Gene therapy for patients with fanconi anemia

Abstract

To provide compositions and methods for restoring FANCA expression in cells with diminished or abolished FANCA gene products, particularly, methods and compositions for the gene therapy of Fanconi anemia.SOLUTION: Provided is an expression cassette comprising a polynucleotide sequence comprising in order from 5' to 3': (a) a human phosphoglycerate kinase (PGK) promoter sequence or a functional homologue or variant thereof; (b) a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof; (c) a woodchuck hepatitis virus regulatory element (WPRE) RNA transport signal sequence or a functional variant or fragment thereof, the sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof being operably linked to the PGK promoter sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims priority to and benefit from U.S. Provisional Application No. 62 / 385185, filed September 8, 2016, and U.S. Provisional Application No. 62 / 412.028, filed October 24, 2016, the contents of which are incorporated herein by reference in their entireties.

[0002] FIELD OF THE INVENTION The present invention relates generally to gene transfer into cells with reduced or eliminated protein activity from one or more FANCA-encoded proteins.

[0003] Sequence Listing Declaration The sequence listing accompanying this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequences is ROPA_002_01WO_ST25.txt. This text file is 46 KB, was created on September 8, 2017, and is submitted electronically via EFS-WEB. [Background technology]

[0004] Background of the Invention Fanconi anemia (FA) is an autosomal recessive disorder (except for the X-linked complementation group FA-B) with a median patient survival of approximately 24 years (Butturini A, et al. (1994) Blood 84: 1650-1655 (Non-Patent Document 1); Kutler DI, et al. (2003) Blood 101: 1249-1256 (Non-Patent Document 2)). At birth, these patients generally have normal blood counts. Macrocytosis is often the first hematologic abnormality detected in these patients. It usually develops along with thrombocytopenia, anemia, and pancytopenia. Bone marrow failure (BMF) is usually observed in these patients after the age of 5 to 10 years, with the average age of onset of hematologic disease being 7 years. Approximately 80% of FA patients will show signs of BMF in the first decade of their life. Based on epidemiological studies to date, in the absence of a malignant episode preceding aplasia, virtually all FA patients will develop BMF by the age of 40 years (Butturini A, et al. (1994) Blood 84: 1650-1655 (Non-Patent Document 1); Kutler DI, et al. (2003) Blood 101: 1249-1256 (Non-Patent Document 2)), which is the leading cause of death in these patients.

[0005] Because the clinical manifestations of FA are complex, management of these patients primarily focuses on improving the following syndromes: bone marrow failure (BMF), myeloid leukemia, and solid tumors.

[0006] Current treatments include androgens, such as fluoxymesterone, oxymesolone, or stanozolol. As recently reviewed by Dufour et al. (Dufour, C. and Svahn, J. (2008). Bone Marrow Transplant 41 Suppl 2: S90-95), FA patients may respond to androgens to a certain extent, unless treatment is initiated at a very advanced stage of the disease. Approximately 75% of patients respond to androgens. The combination of 2 mg / kg / day of prednisolone reduces the risk of liver toxicity. In general, androgens can be considered as an alternative treatment when some residual hematopoietic capacity remains, rather than as a definitive long-term treatment, in the absence of a suitable bone marrow donor.

[0007] Tischkowitz et al. stated that although FA patients may respond to androgens in the early stages of the disease, the majority of FA patients are refractory to these treatments in the long term (Tischkowitz, M. and Dokal, I. (2004). Br J Haematol 126: 176-191).

[0008] There are no specific indications for hematopoietic growth factors in the treatment of FA. However, two pharmacological groups of drugs have been identified that are indicated for the specific manifestations of FA (anemia and neutropenia): 1) erythropoietin and 2) granulocyte-colony-stimulating factor (G-CSF). Various erythropoietic drugs (e.g., Aranesp, Nespo, Exjade) are approved for the treatment of anemia, and G-CSF (e.g., G-CSF analogs such as filgrastim, biogastrin, and Neulasta) are approved for the treatment of neutropenia. Hematopoietic growth factors, such as erythropoietin and granulocyte-colony-stimulating factor, have been tested in a limited number of patients with FA, but responses have been partial and transient (Dufour et al., 2008). Currently, these treatments are not good long-term options. As a short-term treatment for neutropenic patients, G-CSF can be used to increase peripheral neutrophil counts and enhance the effectiveness of antibiotics against acute infections, but these medications are far from definitive management for patients with FA.

[0009] Currently, the only curative treatment for the hematologic manifestations of this disease is based on allogeneic hematopoietic cell transplantation. Although outcomes are generally satisfactory for FA patients who receive grafts from HLA-identical sibling donors, only approximately 20% of FA patients have an HLA-identical sibling. A significant number of FA patients who do not have a sibling donor can receive transplants from alternative donors, but these transplants are associated with high morbidity and mortality. For the remaining FA patients, no alternative treatments are currently available.

[0010] Thus, there remains a great need for effective treatments for FA. The present invention meets this need and more. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Butturini A, et al. (1994) Blood 84: 1650-1655 [Non-patent document 2] Kutler DI, et al. (2003) Blood 101: 1249-1256 [Non-patent document 3] Dufour, C. and Svahn, J. (2008). Bone Marrow Transplant 41 Suppl 2: S90-95 [Non-patent document 4] Tischkowitz, M. and Dokal, I. (2004). Br J Haematol 126: 176-191 Summary of the Invention

[0012] Embodiments of the present invention include polynucleotide cassettes for increased expression of FANCA. In some embodiments, the polynucleotide cassettes include sequences encoding a codon-optimized human FANCA cDNA that enhances mRNA stability during transcription.

[0013] In one embodiment, the present invention provides an expression cassette comprising a polynucleotide sequence comprising, in 5' to 3' order: (a) a human phosphoglycerate kinase (PGK) promoter sequence or a functional homolog or variant thereof, (b) a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof, and (c) a woodchuck hepatitis virus regulatory element (WPRE) RNA export signal sequence or a functional variant or fragment thereof, wherein the sequence encoding the human FANCA polypeptide or a functional fragment or variant thereof is operably linked to the PGK promoter sequence. In a specific embodiment, the FANCA polypeptide or functional fragment or variant thereof comprises the sequence set forth in SEQ ID NO:25; the sequence encoding the FANCA polypeptide or functional fragment or variant thereof comprises the sequence set forth in SEQ ID NO:8; the PGK promoter comprises the nucleotide sequence of SEQ ID NO:7; and / or the WPRE element comprises the nucleotide sequence of SEQ ID NO:23. In a specific embodiment, the cassette comprises a region of the nucleotide sequence of SEQ ID NO:24. In some embodiments, the cassette further comprises one or more enhancer sequences, a polypurine tract (PPT) or polyadenylation (polyA) signal sequence, a packaging signal sequence, a truncated Gag sequence, a Rev response element (RRE), a central polypurine tract (cPPT), a central terminal sequence (CTS), and / or an upstream sequence element (USE), optionally from simian virus 40 (SV40-USE).

[0014] In one embodiment, the present invention provides an expression cassette comprising a polynucleotide sequence comprising: a) a promoter sequence; b) a polypeptide-encoding sequence; and c) a ribonucleic acid (RNA) export signal, wherein the promoter sequence is operably linked to the sequence encoding a FANCA polypeptide (SEQ ID NO:25), and optionally, a)-c) are present in 5'-3' order within the expression cassette. In one embodiment, the promoter is a phosphoglycerate kinase (PGK) promoter. In some embodiments, the polypeptide-encoding sequence is codon-optimized. In some embodiments, the polypeptide-encoding sequence is a codon-optimized version of a human FANCA cDNA having at least 85% identity to SEQ ID NO:8. In a specific embodiment, the RNA export signal is a mutated posttranscriptional regulatory element (wPRE) of woodchuck hepatitis virus.

[0015] In some embodiments, the mutant wPRE is a chimeric wPRE comprising a sequence having at least 80% identity with SEQ ID NO:23. In some embodiments, the expression cassette further comprises one or more enhancer sequences. In some embodiments, the expression cassette further comprises a polypurine tract (PPT) or a polyadenylation (polyA) signal sequence. In some embodiments, the expression cassette further comprises one or more of the following sequences: (i) a packing signal sequence; (ii) a truncated Gag sequence; (iii) a Rev response element (RRE); (iv) a central polypurine tract (cPPT); (v) a central terminal sequence (CTS); and (vi) an upstream sequence element (USE), optionally derived from simian virus 40 (SV40-USE). In some embodiments, the expression cassette further comprises 5' and 3' long terminal repeat (LTR) sequences.

[0016] In a related aspect, the present invention provides a recombinant gene delivery vector comprising an expression cassette disclosed herein. In some embodiments, the recombinant gene delivery vector is a virus or viral vector. In some embodiments, the virus or viral vector is a lentivirus (LV).

[0017] In another related embodiment, the present invention provides a cell comprising an expression cassette or gene delivery vector disclosed herein. In some embodiments, the cell is a blood cell. In some embodiments, the cell is an erythroid cell. In some embodiments, the cell is a bone marrow cell, e.g., a lineage-depleted bone marrow cell. In a specific embodiment, the cell is a hematopoietic stem cell or a CD34 + In some embodiments, the cells are hematopoietic stem cells. In some embodiments, the cells are CD34+ hematopoietic stem cells. In some embodiments, the cells are committed hematopoietic erythroid progenitor cells.

[0018] In a related embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the recombinant gene delivery vector or cell disclosed herein. In a specific aspect of the present invention, a pharmaceutical composition is provided comprising a polynucleotide cassette of the present invention and a pharmaceutical excipient. In another embodiment, the pharmaceutical composition comprises a gene delivery vector of the present invention and a pharmaceutical excipient.

[0019] Methods and compositions are provided for the use of gene therapy vector compositions, e.g., viral vectors, containing these gene expression cassettes for use in the manufacture of medicaments useful for central and targeted gene therapy of diseases, disorders and dysfunctions in animals and particularly humans.

[0020] In another aspect, the present invention provides a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising providing to the subject an expression cassette, gene delivery vector or pharmaceutical composition disclosed herein.

[0021] In another aspect, the present invention includes a method of treating Fanconi anemia in a subject in need thereof, comprising providing to the subject a pharmaceutical composition disclosed herein.

[0022] In a related aspect, the present invention provides a method for treating Fanconi anemia in a subject in need thereof, comprising administering to a subject a CD34 + In one embodiment, the method comprises providing a cell to a subject, wherein the expression cassette comprises a polynucleotide sequence comprising, in 5' to 3' order: (a) a human phosphoglycerate kinase (PGK) promoter sequence or a functional homolog or variant thereof, (b) a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof, and (c) a woodchuck hepatitis virus regulatory element (WPRE) RNA export signal sequence or a functional variant or fragment thereof, wherein the sequence encoding the human FANCA polypeptide or a functional fragment or variant thereof is operably linked to the PGK promoter sequence. + The cells were obtained from a subject. In a specific embodiment, the CD34+ cells were obtained from a subject after the subject was treated with a combination of (i) G-CSF or filgrastim and (ii) plerixafor. In a specific embodiment, the CD34 + The cells were transduced with a recombinant gene delivery vector containing the expression cassette. + The cells were CD34 + Cells were transduced by contacting them with the recombinant gene delivery vector for approximately 24 hours.

[0023] In another embodiment, the present invention provides a method for treating Fanconi anemia in a subject in need thereof, comprising the steps of: (a) providing to the subject a combination of (i) G-CSF or filgrastim and (ii) plerixafor to mobilize CD34+ cells in the subject; (b) mobilizing CD34+ cells from the subject; + (c) preparing a cell population enriched for CD34+ cells from the biological sample; and (d) transducing the cell population enriched for CD34+ cells with a recombinant gene delivery vector comprising an expression cassette comprising a polynucleotide sequence comprising, in 5' to 3' order: (i) a promoter sequence or a functional homolog or variant thereof, and (ii) a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof, wherein the sequence encoding the human FANCA polypeptide or a functional fragment or variant thereof is operably linked to a PGK promoter sequence, and the transduction results in the enrichment of CD34+ cells. + and (e) providing to a subject the lentiviral vector-transduced cell population resulting from step (d). In one embodiment, the step of preparing the cell population comprises removing red blood cells and / or positively selecting, negatively selecting, or a combination thereof, CD34 + The method comprises concentrating cells. In a specific embodiment, the method inhibits the development of Fanconi anemia in a subject, stops the progression of Fanconi anemia, and / or reverses the progression of hematological symptoms of Fanconi anemia. In a specific embodiment, the hematological symptoms of Fanconi anemia are selected from one or more of BMF, thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia.

[0024] [The present invention 1001] In order from 5' to 3': (a) a human phosphoglycerate kinase (PGK) promoter sequence or a functional homolog or variant thereof; (b) a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof; (c) a woodchuck hepatitis virus regulatory element (WPRE) RNA export signal sequence or a functional variant or fragment thereof; 1. An expression cassette comprising a polynucleotide sequence comprising: wherein a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof is operably linked to a PGK promoter sequence. [The present invention 1002] 1001. An expression cassette of the present invention, wherein the FANCA polypeptide or a functional fragment or variant thereof comprises the sequence shown in SEQ ID NO:25. [The present invention 1003] 1001. An expression cassette of the present invention, wherein the sequence encoding a FANCA polypeptide or a functional fragment or variant thereof comprises the sequence shown in SEQ ID NO:8. [The present invention 1004] 1001. An expression cassette of the present invention, wherein the PGK promoter comprises the nucleotide sequence of SEQ ID NO:7. [The present invention 1005] 1001. An expression cassette of the present invention, wherein the WPRE element comprises the nucleotide sequence of SEQ ID NO:23. [The present invention 1006] 1001. An expression cassette of the present invention comprising the nucleotide sequence of SEQ ID NO:24. [The present invention 1007] The expression cassette of any of claims 1001 to 1008, further comprising one or more enhancer sequences. [The present invention 1008] (d) polypurine tract (PPT) or polyadenylation (polyA) signal sequence 1001. An expression cassette of the present invention further comprising: [The present invention 1009] The following array: (e) packaging signal sequence; (f) truncated Gag sequence; (g) Rev response element (RRE), (h) central polypurine tract (cPPT), (i) the central terminal sequence (CTS), and (j) an upstream sequence element (USE), optionally derived from simian virus 40 (SV40-USE); 1001. An expression cassette of the present invention further comprising one or more of: [The present invention 1010] A recombinant gene delivery vector comprising any one of the expression cassettes of the present inventions 1001 to 1009. [The present invention 1011] The recombinant gene delivery vector of the present invention 1010, which is a virus or viral vector. [The present invention 1012] The recombinant gene delivery vector of the present invention, wherein the virus or viral vector is a lentivirus (LV). [The present invention 1013] A cell comprising an expression cassette of the present invention 1001 or a recombinant gene delivery vector of the present invention 1011 or 1012. [The present invention 1014] The cell of the present invention 1013, which is a hematopoietic stem cell. [The present invention 1015] CD34 + The cell of the present invention is a cell. [The present invention 1016] A pharmaceutical composition comprising a pharmaceutically acceptable excipient and any one of the recombinant gene delivery vectors of the present invention. [The present invention 1017] A pharmaceutical composition comprising a pharmaceutically acceptable excipient and any one of 10 13 to 10 15 cells of the present invention. [The present invention 1018] A method of treating Fanconi anemia in a subject in need thereof, comprising providing to said subject 1017 of the present invention or a pharmaceutical composition of 1017 of the present invention. [The present invention 1019] 1. A method for treating Fanconi anemia in a subject in need thereof, comprising administering to a subject a CD34 + providing a cell to the subject, wherein the expression cassette comprises, in 5' to 3' order: (a) a human phosphoglycerate kinase (PGK) promoter sequence or a functional homolog or variant thereof; (b) a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof; (c) a woodchuck hepatitis virus regulatory element (WPRE) RNA export signal sequence or a functional variant or fragment thereof; wherein the sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof is operably linked to a PGK promoter sequence. [The present invention 1020] CD34 + The method of claim 1019, wherein the cell is obtained from said subject. [The present invention 1021] 1020. The method of claim 1020, wherein the CD34+ cells are obtained from the subject after treating said subject with a combination of (i) G-CSF or filgrastim and (ii) plerixafor. [The present invention 1021] CD34 + The method of claim 1020, wherein the cells are transduced with a recombinant gene delivery vector comprising said expression cassette. [The present invention 1022] CD34 + The cells are + The method of claim 1021, wherein the cells are transduced by contacting the cells with said recombinant gene delivery vector for about 24 hours. [The present invention 1023] 1. A method for treating Fanconi anemia in a subject in need thereof, comprising: (a) providing to the subject a combination of (i) G-CSF or filgrastim and (ii) plerixafor to mobilize CD34+ cells in the subject; (b) CD34 from the subject + obtaining a biological sample comprising cells, the biological sample optionally being peripheral blood or bone marrow; (c) preparing a cell population enriched in CD34+ cells from the biological sample; (d) In order from 5' to 3': (i) a promoter sequence or a functional homolog or variant thereof; (ii) a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof; wherein a sequence encoding a human FANCA polypeptide, or a functional fragment or variant thereof, is operably linked to a PGK promoter sequence, and said transduction comprises contacting said CD34+ cell-enriched cell population with the lentiviral vector for about 24 hours; and (e) providing the lentiviral vector-transduced cell population resulting from step (d) to the subject. A method comprising: [The present invention 1024] The method of claim 1023, wherein the step of preparing the cell population comprises a step of removing red blood cells. [The present invention 1025] The step of preparing the cell population comprises selecting CD34 by positive selection, negative selection, or a combination thereof. + The method of claim 1023, further comprising a step of concentrating the cells. [The present invention 1026] The method of claim 1023, wherein the onset of Fanconi anemia in said subject is inhibited, the progression of Fanconi anemia is halted, and / or the progression of the hematological symptoms of Fanconi anemia is reversed. [The present invention 1027] 1026. The method of claim 1026, wherein the hematological symptoms of Fanconi anemia are selected from one or more of BMF, thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia. Other features and advantages of the present invention will be apparent from and are encompassed by the following detailed description and claims. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 is a schematic diagram of an exemplary construct PGK-FANCA.WPRE*LV. [Figure 2] Figure 2A is a schematic diagram of LVs expressing FANCA under the control of different internal promoters. Figure 2B shows Western blot analysis of FANCA in FA-A cells transduced with the vectors shown in panel A. Figure 2B shows the correction of MMC hypersensitivity in hematopoietic precursors from FA-A mice subjected to gene therapy. FA-A bone marrow (BM) cells were transduced with PGK_FANCA-WPRE* or control SF1-EGFP LVs and transplanted into irradiated FA-A mice. Seven months after transplantation, BM samples were collected and cultured in methylcellulose in the presence of increasing concentrations of mitomycin C (MMC). [Figure 3] Figure 3 presents data demonstrating functional analysis of lentiviral vectors expressing FANCA under the control of different internal promoters. Figure 3A shows the reversal of MMC sensitivity in FA-A lymphoblastoid cell line (LCL) cells transduced with LV. Average values ​​from three separate experiments are shown. Figure 3B shows the restoration of nuclear FANCD2 foci formation in FA-A LCLs transduced with the vector and exposed to mitomycin C (MMC). [Figure 4]Figure 4 presents data demonstrating the in vivo efficacy and safety of FA gene therapy using PGK-FANCA.Wpre* LV. Figure 4A shows the construct. Figure 4B shows how bone marrow (BM) cells from FA-A mice were transduced with FANCA LV and then transplanted into irradiated FA-A recipient mice. Figure 4C shows that BM samples from transplanted FA-A mice were cultured in methylcellulose in the absence and presence of MMC. [Figure 5] Figure 5 shows proviral copy numbers in FANCA mice transplanted with syngeneic bone marrow cells previously transduced with a lentiviral vector carrying the therapeutic FANCA gene under the control of the PGK promoter. PB = peripheral blood; BM = bone marrow. [Figure 6] Figure 6 presents data demonstrating the correction of MMC hypersensitivity in hematopoietic precursors from FAA mice subjected to gene therapy using this medical device. FA-A bone marrow (BM) cells were transduced with PGK_FANCA-Wpre* or SF1-EGFP LVs and transplanted into irradiated FA-A mice. Seven months after transplantation, BM samples were collected and cultured in methylcellulose in the presence of increasing concentrations of MMC. [Figure 7] Figure 7 shows the improved transduction efficiency of cryopreserved bone marrow precursors from three Fanconi anemia patients. Samples were subjected to standard transduction (white bars; 1xS), consisting of one transduction cycle (16 h) after a 2 h static preload, or improved transduction (gray bars; 3xD), consisting of three transduction cycles (2 h + 2 h + 12 h) with lentiviral vectors. [Figure 8]Figure 8 shows the relevance of the WPRE sequence to the functional properties of lentiviral vectors expressing FANCA under the control of the PGK promoter. Figure 8A: Reversal of (MMC) sensitivity of FA-A LCLs transduced with PGK-FANCA and PGK-FANCA-WPRE LVs. Average values ​​from three separate experiments are shown. Figure 8B: Reversal of MMC sensitivity of FA-A hematopoietic precursors (colony-forming cells, CFCs) transduced with SFFV-FANCA LV and PGK-FANCA LV ("Expt 1") and PGK-FANCA and PGK-FANCA-WPRE LV ("Expt 2"). White bars = no MMC; black bars = 10 nM MMC. MMC = mitomycin C. [Figure 9] FIG. 9 shows the efficacy of GALV-TR and VSV-G pseudotyped lentiviral vectors in transducing hematopoietic precursors from the bone marrow of Fanconi anemia patients with EGFP-LV. [Figure 10] Figure 10 shows the low in vitro transformation potential of lentiviral vectors carrying the hPGK promoter. Figure 10A: Overview of the vector. Figure 10B: Transformation potential measured by replating times / copy number. [Figure 11] FIG. 11 is an overview of an exemplary hematopoietic stem cell (HSC) collection and gene therapy trial for FA-A patients. [Figure 12] Figure 12 shows hematological parameters of recruited patients in the study described in the Examples. Figure 12A shows results for hemoglobin, Figure 12B shows results for neutrophils, Figure 12C shows results for platelets, and Figure 12D shows results for CD34+ cells. [Figure 13] Figure 13 shows a Fancostem protocol phase II study designed to evaluate the safety and efficacy of CD34+ cell mobilization and collection following treatment with plerixafor (Mozobil) and filgrastim (also known as G-CSF) (Neupogen) in 10 patients with Fanconi anemia. [Figure 14] FIG. 14 shows the mobilization of CD34+ cells in FA-A patients via G-CSF / plelixafor. [Figure 15] FIG. 15 shows G-CSF / plerixafor-mediated mobilization of CFCs in FA-A patients. [Figure 16] Figure 16 is a summary of CD34+ cell collection in G-CSF / plelixafor mobilized FA-A patients. Figure 16A shows CD34+ cell collection in FANCOSTEM, and Figure 16B shows a comparison with previous studies. [Figure 17] FIG. 17 is a chart showing a comparison between estimated CD34+ cell counts in bone marrow (BM) and actual counts in mobilized peripheral blood (mPB). [Figure 18] FIG. 18 is a chart of the collection and purification of mobilized peripheral blood (mPB) FA-A CD34+ cells. [Figure 19] FIG. 19 shows CD34 expression in mobilized peripheral blood (mPB) CD34+ cells from healthy donors (HD) and FA patients before and after immune selection. [Figure 20] Figure 20 shows that patient FA 02005 meets the criteria for both the FANCOSTEM and FANCOLEN studies. Figure 20A shows cell counts. Figure 20B shows hematopoietic stem cell (HSC) mass versus age. [Figure 21] FIG. 21 (A-E) depicts test results showing the FA diagnosis of patient FA-02005 prior to gene therapy. [Figure 22] FIG. 22 shows the follow-up parameters of the cell manufacturing process for FA-A patient 02005. [Figure 23] FIG. 23 is a graph showing vector copy numbers before gene therapy and 2 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, and 5 months after gene therapy in patient FA-02005. [Figure 24] FIG. 24 depicts the follow-up of the first unadjusted FA-A patient (FA-02005) before and after gene therapy, as measured by hemoglobin levels. [Figure 25] FIG. 25 depicts the follow-up of the first unadjusted FA-A patient (FA-02005) before and after gene therapy, as measured by neutrophil load. [Figure 26] FIG. 26 depicts the follow-up of the first unadjusted FA-A patient (FA-02005) before and after gene therapy, as measured by platelet count. [Figure 27] FIG. 27 is a chart of the hematological evaluation of patient FA-A02002. [Figure 28] Figure 28 shows the diagnosis of FA 02002 as non-mosaic; homozygous FANCA c.239 C>T p.Gln99*MMC hypersusceptible; complemented by FANC. [Figure 29] FIG. 29 shows the cell manufacturing process in patient FA-A02002. [Figure 30] FIG. 30 represents the analysis of CD34 expression in healthy donor (HD) and FA-mobilized peripheral blood (mPB) during the different steps required for LV transduction in patient FA 02002. [Figure 31] FIG. 31 is a graph showing vector copy numbers before and after gene therapy in patient FA 02002. [Figure 32] FIG. 32 presents follow-up data for patient FA-A 02002, who was transfused with cryopreserved cells, as measured by hemoglobin content. [Figure 33] FIG. 33 presents follow-up data for patient FA-A 02002, who was transfused with cryopreserved cells, as measured by neutrophil content. [Figure 34] FIG. 34 presents follow-up data for patient FA-A 02005, who was transfused with cryopreserved cells, as measured by platelet volume. [Figure 35] Figure 35 shows the transduction of freshly mobilized peripheral blood (mPB) CD34+ cells from an FA-A patient using the validated conditions. Figure 35A depicts the protocol. Figure 35B shows a graph of results from patient 02002. Figure 35C shows a graph of results from patient 02003. Figure 35D shows a graph of results from patient 02004. [Figure 36]Figure 36 shows data on engraftment of corrected FA-A mPB CD34+ cells in NSG mice. Figure 36A shows the protocol. Figure 36B shows a graph of results from patient 02002. Panel C shows a graph of results from patient 02003. Figure 36D shows a graph of results from patient 02004. mPB = mobilized peripheral blood. [Figure 37] Figure 37 shows in vivo selection of corrected FA HPCs from patient 02002 in NOD-Skid gamma (NSG) mice. Figure 37A shows the protocol. Figure 37B shows a graph of CFCs before transplantation. Figure 37C shows a graph of hCFCs 30 days after transplantation. [Figure 38] Figure 38 is a map of a 5.7 kb plasmid encoding the envelope G glycoprotein of VSV under the control of the CMV promoter and carrying a kanamycin resistance gene for selection purposes. [Figure 39] Figure 39 is a map of a 3.5 kb plasmid encoding the HIV-1 rev gene under the control of the CMV promoter and carrying a kanamycin resistance gene for selection purposes. [Figure 40] Figure 40 is a map of the 8.8 kb plasmid containing the HIV-1 gag and pol genes encoding HIV-1 structural and enzymatic proteins under the control of the CMV promoter. It also contains intron 2 of human beta globin (HBB2), the HIV-1 Rev response element (RRE), and the kanamycin resistance gene. [Figure 41] Figure 41 is a map of the 11,621 base pair transfer cassette pCCL-SIN-cPPT / CTS-hPGK-hFANCA-WPRE. [Figure 42] FIG. 42 depicts LAM-PCR analysis of FANCA-LV insertion sites in FA hematopoietic stem cells (HSCs). [Figure 43] Figure 43 shows LAM-PCR results for tracking of FANCA-LV treated cells. Figure 43A shows the protocol and Figure 43B is a chart of the data. [Figure 44]Figure 44 shows the clonal diversity of Fanca - / - recipients transplanted with LV-corrected HSCs. Figure 44A shows the protocol, and Figure 44B is a graph of the data. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description of the Invention The present invention relates generally to the fields of molecular biology and virology, and in particular to gene expression cassettes and vectors containing them that are useful for delivering nucleic acid segments encoding selected therapeutic constructs (including, for example, peptides, polypeptides, ribozymes, and catalytic RNA molecules) to selected cells and tissues of vertebrates. In particular, these gene constructs are useful in gene therapy for treating mammalian, particularly human, diseases, disorders, and dysfunctions associated with dysfunction of the FANCA gene product.

[0027] In some embodiments, the present invention provides compositions and methods for gene therapy treatment of subjects with Fanconi anemia (FA). In particular, compositions and methods for restoring FANCA gene expression are provided. The specific method disclosed herein relates to the use of lentivirus to deliver human FANCA to hematopoietic progenitor cells of subjects with FA, particularly FA-A.

[0028] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to implement the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0029] As used herein, a "vector" refers to a polymer or polymer-associated entity that contains or is associated with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell. Exemplary vectors include, for example, plasmids, viral vectors (e.g., retroviral vectors such as lentiviral vectors), liposomes, and other gene delivery vehicles.

[0030] The term "LV" is an abbreviation for lentivirus and can be used to refer to the virus itself or its derivatives. The term covers all subtypes, except where required otherwise, and covers both naturally occurring and recombinant forms.

[0031] As used herein, the term "gene" or "coding sequence" refers to an in vitro or in vivo nucleotide sequence that encodes a gene product. In some cases, a gene consists of, or consists essentially of, a coding sequence, i.e., a sequence that encodes a gene product. In other cases, a gene includes additional non-coding sequences. For example, a gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequence and 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0032] As used herein, a "therapeutic gene" refers to a gene that, when expressed, confers a beneficial effect on the cell or tissue in which it is present or on the mammal in which it is expressed. Examples of beneficial effects include ameliorating the signs or symptoms of a condition or disease, preventing or inhibiting a condition or disease, or imparting a desired characteristic. Therapeutic genes include genes that correct a genetic defect in a cell or mammal.

[0033] As used herein, a transgene is a gene that is delivered to a cell by a vector.

[0034] As used herein, the term "gene product" refers to the desired expression product of a polynucleotide sequence, such as a polypeptide, peptide, protein, or interfering RNA, including small interfering RNA (siRNA), miRNA, or short hairpin RNA (shRNA).

[0035] As used herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length. These terms also include modified amino acid polymers, such as those that undergo disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0036] "Comprising" means that the specified element is required, e.g., in a composition, method, kit, etc., but that other elements may be included to form, e.g., a composition, method, kit, etc., within the scope of the claims. For example, an expression cassette "comprising" a gene encoding a therapeutic polypeptide operably linked to a promoter is an expression cassette that may include other elements in addition to the gene and promoter, e.g., a polyadenylation sequence, an enhancer element, other genes, a linker domain, etc.

[0037] "Consisting essentially of" means that the scope of a described, e.g., composition, method, kit, etc., is limited to the specified materials or steps that do not substantially affect the basic novel characteristics of the composition, method, kit, etc. For example, an expression cassette "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter and polyadenylation sequence may include additional sequences, e.g., linker sequences, so long as they do not substantially affect transcription or translation of the gene. As another example, a variant or mutant polypeptide fragment "consisting essentially of" a specified sequence has the amino acid sequence of the specified sequence with about 10 amino acid residues added or removed at the boundaries of the sequence based on the full-length, native polypeptide from which it was derived, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue less than the specified boundary amino acid residue, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues more than the specified boundary amino acid residue.

[0038] "Consisting of" means that any element, step, or ingredient not explicitly stated in the claim is excluded from the composition, method, or kit. For example, an expression cassette "consisting of" a gene encoding a therapeutic polypeptide operably linked to a promoter and post-transcriptional control elements consists only of the promoter, the polynucleotide sequence encoding the therapeutic polypeptide, and the post-transcriptional control elements. As another example, a polypeptide "consisting of a specified sequence" contains only the specified sequence.

[0039] As used herein, "expression vector" includes vectors described above or known in the art, such as plasmids, minicircles, viral vectors, liposomes, etc., that contain a polynucleotide encoding a gene product of interest and are used to achieve expression of the gene product in the intended target cell. Expression vectors also contain regulatory elements operatively linked to the coding region to facilitate expression of the gene product in the target. The combination of regulatory elements, such as promoters, enhancers, UTRs, miRNA targeting sequences, etc., and a gene operably linked for expression is sometimes referred to as an "expression cassette." Many such regulatory elements are known and available in the art or can be easily constructed from components available in the art.

[0040] As used herein, "promoter" includes a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression can be ubiquitous, i.e., highly active in a wide range of cells, tissues, and species, or cell-type, tissue, or species-specific. Promoters can be "constitutive," i.e., constantly active, or "inducible," i.e., activated or inactivated by the presence or absence of biotic or abiotic factors. Enhancer sequences, which may or may not be adjacent to the promoter sequence, are also included in the nucleic acid constructs or vectors of the present invention. Enhancer sequences influence promoter-dependent gene expression and can be located 5' or 3' to the native gene.

[0041] As used herein, "enhancer" includes cis-acting elements that stimulate or inhibit transcription of adjacent genes. Enhancers that inhibit transcription are also called "silencers." Enhancers can function in either orientation (i.e., relative to the coding sequence), at distances up to several kilobase pairs (Kb) downstream of the coding sequence and transcribed region.

[0042] A "termination signal sequence," as used herein, includes a genetic element that causes RNA polymerase to terminate transcription, such as a polyadenylation signal sequence.

[0043] As used herein, the terms "operatively linked" or "operably linked" refer to the juxtaposition of genetic elements, e.g., promoter, enhancer, termination signal sequence, polyadenylation sequence, etc., in a relationship permitting the elements to function in their expected manner. For example, a promoter is operatively linked to a coding region if it helps initiate transcription of the coding sequence. Intervening residues between the promoter and coding region can be present so long as this functional relationship is maintained.

[0044] As used herein, the term "heterologous" means derived from an entity that is genotypically different from the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering into a plasmid or vector derived from a different species is a heterologous polynucleotide. As another example, a promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, an LV vector containing a heterologous nucleic acid encoding a heterologous gene product is an LV vector containing a nucleic acid not normally contained in naturally occurring wild-type LV, and the encoded heterologous gene product is a gene product not normally encoded by naturally occurring wild-type LV.

[0045] As used herein, the term "endogenous" with respect to a nucleotide molecule or gene product refers to a nucleic acid sequence, e.g., a gene or genetic element, or a gene product, e.g., RNA, protein, that is naturally present in or associated with a host virus or host cell.

[0046] The term "native," as used herein, refers to a nucleotide sequence, eg, a gene, or a gene product, eg, RNA, protein, that is present in a wild-type virus or cell.

[0047] As used herein, the term "variant" refers to a variant of a reference polynucleotide sequence or a reference polypeptide sequence, such as a native polynucleotide sequence or a native polypeptide sequence, i.e., a variant having less than 100% sequence identity with a reference polynucleotide sequence or a reference polypeptide sequence. In other words, a variant contains at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) compared to a reference polynucleotide sequence, such as a native polynucleotide sequence or a native polypeptide sequence. For example, a variant can be a polynucleotide having 70% or more sequence identity with a full-length native polynucleotide sequence, such as 75% or 80% or more identity, for example, 85%, 90%, or 95% or more identity, for example, 98% or 99% identity, with a full-length native polynucleotide sequence. As another example, a variant can be a polypeptide having 70% or more sequence identity to a full-length native polypeptide sequence, e.g., 75% or 80% or more, e.g., 85%, 90%, or 95% or more identity, e.g., 98% or 99% identity, to a full-length native polypeptide sequence. Variants can also include variant fragments of a reference sequence, e.g., a native sequence, that share 70% or more sequence identity with a reference sequence, e.g., a fragment of a native sequence, e.g., 75% or 80% or more, e.g., 85%, 90%, or 95% or more identity, e.g., 98% or 99% identity, to the native sequence.

[0048] As used herein, the terms "biological activity" and "having biological activity" refer to activity attributable to a particular biological element in a cell. For example, the "biological activity" of an "immunoglobulin," "antibody," or a fragment or variant thereof, refers to the ability to bind to an antigenic determinant, thereby facilitating immunological function. As another example, the biological activity of a polypeptide or functional fragment or variant thereof refers to the ability of the polypeptide or functional fragment or variant thereof to perform its native function, e.g., binding, enzymatic activity, etc. As a third example, the biological activity of a genetic control element, e.g., a promoter, enhancer, Kozak sequence, etc., refers to the ability of the control element or functional fragment or variant thereof to control the expression of an operably linked gene, i.e., to promote, enhance, or activate its translation, respectively.

[0049] The terms "administration" or "introduction," as used herein, refer to the delivery of a vector for recombinant protein expression to a cell, a cell and / or organ of a subject, or a subject. Such administration or introduction can occur in vivo, in vitro, or ex vivo. A vector for expression of a gene product can be introduced into a cell by transfection, which typically refers to the insertion of heterologous DNA into a cell by physical means (e.g., calcium phosphate transfection, electroporation, microinjection, or lipofection); infection, which typically refers to introduction by an infectious agent, i.e., a virus; or transduction, which typically refers to stable infection of a cell by a virus or the transfer of genetic material from one microorganism to another by a viral agent (e.g., a bacteriophage).

[0050] "Transformation" is typically used to refer to cells, such as bacteria containing heterologous DNA, or tumor cells, that express an oncogene and are therefore converted into a persistent growth mode. The vector used to "transform" a cell can be a plasmid, virus, or other vehicle.

[0051] Typically, cells are referred to as "transduced," "infected," "transfected," or "transformed" depending on the means used to administer, introduce, or insert heterologous DNA (i.e., a vector) into the cells. The terms "transduced," "transfected," and "transformed" can be used interchangeably herein, regardless of the method of introduction of the heterologous DNA.

[0052] The term "host cell," as used herein, refers to a cell that has been transduced, infected, transfected, or transformed with a vector. The vector may be a plasmid, viral particle, phage, etc. Culture conditions such as temperature, pH, etc., will be those conventionally used in the host cell selected for expression and will be apparent to those of skill in the art. It will be understood that the term "host cell" refers to the original transduced, infected, transfected, or transformed cell, and their progeny.

[0053] The terms "treatment," "treating," and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of complete or partial prevention of a disease or its symptoms, e.g., reducing the likelihood of a disease or its symptoms occurring in a subject, and / or therapeutic, in terms of partial or complete treatment of a disease and / or adverse effects resulting from a disease. "Treatment," as used herein, covers the treatment of disease in a mammal and includes: (a) preventing the onset of a disease in a subject predisposed to, but not yet diagnosed with, the disease; (b) inhibiting the disease, i.e., inhibiting its development; or (c) ameliorating the disease, i.e., inducing regression of the disease. Therapeutic agents can be administered before, during, or after the onset of disease or injury. Treatment of ongoing disease is particularly important when treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is desirably performed before complete loss of function in the affected tissue. The treatment of the present invention will desirably be administered during, and in some cases after, the symptomatic stage of the disease.

[0054] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates, e.g., monkeys and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, when the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive, similar to the term "comprising."

[0055] The term "about" or "approximately" means within an acceptable range of error for a particular value, as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, in accordance with practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When particular values ​​are described in this application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable range of error for the particular value.

[0056] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Weir & C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1996), each of which is expressly incorporated herein by reference. al., eds., 1991).

[0057] Various aspects of the present invention are described below with reference to illustrative examples. It should be understood that numerous specific details, relationships, and methods are set forth to provide a thorough understanding of the present invention. However, one of ordinary skill in the relevant art will readily understand that the present invention can be practiced without one or more of these specific details or in other ways. The present invention is not limited by the order of acts or events shown, and some acts may occur in a different order and / or simultaneously with other acts or events. Furthermore, not all shown acts or events are required to practice a method in accordance with the present invention.

[0058] Unless otherwise indicated, all terms used herein have the same meaning as understood by those skilled in the art, and the practice of the present invention employs conventional techniques of microbiology and recombinant DNA technology known to those skilled in the art.

[0059] In some embodiments, the present disclosure provides polynucleotides, polynucleotide cassettes, and expression vectors for expressing genes in cells. Pharmaceutical compositions and methods of using any of the above compositions to promote the expression of genes in cells, e.g., in an individual, for example, to treat or prevent a disorder, are also provided. These and other objects, advantages, and features of the present invention will become apparent to those skilled in the art upon reading the details of the compositions and methods more fully described below.

[0060] In certain aspects, methods and compositions are provided for producing gene therapy vector compositions, e.g., viral vectors, comprising these gene expression cassettes for use in the manufacture of medicaments useful in central and targeted gene therapy of diseases, disorders and dysfunctions in animals, particularly humans.

[0061] In some embodiments, the present invention provides a gene therapy for Fanconi anemia based on an LV vector carrying an hPGK eukaryotic promoter driving the expression of a FANCA cDNA. This therapeutic vector can be used to transduce human hematopoietic stem cells (HSCs), which can then be transplanted into humans with Fanconi anemia.

[0062] In one embodiment, the present invention provides a FANCA LV vector for genetic correction of Fanconi anemia. Overall, the results demonstrate the feasibility of gene therapy for FA using clinically engineered LV.

[0063] The disclosed compositions can be utilized in a variety of research, diagnostic and therapeutic regimens, including the prevention and treatment of a variety of human diseases. Various compositions and methods of the present invention are described below.

[0064] While specific compositions and methods are exemplified herein, it will be understood that any of a number of alternative compositions and methods are applicable and suitable for use in practicing the present invention. It will also be understood that evaluation of the expression constructs and methods of the present invention can be performed using standard methods in the art.

[0065] Fanconi anemia Fanconi anemia (FA) is a rare inherited chromosomal instability syndrome characterized primarily by bone marrow failure (BMF) and a predisposition to cancer (Butturini A et al. Blood. 1994; 84; 1650-1655; Kutler DI et al. Blood. 2003; 101: 1249-1256). The prevalence of FA is estimated to be 1-5 per million, with a heterozygous carrier frequency of 1 in 300 (Tamary H et al. Eur J Haematol. 2004; 72: 330-335).

[0066] FA is genetically and phenotypically heterogeneous. To date, 13 complementation groups have been reported (FA-A, B, C, D1, D2, E, F, G, I, J, L, M, and N) associated with mutations in the 13 corresponding Fanconi anemia complementation group (FANC) genes: FANCA, FANCB, FANCC, FANCD1 / BRCA2, FANCD2, FANCE, FANCF, FANCG / XRCC9, FANCI, BRIP1 / FNACJ, FANCL, FANCM / Hef, and FANCN / PABLB2 (Wang W. Nat Rev Genet. 2007; 8: 735-748). With the exception of FA-B patients (FANCB is located on the X chromosome), FA is autosomal recessive.

[0067] Proteins encoded by FA genes are involved in a biochemical pathway known as the FA / BRCA pathway (see Wang W. Nat Rev Genet. 2007; 8: 735-748). Thirteen FA proteins have been identified in the FA pathway, each of which participates in one of the three FA protein complexes currently characterized in this pathway. The upstream complex, the FA core complex, is composed of eight FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM) and two FA-associated proteins (FAAP24 and FAAP100). The second complex is formed by FANCD2 and FANCI, which both function in the FA-ID complex. Through the E3 ligase activity of the FA core complex (FANCL), FANCD2 and FANCI can be monoubiquitinated and subsequently loaded onto chromatin, forming large nuclear foci in response to DNA damage or replication arrest. Finally, monoubiquitinated FANCD2 / FANCI interacts with downstream FA proteins, such as FANCJ / BRIP1, FANCN / PALB2, and FANCD1 / BRCA2, which form stable complexes with proteins involved in homologous recombination repair (HDR), such as BRCA1 and RAD51.

[0068] FA-A is the most common FA complementation group, accounting for approximately 50%-80% of FA patients (Casado JA et al. J Med Genet. 2007; 44: 241-249; Levitus M et al. Blood. 2004; 103: 2498-2503; Taniguchi T, D'andrea AD. Blood. 2006; 107: 4223-4233). As a result of the absence or null expression of FANCA, the FA core complex cannot form. This prevents activation of the ID complex and, consequently, the translocation of these proteins to chromatin, thereby resulting in the characteristic phenotype of FA cells.

[0069] As reviewed by D'Andrea et al. (Blood. 1997; 90: 1725-1736), FA cells are characterized by a distinct cellular phenotype that is primarily related to defects in cell survival, DNA repair and genomic stability.

[0070] FA is primarily characterized by congenital anomalies, the development of bone marrow failure, and an increased risk of developing acute myeloid leukemia and certain solid tumors. On average, 70% of FA patients have congenital anomalies. Skeletal anomalies (radial ray, hip, scoliosis, ribs) and generalized skin hyperpigmentation, known as café au lait macules, are present in 60-70% of FA patients. Most patients have dwarfism, and approximately one-third of them have microphthalmia and renal abnormalities. Approximately 30% of FA patients have no obvious congenital anomalies (Tischkowitz M, Dokal I. Br J Haematol. 2004; 126: 176-191).

[0071] The most important clinical feature of patients with FA is hematological. Bone marrow failure (BMF) is the primary feature of the disease. It generally develops between the ages of 5 and 10 years. 80% of patients at age 15 develop BMF, and the actuarial risk of BMF exceeds 90% at age 40 years (Butturini et al., 1994, Kutler et al., 2003). Thrombocytopenia or leukopenia typically precedes anemia. Pancytopenia generally worsens over time. Neutropenia is associated with an increased risk of infection.

[0072] FA patients are also susceptible to developing cancer, primarily acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The majority of FA-associated tumors occur after the age of 13, with a mean age of 23. The relative risk of AML is increased 785-fold; the median age of FA patients developing AML is 14 years, and the cumulative incidence of hematologic malignancies is 30-55% by age 40 years (Kutler et al., 2003; Rosenberg PS et al. Blood. 2003; 101: 822-826). Older FA patients also have a high risk of developing solid tumors, primarily squamous cell carcinoma (SCC). The median age at which these patients develop solid tumors is 26 years, with a cumulative incidence of solid tumors of 30% by age 40 years (Kutler et al., 2003, Rosenberg et al., 2003).

[0073] Due to the complex clinical manifestations of FA, management of these patients is primarily focused on reducing symptoms of bone marrow failure (BMF), myeloid leukemia, and solid tumors. The limitations of each of the current treatments for FA patients are reported in an orphan drug document on lentiviral vectors carrying the FANCA gene, sponsored by CIEMAT / CIBER on Rare Disease (Bueren, J. (2010). Center for Biomedical Network Research on Rare Diseases Ref EU / 3 / 10 / 822).

[0074] In certain embodiments, methods and compositions are provided for the production of gene therapy vector compositions, e.g., viral vectors, comprising these gene expression cassettes for use in the manufacture of medicaments useful for central and targeted gene therapy of diseases, disorders and dysfunctions in animals and particularly humans.

[0075] In some embodiments, the present invention provides a gene therapy for Fanconi anemia based on an LV vector carrying an hPGK eukaryotic promoter driving the expression of a FANCA cDNA. This therapeutic vector can be used to transduce human hematopoietic stem cells (HSCs), which can then be transplanted into humans with Fanconi anemia.

[0076] In one embodiment, the present invention provides a FANCA LV vector for genetic correction of Fanconi anemia. Overall, the results demonstrate the feasibility of gene therapy for FA using clinically engineered LV.

[0077] The present disclosure includes gene expression cassettes (e.g., therapeutic cassettes), gene transfer cassettes (e.g., integration cassettes) containing the gene expression cassettes, plasmids containing the gene transfer cassettes, and gene delivery vectors containing the gene transfer cassettes. Gene expression cassettes, gene transfer cassettes, plasmids, and gene delivery vectors contain a polynucleotide sequence encoding a therapeutic gene product operably linked to a promoter sequence. In some embodiments, the polynucleotide sequence is DNA or RNA. In some embodiments, the gene expression cassette is a polynucleotide, the gene transfer cassette is a polynucleotide, and the vector is a virus, such as a lentivirus.

[0078] In certain embodiments, the therapeutic gene product is a FANCA protein or a functional fragment or variant thereof, optionally a wild-type human FANCA protein.

[0079] In specific embodiments, the gene expression cassette comprises a promoter region, a coding sequence, and post-transcriptional regulatory elements. In certain embodiments, the promoter region comprises a promoter sequence or a functional fragment thereof. In one embodiment, the promoter is a human PGK promoter. In some embodiments, the expression cassette also comprises an RNA transport signal. The RNA transport signal may comprise a wPRE sequence. In some embodiments, a mutant wPRE (Schambach, Bohne et al. 2006) without any remaining open reading frame is included to improve the expression level and stability of the therapeutic gene.

[0080] Some embodiments of the present invention include a gene expression cassette for enhancing expression of the FANCA gene product. In some embodiments, the polynucleotide cassette contains a wild-type FANCA cDNA coding sequence or a codon-optimized version of the human FANCA cDNA to increase mRNA stability during transcription. For optimization, GeneArt® software can be used to increase GC content and remove cryptic splice sites to avoid transcriptional silencing and thus increase transgene expression. Alternatively, any optimization method known in the art can be used.

[0081] In some aspects of the present invention, a pharmaceutical composition is provided comprising a gene delivery vector of the present invention and a pharmaceutical excipient. In some embodiments, the pharmaceutical composition comprises a gene delivery vector of the present invention and a pharmaceutical excipient.

[0082] In some aspects of the present invention, a method for expressing a transgene in a mammalian cell is provided. In some embodiments, the method comprises contacting one or more mammalian cells with an effective amount of a polynucleotide cassette or a gene delivery vector of the present invention, whereby the transgene is expressed at a detectable level in one or more mammalian cells. In some embodiments, the method comprises contacting one or more mammalian cells with an effective amount of a polynucleotide cassette or a gene delivery vector of the present invention, whereby the transgene is expressed at a therapeutic level in one or more mammalian cells. In some embodiments, the method is in vitro. In other embodiments, the method is in vivo.

[0083] In some aspects of the present invention, methods are provided for treating or preventing a disease or disorder in a mammal in need thereof, hi some embodiments, the method comprises administering to the mammal an effective amount of a pharmaceutical composition of the present invention, wherein the coding sequence encodes a therapeutic gene product.

[0084] composition In some aspects of the present disclosure, compositions for expression of a FANCA transgene in eukaryotic cells are provided. In some embodiments, the eukaryotic cells are mammalian cells. In one embodiment, the mammalian cells are hematopoietic stem cells (HSCs). In one embodiment, the mammalian cells are hematopoietic progenitors. In one embodiment, the mammalian cells are CD34+. In one embodiment, the mammalian cells are human cells. In a specific embodiment, the cells are human CD34+ cells derived from a subject diagnosed with FA and to be treated with the CD34+ cells after being transduced by gene delivery as disclosed herein, and the cells comprise the disclosed gene expression cassette.

[0085] In one specific embodiment, the present disclosure encompasses a lentiviral vector comprising a gene expression cassette comprising a polynucleotide sequence encoding a therapeutic FANCA protein or its functional fragment or variant. As used herein, a functional variant of a reference polynucleotide or polypeptide comprises one or more amino acid or nucleic acid deletions, additions, or substitutions compared to a reference sequence, and retains at least 50%, at least 80%, at least 90%, or at least 99% of the functional activity of the reference polynucleotide or polypeptide. As used herein, a functional fragment is a fragment of a reference polynucleotide or polypeptide, and retains at least 50%, at least 80%, at least 90%, or at least 99% of the functional activity of the reference polynucleotide or polypeptide.

[0086] In one embodiment, the backbone of the lentiviral vector is the same as that corresponding to the pharmaceutical "Wiskott-Aldrich Syndrome Protein-Containing Lentiviral Vector (WASP-LV)" (Ref 141 / 2000), but for FA treatment, the promoter is the human phosphoglycerate kinase (hPGK) promoter, which is characterized by its stable activity in vivo and improved safety compared to other promoters already used in gene therapy (Modlich U, Navarro S, Zychlinski D et al. Insertional Transformation of Hematopoietic Cells by Self-Inactivating Lentivirus And Gammaretroviral Vectors. Mol Ther. 2009; 17: 1919-1928; Montini E, Cesana D, Schmidt M et al. Hematopoietic Stem Cell Gene Transfer in a Tumor Prone Mouse Model Uncovers Low Genotoxicity Of Lentiviral Vector Integration. Nat Biotechnol. 2006; 24: 687-696).

[0087] In some embodiments of the present disclosure, the composition comprises a polynucleotide cassette. A "polynucleotide cassette" typically refers to a polynucleotide sequence comprising two or more functional polynucleotide sequences, such as a regulatory element, a translation initiation sequence, a coding sequence, a termination sequence, etc., operably linked to each other. Similarly, a "polynucleotide cassette for expression of a transgene in a mammalian cell" refers to a combination of two or more functional polynucleotide sequences, such as a promoter, an enhancer, a 5'UTR, a translation initiation sequence, a coding sequence, a termination sequence, etc., that promotes the expression of a transgene in a cell. Gene expression cassettes and gene transfer cassettes are examples of polynucleotide cassettes.

[0088] In some embodiments, the polynucleotide cassettes of the present disclosure provide enhanced transgene expression in mammalian cells. As evidenced by the examples of the present disclosure, the inventors have discovered numerous polynucleotide elements that individually and collectively provide enhanced transgene expression in mammalian cells, i.e., elements that are improved compared to those known in the art. In certain embodiments, the arrangement of two or more functional polynucleotide sequences within a polynucleotide cassette of the present disclosure provides enhanced transgene expression in mammalian cells. By "enhanced," it is meant that transgene expression is increased, enhanced, or stronger in cells harboring a polynucleotide cassette of the present disclosure compared to cells harboring a transgene operably linked to comparable control elements, e.g., as known in the art. In other words, transgene expression from a polynucleotide cassette of the present disclosure is increased, enhanced, or stronger compared to expression from a polynucleotide cassette that does not contain one or more optimized elements of the present disclosure, i.e., a reference control. In certain embodiments, the enhanced expression is specific to or limited to one or more desired cell types.

[0089] For example, expression of a transgene may be enhanced, or strengthened, or more potent in a cell comprising a polynucleotide cassette including a promoter disclosed herein than in a cell harboring a transgene operably linked to a different promoter, e.g., as known in the art. As another example, expression of a transgene may be enhanced, or increased, enhanced, or more potent in a cell comprising a polynucleotide cassette including an enhancer sequence disclosed herein than in a cell harboring a transgene operably linked to a different enhancer sequence.

[0090] While not wishing to be bound by theory, enhanced transgene expression in cells is believed to be due to faster assembly of the gene product in the cells or a more stable gene product in the cells. Therefore, enhanced transgene expression by the polynucleotide cassette of the present disclosure can be observed in a number of ways. For example, enhanced expression can be observed by detecting transgene expression earlier, e.g., 2 days earlier, 7 days earlier, 2 weeks earlier, 3 weeks earlier, 4 weeks earlier, 8 weeks earlier, 12 weeks earlier, or earlier, after contacting the polynucleotide cassette with the cells than would be detected if the transgene were operably linked to a comparable control element as known in the art. Enhanced expression can also be observed as an increase in the amount of gene product per cell. For example, there can be a 2-fold or greater increase, e.g., a 3-fold or greater increase, a 4-fold or greater increase, a 5-fold or greater increase, or a 10-fold or greater increase in the amount of gene product per mammalian cell. Enhanced expression can also be observed as an increase in the number of mammalian cells expressing detectable levels of the transgene carried by the polynucleotide cassette. For example, there may be a 2-fold or greater increase in the number of mammalian cells expressing detectable levels of the transgene, e.g., a 3-fold or greater increase, a 4-fold or greater increase, a 5-fold or greater increase, or a 10-fold or greater increase.

[0091] In another example, the polynucleotides of the present invention can promote detectable levels of transgene expression in a greater percentage of cells than conventional polynucleotide cassettes; for example, if conventional cassettes promote detectable levels of transgene expression in less than 5% of cells in a certain region, the polynucleotides of the present invention can promote detectable levels of expression in 5% or more of cells in that region; for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 45% or more of the contacted cells, in some cases, 50% or more, 55% or more; 60% or more, 65% or more, 70% or more, or 75% or more, for example, 80% or more, 85% or more, 90% or more, or 95% or more will express detectable levels of gene product. Enhanced expression can also be observed as changes in cell viability and / or function.

[0092] The polynucleotide cassette of the present disclosure typically includes a promoter region. Any suitable promoter region or promoter sequence therein can be used in the polynucleotide cassette of the present invention, as long as the promoter region promotes the expression of the coding sequence in eukaryotic cells. In some embodiments, the promoter region promotes the expression of the coding sequence in mammalian cells. In some cases, the promoter is a ubiquitous promoter, i.e., a promoter that is active in a wide range of cells, tissues, and species. In other cases, the promoter is a human PGK promoter.

[0093] Promoter and enhancer elements can be tissue-specific or stage-specific. For example, a tissue-specific promoter or enhancer drives preferential expression (or higher levels of expression) in one or more specific cell types. Examples of cell types include, but are not limited to, hematopoietic stem cells, long-term hematopoietic stem cells, short-term hematopoietic stem cells, multipotent progenitor cells, hematopoietic CD34+ cells, and cluster differentiation subpopulations within the CD34+ population. A stage-specific promoter or enhancer drives preferential expression (or higher levels of expression) in one or more specific stages of the cell cycle or development. These include, but are not limited to, the beta-globin locus regulatory region, the spectrin promoter, and an erythroid-specific promoter.

[0094] In some embodiments, polynucleotide comprises one or more enhancers.Enhancer is a nucleic acid element known in the art to enhance transcription, and can be located anywhere associated with the gene it controls, for example, upstream, downstream, in intron, etc. Any enhancer element can be used in the polynucleotide cassette and gene therapy vector of the present disclosure, as long as it enhances gene expression when used in combination with a promoter.

[0095] The coding sequence expressed in a cell can be any polynucleotide sequence, e.g., a gene product, e.g., a gene or cDNA encoding a polypeptide, or an RNA-based therapeutic (e.g., siRNA, antisense, ribozyme, shRNA, etc.). The coding sequence can be heterologous to the operably linked promoter sequence, i.e., not naturally associated with it. Alternatively, the coding sequence can be endogenous to the operably linked promoter sequence, i.e., associated with the promoter in nature. The gene product can act endogenously or exogenously in the mammalian cell, e.g., secreted. For example, when the transgene is a therapeutic gene, the coding sequence can be a gene encoding a desired gene product, or a functional fragment or variant thereof, that can be used as a therapeutic to treat a disease or disorder. In various preferred embodiments, the transgene encodes human FANCA, i.e., SEQ ID NO: 25.

[0096] In one embodiment of the present invention, a transgene-encoding sequence is modified, or "codon-optimized," to enhance expression by replacing less frequently represented codons with more frequently represented codons. A coding sequence is a portion of an mRNA sequence that encodes an amino acid for translation. During translation, each of the 61 trinucleotide codons is translated into one of 20 amino acids, resulting in the degeneracy or redundancy of the genetic code. However, different cell types and different animal species utilize tRNAs (each carrying an anticodon) that encode the same amino acid at different frequencies. When a gene sequence contains a codon that is rarely represented by the corresponding tRNA, the ribosomal translation machinery slows down, potentially hindering efficient translation. Expression can be improved through "codon optimization" for a specific species, in which the coding sequence is altered to encode the same protein sequence but utilize codons utilized by highly represented and / or highly expressed human proteins (Cid-Arregui et al., 2003; J. Virol. 77:4928). In one aspect of the present invention, the coding sequence of the transgene is modified to replace codons that are expressed at low frequency in mammals or primates with codons that are expressed at high frequency in primates. For example, in some embodiments, the coding sequence encoded by the transgene encodes a polypeptide having at least 85% sequence identity, e.g., at least 90% sequence identity, e.g., at least 95% sequence identity, at least 98% identity, at least 99% identity, to a polypeptide encoded by a sequence disclosed above or herein, wherein at least one codon of the coding sequence has a higher tRNA frequency in humans than the corresponding codon in the sequence disclosed above or herein.

[0097] In additional embodiments of the present invention, transgene-encoding sequences are modified to enhance expression by terminating or removing open reading frames (ORFs) that do not encode the desired transgene. An open reading frame (ORF) is a nucleic acid sequence that follows a start codon and does not contain a stop codon. ORFs may be in either forward or reverse orientation, and may be "in-frame" or "out-of-frame" relative to the gene of interest. Such open reading frames may be expressed alongside the gene of interest in an expression cassette, potentially resulting in unwanted adverse effects. In one aspect of the present invention, transgene coding sequences are modified to remove open reading frames by further altering codon usage. This was done by eliminating the start codon (ATG) and introducing a stop codon (TAG, TAA, or TGA) into the reversed or out-of-frame ORF, while preserving the amino acid sequence and maintaining highly utilized codons in the gene of interest (i.e., avoiding codons with a frequency of <20%). In the present invention, the transgene-encoding sequence can be optimized by either codon optimization or removal of non-transgene ORFs, or by using both techniques. As will be apparent to those skilled in the art, it is preferable to remove or minimize non-transgene ORFs after codon optimization in order to remove ORFs introduced during codon optimization.

[0098] In some embodiments, the polynucleotide cassette comprises: (i) a phosphoglycerate kinase (PGK) promoter sequence or a functional variant or fragment thereof; (ii) a sequence encoding a human FANCA protein or a functional fragment or variant thereof; and (iii) Woodchuck hepatitis virus sequence posttranscriptional regulatory element (WPRE) Includes.

[0099] In some embodiments, the polynucleotide cassette comprises: (i) a human phosphoglycerate kinase (PGK) promoter sequence; (ii) a sequence encoding the human FANCA protein, and (iii) Mutant WPRE sequence Includes.

[0100] In some embodiments, the polynucleotide cassette comprises: a) a 5'LTR, optionally a modified 5'LTR; b) cPPT sequence, c) a PGK promoter sequence, optionally a human PGK promoter sequence; d) a sequence encoding the human FANCA protein, optionally a cDNA sequence or a codon-optimized sequence; e) the mutated wPRE sequence, and f) 3'LTR, optionally a modified 3'LTR Includes.

[0101] In one embodiment, the modified WPRE is WPRE * It is called WPRE. * is a modified WPRE that lacks an open reading frame (see, e.g., Schambach et al, 2006 Gene Ther. 13: 641-645).

[0102] In some embodiments, the gene transfer cassette comprises one or more additional elements, for example, one or more elements selected from the following: a 5'LTR, a 3'LTR, a cPPT, a CTS, an RRE, an enhancer sequence, and a packaging signal.

[0103] The RRE sequence improves the efficiency of gene transfer. In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the RRE sequence comprises or consists of any of the following sequences or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequences: TIFF2025043346000002.tif32155; or A sequence comprising or consisting of nucleotides 2649 to 2882 of SEQ ID NO:24.

[0104] The retroviral leader region contains a packaging signal (ψ) that is responsible for packaging the retroviral genome into a viral capsid. LV vectors were thought to require approximately 300 bp of the Gag gene in this region. Currently, this Gag sequence has been reduced to only 40 bp (Figure 65). In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the ψ sequence is the HIV-1 ψ sequence, or the ψ sequence comprises or consists of any of the following sequences or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequences: TIFF2025043346000003.tif18155; or A sequence comprising or consisting of polynucleotides 2031 to 2156 of SEQ ID NO:24.

[0105] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the truncated HIV-1 5'LTR comprises or consists of any of the following sequences or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of the following sequences: TIFF2025043346000004.tif25155; or A sequence comprising or consisting of polynucleotides 1586 to 9495 of SEQ ID NO:24.

[0106] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the HIV-1 self-inactivating 3' LTR comprises or consists of any of the following sequences or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequences: TIFF2025043346000005.tif32155; or A sequence comprising or consisting of polynucleotides 9262 to 9495 of SEQ ID NO:24.

[0107] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the human cytomegalovirus (CMV) immediate early promoter comprises or consists of any of the following sequences, functional fragments thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of the following sequences: TIFF2025043346000006.tif25156; or A sequence comprising or consisting of polynucleotides 1586 to 1789 of SEQ ID NO:24.

[0108] The cPPT, which promotes nuclear translocation of the pre-integration complex, along with the CTS, which is involved in the detachment of reverse transcriptase, have been shown to improve viral titers (Zennou, et al. 2000; Follenzi et al. 2000). In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the HIV-1 central polypurine tract and central terminal sequence (cPPT / CTS) comprises or consists of any of the following sequences, functional fragments thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of the following sequences: TIFF2025043346000007.tif25155; or A sequence comprising or consisting of nucleotides 3378 to 3495 of SEQ ID NO:24.

[0109] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the human phosphoglycerate kinase 1 (hPGK) promoter comprises or consists of any of the following sequences, functional fragments thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of the following sequences: TIFF2025043346000008.tif66156; or A sequence comprising or consisting of nucleotides 3541 to 4051 of SEQ ID NO:24.

[0110] Since most FA patients belong to the FA-A complementation group (Casado et al., 2007, Levitus et al., 2004, Taniguchi et al., 2006), in a specific embodiment the encoded therapeutic gene product is FANCA, although the present disclosure also contemplates that FA proteins of other complementation groups may also be delivered and thus may be encoded in the expression cassettes disclosed herein, for example, in place of FANCA.

[0111] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the polynucleotide sequence encoding FANCA is a human FANCA cDNA sequence comprising or consisting of the following sequence, or a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000009.tif72148TIFF2025043346000010.tif216148TIFF2025043346000011.tif216149TIFF2025043346000012.tif45148.

[0112] The present disclosure includes a plasmid comprising the expression cassette or transfer cassette described herein. In a specific embodiment, the plasmid is pCCL-PGK-FANCA-WPRE * (Figure 41; SEQ ID NO:24).

[0113] In certain embodiments, the disclosure includes a cell, e.g., a packaging cell or packaging cell line, e.g., 293 cell, comprising a plasmid disclosed herein. In specific embodiments, the cell comprises the plasmid shown in Figures 38-41.

[0114] In certain embodiments, the transfer cassettes or plasmids disclosed herein further comprise one or more additional elements, such as a CMV promoter and / or enhancer, an SV40 polyA sequence, an origin of replication, such as an SV40 ori sequence, or any element disclosed herein.

[0115] In specific embodiments of any transfer cassette, plasmid, or vector described herein, the human CMV enhancer comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000013.tif52148.

[0116] In specific embodiments of any transfer cassette, plasmid, or vector described herein, the simian virus 40 (SV40) poly(A) signal comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000014.tif18146.

[0117] In specific embodiments of any transfer cassette, plasmid, or vector described herein, the SV40 origin of replication comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000015.tif18148.

[0118] In some embodiments of any transfer cassette, plasmid, or vector described herein, the dNEF signal present in any expression cassette or gene delivery vector described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000016.tif11147.

[0119] In a specific embodiment of any transfer cassette, plasmid or vector described herein, the KanR sequence present in any expression cassette or gene delivery vector described herein comprises or consists of the following sequence: TIFF2025043346000017.tif100149.

[0120] In some embodiments of any of the transfer cassettes, plasmids, or vectors described herein, the rrnG terminator (the transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al, 1991)) present in any of the expression cassettes or gene delivery vectors described herein comprises or consists of the following sequence: TIFF2025043346000018.tif18147.

[0121] In some embodiments of any transfer cassette, plasmid, or vector described herein, the ori (high copy number ColE1 / pMB1 / pBR322 / pUC origin of replication) present in any expression cassette or gene delivery vector described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000019.tif80149.

[0122] In some embodiments of any transfer cassette, plasmid, or vector described herein, the CAP binding site present in any expression cassette or gene delivery vector described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000020.tif4128.

[0123] In some embodiments of any transfer cassette, plasmid, or vector described herein, the E. coli lac promoter present in any expression cassette or gene delivery vector described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000021.tif4128.

[0124] In some embodiments of any transfer cassette, plasmid, or vector described herein, the lac operator present in any expression cassette or gene delivery vector described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000022.tif4128.

[0125] In some embodiments of any transfer cassette, plasmid, or vector described herein, the T3 promoter (promoter of bacteriophage T3 RNA polymerase) present in any expression cassette or gene delivery vector described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000023.tif4128.

[0126] In some embodiments of any transfer cassette, plasmid, or vector described herein, the T7 promoter (promoter of the bacteriophage T7 RNA polymerase) present in any expression cassette or gene delivery vector described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000024.tif4128.

[0127] In some embodiments of any transfer cassette, plasmid, or vector described herein, the f1 ori (f1 bacteriophage origin of replication) present in any expression cassette or gene delivery vector described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000025.tif52151.

[0128] As discussed herein, the polynucleotide cassette of the present invention may contain an RNA export signal. Exemplary RNA export sequences include, but are not limited to, the wPRE. The wPRE significantly increases transgene expression in target cells by improving RNA stability in a transgene-, promoter-, and vector-independent manner (Zuffrey et al., 1999). However, it can express a truncated 60-amino acid protein derived from the WHV X gene, which is involved in liver cancer (Kingsman et al., 2005). Therefore, most preclinical protocols and clinical trials include a mutated version of the wPRE element (Zanta-Boussif et al., 2009). On the other hand, the use of two SV40-USE elements in SIN-LV vectors has been shown to be more effective than the wPRE sequence in suppressing transcriptional readthrough (Schambach et al., 2007). More specifically, the wPRE disclosed herein is a chimeric wPRE having 589 nucleotides (nucleotides 1-589) from the modified WPRE made by Axel Schambach (WO 2008136670A2; [5]) and 88 nucleotides (nucleotides 590-677) from the original wPRE (Zuffrey et al., 1999). The data disclosed herein demonstrate that this chimeric wPRE functions better than the original wPRE. The chimeric wPRE sequence includes the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025043346000026.tif86148.

[0129] In specific embodiments, the mutant WPRE sequence corresponds to nucleotides 8502-9178 of SEQ ID NO:24 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to this region of SEQ ID NO:24. *It comprises or consists of:

[0130] Other combinations of both elements disclosed herein or known in the art will be readily apparent to those skilled in the art.

[0131] Additionally, as will be appreciated by those of skill in the art, the polynucleotide cassette may optionally contain other elements, including, but not limited to, restriction sites to facilitate cloning and control elements for the particular gene expression vector.

[0132] In some aspects of the invention, polynucleotide cassettes of the invention are used to deliver genes to cells, e.g., to determine the effect of the gene on cell viability and / or function, to treat cellular disorders, etc. In various embodiments, delivery of viral vectors to cells by transduction can occur in vitro, ex vivo, or in vitro. Thus, in some aspects of the invention, a composition that provides expression of a transgene in a mammalian cell is a gene delivery vector that includes a polynucleotide cassette of the present disclosure, e.g., a gene transfer cassette.

[0133] Any convenient gene delivery vector useful for delivering polynucleotide sequences to mammalian cells is encompassed by the gene delivery vector of the present disclosure.For example, the vector can contain single-stranded or double-stranded nucleic acid, for example, single-stranded or double-stranded DNA.For example, the gene delivery vector can be DNA, for example, naked DNA, for example, a plasmid, a minicircle, etc.The vector can also contain single-stranded or double-stranded RNA, including modified forms of RNA.In another example, the gene delivery vector can be RNA, for example, mRNA or modified mRNA.

[0134] As another example, the gene delivery vector can be a viral vector derived from a virus, such as an adenovirus, adeno-associated virus, lentivirus (LV), herpesvirus, alphavirus, or retrovirus, such as Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), or Rous sarcoma virus (RSV). While embodiments involving the use of LV are described in more detail below, it is expected that those skilled in the art will understand that similar knowledge and techniques in the art may be applied to non-LV gene therapy vectors.

[0135] In some embodiments, the gene delivery vector is a self-limiting LV. In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the transfer cassette is the pCCL-SIN-cPPT / CTS-hPGK-hFANCA-WPRE (Figure 41) of the present disclosure, which comprises or consists of the following sequence or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:24: SEQ ID NO:24 is the pCCL-PGK-FANCA-WPRE of Figure 41 * Corresponding to the plasmid.

[0136] In one embodiment, the FANCA gene is delivered via a lentiviral vector (LV). The FANCA LV described herein utilizes a self-inactivating lentiviral vector (LV). In one embodiment, the FANCA LV contains the promoter of the human phosphoglycerate (PGK) gene. The safety profile of this vector is significantly improved compared to gamma-retroviral vectors already in clinical use, which have strong viral promoters.

[0137] In one embodiment, the lentiviral vector comprises the gene transfer cassette shown in FIG. 1, which comprises the sequence disclosed in SEQ ID NO:24, PGK-FANCA.WPRE. * LV. PGK-FANCA-WPRE * The LV gene expression cassette contains the human PGK promoter, the coding sequence of FANCA cDNA, and WPRE * and corresponds to nucleotides 3541 to 9178 of SEQ ID NO: 24. * The LV transfer cassette portion comprises approximately the 5' LTR (U5) to approximately the 3' LTR (U5) of the sequence shown in Figure 41. With respect to SEQ ID NO:24, nucleotides 1586-1789 of SEQ ID NO:24 comprise the human CMV immediate early promoter. Nucleotides 2031-2156 of SEQ ID NO:24 comprise the HIV1 psi packaging signal. Nucleotides 2649-2882 of SEQ ID NO:24 comprise the HIV1 RRE element. Nucleotides 3378-3495 of SEQ ID NO:24 comprise the HIV cPPT / CTS element. Nucleotides 3541-4051 of SEQ ID NO:24 comprise the hPGK promoter. Nucleotides 4078-8445 of SEQ ID NO:24 comprise the human FANCA-A cDNA. Nucleotides 8502-9178 of SEQ ID NO:24 comprise a mutated WPRE element. Nucleotides 9262 to 9495 of SEQ ID NO:24 contain the HIV delta U3'LTR.

[0138] In yet another embodiment, the lentiviral vector comprises the following sequence: (i) The lentiviral vector backbone derived from the initial pCCLsin-cppt-hPGK-eGFP-WPRE (Dull et al., 1998; J. Virol 72(11), 9873-9880). The pCCL backbone utilizes a heterologous CMV-HIV 5' LTR to drive high levels of viral RNA transcription in producer cells. Such a heterologous LTR frees the construct from the need to use the HIV Tat protein for the production of rHIV particles, and is therefore a safety feature. The U3 region of the 3' LTR contains a 400 bp deletion described in (Zufferey et al. J Virol, 1998), which confers self-inactivating properties to the vector; (ii) a cDNA of the human FANCA gene (4368 bp GeneBank accession number: X_99226 or as disclosed herein) encoding the FANCA protein (1455 AA) under the control of the human PGK promoter, which has been characterized by its stable activity in vivo and improved safety profile compared to other promoters already used in gene therapy; and (iii) a mutated version of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or WPRE that does not contain the 3' region of the sequence encoding the X protein and any remaining ORFs, as described by Schambach et al. (Gene therapy, 2006; 13, 641-645); * .

[0139] Gene therapy vectors encapsulating the polynucleotide cassettes of the present disclosure can be produced using standard methodologies. For example, in the case of LV virions, an LV expression vector according to the present invention is introduced into producer cells, followed by the introduction of an LV helper construct. The helper construct contains an LV coding region that can be expressed in the producer cells and complements LV helper functions not present in the LV vector. A helper virus and / or additional vectors that provide accessory functions capable of supporting efficient LV virus production are then introduced into the producer cells. The producer cells are then cultured to produce LV. These steps are carried out using standard methodologies. In a specific embodiment, the plasmids shown in Figures 38-41 are used to produce the gene delivery vector.

[0140] Any suitable method can be used to generate viral particles for delivery of the polynucleotide cassettes of the present invention, including, but not limited to, those described in the Examples below. A concentration of viral particles suitable for efficient transduction of mammalian cells can be prepared for contacting mammalian cells in vitro or in vivo. For example, viral particles can be prepared at a concentration of 10 per ml. 8 Vector genomes or more, e.g., 5 x 10 per ml 8 Vector genome: 10 per ml 9 Vector genome: 5 x 10 per ml 9 Vector genome, 10 per ml 10 Vector genome, 5 × 10 per ml 10 Vector genome: 10 per ml 11 Vector genome: 5 x 10 per ml 11 Vector genome: 10 per ml 12 Vector genome: 5 x 10 per ml 12 Vector genome: 10 per ml 13 Vector genome: 1.5 x 10 per ml 13 Vector genome: 3 x 10 per ml 13 Vector genome: 5 x 10 per ml 13Vector genome: 7.5 x 10 per ml 13 Vector genome: 9 x 10 per ml 13 Vector genome: 1 x 10 per ml 14 Vector genome, 5 × 10 per ml 14 vector genomes, or more, typically 1 x 10 per ml 15 It can be formulated at a concentration equal to or less than the vector genome.

[0141] In preparing the LV compositions of the present invention, any host cell can be used to produce LV virions, including, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell can be a packaging cell in which the LV rep and gap genes are stably maintained in the host cell, or a producer cell in which the LV vector genome is stably maintained and packaged. Exemplary packaging and producer cells are derived from SF-9 cells, 293 cells, A549 cells, or HeLa cells. The LV vector is purified and formulated using standard techniques known in the art.

[0142] In certain aspects, the invention includes a cell comprising a gene expression cassette, gene transfer cassette, or gene delivery vector disclosed herein. In related aspects, the cell is transduced by a gene delivery vector comprising an expression cassette disclosed herein, or has an expression cassette disclosed herein integrated into the genome of the cell.

[0143] In some embodiments, the cell is a cell used to produce a viral gene delivery vector, e.g., a packaging cell.

[0144] In other embodiments, the cells are cells delivered to a subject to provide the subject with a gene product encoded by the expression cassette. Thus, in some embodiments, the cells are autologous to or obtained from the subject to be treated. In other embodiments, the cells are allogeneic to the subject to be treated or obtained from a donor other than the subject to be treated. In specific embodiments, the cells are mammalian cells, e.g., human cells. In some embodiments, the cells are blood cells, erythrocytes, hematopoietic progenitor cells, bone marrow cells, e.g., lineage-depleted bone marrow cells, hematopoietic stem cells (e.g., CD34+), or committed hematopoietic erythroid progenitor cells. In specific embodiments, the cells are CD34+ cells obtained from a subject to be treated with CD34+ cells after transduction with a gene delivery vector disclosed herein. In specific embodiments, the cells are CD34+ FA cells obtained from a subject diagnosed with FA.

[0145] The present invention includes pharmaceutical compositions comprising the polynucleotide cassettes, gene delivery vectors, or cells described herein and pharmaceutically acceptable carriers, diluents, or excipients. The polynucleotide cassettes, gene delivery vectors, or cells of the present invention can be combined with pharmaceutically acceptable carriers, diluents, and reagents useful in preparing formulations, including excipients that are generally safe, non-toxic, and, preferably, acceptable for use in primates. Such excipients can be solid, liquid, semisolid, or, in the case of aerosol compositions, gaseous. Examples of such excipients, carriers, or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulation. The solutions or suspensions used for formulation may contain sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetic acid, citric acid, or phosphate; surfactants such as Tween 20 to prevent aggregation; and compounds for adjusting osmotic pressure such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. In a specific embodiment, the pharmaceutical composition is sterile.

[0146] Pharmaceutical compositions suitable for use in the present invention further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.

[0147] Sterile solutions can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the above-listed ingredients, followed by filtration and sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which gives a powder of the active ingredient plus any additional desired ingredients from a previously sterilized-filtered solution.

[0148] In one embodiment, the composition is prepared with a carrier that protects the gene cassette or expression vector from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.Methods for preparing such formulations will be clear to those skilled in the art.Materials may also be commercially available.

[0149] It is particularly advantageous to formulate oral, ophthalmic or parenteral compositions in dosage unit form because of easy administration and uniform dosage.Dosage unit form, as used herein, refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.The specification for the dosage unit form of the present invention is dictated by and directly depends on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and the limitations inherent in the field of preparation of such active compound for individual treatment.

[0150] The pharmaceutical compositions may be included in a container, pack, or dispenser, eg, a syringe, eg, a pre-filled syringe, together with instructions for administration.

[0151] The pharmaceutical compositions of the present invention include pharmaceutically acceptable salts, esters, or salts of such esters, or any other compounds that can provide (directly or indirectly) biologically active metabolites or residues thereof upon administration to an animal, including a human.

[0152] The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of a compound of the present invention; i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects. A variety of pharmaceutically acceptable salts are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and more recent editions), "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J.Pharm.Sci. 66:2 (1977). For a review of suitable salts, see also Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0153] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Metals used as cations include sodium, potassium, magnesium, calcium, and the like. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, e.g., Berge et al., "Pharmaceutical Salts," J. Pharma Sci., 1977, 66, 119). Base addition salts of acidic compounds are prepared in the conventional manner by contacting the free acid form with a sufficient amount of the desired base to produce the salt. The free acid form can be regenerated in the conventional manner by contacting the salt form with an acid and isolating the free acid. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the respective free acids for purposes of the present invention.

[0154] The polynucleotide cassettes, gene delivery vectors, e.g., recombinant viruses (virions), or cells (e.g., transduced by the gene delivery vectors disclosed herein) of the invention can be incorporated into pharmaceutical compositions for administration to mammalian patients, particularly primates, and more particularly humans. The polynucleotide cassettes, gene delivery vectors, e.g., virions, or cells of the invention may be formulated in a non-toxic, inert, pharmaceutically acceptable aqueous carrier, preferably at a pH in the range of 3 to 8, more preferably in the range of 6 to 8. Such sterile compositions will contain vectors or virions containing a nucleic acid encoding a therapeutic molecule dissolved in an aqueous buffer having an acceptable pH upon reconstitution.

[0155] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of the cells, vectors, or virions disclosed herein, mixed with a pharmaceutically acceptable carrier and / or excipient, such as saline, phosphate-buffered saline, phosphate, and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, and sugars include octylphenoxypolyethoxyethanol compounds, monostearate polyethylene glycol compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citric acid, acetic acid, Ringer's solution, Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol. Preferably, the formulation is stable at 4°C for at least 6 months.

[0156] In some embodiments, the pharmaceutical compositions provided herein contain a buffer solution such as phosphate-buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffer solutions known to those skilled in the art, such as those described in Good et al. (1966) Biochemistry 5:467. The pH of the buffer solution in a pharmaceutical composition containing a tumor suppressor gene contained in an adenoviral vector delivery system can be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0157] In one embodiment, the viral vector can be administered in a volume of, but not limited to, 1 x 10 8 Vector genomes or more, e.g., 1 x 10 9 Vector genome, 1 × 10 10 Vector genome, 1 × 10 11 Vector genome, 1 × 10 12 vector genome, or 1 × 10 13vector genome, or more, in certain cases, 1 x 10 14 Vector genomes, typically 4 x 10 15 The vector genome may be formulated into any suitable unit dosage containing less than about 5×10 15 Vector genome, e.g., 1 x 10 14 Vector genome or less, e.g., 1 x 10 13 Vector genome, 1 × 10 12 Vector genome, 1 × 10 11 Vector genome, 1 × 10 10 vector genome, or 1 × 10 9 vector genome, or less, in certain cases, 1 x 10 8 Vector genomes or less, typically 1 x 10 8 In some cases, the unit dosage is 1 x 10 10 ~1×10 11 In some cases, the unit dosage is 1 x 10 10 ~3×10 12 In some cases, the unit dosage is 1 x 10 9 ~3×10 13 In some cases, the unit dosage is 1 x 10 8 ~3×10 14 In one embodiment, the range is about 5 x 10 10 ~Approx. 1×10 11 In some embodiments, the range is about 1 x 10 9 ~Approx. 1×10 10 It is a vector genome.

[0158] In some cases, the unit dosage of the pharmaceutical composition can be measured using the multiplicity of infection (MOI). MOI refers to the ratio or multiple of the vector or virus genome to the cells to which the nucleic acid can be delivered. In some cases, the MOI is 1×10 6 In some cases, the MOI can be 1 x 105 ~1×10 7 In some cases, the MOI can be 1 x 10 4 ~1×10 8 In some cases, the recombinant viruses of the present disclosure may be at least about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 In some cases, the recombinant viruses of the present disclosure are administered at an MOI of 1 x 10 8 ~3×10 14 In some cases, the recombinant viruses of the present disclosure are at most 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 In some embodiments, the range is from about 20 to about 400 MOI.

[0159] In some aspects, the amount of pharmaceutical composition is about 1×10 8~Approx. 1×10 15 Recombinant virus, approximately 1 × 10 9 ~Approx. 1×10 14 Recombinant virus, approximately 1 × 10 10 ~Approx. 1×10 13 recombinant virus, or approximately 1 x 10 11 ~Approx. 3×10 12 Includes recombinant viruses.

[0160] method As described in more detail below, the polynucleotide cassettes and gene delivery vectors of the present invention, collectively referred to herein as "compositions of the present invention," are useful in expressing a transgene, e.g., FANCA, in animal, e.g., mammalian, or human cells. For example, compositions of the present invention can be used in research, e.g., to determine the effect of a gene on cell viability and / or function. As another example, compositions of the present invention can be used in medicine, e.g., to treat disorders such as FA. Thus, in some aspects of the present invention, methods of expressing a gene in a cell are provided, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the contacting is performed in vitro. In some embodiments, the contacting is performed in vivo, i.e., a composition of the present invention is administered to a subject.

[0161] When mammalian cells are contacted in vitro or in vivo with the polynucleotide cassette of the present invention or a gene delivery vector containing the polynucleotide cassette of the present invention, the cells may be derived from any mammalian species, such as rodents (e.g., mice, rats, gerbils, squirrels), rabbits, cats, dogs, goats, sheep, pigs, horses, cows, primates, or humans. Cells may be derived from established cell lines or primary cells, and the terms "primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein to refer to cells and cell cultures derived from a subject and grown in vitro for a limited number of culture passages, i.e., divisions. For example, primary cultures are cultures that may have been passaged 0, 1, 2, 4, 5, 10, or 15 times, a number of times that is not sufficient to cause a crisis. Typically, the primary cell lines of the invention are maintained in vitro for fewer than 10 passages.

[0162] Embodiments of the present invention include mammalian cells (e.g., CD34+ cells) transduced with a viral delivery vector, e.g., an LV vector containing the human FANCA gene. Additionally, the present invention includes methods of transducing mammalian cells, e.g., human hematopoietic stem cells or other cells, described herein, comprising contacting the cells with a gene delivery vector, e.g., an LV vector, comprising an expression cassette disclosed or described herein. In some embodiments, the cells were previously obtained from the subject to be treated or another donor. In a specific embodiment, the subject has been diagnosed with Fanconi anemia, and the cells are transduced with an LV comprising an expression cassette encoding a FANCA coding region or cDNA. It is understood that the disclosed methods, e.g., methods used to deliver a FANCA gene product to a subject using a FANCA cDNA sequence, can also be used to treat Fanconi anemia. In a specific embodiment, the transduced cells are a cell population obtained from a subject with FA to be treated with the cells after they have been transduced. The cells can be obtained from bone marrow or blood. In some embodiments, a subject with FA is treated with a stem cell mobilizing agent, after which blood is drawn from the subject, red blood cells are removed, and CD34+ cells are selected. After selection, the cells are transduced. In specific embodiments, the transduced cells are stored or frozen before use, while in some embodiments, they are provided to the subject immediately or shortly thereafter, for example, within 1 hour, 2 hours, or 4 hours, after transduction.

[0163] In some embodiments, when transducing cells with a gene delivery vector disclosed herein, the cells are contacted with the gene delivery vector for about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 12 hours, about 16 hours, about 18 hours, about 20 hours, about 24 hours, about 36 hours, about 48 hours, or about 60 hours. In some embodiments, the cells are transduced for less than 60 hours, less than 48 hours, less than 36 hours, or less than 24 hours.

[0164] The polynucleotide cassette of the present invention or a gene delivery vector containing the polynucleotide cassette of the present invention can be provided to the cells of the present invention one or more times, for example, once, twice, three times, or more than three times. After each contact event, the cells are incubated with the agent for a certain amount of time, for example, 16 to 24 hours, after which time the medium is replaced with fresh medium and the cells are further cultured. The contacting of the cells can be carried out in any culture medium and under any culture conditions that promote cell survival. The culture can contain a growth factor to which the cells are responsive. A growth factor, as defined herein, is a molecule that can promote cell survival, proliferation, and / or differentiation in either culture or intact tissue through a specific effect on a transmembrane receptor. Growth factors include polypeptide and non-polypeptide factors.

[0165] Typically, an effective amount of the gene delivery vector or transduced cells containing the polynucleotide cassette is provided to cause transgene expression in cells. As described elsewhere herein, an effective amount can be readily determined empirically, for example, by detecting the presence or level of the transgene gene product, detecting effects on cell viability or function, etc. Typically, an effective amount of the polynucleotide cassette of the present invention or a gene delivery vector containing the polynucleotide cassette of the present invention will promote transgene expression in cells greater than the same amount of polynucleotide cassettes known in the art. Typically, expression will be enhanced by 2-fold or more, for example, 3-fold, 4-fold, or 5-fold or more, and in some cases 10-fold, 20-fold, or 50-fold or more, for example, 100-fold, compared to expression from a reference polynucleotide cassette or control polynucleotide cassette known in the art.

[0166] When cells are contacted in vivo with the polynucleotide cassette of the present invention or a gene delivery vector containing the polynucleotide cassette of the present invention, the subject can be any mammal, for example, a rodent (e.g., mouse, rat, gerbil), rabbit, cat, dog, goat, sheep, pig, horse, cow, or primate. In a further preferred embodiment, the primate is a human. In a further embodiment, the cell is a CD34+ cell.

[0167] The methods and compositions of the present disclosure are useful, for example, in the treatment of Fanconi anemia.

[0168] In some embodiments, the method provides a therapeutic benefit, such as inhibiting the onset of a disorder, halting the progression of a disorder, reversing the progression of a disorder, etc. For example, in one embodiment, the disorder is BMF. In one embodiment, the disorder is thrombocytopenia. In another embodiment, the disorder is leukopenia. In one embodiment, the disorder is pancytopenia. In one embodiment, the disorder is neutropenia. In another embodiment, the disorder is anemia. In some embodiments, the method includes detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such measurement of therapeutic effect is applicable to the individual disease being treated, and will find appropriate detection methods to be used to measure the therapeutic effect.

[0169] In another embodiment, the present invention includes a method for treating a disease in a subject in need thereof, comprising providing to the subject an effective amount of cells transduced by a gene delivery vector, e.g., a viral vector, that express a therapeutic gene product in the cells. In a specific embodiment, the cells are autologous to the subject. In some embodiments, the cells are erythroid cells, e.g., hematopoietic stem cells or committed hematopoietic erythroid progenitor cells. In some embodiments, the cells are bone marrow cells, e.g., lineage-depleted bone marrow cells. In a specific embodiment, the method is used to treat FA, and the viral vector is an LV comprising an expression construct disclosed herein that includes a human PGK promoter operably linked to the cDNA or coding sequence of the FANCA gene and a mutant wPRE disclosed herein. In a specific embodiment, the cells are provided to the subject parenterally, e.g., via intravenous injection.

[0170] In another embodiment, the present invention includes a method of treating FA in a subject in need thereof, comprising providing to the subject an effective amount of autologous CD34+ stem cells transduced with an LV vector expressing a FANCA cDNA in the cells, wherein the LV vector comprises a human PGK promoter operably linked to the FANCA cDNA or coding sequence and a mutant wPRE sequence disclosed herein. In a specific embodiment, the cells are hematopoietic stem cells or committed erythropoietic progenitor cells, e.g., bone marrow cells. In a specific embodiment, the cells are provided to the subject parenterally, e.g., via intravenous injection.

[0171] Transgene expression using the transgenes of the present invention is expected to be robust. Thus, in some cases, transgene expression, as detected, for example, by measuring gene product levels, measuring therapeutic efficacy, etc., may be observed within two months after administration of a composition of the present invention, e.g., within four weeks, three weeks, or two weeks after administration, or earlier, e.g., within one week after administration. Transgene expression is expected to persist over time. Thus, in some cases, transgene expression, as detected, for example, by measuring gene product levels, measuring therapeutic efficacy, etc., may be observed two or more months, e.g., four, six, eight, or ten months, or later, in some cases, one year or more, e.g., two, three, four, or five years, after administration of a composition of the present invention, and in certain cases, five years or more.

[0172] In certain embodiments, the method includes detecting expression of the transgene in a cell or subject, wherein expression is enhanced relative to expression from a polynucleotide cassette that does not include one or more improved elements of the present disclosure. Typically, expression will be enhanced by 2-fold or more, e.g., 3-fold, 4-fold, or 5-fold or more, and in some cases 10-fold, 20-fold, or 50-fold or more, e.g., 100-fold, relative to expression from a reference, i.e., control, polynucleotide cassette known in the art, as evidenced, e.g., by earlier detection, higher levels of gene product, a stronger functional effect on the cell, etc.

[0173] Typically, when the composition of the invention is an LV comprising a polynucleotide cassette of the present disclosure, the effective amount to achieve the change is about 1 x 10 8 Vector genomes or more, in some cases, 1 × 10 9 Vector genome, 1 × 10 10 Vector genome, 1 × 10 11 Vector genome, 1 × 10 12 vector genome, or 1 × 10 13vector genome, or more, in certain cases, 1 x 10 14 Vector genome or more, typically 1 x 10 15 In some cases, the amount of vector genome delivered will be at most about 1 x 10 15 Vector genome, e.g., 1 x 10 14 Vector genome or less, e.g., 1 x 10 13 Vector genome, 1 × 10 12 Vector genome, 1 × 10 11 Vector genome, 1 × 10 10 vector genome, or 1 × 10 9 vector genome, or less, in certain cases, 1 x 10 8 Vector genome, typically 1 x 10 8 In some cases, the amount of vector genome delivered is 1×10 or more. 10 ~1×10 11 In some cases, the amount of vector genome delivered is 1×10 10 ~3×10 12 In some cases, the amount of vector genome delivered is 1×10 9 ~3×10 13 In some cases, the amount of vector genome delivered is 1×10 8 ~3×10 14 It is a vector genome.

[0174] In some cases, the amount of pharmaceutical composition administered can be measured using the multiplicity of infection (MOI). In some cases, MOI can refer to the ratio or multiple of vector or viral genome to cells into which the nucleic acid is delivered. In some cases, MOI is 1×10 6 In some cases, the MOI can be 1 x 10 5 ~1×10 7 In some cases, the MOI can be 1 x 10 4 ~1×10 8In some cases, the recombinant viruses of the present disclosure may be at least about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 In some cases, the recombinant viruses of the present disclosure are administered at an MOI of 1 x 10 8 ~3×10 14 In some cases, the recombinant viruses of the present disclosure are at most about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 It's MOI.

[0175] In some aspects, the amount of pharmaceutical composition is about 1×10 8 ~Approx. 1×10 15 recombinant virus particles, approximately 1 x 10 9 ~Approx. 1×10 14 recombinant virus particles, approximately 1 x 10 10 ~Approx. 1×10 13particles of recombinant virus, or approximately 1 x 10 11 ~Approx. 3×10 12 The recombinant virus particles are included.

[0176] Any total number of viral particles suitable to provide adequate transduction of cells to impart a desired effect or treat a disease can be administered to a mammal. In various preferred embodiments, at least 10 8 pieces; 5×10 8 pieces;10 9 pieces; 5×10 9 pieces, 10 10 pieces, 5×10 10 pieces;10 11 pieces; 5×10 11 pieces;10 12 pieces; 5×10 12 pieces;10 13 pieces; 1.5×10 13 pieces; 3×10 13 pieces; 5×10 13 pieces;7.5×10 13 pieces; 9×10 13 pieces, 1×10 14 virus particles, or 5 x 10 14 virus particles, or more, typically 1 x 10 15 No more than 10 viral particles are injected. Any suitable number of administrations of the vector to the mammalian or primate eye can be performed. In one embodiment, the method involves a single administration; in other embodiments, multiple administrations are performed over time as deemed appropriate by the attending physician. In some embodiments, at least 2 x 10 viral particles are injected to provide high transduction efficiency. 8 VG / ml or 5×10 5cells / ml are required in a single administration (24-hour transduction). Individual doses are typically equal to or greater than the amount necessary to produce a measurable effect in a subject and can be determined based on the pharmacokinetics and pharmacology of the absorption, distribution, metabolism, and excretion ("ADME") of the compositions of the invention or their by-products, and thus, the distribution of the composition in the subject. This includes consideration of not only the dosage, but also the route of administration. Effective amounts for doses and / or dosing regimens can be readily determined empirically from preclinical assays, safety and escalation and dose ranging trials, individual physician-patient relationships, and in vitro and in vivo assays such as those described herein and illustrated in the Examples.

[0177] In some embodiments, the dose of cells a patient receives by infusion is the dose resulting from the transduction process. In various preferred embodiments, the dose is at least about 1 x 10 1 , 1x10 2 , 1x10 3 , 1x10 4 , 1x10 5 , 1x10 6 , 1x10 7 , 1x10 8 or more CD34+ cells / KG of patient body weight are infused into the patient. In some embodiments, 1 x 10 6 ~4x10 6 Between 3x10 CD34+ cells / KG of patient body weight are infused into the patient. 5 and 4x10 6 CD34 + Cells / Kg of patient body weight are infused into the patient. In some embodiments, the cells are infused into the patient in a single dose. In other embodiments, the cells are infused into the patient in multiple doses. The transduced cells can be infused immediately after the transduction process is complete.

[0178] After integration, the therapeutic protein (e.g., human FANCA protein) is expressed by the cells. The transduced FA cells are genetically corrected and can therefore activate the FA pathway by monoubiquitinating FANCD2 and FANCI. These proteins, as seen in healthy cells, migrate to areas of DNA damage and cooperate with other DNA repair proteins to promote DNA repair in these cells.

[0179] As described in more detail in the Examples, preclinical in vitro data using BM samples from human FA patients have already demonstrated the efficacy of FANCA LV in correcting the phenotype of these cells.

[0180] Thus, the present invention provides a method for treating hematological symptoms of FA. In one embodiment, the hematological symptom of FA is selected from one or more of BMF, thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia. In a specific embodiment, the hematological symptom is bone marrow failure (BMF), which occurs at a young age in most FA patients. In one embodiment, the hematological symptom is thrombocytopenia. In another embodiment, the hematological symptom is leukopenia. In one embodiment, the hematological symptom is pancytopenia. In one embodiment, the hematological symptom is neutropenia. In another embodiment, the hematological symptom is anemia. In one embodiment, the hematological symptom is a combination of two or more of BMF, thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia.

[0181] FANCA LV does not directly treat solid tumors that may develop in more advanced stages of the disease. However, the improvement of the hematological status of FA patients treated with hematopoietic gene therapy may also improve immunological surveillance for the progression of solid tumors. Therefore, indirect antitumor effects may also occur as a result of treating FA patients with FANCA LV.

[0182] For gene therapy for FA to be successful, it is beneficial to collect a "sufficient" number of hematopoietic stem cells (HSCs) from a subject.

[0183] In one embodiment, HSCs are obtained or collected from a bone marrow sample. In one embodiment, red blood cells are removed from the bone marrow sample. In some embodiments, CD16+ white blood cells are removed from the bone marrow sample. In some embodiments, the cells remaining after the removal technique are washed. In another embodiment, non-specific IgG is added to the washed cells. In some embodiments, the non-specific IgG is flebogamma. CD34+ cells can then be selected from the washed cells. In one embodiment, CD34+ cells can be selected from the bone marrow sample. The method of selecting CD34+ cells can be positive selection, negative selection, or a combination thereof.

[0184] In another embodiment, HSCs are obtained from peripheral blood. In one embodiment, red blood cells are removed from the peripheral blood sample. In some embodiments, CD16+ white blood cells are removed from the blood sample. In some embodiments, blood cells remaining after the removal technique are washed. In another embodiment, non-specific IgG is added to the washed cells. In some embodiments, the non-specific IgG is flebogamma. CD34+ cells can then be selected from the washed cells. In one embodiment, CD34+ cells can be selected from the peripheral blood sample. The method of selecting CD34+ cells can be positive selection, negative selection, or a combination thereof.

[0185] In some embodiments of the present invention, HSCs are obtained from a subject after mobilization. Mobilization can be achieved by treating the subject with a drug or compound that mobilizes stem cells from the bone marrow into the blood. The stem cells can be collected and stored. In some embodiments, mobilization is achieved by treating the subject with G-CSF (filgrastim). In other embodiments, mobilization is achieved by treating the subject with plerixafor. In yet other embodiments, mobilization can be achieved by treating the subject with a combination of filgrastim and plerixafor. (Figures 11 and 14)

[0186] In one embodiment, at least 1-4 x 10 per kilogram of patient body weight 6 CD34 + Corrective cells (e.g., FANCA-transduced HSCs) are administered to restore hematopoietic capacity in unadjusted FA patients. In some embodiments, the transduced cells are infused or administered to the patient immediately after transduction. (Figure 11) In other embodiments, the transduced cells are frozen prior to infusion or administration to the patient. (Figure 11)

[0187] Genetic correction of HSCs from FA patients, followed by autologous transplantation of these cells (hematopoietic gene therapy) is a good alternative for FA patients, especially those who lack HLA-matched siblings. In one embodiment, hematopoietic gene therapy is the preferred treatment regimen for patients who lack HLA-matched siblings. In another embodiment, hematopoietic gene therapy is the preferred treatment regimen for patients who have HLA-matched siblings.

[0188] All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the methods and / or materials described in those publications. In case of conflict, it is understood that the present disclosure supersedes any disclosure of the incorporated publications.

[0189] It should further be noted that each claim may be drafted to exclude any optional element, in which case this declaration is intended to be a predicate to the use of exclusive terminology such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of each claim element.

[0190] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0191] Because FA-A is the most common complementation group in FA patients (Casado et al., 2007, Taniguchi et al., 2006), vectors expressing the FANCA gene and / or the EGFP marker gene are the focus of the examples, but other FANCA genes can be utilized to treat other complement groups as well. [Example]

[0192] Example 1 FANCA lentiviral vector Because the integration pattern of LV is more stable than that of RV (Gonzalez-Murillo et al., 2008; Modlich et al., 2009; Montini et al., 2006; Mitchell RS, Beitzel BF, Schroder AR et al. Plos Biol. 2004;2:E234; Montini E et al. J Clin Invest. 2009;119:964-975; Schroder AR et al. Cell. 2002;110-521-529), we aimed to develop LV as a therapeutic vector to correct the phenotype of FA cells (Gonzalez-Murillo et al., 2009). Furthermore, recent studies have shown that LV, which has a strong internal promoter, can also transactivate neighboring genes (Modlich et al., 2009). LVs have been considered for clinical use as a compromise between therapeutic efficacy and the risk of transactivation of neighboring genes. Therefore, the goal was to define a threshold level of FANCA expression that could be therapeutic in order to suppress the risk of gene transactivation by enhancers / promoters that drive therapeutic gene expression. The effects of FANCA LVs were first tested in vitro in FA-A LCLs, then in primary BM samples from FA-A patients, and finally in vivo in a mouse model of FA-A.

[0193] To this end, we constructed LVs expressing FANCA under the control of different promoters: the vav, PGK, CMV, and SFFV promoters. Figure 1 provides an overview of the medical applications. Figure 2A shows a schematic diagram of LVs expressing FANCA under the control of different internal promoters. Furthermore, we also investigated the influence of the post-transcriptional WPRE element on both the expression level of FANCA and the therapeutic efficacy of LVs. Initially, all LVs were packaged with chimeric GALV-TR envelopes. 1–2 x 10 6Titers of tu / ml were obtained by conventional methods, and transduction was performed at an estimated MOI of 1-2 tu per cell. Figure 2B shows Western blot analysis of FANCA in transduced FA-A cells.

[0194] To determine the levels of FANCA mRNA conferred by each vector, transduced FA-A lymphoblastoid cell lines (LCLs) were selected with 30 nM MMC for 5 days. After the selection process, transduced FA-A LCLs contained 0.81–3.04 copies of each LV per cell (Table 1). Unselected FA-A LCLs transduced with EGFP-LVs and LCLs derived from healthy donors (HDs) were used as controls.

[0195] Total FANCA mRNA levels and relative FANCA mRNA levels per LV copy number were determined in LCLs transduced with different LVs (Table 1). Compared to the FANCA mRNA levels observed in HD LCLs, similar levels of FANCA mRNA / copy were observed in FA-A cells transduced with vav-FANCA and PGK-FANCA LVs. CMV-FANCA and, even more significantly, SFFV-FANCA LVs conferred supraphysiological levels of FANCA mRNA / copy (3.6- and 5.6-fold, respectively). WPRE or mutant WPRE *PGK-FANCA LVs carrying the sequence ( Schambach et al., 2006 ) increased FANCA mRNA levels by 2.3–2.6-fold compared with PGK-LVs without the WPRE. Other studies (Schambach et al., 2006; Zanta-Boussif MA, Charrier S, Brice-Ouzet A Et al., Validation of a Mutated Pre Sequence Allowing High and Sustained Transgene Expression While Abrogating WHV-X Protein Synthesis: Application to the Gene Therapy of WAS. Gene Ther. 2009; 16: 605-619; Zufferey R, Donello JE, Trono D, Hope TJ. Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors. J Virol. 1999; 73: 2886-2892) as well as WPRE or WPRE *Insertion of the sequence significantly increased FANCA mRNA levels in cells transduced with PGK-FANCA LV. Because the wild-type WPRE element encodes a C-terminally truncated version of the hepatitis virus X protein, which may mediate oncogenic effects (Bouchard MJ, Schneider RJ. The Enigmatic X Gene of Hepatitis B Virus. J Virol. 2004;78:12725-12734. 2004; Kingsman SM, Mitrophanous K, Olsen JC. Potential Oncogene Activity of the Woodchuck Hepatitis Post-Transcriptional Regulatory Element (WPRE). Gene Ther. 2005;12:3-4), a mutant WPRE lacking any residual open reading frame was inserted. * LV with sequence (Schambach et al, 2006) is considered more suitable for clinical use.

[0196] Table 1. FANCA expression levels in FA-A LCLs transduced with FANCA-expressing lentiviral vectors. TIFF2025043346000027.tif84165 * To estimate the levels of FANCA mRNA and FANCA protein per FANCA copy, genomic FANCA was considered to be two copies in healthy donor LCLs.

[0197] To test whether the differences in FANCA mRNA expression could be confirmed at the protein level, Western blot analysis (Figure 2B) was performed using the samples shown in Table 1. As seen in the FANCA mRNA determination, FANCA protein values ​​correlated not only with protein loading but also with the proviral copy number determined in each transduced FA-LCL. Compared with the FANCA levels / copy determined in HD-LCL, all tested FANCA-LVs, except for SFFV-FANCA LVs, conferred essentially normal levels of FANCA / copy. In this example, the relative FANCA levels / copy were 3.4-fold higher than those determined in HD-LCLs. Western blots restained with anti-FANCD2 demonstrated that FA-A LCLs transduced with a control vector were unable to monoubiquitinated FANCD2, whereas FA-A LCLs transduced with either type of FANCA-LV expressed both nonubiquitinated and monoubiquitinated forms of FANCD2, consistent with a functional FA pathway in these cells.

[0198] By comparing the FANCA expression levels conferred by different LVs in FA-A LCLs with those observed in healthy donor (HD) LCLs (which contain two copies of FANCA), we concluded that insertion of two LV copies per cell can produce physiological levels of therapeutic protein, except in the case of SFFV-LVs, which conferred supraphysiological levels of FANCA. Achieving this copy number may meet the requirements for clinical trials in FA patients, where a transduction efficiency of at least 50% may be desirable due to the low number of precursors present in the BM of these patients (Gonzalez-Murillo et al., 2009, Jacome et al., 2006, Kelly et al., 2007; Larghero J, Marolleau JP, Soulier J et al. Hematopoietic Progenitor Cell Harvest and Functionality in Fanconi Anemia Patients. Blood. 2002;100:3051).

[0199] To analyze potential differences in the therapeutic effects of different FANCA-expressing LVs, we determined the efficacy of each vector in correcting the MMC hypersensitivity of FA-A lymphoblastoid cell lines (LCLs). To this end, FA-A LCLs were transduced with different LVs (Figure 3A) and then exposed to increasing concentrations of MMC. The viability of transduced cells was then determined. As shown in Figure 3A, all tested FANCA-LVs were equally effective in reversing the hypersensitivity of FA-A LCLs. Similarly, all vectors promoted the generation of nuclear FANCD2 foci in MMC-treated cells, as well as the FA pathway, which was functional in FA-A cells transduced with either FANCA-LV (Figure 3B).

[0200] Example 2 Mouse model studies To assess the replicability of BM samples from either genetically corrected or uncorrected FA patients, several groups have transplanted BM cells from FA patients into immunodeficient mice, but in no instance have significant engraftment been reported, likely due to the low numbers of hematopoietic precursors and HSCs present in the BM of these patients.

[0201] To evaluate the in vivo efficacy of this therapeutic, we used a mouse model of FA-A containing a deletion in the Fanca gene. In contrast to FA patients, these animals do not develop overt hematological disorders. Nevertheless, their BM progenitor cells, similar to those in FA patients, are highly sensitive to MMC (Rio et al., 2002). Therefore, to determine whether FANCA LVs (Figure 4A) stably correct the phenotype of HSCs derived from FA-A mice in vivo, we transduced BM cells from FA-A mice with FANCA LVs and then transplanted them into irradiated FA-A recipients (Figure 4B). To assess whether the hematopoietic precursor phenotype was corrected after transplantation of the genetically corrected cells, BM samples from transplanted FA-A mice were cultured in methylcellulose in the absence and presence of MMC (Figure 4C). Additional data confirming the integration of the therapeutic cassette are shown in Figures 42–44. Linear amplification-augmented (LAM) PCR is a method for investigating the integration sites of different integration vectors into the genome. PCR products starting from a vector of known sequence and extending through the unknown flanking genome are generated and sequenced to identify the genomic location of vector integration. Figure 42 shows LAM-PCR analysis of FANCA-LV insertion sites in FA hematopoietic stem cells (HSCs). Figure 43 shows LAM-PCR results for tracking FANCA-LV-treated cells. Figure 44 shows clonal diversity in Fanca- / - recipients transplanted with LV-corrected HSCs.

[0202] One month after transplantation, normal blood counts were observed in these animals, indicating no overt toxicity of LV. At this time point, the number of proviral FANCA copies per cell varied between 0.5 and 10 copies / cell (similar results were observed 3 and 6 months after transplantation) (Figure 5). To assess whether the hematopoietic precursor phenotype was corrected after transplantation of genetically corrected cells, BM samples from transplanted FA-A mice were cultured in methylcellulose in the absence and presence of MMC.

[0203] As shown in Figure 6, significant correction of MMC hypersensitivity was observed in hematopoietic precursors derived from FA mice subjected to gene therapy with FANCA LVs compared to SF1-EGFP LV controls. * BM samples were collected and cultured in methylcellulose in the presence of increasing concentrations of mitomycin C (MMC).

[0204] These data therefore demonstrate that FANCA LV can reverse the phenotype of hematopoietic precursors derived from FA-A mice after in vivo transplantation. In terms of safety, none of the 30 mice transplanted with BM cells previously transduced with FANCA LV developed symptoms of myeloproliferative disorders or leukemia (data obtained up to 1 year after transplantation).

[0205] Example 3. Efficient transduction of fresh hematopoietic precursors from FA patients using lentiviral vectors Because transduction of FA cryopreserved hematopoietic stem cells (HSCs) has previously been shown to be less efficient than transduction of fresh grafts (Jacome et al., 2009), we attempted to optimize the transduction efficiency of cryopreserved FA BM samples. As shown in Figure 7, performing three transduction cycles significantly increased the transduction efficiency of FA CFCs compared to the value obtained after a single transduction cycle (45.7 ± 4.2% vs. 13.5 ± 5.1%, respectively). Samples were subjected to standard transduction (white bars; 1xS), consisting of one transduction cycle (16 h) after 2 h of static preloading, or to improved transduction (gray bars; 3xD), consisting of three transduction cycles (2 h + 2 h + 12 h) using lentiviral vectors.

[0206] Example 4. PGK-FANCA-WPRE * Lentiviral vectors effectively correct the phenotype of myeloid precursors from FA-A patients Based on the efficacy of LVs to induce FANCA with the PGK promoter, and on previous studies showing the stability (Follenzi A et al. Nat Genet. 2000; 25: 217-222) and low genotoxicity of LVs with the PGK promoter (Modlich et al., 2009, Montini et al., 2006, Montini et al., 2009), we investigated the efficacy of LVs without any WPRE element or with WPRE or WPRE promoters. * Further experiments were performed in LCLs and in bone marrow cells from FA-A patients using PGK-FANCA LV carrying the sequence As shown in Figure 8A, all three vectors similarly reversed the sensitivity of FA-A LCLs to MMC.

[0207] To compare the efficacy of SFFV-FANCA and PGK-FANCA LVs in correcting the phenotype of hematopoietic precursors from FA-A patients, we transduced erythrocyte-depleted BM samples from FA-A patients as described recently (Jacome et al., 2009). LV supernatant was transduced into erythrocyte-depleted BM cells in pre-loaded plates for 16 hours as described recently (Gonzalez-Murillo et al., 2009). After 14 days, the number of colonies grown in the absence and presence of 10 nM MMC was scored, and the proportion of precursors that became resistant to the drug was determined.

[0208] As shown in Figure 8B, when samples were transduced with EGFP-LV, almost no colonies were generated in the presence of MMC. In contrast to this observation, transduction of FA-A BM cells with SFFV-FANCA and PGK-FANCA LVs resulted in 26 and 38% growth of colonies recorded in culture without MMC. * The effects of PGK-FANCA LVs containing the WPRE were compared with those of PGK-FANCA LVs without the WPRE. As shown in Figure 8B, all three LVs provided high and comparable levels of protection against MMC. The post-transcriptional regulatory element WPRE * Although the insertion of ( Schambach et al., 2006 ) was not necessary to improve the efficacy of PGK-FANCA LV, this element would provide a redundant element to maintain long-term therapeutic levels of ectopic FANCA in patients.

[0209] Taken together, the data obtained in these studies support the PGK-FANCA-WPRE * These results strongly suggest that LV confers sufficient levels of FANCA to correct the hematopoietic phenotype in FA-A patients. These results support the stability (Follenzi et al., 2000) and safety characteristics (Modlich et al., 2009, Montini et al., 2006, Montini et al., 2009) of PGK-LV and the mutant WPRE* Together with previous observations showing the effect of posttranscriptional elements ( Schambach et al., 2006 ), PGK-FANCA-WPRE * This strengthens the hypothesis that LV could be an effective and safe vector for gene therapy in FA-A patients.

[0210] Example 5. Analysis of the efficacy of GALV-TR and VSV-G packaged lentiviral vectors to transduce hematopoietic precursors from FA patients Because LVs can be pseudotyped with both GALV-TR and VSV-G envelopes, we performed new experiments in which EGFP-LVs pseudotyped with the two envelopes were used to transduce unselected bone marrow cells from FA patients under optimal conditions.

[0211] For GALV-TR pseudotyped LV, unconcentrated LV (estimated titer: 2x10 5 Two transduction cycles were performed using 100 IU / mL of IgG1 (estimated MOI: 2 IU / cell).

[0212] For VSV-G pseudotyped LVs, concentrated and purified LVs (estimated titer: 10 8 One transduction cycle was performed using 100 IU / mL of IgG1 (estimated MOI: 50 IU / cell).

[0213] As shown in Figure 9, similar transduction of hematopoietic precursors from FA patients was achieved under both conditions, indicating that manufacturing compromises can determine the optimal envelope for packaging.

[0214] Example 6. Safety Study The transformation ability of the lentiviral vectors shown in Figure 10A was measured by the number of replatings per copy number. As shown in Figure 10B, PGK-FANCA-WPRE * The transforming potential of the lentiviral vector backbone corresponding to LV (PGK-derived lentiviral vector) is significantly lower compared to that of vectors carrying viral promoters already used in X1-SCID and CGD clinical trials.

[0215] PGK-FANCA-WPRE * Regarding in vivo studies using mice transplanted with LV-transduced BM cells, 30 mice have been transplanted, and to date (up to 1 year after transplantation), none of the transplanted animals have developed symptoms of myeloproliferative disease or leukemia.

[0216] Example 7. Pharmaceuticals The FANCA lentiviral vector is a third-generation self-inactivating rHIV1-derived vector that encodes the human FANCA cDNA under the control of the human PGK promoter and is regulated at the post-transcriptional level by a mutant WPRE lacking the X protein ORF (see Figure 1, Figure 41, and SEQ ID NO: 24). Such FANCA lentiviral vectors exhibit several advantages over gammaretroviral vectors previously used in FA gene therapy, in particular the ability to transduce cells despite a short pre-activation protocol, which is advantageous for preserving the pluripotency of hematopoietic stem cells.

[0217] For the generation of therapeutic lentiviral vectors, 293T cells were transfected with three additional plasmids (see Figures 38-40) that provide all of the helper proteins required for vector packaging: PGK-FANCA-WPRE * This will include the final packaging of the medical supplies (Figure 41).

[0218] PGK-FANCA-WPRE * The LV therapeutic cassette contains the human PGK promoter, the coding sequence of FANCA cDNA, and the WPRE * It contains an enhancer and comprises nucleotides 3541 to 9178 of SEQ ID NO: 24. It contains the human CMV immediate early promoter, the HIV packaging sequence, the ga and RRE elements, the human PGK promoter, the coding sequence of the FANCA cDNA and the WPRE *The therapeutic cassette, including the enhancer, and the region of the import cassette, including the HIV self-inactivating 3'LTR, are encoded by nucleotides 1586 to 9495 of SEQ ID NO:24.

[0219] Nucleotides 1586-1789 of SEQ ID NO:24 contain the human CMV immediate early promoter. Nucleotides 2031-2156 of SEQ ID NO:24 contain the HIV1 psi packaging signal. Nucleotides 2649-2882 of SEQ ID NO:24 contain the HIV1 RRE element. Nucleotides 3378-3495 of SEQ ID NO:24 contain the HIV cPPT / CTS element. Nucleotides 3541-4051 of SEQ ID NO:24 contain the hPGK promoter. Nucleotides 4078-8445 of SEQ ID NO:24 contain the human FANCA-A cDNA. Nucleotides 8502-9178 of SEQ ID NO:24 contain a mutated WPRE element. Nucleotides 9262-9495 of SEQ ID NO:24 contain the HIV delta U3' LTR.

[0220] Example 8. Clinical Study FANCOSTEM Two gene therapy trials have been conducted in the United States in patients with FA-A and FA-C, but they failed to demonstrate clinical efficacy (Liu, JM, et al. (1999). Engraftment of hematopoietic progenitor cells transduced with the Fanconi anemia group C gene (FANCC). Hum. Gene Ther. 10: 2337-2346; Kelly, PF, et al. (2007). Stem cell collection and gene transfer in Fanconi anemia. Mol Ther 15: 211-219). The efficacy of these trials can be significantly improved through various optimizations. The two clinical trials have not yet demonstrated the feasibility of obtaining sufficient numbers of CD34 +Studies were conducted before and after (FANCOSTEM) to determine the feasibility of the cell harvesting and purification process, and in parallel to evaluate the safety and efficacy of gene therapy in patients with FA complementation group A (FA-A) (FANCOLEN). See Figure 11.

[0221] The main inclusion criteria for FANCOSTEM were a diagnosis of AF confirmed by a chromosomal instability test using diepoxybutane or mitomycin C, age >1 year, and elevated levels of at least one of the following parameters: 1) hemoglobin: 8.0 g / dL, 2) neutrophils: 750 / mm 3 , 3) Platelets: 30,000 / mm 3 Ten patients were enrolled, nine were screened, and two of them were excluded (mosaic patients). Figure 12 shows the hematological parameters of the enrolled patients. Seven patients were treated with G-CSF and plerixafor. Mobilization treatment involved administration of G-CSF (neupogen; 12 μg / kg / 12 hours) and plerixafor (mozobil; 240 μg / kg body weight / day). PGK-FANCA-WPRE was transduced using a short ex vivo transduction protocol under GMP conditions. * Mobilized peripheral blood (mPB) CD34+ cells were transduced with LV (Figure 13). Two patients, aged 15 and 16 years, did not reach the threshold level of peripheral blood CD34+ cells, but apheresis was possible in five patients aged 3 to 5 years. In these patients, the median CD34+ cell count / kg was 6.6 x 10^6 (range: 1.6 x 10^6 to 7.6 x 10^6). After CD34+ cell selection, the number of CD34+ cells / kg was 2.0 x 10^6 (range: 8.5 x 10^5 and 5.1 x 10^6). No serious adverse events related to this mobilization regimen were detected in any of the treated patients followed for up to 2.5 years.

[0222] Figure 14 shows the mobilization of CD34+ cells in FA-A patients using G-SCF / plerixafor, and Figure 15 shows the mobilization of colony-forming cells (CFCs) in FA-A patients using G-SCF / plerixafor. Figure 16A shows the collection of CD34+ cells in FANCOSTEM, and Figure 16B shows a comparison with previous studies.

[0223] Figure 17 shows a comparison between estimated CD34+ cell counts in bone marrow (BM) and actual counts in mobilized peripheral blood (mPB). Figure 18 shows a comparison of CD34+ cell counts in G-CSF / plerixafor-mobilized FA-A patients. + Summary of cell collection. Post-selection CD34+ counts are shown in BM on day 0. + Correlates to the number of cells / µl.

[0224] FIG. 19 shows CD34 expression before and after immune selection of mPB CD34+ cells from healthy donors (HD) and FA patients.

[0225] From these data, we conclude that, compared with other clinical studies, a clear improvement in the collection of CD34+ cells has been observed in patients treated with filgrastim (G-CSF) and plerixafor at this time, and that only FA patients at an early stage of the disease appear to be suitable for the collection of clinically relevant numbers of HSCs.

[0226] Example 9. Clinical Study FANCOLEN The second parallel clinical trial (FANCOLEN) aimed to evaluate the safety and efficacy of gene therapy in patients with Fanconi anemia complementation group A (FA-A). An optimized vector and transduction protocol were developed to restore hematopoietic capacity in FA patients by infusing gene-corrected autologous HSCs. Specifically, this was a phase I / II clinical trial to evaluate the safety and efficacy of infusion of autologous CD34+ cells transduced with a lentiviral vector (an orphan drug) carrying the FANCA gene in patients with Fanconi anemia subtype A.

[0227] The main inclusion criteria were a diagnosis of FA-A, age >1 year, and at least one of the following parameters below the threshold: 1) hemoglobin: 8.0 g / dL, 2) neutrophils: 1,000 / mm 3 , 3) Platelets: 50,000 / mm 3 .

[0228] More specific inclusion criteria were: 1) patients with complementation group FA-A, 2) at least one of the following parameters must be lower than the indicated values: hemoglobin: 8.0 g / dL, neutrophils: 750 / mm 3 , Platelet:30.000 / mm 3 3) Minimum age: 1 year; 4) Maximum age: 21 years; 5) Lansky index > 60%; 6) Moderate organ dysfunction; 7) Informed consent must be provided in accordance with current law; 8) Number of cells to be transduced: at least 3 x 10 5 Purified CD34 + 9) Women of childbearing age must have a negative urine pregnancy test at the baseline visit and must agree to use effective contraception during participation in the study.

[0229] Exclusion criteria included: 1) patients with an HLA-identical family donor; 2) evidence of myelodysplastic syndrome or leukemia, or cytogenetic abnormalities suggestive of these conditions in bone marrow aspirate analysis (this evaluation should be performed by a valid study 2 months before the patient's entry into the clinical trial); 3) evidence of somatic mosaicism in patients with improved hematology; 4) any concurrent disease or condition that, in the investigator's judgment, would render the subject unsuitable for participation in this study; 5) pre-existing sensory or motor impairment >= Grade 2 according to the National Cancer Institute (NCI) criteria; and 6) pregnant or lactating women.

[0230] Route of administration: Patients received cells transduced with the therapeutic vector via intravenous infusion.

[0231] Cell dose: The cell dose administered to patients by infusion is 3x10 cells obtained from transduction. 5 ~4x10 6 Purified CD34 + The dose was in cells / kg patient body weight.

[0232] 3x10 5 CD34 below + The number of cells / kg likely does not allow for the generation of patient grafts from transduced cells, especially considering that this trial will initially infuse unconditioned patients. In a gene therapy trial in Fanconi anemia patients conducted by Dr. Williams (Kelly et al., 2007), the number of cells infused into two patients (FAAGT1001 and 1003) was 4.5 x 10 5 and 3.25x10 5 The nucleated cells / kg patient body weight were 100%. In both patients, transient improvements in Hb and platelet counts were observed, but the presence of the relevant transgene could not be demonstrated. Unlike Dr. Williams's study, in which cells were transduced with a gammaretroviral vector for 4 days, in this clinical study, cells are transduced with a more effective lentiviral vector, and transduction is carried out for a maximum of 48 hours, i.e., a much shorter period than the 4 days used in the previous protocol. Based on this, we are considering a 3x10 5 Purified CD34 + Cells / kg patient weight was considered a reasonable lower limit in this study.

[0233] 4x10 6 Purified CD34 + The upper limit of cells / kg is not based on the need to limit the number of cells infused, as higher cell numbers increase the viability of the graft. Instead, 6 Purified CD34 + The limit of cells / kg of patient body weight is based on the decreased CD34 + This is due to the difficulty of mobilizing and collecting more than this number of cells from patients with Fanconi anemia, which is characterized by cell count (Jacome et al., 2009).

[0234] Dosage regimen: Cells transduced with the therapeutic vector were infused into patients in a single dose. This was for two main reasons: 1) All hematopoietic gene therapy trials conducted to date have been performed using a single infusion of transduced cells (Naldini, 2011). Following this previous experience, we did not consider changing this parameter. 2) A single infusion of all transduced cells increases the likelihood of generating larger grafts compared to infusions of the same fractionated doses.

[0235] Recruitment period: Patients will undergo mobilization treatment for three years. Because patients with subtype FA-A are the most common in the FA patient population (approximately 80% of Spanish patients with FA corresponding to this complementation group (Casado et al. (2007))), the therapeutic vector to be constructed will contain the FANCA gene. Therefore, only FA patients belonging to the subtype FA-A will be able to participate in this study. Patients of this complementation group, whether children or adults, will be included in this study, as long as they meet the specific inclusion and exclusion criteria.

[0236] A detailed description of the primary and, if present, secondary variables evaluated in this clinical trial is as follows: 1) Primary variable: A) Autologous CD34 transduced with therapeutic lentiviral vector in patients with Fanconi anemia subtype A. + To determine the toxicity associated with the infusion of cells, B) autologous CD34 transduced with therapeutic lentiviral vectors in patients with Fanconi anemia subtype A. + To determine the degree of engraftment following cell infusion; 2) secondary variables: autologous CD34 transduced with therapeutic lentiviral vectors in patients with Fanconi anemia subtype A. + and determining the clinical response following cell infusion.

[0237] CD34 mobilized in bone marrow (fresh and / or cryopreserved) and / or peripheral blood from patients with Fanconi anemia subtype A (FA-A) + The cells were transduced ex vivo with a lentiviral vector (an orphan drug) carrying the FANCA gene (Figure 11). After transduction of the cells, patients were infused with these genetically corrected stem cells to restore hematopoietic potential.

[0238] Evaluation: Patients were evaluated prior to initial treatment and provided prior informed consent. Evaluation was performed via standard physical examination (including weight and height), peripheral blood count, basic biochemistry, and bone marrow aspiration in the month prior to infusion of genetically corrected cells.

[0239] Transduction and infusion of genetically corrected CD34+ cells: purified CD34 + The cell population was transduced ex vivo with a therapeutic lentiviral vector. After cell transduction, a quality control assessment of the product was performed, aliquots were cryopreserved for further study, and the product was prepared for infusion into patients.

[0240] If two patients who received an acceptable number of transduced cells (at least 1 million transfused cells / kg body weight in an aliquot with at least 0.3 copies of vector / cell detected after at least 7 days of in vitro culture) did not have at least 0.1 copies of vector / cell observed in either bone marrow or peripheral blood 6 months after transfusion, subsequent patients would undergo a conditioning process before cell infusion.

[0241] To ensure that patients are suitable for any type of preparation, there must be an appropriate method to rescue the potential bone marrow aplasia associated with the preparation and the potential engraftment failure of the transduced cells. Rescue methods include a unit of umbilical cord blood or hematopoietic cells from a haploidentical donor. The cells are infused intravenously.

[0242] The dose of cells administered to patients by infusion is 3x10 obtained from the transduction process.5 ~4x10 6 Purified CD34 + The dose was in cells / kg patient body weight.

[0243] The cells are infused into the patient in a single dose.

[0244] The transduced cells are infused immediately after the transduction process is complete.

[0245] The product to be infused is CD34, which is packed in a sterile bag for infusion by the CLINISTEM GMP laboratory at CIEMAT. + It consists of a suspension of cells.

[0246] Mobilization period: 3 years from the first patient transfusion.

[0247] Follow-up period: 2 years from transduced cell infusion, but patients will be observed outside the clinical trial for 10 years.

[0248] Engraftment of the transduced cells is monitored in peripheral blood and bone marrow samples.

[0249] First 72 hours after infusion: During this period, vital signs were recorded every 8 hours and vital organ function was monitored every 24 hours (electrolyte profile, hematology, renal and liver function).

[0250] Thereafter, the following tests shown in Table 2 were performed.

[0251] (Table 2) TIFF2025043346000028.tif65131

[0252] Patients diagnosed with FA and belonging to complementation group FA-A will be enrolled in this study. Patients will be considered if they demonstrate phenotypic correction of their cells by transduction with a vector carrying the FANCA gene or if they demonstrate biallelic pathogenic mutations in this gene.

[0253] Patient FA-02005 meets the FANCOSTEM and FANCOLEN criteria, as summarized in Figure 20. Figure 21 shows tests demonstrating the FA diagnosis of patient FA-02005 before gene therapy, and Figure 22 shows follow-up of the cell manufacturing process for patient FA-02005. Figure 23 shows the vector copy number in patient FA02005 before gene therapy and 2 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months, and 5 months after gene therapy. Follow-up of patient FA02005 (age 4), the first unadjusted FA-A patient, before and after gene therapy is shown for hemoglobin (Figure 24), neutrophils (Figure 25), and platelets (Figure 26).

[0254] For patient FA02002, the hematological evolution is shown in Figure 27, the diagnosis in Figure 28, and the follow-up of the cell manufacturing process in Figure 29. Figure 30 shows the analysis of CD34 expression in healthy donors and FA mPB during the different steps required for LV transduction for patient FA-02002. Figure 31 shows the vector copy number in patient FA02002 before gene therapy and 2, 4, and 6 weeks after gene therapy. The follow-up of patient FA02002 (transfused with cryopreserved cells) is shown for hemoglobin (Figure 32), neutrophils (Figure 33), and platelets (Figure 34).

[0255] The data for these two patients support the conclusion that the patients received significant numbers of gene-corrected mobilized peripheral blood (mPB) CD34+ cells. No serious adverse events were observed in either of the two treated patients. Gene marking levels of approximately 1-5 copies / 1000 peripheral blood cells were detected in treated patients 15 days to 5 months after GT, with a modest increase over time. These viral copy numbers are approximately 100-fold higher than the highest value detected 3 weeks after GT in a previous study (3 copies / 10^5 cells; Kelly et al. 2007).

[0256] Example 6. Fresh mPB CD34 from an FA-A patient + Transduction of cells Short-term transduction of small mobilized peripheral blood (mPB) CD34+ samples from patients treated with G-CSF / plelixafor showed transduction efficiencies of 17-45% (Figure 35). Small amounts of these samples were transplanted into NSG mice conditioned with 1.5 Gy. The majority of transplanted samples engrafted in NSG mice (1-10% of BM cells were hCD45+ / mCD45-) (Figure 36). Furthermore, a clear selective advantage of corrected CD34+ FA-A cells was observed in engrafted mice (Figure 37).

[0257] These results demonstrate that transduced FA-A mPB CD34 + We demonstrate that cells engraft in NSG mice and that corrected human FA-A repopulating cells exhibit an in vivo expansion advantage in NSG recipient mice.

[0258] Sequence information SEQUENCE LISTING <110> CENTRO DE INVESTIGACIONES ENERGETICAS, MEDIOAMBIENTALES Y TECNOLOGICAS, OA, MP FUNDACION INSTITUTO DE INVESTIGACION SANITARIA FUNDACION JIMENEZ DIAZ CONSORCIO CENTRO DE INVESTIGACION BIOMEDICA EN RED FUNDACION PARA LA INVESTIGACION BIOMEDICA DEL HOSPITAL INFANTIL UNIVERSITARIO NINO JESUS <120> GENE THERAPY FOR PATIENTS WITH FANCONI ANEMIA <150> US 62 / 385,185 <151> 2016-09-08 <150> US 62 / 412,028 <151> 2016-10-24 <160> 25 <170> PatentIn version 3.5 <210> 1 <211> 234 <212> DNA <213> Human immunodeficiency virus 1 (HIV-1) <400> 1 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat 60 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 120 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca 180 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcct 234 <210> 2 <211> 126 <212> DNA <213> Human immunodeficiency virus 1 (HIV-1) <400> 2 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga 60 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tggtgcgag 120 agcgtc 126 <210> 3 <211> 181 <212> DNA <213> Human immunodeficiency virus 1 (HIV-1) <400> 3 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 120 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 180 a 181 <210> 4 <211> 234 <212> DNA <213> Human immunodeficiency virus 1 (HIV-1) <400> 4 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc 60 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 120 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 180 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agca 234 <210> 5 <211> 204 <212> DNA <213> Human herpesvirus-5 <400> 5 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 60 ccaagtctcc accccattga cgtcaatggg agttgtttt ggcaccaaaa tcaacgggac 120 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 180 tgggaggtct atataagcag agct 204 <210> 6 <211> 118 <212> DNA <213> Human immunodeficiency virus 1 (HIV-1) <400> 6 ttttaaaaga aaagggggga ttgggggta cagtgcaggg gaaagaatag tagacataat 60 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaatttt 118 <210> 7 <211> 511 <212> DNA <213> Homo sapiens <400> 7 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc 60 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc 120 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 180 tgctccgccc ctaagtcgggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 240 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 300 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag 360 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 420 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 480 cgttgaccga atcaccgacc tctctcccca g 511 <210> 8 <211> 4368 <212> DNA <213> Homo sapiens <400> 8 atgtccgact cgtgggtccc gaactccgcc tcgggccagg acccagggg ccgccgagg 60 gcctgggccg agctgctggc gggaagggtc agagggaa atataatcc tgaaagggca 120 cagaaattaa aggaatcagc tgtgcgcctc ctgcgaagcc atcaggacct gatgccctt 180 ttgcttgagg tagaggtcc actgtgtaaa aaattgtctc tcagcaagt gattgactgt 240 gabagttctg aggcctatgc taatcattct agttcattct taggctctgc tttgcaggat 300 caagcctcaa ggctgggggt tcccgtgggt attctctcag ccgggatggt tgcctctagc 360 gtgggacaga tctgcacggc tccagcggag accagtcacc ctgtgctgct gactgtggag 420 cagagaaaga agctgtcttc cctgttagag tttgctcagt atttattggc acacagtatg 480 ttctcccgtc tttccttctg tcaagaatta tggaaaatac agagttcttt gttgcttgaa 540 gcggtgtggc atcttcacgt acaaggcatt gtgagcctgc aagagctgct ggaaagccat 600 cccgacatgc atgctgtggg atcgtggctc ttcaggaatc tgtgctgcct ttgtgaacag 660 atggaagcat cctgccagca tgctgacgtc gccagggcca tgctttctga ttttgttcaa 720 atgtttgttt tgaggggatt tcagaaaaac tcagatctga gaagaactgt ggagcctgaa 780 aaaatgccgc aggtcacggt tgatgtactg cagagaatgc tgatttttgc acttgacgct 840 ttggctgctg gagtacagga ggagtcctcc actcacaaga tcgtgaggtg ctggttcgga 900 gtgttcagtg gacacacgct tggcagtgta atttccacag atcctctgaa gaggttcttc 960 agtcataccc tgactcagat actcactcac agccctgtgc tgaaagcatc tgatgctgtt 1020 cagatgcaga gagagtggag ctttgcgcgg acacaccctc tgctcacctc actgtaccgc 1080 aggctctttg tgatgctgag tgcagaggag ttggttggcc atttgcaaga agttctggaa 1140 acgcaggagg ttcactggca gagagtgctc tcctttgtgt ctgccctggt tgtctgcttt 1200 ccagaagcgc agcagctgct tgaagactgg gtggcgcgtt tgatggccca ggcattcgag 1260 agctgccagc tggacagcat ggtcactgcg ttcctggttg tgcgccaggc agcactggag 1320 ggcccctctg cgttcctgtc atatgcagac tggttcaagg cctcctttgg gagcacacga 1380 ggctaccatg gctgcagcaa gaaggccctg gtcttcctgt ttacgttctt gtcagaactc 1440 gtgccttttg agtctccccg gtacctgcag gtgcacattc tccacccacc cctggttccc 1500 agcaagtacc gctccctcct cacagactac atctcattgg ccaagacacg gctggccgac 1560 ctcaaggttt ctatagaaaa catgggactc tacgaggatt tgtcatcagc tggggacatt 1620 actgagcccc acagccaagc tcttcaggat gttgaaaagg ccatcatggt gtttgagcat 1680 acggggaaca tcccagtcac cgtcatggag gccagcatat tcaggaggcc ttactacgtg 1740 tcccacttcc tccccgccct gctcacacct cgagtgctcc ccaaagtccc tgactcccgt 1800 gtggcgttta tagagtctct gaagagagca gataaaatcc ccccatctct gtactccacc 1860 tactgccagg cctgctctgc tgctgaagag aagccagaag atgcagccct gggagtgagg 1920 gcagaaccca actctgctga ggagcccctg ggacagctca cagctgcact gggagagctg 1980 agagcctcca tgacagaccc cagccagcgt gatgttatat cggcacaggt ggcagtgatt 2040 tctgaaagac tgagggctgt cctgggccac aatgaggatg acagcagcgt tgagatatca 2100 aagattcagc tcagcatcaa cacgccgaga ctggagccac gggaacacat tgctgtggac 2160 ctcctgctga cgtctttctg tcagaacctg atggctgcct ccagtgtcgc tcccccggag 2220 aggcagggtc cctgggctgc cctcttcgtg aggaccatgt gtggacgtgt gctccctgca 2280 gtgctcaccc ggctctgcca gctgctccgt caccagggcc cgagcctgag tgccccacat 2340 gtgctggggt tggctgccct ggccgtgcac ctgggtgagt ccaggtctgc gctcccagag 2400 gtggatgtgg gtcctcctgc acctggtgct ggccttcctg tccctgcgct ctttgacagc 2460 ctcctgacct gtaggacgag ggattccttg ttcttctgcc tgaaattttg tacagcagca 2520 atttcttact ctctctgcaa gttttcttcc cagtcacgag atactttgtg cagctgctta 2580 tctccaggcc ttattaaaaa gtttcagttc ctcatgttca gattgttctc agaggcccga 2640 cagcctcttt ctgaggagga cgtagccagc ctttcctgga gacccttgca ccttccttct 2700 gcagactggc agagagctgc cctctctctc tggacacaca gaaccttccg agaggtgttg 2760 aaagaggaag atgttcactt aacttaccaa gactggttac acctggagct ggaaattcaa 2820 cctgaagctg atgctctttc agatactgaa cggcaggact tccaccagtg ggcgatccat 2880 gagcactttc tccctgagtc ctcggcttca gggggctgtg acggagacct gcaggctgcg 2940 tgtaccattc ttgtcaacgc actgatggat ttccaccaaa gctcaaggag ttatgaccac 3000 tcagaaaatt ctgatttggt ctttggtggc cgcacaggaa atgaggatat tatttccaga 3060 ttgcaggaga tggtagctga cctggagctg cagcaagacc tcatagtgcc tctcggccac 3120 accccttccc aggagcactt cctctttgag attttccgca gacggctcca ggctctgaca 3180 agcgggtgga gcgtggctgc cagccttcag agacagaggg agctgctaat gtacaaacgg 3240 atcctcctcc gcctgccttc gtctgtcctc tgcggcagca gcttccaggc agaacagccc 3300 atcactgcca gatgcgagca gttcttccac ttggtcaact ctgagatgag aaacttctgc 3360 tcccacggag gtgccctgac acaggacatc actgcccact tcttcagggg cctcctgaac 3420 gcctgtctgc ggagcagaga cccctccctg atggtcgact tcatactggc caagtgccag 3480 acgaaatgcc ccttaatttt gacctctgct ctggtgtggt ggccgagcct ggagcctgtg 3540 ctgctctgcc ggtggaggag acactgccag agcccgctgc cccgggaact gcagaagcta 3600 caagaaggcc ggcagtttgc cagcgatttc ctctcccctg aggctgcctc cccagcaccc 3660 aacccggact ggctctcagc tgctgcactg cactttgcga ttcaacaagt cagggaagaa 3720 aacatcagga agcagctaaa gaagctggac tgcgagagag aggagctatt ggttttcctt 3780 ttcttcttct ccttgatggg cctgctgtcg tcacatctga cctcaaatag caccacagac 3840 ctgccaaagg ctttccacgt ttgtgcagca atcctcgagt gtttagagaa gaggaagata 3900 tcctggctgg cactctttca gttgacagag agtgacctca ggctggggcg gctcctcctc 3960 cgtgtggccc cggatcagca caccaggctg ctgcctttcg cttttacag tcttctctcc 4020 tacttccatg aagacgcggc catcagggaa gaggccttcc tgcatgttgc tgtggacatg 4080 tacttgaagc tggtccagct cttcgtggct ggggatacaa gcacagtttc acctccagct 4140 ggcaggagcc tggagctcaa gggtcagggc aaccccgtgg aactgataac aaaagctcgt 4200 cttttctgc tgcagttaat acctcggtgc ccgaaaaaga gcttctcaca cgtggcagag 4260 ctgctggctg atcgtgggga ctgcgaccca gaggtgagcg ccgccctcca gagcagacag 4320 caggctgccc ctgacgctga cctgcccag gagcctcatc tcttctga 4368 <210> 9 <211> 380 <212> DNA <213> Human herpesvirus-5 <400> 9 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240 aagtgtatca tatgccaagt acgccccta ttgacgtcaa tgacggtaaa tggcccgcct 300 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360 tagtcatcgc tattaccatg 380 <210> 10 <211> 122 <212> DNA <213> Simian virus 40 <400> 10 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120 is 122 <210> 11 <211> 136 <212> DNA <213> Simian virus 40 <400> 11 atccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 60 tttattatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga 120 ggctttttg gaggcc 136 <210> 12 <211> 28 <212> DNA <213> Human immunodeficiency virus 1 (HIV-1) <400> 12 tttaaaagaa aaggggggat tggggggt 28 <210> 13 <211> 83 <212> DNA <213> Human immunodeficiency virus 1 (HIV-1) <400> 13 gaattcgagc tcggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca 60 ctttttaaaa gaaaaggggg gac 83 <210> 14 <211> 795 <212> DNA <213> Escherichia coli <400> 14 atgattgaac aagatggatt gcacgcaggt tctccggcgg cttgggtgga gaggctattc 60 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120 gcgcaggggc gtccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180 caagacgagg cagcgcggct atcgtggctg gcgacgacgg gcgttccttg cgcggctgtg 240 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgtc tatgcccgac 540 ggcgaggatc tcgtcgtgac ccacggcgat gcctgcttgc cgaatatcat ggtggaaaat 600 ggccgctttt ctggattcat cgactgtggc cgtctgggtg tggcggaccg ctatcaggac 660 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720 cttgtgcttt acggtatcgc cgcgcccgat tcgcagcgca tcgccttcta tcgccttctt 780 gacgagttct tctga 795 <210> 15 <211> 137 <212> DNA <213> Escherichia coli <400> 15 gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc 60 cagattcagc aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac 120 page 137 <210> 16 <211> 589 <212> DNA <213> unknown <220> <223> ColE1 / pMB1 / pBR322 / pUC origin of replication plasmid sequence <400> 16 ttgagatcct ttttttgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 60 agcggtggtt tgtttgccgg atcaagagct accaactctt ttccgaagg taactggctt 120 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 180 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 240 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 300 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 360 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 420 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 480 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggttcgcc acctctgact 540 tgagcgtcga ttttgtgat gctcgtcagg ggggcggagc ctatggaaa 589 <210> 17 <211> 22 <212> DNA <213> Escherichia coli <400> 17 taatgtgagt tagctcactc at 22 <210> 18 <211> 31 <212> DNA <213> Escherichia coli <400> 18 tttacacttt atgcttccgg ctcgtatgtt g 31 <210> 19 <211> 17 <212> DNA <213> Escherichia coli <400> 19 ttgtgagcgg ataacaa 17 <210> 20 <211> 19 <212> DNA <213> Bacteriophage T3 <400> 20 aattaaccct cactaaagg 19 <210> 21 <211> 19 <212> DNA <213> Bacteriophage T7 <400> 21 cctatagtga gtcgtatta 19 <210> 22 <211> 428 <212> DNA <213> F1 bacteriophage <400> 22 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 60 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 120 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 180 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 240 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 300 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 360 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 420 acgcgaat 428 <210> 23 <211> 677 <212> DNA <213> Woodchuck hepatitis virus <400> 23 cgagcatctt accgccattt attcccatat ttgttctgtt tttcttgatt tgggtataca 60 tttaaatgtt aataaaacaa aatggtgggg caatcattta catttttagg gatatgtaat 120 tactagttca ggtgtattgc cacaagacaa acatgttaag aaactttccc gttatttacg 180 ctctgttcct gttaatcaac ctctggatta caaaatttgt gaaagattga ctgatattct 240 taactatgtt gctcctttta cgctgtgtgg atatgctgct ttaatgcctc tgtatcatgc 300 tattgcttcc cgtacggctt tcgttttctc ctccttgtat aaatcctggt tgctgtctct 360 ttatgaggag ttgtggcccg ttgtccgtca acgtggcgtg gtgtgctctg tgtttgctga 420 cgcaaccccc actggctggg gcattgccac cacctgtcaa ctcctttctg ggactttcgc 480 tttccccctc ccgatcgcca cggcagaact catcgccgcc tgccttgccc gctgctggac 540 aggggctagg ttgctgggca ctgataattc cgtggtgttg tcggggaagg gcctgctgcc 600 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg 660 ggccgcctcc ccgcctg 677 <210> 24 <211> 11433 <212> DNA <213> Artificial Sequence <220> <223> Made in lab transfer cassette pCCL-SIN-cPPT / CTS -hPGK-hFANCA-WPRE <400> 24 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 60 gatcaaagga tcttcttgag atccttttt tctgcgcgta atctgctgct tgcaaaaaa 120 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 180 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 240 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 300 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 360 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 420 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 480 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 540 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 600 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 660 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 720 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 780 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 840 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 900 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag 960 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 1020 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa 1080 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt ggccattgca 1140 tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa cattaccgcc 1200 atgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca 1260 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 1320 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 1380 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 1440 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 1500 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 1560 cgtattagtc atcgcatta ccatggtgat gcggttttgg footcatca atgggcgtgg 1620 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 1680 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 1740 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa 1800 ccggggtctc tctgggtaga ccagatctga gcctgggagc tctctggcta actagggaac 1860 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg 1920 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct 1980 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac 2040 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta 2100 cgccaaaaat tttgactagc ggaggctaga aggagaga tggtgcgag agcgtcagta 2160 ttaagcgggg gagaattaga tcgcgatgggg aaaaatttcg gttaaggcca gggggaaga 2220 aaaataata attaaaacat atagtaggg caagcaggga gctagaacga ttcgcagtta 2280 atcctggcct gttagaaca tcagaggct gtagacaat actgggacag ctacaaccat 2340 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt 2400 gtgtgcatca aaggatagag aaaagaca ccaaggaagc tttagacaag atagaggag 2460 agcaaaaaaa agtaagacc accgcacagc aagcggccgc tgatctcag acctgagga 2520 ggagaattga gggacaattg gagagtgaa ttataat aataagtagt aaaattgaa 2580 ccattaggag tagcaccac ccaggchaag agagagtgg tgcagagaga aaaagagca 2640 gtgggaatag gagctttgtt ccttggggttc ttgggagcag caggaagcac tatgggcgca 2700 gcgtcaatga cgctgacggt acaggccaga cattattgt ctggtatagt gcagcagcag 2760 aacaatttgc tgaggctat tgaggcgca cagcatctgt tgcactcac agtctggggc 2820 atcaagcagc tccaggcaag aatcctggct gtggaagat acctaagga tcacagctc 2880 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct 2940 agttggagta ataatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac 3000 agagaaatta acattacac aagcttaata cactccttaa ttgaagaatc gcaaaccag 3060 caghaaaga atgaaga attattgaa ttagataat gggcaagtttt gtggaatttgg 3120 tttaacataa caattggct gtgtatata aattattca taatgatagt aggaggctg 3180 gtaggtttaa gatagtttt tgctgtactt tctatagtga atagagttag gcaggtat 3240 tcaccattat cgtttcagac ccaccccca accccgaggg gaccgacag gcccgaagga 3300 atagagaag aggtggaga gagagacaga gatacca ttcgattagt gaacggatct 3360 cgacggtatc ggttaacttt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 3420 agaatagtag acatatagc aagacata aactaag attaxaaaaaattaca 3480 aaaattcaaa attttatcga tcacgagact agcctcgaga agcttgatat cgaattccac 3540 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc 3600 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc 3660 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 3720 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 3780 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 3840 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag 3900 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 3960 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 4020 cgttgaccga atcaccgacc tctctcccca gggggatccc ccgggctgca ggaattcatg 4080 tccgactcgt gggtcccgaa ctccgcctcg ggccaggacc cagggggccg ccggagggcc 4140 tgggccgagc tgctggcggg aagggtcaag agggaaaaat ataatcctga aagggcacag 4200 aaattaaagg aatcagctgt gcgcctcctg cgaagccatc aggacctgaa tgcccttttg 4260 cttgaggtag aaggtccact gtgtaaaaaa ttgtctctca gcaaagtgat tgactgtgac 4320 agttctgagg cctatgctaa tcattctagt tcatttatag gctctgcttt gcaggatcaa 4380 gcctcaaggc tgggggttcc cgtgggtatt ctctcagccg ggatggttgc ctctagcgtg 4440 ggacagatct gcacggctcc agcggagacc agtcaccctg tgctgctgac tgtggagcag 4500 agaaagaagc tgtcttccct gttagagttt gctcagtatt tattggcaca cagtatgttc 4560 tcccgtcttt ccttctgtca agaattatgg aaaatacaga gttctttgtt gcttgaagcg 4620 gtgtggcatc ttcacgtaca aggcattgtg agcctgcaag agctgctgga aagccatccc 4680 gacatgcatg ctgtgggatc gtggctcttc aggaatctgt gctgcctttg tgaacagatg 4740 gaagcatcct gccagcatgc tgacgtcgcc agggccatgc tttctgattt tgttcaaatg 4800 tttgttttga ggggatttca gaaaaactca gatctgagaa gaactgtgga gcctgaaaaa 4860 atgccgcagg tcacggttga tgtactgcag agaatgctga tttttgcact tgacgctttg 4920 gctgctggag tacaggagga gtcctccact cacaagatcg tgaggtgctg gttcggagtg 4980 ttcagtggac acacgcttgg cagtgtaatt tccacagatc ctctgaagag gttcttcagt 5040 cataccctga ctcagatact cactcacagc cctgtgctga aagcatctga tgctgttcag 5100 atgcagagag agtggagctt tgcgcggaca caccctctgc tcacctcact gtaccgcagg 5160 ctctttgtga tgctgagtgc agaggagttg gttggccatt tgcaagaagt tctggaaacg 5220 caggaggttc actggcagag agtgctctcc tttgtgtctg ccctggttgt ctgctttcca 5280 gaagcgcagc agctgcttga agactgggtg gcgcgtttga tggcccaggc attcgagagc 5340 tgccagctgg acagcatggt cactgcgttc ctggttgtgc gccaggcagc actggagggc 5400 ccctctgcgt tcctgtcata tgcagactgg ttcaaggcct cctttgggag cacacgaggc 5460 taccatggct gcagcaagaa ggccctggtc ttcctgttta cgttcttgtc agaactcgtg 5520 ccttttgagt ctccccggta cctgcaggtg cacattctcc acccacccct ggttcccagc 5580 aagtaccgct ccctcctcac agactacatc tcattggcca agacacggct ggccgacctc 5640 aaggtttcta tagaaaacat gggactctac gaggatttgt catcagctgg ggacattact 5700 gagccccaca gccaagctct tcaggatgtt gaaaaggcca tcatggtgtt tgagcatacg 5760 gggaacatcc cagtcaccgt catggaggcc agcatattca ggaggcctta ctacgtgtcc 5820 cacttcctcc ccgccctgct cacacctcga gtgctcccca aagtccctga ctcccgtgtg 5880 gcgtttatag agtctctgaa gagagcagat aaaatccccc catctctgta ctccacctac 5940 tgccaggcct gctctgctgc tgaagagaag ccagaagatg cagccctggg agtgagggca 6000 gaacccaact ctgctgagga gcccctggga cagctcacag ctgcactggg agagctgaga 6060 gcctccatga cagaccccag ccagcgtgat gttatatcgg cacaggtggc agtgatttct 6120 gaaagactga gggctgtcct gggccacaat gaggatgaca gcagcgttga gatatcaaag 6180 attcagctca gcatcaacac gccgagactg gagccacggg aacacattgc tgtggacctc 6240 ctgctgacgt ctttctgtca gaacctgatg gctgcctcca gtgtcgctcc cccggagagg 6300 cagggtccct gggctgccct cttcgtgagg accatgtgtg gacgtgtgct ccctgcagtg 6360 ctcacccggc tctgccagct gctccgtcac cagggcccga gcctgagtgc cccacatgtg 6420 ctggggttgg ctgccctggc cgtgcacctg ggtgagtcca ggtctgcgct cccagaggtg 6480 gatgtgggtc ctcctgcacc tggtgctggc cttcctgtcc ctgcgctctt tgacagcctc 6540 ctgacctgta ggacgaggga ttccttgttc ttctgcctga aattttgtac agcagcaatt 6600 tcttactctc tctgcaagtt ttcttcccag tcacgagata ctttgtgcag ctgcttatct 6660 ccaggcctta ttaaaaagtt tcagttcctc atgttcagat tgttctcaga ggcccgacag 6720 cctctttctg aggaggacgt agccagcctt tcctggagac ccttgcacct tccttctgca 6780 gactggcaga gagctgccct ctctctctgg acacacagaa ccttccgaga ggtgttgaaa 6840 gaggaagatg ttcacttaac ttaccaagac tggttacacc tggagctgga aattcaacct 6900 gaagctgatg ctctttcaga tactgaacgg caggacttcc accagtgggc gatccatgag 6960 cactttctcc ctgagtcctc ggcttcaggg ggctgtgacg gagacctgca ggctgcgtgt 7020 accattcttg tcaacgcact gatggatttc caccaaagct caaggagtta tgaccactca 7080 gaaaattctg atttggtctt tggtggccgc acaggaaatg aggatattat ttccagattg 7140 caggagatgg tagctgacct ggagctgcag caggacctca tagtgccct cggccacacc 7200 ccttcccagg agcactcct ctttgagat ttcgcagac ggctccaggc tctgacaagc 7260 gggtggagcg tggctgccag ccttcagaga cagagggagc tgctaatgta caaacggatc 7320 ctcctccgcc tgccttcgtc tgtcctctgc ggcagcagct tccaggcaga acagcccatc 7380 actgccagat gcgagcagtt cttccacttg gtcactctg agatgagaa cttctgctcc 7440 cacggaggtg cctgacaca ggacacact gcccactct tcaggggcct cctgaacgcc 7500 tgtctgcgga gcagagaccc ctccctgatg gtcgactca tactggccaa gtgccagacg 7560 aaatgcccct taattttgac ctctgctctg gtgtgtgc cgagcctgga gcctgtgctg 7620 ctctgccggt ggaggagaca ctgccagagc ccgctgcccc gggaactgca gaagctacaa 7680 gaaggccggc agtttgccag cgatttcctc tccctgagg ctgcctcccc agcacccac 7740 ccggactggc tctcagctgc tgcactgcac tttgcgattc aacaagtcag ggaagaaaac 7800 atcaggaagc agctaagaa gctggactgc gagagagagg agctattggt ttccttttc 7860 ttcttctcct tgatgggcct gctgtcgtca catctgacct caaatagcac cacagacctg 7920 ccaaaggctt tccacgtttg tgcagcaatc ctcgagtgtt tagagaagag gaagatatcc 7980 tggctggcac tctttcagtt gacagagagt gacctcaggc tggggcggct cctcctccgt 8040 gtggccccgg atcagcacac caggctgctg cctttcgctt tttacagtct tctctcctac 8100 ttccatgaag acgcggccat cagggaagag gccttcctgc atgttgctgt ggacatgtac 8160 ttgaagctgg tccagctctt cgtggctggg gatacaagca cagtttcacc tccagctggc 8220 aggagcctgg agctcaaggg tcagggcaac cccgtggaac tgataacaaa agctcgtctt 8280 tttctgctgc agttaatacc tcggtgcccg aaaaagagct tctcacacgt ggcagagctg 8340 ctggctgatc gtggggactg cgacccagag gtgagcgccg ccctccagag cagacagcag 8400 gctgcccctg acgctgacct gtcccaggag cctcatctct tctgatgaga attcgatatc 8460 aagcttatcg ataccgtcga atcccccggg ctgcaggaat tcgagcatct taccgccatt 8520 tattcccata tttgttctgt ttttcttgat ttgggtatac atttaaatgt taataaaaca 8580 aaatggtggg gcaatcattt acatttttag ggatatgtaa ttactagttc aggtgtattg 8640 ccacaagaca aacatgttaa gaaactttcc cgttatttac gctctgttcc tgttaatcaa 8700 cctctggatt acaaaatttg tgaaagattg actgatattc ttaactatgt tgctcctttt 8760 acgctgtgtg gatatgctgc tttaatgcct ctgtatcatg ctattgcttc ccgtacggct 8820 ttcgttttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc 8880 gttgtccgtc aacgtggcgt ggtgtgctct gtgtttgctg acgcaacccc cactggctgg 8940 ggcattgcca ccacctgtca actcctttct gggactttcg ctttccccct cccgatcgcc 9000 acggcagaac tcatcgccgc ctgccttgcc cgctgctgga caggggctag gttgctgggc 9060 actgataatt ccgtggtgtt gtcggggaag ggcctgctgc cggctctgcg gcctcttccg 9120 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgga 9180 attcgagctc ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact 9240 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc 9300 tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct 9360 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt 9420 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt 9480 ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta taacttgcaa 9540 agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat 9600 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 9660 gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac 9720 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 9780 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta 9840 gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat 9900 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 9960 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 10020 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt 10080 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 10140 agcgccctag cgcccgctcc ttcgctttc ttcccttcct ttctcgccac gttcgccggc 10200 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 10260 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 10320 tagacggtt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 10380 caaactggaa caacactcaa ccctatctcg gtctattcttt ttgatttata agggattttg 10440 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattttt 10500 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc 10560 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 10620 gataaatgct tcaataatag cacctagatc aagagacagg atgaggatcg tttcgcatga 10680 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 10740 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 10800 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag 10860 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 10920 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 10980 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 11040 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 11100 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 11160 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg 11220 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 11280 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 11340 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 11400 tgctttacgg tatcgccgct cccgattcgc agc 11433 <210> 25 <211> 1455 <212> PRT <213> Homo sapiens <400> 25 Met Ser Asp Ser Trp Val Pro Asn Ser Ala Ser Gly Gln Asp Pro Gly 1 5 10 15 Gly Arg Arg Arg Ala Trp Ala Glu Leu Leu Ala Gly Arg Val Lys Arg 20 25 30 Glu Lys Tyr Asn Pro Glu Arg Ala Gln Lys Leu Lys Glu Ser Ala Val 35 40 45 Arg Leu Leu Arg Ser His Gln Asp Leu Asn Ala Leu Leu Leu Leu Glu Val 50 55 60 Glu Gly Pro Leu Cys Lys Lys Leu Ser Leu Ser Lys Val Ile Asp Cys 65 70 75 80 Asp Ser Ser Glu Ala Tyr Ala Asn His Ser Ser Ser Phe Ile Gly Ser 85 90 95 Ala Leu Gln Asp Gln Ala Ser Arg Leu Gly Val Pro Val Gly Ile Leu 100 105 110 Ser Ala Gly Met Val Ala Ser Ser Val Gly Gln Ile Cys Thr Ala Pro 115 120 125 Ala Glu Thr Ser His Pro Val Leu Leu Thr Val Glu Gln Arg Lys Lys 130 135 140 Leu Ser Ser Leu Leu Glu Phe Ala Gln Tyr Leu Leu Ala His Ser Met 145 150 155 160 Phe Ser Arg Leu Ser Phe Cys Gln Glu Leu Trp Lys Ile Gln Ser Ser 165 170 175 Leu Leu Leu Glu Ala Val Trp His Leu His Val Gln Gly Ile Val Ser 180 185 190 Leu Gln Glu Leu Leu Glu Ser His Pro Asp Met His Ala Val Gly Ser 195 200 205 Trp Leu Phe Arg Asn Leu Cys Cys Leu Cys Glu Gln Met Glu Ala Ser 210 215 220 Cys Gln His Ala Asp Val Ala Arg Ala Met Leu Ser Asp Phe Val Gln 225 230 235 240 Met Phe Val Leu Arg Gly Phe Gln Lys Asn Ser Asp Leu Arg Arg Thr 245 250 255 Val Glu Pro Glu Lys Met Pro Gln Val Thr Val Asp Val Leu Gln Arg 260 265 270 Met Leu Ile Phe Ala Leu Asp Ala Leu Ala Ala Gly Val Gln Glu Glu 275 280 285 Ser Ser Thr His Lys Ile Val Arg Cys Trp Phe Gly Val Phe Ser Gly 290 295 300 His Thr Leu Gly Ser Val Ile Ser Thr Asp Pro Leu Lys Arg Phe Phe 305 310 315 320 Ser His Thr Leu Thr Gln Ile Leu Thr His Ser Pro Val Leu Lys Ala 325 330 335 Ser Asp Ala Val Gln Met Gln Arg Glu Trp Ser Phe Ala Arg Thr His 340 345 350 Pro Leu Leu Thr Ser Leu Tyr Arg Arg Leu Phe Val Met Leu Ser Ala 355 360 365 Glu Glu Leu Val Gly His Leu Gln Glu Val Leu Glu Thr Gln Glu Val 370 375 380 His Trp Gln Arg Val Leu Ser Phe Val Ser Ala Leu Val Val Cys Phe 385 390 395 400 Pro Glu Ala Gln Gln Leu Leu Glu Asp Trp Val Ala Arg Leu Met Ala 405 410 415 Gln Ala Phe Glu Ser Cys Gln Leu Asp Ser Met Val Thr Ala Phe Leu 420 425 430 Val Val Arg Gln Ala Ala Leu Glu Gly Pro Ser Ala Phe Leu Ser Tyr 435 440 445 Ala Asp Trp Phe Lys Ala Ser Phe Gly Ser Thr Arg Gly Tyr His Gly 450 455 460 Cys Ser Lys Lys Ala Leu Val Phe Leu Phe Thr Phe Leu Ser Glu Leu 465 470 475 480 Val Pro Phe Glu Ser Pro Arg Tyr Leu Gln Val His Ile Leu His Pro 485 490 495 Pro Leu Val Pro Ser Lys Tyr Arg Ser Leu Leu Thr Asp Tyr Ile Ser 500 505 510 Leu Ala Lys Thr Arg Leu Ala Asp Leu Lys Val Ser Ile Glu Asn Met 515 520 525 Gly Leu Tyr Glu Asp Leu Ser Ser Ala Gly Asp Ile Thr Glu Pro His 530 535 540 Ser Gln Ala Leu Gln Asp Val Glu Lys Ala Ile Met Val Phe Glu His 545 550 555 560 Thr Gly Asn Ile Pro Val Thr Val Met Glu Ala Ser Ile Phe Arg Arg 565 570 575 Pro Tyr Tyr Val Ser His Phe Leu Pro Ala Leu Leu Thr Pro Arg Val 580 585 590 Leu Pro Lys Val Pro Asp Ser Arg Val Ala Phe Ile Glu Ser Leu Lys 595 600 605 Arg Ala Asp Lys Ile Pro Ser Leu Tyr Ser Thr Tyr Cys Gln Ala 610 615 620 Cys Ser Ala Ala Glu Glu Lys Pro Glu Asp Ala Ala Leu Gly Val Arg 625 630 635 640 Ala Glu Pro Asn Ser Ala Glu Glu Pro Leu Gly Gln Leu Thr Ala Ala 645 650 655 Leu Gly Glu Leu Arg Ala Ser Met Thr Asp Pro Ser Gln Arg Asp Val 660 665 670 Ile Ser Ala Gln Val Ala Val Ile Ser Glu Arg Leu Arg Ala Val Leu 675 680 685 Gly His Asn Glu Asp Asp Ser Ser Val Glu Ile Ser Lys Ile Gln Leu 690 695 700 Ser Ile Asn Thr Pro Arg Leu Glu Pro Arg Glu His Ile Ala Val Asp 705 710 715 720 Leu Leu Leu Thr Ser Phe Cys Gln Asn Leu Met Ala Ala Ser Ser Val 725 730 735 Ala Pro Pro Glu Arg Gln Gly Pro Trp Ala Ala Leu Phe Val Arg Thr 740 745 750 Met Cys Gly Arg Val Leu Pro Ala Val Leu Thr Arg Leu Cys Gln Leu 755 760 765 Leu Arg His Gln Gly Pro Ser Leu Ser Ala Pro His Val Leu Gly Leu 770 775 780 Ala Ala Leu Ala Val His Leu Gly Glu Ser Arg Ser Ala Leu Pro Glu 785 790 795 800 Val Asp Val Gly Pro Pro Ala Pro Gly Ala Gly Leu Pro Val Pro Ala 805 810 815 Leu Phe Asp Ser Leu Leu Thr Cys Arg Thr Arg Asp Ser Leu Phe Phe 820 825 830 Cys Leu Lys Phe Cys Thr Ala Ala Ile Ser Tyr Ser Leu Cys Lys Phe 835 840 845 Ser Ser Gln Ser Arg Asp Thr Leu Cys Ser Cys Leu Ser Pro Gly Leu 850 855 860 Ile Lys Lys Phe Gln Phe Leu Met Phe Arg Leu Phe Ser Glu Ala Arg 865 870 875 880 Gln Pro Leu Ser Glu Glu Asp Val Ala Ser Leu Ser Trp Arg Pro Leu 885 890 895 His Leu Pro Ser Ala Asp Trp Gln Arg Ala Ala Leu Ser Leu Trp Thr 900 905 910 His Arg Thr Phe Arg Glu Val Leu Lys Glu Glu Asp Val His Leu Thr 915 920 925 Tyr Gln Asp Trp Leu His Leu Glu Leu Glu Ile Gln Pro Glu Ala Asp 930 935 940 Ala Leu Ser Asp Thr Glu Arg Gln Asp Phe His Gln Trp Ala Ile His 945 950 955 960 Glu His Phe Leu Pro Glu Ser Ser Ala Ser Gly Gly Cys Asp Gly Asp 965 970 975 Leu Gln Ala Ala Cys Thr Ile Leu Val Asn Ala Leu Met Asp Phe His 980 985 990 Gln Ser Ser Arg Ser Tyr Asp His Ser Glu Asn Ser Asp Leu Val Phe 995 1000 1005 Gly Gly Arg Thr Gly Asn Glu Asp Ile Ile Ser Arg Leu Gln Glu 1010 1015 1020 Met Val Ala Asp Leu Glu Leu Gln Gln Asp Leu Ile Val Pro Leu 1025 1030 1035 Gly His Thr Pro Ser Gln Glu His Phe Leu Phe Glu Ile Phe Arg 1040 1045 1050 Arg Arg Leu Gln Ala Leu Thr Ser Gly Trp Ser Val Ala Ala Ser 1055 1060 1065 Leu Gln Arg Gln Arg Glu Leu Leu Met Tyr Lys Arg Ile Leu Leu 1070 1075 1080 Arg Leu Pro Ser Ser Val Leu Cys Gly Ser Ser Phe Gln Ala Glu 1085 1090 1095 Gln Pro Ile Thr Ala Arg Cys Glu Gln Phe Phe His Leu Val Asn 1100 1105 1110 Ser Glu Met Arg Asn Phe Cys Ser His Gly Gly Ala Leu Thr Gln 1115 1120 1125 Asp Ile Thr Ala His Phe Phe Arg Gly Leu Leu Asn Ala Cys Leu 1130 1135 1140 Arg Ser Arg Asp Pro Ser Leu Met Val Asp Phe Ile Leu Ala Lys 1145 1150 1155 Cys Gln Thr Lys Cys Pro Leu Ile Leu Thr Ser Ala Leu Val Trp 1160 1165 1170 Trp Pro Ser Leu Glu Pro Val Leu Leu Cys Arg Trp Arg Arg His 1175 1180 1185 Cys Gln Ser Pro Leu Pro Arg Glu Leu Gln Lys Leu Gln Glu Gly 1190 1195 1200 Arg Gln Phe Ala Ser Asp Phe Leu Ser Pro Glu Ala Ala Ser Pro 1205 1210 1215 Ala Pro Asn Pro Asp Trp Leu Ser Ala Ala Ala Leu His Phe Ala 1220 1225 1230 Ile Gln Gln Val Arg Glu Glu Asn Ile Arg Lys Gln Leu Lys Lys 1235 1240 1245 Leu Asp Cys Glu Arg Glu Glu Leu Leu Val Phe Leu Phe Phe Phe 1250 1255 1260 Ser Leu Met Gly Leu Leu Ser Ser His Leu Thr Ser Asn Ser Thr 1265 1270 1275 Thr Asp Leu Pro Lys Ala Phe His Val Cys Ala Ala Ile Leu Glu 1280 1285 1290 Cys Leu Glu Lys Arg Lys Ile Ser Trp Leu Ala Leu Phe Gln Leu 1295 1300 1305 Thr Glu Ser Asp Leu Arg Leu Gly Arg Leu Leu Leu Arg Val Ala 1310 1315 1320 Pro Asp Gln His Thr Arg Leu Leu Pro Phe Ala Phe Tyr Ser Leu 1325 1330 1335 Leu Ser Tyr Phe His Glu Asp Ala Ala Ile Arg Glu Glu Ala Phe 1340 1345 1350 Leu His Val Ala Val Asp Met Tyr Leu Lys Leu Val Gln Leu Phe 1355 1360 1365 Val Ala Gly Asp Thr Ser Thr Val Ser Pro Pro Ala Gly Arg Ser 1370 1375 1380 Leu Glu Leu Lys Gly Gln Gly Asn Pro Val Glu Leu Ile Thr Lys 1385 1390 1395 Ala Arg Leu Phe Leu Leu Gln Leu Ile Pro Arg Cys Pro Lys Lys 1400 1405 1410 Ser Phe Ser His Val Ala Glu Leu Leu Ala Asp Arg Gly Asp Cys 1415 1420 1425 Asp Pro Glu Val Ser Ala Ala Leu Gln Ser Arg Gln Gln Ala Ala Ala 1430 1435 1440 Pro Asp Ala Asp Leu Ser Gln Glu Pro His Leu Phe 1445 1450 1455

Claims

1. In order from 5' to 3': (a) a human phosphoglycerate kinase (PGK) promoter sequence or a functional homologue or variant thereof; (b) a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof; (c) the woodchuck hepatitis virus regulatory element (WPRE) RNA export signal sequence or a functional variant or fragment thereof; 1. An expression cassette comprising a polynucleotide sequence comprising:

2. 2. The expression cassette of claim 1, wherein the FANCA polypeptide or a functional fragment or variant thereof comprises the sequence shown in SEQ ID NO:

25.

3. 2. The expression cassette of claim 1, wherein the sequence encoding the FANCA polypeptide or a functional fragment or variant thereof comprises the sequence shown in SEQ ID NO:

8.

4. 2. The expression cassette of claim 1, wherein the PGK promoter comprises the nucleotide sequence of SEQ ID NO:

7.

5. 2. The expression cassette of claim 1, wherein the WPRE element comprises the nucleotide sequence of SEQ ID NO:

23.

6. 2. The expression cassette of claim 1, comprising the nucleotide sequence of SEQ ID NO:

24.

7. 9. The expression cassette of claim 1, further comprising one or more enhancer sequences.

8. (d) polypurine tract (PPT) or polyadenylation (polyA) signal sequence 2. The expression cassette of claim 1, further comprising:

9. The following sequence: (e) a packaging signal sequence, (f) a truncated Gag sequence; (g) Rev response element (RRE), (h) central polypurine tract (cPPT), (i) the central terminal sequence (CTS), and (j) an upstream sequence element (USE), optionally derived from simian virus 40 (SV40-USE); 2. The expression cassette of claim 1, further comprising one or more of:

10. A recombinant gene delivery vector comprising the expression cassette of any one of claims 1 to 9.

11. 11. The recombinant gene delivery vector of claim 10, which is a virus or a viral vector.

12. The recombinant gene delivery vector of claim 11, wherein the virus or viral vector is a lentivirus (LV).

13. 13. A cell comprising the expression cassette of claim 1 or the recombinant gene delivery vector of claim 11 or claim 12.

14. The cell of claim 13, which is a hematopoietic stem cell.

15. CD34 + The cell of claim 13, which is a cell.

16. A pharmaceutical composition comprising a pharma- ceutically acceptable excipient and the recombinant gene delivery vector of any one of claims 10 to 12.

17. A pharmaceutical composition comprising a pharma- ceutically acceptable excipient and the cell of any one of claims 13 to 15.

18. 18. A method of treating Fanconi anemia in a subject in need thereof, comprising providing to the subject a pharmaceutical composition according to claim 17 or claim 17.

19. A method for treating Fanconi anemia in a subject in need thereof, comprising administering to said subject a CD34 + providing a cell to the subject, the expression cassette comprising, in 5' to 3' order: (a) a human phosphoglycerate kinase (PGK) promoter sequence or a functional homologue or variant thereof; (b) a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof; (c) the woodchuck hepatitis virus regulatory element (WPRE) RNA export signal sequence or a functional variant or fragment thereof; wherein the sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof is operably linked to a PGK promoter sequence.

20. CD34 + 20. The method of claim 19, wherein the cells are obtained from the subject.

21. 21. The method of claim 20, wherein the CD34+ cells are obtained from the subject after treating the subject with a combination of (i) G-CSF or filgrastim and (ii) plerixafor.

21. CD34 + The method of claim 20, wherein the cells are transduced with a recombinant gene delivery vector comprising the expression cassette.

22. CD34 + The cells are + 22. The method of claim 21, wherein the cells are transduced by contacting the recombinant gene delivery vector with the cells for about 24 hours.

23. 1. A method for treating Fanconi anemia in a subject in need thereof, comprising: (a) providing to said subject a combination of (i) G-CSF or filgrastim and (ii) plerixafor to mobilize CD34+ cells in said subject; (b) isoform of CD34 + Obtaining a biological sample comprising cells, the biological sample optionally being peripheral blood or bone marrow; (c) preparing a cell population enriched for CD34+ cells from the biological sample; (d) the following, in 5' to 3' order: (i) a promoter sequence or a functional homologue or variant thereof; (ii) a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof; wherein a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof is operably linked to a PGK promoter sequence, said transduction comprising contacting said CD34+ cell enriched cell population with the lentiviral vector for about 24 hours; and (e) providing to the subject the lentiviral vector-transduced cell population resulting from step (d). A method comprising:

24. 24. The method of claim 23, wherein preparing the cell population comprises removing red blood cells.

25. The step of preparing the cell population comprises selecting CD34 + 24. The method of claim 23, further comprising concentrating the cells.

26. 24. The method of claim 23, wherein the method inhibits the development of Fanconi anemia in the subject, halts the progression of Fanconi anemia, and / or reverses the progression of the hematological symptoms of Fanconi anemia.

27. 27. The method of claim 26, wherein the hematological symptoms of Fanconi anemia are selected from one or more of BMF, thrombocytopenia, leukopenia, pancytopenia, neutropenia and anemia.

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