BR112019004594B1 - Use of CD34+ cells comprising an expression cassette to treat Fanconi anemia. - Google Patents

Abstract

The present invention provides compositions and methods for restoring FANCA expression in cells with decreased or absent FANCA gene product. In particular, methods and compositions for gene therapy of Fanconi anemia are disclosed.

Description

RELATED ORDERS

[001]This application claims priority to, and the benefit of, U.S. Provisional Application No. 62/385185, filed September 8, 2016, and U.S. Provisional Application 62/412028, filed October 24, 2016, the contents of which are incorporated in full by reference herein.

FIELD OF THE INVENTION

[002]The present invention generally relates to gene transfer into cells with decreased or absent protein activity from one or more proteins encoded by FANCA.

STATEMENT REGARDING THE LISTING OF SEQUENCES

[003]The Sequence Listing associated with this request is provided in text format instead of a paper copy and is incorporated as a reference in the descriptive report. The name of the text file containing the sequence is ROPA_002_01WO_ST25.txt. The text file is 46KB in size, was created on September 8, 2017, and is being submitted electronically via EFS-WEB.

FUNDAMENTALS OF THE INVENTION

[004] Fanconi anemia (FA) is an autosomal recessive disease (except for the FA-B complementation group, which is X-linked), where the average survival of patients is around 24 years (Butturini A, et al. (1994) Blood 84:1650 to 1655; Kutler DI, et al. (2003) Blood 101:1249 to 1256). At birth, the blood count of these patients is generally normal. Macrocytosis is often the first hematological abnormality detected in these patients. Usually, this develops with thrombocytopenia, anemia, and pancytopenia. Bone marrow failure (BMF) is usually observed in these patients after 5 to 10 years, with an average age of onset of the hematological disease of 7 years. About 80% of patients with FA will develop evidence of BMF in the first decade of life. Based on epidemiological studies to date, if malignant episodes do not appear before aplasia, virtually all patients with AF will develop BMF by age 40 (Butturini A, et al. (1994) Blood 84:1650 to 1655; Kutler DI, et al. (2003) Blood 101:1249 to 1256), this being the main cause of mortality in these patients.

[005]Due to the complex clinical manifestations of AF, the management of these patients is focused primarily on improving the following syndromes: bone marrow failure (BMF), myeloid leukemia, and solid tumors.

[006]Current treatments include androgens, such as those composed of fluoxymesterone, oxymetholone, or stanozolol. As recently reviewed by Dufour et al. (Dufour, C. and Svahn, J. (2008). Bone Marrow Transplant 41 Suppl 2: S90 to 95), AF patients may show some response to androgens, provided that this treatment is not initiated at a very advanced stage of the disease. Approximately 75% of patients respond to androgens. A combination with 2 mg/kg/day of prednisolone reduces the risk of liver toxicity. In general, in the absence of a suitable bone marrow donor, androgens may be considered as an alternative treatment when there is some residual hematopoiesis, but not as a definitive long-term treatment.

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

[008]There are no specific indications for hematopoietic growth factors in the treatment of AF. However, two pharmacological groups of drugs with indications for the specific symptoms of AF (anemia and neutropenia) have been identified: 1) erythropoietin and 2) granulocyte colony-stimulating factors (G-CSFs). Several erythropoietins (e.g., Aranesp, Nespo, Exjade) are approved for the treatment of anemia, and G-CSFs (e.g., G-CSF analogs such as filgrastim, biogastrin, neulasta) are approved for the treatment of neutropenia. Although hematopoietic growth factors, such as erythropoietin and granulocyte colony-stimulating factors, have been tested in a limited number of patients with AF, the responses have been partial and transient (Dufour et al., 2008). Currently, these treatments do not represent satisfactory long-term options. For short-term treatment in neutropenic patients, G-CSF can be used for acute infections to increase the number of peripheral neutrophils, potentiating antibiotics. However, these drug treatments are far from being a definitive treatment for patients with AF.

[009]Currently, the only curative treatment for the hematological manifestations of the disease is based on allogeneic hematopoietic transplantation. Although the outcome for AF patients transplanted with grafts from HLA-identical sibling donors is generally satisfactory, only about 20% of AF patients will have an HLA-identical sibling. A significant proportion of AF patients without a sibling donor can be transplanted from alternative donors, although these transplants are associated with higher morbidity and mortality. In the remaining AF patients, alternative therapies are not currently available.

[010]Consequently, there is a critical need for an effective treatment regimen for AF. The present invention addresses this need, among others.

SUMMARY OF THE INVENTION

[011] Embodiments of the present invention comprise polynucleotide cassettes for enhanced FANCA expression. In some embodiments, the polynucleotide cassette comprises a sequence encoding a human FANCA cDNA optimized at the codon to increase mRNA stability after transcription.

[012] In one embodiment, the present invention includes an expression cassette comprising a polynucleotide sequence comprising, in the following order 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 marmot hepatitis virus (WPRE) regulatory element 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 particular embodiments, the FANCA polypeptide or a functional fragment or variant thereof comprises the sequence presented in SEQ ID NO: 25; the sequence encoding the FANCA polypeptide or a functional fragment or variant thereof comprises the sequence presented in SEQ ID NO: 8; The PGK promoter comprises a nucleotide sequence of SEQ ID NO: 7; and/or the WPRE element comprises a nucleotide sequence of SEQ ID NO: 23. In particular embodiments, the cassette comprises a region of the nucleotide sequence of SEQ ID NO: 24. In certain 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).

[013] In one embodiment, the present invention provides an expression cassette comprising a polynucleotide sequence comprising: a) a promoter sequence; b) a sequence encoding a polypeptide; and c) a ribonucleic acid (RNA) export signal, wherein the promoter sequence is operably linked to the sequence encoding the FANCA polypeptide (SEQ ID NO: 25), and optionally wherein a) to c) are present in the expression cassette in the 5’ to 3’ order. In certain embodiments, the promoter is a phosphoglycerate kinase (PGK) promoter. In certain embodiments, the sequence encoding the polypeptide is optimized at the codon. In some embodiments, the sequence encoding the polypeptide is an optimized version in the human FANCA cDNA codon that exhibits at least 85% identity to SEQ ID NO: 8. In particular embodiments, the RNA export signal is a modified post-transcriptional regulatory element from marmot hepatitis virus (wPRE).

[014] In certain embodiments, the modified wPRE is a chimeric wPRE comprising a sequence that exhibits at least 80% identity to 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 polyadenylation (polyA) signal sequence. In some embodiments, the expression cassette further comprises one or more of the following sequences: i) a packaging 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 from simian virus 40 (SV40-USE). In some embodiments, the expression cassette further comprises 5’ and 3’ long terminal repeat (LTR) sequences.

[015]In a related embodiment, the present invention provides a recombinant gene delivery vector comprising an expression cassette disclosed in this report. In certain embodiments, the recombinant gene delivery vector is a virus or viral vector. In certain embodiments, the virus or viral vector is a lentivirus (LV).

[016] In another related embodiment, the present invention provides a cell comprising an expression cassette or gene delivery vector disclosed in this report. 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, for example, a bone marrow cell depleted in the lineage. In particular embodiments, the cell is a hematopoietic stem cell or a CD34+ cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a CD34+ hematopoietic stem cell. In some embodiments, the cell is a committed hematopoietic erythroid progenitor cell.

[017] In a related embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and recombinant gene or cell delivery vector disclosed in this report. In certain aspects of the invention, pharmaceutical compositions are provided comprising a polynucleotide cassette of the invention and a pharmaceutical excipient. In other embodiments, the pharmaceutical composition comprises a gene delivery vector of the invention and a pharmaceutical excipient.

[018]Methods and compositions are provided for the use of vector compositions for gene therapy, for example, viral vectors, comprising such gene expression cassettes for use in the preparation of drugs useful in central and targeted gene therapy of diseases, disorders and dysfunctions in an animal and, in particular, in humans.

[019]In another embodiment, the present invention provides a method for treating or preventing a disease or disorder in an individual in need thereof, comprising providing the individual with an expression cassette, gene delivery vector or pharmaceutical composition disclosed in this report.

[020]In another embodiment, the present invention includes a method for treating Fanconi anemia in an individual in need thereof, comprising providing the individual with a pharmaceutical composition disclosed in this report.

[021] In a related embodiment, the present invention includes a method for treating Fanconi anemia in an individual in need thereof, comprising providing the individual with CD34+ cells comprising an expression cassette, wherein the expression cassette comprises a polynucleotide sequence comprising in the following 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 marmot hepatitis virus (WPRE) regulatory element 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 certain embodiments, the CD34+ cells have been obtained from the individual. In particular embodiments, CD34+ cells were obtained from the individual after the individual was treated with a combination of: (i) G-CSF or Filgrastine; and (ii) Plerifaxor. In particular embodiments, the CD34+ cells were transduced with the recombinant gene delivery vector comprising the expression cassette. In one embodiment, the CD34+ cells were transduced by contacting the CD34+ cells with the recombinant gene delivery vector for approximately 24 hours.

[022] In another embodiment, the present invention provides a method for treating Fanconi anemia in an individual in need thereof, comprising: (a) providing the individual with a combination of: (i) G-CSF or Filgrastine; and (ii) Plerifaxor to mobilize CD34+ cells in the individual; (b) obtaining a biological sample comprising CD34+ cells from the individual, wherein the biological sample is optionally peripheral blood or bone marrow; (c) preparing a cell population enriched with CD34+ cells from the biological sample; (d) transducing the cell population enriched with CD34+ cells with a recombinant gene delivery vector comprising an expression cassette comprising a polynucleotide sequence comprising in the following order 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 the sequence encoding the human FANCA polypeptide or functional fragment or variant thereof is operably linked to the PGK promoter sequence, wherein transduction comprises contacting the CD34+ cell-enriched cell population with the lentiviral vector for approximately 24 hours; and (e) delivering the lentiviral vector-transduced cell population resulting from step (d) to the individual. In certain embodiments, cell population preparation comprises depleting erythrocytes and/or enriching CD34+ cells by positive selection, negative selection, or a combination thereof. In particular embodiments, the method inhibits the development, halts the progression, and/or reverses the progression of a hematological manifestation of Fanconi anemia in the individual. In particular embodiments, the hematological manifestation of Fanconi anemia is selected from one or more of the following: BMF, thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia.

[023]Other features and advantages of the invention will be evident from, and covered by, the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[024]Figure 1 is a schematic diagram of an exemplary construct, PGK-FANCA.WPRE*LV.

[025] Figure 2A shows a schematic representation of LVs expressing FANCA under the control of different internal promoters. Figure 2B shows a Western blot analysis of FANCA in FA-A cells transduced with vectors shown in panel A. The correction of MMC hypersensitivity from hematopoietic progenitors from FA-A mice subjected to gene therapy is shown. Bone marrow (BM) cells from FA-A were transduced with PGK_FANCA-WPRE* or control SF1-EGFP LVs and transplanted into irradiated FA-A mice. At 7 months post-transplant, BM samples were collected and cultured on methylcellulose in the presence of increased concentrations of mitomycin C (MMC).

[026] Figure 3 presents data showing the 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 lymphoblast cell line (LCL) cells transduced with LVs. Mean values from 3 different experiments are shown. Figure 3B shows the restored formation of FANCD2 nuclear foci in vector-transduced FA-A LCLs exposed to mitomycin C (MMC).

[027] Figure 4 presents data showing the in vivo efficacy and safety of gene therapy for FA with PGK-FANCA.Wpre* LV. Figure 4A shows the construct. Figure 4B represents the methodology by which 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 BM samples from transplanted FA-A mice cultured on methylcellulose in the absence and presence of MMC.

[028]Figure 5 shows the number of proviral copies in FANCA-/- mice transplanted with syngeneic bone marrow cells previously transduced with lentiviral vectors carrying the therapeutic FANCA gene under the control of the PGK promoter. PB = peripheral blood; BM = bone marrow.

[029]Figure 6 presents data showing the correction of MMC hypersensitivity from hematopoietic progenitors from FAA mice subjected to gene therapy with the medicinal product. Bone marrow (BM) cells from FA-A were transduced with PGK_FANCA-Wpre* or SF1-EGFP LVs and transplanted into irradiated FA-A mice. Seven (7) months post-transplant, BM samples were collected and cultured on methylcellulose in the presence of increased concentrations of MMC.

[030]Figure 7 represents enhanced transduction efficacy of cryopreserved bone marrow progenitors from three patients with Fanconi anemia. Samples were subjected to standard transductions consisting of a single transduction cycle (16 h) after 2 h of static preloading (white bars; 1xS) or enhanced transduction consisting of three transduction cycles (2 h + 2 h + 12 h) with lentiviral vectors (gray bars; 3xD).

[031] Figure 8 shows the relevance of the WPRE sequence on the functional properties of lentiviral vectors expressing FANCA under the control of the PGK promoter. Figure 8A: Reversal of sensitivity (MMC) of FA-A LCLs transduced with PGK-FANCA and PGK-FANCA-WPRE LVs. Mean values from 3 different experiments are shown. Figure 8B: Reversal of sensitivity MMC of FA-A hematopoietic progenitors (colony-forming cells, CFCs) transduced with SFFV-FANCA LV and PGK-FANCA LVs (“Expt 1”) and with PGK-FANCA and PGK-FANCA-WPRE LVs (“Expt 2”). White bars = no MMC; Black bars = 10 nM MMC. MMC = mitomycin C

[032]Figure 9 shows the effectiveness of GALV-TR and VSV-G pseudotyped lentiviral vectors in transducing hematopoietic progenitors from the bone marrow of patients with Fanconi anemia with EGFP-LVs.

[033] Figure 10 shows low in vitro transformation potential of lentiviral vectors that host the hPGK promoter. Figure 10A: representation of the vectors. Figure 10B: transformation capacity measured in replication frequency relative to the number of copies.

[034]Figure 11 is a representation of a hematopoietic stem cell (HSC) collection and gene therapy experiment illustrative of FA-A patients.

[035] Figure 12 shows the hematological parameters of patients recruited in the study described in the Examples. Figure 12A shows the results for hemoglobin. Figure 12B shows the results for neutrophils. Figure 12C shows the results for platelets. Figure 12D shows the results for CD34+ cells.

[036]Figure 13 illustrates the phase II study of the Fancostem protocol aimed at evaluating the safety and efficacy of CD34+ cell mobilization and collection after treatment with Plerixafor (MOZOBIL) and Filgrastim (also known as G-CSF) (NEUPOGENE) in patients with Fanconi anemia. The number of patients is 10.

[037]Figure 14 shows the G-CSF/Plerixafor-mediated mobilization of CD34+ cells in FA-A patients.

[038]Figure 15 shows G-CSF/Plerixafor-mediated mobilization of CFCs in AF-A patients.

[039]Figure 16 is a summary of CD34+ cells collected from FA-A patients mobilized with G-CSF/Plerixafor. Figure 16A shows the collection of CD34+ cells in FANCOSTEM and Figure 16B shows a comparison to previous studies.

[040]Figure 17 is a graph showing the comparison between predicted CD34+ cell numbers in bone marrow (BM) versus actual numbers in mobilized peripheral blood (mPB).

[041]Figure 18 is a graph of the collection and purification of CD34+ FA- A cells from mobilized peripheral blood (mPB).

[042]Figure 19 shows the expression of CD34 before and after immunoselection of mobilized peripheral blood (mPB) CD34+ cells from healthy donors (HD) and FA patients.

[043]Figure 20 shows patient FA 02005 meeting the criteria for the FANCOSTEM and FANCOLEN studies. Figure 20A shows cell counts; Figure 20B shows hematopoietic stem cell (HSC) content versus age.

[044]Figure 21 (A to E) presents test results showing the FA diagnosis of patient FA-02005 before gene therapy.

[045]Figure 22 shows the monitoring parameter of the cell preparation process for Patient FA-A 02005.

[046]Figure 23 is a graph representing the number of vector copies before and at 2 weeks, 4 weeks, 6 weeks, 2 months, 3 months, 4 months and 5 months after gene therapy in patient FA-02005.

[047]Figure 24 shows the follow-up of the first unconditioned FA-A patient (FA-02005) before and after gene therapy, as measured by hemoglobin levels.

[048]Figure 25 shows the follow-up of the first unconditioned FA-A patient (FA-02005) before and after gene therapy, as measured by neutrophil counts.

[049]Figure 26 shows the follow-up of the first unconditioned FA-A patient (FA-02005) before and after gene therapy, as measured by platelet counts.

[050]Figure 27 is a graph of the hematological evolution of patient FA-A 02002.

[051]Figure 28 shows the diagnosis of FA 02002 as Non-mosaic; Homozygous FANCA c.239 C>T p.Gln99*MMC Hypersensitive; Supplemented by FANC.

[052]Figure 29 shows the cell preparation process in patient FA- A 02002.

[053]Figure 30 presents the analysis of CD34 expression in a healthy donor (HD) and mobilized peripheral blood (mPB) FA during the different steps required for LV transduction in patient FA 02002.

[054]Figure 31 is a graph representing the number of vector copies before and after gene therapy in patient FA 02002.

[055]Figure 32 presents the follow-up data for patient FA-A 02002 infused with cryopreserved cells, as measured by the amount of hemoglobin.

[056]Figure 33 presents the follow-up data for patient FA-A 02002 infused with cryopreserved cells, as measured by the number of neutrophils.

[057]Figure 34 presents the follow-up data for patient FA-A 02005 infused with cryopreserved cells, as measured by the amount of platelets.

[058] Figure 35 shows the transduction of fresh mobilized peripheral blood (mPB) CD34+ cells from FA-A patients using validated conditions. Figure 35A presents 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.

[059] Figure 36 shows the data for the grafting of CD34+ FA-A mPB-corrected cells in NSG mice. Figure 36A shows the protocol. Figure 36B shows the results for patient 02002. Panel C shows the results for patient 02003. Figure 36D shows the results for patient 02004. mPB = mobilized peripheral blood.

[060] Figure 37 represents the in vivo selection of FA-corrected HPCs from patient 02002 in NOD scid gamma (NSG) mice. Figure 37A shows the protocol. Figure 37B is a graph of pre-transplant CFCs. Figure 37C is a graph of hCFCs 30 days post-transplant.

[061]Figure 38 is a map of the 5.7 kb plasmid that encodes the VSV envelope glycoprotein G under the control of the CMV promoter and carries the kanamycin resistance gene for selection purposes.

[062]Figure 39 is a map of the 3.5 kb plasmid that encodes the HIV-1 rev gene under the control of the CMV promoter and carries the kanamycin resistance gene for selection purposes.

[063]Figure 40 is a map of the 8.8 kb plasmid containing the HIV-1 gag and pol genes that encode for HIV-1 structural and enzymatic proteins under the control of the CMV promoter. It also contains human beta-globin intron 2 (HBB2), the HIV-1 response element to Rev (RRE), and the kanamycin resistance gene.

[064]Figure 41 is a map of the 11621 base pair pCCL-SIN-cPPT/CTS-hPGK-hFANCA-WPRE transfer cassette.

[065]Figure 42 represents the LAM-PCR analysis of FANCA-LV insertion sites in FA hematopoietic stem cells (HSCs).

[066]Figure 43 represents the LAM-PCR results for screening cells treated with FANCA-LV. Figure 43A represents the protocol and Figure 43B is a graph of the data.

[067]Figure 44 shows the clonal diversity of Fanca -/- transplanted HSCs corrected with LV. Figure 4A shows the protocol and Figure 44B presents a graph of the data.

DETAILED DESCRIPTION OF THE INVENTION

[068]The present invention generally relates to the fields of molecular biology and virology and, in particular, to gene expression cassettes and vectors comprising the same useful for the delivery of nucleic acid segments encoding selected therapeutic constructs (including, for example, peptides, polypeptides, ribozymes and catalytic RNA molecules) to selected cells and tissues of vertebrate animals. In particular, these genetic constructs are useful in gene therapy for the treatment of mammals and, in particular, human diseases, disorders and dysfunctions related to the dysregulation of the FANCA gene product.

[069] In certain embodiments, the invention provides compositions and methods for gene therapy treatment of individuals with Fanconi Anemia (FA). In particular, compositions and methods for restoring FANCA gene expression are provided. Specific methods disclosed in this report relate to the use of lentiviral vectors to deliver human FANCA to hematopoietic progenitor cells of an individual with FA, particularly FA-A.

DEFINITIONS

[070] Unless otherwise defined, all technical and scientific terms used in this report have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described in this report may be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned in this report are expressly and fully incorporated by reference in this report. In case of conflict, this descriptive report, including definitions, shall prevail. Furthermore, the materials, methods and examples described in this report are for illustrative purposes only and are not limiting.

[071]A “vector”, as used in this report, refers to a macromolecule or association of macromolecules comprising or associating with a polynucleotide and which can be used to mediate the release of the polynucleotide to a cell. Illustrative vectors include, for example, plasmids, viral vectors (e.g., retroviral vectors such as lentiviral vectors), liposomes, and other gene delivery vehicles.

[072]The term “LV” is an abbreviation for lentivirus and may be used to refer to the virus itself or derivatives thereof. The term covers all naturally occurring and recombinant subtypes and forms, except where otherwise required.

[073]As used in this report, the term “gene” or “coding sequence” refers to a nucleotide sequence in vitro or in vivo that codes for a gene product. In some instances, the gene consists of, or essentially consists of, the coding sequence, that is, the sequence that codes for the gene product. In other instances, the gene comprises an additional, non-coding sequence. For example, the gene may or may not include regions that precede and follow the coding region, for example, 5’ untranslated (5’ UTR) or “leader” sequences and 3’ UTR or “tracker” sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[074]As used in this report, 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 a mammal in which the gene is expressed. Examples of beneficial effects include improving a sign or symptom of a condition or disease, preventing or inhibiting a condition or disease, or conferring a desired characteristic. Therapeutic genes include genes that correct a genetic deficiency in a cell or mammal.

[075]As used in this report, a transgene is a gene that is delivered to a cell by a vector.

[076]As used in this report, the term “gene product” refers to the desired expression product of a polynucleotide sequence, such as a polypeptide, peptide, protein, or interfering RNA, including short interfering RNA (siRNA), miRNA, or small loop RNA (shRNA).

[077]As used in this report, the terms “polypeptide”, “peptide” and “protein” refer to amino acid polymers of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation or conjugation with a labeling component.

[078]“Comprising” means that the cited elements are required, for example, in the composition, method, kit, etc., but other elements may be included to form, for example, the composition, method, kit, etc. within the scope of the claim. 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 besides the gene and promoter, for example, polyadenylation sequence, enhancer elements, other genes, linker domains, etc.

[079]“Essentially consisting of” means a limitation of the scope, for example, of the composition, method, kit, etc., described for the specified materials or steps that do not materially affect the basic and novel characteristics, for example, of the composition, method, kit, etc. For example, an expression cassette “essentially consisting of” a gene encoding a therapeutic polypeptide operably linked to a promoter and a polyadenylation sequence may include additional sequences, for example, linker sequences, provided that it does not materially affect the transcription or translation of the gene. As another example, a variant, or mutant, polypeptide fragment “consisting essentially of” a quoted sequence exhibits the amino acid sequence of the quoted sequence plus or minus about 10 amino acid residues at the limits of the sequence based on the full-length naive polypeptide from which it was derived, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue shorter than the quoted limit amino acid residue, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues longer than the quoted limit amino acid residue.

[080]“Consisting of” means the exclusion from the composition, method, or kit of any element, step, or ingredient not specified in the claim. For example, an expression cassette “consisting of” a gene encoding a therapeutic polypeptide operably linked to a promoter, and a post-transcriptional regulatory element consists only of the promoter, polynucleotide sequence encoding the therapeutic polypeptide, and post-transcriptional regulatory element. As another example, a polypeptide “consisting of” a quoted sequence contains only the quoted sequence.

[081]An “expression vector,” as used in this report, comprises a vector, for example, a plasmid, minicircle, viral vector, liposome, and the like, as discussed above or as known in the art, comprising a polynucleotide encoding a gene product of interest, and is used to effect the expression of a gene product in an intended target cell. An expression vector also comprises control elements operably linked to the coding region to facilitate the expression of the gene product in the target. The combination of control elements, for example, promoters, enhancers, UTRs, miRNA bleaching sequences, etc., and a gene or genes to which they are operably linked for expression is sometimes referred to as an “expression cassette.” Many such control elements are known and available in the art or can be readily constructed from components that are available in the art.

[082]A “promoter,” as used in this report, comprises a DNA sequence that directs RNA polymerase binding and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning they are strongly active in a wide range of cells, tissues, and species, or cell-type specific, tissue-specific, or species-specific. Promoters may be “constitutive,” meaning they are continuously active, or “inducible,” meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors. Also included in the nucleic acid constructs or vectors of the invention are enhancer sequences that may or may not be continuous with the promoter sequence. Enhancer sequences influence promoter-dependent gene expression and may be located in the 5’ or 3’ regions of the native gene.

[083]An “enhancer,” as used in this report, encompasses a cis-acting element that stimulates or inhibits the transcription of adjacent genes. An enhancer that inhibits transcription is also termed a “silencer.” Enhancers can function (i.e., can be associated with a coding sequence) in any orientation, at distances of up to several kilobase pairs (kb) from the coding sequence and from a downstream position of a transcribed region.

[084]A “termination signal sequence” used in this report covers any genetic element that causes RNA polymerase to terminate transcription, such as, for example, a polyadenylation signal sequence.

[085]As used in this report, the term “operably linked” refers to a juxtaposition of genetic elements, for example, promoter, enhancer, termination signal sequence, polyadenylation sequence, etc., in which the elements are in a relationship that allows them to operate in the expected manner. For example, a promoter is operably linked to a coding region if the promoter helps to initiate transcription of the coding sequence. There may be intervening residues between the promoter and the coding region, as long as their functional relationship is maintained.

[086]As used in this report, the term “heterologous” means derived from an entity genotypically distinct from that of the remainder of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques 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 operably linked to a coding sequence to which it is not naturally linked is a heterologous promoter. Thus, for example, a LV vector that includes a heterologous nucleic acid encoding a heterologous gene product is a LV vector that includes a nucleic acid not normally included in a naturally occurring wild-type LV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally occurring wild-type LV.

[087]The term “endogenous”, as used in this report with reference to a nucleotide molecule or gene product, refers to a nucleic acid sequence, for example, a gene or genetic element, or gene product, for example, RNA, protein, that is naturally occurring in, or associated with, a virus or host cell.

[088]The term “native”, as used in this report, refers to a nucleotide sequence, for example, a gene, or gene product, for example, RNA, protein, that is present in a wild-type virus or cell.

[089]The term “variant”, as used in this report, refers to a mutant of a reference polynucleotide or polypeptide sequence, for example, a native polynucleotide or polypeptide sequence, that is, exhibiting less than 100% sequence identity with the reference polynucleotide or polypeptide sequence. In other words, a variant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) from a reference polynucleotide sequence, for example, a native polynucleotide or polypeptide sequence. For example, a variant can be a polynucleotide that exhibits a sequence identity of 70% or more with a full-length native polynucleotide sequence, for example, an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full-length native polynucleotide sequence. As another example, a variant can be a polypeptide that exhibits a sequence identity of 70% or more with a full-length native polypeptide sequence, for example, an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the full-length native polypeptide sequence. Variants may also include variant fragments of a reference, for example, a native sequence that shares a sequence identity of 70% or more with a fragment of the reference, for example, a native sequence, for example, an identity of 75% or 80% or more, such as 85%, 90%, or 95% or more, for example, 98% or 99% identity with the native sequence.

[090]As used in this report, the terms “biological activity” and “biologically active” refer to the activity attributed to a particular biological element in a cell. For example, the “biological activity” of an “immunoglobulin,” “antibody,” or fragment or variant thereof refers to the ability to bind to an antigenic determinant and thereby facilitate immune 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 functions, e.g., binding, enzymatic activity, etc. As a third example, the biological activity of a gene regulatory element, e.g., promoter, enhancer, Kozak sequence, and the like, refers to the ability of the regulatory element or functional fragment or variant thereof to regulate, i.e., promote, enhance, or activate the translation, respectively, of the expression of the gene to which it is operably bound.

[091]The terms “administer” or “introduce” used in this report refer to the release of a vector for the expression of recombinant protein into a cell, cells and/or organs of an individual or an individual. Such administration or introduction may occur in vivo, in vitro or ex vivo. A vector for the expression of a gene product may be introduced into a cell by transfection, which typically means insertion of heterologous DNA into a cell by physical means (e.g., transfection with calcium phosphate, electroporation, microinjection or lipofection); infection, which typically refers to introduction via an infectious agent, i.e., a virus; or transduction, which typically means stable infection of a cell with a virus or the transfer of genetic material from one microorganism to another via a viral agent (e.g., a bacteriophage).

[092]“Transformation” is typically used to refer to bacteria comprising heterologous DNA or cells expressing an oncogene and therefore converted into a continuous growth mode, such as tumor cells. A vector used to “transform” a cell can be a plasmid, virus, or other carrier.

[093]Typically, a cell is referred to as “transduced”, “infected”, or “transformed” depending on the means used for the administration, introduction, or insertion of heterologous DNA (i.e., the vector) into the cell. The terms “transduced”, “transfected”, and “transformed” may be used interchangeably in this report regardless of the method of heterologous DNA introduction.

[094]The term “host cell,” as used in this report, refers to a cell that has been transduced, infected, transfected, or transformed with a vector. The vector may be a plasmid, a viral particle, a phage, etc. Culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression and will be evident to those skilled in the art. It will be assessed that the term “host cell” refers to the original transduced, infected, transfected, or transformed cell and its progeny.

[095]The terms “treatment,” “treat,” and similar terms are used in this report to generally mean the attainment of a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, for example, reducing the likelihood that the disease or symptom thereof will occur in the individual, and/or it may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used in this report, covers any treatment of a disease in a mammal, and includes: (a) preventing the occurrence of the disease in an individual who may be predisposed to the disease but has not yet been diagnosed with it; (b) inhibiting the disease, that is, halting its development; or (c) alleviating the disease, that is, causing the disease to regress. The therapeutic agent may be administered before, during, or after the onset of the disease or injury. Treatment of the ongoing disease, where treatment stabilizes or reduces the patient's undesirable clinical symptoms, is of particular interest. Such treatment is ideally carried out before complete loss of function in the affected tissues. Therapy will ideally be administered to the individual during the symptomatic stage of the disease and, in some cases, after the symptomatic stage of the disease.

[096]The terms “individual”, “host”, “individual” and “patient” are used interchangeably in this report and refer to a mammal, including but not limited to human and non-human primates, including apes and humans; mammalian animals for sport (e.g., horses); farmed mammalian animals (e.g., sheep, goats, etc.); pet mammalian animals (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). The terminology used in this report is for the purpose of describing only particular embodiments and is not limiting of the invention. As used in this report, the singular forms “a”, “an” and “the” also include the plural forms, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “presenting”, “presents”, “with”, or variants thereof are used in the detailed description and/or claims, such terms are inclusive in a manner similar to the term “comprising”.

[097]The term “about” or “approximately” means within an acceptable range of error for the particular value, as determined by a person skilled in the art, which will depend, in part, on how the value is measured or determined, i.e., the limitations of the measuring system. For example, “about” may mean within 1 or more than 1 standard deviation, according to practice in the art. Alternatively, “about” may mean a range up to 20%, preferably up to 10%, more preferably up to 5%, and most preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times, and most preferably within 2 times, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term "approximately" means that within an acceptable range of error, the particular value should be considered.

[098] Unless otherwise indicated, the practice of the present invention utilizes conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the scope of the skilled person. Such techniques are fully explained in the literature, such as, “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., 1991), each of which is expressly incorporated by reference in this report.

[099]Several aspects of the invention are described below with reference to exemplary applications for illustration. It should be understood that various specific details, relationships, and methods are presented to provide a complete understanding of the invention. A person having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated order of actions or events, as some actions may occur in different orders and/or concurrently with other actions or events. Furthermore, not all illustrated actions or events are required to implement a methodology according to the present invention.

[0100]Unless otherwise indicated, all terms used in this report have the same meaning as a person skilled in the art and practicing the present invention would use conventional microbiology techniques and recombinant DNA technology that are within the knowledge of such a person skilled in the art.

[0101] In certain embodiments, the present disclosure provides polynucleotides, polynucleotide cassettes, and expression vectors for the expression of a gene in cells. Also provided are pharmaceutical compositions and methods for the use of any one of the compositions in promoting the expression of a gene in cells, for example, in an individual, for example, for the treatment or prophylaxis of a disorder. These and other objects, advantages, and features of the invention will become apparent to those skilled in the art after reading the details of the compositions and methods, as more fully described below.

[0102]In certain embodiments, methods and compositions are provided for the preparation of vector compositions for gene therapy, for example, viral vectors, these comprising gene expression cassettes for use in the preparation of drugs useful in central and targeted gene therapy of diseases, disorders and dysfunctions in an animal and, in particular, in humans.

[0103] In some embodiments, the present invention provides gene therapy for Fanconi Anemia based on an LV vector harboring the eukaryotic hPGK promoter that activates FANCA cDNA expression. This therapeutic vector can be used to transduce human hematopoietic stem cells (HSCs), which can subsequently be transplanted into humans with Fanconi Anemia.

[0104] In certain embodiments, the present invention provides a FANCA LV vector for the genetic correction of Fanconi Anemia. In general, the results demonstrate the feasibility of gene therapy for FA with a LV designed for clinical application.

[0105]The disclosed compositions can be used in a variety of investigative, diagnostic and therapeutic regimens, including the prevention and treatment of a variety of human diseases. The various compositions and methods of the invention are described below.

[0106]Although particular compositions and methods are exemplified in this report, it should be understood that any alternative compositions and methods are applicable and suitable for use in the practice of the invention. It should also be understood that an evaluation of the constructs and methods of expression of the invention can be performed using standard procedures in the art.

Fanconi anemia

[0107]Fanconi anemia (FA) is a rare inherited chromosomal instability syndrome primarily characterized by bone marrow failure (BMF) and a predisposition to cancer (Butturini A et al. Blood. 1994;84:1650 to 1655; Kutler DI et al. Blood. 2003; 101:1249 to 1256.). The prevalence of FA is 1 to 5 per million, and the frequency of heterozygous carriers is estimated at 1 in 300 (Tamary H et al. Eur J Haematol. 2004;72:330 to 335).

[0108]FA is both genetically and phenotypically heterogeneous. To date, thirteen (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 genes of the 13 corresponding Fanconia Anemia (FANC) complementation groups: FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG/XRCC9, FANCI, BRIP1/FANCJ, FANCL, FANCM/Hef and FANCN/PABLB2 (Wang W. Nat Rev Genet. 2007;8:735 to 748). Except in the case of FA-B patients (FANCB is located on the X chromosome), FA is autosomal recessive.

[0109] Proteins encoded by FA genes participate 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 participating in one of the three FA protein complexes characterized so far in this pathway. The upstream complex, the FA core complex, is comprised of eight FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, FANCM) and two FA-associated proteins (FAAP24 and FAAP100). A second complex is formed by FANCD2 and FANCI, which work together in the FA-ID complex. Due to the activity of E3 ligase (FANCL) in the FA core complex, FANCD2 and FANCI can be mono-ubiquitinated and then loaded into chromatin, forming large nuclear foci in response to DNA damage or replication arrest. Finally, mono-ubiquitinated FANCD2/FANCI interact with downstream FA proteins, such as FANCJ/BRIP1, FANCN/PALB2, and FANCD1/BRCA2, forming stable complexes with proteins involved in homology-directed repair (HDR), such as BRCA1 and RAD51.

[0110]FA-A is the most frequent FA complementation group, with approximately 50% to 80% of FA patients corresponding to this complementation group (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 consequence of deficient or absent FANCA expression, the FA core complex cannot be formed. This prevents the activation of the ID complex and, consequently, the migration of these proteins to the chromatin, thus resulting in the characteristic phenotype of FA cells.

[0111]As reviewed by D’Andrea et al. (Blood. 1997;90:1725 to 1736), FA cells are characterized by different cellular phenotypes, mainly related to defects in cell survival, DNA repair and genomic stability.

[0112] FA is primarily characterized by congenital abnormalities, development of bone marrow failure, and a high risk of developing acute myeloid leukemia and certain solid tumors. On average, 70% of FA patients have congenital defects. Skeletal abnormalities (radial radius, hip, vertebral scoliosis, rib) and generalized skin hyperpigmentation, café-au-lait spots, are present in 60 to 70% of FA patients. Most patients have short stature, and around one-third of them have microphthalmia and renal abnormalities. In about 30% of FA patients, no obvious congenital abnormalities are observed (Tischkowitz M, Dokal I. Br J Haematol. 2004;126:176-191).

[0113]The most important clinical features of AF patients are hematological. Bone marrow failure (BMF) is the main feature of the disease. It usually appears between the ages of 5 and 10 years. Eight percent of 15-year-old patients develop BMF, with the actuarial risk of BMF exceeding 90% by age 40 (Butturini et al., 1994, Kutler et al., 2003). Thrombocytopenia or leukopenia typically precedes anemia. Pancytopenia usually worsens over time. Neutropenia is associated with an increased risk of infections.

[0114] FA patients are also prone to developing cancer, primarily acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Most tumors associated with FA develop after age 13, with a median age of 23 years. The relative risk for AML is increased 785-fold, with the median age of FA patients who developed AML being 14 years and the cumulative incidence of hematologic malignancy being 30 to 55% by age 40 (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 carcinomas (SCC). The average age at which these patients develop solid tumors is 26 years, with a cumulative incidence of solid tumors of 30% by age 40 (Kutler et al., 2003, Rosenberg et al., 2003).

[0115]Due to the complex clinical manifestations of FA, the 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 therapies for FA patients are reported in the documentation of orphan medicinal products for the lentiviral vector carrying the FANCA gene, sponsored by CIEMAT/CIBER in Rare Diseases (Bueren, J. (2010). Center for Biomedical Network Research on Rare Diseases Ref EU/3/10/822).

[0116]In certain embodiments, methods and compositions are provided for the preparation of vector compositions for gene therapy, for example, viral vectors, these comprising gene expression cassettes for use in the preparation of drugs useful in central and targeted gene therapy of diseases, disorders and dysfunctions in an animal and, in particular, in humans.

[0117] In some embodiments, the present invention provides gene therapy for Fanconi Anemia based on an LV vector harboring the eukaryotic hPGK promoter that activates FANCA cDNA expression. This therapeutic vector can be used to transduce human hematopoietic stem cells (HSCs), which can subsequently be transplanted into humans with Fanconi Anemia.

[0118] In certain embodiments, the present invention provides a FANCA LV vector for the genetic correction of Fanconi Anemia. In general, the results demonstrate the feasibility of gene therapy for FA with a LV designed for clinical application.

[0119] This disclosure includes gene expression cassettes (e.g., therapeutic cassettes), gene transfer cassettes comprising gene expression cassettes (e.g., integration cassettes), plasmids comprising gene transfer cassettes, and gene delivery vectors comprising gene transfer cassettes. The gene expression cassettes, gene transfer cassettes, plasmids, and gene delivery vectors comprise a polynucleotide sequence encoding a therapeutic gene product operably linked to a promoter sequence. In certain embodiments, the polynucleotide sequence is DNA or RNA. In certain embodiments, the gene expression cassette is a polynucleotide, the gene transfer cassette is a polynucleotide, and the vector is a virus, for example, a lentivirus.

[0120]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.

[0121]In particular embodiments, a gene expression cassette comprises a promoter region, a coding sequence, and a post-transcriptional regulatory element. 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 export signal. The RNA export signal may comprise a wPRE sequence. In some embodiments, a modified wPRE, devoid of any residual open reading frame (Schambach, Bohne et al. 2006), is included to enhance the expression level and stability of the therapeutic gene.

[0122] Some embodiments of the present invention comprise gene expression cassettes for enhanced expression of a FANCA gene product. In some embodiments, the polynucleotide cassette comprises a wild-type FANCA cDNA coding sequence or a codon-optimized version of human FANCA cDNA to increase mRNA stability after transcription. For optimization, GeneArt® software can be used, increasing the GC content and removing hidden binding sites so as to avoid transcriptional silencing and thus increase transgene expression. Alternatively, any optimization method known in the art can be used.

[0123] In some aspects of the invention, the pharmaceutical compositions are provided comprising a gene delivery vector of the invention and a pharmaceutical excipient. In some embodiments, the pharmaceutical composition comprises a gene delivery vector of the invention and a pharmaceutical excipient.

[0124] In some aspects of the invention, methods are provided for expressing a transgene in mammalian cells. In some embodiments, the method comprises contacting one or more mammalian cells with an effective amount of a polynucleotide cassette of the invention or a gene delivery vector of the invention, wherein the transgene is expressed at detectable levels 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 of the invention or a gene delivery vector of the invention, wherein the transgene is expressed at therapeutic levels in one or more mammalian cells. In some embodiments, the method occurs in vitro. In other embodiments, the method occurs in vivo.

[0125] In some aspects of the invention, methods are provided for the treatment or prophylaxis of a disease or disorder in a mammal in need of treatment or prophylaxis for a disease or disorder. In some embodiments, the method comprises administering to the mammal an effective amount of a pharmaceutical composition of the invention, wherein the coding sequence encodes a therapeutic gene product.

COMPOSITIONS

[0126]In some aspects of the disclosure, compositions are provided for the expression of a FANCA transgene in eukaryotic cells. In certain embodiments, the eukaryotic cell is a mammalian cell. In one embodiment, the mammalian cell is a hematopoietic stem cell (HSC). In one embodiment, the mammalian cell is a hematopoietic progenitor. In one embodiment, the mammalian cell is CD34+. In one embodiment, the mammalian cell is a human cell. In particular embodiments, the cell is a human CD34+ cell derived from an individual diagnosed with FA who is to be treated with a CD34+ cell after the same has been transduced with a gene release disclosed in this report and comprises a disclosed gene expression cassette.

[0127]In a specific embodiment, the present disclosure includes a lentiviral vector comprising a gene expression cassette comprising a polynucleotide sequence encoding a therapeutic FANCA protein or a functional fragment or variant thereof. As used in this report, a functional variant of a reference polynucleotide or polypeptide comprises one or more deletions, additions, or substitutions of amino acids or nucleic acids compared to the reference sequence and conserves 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 in this report, a functional fragment is a fragment of a reference polynucleotide or polypeptide and conserves at least 50%, at least 80%, at least 90%, or at least 99% of the functional activity of the reference polynucleotide or polypeptide.

[0128] In one embodiment, the lentiviral vector main chain is the same as that corresponding to the medicinal product “Wiscott-Aldrich Syndrome Protein-Carrying Lentiviral Vector (WASP-LV)” (Ref141/2000), although for FA treatment, the promoter is the human phosphoglycerate kinase (hPGK) promoter, characterized by its stable in vivo activity and improved safety properties 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 Lentiviral And Gammaretroviral Vectors. Mol Ther. 2009;17:1919 to 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 to 696.).

[0129] In some embodiments of disclosure, the composition comprises a polynucleotide cassette. A “polynucleotide cassette” means a polynucleotide sequence comprising two or more functional polynucleotide sequences, for example, regulatory elements, translation initiation sequences, coding sequences, termination sequences, etc., typically in operable linkage to each other. Similarly, a “polynucleotide cassette for the expression of a transgene in a mammalian cell” means a combination of two or more functional polynucleotide sequences, for example, promoter, enhancer, 5’UTR, translation initiation sequence, coding sequence, termination sequences, etc., that promotes the expression of the transgene in a cell. Gene expression cassettes and gene transfer cassettes are examples of polynucleotide cassettes.

[0130] In some embodiments, the polynucleotide cassettes of the present disclosure provide enhanced expression of a transgene in mammalian cells. As demonstrated by the working examples of the present disclosure, the present inventors have discovered several polynucleotide elements, that is, improved elements compared to those known in the art, which individually and synergistically provide enhanced expression of transgenes in mammalian cells. In certain embodiments, the arrangement of two or more functional polynucleotide sequences within the polynucleotide cassettes of the present disclosure provides enhanced expression of a transgene in mammalian cells. “Enhanced” means that the expression of the transgene is enhanced or increased or stronger in cells carrying the polynucleotide cassettes of the present disclosure compared to cells carrying the transgene operably linked to comparable regulatory elements, for example, as known in the art. In other words, transgene expression is enhanced or increased or stronger from the polynucleotide cassettes of the present disclosure compared to expression from a polynucleotide cassette not comprising 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.

[0131]For example, transgene expression may be accentuated or increased or stronger in cells comprising a polynucleotide cassette comprising a promoter disclosed in this report than in cells carrying the transgene operably linked to a different promoter, for example, as known in the art. As another example, transgene expression may be accentuated or increased or stronger in cells comprising a polynucleotide cassette comprising an enhancer sequence disclosed in this report than in cells carrying the transgene operably linked to a different enhancer sequence.

[0132]Without limiting the invention to a theory, it is believed that enhanced expression of a transgene in cells occurs due to a faster construction of the gene product in the cells or a more stable gene product in the cells. Thus, enhanced expression of a transgene by the disclosure polynucleotide cassettes can be observed in several ways. For example, enhanced expression can be observed by detecting transgene expression after contact of the polynucleotide cassette with cells more rapidly, for example, 2 days earlier, 7 days earlier, 2 weeks earlier, 3 weeks earlier, 4 weeks earlier, 8 weeks earlier, 12 weeks earlier or more, than expression would be detected if the transgene were operably linked to comparable regulatory elements, for example, as known in the art. Enhanced expression can also be observed as an increase in the amount of gene product per cell. For example, it could be a 2-fold or greater increase, for example, 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. Increased 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, it could be a 2-fold or greater increase, for example, 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 number of mammalian cells expressing detectable levels of the transgene.

[0133]As another example, the polynucleotide of the present invention can promote detectable levels of the transgene in a higher percentage of cells compared to a conventional polynucleotide cassette; for example, where a conventional cassette can promote detectable levels of transgene expression, for example, in less than 5% of cells in a given region, the polynucleotide of the present invention promotes detectable levels of expression in 5% or more of cells in such a 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, in some examples, 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 of the cells that are contacted will express detectable levels of the gene product. Increased expression may also be observed as an alteration in cell viability and/or function.

[0134]The polynucleotide cassettes of the present disclosure typically comprise a promoter region. Any suitable promoter region or promoter sequence therein may be used in the polynucleotide cassettes, provided that the promoter region promotes the expression of a coding sequence in eukaryotic cells. In certain embodiments, the promoter region promotes the expression of a coding sequence in mammalian cells. In some examples, the promoter is a ubiquitous promoter, that is, it is a promoter that is active in a wide range of cells, tissues, and species. In other examples, the promoter is a human PGK promoter.

[0135] Promoters and enhancers can be tissue-specific or stage-specific. For example, a tissue-specific promoter or enhancer preferentially activates expression (or a higher level of expression) in one or more particular 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 progenitors, CD34+ hematopoietic cells, and any cluster differentiation subpopulation within the CD34+ population. A stage-specific promoter or enhancer preferentially activates expression (or a higher level of expression) during one or more specific stages of the cell cycle or development. These include, but are not limited to, the beta-globin locus control region, spectrin promoter, and an erythroid-specific promoter.

[0136]In some embodiments, the polynucleotide comprises one or more enhancers. Enhancers are nucleic acid elements known in the art to enhance transcription and may be located anywhere in association with the gene they regulate, for example, upstream, downstream, within an intron, etc. Any enhancer element may be used in the polynucleotide cassettes and gene therapy vectors of the present disclosure, provided that it enhances gene expression when used in combination with the promoter.

[0137]The coding sequence that will be expressed in cells can be any polynucleotide sequence, for example, a gene or cDNA that encodes a gene product, for example, a polypeptide or RNA-based therapeutic product (siRNA, antisense, ribozyme, shRNA, etc.). The coding sequence can be heterologous to the promoter sequence to which it is operably linked, that is, not naturally operably associated with it. Alternatively, the coding sequence can be endogenous to the promoter sequence to which it is operably linked, that is, it is naturally associated with that promoter. The gene product can act intrinsically in the mammalian cell or it can act extrinsically, for example, it can be secreted. For example, when the transgene is a therapeutic gene, the coding sequence can be any gene that encodes a desired gene product or functional fragment or variant thereof that can be used as a therapeutic product to treat a disease or disorder. In several preferred embodiments, the transgene encodes human FANCA, that is, SEQ ID NO: 25.

[0138] In one embodiment of the invention, the coding sequence of the transgene is modified or “codon-optimized” to enhance expression by replacing infrequently represented codons with more frequently represented codons. The coding sequence is the portion of the mRNA sequence that codes for amino acids for translation. During translation, each of the 61 trinucleotide codons is translated into that of 20 amino acids, leading to degeneracy or redundancy in the genetic code. However, different cell types and different animal species utilize tRNAs (carriers of an anticodon) that code for the same amino acids at different frequencies. When a gene sequence contains codons that are infrequently represented by the corresponding tRNA, the ribosomal translation machinery may slow down, preventing effective translation. The expression can be improved through “codon optimization” for a particular species, where the coding sequence is altered to encode the same protein sequence, but using codons that are highly represented and/or used by highly expressed human proteins (Cid-Arregui et al., 2003; J. Virol. 77: 4928). In one aspect of the present invention, the transgene coding sequence is modified to replace codons infrequently expressed in mammals or primates with codons frequently expressed in primates. For example, in some embodiments, the coding sequence encoded by the transgene encodes a polypeptide that exhibits at least 85% sequence identity to a polypeptide encoded by a sequence disclosed above or in this report, for example, at least 90% sequence identity, for example, at least 95% sequence identity, at least 98% identity, at least 99% identity, wherein at least one codon of the coding sequence exhibits a higher tRNA frequency in humans than the corresponding codon in the sequence disclosed above or in this report.

[0139] In a further embodiment of the invention, the transgene coding sequence is 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 the nucleic acid sequence that follows a start codon and does not contain a stop codon. ORFs can be forward or reverse oriented and can be “in-frame” or “out-of-frame” relative to the gene of interest. Such open reading frames have the potential to be expressed in an expression cassette along the gene of interest and may lead to undesirable adverse effects. In one aspect of the present invention, the transgene coding sequence has been modified to remove open reading frames by further altering the preferential use of codons. This was done by eliminating start codons (ATG) and introducing stop codons (TAG, TAA, or TGA) into reverse-oriented or out-of-frame ORFs, preserving the amino acid sequence and maintaining highly used codons in the gene of interest (i.e., avoiding codons with a frequency < 20%). In the present invention, the coding sequence of the transgene can be optimized by codon optimization and removal of non-transgene ORFs or by using both techniques. As will be evident to one skilled in the art, it is preferable to remove or minimize non-transgene ORFs after codon optimization, so as to remove ORFs introduced during codon optimization.

[0140]In some embodiments, a 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) a post-transcriptional regulatory element of the marmot hepatitis virus (WPRE) sequence.

[0141]In some embodiments, a polynucleotide cassette comprises:(i) a human phosphoglycerate kinase (PGK) promoter sequence;(ii) a sequence encoding a human FANCA protein; and:(iii) a mutant WPRE sequence.

[0142]In some embodiments, a polynucleotide cassette comprises: a) a 5’ LTR, optionally a modified 5’ LTR; b) a cPPT sequence; c) a PGK promoter sequence, optionally a human PGK promoter sequence; d) a sequence encoding a human FANCA protein, optionally a cDNA sequence or an optimized sequence at the codon; e) a mutant wPRE sequence; and f) a 3’ LTR, optionally a modified 3’ LTR.

[0143]In one embodiment, the modified WPRE is referred to as WPRE*. WPRE* is a modified WPRE that is devoid of an open reading frame (see, for example, Schambach et al., 2006 Gene Ther. 13:641-645).

[0144]In certain embodiments, a gene transfer cassette comprises one or more additional elements, for example, one or more elements selected from the following: 5’ LTR, 3’ LTR, cPPT, CTS, RRE, enhancer sequences and packaging signals.

[0145]The RRE sequence improves gene transfer efficiency. In particular embodiments of any of the gene expression cassettes and gene delivery vectors described in this report, the RRE sequence comprises or consists of any of the following sequences, or sequences that exhibit at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequences: (AGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAG CGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGC AGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCA CAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATAC CTAAAGGATCAACAGCTCCT (SEQ ID NO: 1); or a sequence comprising or consisting of the nucleotides 2649 to 2882 or SEQ ID NO: 24.

[0146]The retroviral leader region contains the packaging signal (^), which is involved in packaging the retroviral genome into the viral capsid. It is thought that LV vectors 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 particular embodiments of any of the gene expression cassettes and delivery vectors described in this report, the Φ sequence is an HIV-1 Φ sequence or the Φ sequence comprises or consists of any of the following sequences, or sequences that exhibit at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the following sequences: CTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGG GGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAG AGAGATGGGTGCGAGAGCGTC (SEQ ID NO: 2); or a sequence comprising or consisting of polynucleotides 2031 to 2156 of SEQ ID NO: 24.

[0147]In particular embodiments of any of the gene expression cassettes and delivery vectors described in this report, the truncated HIV-1 5’ LTR comprises or consists of any of the following sequences, or sequences that exhibit 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:GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGG GAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTG TGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTC AGTGTGGAAAATCTCTAGCA (SEQ ID NO: 3); or a sequence comprising or consisting of polynucleotides 1586 to 9495 of SEQ ID NO:24.

[0148]In particular embodiments of any of the gene expression cassettes and delivery vectors described in this report, the HIV-1 autoinactivation 3’ LTR comprises or consists of any of the following sequences, or sequences that exhibit at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the following sequences:TGGAAGGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTG GGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGG AACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGT GCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCA GTGTGGAAAATCTCTAGCA (SEQ ID NO: 4); or a sequence comprising or consisting of polynucleotides 9262 to 9495 of SEQ ID NO: 24.

[0149]In particular embodiments of any of the gene expression cassettes and delivery vectors described in this report, the immediate early promoter of human cytomegalovirus (CMV) comprises or consists of any of the following sequences, a functional fragment thereof, or a sequence that exhibits 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: GTGATGCGGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACG GGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCA AAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAAT GGGCGGTAGGCGTTACGTGGGAGGTCTATATAAGCAGAGCT (SEQ ID NO: 5); or a sequence comprising or consisting of polynucleotides 1586 to 1789 of SEQ ID NO:24.

[0150]cPPT, which facilitates nuclear translocation of preintegration complexes, along with CTS involved in reverse transcriptase separation, has been observed to enhance viral titer (Zennou, et al. 2000; Follenzi et al. 2000). In particular embodiments of any of the gene expression cassettes and delivery vectors described in this report, the HIV-1 central polypurine tract and central termination sequence (cPPT/CTS) comprise or consist of any of the following sequences, a functional fragment thereof, or a sequence that exhibits 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: TTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAG ACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATT CAAAATTTT (SEQ ID NO: 6);TTTAAAAGAAAAGGGGGGATTGGGGGGT (SEQ ID NO: 12); or a sequence comprising or consisting of nucleotides 3378 to 3495 of SEQ ID NO:24.

[0151]In particular embodiments of any of the gene expression cassettes and gene delivery vectors described in this report, the human phosphoglycerate kinase 1 (hPGK) promoter comprises or consists of any of the following sequences, a functional fragment thereof, or a sequence that exhibits 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:GGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGC GGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTC GCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTG TGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTG CGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTAC CCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGG GCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGG GAAGGGGCGGTGCGGGAGGCGGGGTGGGGCGGTAGTGTGGGCCCTGTTC CTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAG (SEQ ID NO: 7); or a sequence comprising or consisting of nucleotides 3541 to 4051 of SEQ ID NO: 24.

[0152]Since most FA patients belong to the FA-A complementation group (Casado et al., 2007, Levitus et al., 2004, Taniguchi et al., 2006), in particular embodiments, the encoded therapeutic gene product is FANCA, although the disclosure considers that FA proteins from other complementation groups may also be released and thus encoded in the expression cassettes disclosed in this report, for example, instead of FANCA.

[0153]In particular embodiments of any of the gene expression cassettes and gene delivery vectors described in this report, 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 exhibiting at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the following sequence:ATGTCCGACTCGTGGGTCCCGAACTCCGCCTCGGGCCAGGACCCAGGGGGCC GCCGGAGGGCCTGGGCCGAGCTGCTGGCGGGAAGGGTCAAGAGGGGAAAATA TAATCCTGAAAGGGCACAGAAATTAAAGGAATCAGCTGTGCGCCTCCTGCGAAG CCATCAGGACCTGAATGCCCTTTTGCTTGAGGTAGAAGGTCCACTGTGTAAAAA ATTGTCTCTCAGCAAAGTGATTGACTGTGACAGTTCTGAGGCCTATGCTAATCAT TCTAGTTCATTTATAGGCTCTGCTTTGCAGGATCAAGCCTCAAGGCTGGGGTT CCCGTGGGTATTCTCTCAGCCGGGATGGTTGCCTCTAGCGTGGGACAGATCTG CACGGCTCCAGCGGAGACCAGTCACCCTGTGCTGCTGACTGTGGAGCAGAGAA AGAAGCTGTCTTCCCTGTTAGAGTTTGCTCAGTATTTATTGGCACACAGTATGTT CTCCCGTCTTTCCTTCTGTCAAGAATTATGGAAAATACAGAGTTCTTTGTTGCTT GAAGCGGTGTGGCATCTTCACGTACAAGGCATTGTGAGCCTGCAAGAGCTGCT GGAAAGCCATCCCGACATGCATGCTGTGGGATCGTGGCTCTTCAGGAATCTGT GCTGCCTTTGTGAACAGATGGAAGCATCCTGCCAGCATGCTGACGTCGCCAGG GCCATGCTTTCTGATTTTGTTCAAATGTTTGTTTTGAGGGGATTTCAGAAAAACT CAGATCTGAGAAGAACTGTGGAGCCTGAAAAAATGCCGCAGGTCACGGTTGAT GTACTGCAGAGAATGCTGATTTTTGCACTTGACGCTTTGGCTGCTGGAGTACAG GAGGAGTCCTCCACTCACAAGATCGTGAGGTGCTGGTTCGGAGTGTTCAGTGG ACACACGCTTGGCAGTGTAATTTCCACAGATCCTCTGAAGAGGTTCTTCAGTCAT ACCCTGACTCAGATACTCACTCACAGCCCTGTGCTGAAAGCATCTGATGCTGTT CAGATGCAGAGAGAGTGGAGCTTTGCGCGGACACACCCTCTGCTCACCTCACT GTACCGCAGGCTCTTTGTGATGCTGAGTGCAGAGGAGTTGGTTGGCCATTTGCA AGAAGTTCTGGAAACGCAGGAGGTTCACTGGCAGAGAGTGCTCTCCTTTTGTC TGCCCTGGTTGTCTGCTTTCCAGAAGCGCAGCAGCTGCTTGAAGACTGGGTGG CGCGTTTGATGGCCCAGGCATTCGAGAGCTGCCAGCTGGACAGCATGGTCACT GCGTTCCTGGTTGTGCGCCAGGCAGCACTGGAGGGCCCCTCTGCGTTCCTGTC ATATGCAGACTGGTTCAAGGCCTCCTTTGGGAGCACACGAGGCTACCATGGCT GCAGCAAGAAGGCCCTGGTCTTCCTGTTTACGTTCTTGTCAGAACTCGTGCCTT TTGAGTCTCCCCGGTACCTGCAGGTGCACATTCTCCACCCACCCCTGGTTCCCA GCAAGTACCGCTCCCTCCTCACAGACTACATCTCATTGGCCAAGACACGGCTGG CCGACCTCAAGGTTTCTAGAAAACATGGGACTCTACGAGGATTTGTCATCAG CTGGGGACATTACTGAGCCCCACAGCCAAGCTCTTCAGGATGTTGAAAAGGCC ATCATGGTGTTTGAGCATACGGGGAACATCCCAGTCACCGTCATGGAGGCCAG CATATTCAGGAGGCCTTACTACGTGTCCCACTTCCTCCCCGCCCTGCTCACACC TCGAGTGCTCCCCAAAGTCCCTGACTCCCGTGTGGCGTTTATAGAGTCTCTGAA GAGAGCAGATAAAATCCCCCCATCTCTGTACTCCACCTACTGCCAGGCCTGCTC TGCTGCTGAAGAGAAGCCAGAAGATGCAGCCCTGGGAGTGAGGGCAGAACCCA ACTCTGCTGAGGAGCCCCTGGGACAGCTCACAGCTGCACTGGGAGAGCTGAGA GCCTCCATGACAGACCCCAGCCAGCGTGATGTTATATCGGCACAGGTGGCAGT GATTTCTGAAAGACTGAGGGCTGTCCTGGGCCACAATGAGGATGACAGCAGCG TTGAGATATCAAAGATTCAGCTCAGCATCAACACGCCGAGACTGGAGCCACGG GAACACATTGCTGTGGACCTCCTGCTGACGTCTTTCTGTCAGAACCTGATGGCT GCCTCCAGTGTCGCTCCCCCGGAGAGGCAGGGTCCCTGGGCTGCCCTCTTCGT GAGGACCATGTGTGGACGTGTGCTCCCTGCAGTGCTCACCCGGCTCTGCCAGC TGCTCCGTCACCAGGGCCCGAGCCTGAGTGCCCCACATGTGCTGGGGTTGGCT GCCCTGGCCGTGCACCTGGGTGAGTCCAGGTCTGCGCTCCCAGAGGTGGATG TGGGTCCTCCTGCACCTGGTGCTGGCCTTCCTGTCCCTGCGCTCTTTGACAGC CTCCTGACCTGTAGGACGAGGGATTCCTTGTTCTTCTGCCTGAAATTTTGTACAG CAGCAATTTCTTACTCTCTCTGCAAGTTTTCTTCCCAGTCACGAGATACTTTGTG CAGCTGCTTATCTCCAGGCCTTATTAAAAGTTTCAGTTCCTCATGTTCAGATTG TTCTCAGAGGCCCGACAGCCTCTTTCTGAGGAGGACGTAGCCAGCCTTTCCTG GAGACCCTTGCACCTTCCTTCTGCAGACTGGCAGAGAGCTGCCCTCTCTCTCTG GACACAGAACCTTCCGAGAGGTGTTGAAAGAGGAAGATGTTCACTTAACTTA CCAAGACTGGTTACACCTGGAGCTGGAAATTCAACCTGAAGCTGATGCTCTTTC AGATACTGAACGGCAGGACTTCCACCAGTGGGCGATCCATGAGCACTTTCTCCC TGAGTCCTCGGCTTCAGGGGGCTGTGACGGAGACCTGCAGGCTGCGTGTACCA TTCTTGTCAACGCACTGATGGATTTCCACCAAAGCTCAAGGAGTTATGACCACTC AGAAAATTCTGATTTGGTCTTTGGTGGCCGCACAGGAAATGAGGATATTATTTCC AGATTGCAGGAGATGGTAGCTGACCTGGAGCTGCAGCAAGACCTCATAGTGCC TCTCGGCCACACCCCTTCCCAGGAGCACTTCCTCTTTGAGATTTTCCGCAGACG GCTCCAGGCTCTGACAAGCGGGTGGAGCGTGGCTGCCAGCCTTCAGAGACAG AGGGAGCTGCTAATGTACAAACGGATCCTCCTCCGCCTGCCTTCGTCTGTCCTC TGCGGCAGCAGCTTCCAGGCAGAACAGCCCATCACTGCCAGATGCGAGCAGTT CTTCCACTTGGTCAACTCTGAGATGAGAAACTTCTGCTCCCACGGAGGTGCCCT GACACAGGACATCACTGCCCACTTCTTCAGGGGCCTCCTGAACGCCTGTCTGC GGAGCAGAGACCCCTCCCTGATGGTCGACTTCATACTGGCCAAGTGCCAGACG AAATGCCCCTTAATTTTGACCTCTGCTCTGGTGTGGTGGCCGAGCCTGGAGCCT GTGCTGCTCTGCCGGTGGAGGAGACACTGCCAGAGCCCGCTGCCCCGGGAAC TGCAGAAGCTACAAGAAGGCCGGCAGTTTGCCAGCGATTTCCTCTCCCCTGAG GCTGCCTCCCCAGCACCCAACCGGACTGGCTCTCAGCTGCTGCACTGCACTT TGCGATTCAACAAGTCAGGGAAGAAAACATCAGGAAGCAGCTAAAGAAGCTGGA CTGCGAGAGAGAGGAGCTATTGGTTTTCCTTTTCTTCTTCTCCTTGATGGGCCT GCTGTCGTCACATCTGACCTCAAATAGCACCACAGACCTGCCAAAGGCTTTCCA CGTTTGTGCAGCAATCCTCGAGTGTTTAGAGAAGAGGAAGATATCCTGGCTGGC ACTCTTTCAGTTGACAGAGAGTGACCTCAGGCTGGGGCGGCTCCTCCTCCGTG TGGCCCCGGATCAGCACACCAGGCTGCTGCCTTTCGCTTTTTACAGTCTTCTCT CCTACTTCCATGAAGACGCGGCCATCAGGGAAGAGGCCTTCCTGCATGTTGCT GTGGACATGTACTTGAAGCTGGTCCAGCTCTTCGTGGCTGGGGATACAAGCAC AGTTTCACCTCCAGCTGGCAGGAGCCTGGAGCTCAAGGGTCAGGGCAACCCCG TGGAACTGATAACAAAAGCTCGTCTTTTTCTGCTGCAGTTAATACCTCGGTGCCC GAAAAAGAGCTTCTCACACGTGGCAGAGCTGCTGGCTGATCGTGGGGACTGCG ACCCAGAGGTGAGCGCCGCCCTCCAGAGCAGACAGCAGGCTGCCCCTGACGC TGACCTGTCCCAGGAGCCTCATCTCTTCTGA(SEQ ID NO: 8).

[0154]This disclosure includes plasmids comprising an expression cassette or transfer cassette described in this report. In particular embodiments, the plasmid is pCCL-PGK-FANCA-WPRE* (Figure 41; SEQ ID NO: 24).

[0155]In certain embodiments, the disclosure includes a cell, for example, a packaging cell or packaging cell lines, for example, cells 293, comprising a plasmid disclosed in this report. In particular embodiments, the cell comprises the plasmids represented in Figures 38 to 41.

[0156]In certain embodiments, a transfer cassette or plasmid disclosed in this report further comprises one or more additional elements, for example, a CMV promoter and/or enhancer, an SV40 polyA sequence, an origin of replication, for example, an SV40 ori sequence, or any of the elements disclosed in this report.

[0157]In particular embodiments of any of the transfer cassettes, plasmids or vectors described in this report, the human CMV enhancer comprises or consists of the following sequence, a functional fragment thereof, or a sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the following sequence: GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCAT AGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGC TGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATA GTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAA ACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTA TGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG (SEQ ID NO: 9).

[0158]In particular embodiments of any of the transfer cassettes, plasmids or vectors described in this report, the simian virus 40 (SV40) poly(a) signal comprises or consists of the following sequence, a functional fragment thereof, or a sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the following sequence: AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTT CACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCA ATGTATCTTA (SEQ ID NO: 10).

[0159]In particular embodiments of any of the transfer cassettes, plasmids or vectors described in this report, the SV40 origin of replication comprises or consists of the following sequence, a functional fragment thereof, or a sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the following sequence:ATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTA ATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAG AAGTAGTGAGGAGGCTTTTTTGGAGGCC (SEQ ID NO: 11).

[0160]In some embodiments of any of the transfer cassettes, plasmids, or vectors described in this report, the dNEF signal present in any of the gene expression cassettes or delivery vectors described in this report comprises or consists of the following sequence, a functional fragment thereof, or a sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: GAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCT TAGCCACTTTTTAAAAGAAAAGGGGGGAC (SEQ ID NO: 13).

[0161] In particular embodiments of any of the transfer cassettes, plasmids, or vectors described in this report, the KanR sequence present in any of the gene expression cassettes or delivery vectors described in this report comprises or consists of the following sequence: ATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCGGCTTGGGTGGAGAG GCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCG TGTTCCGGCTGTCAGCGCAGGGGCGTCCGGTTCTTTTTGTCAAGACCGACCTG TCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGC GACGACGGGCGTTCCTTGCGCGGCTGTGCTCGACGTTGTCACTGAAGCGGGAA GGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCAC CTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCAT ACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGA GCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACG AAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGTCT ATGCCCGACGGCGAGGATCTCGTCGTGACCCACGGCGATGCCTGCTTGCCGAA TATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGTCTGGG TGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAG AGCTTGGCGGCGAATGGGCTGACCGCTTCCTTGTGCTTTACGGTATCGCCGCG CCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGA (SEQ ID NO: 14)

[0162]In some embodiments of any of the transfer cassettes, plasmids, or vectors described in this report, the rrnG terminator (transcription terminator from the rrnG operon of E. coli ribosomal RNA (Albrechtsen et al., 1991) present in any of the gene expression cassettes or delivery vectors described in this report comprises or consists of the following sequence: GCATTGGCGCAGAAAAAAATGCCTGATGCGACGCTGCGCGTCTTATACTCCCAC ATATGCCAGATTCAGCAACGGATACGGCTTCCCCAACTTGCCCACTTCCATACG TGTCCTCCTTACCAGAAATTTATCCTTAA (SEQ ID NO: 15)

[0163] In some embodiments of any of the transfer cassettes, plasmids, or vectors described in this report, the ori (high copy number of ColE1/pMB1/pBR322/pUC origin replicators) present in any of the gene expression cassettes or delivery vectors described in this report comprises or consists of the following sequence, a functional fragment thereof, or a sequence that exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCG CTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAG GTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCG TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTG CTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGG TTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGG GGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGAT ACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCG GACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGC TTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCT GACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA (SEQ ID NO: 16).

[0164]In some embodiments of any of the transfer cassettes, plasmids or vectors described in this report, the CAP binding site present in any of the gene expression cassettes or gene delivery vectors described in this report comprises or consists of the following sequence, a functional fragment thereof, or a sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to the following sequence:TAATGTGAGTTAGCTCACTCAT (SEQ ID NO: 17).

[0165]In some embodiments of any of the transfer cassettes, plasmids or vectors described in this report, the E.coli lac promoter present in any of the gene expression cassettes or delivery vectors described in this report comprises or consists of the following sequence, a functional fragment thereof, or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the following sequence: TTTACACTTTATGCTTCCGGCTCGTATGTTG (SEQ ID NO: 18).

[0166]In some embodiments of any of the transfer cassettes, plasmids, or vectors described in this report, the lac operator present in any of the gene expression cassettes or gene delivery vectors described in this report comprises or consists of the following sequence, a functional fragment thereof, or a sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TTGTGAGCGGATAACAA (SEQ ID NO: 19).

[0167]In some embodiments of any of the transfer cassettes, plasmids, or vectors described in this report, the T3 promoter (promoter for bacteriophage T3 RNA polymerase) present in any of the gene expression cassettes or delivery vectors described in this report comprises or consists of the following sequence, a functional fragment thereof, or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the following sequence: AATTAACCCTCACTAAAGG (SEQ ID NO: 20).

[0168]In some embodiments of any of the transfer cassettes, plasmids, or vectors described in this report, the T7 promoter (promoter for bacteriophage T7 RNA polymerase) present in any of the gene expression cassettes or delivery vectors described in this report comprises or consists of the following sequence, a functional fragment thereof, or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the following sequence: CCTATAGTGAGTCGTATTA (SEQ ID NO: 21).

[0169] In some embodiments of any of the transfer cassettes, plasmids, or vectors described in this report, the f1 ori (replication bacteriophage f1 origin) present in any of the gene expression cassettes or delivery vectors described in this report comprises or consists of the following sequence, a functional fragment thereof, or a sequence that exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAG CGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCTTTCGCTTTCTTCCC TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCT CCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGAT TAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCT TTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAA CACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCG GCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATT (SEQ ID NO: 22).

[0170] As discussed in this report, the polynucleotide cassettes of the present invention may comprise an RNA export signal. Exemplary RNA export sequences include, but are not limited to, wPRE. wPRE significantly increases transgene expression in target cells, increasing RNA stability in a transgene-, promoter-, and vector-independent manner (Zuffrey et al., 1999). However, it can also express a truncated 60-amino acid protein derived from the WHV X gene involved in liver cancer (Kingsman et al., 2005). Therefore, most preclinical protocols and clinical trials include a modified version of the wPRE element (Zanta-Boussif et al., 2009). On the other hand, it has been observed that the use of two SV40-USE elements in SIN-LV vectors is more effective than the wPRE sequence in suppressing transcriptional reading (Schambach et al., 2007). More precisely, the wPRE disclosed in this report is a chimeric wPRE that carries 589 nucleotides from the modified wPRE presented by Axel Schambach (nucleotides 1 to 589) (WO 2008136670 A2; [5]) and 88 from a previous wPRE (nucleotides 590 to 677) (Zuffrey et al., 1999). The data disclosed in this report show that this chimeric wPRE operates better than the previous wPRE. The chimeric wPRE sequence comprises the following sequence, a functional fragment thereof, or a sequence exhibiting at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: CGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTA TACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGA TATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTT CCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAA AGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTG CTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTC CTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGT CAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCACTGGCTGGGG CATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGAT CGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCT AGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGGGGAAGGGCCTGCTGCC GGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCT CCCTTTGGGCCGCCTCCCCGCCTG (SEQ ID NO:23).

[0171]In particular embodiments, the modified WPRE sequence comprises or consists of WPRE*, which corresponds to nucleotides 8502 to 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.

[0172]Other combinations of the elements disclosed in this report or known in the art will be easily evaluated by a person skilled in the art.

[0173]Additionally, as will be recognized by a person skilled in the art, polynucleotide cassettes may optionally contain other elements, including, but not limited to, restriction sites to facilitate cloning and regulatory elements for a particular gene expression vector.

[0174] In some aspects of the present invention, polynucleotide cassettes are used to deliver a gene to cells, for example, to determine the effect that the gene has on cell viability and/or function, to treat a cellular disorder, etc. In various embodiments, the delivery of a viral vector to cells by transduction can occur in vitro, ex vivo, or in vitro. Consequently, in some aspects of the invention, the composition that provides the expression of a transgene in mammalian cells is a gene delivery vector, wherein the gene delivery vector comprises a polynucleotide cassette, for example, a gene transfer cassette, of the present disclosure.

[0175]Any convenient gene delivery vector that finds use in delivering polynucleotide sequences to mammalian cells is covered by the gene delivery vectors of this disclosure. For example, the vector may comprise single-stranded or double-stranded nucleic acid, for example, single-stranded or double-stranded DNA. For example, the gene delivery vector may be DNA, for example, a discovered DNA, for example, a plasmid, a minicircle, etc. The vector may comprise single-stranded or double-stranded RNA, including modified forms of RNA. In another example, the gene delivery vector may be RNA, for example, mRNA or modified mRNA.

[0176]As another example, the gene delivery vector can be a viral vector derived from a virus, for example, an adenovirus, an adeno-associated virus, a lentivirus (LV), a herpesvirus, an alphavirus or a retrovirus, for example, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon monkey leukemia virus (GaLV), feline leukemia virus (FLV), foam virus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) or Rous Sarcoma Virus (RSV). Although embodiments involving the use of LV are described in more detail below, it is expected that the person skilled in the art will be able to assess that similar knowledge and skill in the technique can also be used in gene therapy vectors without LV.

[0177]In some embodiments, the gene delivery vector is a self-limiting LV. In one specific embodiment of any of the expression cassettes and gene delivery vectors described in this report, the release cassette is a pCCL-SIN-cPPT/CTS-hPGK-hFANCA-WPRE (Figure 41) comprising or consisting of the following sequence or a sequence that exhibits 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 corresponds to the pCCL-PGK-FANCA-WPRE* plasmid of Figure 41.

[0178]In one embodiment, a FANCA gene is delivered via a lentiviral vector (LV). The FANCA LVs described in this report utilize a self-inactivating lentiviral vector (LV). In one embodiment, the FANCA LV comprises a human phosphoglycerate (PGK) gene promoter. The safety properties of this vector have been markedly improved compared to gamma-retroviral vectors already used in the clinic, which harbored strong viral promoters.

[0179] In certain embodiments, the lentiviral vector is PGK-FANCA.WPRE*LV, which comprises the gene transfer cassette represented in Figure 1, comprising the sequences disclosed in SEQ ID NO: 24. The gene expression cassette portion of PGK-FANCA-WPRE*LV comprises the human PGK promoter, the coding sequence for FANCA cDNA, and WPRE*; and corresponds to nucleotides 3541 to 9178 of SEQ ID NO: 24. The transfer cassette portion of PGK-FANCA-WPRE*LV comprises approximately LTR 5’ (U5) to approximately LTR 3’ (U5) of the sequence shown in Figure 41. With respect to SEQ ID NO: 24, nucleotides 1586 to 1789 of SEQ ID NO: 24 comprise the immediate early promoter of human CMV. Nucleotides 2031 to 2156 of SEQ ID NO: 24 comprise the HIV1 psi packaging signal. Nucleotides 2649 to 2882 of SEQ ID NO: 24 comprise the HIV1 RRE element. Nucleotides 3378 to 3495 of SEQ ID NO: 24 comprise the HIV cPPT/CTS element. Nucleotides 3541 to 4051 of SEQ ID NO: 24 comprise the hPGK promoter. Nucleotides 4078 to 8445 of SEQ ID NO: 24 comprise human FANCA-A cDNA. Nucleotides 8502 to 9178 of SEQ ID NO: 24 comprise the modified WPRE element. Nucleotides 9262 to 9495 of SEQ ID NO: 24 comprise the HIV 3’ delta U LTR.

[0180]In yet another embodiment, the lentiviral vector contains the following elements: (i) the lentiviral vector backbone derived from the initial pCCLsin-cppt-hPGK-eGFP-WPRE (Dull et al., 1998; J.Virol 72 (11), 9873 to 9880). The pCCL backbone utilizes a 5’ LTR from heterologous CMV-HIV to achieve high levels of viral RNA transcription in the producing cells. This heterologous LTR makes the construct independent of 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 LTR 3’ contains a 400 bp deletion, as described in (Zufferey et al. J Virol, 1998), which confers self-inactivation properties to the vector; (ii) the cDNA of the human FANCA gene (GenBank accession number 4368 bp: X_99226 or as disclosed in this report) encoding the FANCA protein (1455 AA) under the control of the human PGK promoter. The promoter has already been characterized for its stable in vivo activity and improved safety properties compared to other promoters already used in gene therapy; and (iii) a modified version of the post-transcriptional regulatory element of the marmot hepatitis virus (WPRE) that is deleted in the 3’ region of a sequence encoding protein X and any residual ORF described by Schambach et al. (Gene Therapy, 2006; 13, 641-645) or WPRE*.

[0181]Gene therapy vectors encapsulating the polynucleotide cassettes of the present disclosure can be produced using standard methodology. For example, in the case of LV virions, an LV expression vector according to the invention can be introduced into a producer cell, followed by the introduction of an LV helper construct, wherein the helper construct includes LV coding regions capable of being expressed in the producer cell and which complement the LV helper functions absent in the LV vector. This is followed by the introduction of additional helper viruses and/or vectors into the producer cell, wherein the additional helper viruses and/or vectors provide additional functions capable of supporting effective LV virus production. The producer cells are then cultured to produce LV. These steps are performed using standard methodology. In particular embodiments, the plasmids represented in Figures 38 to 41 are used to produce the gene delivery vectors.

[0182]Any suitable method for producing viral particles for the release of polynucleotide cassettes may be used, including, but not limited to, those described in the following examples. Any concentration of viral particles suitable for effectively transducing mammalian cells may be prepared for contacting mammalian cells in vitro or in vivo. For example, viral particles may be formulated at a concentration of 10⁸ vector genomes per mL or more, for example, 5 x 10⁸ vector genomes per mL; 10⁹ vector genomes per mL; 5 x 10⁹ vector genomes per mL; 10¹⁰ vector genomes per mL; 5 x 10¹⁰ vector genomes per mL; 10¹¹ vector genomes per mL; 5 x 10¹¹ vector genomes per mL; 10¹² vector genomes per mL; 5 x 10¹² vector genomes per mL; 10¹³ vector genomes per mL; 1.5 x 10¹³ vector genomes per mL; 3 x 10¹³ vector genomes per mL; 5 x 10¹³ vector genomes per mL; 7.5 x 10¹³ vector genomes per mL; 9 x 10¹³ vector genomes per mL; 1 x 10¹⁴ vector genomes per mL, 5 x 10¹⁴ vector genomes per mL or more, but typically not more than 1 x 10¹⁵ vector genomes per mL.

[0183] In the preparation of LV compositions, any host cells to produce LV virions can be used, including, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. Host cells can also be packaging cells in which the rep and cap genes of LV are stably maintained in the host cell or producer cells in which the LV vector genome is stably maintained and packaged. Exemplary packaging and producer cells are derived from SF-9, 293, A549, or HeLa cells. LV vectors are purified and formulated using standard techniques known in the field.

[0184] In certain embodiments, the present invention includes a cell comprising a gene expression cassette, gene transfer cassette, or gene delivery vector disclosed in this report. In related embodiments, the cell is transduced with a gene delivery vector comprising an expression cassette disclosed in this report or features an expression cassette disclosed in this report integrated into the cell's genome.

[0185]In certain embodiments, the cell is a cell used to produce a viral gene release vector, for example, a packaging cell.

[0186]In other embodiments, the cell is a cell that will be released to an individual in order to provide the individual with the gene product encoded by the expression cassette. Thus, in certain embodiments, the cell is autologous to the individual to be treated or was obtained from the individual to be treated. In other embodiments, the cell is allogeneic to the individual to be treated or was obtained from a donor other than the individual to be treated. In particular embodiments, the cell is a mammalian cell, for example, a human cell. In certain embodiments, the cell is a blood cell, an erythrocyte, a hematopoietic progenitor cell, a bone marrow cell, for example, a lineage-depleted bone marrow cell, a hematopoietic stem cell (e.g., CD34+), or a compromised hematopoietic erythroid progenitor cell. In particular embodiments, the cell is a CD34+ cell obtained from an individual who will be treated with the cell after it is transduced by a gene delivery vector disclosed in this report. In one particular embodiment, the cell is a CD34+ FA cell obtained from an individual diagnosed with FA.

[0187]The present invention includes pharmaceutical compositions comprising a polynucleotide cassette, gene or cell delivery vector described in this report and a pharmaceutically acceptable carrier, diluent or excipient. The polynucleotide cassette, gene or cell delivery vector may be combined with pharmaceutically acceptable carriers, diluents and reagents useful in preparing a formulation that is generally safe, non-toxic and desirable, and includes excipients that are acceptable for use in primates. Such excipients may be solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous. Examples of such excipients, carriers or diluents include, but are not limited to, water, saline solution, Ringer's solutions, dextrose solution and 5% human serum albumin. Supplemental active compounds may also be incorporated into the formulations. Solutions or suspensions used for the formulations may include a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating compounds, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates; detergents, such as Tween 20 to prevent clumping; and compounds for adjusting tonicity, such as sodium chloride or dextrose. The pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In particular embodiments, the pharmaceutical compositions are sterile.

[0188]Pharmaceutical compositions suitable for use in the present invention also include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of injectable solutions or sterile dispersions.

[0189] Sterile solutions can be prepared by incorporating the active compound in the required quantity into an appropriate solvent with one or a combination of the ingredients listed above, as required, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and lyophilization, which produces a powder of the active ingredient plus any desired additional ingredient from a previously sterile filtered solution thereof.

[0190] In one embodiment, the compositions are prepared with carriers that will protect the gene expression cassette or vector against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be evident to those skilled in the art. The materials may also be commercially obtained.

[0191] It is especially advantageous to formulate oral, ocular, or parenteral compositions in unit dosage form because of the ease of administration and uniformity of dosage. The unit dosage form, as used in this report, refers to physically distinct units suitable as unit dosages for the individual to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification of the unit dosage forms of the invention is dictated by, and directly dependent on, the unique characteristics of the active compound and the particular therapeutic effect to be obtained, and the inherent limitations in the technique of composing such active compound for the treatment of individuals.

[0192]Pharmaceutical compositions may be included in a container, package or dispenser, for example, a syringe, for example, a pre-filled syringe, together with instructions for administration.

[0193]The pharmaceutical compositions of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, after administration to an animal, including a human being, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.

[0194]The term “pharmaceutically acceptable salt” refers to the physiologically and pharmaceutically acceptable salts of the compounds of the invention: that is, salts that preserve the desired biological activity of the parent compound and do not impart undesirable toxicological effects to it. A variety of pharmaceutically acceptable salts are known in the art and 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 thereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2 (1977). In addition, for a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0195] 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, for example, Berge et al., “Pharmaceutical Salts”, J. Pharma Sci., 1977, 66, 119). Base addition salts of said acid compounds are prepared by contacting the free acid form with a sufficient quantity of the desired base to produce the salt in the conventional manner. The free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. 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 their respective free acid for the purposes of the present invention.

[0196]The polynucleotide cassette, gene delivery vector, for example, recombinant virus (virions), or cell (for example, transduced with a gene delivery vector disclosed in this report) may be incorporated into pharmaceutical compositions for administration to mammalian patients, particularly primates, and more particularly humans. The polynucleotide cassette, gene delivery vector, for example, virions, or cell may be formulated in non-toxic, inert, and pharmaceutically acceptable aqueous carriers, preferably at a pH ranging from 3 to 8, more preferably ranging from 6 to 8. Such sterile compositions will comprise the vector or virion containing the nucleic acid encoding the therapeutic molecule dissolved in an aqueous buffer exhibiting an acceptable pH after reconstitution.

[0197]In some embodiments, the pharmaceutical composition provided in this report comprises a therapeutically effective amount of a cell, vector or virion disclosed in this report in a mixture with a pharmaceutically acceptable carrier and/or excipient, for example, saline solution, phosphate-buffered saline solution, phosphate and amino acids, polymers, polyols, sugar, buffers, preservatives and other proteins. Exemplary amino acids, polymers, and sugars are composed of octylphenoxy polyethoxy ethanol, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol. Preferably, this formulation is stable for at least six months at 4 °C.

[0198]In some embodiments, the pharmaceutical composition provided in this report comprises a buffer, such as phosphate-buffered saline (PBS) or sodium phosphate/sodium sulfate, tris buffer, glycine buffer, sterile water and other buffers known to those skilled in the art, such as those described by Good et al. (1966) Biochemistry 5:467. The pH of the buffer in which the pharmaceutical composition comprising the tumor suppressor gene contained in the adenoviral vector delivery system may be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0199]In certain embodiments, viral vectors may be formulated in any suitable unit dosage, including, without limitation, 1 x 108 vector genomes or more, for example, 1 x 109, 1 x 1010, 1 x 1011, 1 x 1012 or 1 x 1013 vector genomes or more, in certain examples, 1 x 1014 vector genomes, but usually not more than 4 x 1015 vector genomes. In some cases, the unit dosage is at most about 5 x 10¹⁵ vector genomes, for example, 1 x 10¹⁴ vector genomes or less, for example 1 x 10¹³, 1 x 10¹², 1 x 10¹¹, 1 x 10¹⁰ or 1 x 10⁹ vector genomes or less, in certain examples, 1 x 10⁸ vector genomes or less, and typically not less than 1 x 10⁸ vector genomes. In some cases, the unit dosage is 1 x 10¹⁰ to 1 x 10¹¹ vector genomes. In some cases, the unit dosage is 1 x 10¹⁰ to 3 x 10¹² vector genomes. In some cases, the unit dosage is 1 x 10⁹ to 3 x 10¹³ vector genomes. In some cases, the unit dosage is 1 x 10⁸ to 3 x 10¹⁴ vector genomes. In one embodiment, the range is about 5 x 10¹⁰ to about 1 x 10¹¹ vector genomes. In some embodiments, the range is about 1 x 10⁹ to about 1 x 10¹⁰ vector genomes.

[0200]In some cases, the unit dosage of a pharmaceutical composition can be measured using multiplicity of infection (MOI). MOI means the ratio, or multiple, of vector or viral genomes to the cells to which the nucleic acid can be released. In some cases, the MOI may be 1 x 10⁶. In some cases, the MOI may be 1 x 10⁵ to 1 x 10⁷. In some cases, the MOI may be 1 x 10⁴ to 1 x 10⁸. In some cases, the recombinant viruses from the disclosure exhibit at least approximately 1 x 10¹, 1 x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x 10¹⁰, 1 x 10¹¹, 1 x 10¹², 1 x 10¹³, 1 x 10¹⁴, 1 x 10¹⁵, 1 x 10¹⁶, 1 x 10¹⁷, and 1 x 10¹⁸ MOI. In some cases, the recombinant viruses in this disclosure have MOIs ranging from 1 x 10⁸ to 3 x 10¹⁴. In some cases, the recombinant viruses in the disclosure have maximum MOIs of approximately 1 x 10¹, 1 x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x 10¹⁰, 1 x 10¹¹, 1 x 10¹², 1 x 10¹³, 1 x 10¹⁴, 1 x 10¹⁵, 1 x 10¹⁶, 1 x 10¹⁷, and 1 x 10¹⁸. In some embodiments, the range is from approximately 20 to approximately 400 MOI.

[0201]In some aspects, the amount of pharmaceutical composition comprises about 1 x 108 to about 1 x 1015 recombinant viruses, about 1 x 109 to about 1 x 1014 recombinant viruses, about 1 x 1010 to about 1 x 1013 recombinant viruses or about 1 x 1011 to about 3 x 1012 recombinant viruses.

METHODS

[0202] As discussed in more detail below, the polynucleotide cassettes and gene delivery vectors collectively referred to in this report as the “compositions” find use in the expression of a transgene, for example, FANCA, in cells of an animal, for example, a mammal or human. For example, the compositions can be used in research, for example, to determine the effect that the gene has on cell viability and/or function. As another example, the compositions can be used in medicine, for example, to treat a disorder, such as FA. Thus, in some aspects of the invention, methods are provided for the expression of a gene in cells, the method comprising contacting the cells with a composition of the present disclosure. In some embodiments, the contact occurs in vitro. In some embodiments, the contact occurs in vivo, i.e., the composition is administered to an individual.

[0203]For examples where mammalian cells are to be contacted in vitro or in vivo with a polynucleotide cassette or gene delivery vector comprising a polynucleotide cassette, the cells may be presented from any mammalian species, for example, rodent (e.g., mice, rats, gerbils, squirrels), rabbit, feline, canine, goat, sheep, pig, equine, bovine, primate, human. The cells may be presented from established cell lines or may be primary cells, wherein “primary cells”, “primary cell lines” and “primary cultures” are used interchangeably in this report to refer to cells and cell cultures that have been derived from an individual and allowed to grow in vitro for a limited number of passages, i.e., divisions, of the culture. For example, primary cultures are cultures that have been passed 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough to pass the crisis stage. Typically, the primary cell lines of the present invention are maintained for fewer than 10 passages in vitro.

[0204] Embodiments of the present invention comprise mammalian cells (e.g., CD34+ cells) transduced with a viral delivery vector, for example, a LV vector containing the human FANCA gene. Furthermore, the present invention includes a method for transducing a mammalian cell, for example, a human hematopoietic stem cell or other cells described in this report, comprising contacting the cell with a gene delivery vector, for example, an LV vector, disclosed in this report or comprising an expression cassette described in this report. In certain embodiments, the cell has been previously obtained from an individual to be treated or from another donor. In particular embodiments, the individual has been diagnosed with Fanconi anemia and the cell is transduced with an LV comprising an expression cassette encoding a coding region or cDNA of FANCA. It should be understood that the disclosed methods, for example, those used to deliver a FANCA gene product, for instance, using a FANCA cDNA sequence, to an individual can also be used to treat Fanconi anemia. In particular embodiments, the transduced cells are a population of cells obtained from an individual with FA, who must be treated with the cells once they have been transduced. The cells can be obtained from bone marrow or blood. In certain embodiments, the individual with FA is treated with agents to mobilize stem cells, then blood is collected from the individual, red blood cells are removed, and CD34+ cells are selected. After selection, the cells are then transduced. In particular embodiments, the transduced cells are stored or frozen before use, whereas in certain embodiments, they are delivered to the individual immediately or immediately after they are transduced, for example, within one hour, two hours, or four hours.

[0205]In certain embodiments, during the transduction of a cell with a gene delivery vector disclosed in this report, the cells are contacted with the gene delivery vector for approximately 30 minutes, approximately 1 hour, approximately 1.5 hours, approximately 2 hours, approximately 2.5 hours, approximately 3 hours, approximately 3.5 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 12 hours, approximately 16 hours, approximately 18 hours, approximately 20 hours, approximately 24 hours, approximately 36 hours, approximately 48 hours, approximately 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.

[0206]The polynucleotide cassette or gene delivery vector comprising a polynucleotide cassette may be delivered to cells one or more times, for example, once, twice, three times or more than three times, and the cells incubated with the agents for some time after each contact event, for example, 16 to 24 hours, after which the medium is replaced with fresh medium and the cells are further cultured. Cell contact may occur in any culture medium and under any culture conditions that promote cell survival. The culture may contain growth factors to which the cells are responsive. Growth factors, as defined in this report, are molecules capable of promoting cell survival, growth and/or differentiation, in culture or intact tissue, through specific effects on a transmembrane receptor. Growth factors include polypeptide and non-polypeptide factors.

[0207]Typically, an effective amount of gene delivery vector or transduced cells comprising a polynucleotide cassette is provided to produce transgene expression in cells. As discussed elsewhere in this report, the effective amount can be easily determined empirically, for example, by detecting the presence or levels of the transgene gene product, by detecting an effect on cell viability or function, etc. Typically, an effective amount of the polynucleotide cassette or gene delivery vector comprising a polynucleotide cassette will promote greater transgene expression in cells than the same amount of a polynucleotide cassette, as known in the art. Typically, the expression will be accented 2 times or more relative to the expression from a reference, or control, polynucleotide cassette, for example, as known in the art, for example 3 times, 4 times or 5 times or more, in some examples 10 times, 20 times or 50 times or more, for example, 100 times.

[0208]For examples where cells are to be contacted in vivo with a polynucleotide cassette or gene delivery vector comprising a polynucleotide cassette, the individual may be any mammal, for example, rodent (e.g., mice, rats, gerbils), rabbit, feline, canine, goat, sheep, pig, equine, bovine, or primate. In a further preferred embodiment, the primate is a human being. In another embodiment, the cells are CD34+ cells.

[0209]The methods and compositions of the present disclosure are used, for example, in the treatment of Fanconi anemia.

[0210] In some embodiments, the method results in a therapeutic benefit, for example, preventing the development 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 comprises the step of detecting that a therapeutic benefit has been obtained. The person skilled in the art will assess that such measurements of therapeutic efficacy will be applicable to the particular disease to be modified and will recognize the appropriate detection methods for measuring therapeutic efficacy.

[0211] In another embodiment, the present invention includes a method for treating a disease in an individual in need thereof comprising providing the individual with an effective quantity of cells transduced with a gene delivery vector, for example, a viral vector, which expresses a therapeutic gene product in the cells. In particular embodiments, the cells are autologous to the individual. In certain embodiments, the cells are erythroid cells, for example, hematopoietic stem cells or committed hematopoietic erythroid progenitor cells. In some embodiments, the cell is a bone marrow cell, for example, a bone marrow cell depleted in the lineage. In particular embodiments, the method is used to treat FA and the viral vector is a LV comprising an expression construct disclosed in this report comprising a human PGK promoter operably linked to a cDNA or coding sequence of the FANCA gene and a modified wPRE disclosed in this report. In particular embodiments, the cells are delivered to the individual parenterally, for example, by intravenous injection.

[0212] In another embodiment, the present invention includes a method for treating FA in an individual in need thereof, comprising providing the individual with an effective quantity of autologous CD34+ stem cells transduced with a LV vector expressing a FANCA cDNA on the cells, wherein the LV vector comprises a human PGK promoter operably linked to the FANCA cDNA or coding sequence and a modified wPRE sequence disclosed in this report. In particular embodiments, the cells are hematopoietic stem cells or committed hematopoietic erythroid progenitor cells, for example, bone marrow cells. In particular embodiments, the cells are provided to the individual parenterally, for example, by intravenous injection.

[0213] Transgene expression using the target transgene is expected to be robust. Consequently, in some examples, transgene expression, for example, as detected by measuring gene product levels, by measuring therapeutic efficacy, etc., may be observed two months or less after administration, for example, 4, 3 or 2 weeks or less after administration, for example, 1 week after administration of the composition. Transgene expression is also expected to persist over time. Consequently, in some examples, transgene expression, for example, as detected by measuring gene product levels, by measuring therapeutic efficacy, etc., may be observed 2 months or more after administration of the composition, for example, 4, 6, 8 or 10 months or more, in some examples, 1 year or more, for example 2, 3, 4 or 5 years, in certain examples, more than 5 years.

[0214] In certain embodiments, the method comprises the step of detecting transgene expression in cells or in the individual, wherein the expression is enhanced relative to the expression from a polynucleotide cassette not comprising one or more enhanced elements of the present disclosure. Typically, the expression will be enhanced 2 times or more relative to the expression from a reference, i.e., a control polynucleotide cassette, for example, as known in the art, for example, 3 times, 4 times or 5 times or more, in some examples, 10 times, 20 times or 50 times or more, for example, 100 times, as evidenced, for example, by early detection, higher levels of the gene product, a stronger functional impact on cells, etc.

[0215]Typically, if the composition is an LV comprising the polynucleotide cassette of the present disclosure, an effective amount to achieve a change will be about 1 x 108 vector genomes or more, in some cases 1 x 109, 1 x 1010, 1 x 1011, 1 x 1012 or 1 x 1013 vector genomes or more, in certain examples 1 x 1014 vector genomes or more, and usually not more than 1 x 1015 vector genomes. In some cases, the amount of vector genomes released is at most about 1 x 10¹⁵ vector genomes, for example, 1 x 10¹⁴ vector genomes, or less, for example 1 x 10¹³, 1 x 10¹², 1 x 10¹¹, 1 x 10¹⁰ or 1 x 10⁹ vector genomes, or less, in certain examples, 1 x 10⁸ vector genomes, and typically not less than 1 x 10⁸ vector genomes. In some cases, the amount of vector genomes released is 1 x 10¹⁰ to 1 x 10¹¹ vector genomes. In some cases, the amount of vector genomes released is 1 x 10¹⁰ to 3 x 10¹² vector genomes. In some cases, the number of vector genomes released is 1 x 10⁹ to 3 x 10¹³ vector genomes. In some cases, the number of vector genomes released is 1 x 10⁸ to 3 x 10¹⁴ vector genomes.

[0216]In some cases, the amount of pharmaceutical composition to be administered can be measured using multiplicity of infection (MOI). In some cases, MOI may refer to the ratio, or multiple, of vector or viral genomes to the cells to which nucleic acid can be released. In some cases, the MOI may be 1 x 10⁶. In some cases, the MOI may be 1 x 10⁵ to 1 x 10⁷. In some cases, the MOI may be 1 x 10⁴ to 1 x 10⁸. In some cases, the recombinant viruses of the disclosure are at least approximately 1 x 10¹, 1 x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x 10¹⁰, 1 x 10¹¹, 1 x 10¹², 1 x 10¹³, 1 x 10¹⁴, 1 x 10¹⁵, 1 x 10¹⁶, 1 x 10¹⁷, and 1 x 10¹⁸ MOI. In some cases, the recombinant viruses in this disclosure are 1 x 10⁸ to 3 x 10¹⁴ MOI. In some cases, the recombinant viruses in the disclosure are at most about 1 x 10¹, 1 x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x 10¹⁰, 1 x 10¹¹, 1 x 10¹², 1 x 10¹³, 1 x 10¹⁴, 1 x 10¹⁵, 1 x 10¹⁶, 1 x 10¹⁷, and 1 x 10¹⁸ MOI.

[0217]In some aspects, the amount of pharmaceutical composition comprises about 1 x 108 to about 1 x 1015 recombinant virus particles, about 1 x 109 to about 1 x 1014 recombinant virus particles, about 1 x 1010 to about 1 x 1013 recombinant virus particles or about 1 x 1011 to about 3 x 1012 recombinant virus particles.

[0218]Any total number of viral particles suitable to provide appropriate cell transduction to confer the desired effect or treat the disease may be administered to the mammal. In various preferred embodiments, at least 10⁸; 5 x 10⁸; 10⁹; 5 x 10⁹, 10¹⁰, 5 x 10¹⁰; 10¹¹; 5 x 10¹¹; 10¹²; 5 x 10¹²; 10¹³; 1.5 x 10¹³; 3 x 10¹³; 5 x 10¹³; 7.5 x 10¹³; 9 x 10¹³, 1 x 10¹⁴ viral particles, or 5 x 10¹⁴ viral particles or more, but typically no more than 1 x 10¹⁵ viral particles are injected. Any suitable number of vector administrations to the eye of the mammal or primate may be made. In one embodiment, the methods comprise a single administration; in other embodiments, multiple administrations are made over time as deemed appropriate by a physician. In some embodiments, at least 2 x 10⁸ VG/ml of 5 x 10⁵ cells/ml is required in a single administration (24-hour transduction) to result in high transduction efficacies. Individual doses are typically not less than the amount required to produce a measurable effect in the individual and can be determined based on the pharmacokinetics and pharmacology for absorption, distribution, metabolism, and excretion (“ADME”) of the composition or its byproducts and thus based on the disposition of the composition in the individual. This includes consideration of the route of administration as well as dosage amount. Effective dose amounts and/or dosing regimens can be readily determined empirically from preclinical trials, clinical safety and dose escalation and range trials, individual physician-patient relationships, as well as in vitro and in vivo trials, such as those described in this report and illustrated in the Examples.

[0219] In some embodiments, the dose of cells that patients receive by infusion will be that which is obtained from the transduction process. In several preferred embodiments, at least about 1 x 10¹, 1 x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸ or more CD34+ cells/kg of patient weight are infused into the patient. In some embodiments, between 1 x 10⁶ and 4 x 10⁶ CD34+ cells/kg of patient weight are infused into the patient. In other embodiments, 3 x 10⁵ and 4 x 10⁶ CD34+ cells/kg of patient weight are infused into the patient. In some embodiments, the cells will be infused into the patient in a single dose. In other embodiments, the cells will be infused into the patient in multiple doses. Transduced cells can be infused immediately after the transduction process is complete.

[0220]Once integrated, the therapeutic protein (e.g., human FANCA protein) is expressed by the cells. The transduced FA cells are genetically modified and thus able to activate the FA pathway by mono-ubiquitination of FANCD2 and FANCI. These proteins migrate to areas of DNA damage and, in cooperation with other DNA repair proteins, promote DNA repair in these cells, as occurs in healthy cells.

[0221]As described in more detail in the Examples, preclinical in vitro data with BM samples from human FA patients have already shown the efficacy of a FANCA LV in correcting the phenotype of these cells.

[0222] Consequently, the present invention provides methods for treating the hematological manifestations of AF. In one embodiment, the hematological manifestation of AF is selected from one or more of BMF, thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia. In a particular embodiment, the hematological manifestation is bone marrow failure (BMF), which appears in pediatric ages in most AF patients. In one embodiment, the hematological manifestation is thrombocytopenia. In another embodiment, the hematological manifestation is leukopenia. In one embodiment, the hematological manifestation is pancytopenia. In one embodiment, the hematological manifestation is neutropenia. In another embodiment, the hematological manifestation is anemia. In one embodiment, the hematological manifestation is a combination of two or more of BMF, thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia.

[0223] A FANCA LV does not directly treat solid tumors that may develop in later stages of the disease. However, the improved hematological status of FA patients treated with hematopoietic gene therapy may also improve immune surveillance against the development of solid tumors. Therefore, an indirect antitumor effect may also be generated as a consequence of treating FA patients with a FANCA LV.

[0224]In order to obtain successful gene therapy in FA, it is beneficial to collect a “sufficient” number of hematopoietic stem cells (HSCs) from an individual.

[0225] In one embodiment, HSCs are obtained or collected from a bone marrow sample. In one embodiment, the bone marrow sample is depleted of erythrocytes. In some embodiments, the bone marrow sample is depleted of CD16+ white blood cells. In some embodiments, the remaining cells after the depletion techniques are washed. In another embodiment, non-specific IgG is added to the washed cells. In some embodiments, the non-specific IgG is phlebogamma. Subsequently, CD34+ cells can be selected from the washed cells. In one embodiment, CD34+ cells are selected from the bone marrow sample. Selection methods for CD34+ cells can be positive selection, negative selection, or a combination thereof.

[0226] In another embodiment, HSCs are obtained from peripheral blood. In one embodiment, the peripheral blood sample is depleted of erythrocytes. In some embodiments, the blood sample is depleted of CD16+ white blood cells. In some embodiments, the blood cells remaining after the depletion techniques are washed. In another embodiment, non-specific IgG is added to the washed cells. In some embodiments, the non-specific IgG is phlebogamma. Subsequently, CD34+ cells can be selected from the washed cells. In one embodiment, CD34+ cells are selected from the peripheral blood sample. The selection methods for CD34+ cells can be positive selection, negative selection, or a combination thereof.

[0227] In some embodiments of the present invention, HSCs are obtained from an individual after mobilization. Mobilization can be achieved by treating the individual with drugs or compounds that cause the movement of 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 individual with G-CSF (filgrastine). In other embodiments, mobilization is achieved by treating the individual with plerixafor. Still in other embodiments, mobilization is achieved by treating the individual with a combination of filgrastine and plerixafor. (Figure 11 and Figure 14)

[0228]In one embodiment, at least 1 to 4 x 106 corrected CD34+ cells (e.g., HSCs transduced with FANCA) per kilogram of patient weight are administered to restore hematopoiesis in an unconditioned FA patient. 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 before infusion or administration to the patient (Figure 11).

[0229] Genetic correction of HSCs from AF patients, followed by autologous transplantation of these cells (hematopoietic gene therapy), is a satisfactory alternative for AF patients, particularly those lacking an HLA-identical sibling. In one embodiment, hematopoietic gene therapy is the preferred treatment regimen for a patient lacking an HLA-identical sibling. In another embodiment, hematopoietic gene therapy is the preferred treatment regimen for a patient who has an HLA-identical sibling.

[0230]All publications mentioned in this report are incorporated herein by reference to disclose and describe the methods and/or materials in relation to which the publications are cited. It should be understood that this disclosure supersedes any disclosure of an incorporated publication to the extent that there is a contradiction.

[0231] It should also be noted that claims may be worded to exclude any optional element. As such, this statement is intended to serve as a precedent for the use of such exclusive terminology as “only”, “just” and the like in relation to the description, according to the claim elements, or the use of a “negative” limitation.

[0232]The publications discussed in this report are provided for your information only prior to the filing date of this application. Furthermore, the publication dates provided may differ from the actual publication dates, which may need to be independently verified.

[0233]Because FA-A is the most frequent 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; however, other FANCA genes can be used to similarly treat other complementation groups.

EXAMPLES Example 1 - Lentiviral vectors of FANCA

[0234]Because of the safer integration pattern of LVs compared to RVs (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 to 975; Schroder AR et al. Cell. 2002;110:521 to 529), it was an objective to develop LVs as therapeutic vectors to correct the FA cell phenotype (Gonzalez-Murillo et al., 2009). Additionally, since recent studies have shown that LVs hosting potent internal promoters can also transactivate neighboring genes (Modlich et al., 2009), LVs were considered for clinical use as a compromise between therapeutic efficacy and risks of transactivating neighboring genes. The aim was, therefore, to define the threshold levels of FANCA expression that can be therapeutic, in order to limit the risks of gene transactivation by the enhancer/promoter activating the expression of the therapeutic gene. The efficacy of FANCA LVs was first verified in vitro in FA-A LCLs, subsequently in primary BM samples from FA-A patients, and finally in vivo in a mouse model of FA-A.

[0235] For this purpose, LVs expressing FANCA under the control of different promoters—vav, PGK, CMV, and SFFV promoters—were constructed. Figure 1 is a schematic of the medicinal product. Figure 2A shows a schematic representation of LVs expressing FANCA under the control of different internal promoters. Additionally, we also investigated the influence of post-transcriptional WPRE elements on both the level of FANCA expression and the therapeutic efficacy of the LVs. Initially, all LVs were packaged with the chimeric GALV-TR envelope. Titers of 1 to 2 x 10⁶ tu/ml were routinely obtained, and transductions were conducted in estimated MOIs of 1 to 2 tu/cell. Figure 2B shows a Western blot analysis of FANCA in transduced FA-A cells.

[0236] In order to determine the level of FANCA mRNA conferred by each vector, FA-A transduced lymphoblast cell lines (LCLs) were screened with 30 nM MMC for 5 days. After the screening process, transduced FA-A LCLs contained 0.81 to 3.04 copies of the respective LV per cell (Table 1). Unscreened FA-A LCLs transduced with EGFP-LVs and LCLs from a healthy donor (HD) were used as controls.

[0237]Total FANCA mRNA levels, as well as relative FANCA mRNA levels per LV copy number, were determined in LCLs transduced with different LVs (Table 1). Compared to FANCA mRNA levels observed in HD LCLs, similar FANCA mRNA/copy levels were observed in FA-A cells transduced with vav-FANCA and PGK-FANCA LVs. CMV-FANCA and, more significantly, SFFV-FANCA LVs conferred supraphysiological FANCA mRNA/copy levels (3.6 and 5.6 times, respectively). PGK-FANCA LVs hosting WPRE or modified WPRE* sequences (Schambach et al., 2006) increased FANCA mRNA levels 2.3 to 2.6 times compared to PGK-LVs without WPRE. Consistent with 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), the insertion of WPRE or WPRE* sequences significantly increased FANCA mRNA levels in cells transduced with PGK-FANCA LVs. Since the wild-type WPRE element encodes for a truncated C-terminal version of the hepatitis B virus X protein that can mediate a tumorigenic effect (Bouchard MJ, Schneider RJ. The Enigmatic X Gene Of Hepatitis B Virus. J Virol. 2004;78:12725 to 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 to 4), the LV with the modified WPRE* sequence (Schambach et al., 2006), which is devoid of any residual open reading frames, is considered more suitable for clinical uses. Table 1. Expression levels of FANCA in FA-A LCLS transduced with lentiviral vectors expressing FANCA. *To estimate FANCA mRNA and FANCA protein levels per FANCA copy, 2 copies of genomic FANCA were considered in healthy donor LCLs.

[0238] To test whether the differences in FANCA mRNA expression were confirmed at the protein level, Western blot analyses (Figure 2B) were conducted with the samples shown in Table 1. As with FANCA mRNA determinations, FANCA protein values were related not only to protein loadings but also to the number of provirus copies determined in each transduced FA-LCL. Compared to FANCA/copy levels determined in HD-LCLs, essentially normal FANCA/copy levels were observed for all FANCA-LVs tested, except for the SFFV-FANCA LV. In this case, relative FANCA/copy levels were 3.4 times higher than the levels determined in HD-LCLs. Western blots repigmented with anti-FANCD2 showed that, while FA-A LCLs transduced with the control vector were unable to mono-ubiquitinate FANCD2, FA-A LCLs transduced with any type of FANCA-LVs expressed both non-ubiquitinated and mono-ubiquitinated forms of FANCD2, consistent with a functional FA pathway in these cells.

[0239]When the FANCA expression levels conferred by different LVs in FA-A LCLs were compared with those observed in healthy donor (HD) LCLs (with two copies of FANCA), we concluded that the insertion of two copies of LV per cell can result in physiological levels of the therapeutic protein, except in the case of SFFV-LVs, which would confer supraphysiological levels of FANCA. Obtaining this number of copies can meet the requirements of a clinical trial in FA patients, where transduction efficiencies of at least 50% may be desired because of the low number of progenitors 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).

[0240] In order to analyze the possible differences in the therapeutic efficacy of LVs expressing different FANCA, the efficacy of each vector in correcting MMC hypersensitivity in FA-A lymphoblast cell lines (LCLs) was determined. For this purpose, FA-A LCLs were transduced with the different LVs (Figure 3A) and then exposed to increased concentrations of MMC. Subsequently, the viability of transduced cells was determined. As shown in Figure 3A, all FANCA-LVs tested were equally effective in reversing the hypersensitivity of FA-A LCLs. Similarly, all vectors promoted the generation of FANCD2 nuclear foci in MMC-treated cells (Figure 3B), consistent with a functional FA pathway in FA-A cells transduced with FANCA-LV.

Example 2 - Mouse model studies

[0241]To evaluate the repopulation of BM samples from FA patients, genetically corrected or not, several groups have transplanted BM cells from FA patients into immunodeficient mice. However, in no example were significant grafts reported, most likely due to the reduced number of hematopoietic progenitors and HSCs present in the BM of these patients.

[0242] To evaluate the in vivo effects of the medicinal product, a mouse model for FA-A, which contains the deletion in the Fanca gene, was used. Unlike FA patients, these animals do not develop evident hematological defects. Nevertheless, their BM progenitor cells are highly sensitive to MMC (Rio et al., 2002), as is also the case in FA patients. Therefore, to determine whether FANCA LV (Figure 4A) stably corrected the HSC phenotype from FA-A mice in vivo, BM cells from FA-A mice were transduced with FANCA LV and then transplanted into irradiated FA-A recipients (Figure 4B). To evaluate whether the hematopoietic progenitor phenotype was corrected after transplantation of genetically corrected cells, BM samples from transplanted FA-A mice were cultured on methylcellulose in the absence and presence of MMC (Figure 4C). Additional data confirming the integration of the therapeutic cassette are presented in Figures 42 to 44. Linear Amplification-Mediated PCR (LAM) is a method for restoring the integration sites of different integration vectors in the genome. A PCR product that starts from the known vector sequence and extends through the unknown flanking genome is generated and sequenced to identify the position within the genome of the integration vector. Figure 42 represents the LAM-PCR analysis of FANCA-LV insertion sites in FA hematopoietic stem cells (HSCs). Figure 43 represents the LAM-PCR results for screening FANCA-LV treated cells. Figure 44 shows the clonal diversity of FANCA-/transplant recipients with LV-corrected HSCs.

[0243]One month post-transplant, normal hematological counts were observed in these animals, showing no evident toxicity of LV. At this time, the number of copies of proviral FANCA per cell ranged from 0.5 to 10 copies/cell (similar results were observed at 3 and 6 months post-transplant) (Figure 5). To assess whether the phenotype of the hematopoietic progenitors was corrected after transplantation of genetically corrected cells, BM samples from transplanted FA-A mice were cultured on methylcellulose in the absence and presence of MMC.

[0244]As shown in Figure 6, a significant correction of MMC hypersensitivity was observed in hematopoietic progenitors from FA mice that underwent gene therapy with FANCA LV compared to the SF1-EGFP LV control. 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 post-transplant, BM samples were collected and cultured on methylcellulose in the presence of increased concentrations of mitomycin C (MMC).

[0245]These data, therefore, indicate that FANCA LV can reverse the phenotype of hematopoietic progenitors from FA-A mice after in vivo transplantation. In terms of safety, none of the 30 mice that were transplanted with BM cells previously transduced with FANCA LV developed symptoms of myeloproliferative disorders or leukemia (data obtained up to 1 year post-transplant).

Example 3. Effective transduction of fresh hematopoietic progenitors from FA patients with lentiviral vectors.

[0246]Given that it has been previously shown that transduction of cryopreserved hematopoietic stem cells (HSCs) FA is less effective compared to transduction of fresh grafts (Jacome et al., 2009), an effort to optimize the transduction efficacy of cryopreserved BM FA samples was undertaken. As shown in Figure 7, conducting three transduction cycles significantly increased the transduction efficacy of CFCs FA compared to the values obtained after a single transduction cycle (45.7±4.2% versus 13.5±5.1%, respectively). Samples were subjected to standard transductions consisting of a single transduction cycle (16 h) after 2 h of static preloading (white bars; 1xS) or enhanced transduction consisting of three transduction cycles (2 h + 2 h + 12 h) with lentiviral vectors (gray bars; 3xD).

Example 4. The lentiviral vector PGK-FANCA-WPRE* effectively corrects the phenotype of bone marrow progenitors from FA-A patients.

[0247] Because of the efficacy of LVs in which FANCA was activated by the PGK promoter, and based on previous studies showing the stability (Follenzi A et al. Nat Genet, 2000; 25:217-222) and low genotoxic properties of LVs carrying the PGK promoter (Modlich et al., 2009, Montini et al., 2006, Montini et al., 2009), further experiments were conducted in LCLs and also in bone marrow cells from FA-A patients using PGK-FANCA LVs, free of any WPRE element or hosting WPRE or WPRE sequences*. As shown in Figure 8A, all three of these vectors conferred the same reversal in hypersensitivity of FA-A LCLs in MMC.

[0248]To compare the efficacy of SFFV-FANCA and PGK-FANCA LVs in correcting the phenotype of hematopoietic progenitors from FA-A patients, erythrocyte-depleted BM samples from FA-A patients were transduced, as recently described (Jacome et al., 2009). Erythrocyte-depleted BM cells were transduced for 16 h in plates pre-loaded with LV supernatants, as recently described (Gonzalez-Murillo et al., 2009). Fourteen days later, the number of colonies cultured in the absence and presence of 10 nM MMC was recorded to determine the proportion of progenitors that became resistant to the drug.

[0249] As shown in Figure 8B, when samples were transduced with EGFP-LVs, almost no colonies were generated in the presence of MMC. In contrast to this observation, transduction of BM FA-A cells with SFFV-FANCA and PGK-FANCA LVs enabled the growth of 26 and 38% of the colonies recorded in cultures without MMC. The efficacy of PGK-FANCA LVs hosting the WPRE and WPRE* sequences was compared to WPRE-free PGK-FANCA LVs. As shown in Figure 8B, all three LVs mediated a high level of protection similar to MMC. Although the insertion of the post-transcriptional regulatory element WPRE* (Schambach et al., 2006) is not necessary to improve the efficacy of PGK-FANCA LVs, this element will offer a redundant element to maintain long-term therapeutic levels of ectopic FANCA in the patient.

[0250]Taken together, the data obtained in these studies strongly suggest that PGK-FANCA-WPRE* LVs confer sufficient levels of FANCA expression to correct the hematopoietic phenotype of FA-A patients. These results, together with previous observations showing the stability (Follenzi et al., 2000) and safety properties of PGK-LVs (Modlich et al., 2009, Montini et al., 2006, Montini et al., 2009) and the efficacy of the modified post-transcriptional WPRE* element (Schambach et al., 2006), reinforce the hypothesis that PGK-FANCA-WPRE* LV may constitute an effective and safe vector for gene therapy in FA-A patients.

Example 5. Analysis of the effectiveness of packaged lentiviral vectors GALV-TR and VSV-G to transduce hematopoietic progenitors from FA patients.

[0251]Because LVs can be pseudotyped with GALV-TR and VSV-G envelopes, new experiments were conducted in which unselected bone marrow cells from AF patients were transduced under optimized conditions with EGFP-LVs pseudotyped in both envelopes.

[0252]For GALV-TR pseudotyped LVs, two transduction cycles with non-concentrated LVs (estimated titer: 2 x 105 IUs/mL; estimated MOI: 2 IUs/cell) were conducted.

[0253]For VSV-G pseudotyped LVs, a transduction cycle with concentrated and purified LVs (estimated titer: 108 IUs/mL; estimated MOI: 50 IUs/cell).

[0254]As shown in Figure 9, under similar conditions, transductions of hematopoietic progenitors from FA patients were obtained, indicating that manufacturing compromises may determine the best envelope for packaging.

Example 6. Safety studies.

[0255]The transformation potential of the lentiviral vectors shown in Figure 10A was measured in replication frequency over copy number. As shown in Figure 10B, the transformation potential of the lentiviral main strand corresponding to PGK-FANCA-WPRE* LV (a PGK-derived lentiviral vector) is considerably lower compared to the transformation capacity of vectors that host viral promoters, which are those already used in the X1-SCID and CGD clinical trials.

[0256]With respect to in vivo studies with mice transplanted with BM cells transduced with PGK-FANCA-WPRE* LV, 30 mice were transplanted, and to date (up to 1 year post-transplant) none of the transplanted animals developed symptoms of myeloproliferative disorders or leukemia.

Example 7. Pharmaceutical product

[0257]The FANCA lentiviral vector is a third-generation self-inactivated rHIV1-derived vector that encodes human FANCA cDNA under the control of the human PGK promoter and regulated post-transcriptionally by a modified WPRE devoid of the X protein ORF (See Figure 1, Figure 41 and SEQ ID NO: 24). This FANCA lentiviral vector presents several advantages over previously used gamma-retroviral vectors in gene therapy for FA, notably the ability to transduce cells despite short pre-activation protocols, which is advantageous for preserving the potential of multiple hematopoietic stem cell lines.

[0258]For the generation of the lentiviral therapeutic vector, 293T cells transfected with three additional plasmids, providing all the auxiliary proteins required for vector packaging (See Figures 38 to 40) will comprise the final packaging of the medicinal product PGK-FANCA-WPRE* (Figure 41).

[0259]The PGK-FANCA-WPRE*LV therapeutic cassette comprises the human PGK promoter, the coding sequence for FANCA cDNA and the WPRE* enhancer and comprises nucleotides 3541 to 9178 of SEQ ID NO: 24. The transfer cassette region comprising the human CMV immediate early promoter, the HIV packaging sequence, the ga and RRE elements, the therapeutic cassette and the HIV autoinactivation 3’ LTR, wherein the therapeutic cassette comprises the human PGK promoter, the coding sequence for FANCA cDNA and the WPRE* enhancer is encoded by nucleotides 1586 to 9495 of SEQ ID NO: 24.

[0260]Nucleotides 1586 to 1789 of SEQ ID NO: 24 comprise the immediate early promoter of human CMV. Nucleotides 2031 to 2156 of SEQ ID NO: 24 comprise the HIV1 psi packaging signal. Nucleotides 2649 to 2882 of SEQ ID NO: 24 comprise the HIV1 RRE element. Nucleotides 3378 to 3495 of SEQ ID NO: 24 comprise the HIV cPPT/CTS element. Nucleotides 3541 to 4051 of SEQ ID NO: 24 comprise the hPGK promoter. Nucleotides 4078 to 8445 of SEQ ID NO: 24 comprise human FANCA-A cDNA. Nucleotides 8502 to 9178 of SEQ ID NO: 24 comprise the modified WPRE element. Nucleotides 9262 to 9495 of SEQ ID NO: 24 comprise the HIV 3’ delta U LTR.

Example 8. FANCOSTEM Clinical Study

[0261] In the US, two gene therapy experiments were conducted in FA-A and FA-C patients, which did not show clinical efficacy (Liu, J. M., et al. (1999). Hematopoietic stem cell graft transduced with the Fanconi anemia group C (FANCC) gene. Hum. Gene Ther. 10: 2337-2346; Kelly, P. F., et al. (2007). Stem cell collection and gene transfer in fanconi anemia. Mol Ther 15: 211-219). The efficacy of the aforementioned experiments can be significantly improved through various optimizations. Two clinical trials were conducted in tandem to determine the feasibility of a process for collecting and purifying a sufficient number of CD34+ cells for future clinical use (FANCOSTEM) 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.

[0262]The main inclusion criteria for FANCOSTEM were Patients with a diagnosis of AF, confirmed by a chromosomal instability test with diepoxybutane or mitomycin C, Age > 1 year, and_At least one of the following parameters must be as high as:_ 1) Hemoglobin: 8.0 g/dL, 2) Neutrophils: 750/mm3, 3) Platelets: 30,000/mm3. Ten patients were recruited, nine were selected, of which 2 failed (mosaic patients). Figure 12 shows the hematological parameters of the recruited patients. Seven (7) patients were treated with G-CSF and Plerixafor. The mobilization regimen used the administration of G-CSF (neupogen; 12 μg/Kg/12 hours) and plerixafor (mozobil; 240 μg/kg body weight/day). Mobilized peripheral blood (mPB) CD34+ cells were transduced under GMP conditions with a PGK-FANCA-WPRE* LV using a short ex vivo transduction protocol (Figure 13). While two patients aged 15 and 16 years did not reach the threshold level of peripheral blood CD34+ cells, apheresis could be conducted in five patients aged 3 to 5 years. In these patients, the mean number of CD34+ cells/kg was 6.6 x 10A6 (range: 1.6 x 10A6 to 7.6 x 10A6). After selection of CD34+ cells, the number of CD34+ cells/kg was 2.0 x 10A6 (range: 8.5 x 10A5 and 5.1 x 10A6). No severe adverse events related to the mobilization regimen were detected in any treated patient followed for up to 2.5 years.

[0263] Figure 14 shows G-SCF/Plerifaxor-mediated mobilization of CD34+ cells in FA-A patients and Figure 15 shows G-SCF/Plerifaxor-mediated mobilization of colony-forming cells (CFCs) in FA-A patients. Figure 16A shows CD34+ cell collection in FANCOSTEM and Figure 16B shows a comparison to previous studies.

[0264]Figure 17 shows the comparison between predicted CD34+ cell counts in bone marrow (BM) vs. actual counts in mobilized peripheral blood (mPB). Figure 18 is a summary of CD34+ cell collection in FA-A patients mobilized with G-CSF/Plerixafor. The CD34+ count after screening correlates with the CD34+ cell count/μl in BM on day 0 (data not shown).

[0265]Figure 19 represents the expression of CD34 before and after immunoselection of CD34+ mPB cells from healthy donor (HD) and FA patients.

[0266] Based on these data, we conclude that compared to other clinical studies, evident improvements in CD34+ cell collection have been observed so far in patients treated with Filgrastine (G-CSF) and Plerixafor, and only AF patients in early stages of the disease appear to be suitable for collecting clinically relevant numbers of HSCs. The patient is ineligible to participate in the clinical trial; 3) Evidence that the patient presents signs of somatic mosaicism with improved hematology; 4) Any concomitant disease or condition that, in the investigator's opinion, renders the individual unsuitable for participation in the study; 5) Pre-existing sensory or motor impairment >= grade 2, according to the National Cancer Institute (NCI) criteria; 6) Pregnant or breastfeeding women.

[0271]Route of administration: Patients received the transduced cells with therapeutic vector by intravenous infusion.

[0272]Cell dose: The cell dose that patients received by transfusion was that obtained from transduction, between 3 x 105 and 4 x 106 purified CD34+ cells/kg of patient weight.

[0273]Below 3 x 10⁵ CD34+ cells/kg it is highly unlikely to produce a patient graft from transduced cells, especially considering that this clinical trial will initially infuse unconditioned patients. In the gene therapy trial in patients with Fanconi anemia conducted by Dr. Williams (Kelly et al., 2007), the number of cells infused in 2 patients (FAAGT1001 and 1003) was 4.5 x 10⁵ and 3.25 x 10⁵ nucleated cells/kg of patient weight, respectively. In both patients, a transient improvement in Hb and platelet count was observed, but without being able to demonstrate an associated presence of the transgene. Unlike Dr. Williams' experiment where cells were transduced for 4 days with a gamma-retroviral vector, in this clinical trial, cells were transduced with a more effective lentiviral vector and the transduction was conducted for a maximum of 48 hours, that is, a shorter time than the 4 days used in the previous protocol. Therefore, we considered 3 x 10⁵ purified CD34+ cells/kg of the patient's body weight as a reasonable lower limit for this experiment.

[0274]The upper limit of 4 x 106 purified CD34+ cells/kg is not presented based on the need to limit the number of cells infused, since a higher number of cells increases the probability of engraftment. On the contrary, the limit of 4 x 106 purified CD34+ cells/kg of the patient's body weight stems from the difficulty in mobilizing and collecting cells that exceed this number from patients with Fanconi anemia, characterized by having a reduced CD34+ cell count in their bone marrow (Jacome et al., 2009).

[0275] Dosage Regimen: Cells transduced with the therapeutic vector were infused into the patient in a single dose. This was done for two main reasons: 1) All hematopoietic gene therapy experiments conducted to date have been performed using a single infusion of transduced cells (Naldini, 2011). Following this previous experience, we were not inclined to vary this parameter. 2) A single infusion of all transduced cells would increase the likelihood of a larger graft compared to infusion of the same fractionated dose.

[0276]Recruitment Period: Patients were recruited over a 3-year period. As FA-A subtype patients are the most frequent in the FA patient population (around 80% of Spanish FA patients correspond to this complementation group (Casado et al. (2007))), the constructed therapeutic vector carries the FANCA gene. Therefore, of the FA patients, only those belonging to the FA-A subtype participated in this study. Any patients from this complementation group, pediatric or adult, provided they met the defined inclusion and exclusion criteria, were included in the study.

[0277]The specific description of the primary and, if any, secondary variables that will be evaluated in the clinical trial includes: 1) Primary variable: A) To determine the toxicity associated with the infusion of autologous CD34+ cells transduced with the lentiviral therapeutic vector in patients with Fanconi anemia subtype A. B) To determine the degree of engraftment associated with the infusion of autologous CD34+ cells transduced with the lentiviral therapeutic vector in patients with Fanconi anemia subtype A. 2) Secondary variables: To determine the clinical response associated with the infusion of autologous CD34+ cells transduced with the lentiviral therapeutic vector in patients with Fanconi anemia subtype A.

[0278]CD34+ cells from bone marrow and/or mobilized in peripheral blood (fresh and/or cryopreserved) from patients with Fanconi anemia subtype A (FA-A) were transduced ex vivo with a lentiviral vector carrying the FANCA gene (orphan drug) (Figure 11). After cell transduction, patients received an infusion of these genetically corrected stem cells in order to restore hematopoiesis.

[0279]Assessment: Patients were assessed prior to treatment initiation, obtaining prior informed consent. The assessment was performed one month before the infusion of genetically corrected cells, through a standard physical examination (including weight and height), peripheral blood cell counts, basic biochemistry, and a bone marrow aspirate.

[0280]Transduction and infusion of genetically corrected CD34+ cells: The purified CD34+ cell population was transduced ex vivo with the therapeutic lentiviral vector. After cell transduction, product quality control assessments were performed, aliquots were cryopreserved for further study, and the product was prepared for infusion into patients.

[0281]If in two patients infused with an acceptable number of transduced cells (at least 1 million cells infused/kg of body weight, in an aliquot in which at least 0.3 vector copy/cell is detected after at least 7 days in in vitro culture) AT LEAST 0.1 COPY of VECTOR/CELL is not observed in the bone marrow or peripheral blood 6 MONTHS POST-INFUSION, subsequent patients will be subjects for process conditioning prior to cell infusion.

[0282]For a patient to be eligible for conditioning of any type, there must be an adequate method of rescuing the potential bone marrow aplasia associated with conditioning and possible failure of the transduced cell implant. Rescue methods include a unit of umbilical cord blood or hematopoietic cells from a haploidentical donor. The cells were intravenously infused.

[0283]The dose of cells that patients received by infusion was that obtained from the transduction process, between 3 x 105 and 4 x 106 CD34+ cells/Kg of patient weight.

[0284]The cells were infused into the patient in a single dose.

[0285]The transduced cells were infused immediately after the transduction process was completed.

[0286]The infused product consists of a suspension of CD34+ cells that has been packaged in a sterile infusion bag by the CLINISTEM GMP laboratory at CIEMAT.

[0287]Recruitment Period: 3 years from the first patient infusion.

[0288]Follow-up period: two years from the infusion of transduced cells. However, patients are monitored outside the clinical trial for a period of 10 years.

[0289]Monitoring of transduced cell grafting was performed on peripheral blood and bone marrow samples.

[0290]First 72 hours after infusion: During this period, vital signs were recorded every 8 hours and vital organ functions were monitored (electrolyte profile, hematology, renal and hepatic function) every 24 hours.

[0291]Subsequently, the following checks were performed, as shown in Table 2.Table 2

[0292] Patients diagnosed with FA and belonging to the FA-A complementation group were included in the study. Patients were considered if correction of the cellular phenotype was demonstrated by transduction with vectors carrying the FANCA gene or if pathogenic biallelic mutations in this gene are demonstrated.

[0293] Patient FA 02005 meets the criteria for FANCOSTEM and FANCOLEN, as summarized in Figure 20. Figure 21 shows the tests that show the FA diagnosis of patient FA-02005 before gene therapy, and Figure 22 shows the follow-up of the cell preparation process for patient FA-02005. Figure 23 shows the number of vector copies 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. The follow-up of the first unconditioned FA-A patient before and after gene therapy, patient FA02005 (4 years old), is shown for hemoglobin (Figure 24), neutrophils (Figure 25), and platelets (Figure 26).

[0294] For patient FA02002, the hematological evolution is presented in Figure 27, the diagnosis is presented in Figure 28, and the follow-up of the cell preparation process is presented in Figure 29. Figure 30 shows the analysis of CD34 expression in the healthy donor and FA mPB during the different stages required for LV transduction for patient FA-02002. Figure 31 shows the number of vector copies in patient FA02002 before gene therapy and 2 weeks, 4 weeks, and 6 weeks after gene therapy. The follow-up of patient FA02002 (infused with cryopreserved cells) is presented for hemoglobin (Figure 32), neutrophils (Figure 33), and platelets (Figure 34).

[0295]The data for these two patients support the conclusion that the patients were infused with a significant number of mobilized peripheral blood CD34+ cells (mPB) corrected for the gene. No serious adverse events were observed in either of the two treated patients. Gene labeling levels around 1 to 5 copies/1000 peripheral blood cells were detected in treated patients 15 days to 5 months post-GT, with moderate increases over time. These viral copy numbers were around 100x higher compared to the highest value detected at 3 weeks post-GT in previous experiments (3 copies/10A5 cells; Kelly et al. 2007).

Example 6. Transduction of Fresh mPB CD34+ Cells from FA-A Patients

[0296]Short transduction with the therapeutic vector of small aliquots of mobilized peripheral blood (mPB) CD34+ cell samples from patients treated with the G-CSF/Plerixafor regimen showed transduction efficiencies between 17 and 45% (Figure 35). Small aliquots of these samples were transplanted into NSG mice conditioned with 1.5 Gy. Most of the transplanted samples engrafted in NSG mice (1 to 10% of BM cells were hCD45+/mCD45-) (Figure 36). Furthermore, a clear selection advantage of FA-A corrected CD34+ cells was observed in engrafted mice (Figure 37).

[0297]These results show the grafting of CD34+ FA-A transduced mPB cells into NSG mice and that an in vivo proliferation advantage of the corrected human FA-A repopulation cells occurs in NSG recipient mice.

Claims (6)

1. Use of CD34+ cells comprising an expression cassette, CHARACTERIZED in that it is for the preparation of a medicament to treat Fanconi anemia, wherein the expression cassette comprises a polynucleotide sequence comprising in the following order 5’ to 3’:(a) a human phosphoglycerate kinase (PGK) promoter sequence, wherein the PGK promoter sequence consists of the nucleotide sequence presented in SEQ ID NO: 7;(b) a sequence encoding a human FANCA polypeptide, wherein the sequence encoding a human FANCA polypeptide consists of the nucleotide sequence presented in SEQ ID NO: 8; (c) a sequence of the marmot hepatitis virus regulatory element (WPRE), wherein the WPRE element consists of the nucleotide sequence presented in SEQ ID NO: 23, wherein the sequence encoding the human FANCA polypeptide is operably linked to the PGK promoter sequence, and wherein the cells are autologous to the individual. 2. Use, according to claim 1, CHARACTERIZED in that the FANCA polypeptide consists of the sequence presented in SEQ ID NO: 25. 3. Use, according to claim 1 or 2, CHARACTERIZED in that the cassette consists of the nucleotide sequence presented in nucleotides 3541 to 9178 of SEQ ID NO: 24. 4. Use, according to any one of claims 1 to 3, CHARACTERIZED in that the cassette further comprises one or more accentuator sequences. 5. Use, according to any one of claims 1 to 4, CHARACTERIZED in that the cassette further comprises: (d) a polypurine (PPT) or polyadenylation (polyA) tract signal sequence. 6. Use, according to any one of claims 1 to 5, CHARACTERIZED in that the cassette further comprises one or more of the following sequences: (e) a packing signal sequence; (f) a truncated Gag sequence; (g) a Rev response element (RRE); (h) a central polypurine tract (cPPT); and (i) a central terminal sequence (CTS).