545). For use herein, the term "thalassemia" encompasses hereditary anemias that occur due to mutations that affect hemoglobin synthesis. Thus, the term includes any symptomatic anemia that results from thalassemia conditions, such as β or severe thalassemia, thalassemia major, thalassemia intermedia, α thalassemias, such as hemoglobin H disease.
[00288] For use herein, "thalassemia" refers to an inherited disorder characterized by defective hemoglobin production. Examples of thalassemias include α and β thalassemia. β-thalassemias are caused by a mutation in the beta globin chain and can occur in a greater or lesser form. In the major form of β-thalassemia, children are normal at birth but develop anemia during the first year of life. The minor form of β-thalassemia produces small red blood cells. Thalassemia minor occurs if the individual receives the defective gene from only one relative. People with this form of the disorder carry the disease and usually have no symptoms.
[00289] α-thalassemia typically results from deletions involving the HBA1 and HBA2 genes. Both of these genes encode α-globin, which is a component (subunit) of hemoglobin. There are two copies of the HBA1 gene and two copies of the HBA2 gene in each cell genome. As a consequence of this, there are four alleles that produce α-globin. Different types of α-thalassemia result from the loss of some or all of these alleles. Hb Bart syndrome, the most severe form of α-thalassemia, results from the loss of all four α-globin alleles. HbH disease is caused by a loss of three out of four α-globin alleles. In these two conditions, a shortage of α-globin prevents cells from making normal hemoglobin. Instead, the cells produce abnormal forms of hemoglobin called hemoglobin Bart (Hb Bart) or hemoglobin H (HbH). These abnormal hemoglobin molecules cannot effectively carry oxygen to the body's tissues. Substituting Hb Bart or HbH for normal hemoglobin causes anemia and other serious health problems associated with α-thalassemia.
[00290] In a preferred embodiment, the gene therapy methods of the invention are used to treat, prevent or ameliorate a hemoglobinopathy that is selected from the group consisting of: hemoglobin C disease, hemoglobin sickle cell disease (SCD) ), sickle cell anemia, hereditary anemia, thalassemia, β-thalassemia, thalassemia major, thalassemia intermedia, α-thalassemia and hemoglobin H disease.
[00291] In several modalities, retroviral vectors are administered through direct injection to a cell, tissue or organ of an individual in need of gene therapy, in vivo. In several other embodiments, cells are transduced in vitro or ex vivo with vectors of the invention and optionally expanded ex vivo. The transduced cells are then administered to an individual in need of gene therapy.
Cells suitable for transduction and administration in the gene therapy methods of the invention include, but are not limited to, stem cells, progenitor cells and differentiated cells as described elsewhere herein. In certain modalities, transduced cells are embryonic stem cells, induced pluripotent stem cells, bone marrow stem cells, umbilical cord stem cells, placental stem cells, mesenchymal stem cells, neural stem cells, cells - liver stem cells, pancreatic stem cells, cardiac stem cells, kidney stem cells, hematopoietic stem cells, as described elsewhere herein.
In preferred embodiments, the transduced cells are progenitor and/or hematopoietic stem cells isolated from bone marrow, umbilical cord blood, or peripheral circulation. In particular preferred embodiments, the transduced cells are hematopoietic stem cells isolated from bone marrow, umbilical cord blood or peripheral circulation.
[00294] HSCs can be identified according to certain phenotypic or genotypic markers. For example, HSCs can be identified by their small size, lack of lineage markers (lin), low labeling (lateral population) with vital dyes such as rhodamine 123 (rhodamineDULL, also called rholo) or Hoechst 33342, and presence of several antigenic markers on its surface, many of which belong to the differentiation series cluster (eg, CD34, CD38, CD90, CD133, CD105, CD45, Ter119 and c-kit, the receptor for stem cell factor) . HSCs are mostly negative for the markers that are typically used to detect lineage compromise and thus are often referred to as Lin(-) cells.
[00295] In one embodiment, human HSCs can be characterized as CD34+, CD59+, Thy1/CD90+, CD38lo/-, C-kit/CD117+ and Lin(-). However, not all stem cells are covered by these combinations, as certain HSCs are CD34-/CD38-. Furthermore, some studies suggest that early stem cells may lack c-kit on the cell surface. For human HSCs, CD133 may represent an early marker, as both CD34+ and CD34-HSCs have been shown to be CD133+. It is known in the art that CD34+ and Lin(-) cells also include hematopoietic progenitor cells.
[00296] In another modality, the hematopoietic hierarchy is determined by a SLAM code. The SLAM (Lymphocyte Signaling Activation Molecule) family is a group of >10 molecules whose genes are located predominantly in tandem at a single location on chromosome 1 (mouse), all belonging to a subset of the immunoglobulin gene superfamily, and it is believed to be originally in T cell stimulation. This family includes CD48, CD150, CD244, etc., CD150 being the founding element and thus also called slamF1, ie a member of the SLAM 1 family. of SLAM signature for the hematopoietic hierarchy consists of hematopoietic stem cells (HSC) - CD150+CD48-CD244-; multipotent progenitor cells (MPPs) - CD150-CD48-CD244+; lineage-restricted progenitor cells (LRPs) - CD150-CD48+CD244+; common myeloid progenitor (CMP) - lin-SCA-1-c-kit+CD34+CD16/32mid; granulocyte-macrophage progenitor (GMP) - lin-SCA-1-c-kit+CD34+CD16/32hi; and megakaryocyte-erythroid (MEP) progenitor - lin-SCA-1-c-kit+CD34-CD16/32low.
[00297] In mice, Irving Weissman's group at Stanford University was the first to isolate mouse hematopoietic stem cells in 1988 and was also the first to determine markers to distinguish the mouse hematopoietic hierarchy. Markers for the hematopoietic hierarchy consist of long-term hematopoietic stem cells (LT-HSC) - CD34-, SCA-1+ , Thy1.1+/lo, C-kit+, lin-, CD135-, Slamf1/CD150+; short-term hematopoietic stem cells (ST-HSC) - CD34+, SCA-1+ , Thy1.1+/lo, C-kit+, lin-, CD135-, Slamf1/CD150+, Mac-1 (CD11b)lo; early multipotent progenitors - (early MPP) - CD34+, SCA-1+ , Thy1.1-, C-kit+, lin-, CD135+, Slamf1/CD150-, Mac-1 (CD11b)lo, CD4lo; and late multipotent parents (late MPP) - CD34+, SCA-1+ , Thy1.1-, C-kit+, lin-, CD135high, Slamf1/CD150-, Mac-1 (CD11b)lo, CD4lo.
[00298] In one embodiment, the hematopoietic cells are CD105+ Sca1+ cells.
[00299] The cells of the invention can be autologous/autogenic ("auto") or non-autologous ("non-auto", for example, allogeneic, syngenic or xenogenic). "Autologous" for use in the present invention refers to cells from the same individual. "Allogeneic" for use in the present invention refers to cells of the same species that differ genetically from the cell being compared. "Syngenic" for use in the present invention refers to cells from a different individual that are genetically identical to the cell being compared. "Xenogenic" for use in the present invention refers to cells of a species other than the cell being compared. In preferred embodiments, the cells of the invention are allogeneic.
[00300] An "individual", for use in the present invention, includes any animal that exhibits a symptom of a monogenic disease, disorder or condition that can be treated with the gene therapy vectors, cell-based therapeutic products and methods disclosed elsewhere in this document. In preferred embodiments, an individual includes any animal that exhibits symptoms of a disease, disorder or condition of the hematopoietic system, e.g., a hemoglobinopathy, which can be treated with the gene therapy vectors, cell-based therapeutics and methods disclosed. elsewhere in this document. Suitable individuals (for example, patients) include laboratory animals (such as a mouse, rat, rabbit or guinea pig), farm animals and domestic animals or pets (such as a cat or dog). Non-human primates, and preferably human patients, are included. Typical individuals include animals that exhibit abnormal amounts (smaller or greater amounts than a "normal" or "healthy" individual) of one or more physiological activities that can be modulated by gene therapy.
[00301] For use in the present invention, "treatment" or "treating" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition that is treated. Treatment may optionally involve reducing or ameliorating symptoms of this disease or condition, or delaying the progress of the disease or condition. “Treatment” does not necessarily indicate the eradication or complete cure of the disease or condition or associated symptoms.
[00302] For use in the present invention, "prevent", and similar words such as "prevented", "preventing", etc., indicates an approach to prevent, inhibit or reduce the likelihood of the occurrence or recurrence of a disease or condition . It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of symptoms of a disease or condition. For use herein, "prevention", and similar words, also include reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to the onset or recurrence of the disease or condition.
[00303] For use herein, the term "amount" refers to "an effective amount" or "an effective amount" of a virus or transduced therapeutic cell to achieve a desired or beneficial therapeutic or prophylactic result, which includes clinical results .
A "prophylactically effective amount" refers to an amount of a transduced therapeutic virus or cell effective to achieve the desired prophylactic results. Typically, but not necessarily, since the prophylactic dose is used in individuals before or at an early stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.
[00305] A "therapeutically effective amount" of a transduced therapeutic virus or cell may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of the stem and progenitor cells to produce a desired response in the individual. A therapeutically effective amount is also one in which any toxic or harmful effects of the transduced therapeutic virus or cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" an individual (e.g., a patient).
[00306] Without sticking to any particular theory, an important advantage provided by the vectors, compositions and methods of the present invention is the high efficacy of gene therapy that can be achieved by administering cell populations that comprise high percentages of transduced cells compared to existing methods.
The transduced cells can be administered as part of a bone marrow or umbilical cord blood transplant in an individual who has or has not undergone bone marrow ablative therapy. In one embodiment, the transduced cells of the invention are administered in a bone marrow transplant to an individual who has undergone chemoablative or radioablative bone marrow therapy.
[00308] In one embodiment, a dose of transduced cells is delivered to an individual intravenously. In preferred embodiments, the transduced hematopoietic stem cells are administered intravenously to a subject.
[00309] In an illustrative modality, the effective amount of transduced cells provided to an individual is less than 1 x 1012 cells per 100 kg, less than 1 x 1011 cells per 100 kg, less than 1 x 1010 cells per 100 kg, less than 1 x 109 cells per 100 kg, less than 1 x 108 cells per 100 kg, less than 1 x 107 cells per 100 kg, less than 5 x 106 cells per 100 kg, less than 4 x 106 cells per 100 kg , less than 3 x 106 cells per 100 kg, less than 2 x 106 cells per 100 kg, less than 1 x 106 cells per 100 kg, less than 5 x 105 cells per 100 kg, less than 4 x 105 cells per 100 kg, less than 3 x 105 cells per 100 kg, less than 2 x 105 cells per 100 kg, less than 1 x 105 cells per 100 kg, less than 5 x 104 cells per 100 kg or less than 1 x 104cells per 100 kg of individual body weight.
[00310] In another illustrative embodiment, the effective amount of transduced cells provided to an individual is about 1 x 1012 cells per 100 kg, about 1 x 1011 cells per 100 kg, about 1 x 1010 cells per 100 kg, about 1 x 109 cells per 100 kg, about 1 x 108 cells per 100 kg, about 1 x 107 cells per 100 kg, about 5 x 106 cells per 100 kg, about 4 x 106 cells per 100 kg, about 3 x 106 cells per 100 kg, about 2 x 106 cells per 100 kg, about 1 x 106 cells per 100 kg, about 5 x 105 cells per 100 kg, about 4 x 105 cells per 100 kg, about 3 x 105 cells per 100 kg, about 2 x 105 cells per 100 kg, about 1 x 105 cells per 100 kg, about 5 x 104 cells per 100 kg or about 1 x 104 cells per 100 kg.
[00311] In another illustrative embodiment, the effective amount of transduced cells provided to an individual is from about 1 x 101 cells per 100 kg to about 1 x 1012 cells per 100 kg, from about 1 x 102 cells per 100 kg to about 1 x 1011 cells per 100 kg, from about 1 x 103 cells per 100 kg to about 1 x 1010 cells per 100 kg, from about 1 x 104 cells per 100 kg to about 1 x 109 cells per 100 kg, from about 1 x 105 cells per 100 kg to about 1 x 108 cells per 100 kg, from about 1 x 106 cells per 100 kg to about 1 x 107 cells per 100 kg or any intermediate ranges of cells per 100 kg.
[00312] In various embodiments, the methods of the invention provide more robust and safer gene therapy than existing methods and comprise administering a population or dose of cells comprising about 5% transduced cells, about 10% cells transduced, about 15% transduced cells, about 20% transduced cells, about 25% transduced cells, about 30% transduced cells, about 35% transduced cells, about 40% transduced cells, about 45% transduced cells, about 50% transduced cells, about 55% transduced cells, about 60% transduced cells, about 65% transduced cells, about 70% transduced cells, about 75% transduced cells, about 80% transduced cells, about 85% transduced cells, about 90% transduced cells, about 95% transduced cells, about 98% transduced cells or about 100% of tr cells ansduced, to an individual.
[00313] In various embodiments, the vectors, compositions and methods of the present invention offer improved methods of gene therapy using ex vivo gene therapy and autologous transplantation. In a preferred embodiment, the invention provides transduced cells, such as a stem cell, e.g., hematopoietic stem cell. In particular embodiments, hematopoietic stem cells are transduced with a vector of the invention and administered to an individual in need of therapy for a hemoglobinopathy.
In particular embodiments, hematopoietic stem cells are transduced with a vector of the invention and administered to an individual in need of therapy for an adrenoleukodystrophy or an adrenomyeloneuropathy.
[00315] In a preferred embodiment, the invention provides improved viral vector systems optimized to express high levels of one or more therapeutic proteins in erythroid cells or erythroid precursor cells. Retroviral vectors, which include lentiviral vectors, of the invention further comprise a polynucleotide of interest, which includes, for example, a globin gene or a gene encoding an antisickling protein. In one embodiment, the globin gene expressed in the retroviral vector of the invention is β-globin, δ-globin or Y-globin. In another embodiment, the human β-globin gene is the wild-type human β-globin gene or human βA-globin gene. In another embodiment, the human β-globin gene comprises one or more intron sequence deletions or is a mutant human β-globin gene that encodes at least one antisickling amino acid residue. Anti-sickling amino acids can be derived from human δ-globin or human Y-globin. In another embodiment, the mutant human β-globin gene encodes a threonine to glutamine mutation at codon 87 (βA-T87Q).
The retroviral vectors, which include lentiviral vectors, of the invention can be used in gene therapy, which include for the treatment of hemoglobinopathies. In particular embodiments, the invention provides methods for using the aforementioned vectors to achieve stable high levels of gene expression in erythroid cells, for example, in order to treat specific erythroid diseases. In a particular embodiment, gene therapy vectors are used to treat hemoglobinopathies, which include, for example, sickle cell disease (SCD). In another preferred embodiment, gene therapy vectors are used for the treatment of thalassemias, which include, but are not limited to, β-thalassemia.
[00317] In another preferred embodiment, hematopoietic stem cells are subjected to transduction with vectors of the invention that comprise an ABCD1 gene for the treatment of adrenoleukodystrophies and/or adrenomyeloneuropathies.
[00318] The present invention will now be described in greater detail by the following examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments presented herein; preferably, these embodiments are provided so that this description will be detailed and complete, and will fully convey the scope of the invention to those skilled in the art. EXAMPLES EXAMPLE 1 PRE-STIMULATION OF CELLS FOR TRANSDUCTION
[00319] A vial of CD34+ cells (AllCells) was thawed by incubating at 37°C for 1 to 2 minutes and the contents were transferred to 10 ml of stem cell growth medium (referred to later in this document as SCGM ) in a 15-mL conical tube. Cells were centrifuged for 5 minutes at 1500 RPM in a standard tabletop centrifuge, resuspended in 10 mL SCGM and counted in a hemacytometer. A volume correlated with an adequate number of cells was transferred to a fresh 15-mL conical tube and centrifuged again for 5 minutes at 1500 RPM. Cells were resuspended at the desired cell concentration in SCGM + 1x cytokines (100 ng/ml SCF, 100 ng/ml TPO, 100 ng/ml FltL and 30 ng/ml IL-3), and distributed in a sterile non-stick surface at 37°C in a standard humidified tissue culture incubator (5% CO2).
A screening for compounds that increase the viral transduction efficiency of CD34+ cells was conducted using varying concentrations of soluble compounds from a number of classes (Table 1). The screening results are shown in Figure 1. Table 1
Table 1 (Cont.)
EXAMPLE 2 TRANSDUCTION
Prestimulated cells (Example 1) were counted after 18 to 24 hours of culture. Cells were collected and centrifuged for 5 minutes at 1500 RPM. 1.2 x 106 prestimulated CD34+ cells were resuspended in 60 uL 10x cytokines (1000 ng/ml SCF, 1000 ng/ml TPO, 1000 ng/ml FltL and 300 ng/ml IL-3), 7 .8 uL of protamine sulfate, 111 uL of viral supernatant and 361.2 uL of SCGM. 90 µl of cell/virus suspension (about 200,000 cells) was added to each well of a standard non-stick 96-well plate. dmPGE2 was added during this viral transduction step at a final concentration of 100 uM, 50 uM, 25 uM, 12.5 uM, 1 uM or 0 uM. The viral stock had a titer of 2.7 x 108 TU/ml and the multiplicity of infection (MOI) was about 25.
Cells were incubated at 37°C in a standard humidified tissue culture incubator (5% CO2). EXAMPLE 3 STIMULATION OF CELL DMPGE2
10 mM aliquots of dmPGE2 in DMSO were prepared from 1 mg of previously processed dmPGE2 (Cayman Chemicals). Briefly, air was pipetted into the dmPGE2 vial until methyl acetate was evaporated. 263 uL of DMSO was added to the PGE2 remaining in the vial, and the 25 uL aliquots were added to 1.5 mL Eppendorf tubes and stored at -80°C. The 10x working stock solutions were prepared by serially diluting dmPGE2 in SCGM, and were then added to cells at appropriate working concentrations as per table 2. The cells were then incubated at 37°C in a standard humidified tissue culture incubator (5% CO2). Table 2: Serial Dilutions of dmPGE2 and Addition of dmPGE2 to Cells

EXAMPLE 4 VALIDATION TESTS Cell Preparation for Validation Tests
[00324] After 24 hours of culture with virus and dmPGE2, cells were washed before subsequent functional validation assays. Washing was performed by transferring the cells to a 96-well U-bottom plate and centrifuging for 5 minutes at 1500 RPM in a standard tabletop centrifuge. The medium was aspirated and cells were resuspended in 200 µl of SCGM. Cells were centrifuged again for 5 minutes at 1500 RPM and the medium was aspirated. Cells were resuspended again in 200 uL of SCGM, then centrifuged for 5 minutes at 1500 RPM, and again the medium was aspirated. Particular functional validation assays are described below. 7 days of liquid culture
Washed cells were resuspended in 200 ul SCGM + 1x cytokines (as described in Example 1) and transferred to a standard 12-well non-adherent tissue culture plate containing 800 ul SCGM + 1x additional cytokines. Cells were kept for an additional 6 days in a standard humidified tissue culture incubator (5% CO2) and then subjected to vector copy number analysis (Example 5) and FACS analysis. For FACS analysis, cells were analyzed for the presence of a virally encoded transgene, green fluorescent protein (GFP). The frequency of virally labeled cells within the cultured cell pool was quantified as the frequency of GFP+ cells within the population. The mean fluorescence intensity of labeled cells was quantified. The results for the 7-day liquid culture trial with varying concentrations of dmPGE2 are shown in Table 3. Evaluation of colony formation activity on Methylcellulose
The washed cells were resuspended in 200 uL of SCGM and then transferred to 3 ml aliquots of cytokine-supplemented methylcellulose (eg, Methocult M4434 Classic). 1.5 mL was then transferred to parallel 35-mm tissue culture dishes using a 16 gauge blunt needle. Dishes were kept in a standard humidified tissue culture incubator for 14 to 16 days and colonies were marked for size, morphology and cell composition. Individual colonies were then selected for subsequent vector copy number analysis (Example 5) or contents of an entire 35-mm dish were grouped and then subjected to vector copy number analysis (Example 5 ). Long-term culture initiation cells (LTC-IC)
[00327] Cells were resuspended in 200 uL of SCGM, counted and then transferred to stromal layer of MS-5 pre-distributed in plate at various dilutions (2000; 1000; 500; 250; 125; 62; 31; 16 cells per well in 200μL of StemSpan SFEM (StemCell Technologies, cat no. 09650), supplemented with Pen-Strep 100U/mL-100μg/mL) and 24 replicates per dilution. At weekly intervals, 100μL was replaced by 100μL of fresh medium. Within 5 weeks, cultures were collected. 100μL were discarded, cells were washed with the remaining 100μL and the well was rinsed with 50μL of fresh medium, and all contents were seeded in Methocult™ H4434; 150μL of cell suspension was homogenized with 600μL of Methocult H4434 and placed in a well of a 12-well plate for 14 days. The colonies were then counted. The number of wells containing at least one colony (>40 cells) and the total number of wells analyzed for each dilution were used to calculate the frequency of LTC-ICs and 95% confidence interval using the L-calc software (Stem Cell Technologies). 100 colonies from each treatment group were selected from 100 different wells and individually scored for the presence of the vector. 100 colonies from each treatment group were pooled, genomic DNA was extracted, and the mean vector copy number was assessed by qPCR (Example 5). Transplantation in NOD/SCID Gamma (NSG) mice
[00328] To determine whether dmPGE2 promotes long-term viral transduction of human hematopoietic stem cells with minimal residual toxicity, transduced cells were washed and resuspended in phosphate buffered saline (PBS) and transplanted into the tail vein of adult NSG mice irradiated. The mice were housed in a pathogen-free environment per standard IACUC animal care guidelines. At gradual time points, the human donor-derived contribution to peripheral blood was quantified by collecting from mice using standard protocols. Briefly, red blood cells were pelleted with 2% Dextran and then the supernatant was further cleaned by treatment with red blood cell lysis buffer. Mononuclear cells were then labeled with fluorophore-conjugated antibodies as described by Majeti, et al., Cell Stem-Cell 2007, and analyzed by flow cytometry on an LSR-II (Becton Dickinson). Integration Site Analysis
[00329] To determine whether dmPGE2 alters the lentiviral vector integration site preference, bone marrow samples from mice transplanted with stem cells and virally transduced human hematopoietic progenitors treated with dmPGE2 and treated with mock were subjected to Linear amplification-mediated PCR ( Cartier, (2009) Science 326(5954):818-23). In summary, 1 to 1000 ng of DNA served as a template for linear PCR using retroviral LTR-specific biotinylated primers. Linear PCR products were separated with paramagnetic beads. Additionally, second strand DNA synthesis, restriction digestion (Tsp509I, NlaIII or HpyCH4IV) and ligation of a linker cassette were performed in semi-solid phase, followed by two additional exponential PCR steps. The resulting LAM-PCR amplicons were further prepared for 454 pyrosequencing (GS Flx; Roche Diagnostics) by performing an additional exponential PCR to add the GS Flx specific amplification and sequencing primers A and B to both ends of the amplicons. LAM-PCR. Primer design was done as suggested by the manufacturer. A 6-base recognition sequence was incorporated into primer A to simultaneously analyze different samples in a single sequencing procedure. 40ng of purified LAM-PCR products were used. PCR conditions were as follows: initial denaturation for 120 s at 95 °C; 12 cycles at 95 °C for 45 s, 60 °C for 45 s and 72 °C for 60 s; final elongation 300 s at 72 °C. Amplicon sequences of LAM-PCR were prepared and aligned using BLAST. EXAMPLE 5 VECTOR COPY NUMBER ANALYSIS
[00330] In summary, total genomic DNA was isolated from cells using standard protocols (eg, using DNEasy columns available from Qiagen). Genomic DNA was subjected to real-time quantitative polymerase chain reaction (qRT-PCR) with TaqMan probes for viral LTR and human beta-actin. Ct values for viral signal and beta-actin signal were normalized to a standardized control, and the number of viral copies per beta actin copy was calculated. A linear relationship between vector copy number and mean fluorescence intensity (Example 4) was observed when a viral construct encoding GFP was used. Results for vector copy number (VCN) analysis with varying concentrations of dmPGE2 are shown in Tables 3A-C.
Tables 3A-C indicate the dose response of dmPGE2 in promoting viral transduction of CD34+ cells for three separate experiments. CD34+ cells were thawed and prestimulated with SCF, TPO, Flt L and IL3, then transduced (A) with GFP+ lentivirus at a multiplicity of infection of 25, (B) with GFP+ lentivirus at a multiplicity of infection of 5, or (C) transduced with an ALD expressing lentivirus (ABCD1) at a multiplicity of infection of 25. Cells were exposed to dmPGE2 during the viral transduction step (24 to 48 hours of culture). Cells were then washed and analyzed by flow cytometry and PCR after approximately 1 week in culture. The percentage of cells positive for GFP (A, B) or ALD (C) by FACS labeling is indicated, along with the mean fluorescence intensity (MFI) and vector copy number (VCN) (A, B). TABLE 3A: GFP MOI 25
TABLE 3B: GFP MOI 5

TABLE 3C: ABCD1 MOI 25
EXAMPLE 6 EVOLUTION AND DOSE RESPONSE OF DMPGE2 IN THE PROMOTION OF VIRAL TRANSDUCTION OF CD34+ CELLS
[00332] MCD-34+ cells were thawed and prestimulated with SCF, TPO, FltL and IL3, then transduced with GFP+ lentivirus at a multiplicity of infection of 25. Cells were exposed to dmPGE2 during the viral transduction step (24 to 25 hours of culture; 24 to 26 hours of culture; 24 to 28 hours of culture; or 24 to 48 hours of culture) and then washed and analyzed by flow cytometry after approximately 1 week in culture. Alternatively, cells were exposed to dmPGE2 during the prestimulation step (22 to 24 hours of culture; 23 to 24 hours of culture). The percentage of GFP positive cells is indicated in Table 4. TABLE 4

EXAMPLE 7 CORRECTION OF BETA-THALASSEMIA OR SICKLE CELL DISEASE AFTER TRANSDUCTION OF HSC WITH LENTIVIRAL VECTORS IN THE PRESENCE OF DMPGE2
[00333] Mobilized peripheral blood should be collected by apheresis from patients with informed consent and in accordance with approved institutional review committee (IRB) protocols. A Ficoll gradient will be used to remove erythrocytes and CD34-enriched cells obtained after CD34+ selection using the Miltenyi CliniMACS system (Miltenyi Biotec). Cells should be prestimulated with SCF, FltL, TPO and human IL3 at a concentration of approximately 4E6 cells/mL for 18 to 24 hours. Cells are then transduced with Lentiglobin GTP, containing a human β-globinA-T87Q gene, in a multiplicity of infection of 25 for 18 to 24 hours in the presence of SCF, FltL, TPO, IL3, protamine sulfate and dmPGE2.
[00334] After transduction, a portion of cells is removed for the release test, and the remainder cryopreserved and stored at -80°C. As part of the release test, cells transduced to an individual are then subjected to 7-day culture and VCN analysis (Example 1) to verify 0.5 to 3 copies per cell average, as well as >50% efficiency of transduction. Under successful release testing, patients will undergo treatment with busulfan and cyclophosphamide.
The autologous CD34+ cell dose is then administered intravenously to the subject in a single intravenous dose of >3 x 106 CD34+ cells/kg. Patients are monitored daily in the transplant unit for adverse events and laboratory parameters to monitor the bone marrow graft.
[00336] Once the graft occurs and patients are stable, they are released from the hospital and followed monthly for 6 months and at least every 3 months for a total of 24 months. Assessments will include routine hematology and chemical safety laboratory assessment and special hematology testing, bone marrow examination, collection of adverse events and concomitant medications, and assessment of disease-specific clinical and hematological parameters.
[00337] The main criteria are safety and tolerance of cell infusion transduced by lentiglobin and time to engraftment of autologous manipulated CD34+ cells. Additional criteria include biological and biochemical measures of the presence of transduced gene and gene product in blood and hematopoietic cells, transfusion requirements, and the number of hospitalizations and clinical events that occur at various times during the course of the 2-year follow-up period. All patients will be followed at least annually for a total of 15 years post-transplant for adverse events, RCL testing, and blood cell banking for insertional mutagenesis testing in the event that a malignancy develops. EXAMPLE 8 CORRECTION OF ADRENOLEUKODYSTROPHY AFTER TRANSDUCTION OF HSC WITH LENTIVIRAL VECTORS IN THE PRESENCE OF DMPGE2
[00338] Mobilized peripheral blood should be collected by apheresis from patients with informed consent and in accordance with approved institutional review committee (IRB) protocols. A Ficoll gradient will be used to remove erythrocytes and CD34-enriched cells obtained after CD34+ selection using the Miltenyi CliniMACS system (Miltenyi Biotec). Cells should be prestimulated with SCF, FltL, TPO and human IL3 at a concentration of approximately 4E6 cells/mL for 18 to 24 hours. Cells are then transduced with Lenti-D GTP, containing a human ABCD1 gene, at a multiplicity of infection of 25 for 18 to 24 hours in the presence of SCF, FltL, TPO, IL3, protamine sulfate and dmPGE2.
[00339] After transduction, a portion of cells is removed for the release test, and the remainder cryopreserved and stored at -80°C. As part of the release test, cells transduced to an individual are then subjected to 7-day culture and VCN analysis (Example 1) to verify 0.5 to 3 copies per cell average, as well as >50% efficiency of transduction. Under successful release testing, patients will undergo treatment with busulfan and cyclophosphamide.
The dose of autologous CD34+ cells is then administered intravenously to the subject in a single intravenous dose of >3 x 106 CD34+ cells/kg. Patients are monitored daily in the transplant unit for adverse events and laboratory parameters to monitor the bone marrow graft.
[00341] Once engraftment occurs and patients are stable, they are released from the hospital and followed monthly for 6 months and at least every 3 months for a total of 24 months. Assessments will include routine hematology and chemical safety laboratory assessment and special hematology testing, bone marrow examination, collection of adverse events and concomitant medications, and assessment of disease-specific clinical and hematological parameters.
The main criteria are safety and tolerance of cell infusion transduced by Lenti-D and time to engraftment of the manipulated autologous CD34+ cells. Additional criteria include biological and biochemical measures of the presence of transduced gene and gene product in blood and hematopoietic cells, brain MRI and cognitive studies, and the number of hospitalizations and clinical events that occur at various times during the course of the follow-up period 2 years. All patients will be followed at least annually for a total of 15 years post-transplant for adverse events, RCL testing, and blood cell banking for insertional mutagenesis testing in the event that a malignancy develops.
[00343] As one skilled in the art will readily recognize upon reading the present description, numerous modifications can be made to the modalities in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit claims to the specific embodiments set out in the specification and claims, but should be interpreted to include all possible embodiments along with the full scope of equivalents to which such claims are designated. Consequently, the claims are not limited by the description.

权利要求:
Claims (13)
[0001]
1. Method to increase the lentiviral transduction efficiency of CD34+ hematopoietic stem cells or progenitor cells CHARACTERIZED by the fact that it comprises: cultivating CD34+ hematopoietic stem cells or progenitor cells in a culture medium comprising a lentiviral vector and prostaglandin E2 ( PGE2), 16,16-dimethyl PGE2, or an analogue thereof.
[0002]
2. Method, according to claim 1, CHARACTERIZED by the fact that, optionally: (a) the progenitor cells or CD34+ hematopoietic stem cells are progenitor cells and CD34+ hematopoietic stem cells; (b) the progenitor cells or CD34+ hematopoietic stem cells are CD34+ hematopoietic stem cells; or (c) the progenitor cells or CD34+ hematopoietic stem cells are CD34+ hematopoietic progenitor cells.
[0003]
3. Method, according to claim 2, CHARACTERIZED by the fact that, optionally: (a) at least about 50% of the progenitor cells or CD34+ hematopoietic stem cells are transduced; (b) at least about 75% of the progenitor cells or CD34+ hematopoietic stem cells are transduced; or (c) at least about 90% of the progenitor cells or CD34+ hematopoietic stem cells are transduced.
[0004]
4. Method according to any one of claims 1 to 3, CHARACTERIZED by the fact that it additionally comprises cultivating the cells and the lentiviral vector in the presence of a histone deacetylase (HDAC) inhibitor, in which the HDAC inhibitor is optionally selected from the group consisting of: Trichostatin A (TSA), valproic acid (VPA), sodium butyrate, suberoylanilide hydroxamic acid (SAHA), sodium phenylbutyrate, depsipeptide, trapoxin (TPX), peptide containing cyclic hydroxamic acid 1 (CHAP1), MS-275, LBH589 and PXD-101.
[0005]
5. Method according to any one of claims 1 to 4, CHARACTERIZED by the fact that the lentiviral vector is derived from a human immunodeficiency virus (HIV) and/or in which the lentiviral vector is optionally pseudotyped with an envelope protein of the vesicular stomatitis virus G protein (VSV-G).
[0006]
6. Method, according to any one of claims 1 to 5, CHARACTERIZED by the fact that progenitor cells or CD34+ hematopoietic stem cells are cultured in the presence of prostaglandin E2 (PGE2), 16,16-dimethyl PGE2, or an analogue of it during transduction, optionally for at least about twenty-four hours.
[0007]
7. Method according to claim 6, CHARACTERIZED by the fact that progenitor cells or CD34+ hematopoietic stem cells are cultured in the presence of prostaglandin E2 (PGE2), 16,16-dimethyl PGE2, or an analogue thereof during the first twenty-four hours of transduction or during the first forty-eight hours of transduction.
[0008]
8. Method, according to any one of claims 1 to 7, CHARACTERIZED by the fact that the lentiviral vector comprises: a) a left (5') lentiviral LTR; b) an expression control sequence operably linked to a gene of interest; and c) a right lentiviral LTR (3').
[0009]
9. Method, according to any one of claims 1 to 8, CHARACTERIZED by the fact that, optionally: (I) the lentiviral vector comprises: a) a left HIV-1 (5') LTR; b) a Psi packing sequence (Φ+); c) a central polypurine/HIV-1 flap DNA tract (cPPT/FLAP); d) a rev response element (RRE); e) a β-globin promoter and a β-globin locus control region (LCR) operably linked to a gene of interest; and f) a right HIV-1 (3') LTR comprising: i) one or more insulating elements, or ii) a rabbit β-globin polyA (rβgpA) sequence; (II) the progenitor cells or CD34+ hematopoietic stem cells are formulated for administration to a patient suffering from a hemoglobinopathy, wherein the hemoglobinopathy is, optionally, β-thalassemia or sickle cell disease; or (III) the lentiviral vector comprises: (a) a left (5') HIV-1 LTR; (b) a Psi packing signal (^); (c) a cPPT/FLAP; (d) an RRE; (e) an MND promoter operably linked to a polynucleotide encoding a human ABCD1 polypeptide; (f) a right (3') HIV-1 LTR; and (g) a rabbit β-globin polyadenylation sequence.
[0010]
10. Method according to any one of claims 1 to 9, CHARACTERIZED by the fact that the lentiviral vector has imperfect replication.
[0011]
11. Method according to any one of claims 8 to 10, CHARACTERIZED by the fact that the 3’ LTR lentiviral vector is a self-inactivating LTR (SIN).
[0012]
12. Use of a composition comprising CD34+ progenitor cells or hematopoietic stem cells, as defined in item (III) of claim 9, CHARACTERIZED by the fact that it is for the manufacture of a drug to treat a patient suffering from an adrenoleukodystrophy or an adrenomyeloneuropathy.
[0013]
13. Use of prostaglandin E2 (PGE2), 16,16-dimethyl PGE2, or an analogue thereof, CHARACTERIZED by the fact that it is for the manufacture of a drug to increase the transduction efficiency of progenitor cells and/or cells. CD34+ hematopoietic trunks cultured with a lentiviral vector.

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法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-06-04| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2020-01-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-02-27| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-04-07| B25G| Requested change of headquarter approved|Owner name: BLUEBIRD BIO, INC (US) |

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2021-03-30| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-06-01| 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 28/09/2012, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-07-20| B25K| Entry of change of name and/or headquarter and transfer of application, patent and certificate of addition of invention: republication|Owner name: BLUEBIRD BIO, INC. (US) Free format text: RETIFICACAO DO DESPACHO 25.7 ? ALTERACAO DE SEDE PUBLICADO NA RPI NO 2570, DE 07/04/2020.ONDE SE LE: 60 BINNEY ST. CAMBRIDGE, MA 02141, EUA. LEIA-SE: 60 BINNEY ST. CAMBRIDGE, MA 02142, EUA. |

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US201161541736P| true| 2011-09-30|2011-09-30|

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US61/541,736|2011-09-30|

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