Compositions and methods for stem cell transplant

Abstract

To provide systems and methods for manufacturing and using gene corrected stem cells for gene therapy.SOLUTION: Provided are methods for treating Fanconi anemia in which a subject's stem cells are selected by a combination of high stringency CD34+ selection and low stringency CD34+ selection, genetically modified using a gene therapy vector encoding a FANC protein or a gene editing system, and administered to the subject.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates generally to methods for preparing populations of hematopoietic cells, including genetically modified hematopoietic stem cells (HSCs), for use in HSC transplantation, including the treatment of Fanconi Anemia (FA).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 656,292, filed April 11, 2018, which is incorporated by reference in its entirety.

[0003] Description of electronically submitted text files The contents of the text files submitted electronically herein are incorporated herein by reference in their entirety. Computer-readable copy of the sequence listing (file name: ROPA_008_01WO_SeqList_ST25, date of record: April 11, 2019, file size approximately 52 kilobytes). [Background technology]

[0004] 2. Background of the Invention Gene transfer into target cells via ex vivo is a method that is clinically applied in cell and gene therapy. Isolation of CD34 enriched cell populations including HSCs and genetic modification ex vivo provides two major advantages: reduced gene transfer into non-target cells, and thus reduced need / amount of genetic modifiers, such as gene therapy vectors, thereby reducing the cost associated with clinical production of genetically modified HSCs. The model system for transplantation of genetically modified HSCs is FA. The method developed in the context of FA can also be applied to other disorders and conditions.

[0005] FA is an autosomal recessive disorder (except for complement group FA-B, which is X-linked), and the median survival of patients is about 24 years (Butturini A, et al. (1994) Blood 84:1650-1655 (Non-Patent Document 1); Kutler DI, et al. (2003) Blood 101:1249-1256 (Non-Patent Document 2)). At birth, these patients generally have normal blood counts. Macrocytosis is often the first hematological abnormality detected in these patients. It usually occurs along with thrombocytopenia, anemia, and pancytopenia. Bone marrow failure (BMF) is usually observed in these patients after 5 to 10 years, with the average age of onset of hematological disease being 7 years. Approximately 80% of patients with FA will develop evidence of BMF during the first decade of life. Based on previous epidemiological studies, if no malignant episodes appear before aplasia, virtually all patients with FA develop BMF by the age of 40 (Butturini A, et al. (1994) Blood 84:1650-1655; Kutler DI, et al. (2003) Blood 101:1249-1256), which is the main cause of death in these patients. Due to the complex clinical manifestations of FA, the management of these patients is mainly focused on improving the clinical symptoms of bone marrow failure (BMF), myeloid leukemia, and solid tumors.

[0006] The treatment of FA and other diseases depends on efficient engraftment of genetically modified HSCs. Thus, there is a need for a method of preparing a cell population comprising genetically modified HSCs that achieves high levels of engraftment in patients, including FA patients. The present invention addresses this need and others. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Butturini A, et al. (1994)Blood 84:1650-1655 [Non-Patent Document 2] Kutler DI,et al.(2003)Blood 101:1249-1256 Summary of the Invention

[0008] The present invention relates generally to the fields of hematological malignancies and stem cell transplantation, and in particular to the use of CD34 for use in gene therapy, including both gene delivery and gene repair. + The present disclosure relates to the production and use of enriched stem cell populations of cells.In particular, these CD34 enriched cell populations are useful in gene therapy for treating mammalian, particularly human diseases, disorders and dysfunctions associated with Fanconi anemia complementation group A (FANCA), group C (FANCC) or group G (FANCG) gene product dysregulation.In certain embodiments, the method of the present disclosure is carried out ex vivo and not in the human body itself.

[0009] In one embodiment, the present disclosure provides a method of treating Fanconi anemia in a subject in need thereof, comprising: (i) detecting CD34 under high stringency conditions from a first biological sample obtained from said subject; + and (ii) a high stringency CD34 enriched cell population prepared by selecting cells from a second biological sample obtained from said subject under low stringency conditions. + The present invention provides a method of treating Fanconi anemia, comprising providing to a subject a combination of low stringency CD34 enriched cell populations prepared by selecting cells, wherein one or both of the high stringency CD34 enriched cell population and / or the low stringency CD34 enriched cell population have been transduced with a recombinant gene therapy vector encoding a FANC polypeptide, including a functional variant or fragment or a naturally occurring FANC protein (e.g., FANCA or FANCC or FANCG), and the first biological sample and the second live biological sample are optionally the same biological sample.

[0010] In one embodiment, the method comprises preparing a CD34 enriched cell population under high stringency conditions, preparing another CD34 enriched cell population under low stringency conditions, contacting one or both of the CD34 enriched cell populations with a recombinant gene therapy vector, and administering the two CD34 enriched cell populations to the subject, either sequentially or simultaneously, thereby treating Fanconi anemia, thereby treating Fanconi anemia. In certain embodiments, the cell population(s) contacted with the gene therapy vector are subsequently transduced and thus contain a polynucleotide encoding a therapeutic nucleic acid or polypeptide for the treatment of Fanconi anemia. In one embodiment, the recombinant gene therapy vector comprises a self-inactivating lentiviral vector encoding a therapeutic FANC (e.g., FANCA or FANCC or FANCG) gene segment or protein, such as the vector described in International Patent Application No. PCT / US2017 / 050837. In one embodiment, a recombinant gene therapy vector is configured to repair an endogenous FANC gene (e.g., FANCA or FANCC or FANCG) by delivering a Cas protein or a nucleic acid encoding a Cas protein, a CRISPR / Cas system comprising a gRNA or sgRNA, and a repair template comprising the FANC gene or a fragment of the FANC gene where one or more mutations overlap with the endogenous FANC gene.

[0011] In one embodiment, the present disclosure provides a method for treating Fanconi anemia in a subject in need thereof, comprising: + preparing a high stringency CD34 enriched cell population from a first biological sample obtained from a subject by selecting cells that are CD34 enriched under low stringency conditions; +The method includes preparing a low stringency CD34 enriched cell population from a second biological sample obtained from the subject by selecting cells, contacting one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population with a recombinant gene therapy vector for Fanconi anemia, and administering the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population to the subject, wherein one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population are contacted or transduced with the recombinant gene therapy vector, thereby treating Fanconi anemia.

[0012] In one embodiment of the present invention, the first biological sample and the second biological sample are each independently peripheral blood or bone marrow.In one embodiment, the first biological sample and the second biological sample are peripheral blood obtained after the subject is treated with granulocyte macrophage colony stimulating factor (G-CSF), plerixafor, or a combination of G-CSF and plerixafor.

[0013] In one embodiment, any of the methods comprises the step of: + The method further comprises selecting the cells, + The method may include applying a first biological sample to a capture matrix that binds the cells, washing the capture matrix one or more times using a wash buffer, and eluting the high stringency CD34 enriched cell population from the capture matrix using an elution buffer. In one embodiment, the applying step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 20-25 mL / min, or 25-30 mL / min. In one embodiment, the elution step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 20-25 mL / min, or 25-30 mL / min. In one embodiment, the applying step is performed at 10-20 mL / min. In one embodiment, the elution step is performed at 20 mL / min.

[0014] In one embodiment, any of the methods comprises the step of: + The method further comprises selecting the cells, + The method may include applying a second biological sample to a capture matrix that binds the cells, allowing the unbound fraction of the second biological sample to flow through the capture matrix, and eluting the low stringency CD34 enriched cell population from the capture matrix using an elution buffer. In one embodiment, the applying step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 20-25 mL / min, or 25-30 mL / min. In one embodiment, the elution step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 20-25 mL / min, or 25-30 mL / min. In one embodiment, the applying step is performed at 10-20 mL / min. In one embodiment, the elution step is performed at 20 mL / min.

[0015] In one aspect of the invention, the high stringency CD34 enriched cell population and / or the low stringency CD34 enriched cell population are contacted with a recombinant gene therapy vector. In some embodiments, the percentage of CD34+ cells in the high stringency CD34 enriched cell population is 2-4 times greater than the percentage of CD34+ cells in the low stringency CD34 enriched cell population. In one embodiment, the percentage of CD34+ cells in the high stringency CD34 enriched cell population is about 60%, and the percentage of CD34+ cells in the low stringency CD34 enriched cell population is less than 60%, or 15-60%, or 15-30%. In one embodiment, the percentage of CD34+ cells in the high stringency CD34 enriched cell population is about 70% or more, and the percentage of CD34+ cells in the low stringency CD34 enriched cell population is 17.5-35%. In one embodiment, the percentage of CD34+ cells in the high stringency CD34 enriched cell population is about 80% or more, and the percentage of CD34+ cells in the low stringency CD34 enriched cell population is less than 40% or between 20% and 40%. In one embodiment, the percentage of CD34+ cells in the high stringency CD34 enriched cell population is about 90% or more, and the percentage of CD34+ cells in the low stringency CD34 enriched cell population is less than 40% or between 22.5% and 40%.

[0016] In one embodiment of the present invention, a recombinant gene therapy vector for the treatment of Fanconi anemia comprises a polynucleotide sequence comprising, in 5' to 3' order, (a) a promoter sequence active in eukaryotes, and (b) a sequence encoding a human FANC gene or polypeptide, including functional fragments and variants thereof, wherein the sequence encoding the human FANC gene or polypeptide, or a functional fragment or variant thereof, is operably linked to the eukaryotically active promoter sequence, and the FANC gene or polypeptide is selected from FANCA, FANCC, and FANCG, e.g., native human FANCA, FANCC, and FANCG, or functional fragments or variants thereof. In certain embodiments, the functional fragment or functional variant of the native FANC protein has substantially similar biological activity as the native FANC protein.

[0017] In one embodiment of the invention, a recombinant gene therapy vector for Fanconi anemia comprises a gene editing system capable of directed repair of an endogenous FANC gene, the gene editing system comprising a Cas protein or a polynucleotide encoding a Cas protein, a gRNA, and a repair template comprising a sequence comprising a FANC gene or a fragment thereof that overlaps with one or more mutations in the endogenous FANC gene, wherein the sgRNA is configured to guide the repair template to the FANC gene, wherein the FANC gene is selected from FANCA, FANCC, and FANCG.

[0018] In one embodiment, the selection method is carried out by bead-based magnetic selection.

[0019] In one embodiment, the methods disclosed herein further comprise performing one or more apheresis on the peripheral blood.

[0020] In one aspect, the methods disclosed herein result in the expansion of genetically modified Fanconi anemia cells over time.

[0021] In one aspect, the therapeutic methods disclosed herein inhibit the development of, halt the progression of, and / or reverse the progression of hematological manifestations of Fanconi anemia in a subject, such as, for example, but not limited to, one or more of bone marrow failure, thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia.

[0022] In one aspect, the methods disclosed herein result in the restoration of one or more hematopoietic lineages that were decreased in the subject prior to administration of the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population to the subject, such as, for example, but not limited to, one or more of lymphocytes, eosinophils, neutrophils, erythrocytes, and platelets. In certain embodiments, the methods result in a delayed or reduced decline of one or more hematopoietic lineages, or a stabilization of one or more populations of one or more hematopoietic lineages, such as one or more of lymphocytes, eosinophils, neutrophils, erythrocytes, and platelets.

[0023] In one aspect, the method results in the stabilization or restoration of one or more hematological parameters, such as hemoglobin, that were decreased in the subject prior to administration of the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population to the subject.

[0024] In one embodiment, the present disclosure provides a method for preparing genetically modified cells for the treatment of Fanconi anemia, comprising: +The method includes preparing a high stringency CD34 enriched cell population from a first biological sample obtained from a subject by selecting cells, preparing a low stringency CD34 enriched cell population from a second biological sample obtained from a subject by selecting CD34+ cells under low stringency conditions, and contacting one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population with a recombinant gene therapy vector for Fanconi anemia. In certain embodiments, the cell population(s) contacted with the gene therapy vector are transduced by the gene therapy vector, resulting in a cell population comprising transduced cells comprising a nucleotide sequence encoding a therapeutic nucleic acid or polypeptide (e.g., a FANC polypeptide, which may be a wild-type or native FANC polypeptide or a functional fragment or variant thereof).

[0025] In one embodiment, the selection of either or both of the low stringency and high stringency CD34 enriched cell populations is carried out using an antibody or functional fragment thereof that specifically binds to CD34. In one embodiment, the selection is carried out using a flow rate of 10-20 mL / min.

[0026] In one embodiment, the present invention provides a method for isolating CD34 from a first biological sample under high stringency conditions. +The present invention provides a system comprising a high stringency CD34 enriched cell population prepared by selecting CD34+ cells from a second biological sample under low stringency conditions, and a low stringency CD34 enriched cell population prepared by selecting CD34+ cells from a second biological sample under low stringency conditions, wherein one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population are contacted with or transduced by a recombinant gene therapy vector for Fanconi anemia. In certain embodiments, the high stringency CD34 enriched cell population is present in a first pharmaceutical composition comprising one or more pharma- ceutically acceptable carriers, diluents, or excipients, and the low stringency CD34 enriched cell population is present in a second pharmaceutical composition comprising one or more pharma-ceutically acceptable carriers, diluents, or excipients. In some embodiments, both the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population are present in the same pharmaceutical composition comprising one or more pharma-ceutically acceptable carriers, diluents, or excipients. In certain embodiments, both the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population are transduced with a gene therapy vector for the treatment of Fanconi anemia. In certain embodiments, the gene therapy vector and / or the transduced cells comprise a nucleotide sequence encoding a therapeutic nucleic acid or polypeptide (e.g., a FANC polypeptide, which may be a wild-type or native FANC polypeptide or a functional fragment or variant thereof).

[0027] In another embodiment, the present invention provides a method for isolating CD34 from a first biological sample under high stringency conditions. +The present invention provides a pharmaceutical composition comprising a high stringency CD34 enriched cell population prepared by selecting CD34+ cells from a second biological sample under low stringency conditions, and a low stringency CD34 enriched cell population prepared by selecting CD34+ cells from a second biological sample under low stringency conditions, wherein one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population are transduced with a recombinant gene therapy vector. In certain embodiments, the gene therapy vector is a lentivirus. In certain embodiments, the gene therapy vector encodes a therapeutic FANC (e.g., FANCA or FANCC or FANCG) gene segment or protein or a functional variant or fragment thereof. The pharmaceutical composition may include one or more pharma- ceutical acceptable excipients, diluents, or carriers.

[0028] [The present invention 1001] 1. A method of treating Fanconi anemia in a subject in need thereof, comprising: (i) detecting CD34 under high stringency conditions from a first biological sample obtained from the subject; + A high stringency CD34 enriched cell population prepared by cell selection, and (ii) detecting CD34 under low stringency conditions from a second biological sample obtained from the subject; + Low stringency CD34 enriched cell populations prepared by cell selection providing to said subject one or both of the high stringency CD34 enriched cell population and / or the low stringency CD34 enriched cell population (i) a recombinant gene therapy vector comprising a polynucleotide sequence encoding a Fanconi Anemia Complementation Group (FANC) polypeptide or a functional variant or fragment thereof; or (ii) A gene editing system that targets the direct repair of endogenous mutant FANC genes genetically modified by, and The method, wherein the first biological sample and the second biological sample are optionally the same biological sample. [The present invention 1002] (a) CD34 under high stringency conditions + preparing a high stringency CD34 enriched cell population from a first biological sample obtained from said subject by selecting cells; (b) CD34 under low stringency conditions + preparing a low stringency CD34 enriched cell population from a second biological sample obtained from said subject by selecting cells; (c) subjecting one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population to (i) a recombinant gene therapy vector comprising a polynucleotide sequence encoding a Fanconi anemia complementation group A (FANCA) polypeptide or a functional variant or fragment thereof; or (ii) the gene editing system that targets the direct repair of the endogenous mutant FANC gene; and genetically modifying the (d) providing said high stringency CD34 enriched cell population and said low stringency CD34 enriched cell population to said subject. Further comprising: Thereby, the method of the present invention 1001 treats Fanconi anemia. [The present invention 1003] The method of any one of claims 1001 to 1002, wherein said first biological sample and said second biological sample are each independently peripheral blood or bone marrow. [The present invention 1004] The method of any one of claims 1001 to 1002, wherein the first biological sample and the second biological sample are peripheral blood obtained after the subject has been treated with G-CSF, plerixafor, or a combination of G-CSF and plerixafor. [The present invention 1005] Under the high stringency conditions, CD34 + Selecting cells is a step to identify CD34 +The method of claim 1002, comprising applying the first biological sample to a capture matrix that binds cells, washing the capture matrix one or more times using a wash buffer, and eluting the high stringency CD34 enriched cell population from the capture matrix using an elution buffer. [The present invention 1006] Under the low stringency conditions, CD34 + Selecting cells is a step to identify CD34 + The method of claim 1002, comprising applying the second biological sample to a capture matrix that binds cells, allowing an unbound fraction of the second biological sample to flow through the capture matrix, and eluting the low stringency CD34 enriched cell population from the capture matrix using an elution buffer. [The present invention 1007] The method of any one of claims 1001 to 1002, wherein said high stringency CD34 enriched cell population is genetically modified. [The present invention 1008] The method of any one of claims 1001, 1002, 1006, or 1007, wherein said low stringency CD34 enriched cell population is genetically modified. [The present invention 1009] The method according to any one of claims 1001 to 1008, wherein the proportion of CD34+ cells in the high stringency CD34 enriched cell population is 2 to 4 times greater than the proportion of CD34+ cells in the low stringency CD34 enriched cell population. [The present invention 1010] The method of any of claims 1001 to 1009, wherein said high stringency CD34 enriched cell population comprises CD34+ cells at a purity of more than 20% or more than 30%. [The present invention 1011] The method of any one of claims 1001 to 1010, wherein the low stringency CD34 enriched cell population comprises CD34+ cells at a purity of less than 30%. [The present invention 1012] The method of any of claims 1001 to 1011, wherein the high stringency CD34 enriched cell population comprises CD34+ cells at a yield of more than 20%. [The present invention 1013] The method of any of claims 1001 to 1012, wherein the low stringency CD34 enriched cell population comprises CD34+ cells at a yield of more than 35%. [The present invention 1014] The recombinant gene therapy vector comprises: (a) a promoter sequence active in eukaryotes, and (b) a sequence encoding a human FANC gene polypeptide or a functional fragment or variant thereof In the 5' to 3' order, Any of the methods of claims 1001 to 1003, wherein the sequence encoding the human FANC gene polypeptide or a functional fragment or variant thereof is operably linked to a promoter sequence active in the eukaryote, and the FANC gene is selected from FANCA, FANCC, and FANCG. [The present invention 1015] The gene editing system comprises: (a) a Cas protein or a polynucleotide encoding a Cas protein; (b) gRNA, and (c) a repair template comprising a sequence comprising an endogenous FANC gene or a fragment thereof that overlaps with one or more mutations in the FANC gene. Including, The method of claim 1001, wherein the sgRNA is configured to guide the repair template to the FANC gene, the FANC gene is selected from FANCA, FANCC, and FANCG, and the gene editing system is capable of direct repair of the endogenous FANC gene. [The present invention 1016] Any of the methods of claims 1001 to 1015, comprising inhibiting the development, halting the progression, and / or reversing the progression of hematological signs of Fanconi anemia in said subject, wherein said hematological signs of Fanconi anemia are arbitrarily selected from one or more of bone marrow failure, thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia. [The present invention 1017] The method of claim 1002, wherein said selection is carried out by bead-based magnetic selection. [The present invention 1018] The method of claim 1004, further comprising performing one or more apheresis procedures on said peripheral blood. [The present invention 1019] The method of any of claims 1001 to 1018, which results in a progressive increase in genetically modified Franconia anemia cells over time. [The present invention 1020] Any of the methods of claims 1001 to 1019, which results in the restoration of one or more hematopoietic lineages that were reduced in the subject prior to administering to the subject the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population. [The present invention 1021] The method of claim 1020, wherein said one or more hematopoietic lineages include one or more of lymphocytes, eosinophils, neutrophils, erythrocytes, and platelets. [The present invention 1022] Any of the methods of claims 1001 to 1019, which results in the restoration of one or more hematological parameters that were decreased in the subject prior to administering the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population to the subject. [The present invention 1023] The method of claim 1022, wherein the hematological parameter is hemoglobin. [The present invention 1024] 1. A method for preparing a genetically modified cell for the treatment of Fanconi anemia, comprising: (a) CD34 under high stringency conditions +preparing a high stringency CD34 enriched cell population from a first biological sample obtained from a subject by selecting cells; (b) preparing a low stringency CD34 enriched cell population from a second biological sample obtained from the subject by selecting CD34+ cells under low stringency conditions; and (c) genetically modifying one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population with a recombinant gene therapy vector for Fanconi anemia, wherein the gene therapy vector optionally comprises a polynucleotide encoding a Fanconi anemia complementation group (FANC) polypeptide or a functional variant or fragment thereof. The method comprising: [The present invention 1025] The method of claim 1024, wherein said first biological sample and said second biological sample are each independently peripheral blood or bone marrow. [The present invention 1026] The method of claim 1025, wherein said first biological sample and said second biological sample are peripheral blood obtained after said subject has been treated with G-CSF, plerixafor, or a combination of G-CSF and plerixafor. [The present invention 1027] Under the high stringency conditions, CD34 + Selecting cells is a step to identify CD34 + The method of claim 1024, comprising applying the first biological sample to a capture matrix that binds cells, washing the capture matrix one or more times using a wash buffer, and eluting the high stringency CD34 enriched cell population from the capture matrix using an elution buffer. [The present invention 1028] Under the low stringency conditions, CD34 + Selecting cells is a step to identify CD34 +The method of the present invention 1024, comprising applying the second biological sample to a capture matrix that binds cells, allowing an unbound fraction of the second biological sample to flow through the capture matrix, and eluting the low stringency CD34 enriched cell population from the capture matrix using an elution buffer. [The present invention 1029] The method of claim 1024, wherein said high stringency CD34 enriched cell population is contacted with said recombinant gene therapy vector. [The present invention 1030] 1029. The method of any of claims 1024 to 1029, wherein said low stringency CD34 enriched cell population is contacted with said recombinant gene therapy vector. [The present invention 1031] The method according to any one of claims 1240 to 1030, wherein the percentage of CD34+ cells in the high stringency CD34 enriched cell population is 2 to 4 times higher than the percentage of CD34+ cells in the low stringency CD34 enriched cell population. [The present invention 1032] 10. The method of any of claims 1024 to 1031, wherein said high stringency CD34 enriched cell population comprises CD34+ cells at a purity of more than 20% or more than 30%. [The present invention 1033] The method of any of claims 1024 to 1032, wherein the low stringency CD34 enriched cell population comprises CD34+ cells at a purity of less than 30%. [The present invention 1034] The method of any of claims 1024 to 1033, wherein the high stringency CD34 enriched cell population comprises CD34+ cells at a yield of more than 20%. [The present invention 1035] 15. The method of any one of claims 1024 to 1034, wherein the low stringency CD34 enriched cell population comprises CD34+ cells at a yield of more than 35%. [The present invention 1036] The recombinant gene therapy vector for Fanconi anemia comprises (a) a promoter sequence active in eukaryotes, and (b) a sequence encoding a human FANC gene polypeptide or a functional fragment or variant thereof In the 5' to 3' order, Any of the methods of claims 1024 to 1035, wherein the sequence encoding the human FANC gene polypeptide or a functional fragment or variant thereof is operably linked to a promoter sequence active in the eukaryote, and the FANC gene is selected from FANCA, FANCC, and FANCG. [The present invention 1037] The recombinant gene therapy vector for Fanconi anemia comprises a gene editing system capable of directly repairing an endogenous FANC gene, the gene editing system comprising: (a) a Cas protein or a polynucleotide encoding a Cas protein; (b) gRNA, and (c) a repair template comprising a sequence comprising the FANC gene or a fragment thereof that overlaps with one or more mutations in the endogenous FANC gene. Including, Any of the methods of claims 1024 to 1035, wherein the sgRNA is configured to guide the repair template to the FANC gene, and the FANC gene is selected from FANCA, FANCC, and FANCG. [The present invention 1038] 1025. The method of claim 1024, wherein the selection of (a) and / or (b) is carried out by bead-based magnetic selection. [The present invention 1039] The method of claim 1024 or 1038, wherein the selection of (a) and / or (b) is carried out using an antibody or a functional fragment thereof that specifically binds to CD34. [The present invention 1040] The method of any one of claims 1024 to 1038, wherein the selection of (a) and / or (b) is carried out using a flow rate of 10 to 20 mL / min. [The present invention 1041] (a) CD34 from a first biological sample under high stringency conditions +A high stringency CD34 enriched cell population prepared by cell selection, and (b) a low stringency CD34 enriched cell population prepared by selecting CD34+ cells under low stringency conditions from the second biological sample. Including, The composition, wherein one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population are contacted with a recombinant gene therapy vector for Fanconi anemia. [The present invention 1042] The composition of claim 1041, wherein said first biological sample and said second biological sample are each independently peripheral blood or bone marrow. [The present invention 1043] The composition of the present invention 1041, wherein the first biological sample and the second biological sample are peripheral blood obtained from the subject after the subject has been treated with G-CSF, plerixafor, or a composition of G-CSF and plerixafor. [The present invention 1044] Any of the compositions of claims 1041 to 1043, wherein the percentage of CD34+ cells in the high stringency CD34 enriched cell population is 2 to 4 times higher than the percentage of CD34+ cells in the low stringency CD34 enriched cell population. [The present invention 1045] The composition of any of claims 1041 to 1044, wherein the high stringency CD34 enriched cell population comprises CD34+ cells at a purity of more than 20% or more than 30%. [The present invention 1046] The composition of any of claims 1041 to 1045, wherein the low stringency CD34 enriched cell population comprises CD34+ cells at a purity of less than 30%. [The present invention 1047] The composition of any of claims 1041 to 1046, wherein the high stringency CD34 enriched cell population comprises CD34+ cells at a yield of more than 20%. [The present invention 1048] The composition of any of claims 1041 to 1047, wherein the low stringency CD34 enriched cell population comprises CD34+ cells at a yield of more than 35%. [The present invention 1049] The composition of any of claims 1041 to 1048, wherein said high stringency CD34 enriched cell population is contacted with said recombinant gene therapy vector. [The present invention 1050] The composition of any of claims 1041 to 1049, wherein said low stringency CD34 enriched cell population is contacted with said recombinant gene therapy vector. [The present invention 1051] The recombinant gene delivery vector comprises: (a) a promoter sequence active in eukaryotes, and (b) a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof In the 5' to 3' order, Any of the compositions of claims 1041 to 1050, wherein the sequence encoding the human FANC polypeptide or a functional fragment or variant thereof is operably linked to a promoter sequence active in the eukaryote, and the FANC gene is selected from FANCA, FANCC, and FANCG. [The present invention 1052] A pharmaceutical composition comprising any one of the compositions of the present inventions 1041 to 1051 and a pharma- ceutically acceptable carrier. Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims. [Brief description of the drawings]

[0029] [Figure 1] 1 shows a map of an exemplary recombinant gene therapy plasmid vector, pCCL-PGK-FANCAW-82-RO. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description Fanconi anemia (FA) presents several unique challenges for drug product manufacturing. For FA, as with other ex vivo gene therapy applications, the target cell population for gene transfer expresses the CD34 cell surface protein. For FA patients, when CD34+ cells are analyzed by flow cytometry, a lower percentage of BM cells are CD34+ compared to healthy individuals, 0.1-1.5% compared to 1-3%, respectively. The lower absolute CD34+ cells from drug material manufactured from FA patient mPB starting material was not surprising given the established feature of limited CD34+ cells in FA patients, which declines with age. However, reduced CD34+ yields and poor purity from FA patients were of concern, as this directly impacts the potential efficacy of the drug product being manufactured.

[0031] In standard CD34+ cell enrichment, a low percentage of CD34+ cells (compared to total cells) are bound by immunomagnetic beads, and therefore, since purity is the primary objective, columns are loaded relatively rapidly (mL / min) and washing is rigorous. The inventors recognized a problem when using mPB from FA patients, where this approach results in suboptimal results with low CD34 yields.

[0032] To address the limitations of standard CD34+ cell enrichment protocols for FA patients, the inventors have developed new methods for preparing CD34+ cell populations from FA patients that result in higher yields and effective preparations. These methods involve preparing two cell populations, one selected under standard CD34+ cell enrichment conditions ("high stringency" conditions) and the other selected under "low stringency" conditions. One or both of these two cell populations are transduced with a suitable gene therapy vector and administered to the FA patient. As shown herein, the new methods show advantageous therapeutic effects. Without being bound by theory, it is believed that either the presence of a higher number of CD34+ cells or other cells or other factors present in the preparation of cells with lower CD34+ cell purity contribute to the surprising efficacy of the compositions prepared according to the methods disclosed herein.

[0033] Thus, the present disclosure provides systems and methods for producing and using genetically modified or corrected stem cells for gene therapy.In particular, a method is provided herein for treating a disease or disorder (e.g., Fanconi anemia) in which the stem cells of a subject are selected by a combination of high stringency CD34+ selection and low stringency CD34+ selection, transduced with a vector encoding a therapeutic agent (e.g., FANC protein), and administered to the subject.Unexpectedly, treatment with a combination of high stringency selected CD34+ cells and low stringency selected CD34+ cells transduced with a therapeutic vector has a lower CD34+ cell purity than conventional high stringency preparations, and the therapeutic effect is improved.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.Methods and materials similar or equivalent to those described herein can be used to implement the present invention, and suitable methods and materials are described below.All publications, patent applications, patents, and other references referred to herein are expressly incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.In addition, the materials, methods, and examples described herein are illustrative and are not intended to be limiting.

[0035] In one aspect, provided is the production of high stringency and low stringency CD34 enriched cell populations (e.g., by bead-based magnetic selection using an antibody or functional fragment thereof that specifically binds to CD34) from biological samples under high stringency and / or low stringency conditions, and the use of these CD34 enriched cell populations in the preparation of medicaments useful for targeted gene therapy of diseases, disorders, and dysfunctions in mammals, particularly humans.

[0036] In another aspect, the invention provides HSC transplantation regimens for treating Fanconia anemia and FANC gene product associated disorders based on administering high stringency and low stringency CD34 enriched cell populations, where either or both of the CD34 enriched cell populations are contacted with a gene therapy vector (e.g., transduced with a lentiviral vector carrying a FANCA, FANCC, or FANCG gene fragment) or contacted with a gene therapy vector that induces site-specific repair of the FANC gene (e.g., CRISP-Cas).

[0037] In some embodiments, the biological sample is peripheral blood or bone marrow obtained from a subject, for example, a subject to be treated. In certain embodiments, the biological sample is peripheral blood obtained after the subject is treated with G-CSF, plerixafor, or a combination of G-CSF and plerixafor. In one embodiment, one or both of the biological samples are prepared by performing one or more apheresis on peripheral blood. The present disclosure collectively refers to these methods as "mobilized leukocyte apheresis."

[0038] In some embodiments, CD34 under high stringency conditions + Selecting cells is done by using CD34 +The method includes applying a biological sample to a capture matrix that binds the cells, washing the capture matrix one or more times with a wash buffer, and eluting the high stringency CD34 enriched cell population from the capture matrix with an elution buffer. In some cases, the biological sample is reapplied to the capture matrix one or more times, such as 2, 3, 4, 5, or 6 times. In one embodiment, the applying step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 20-25 mL / min, or 25-30 mL / min. In certain embodiments, the elution step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 25-25 mL / min, or 25-30 mL / min. In one embodiment, the applying step is performed at 10-20 mL / min. In one embodiment, the elution step is performed at 20 mL / min. In some embodiments, the washing step includes washing the capture matrix one or more times with a wash buffer. In some cases, the wash buffer comprises a volume of at least 100 mL, at least 200 mL, at least 300 mL, at least 400 mL, at least 500 mL or more. In one embodiment, the wash step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 20-25 mL / min, or 25-30 mL / min. In one embodiment, the wash step is performed at 10-20 mL / min. In some embodiments, the wash buffer is phosphate buffered saline (PBS), optionally containing ethylenediaminetetraacetic acid (EDTA), and / or optionally containing human serum albumin, for example, at a concentration of about 2.5% w / v. In some embodiments, the elution buffer is phosphate buffered saline (PBS), optionally containing ethylenediaminetetraacetic acid (EDTA), and / or optionally containing human serum albumin, for example, at a concentration of about 2.5% w / v. In some embodiments, the wash buffer and the elution buffer are hypotonic.

[0039] In some embodiments, CD34 under low stringency conditions + Selecting for cells is done by CD34 +The method includes applying a biological sample to a capture matrix that binds cells, allowing the unbound fraction of the biological sample to flow through the capture matrix, and eluting the low stringency CD34 enriched cell population from the capture matrix using an elution buffer. In one embodiment, the applying step is performed at 1-2 mL / min, 2-3 mL / min, 1-2 mL / min, 1-2 mL / min, 5-10 mL / min, 5-15 mL / min, 15-20 mL / min. In one embodiment, the elution step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 25-25 mL / min, or 25-30 mL / min. In one embodiment, the applying step is performed at 10-20 mL / min. In some embodiments, the application step is carried out at a flow rate that is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or lower than the flow rate under high stringency conditions, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than the flow rate used under high stringency conditions. In some embodiments, the wash step comprises washing the capture matrix one or more times with a wash buffer. In some cases, the wash buffer comprises a volume of 500 mL, 400 mL, 300 mL, 200 mL, 100 mL or less, for example, less than 500 mL, less than 400 mL, less than 300 mL, less than 200 mL, or less than 100 mL. In some cases, the wash step is omitted. In one embodiment, the wash step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 20-25 mL / min, or 25-30 mL / min. In one embodiment, the wash step is performed at 10-20 mL / min. In some embodiments, the wash buffer is phosphate buffered saline (PBS), optionally containing ethylenediaminetetraacetic acid (EDTA), and / or optionally human serum albumin, for example, at a concentration of about 2.5% w / v.In some embodiments, the elution buffer is phosphate buffered saline (PBS), optionally containing ethylenediaminetetraacetic acid (EDTA), and / or optionally human serum albumin, e.g., at a concentration of about 2.5% w / v. In some embodiments, the wash buffer and elution buffer are hypotonic.

[0040] In some embodiments, the selection is performed by bead-based magnetic selection. In one embodiment, an antibody or functional fragment thereof that specifically binds to CD34 is used for the selection, e.g., bead-based magnetic selection. The selection is performed, in some cases, using a flow rate of 10-20 mL / min. In some cases, the flow rate is 1, 5, 10, 15, 20, 25, 30, or more mL / min, or any value in between.

[0041] In some embodiments, high stringency CD34 enriched cell population is contacted with recombinant gene therapy vector, low stringency CD34 enriched cell population is contacted with recombinant gene therapy vector, or high stringency and low stringency CD34 enriched cell population are both contacted with recombinant gene therapy vector.In some embodiments, the vector is a virus, a liposome, or a lipid or lipid-like nanoparticle.In some cases, the virus is a lentivirus, an adeno-associated virus, an adenovirus, or an effusive virus.

[0042] In some embodiments, the percentage of CD34+ cells in a high stringency CD34 enriched cell population is 2-4 times greater than the percentage of CD34+ cells in a low stringency CD34 enriched cell population.

[0043] In embodiments of the methods or systems of the present invention, the recombinant gene therapy vector comprises a polynucleotide sequence comprising, in 5' to 3' order, (a) a promoter sequence active in eukaryotes, and (b) a sequence encoding a human FANC gene polypeptide or a functional fragment or variant thereof, wherein the sequence encoding the human FANC gene polypeptide or a functional fragment or variant thereof is operably linked to the promoter sequence active in eukaryotes. In some embodiments, the FANC gene is selected from FANCA, FANCC, and FANCG.

[0044] In one embodiment, the method inhibits the development, stops the progression, and / or reverses the progression of hematological manifestations of a disease or disorder (e.g., Fanconi anemia) in a subject, and optionally, the hematological manifestations of Fanconi anemia are selected from one or more of bone marrow failure (BMF), thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia. In one embodiment, the method results in an increase in genetically modified Franconian anemia (or cells of a subject suffering from another disease or disorder, such as a myeloproliferative disorder or an immunodeficiency disorder) cells over time. In one embodiment, the method results in the restoration of one or more hematological parameters (e.g., hemoglobin) that are decreased in the subject before administering the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population to the subject.

[0045] In one embodiment, the method results in the recovery of one or more hematopoietic lineages that are decreased in the subject before administering the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population to the subject, and the one or more hematopoietic lineages may include one or more of lymphocytes, eosinophils, neutrophils, erythrocytes, and platelets. Recovery of specific cell populations is achieved in some cases. Recovery can be monitored by various methods known in the art, such as flow-activated cell sorting, cytometry, or microscopy.

[0046] The invention further provides compositions and systems for use in any of these method embodiments.The invention provides CD34 enriched cell populations for use in medicine, including, but not limited to, CD34 enriched cell populations transduced with a gene therapy vector, for example, a gene therapy vector comprising a polynucleotide sequence encoding a human FANC protein or a functional variant or fragment thereof.

[0047] As used herein, "high stringency" or "high stringency conditions" refers to an enrichment method for a cell population intended to result in substantial enrichment of cells for cells expressing a particular biological marker, e.g., CD34. For example, a clinically used "high stringency" CD34 enrichment results in a mean: 61.6% and median: 65.7% yield of CD34. +cells, yielding mean: 88.5%, median: 95.9% relative purity (N=166) (Clin Lab. 2016 Jul 1;62(7):1243-1248 (PMID:28164638)). "High stringency" refers to a process aimed at substantial enrichment of the relatively rare cell type, CD34+, which typically constitutes 0.2-2% of the cell product in mobilized leukapheresis or bone marrow collection. High stringency enrichment of CD34+ cells from mobilized leukapheresis or bone marrow collection targets a final CD34+ percentage that is increased from 0.2-2% to >80%. To achieve this, after the initial application of the biological sample to the capture matrix, repeated buffer exchanges, referred to herein as "washing", are performed with the goal of removing cells that are weakly or non-specifically bound to the capture matrix. Generally, cells are removed from the capture matrix and reapplied with each wash cycle. Removal and reapplication can be accomplished manually by pipetting from the tube or automatically using a pump and tube system. For example, using the Quad Technologies MagCloudz® coupled with the Dynabeads® magnetic cell separation system, the cell-magnetic particle complexes are separated in a tube on a magnetic stand and washed manually. Using the Miltenyi Biotec CliniMACS® system, a pre-set automated program applies the cell-magnetic particle complexes to a magnetic column in a tube set, and washing / reapplication is performed using a valve pump system.In certain embodiments, the selection under high stringency conditions can be performed on various instruments, including, but not limited to, Miltenyi Biotec MACSQuant Tyto®, Quad Technologies MagCloudz®, GE Sepax® cell separation system, Terumo Elutra® cell separation system, COBE Spectra® cell separator, SynGen LAB® or WASH® system, Fresenius-Kabi Lovo®, Miltenyi Biotec CliniMACS® system, or CliniMACS Prodigy® system. Selection can be performed in the laboratory or at the point of care. Detailed methods for the preparation and enrichment of cells and cell populations, including exemplary methods for selecting CD34+ cells under high stringency conditions, are described, for example, in International Patent Publication No. WO 2016 / 118780. Exemplary selection methods useful for high stringency selection are provided in U.S. Pat. No. 8,727,132.

[0048] In a high stringency enrichment protocol, a biological sample containing CD34+ cells is labeled with a CD34 labeling reagent, for example, directly coupled immunomagnetic beads. The biological sample may be suspended in any suitable fluid, such as, but not limited to, phosphate buffered saline (PBS) optionally containing ethylenediaminetetraacetic acid (EDTA) at a buffer pH and isotonicity compatible with cell viability. In some cases, the fluid used also contains human serum albumin at a suitable concentration, such as about 2.5%. Using magnetic activated cell sorting (MACS) technology, the biological sample is applied to a column containing a magnetically susceptible or ferromagnetic material after being labeled. Using the MACS system, the magnetically susceptible or ferromagnetic material of the column retains the target cells without affecting the ability of non-target cells to flow through the column. Such magnetically susceptible or ferromagnetic materials include iron, steel, cobalt nickel, and other ferromagnetic rare earth metals in their alloys. It will be understood by those skilled in the art that such materials can be easily magnetized and demagnetized. In some embodiments, the biological sample is recirculated one or more times over the magnetically susceptible or ferromagnetic material. After loading the column, the bound cells are washed, eluted, and / or reloaded on the column at a slower rate to increase the purity of the enriched fraction. Suitable washing buffers include PBS containing (optionally) EDTA and (optionally) human serum albumin. Any components of the labeled biological sample that are removed during the washing step are collected in a waste or "non-target" bag. After a suitable washing step, the high stringency enriched cells are eluted into a target cell bag.

[0049] As used herein, "low stringency" or "low stringency conditions" refers to a method of enriching a cell population intended to result in enrichment of cells expressing a particular biological marker, e.g., cells expressing CD34, in a manner that maintains a higher yield of the enriched cell population than achieved by high stringency selection, at the expense of enrichment of cells expressing the biological marker, i.e., reduced enrichment, relative to other cells in a biological sample. By definition, the fold enrichment under high stringency conditions is greater than the fold enrichment under low stringency conditions. The fold enrichment of, e.g., CD34+ cells in a high stringency (CD34 or other marker)-enriched cell population is, in some cases, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4 times the fold enrichment of CD34+ cells in a low stringency (CD34 or other marker)-enriched cell population. In one embodiment, the fold enrichment of, for example, CD34+ cells in a high stringency (CD34 or other marker) enriched cell population is 2-4 times the fold enrichment of CD34+ cells in a low stringency (CD34 or other marker) enriched cell population. In certain embodiments, selection under low stringency conditions can be performed on a variety of instruments, including, but not limited to, Miltenyi Biotec MACSQuant Tyto®, Quad Technologies MagCloudz®, GE Sepax® Cell Separation System, Terumo Elutra® Cell Separation System, COBE Spectra® Cell Separator, SynGen LAB® or WASH® System, Fresenius-Kabi Lovo®, Miltenyi Biotec CliniMACS® System, or CliniMACS Prodigy® System. Selection can be performed in the laboratory or at the point of care.

[0050] In low stringency enrichment protocol, a biological sample containing CD34+ cells is labeled with a CD34 labeling reagent, for example, directly bound immunomagnetic beads. Using magnetic activated cell sorting (MACS) technology, the biological sample is labeled and then applied to a column containing magnetically susceptible or ferromagnetic material at a lower flow rate than high stringency enrichment. As with high stringency enrichment, the magnetically susceptible or ferromagnetic material retains the target cells without affecting the ability of non-target cells to flow through the column. In some embodiments, the biological sample is recirculated over the magnetically susceptible or ferromagnetic material one or more times. After column loading, the bound cells are washed at a lower stringency for low stringency enrichment. The bound cells are then eluted into a collection bag.

[0051] In an exemplary embodiment, low stringency enrichment is performed by modifying the standard operating procedure of the MACS system so that the "depletion mode" software program, which is intended to achieve high stringency depletion (i.e., removal of target cells), instead results in low stringency enrichment. Operation of the MACS system in depletion mode causes the target cells of the biological sample to be bound to the magnetically susceptible or ferromagnetic material in the column, using slower column loading and lower stringency washing steps than operation in enrichment mode. Non-target cells are flushed by the MACS system into a wash bag or so-called "target" bag. The depletion mode program then switches the output valve to direct the fluid into the so-called "non-target" bag, and then demagnetizes the column. Continuing to flow the liquid through the demagnetized column causes the CD34+ enriched cell population, enriched under low stringency conditions, to elute into the so-called "non-target" bag, and the target cells are collected using this method.

[0052] Those skilled in the art will recognize that this low stringency enrichment method can be performed on a variety of instruments, including, but not limited to, Miltenyi Biotec MACSQuant Tyto®, Quad Technologies MagCloudz®, GE Sepax® Cell Separation System, Terumo Elutra® Cell Separation System, COBE Spectra® Cell Separator, SynGen LAB® or WASH® System, Fresenius-Kabi Lovo®, Miltenyi Biotec CliniMACS® System, or CliniMACS Prodigy® System. Those skilled in the art will be able to reprogram the software of such MAC systems without undue experimentation so that the output valve directs the flow-through of the initial binding step to waste or a "non-target" bag (rather than the target bag) and directs the eluted low stringency CD34 enriched population to the "target" bag. In effect, the low stringency enrichment is then performed in separation mode without the usual wash steps of conventional MAC programs.

[0053] As used herein, "vector" refers to a polymer or an association of polymers that contains or associates with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell. Exemplary vectors include, for example, plasmids, viral vectors (e.g., retroviral vectors such as lentiviral vectors), liposomes, and other gene delivery vehicles.

[0054] The term "LV" is an abbreviation for lentivirus and can be used to refer to the virus itself or its derivatives. The term includes all subtypes, and both naturally occurring and recombinant forms, unless otherwise required.

[0055] As used herein, the term "gene" or "coding sequence" refers to an in vitro or in vivo nucleotide sequence that codes for a gene product. In some cases, a gene consists of or consists essentially of a coding sequence, i.e., a sequence that codes for a gene product. In other cases, a gene includes additional non-coding sequences. For example, a gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequences, and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0056] As used herein, a "therapeutic gene" refers to a gene that, when expressed, confers a beneficial effect on the cell or tissue in which it resides, or on the mammal in which it is expressed. Examples of beneficial effects include ameliorating a sign or symptom of a condition or disease, preventing or inhibiting a condition or disease, or imparting a desired characteristic. Therapeutic genes include genes that correct a genetic deficiency in a cell or mammal.

[0057] As used herein, a transgene is a gene that is delivered to a cell by a vector.

[0058] As used herein, the term "gene product" refers to the desired expression product of a polynucleotide sequence, such as a polypeptide, peptide, protein, or interfering RNA, including small interfering RNA (siRNA), miRNA, or small hairpin RNA (shRNA). In certain embodiments, the gene product is a therapeutic gene product, which upon expression confers a beneficial effect on the cell or tissue in which it is present, or on the mammal in which the gene is expressed.

[0059] As used herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length. The terms also encompass amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, pegylation, phosphorylation, or conjugation with a labeling moiety.

[0060] "Comprising" means that the recited elements are required, for example, in a composition, method, kit, etc., but other elements may be included, for example, to form a composition, method, kit, etc., within the scope of the claims. For example, an expression cassette "comprising" a gene encoding a therapeutic polypeptide operably linked to a promoter is an expression cassette that may include other elements in addition to the gene and promoter, for example, polyadenylation sequences, enhancer elements, other genes, linker domains, etc. For example, a preparation or treatment method "comprising" preparing a CD34-enriched cell population may include another step for preparing the CD34-enriched cell population, for example, administering an agent for mobilizing stem cells, administering an induction therapy, or co-administering a drug in combination therewith.

[0061] The term "comprising" or "comprises" as used herein with respect to low stringency conditions should not be construed as allowing additional high stringency selection to be applied to the same sample during the same selection step. It should be understood that performing selection under both low stringency and high stringency conditions results in high stringency selection, while low stringency selection is expressly defined herein as excluding selection under high stringency conditions. CD34 under low stringency conditions + Preparing a low stringency CD34 enriched cell population by selection for cells may include other method steps in which the column is not washed under high stringency conditions between loading the biological sample onto the column and eluting the low stringency CD34 enriched cell population from the column.

[0062] "Consisting essentially of" refers to the limitation of the scope of, e.g., a composition, method, kit, etc., described to specific materials or steps that do not substantially affect the basic and novel feature(s) of, e.g., the composition, method, kit, etc. For example, "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter, and a polyadenylation sequence may include additional sequences, e.g., linker sequences, so long as they do not substantially affect the transcription or translation of the gene. As another example, a variant, or mutant, polypeptide fragment "consisting essentially of" a recited sequence has the amino acid sequence of the recited sequence plus or minus about 10 amino acid residues at the border of the sequence based on the full-length native polypeptide from which it is derived, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue less than the recited binding amino acid residues, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues more than the recited binding amino acid residues.

[0063] "Consisting of" means that the composition, method, or kit excludes 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, a polynucleotide sequence encoding the therapeutic polypeptide, and a post-transcriptional regulatory element. As another example, a polypeptide "consisting of" a recited sequence contains only the recited sequence.

[0064] As used herein, "bone marrow cells" or "bone marrow stem cells" refer herein to both cells obtained directly from the bone marrow, such as by biopsy, and to cells obtained from peripheral blood derived from the bone marrow, for example after mobilization.

[0065] As used herein, "expression vector" includes vectors, such as plasmids, minicircles, viral vectors, liposomes, etc., that contain a polynucleotide encoding a gene product of interest, as discussed above or known in the art, and are used to induce expression of the gene product in a target cell of interest. Expression vectors also include control elements that are operably linked to the coding region to promote expression of the gene product in the target. The combination of control elements, such as promoters, enhancers, UTRs, miRNA targeting sequences, etc., and one or more genes that are operably linked for expression, may be referred to as "expression cassettes". Many such control elements are known and available in the art, or can be easily constructed from components available in the art.

[0066] As used herein, a "promoter" encompasses a DNA sequence that induces the binding of RNA polymerase, thereby facilitating RNA synthesis, i.e., a minimal sequence sufficient to induce transcription. The expression of a promoter and the corresponding protein or polypeptide can be ubiquitous, meaning highly active in a wide range of cells, tissues, and species, or cell type-specific, tissue-specific, or species-specific. A promoter can be "constitutive," meaning continuously active, or "inducible," meaning that the promoter can be activated or deactivated by the presence or absence of a biotic or abiotic factor. The nucleic acid construct or vector of the present invention also includes enhancer sequences, which may or may not be adjacent to the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and can be located in the 5' or 3' region of the native gene.

[0067] As used herein, "enhancer" includes cis-acting elements that stimulate or inhibit the transcription of adjacent genes. Enhancers that inhibit transcription are also called "silencers." Enhancers can function (i.e., associate with coding sequences) in either direction, over distances of up to several kilobase pairs (kb) from the coding sequence and downstream of the transcribed region.

[0068] As used herein, a "termination signal sequence" encompasses any genetic element that causes RNA polymerase to terminate transcription, such as, for example, a polyadenylation signal sequence.

[0069] As used herein, the term "operably linked" or "operably linked" refers to genetic elements, such as promoters, enhancers, termination signal sequences, polyadenylation sequences, etc., being juxtaposed and in a relationship that allows the elements to operate in a predicted manner. For example, a promoter is operably linked to a coding region if it helps initiate the transcription or expression of the coding sequence. Intervening residues may be present between the promoter and the coding region, so long as this functional relationship is maintained.

[0070] As used herein, the term "heterologous" means that it is derived from an entity that is genotypically different from the rest of the entity to which it is compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide. As another example, a promoter that is removed from its native coding sequence and operably linked to a coding sequence that is not found to be linked in nature is a heterologous promoter. Thus, for example, an LV vector that contains a heterologous nucleic acid encoding a heterologous gene product is an LV vector that contains a nucleic acid that is not normally included in naturally occurring wild-type LV, and the encoded heterologous gene product is a gene product that is not normally encoded by naturally occurring wild-type LV.

[0071] The term "endogenous" as used herein with respect to a nucleotide molecule or gene product refers to a nucleic acid sequence, e.g., a gene or genetic element, or a gene product, e.g., RNA, protein, that is naturally present in or associated with a host virus or cell.

[0072] The term "natural" as used herein refers to a nucleotide sequence, e.g., a gene, or a gene product, e.g., RNA, protein, present in a wild-type virus or cell. The term "variant" as used herein refers to a mutant or variant of a reference polynucleotide or polypeptide sequence, e.g., a natural polynucleotide or polypeptide sequence, i.e., having 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) with respect to a reference polynucleotide sequence, e.g., a natural polynucleotide or polypeptide sequence. For example, a variant can be a polynucleotide having 70% or more sequence identity with a full-length natural polynucleotide sequence, e.g., 75% or 80% or more, such as 85%, 90%, or 95% or more, e.g., 98% or 99% identity with a full-length natural polynucleotide sequence. As another example, a variant can be a polypeptide having 70% or more sequence identity with a full-length native polypeptide sequence, e.g., 75% or 80% or more, such as 85%, 90%, or 95% or more, e.g., 98% or 99% identity with a full-length native polypeptide sequence. A variant can also include a variant fragment of a reference, e.g., a native sequence, that shares 70% or more sequence identity with a fragment of the reference, e.g., a native sequence, e.g., 75% or 80% or more identity, such as 85%, 90%, or 95% or more, e.g., 98% or 99% identity with the native sequence.

[0073] As used herein, the terms "biological activity" and "biologically active" refer to activity resulting from a particular biological element in a cell. For example, the "biological activity" of an "immunoglobulin", "antibody" or a fragment or variant thereof refers to the ability to bind to an antigenic determinant and thereby promote immunological function. As another example, the biological activity of a polypeptide or a functional fragment or variant thereof refers to the ability of the polypeptide or a functional fragment or variant thereof to perform a natural function, such as, for example, binding, enzymatic activity, etc. As a third example, the biological activity of a gene regulatory element, such as a promoter, enhancer, Kozak sequence, etc., refers to the ability of the regulatory element or a functional fragment or variant thereof to regulate, i.e., promote, enhance, or activate, the translation of a gene to which it is operably linked, respectively.

[0074] The terms "administer" or "introduce" as used herein refer to delivering a cell population to a subject, for example, by intraarterial or intravenous delivery of the cell population to the subject's blood. The cell population may be administered in a variety of solutions, such as saline. In some embodiments, the solution used is isotonic with respect to the subject's blood and pH buffered.

[0075] "Transformation" is typically used to refer to the process of introducing heterologous DNA into a bacterium or another cell, or to a cell that has expressed an oncogene and is thus converted into a continuous growth mode, such as a tumor cell. The vector used to "transform" a cell can be a plasmid, virus, or other vehicle.

[0076] Typically, depending on the means used to administer, introduce, or insert heterologous DNA (i.e., a vector) into a cell, the cell is referred to as being "transduced," "infected," "transfected," or "transformed." The terms "transduced," "transfected," and "transformed" may be used interchangeably herein regardless of the method of introduction of heterologous DNA.

[0077] The term "host cell", as used herein, refers to a cell that has been transduced, infected, transfected, or transformed with a vector. The vector may be a plasmid, a viral particle, a phage, etc. The culture conditions, such as temperature, pH, etc., will be those previously used for the host cell selected for expression and will be apparent to one of skill in the art. It is to be understood that the term "host cell" refers to the original transduced, infected, transfected, or transformed cell, and its progeny.

[0078] The terms "treatment", "treating" and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, e.g., reduces the likelihood of the disease or its symptoms occurring in a subject, and / or it may be therapeutic in that it partially or completely cures the disease and / or slows the adverse effects caused by the disease or the progression of the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, (b) inhibiting the disease, i.e., arresting its development, or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of the disease or injury. Of particular interest is the treatment of an ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient. It is desirable for such treatment to occur before complete loss of function in the affected tissue. The subject may be treated during the symptomatic stage of the disease, or in some cases after the symptomatic stage of the disease.In certain embodiments, the subject is treated following a genetic test that identifies the subject as having a mutation associated with or causing the disease, e.g., FA.

[0079] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates, including monkeys and humans; mammalian sports animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pet animals (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0080] As used herein, a "fragment" as applied to a polypeptide will usually be at least 10 amino acid residues in length, more typically at least 20 residues, preferably at least 30 (e.g., 50) residues, but shorter than the entire intact sequence. Fragments can be generated by methods known to those of skill in the art, for example, by enzymatic digestion of natural or recombinant proteins, by recombinant DNA techniques using expression vectors encoding defined fragments, or by chemical synthesis. The ability of candidate fragments to bind to a particular DNA sequence can be evaluated by methods described herein. Purified or antigenic fragments can be used to isolate regulatory regions or to generate new regulatory enzymes (e.g., using multiple functional fragments from different enzymes), and to generate antibodies, all using standard protocols known to those of skill in the art. As used herein, "functional fragment" is meant to encompass peptide fragments that retain biological activity as well as peptide fragments that retain binding specificity with a particular nucleotide sequence.

[0081] A "functional fragment" of an anti-CD34 antibody is one that is capable of binding to a CD34 molecule on the cell surface with sufficient affinity and specificity to allow selection or enrichment of CD34+ cells.

[0082] The term "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotides or amino acid residues when compared and aligned for maximum correspondence. To determine percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In some embodiments, the two sequences are the same length.

[0083] An example of a computer program for performing optimal alignment taking into account gap penalties is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software that can perform sequence comparison include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid-Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), the GENEWORKS suite of comparison tools, the GCG Bestfit program can be used, and BLAST 2 Sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8). A scaled similarity score matrix can be used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix is ​​the BLOSUM62 matrix, the default matrix for the BLAST suite of programs. GCG Wisconsin programs can use either the public default values ​​or custom symbol comparison tables, if provided (see user manual for details).

[0084] The term "substantially identical," in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity (as determined, for example, using one of the methods described below).

[0085] Unless otherwise indicated by context, a "variant" is a polypeptide or fragment thereof that has one or more non-conservative or conservative amino acid substitutions compared to a second polypeptide (also referred to as a "derivative"), or a polypeptide or fragment thereof that is modified, e.g., by the attachment of a heterologous polypeptide or the covalent attachment of a second molecule by glycosylation, acetylation, phosphorylation, etc. Further included within the definition of "variant" are, for example, polypeptides that contain one or more analogs of an amino acid (such as, for example, unnatural amino acids), polypeptides that have unsubstituted linkages, and other modifications known in the art, both naturally and non-naturally occurring.

[0086] Various compositions and methods of the present invention are described below. Although certain compositions and methods are exemplified herein, it is understood that any of many alternative compositions and methods are applicable and suitable for use in carrying out the present invention. It is also understood that evaluation of the expression constructs and methods of the present invention can be performed using standard procedures in the art.

[0087] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described, for example, in "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" (RIFreshney, ed., 1987); Immunology" (DMWeir & C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (JMMiller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (FMAusubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al. al., eds., 1994); and “Current Protocols in Immunology” (JEColigan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0088] Some aspects of the present invention are described below with reference to exemplary applications for illustration. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the present invention. However, it will be readily understood by those skilled in the relevant art that the present 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 acts or events, and some acts may be performed in a different order and / or simultaneously with other acts or events. Moreover, not all illustrated acts or events are necessary to perform the method according to the present invention.

[0089] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in any of the detailed description and / or claims, such terms are intended to be inclusive, as is the term "comprising."

[0090] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one skilled in the art, which depends in part on how the value is measured or determined, i.e., the limits of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, according to the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in this application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0091] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication in the event of a conflict.

[0092] It is further noted that the claims may be drafted to exclude any optional element, and as such, this statement is intended to serve as a predicate to the use of exclusive terminology such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0093] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0094] Unless otherwise indicated, all terms used herein have the same meaning as they would to one of ordinary skill in the art and the practice of the present invention employs conventional techniques of microbiology and recombinant DNA technology that are within the knowledge of those skilled in the art.

[0095] Methods for preparing, transferring, and using CD34+ enriched cell populations In one aspect, the present disclosure provides a method for preparing a population of cells enriched in CD34+ cells, comprising genetically modified CD34+ cells.In certain embodiments, the method comprises preparing two CD34-enriched cell populations, one using low stringency conditions and the other using high stringency conditions.As shown herein, the use of a combination of low stringency CD34-enriched cell population and high stringency CD34-enriched cell population advantageously results in the unexpectedly effective reconstitution of CD34+ stem cells in subjects.

[0096] In another aspect, the present disclosure provides a method for treating a subject having a disease or disorder by providing a therapeutically effective amount of one or more cell populations enriched in CD34+ cells to the subject, comprising genetically modified CD34+ cells, wherein the genetically modified CD34+ cells encode a therapeutic agent effective for treating the disease or disorder. In certain embodiments, the therapeutic agent is a polypeptide or polynucleotide, e.g., RNA. In certain embodiments, the CD34 enriched cell population is prepared from CD34 cells obtained from the subject to be treated.

[0097] In certain embodiments of the method disclosed herein, two cell populations are produced that are enriched in CD34+ cells, including a high stringency CD34 enriched cell population and a low stringency CD34 enriched cell population.These two cell populations can be kept separate, or they can be combined to produce a mixed cell population that includes both high stringency CD34 enriched cells and low stringency CD34 enriched cells.In certain embodiments, either or both of the two cell populations are transduced with a gene therapy vector, for example, the LV vector that codes for FANCA or its functional fragment or variant.

[0098] In certain embodiments, the cells may be derived from any mammalian species, such as rodents (e.g., mice, rats, gerbils, squirrels), rabbits, cats, dogs, goats, sheep, pigs, horses, cows, primates, humans. In certain embodiments, the cells may be derived from established cell lines or may be primary cells, and "primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein to refer to cells and cell cultures that are derived from a subject and can be propagated in vitro for a limited number of culture passages, i.e., division. For example, a primary culture is a culture that has been passaged 0, 1, 2, 4, 5, 10, or 15 times, but not enough times to go through a crisis phase. Typically, the primary cell lines of the present invention are maintained in vitro for less than 10 passages.

[0099] In certain embodiments, the method comprises preparing a CD34 enriched cell population from a biological sample obtained from a subject. In one embodiment, the biological sample is a bone marrow sample. In another embodiment, the biological sample is peripheral blood.

[0100] In certain embodiments, the biological sample, for example, peripheral blood, is obtained from a subject after mobilization of hematopoietic stem cells (HSCs). In one embodiment, HSCs and / or progenitor cells are mobilized, for example, by treating the subject with G-CSF or an analog thereof in an amount and for a time sufficient to cause mobilization of HSCs in the patient. HSCs and progenitor cells (HSPCs) in peripheral blood can be mobilized before collection of the biological sample. Peripheral blood HSCs and HSPCs can be mobilized by any method known in the art. Peripheral blood HSCs and HSPCs can be mobilized by treating the subject with any agent(s) described herein or known in the art that increases the number of HSCs and / or HSPCs circulating in the peripheral blood of the subject. Reference to either HSCs or HSPCs is intended to encompass both HSCs and HSPCs, unless otherwise indicated. For example, in certain embodiments, peripheral blood is mobilized by treating the subject with one or more cytokines or growth factors (e.g., G-CSF, Kit Ligand (KL), IL-1, IL-7, IL-8, IL-11, Flt3 Ligand, SCF, thrombopoietin, or GM-CSF (such as sargramostim)). Various types of G-CSF that can be used in methods to mobilize peripheral blood include filgrastim and the longer acting G-CSF: pegfilgrastim. In certain embodiments, peripheral blood is mobilized by treating the subject with one or more chemokines (e.g., macrophage inflammatory protein-1a (MIP1a / CCL3)), chemokine receptor ligands (e.g., chemokine receptor 2 ligands GROL3 and GR013M), chemokine receptor analogs (e.g., stromal cell-derived factor-1a (SDF-1a) protein analogs, e.g., CTCE-0021, CTCE-0214, or SDF-1a, e.g., Met-SDF-113), or chemokine receptor antagonists (e.g., chemokine (C-X-C motif) receptor 4 (CXCR4) antagonists, e.g., AMD3100).In certain embodiments, peripheral blood is mobilized by treating the subject with one or more anti-integrin signaling agents (e.g., function-blocking anti-very late antigen 4 (VLA-4) antibodies, or anti-vascular cell adhesion molecule 1 (VCAM-1)). In certain embodiments, peripheral blood is mobilized by treating the subject with one or more cytotoxic drugs, such as cyclophosphamide, etoposide, or paclitaxel. In certain embodiments, peripheral blood can be mobilized by administering to the subject one or more of the agents listed above for a period of time. For example, the subject can be treated with one or more agents (e.g., G-CSF) via once-daily or twice-daily infusions (e.g., subcutaneous, intravenous, or intraperitoneal) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to collection of HSPCs. In certain embodiments, HSPCs are collected within 1, 2, 3, 4, 5, 6, 7, 8, 12, 14, 16, 18, 20, or 24 hours after the last dose of the agent used to mobilize HSPCs into peripheral blood. In certain embodiments, HSCs and HSPCs are mobilized by treating the subject with two or more different types of agents as described above or known in the art, such as a growth factor (e.g., G-CSF) and a chemokine receptor antagonist (e.g., a CXCR4 receptor antagonist, e.g., AMD3100), or a growth factor (e.g., G-CSF or KL) and an anti-integrin agent (e.g., a function blocking VLA-4 antibody). In one embodiment, HSCs and / or progenitor cells are mobilized by treating the subject with G-CSF or an analog thereof. In one embodiment, the G-CSF is filgrastim. In one embodiment, HSCs and / or progenitor cells are mobilized by treating the subject with plerixafor. In certain embodiments, HSCs and / or progenitor cells are mobilized using a combination of filgrastim and plerixafor, filgrastim alone, or plerixafor alone, In certain embodiments, different types of mobilizing agents are administered simultaneously or sequentially.For additional information regarding methods of peripheral blood mobilization, see, e.g., Craddock et al., 1997, Blood 90(12):4779-4788; Jin et al., 2008, Journal of Translational Medicine 6:39; Pelus, 2008, Curr. Opin. Hematol. 15(4):285-292; Papayannopoulou et al., 1998, Blood 91(7):2231-2239; Tricot et al., 2008, Haematologica 93(11):1739-1742; and Weaver et al., 2001, Bone Marrow Transplantation 27(2):S23-S29).

[0101] In certain embodiments, peripheral blood is obtained through a syringe or catheter inserted into a vein of the subject. For example, peripheral blood can be collected using an apheresis machine. Blood flows from the vein through a catheter to the apheresis machine, which separates the white blood cells, including HSPCs, from the rest of the blood, which is then returned to the subject's body. Apheresis can be performed for several days (e.g., 1-5 days) until sufficient HSPCs are collected.

[0102] In certain embodiments, bone marrow is obtained from the subject's posterior iliac crest by needle aspiration (see, eg, Koda et al., 1984, J. Clin Invest. 73:1377-1384).

[0103] In certain embodiments, the hematocrit level of a biological sample may be determined. The hematocrit level may be determined by centrifuging the sample in a treatment chamber to separate the RBCs of the sample into layers, and thereby determining the volume of packed cells. It is understood that the sample may be combined with an anticoagulant, and such an anticoagulant may be added to the treatment chamber before or during centrifugation, to aid in determining the hematocrit level. Alternatively, the hematocrit level may be determined by measuring an optical property of the sample. For example, a spectrometer may be used to analyze the sample. It is understood that any type of known spectroscopic method for determining the hematocrit level may be used, such as, for example, Raman spectroscopy and / or light scattering techniques.

[0104] In certain embodiments, the biological sample is depleted of red blood cells, for example, prior to preparing one or more cell populations enriched for CD34+ cells from the biological sample. In some embodiments, the cells remaining after the depletion technique are washed. In another embodiment, non-specific IgG is added to the washed cells. In some embodiments, the non-specific IgG is phlebogamma.

[0105] The two cell populations enriched for CD34+ cells can be produced by selecting a cell population of CD34+ cells under high stringency conditions and selecting another cell population of CD34+ cells under low stringency conditions, thereby producing two cell populations, one being a high stringency CD34 enriched cell population and the other being a low stringency CD34 enriched cell population. The method used to select CD34+ cells can be positive selection, negative selection, or a combination thereof. In certain embodiments, the biological sample obtained from the subject is divided into two samples, one sample is used to prepare a high stringency CD34 enriched cell population and the other sample is used to prepare a low stringency CD34 enriched cell population.

[0106] In other embodiments, a biological sample obtained from a subject is first subjected to low stringency CD34+ selection to prepare a low stringency CD34 enriched cell population, and then a portion of the low stringency CD34 enriched population is subjected to high stringency CD34+ selection to prepare a high stringency CD34 enriched cell population. Selections can be applied sequentially, for example, selection of CD34 enriched cells under low stringency conditions can be applied first, followed by selection from the resulting population of additional CD34 enriched cells under high stringency conditions.

[0107] In other cases, the selection of CD34 enriched cells under high stringency conditions can be applied first, followed by the selection of the remaining population of CD34 enriched cells under low stringency conditions. In some cases, the cell population can be divided so that low stringency or high stringency selection is applied to the fraction of cells that have previously been subjected to high stringency or low stringency selection. In some cases, a biological sample is divided into two or more samples before selecting CD34 enriched cells under low stringency or high stringency conditions.

[0108] In some cases, two or more biological samples are mixed together before selection, including mobilized bone marrow samples obtained at different time periods, such as 1, 2, 3, 4 days or more apart, or 1, 2 or 3 weeks apart, or 1, 2 or 3 months apart, or years apart, including other time increments. In cases, biological samples from different subjects are mixed, such as an autologous biological sample and a sample from an allogeneic donor. In all cases, high stringency or low stringency selection before or after mixing or splitting biological samples or enriched cell populations is contemplated in all possible permutations. In certain embodiments, the method comprises the steps of: (a) detecting CD34 IgG under high stringency conditions; (b) detecting CD34 IgG under high stringency conditions; and (c) detecting CD34 IgG under high stringency conditions. + preparing a high stringency CD34 enriched cell population from a first biological sample obtained from the subject by selecting cells, and selecting CD34 cells under low stringency conditions. +The method includes preparing a low stringency CD34 enriched cell population from a second biological sample obtained from the subject by selecting the cells.

[0109] In some embodiments, the percentage of CD34+ cells in a high stringency CD34 enriched cell population is 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, or in some cases, 5-fold, 6-fold, 7-fold, or 8-fold greater than the percentage of CD34+ cells in a low stringency CD34 enriched cell population.

[0110] Various yields and purities of CD34+ cells can be achieved by the disclosed methods. In some cases, selection of CD34+ cells under high stringency conditions results in a high stringency CD34 enriched cell population having a yield of CD34+ cells of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more, or any value therebetween, compared to the input biological sample, and / or a purity of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, or any value therebetween, compared to the total number of cells in the enriched sample. In some cases, selection of CD34+ cells under high stringency conditions results in a high stringency CD34 enriched cell population having a yield of CD34+ cells of about 10%-20%, 20%-30%, or 30%-40%, or any value therebetween, compared to the input biological sample, and / or a purity of at least about 20%, 30%, 40%, 50%, or any value therebetween, compared to the total number of cells in the enriched sample. In certain embodiments, selection of CD34+ cells under high stringency conditions results in a high stringency CD34 enriched cell population having a yield of CD34+ cells of at least about 20% compared to the input biological sample, and / or a purity of at least about 20% or at least about 30% greater, compared to the total number of cells in the enriched sample.

[0111] In some cases, selection of CD34+ cells under low stringency conditions results in a low stringency CD34 enriched cell population having a yield of CD34+ cells of at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, or any value therebetween, relative to the input biological sample, and a purity of at least about 5%, 10%, 15%, 25%, 40%, 50% or more, or any value therebetween, relative to the total number of cells in the enriched sample. In some cases, selection of CD34+ cells under low stringency conditions results in a low stringency CD34 enriched cell population having a yield of CD34+ cells of more than 30%, about 30%-40%, 40%-50%, or 50%-60%, or any value therebetween, relative to the input biological sample, and a purity of at least about 3%, 5%, 8%, 10%, or any value therebetween, relative to the total number of cells in the enriched sample. In certain embodiments, selection of CD34+ cells under low stringency conditions results in a high stringency CD34 enriched cell population with a yield of more than 35% CD34+ cells compared to the input biological sample, and / or a purity of less than 30%, less than 25%, less than 20%, less than 10%, or less than 5% compared to the total number of cells in the enriched sample. In certain embodiments, selection of CD34+ cells under low stringency conditions results in a low stringency CD34 enriched cell population with a purity of 1-30%, 1-20%, 1-10%, 10-30%, 10-20%, or 20-30% compared to the total number of cells in the enriched sample. In some embodiments, the low stringency CD34 enriched cell population further comprises at least neutrophils and B cells.

[0112] In certain embodiments, both high stringency and low stringency selection are carried out by positive selection, for example, using an agent that binds to CD34+ cells.In certain embodiments, the agent is in solution, while in other embodiments, the agent is immobilized on a solid surface.In certain embodiments, the agent is bound to the surface of magnetic beads.In some embodiments, the agent is an anti-CD34 antibody or a functional fragment thereof.

[0113] In certain embodiments, selection under high stringency and / or low stringency conditions can be performed on a variety of instruments, including, but not limited to, Miltenyi Biotec MACSQuant Tyto®, Quad Technologies MagCloudz®, GE Sepax® cell separation system, Terumo Elutra® cell separation system, COBE Spectra® cell separator, SynGen LAB® or WASH® system, Fresenius-Kabi Lovo®, Miltenyi Biotec CliniMACS® system, or CliniMACS Prodigy® system. Selection can be performed in the laboratory or at the point of care. Detailed methods for the preparation and enrichment of cells and cell populations, including exemplary methods for selecting CD34+ cells under high stringency conditions, are described, for example, in International Patent Publication No. WO 2016 / 118790. Exemplary selection methods useful for high stringency selection are provided in U.S. Pat. No. 8,727,132.

[0114] In one embodiment, the instrument is a closed system device that includes at least one processing chamber with centrifugation and culture capabilities, a closed tubing set, a pump, and target cell selector material inputs (e.g., sample, buffer, gas). The control software allows the device to isolate, genetically modify, and formulate target cells ex vivo directly from a subject sample in certain embodiments. In certain embodiments, the selection process can be completed within 30 hours, 25 hours, or 20 hours with minimal or no user input. In certain embodiments, the entire process is completed within 72 hours or 64 hours. In certain embodiments, minimal user input means that the user interacts with the device no more than 20, 15, 10, or 5 times, and / or no more than 12, 10, 8, 5, 4, or 3 hours between sample input into the device and recovery of genetically modified cells formulated for administration to a subject. Exemplary interactions from the user can include one or more of: connecting a sterile tubing set, verifying maintenance of a closed sterile system, determining that a stage should be repeated (e.g., precipitation), verifying that a stage was completed successfully, allowing a new stage to be initiated following a process quality check, providing reagents for device input, and determining and / or calculating doses for addition or removal. Interactions can be timed by the time the user is adjusting the device or actually interacting with the device after sample receipt.

[0115] In certain embodiments, one or both of the high stringency CD34 enriched cell population and / or the low stringency CD34 enriched cell population are purified using CliniMACS®, a software-controlled instrument that processes HSC apheresis. plus The preparation is carried out using a CliniMACS® instrument such as the CliniMACS® instrument. plus(The instrument and associated equipment and reagents are commercially available from MiltenyiBiotec GmbH, Bergisch Gladbach, Germany.) The instruments or reagents used include CliniMACS® CD34 antigen / reagent, which is a solution containing an antibody conjugate consisting of a monoclonal antibody, mouse IgG, directed against a class II epitope of the human CD34 antigen, which is chemically bound to dextran beads with an iron oxide / hydroxide core, and CliniMACS® tubes are used for the selection or enrichment of CD34+ cells, which are 60×10 9 Of the total cell counts not exceeding .6x10 white blood cells (standard scale), 9 of CD34+ cells, or 120x10 9 (Large scale) White blood cells, with a total cell count not exceeding 1.2x10 9A single-use sterile disposable tubing set with two cell separation columns for processing CD34+ cells, and CliniMACS® PBS / EDTA buffer, which is a sterile isotonic phosphate buffer, 1 mM EDTA, saline, used as external washing and transport fluid for in vitro processing of HSCs, apheresis. Washing can be performed using CliniMACS buffer containing PBS and 2.5% human serum albumin. Instructions for using the CliniMACS® system are provided in the CliniMACS User Manual on the Miltenyi website, including, for example, the CliniMACS® User Manual for the CliniMACS® CD34 Reagent System, available at https: / / www.miltenyibiotec.com / _Resources / Persistent / 2c28c84939f4ce793b29c9f2e897c0bb1a334c47 / User%20Manual%20for%20the%20CliniMACS%20CD34%20Reagent%20System.pdf on April 8, 2019. In one embodiment, a high stringency CD34 enriched cell population is prepared by labeling cells using a CD34 reagent, and then CD34+ cells are selected using a standard CliniMACS® CD34 enrichment program. In one embodiment, the low stringency CD34 enriched cell population is prepared using a modified version of the CliniMACS® depletion program. According to this embodiment, cells are labeled using CD34 reagents, but a modified depletion program is performed. After the magnet is turned on and the labeled cells are loaded, the Cell Collection bag is removed and the collected cells are not used for transplantation. Instead, the magnet is turned off and an elution buffer is applied to the instrument to elute and collect the CD34+ cells. In a particular embodiment, the instrument is loaded more slowly (mL / min) and the wash is less stringent under the depletion mode compared to the selection mode.

[0116] In certain embodiments, one or both of the high stringency CD34 enriched cell population and / or the low stringency CD34 enriched cell population are transduced with a vector encoding a therapeutic agent, e.g., a gene therapy vector, for example, by contacting one or both of the high stringency CD34 enriched cell population and / or the low stringency CD34 enriched cell population with the vector. In certain embodiments, the cells are contacted with the vector for about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 12 hours, about 16 hours, about 18 hours, about 20 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours. In some embodiments, the cells are transduced for less than 60 hours, less than 48 hours, less than 36 hours, or less than 24 hours. In certain embodiments, the cells may be contacted with the vector one or more times, e.g., one, two, three, or more than three times, and the cells may be incubated with the agent(s) for some time after each contacting event, e.g., 16-24 hours, after which time the medium is replaced with fresh medium and the cells are further cultured. Contacting the cells with the vector may be performed in any culture medium and under any culture conditions that promote cell survival. The culture may contain growth factors to which the cells respond. Growth factors, as defined herein, are molecules capable of promoting cell survival, growth and / or differentiation, either in culture or in intact tissues, through specific effects on transmembrane receptors. Growth factors include polypeptide and non-polypeptide factors.

[0117] In certain embodiments, the cells are contacted with an effective amount of the gene therapy vector sufficient to achieve a detectable level of the encoded therapeutic agent. In certain embodiments, the therapeutic agent is a polypeptide or polynucleotide useful or effective in treating a disease or disorder. In certain embodiments, the therapeutic agent is a polypeptide that is deregulated or mutated in cells obtained from a subject, such as a FANC polypeptide for the treatment of FA. An effective amount can be easily determined empirically, such as by detecting the presence or level of the therapeutic agent, and detecting an effect on cell viability or function. Typically, expression of the therapeutic agent is enhanced 2-fold or more, such as 3-fold, 4-fold, or 5-fold or more, in some cases 10-fold, 20-fold, or 50-fold or more, such as 100-fold, in transduced cells compared to non-transduced cells.

[0118] Any convenient gene therapy vector used to deliver polynucleotide sequence to mammalian cells is included in the gene therapy vector of the present disclosure.For example, the vector can comprise single-stranded or double-stranded nucleic acid, for example, single-stranded or double-stranded DNA.For example, the gene therapy vector can be DNA, for example, naked DNA, for example, plasmid, minicircle, etc.The vector can comprise single-stranded or double-stranded RNA, including modified forms of RNA.In another example, the gene therapy vector can be RNA, for example, mRNA or modified mRNA.

[0119] In certain embodiments, the gene therapy vector can be a viral vector derived from a virus, such as adenovirus, adeno-associated virus, lentivirus (LV), herpes virus, alphavirus, or retrovirus, such as Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend murine leukemia virus, murine stem cell virus (MSCV), or Rous sarcoma virus (RSV). Although embodiments that include the use of LV are described in more detail below, it is expected that those skilled in the art will understand that similar knowledge and techniques in the art can be applied to non-LV gene therapy vectors as well. In some embodiments, the gene therapy vector is a self-limiting LV.

[0120] In particular, certain methods disclosed herein relate to transducing one or both of a population of stem or progenitor cells, e.g., hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (also referred to herein as "hematopoietic precursors"), with a gene therapy vector encoding and / or expressing a therapeutic polypeptide, e.g., FANCA, where one population is prepared by selection under high stringency conditions and the other population is prepared by selection under low stringency conditions, and in one embodiment, the cell population is enriched for CD34+ cells. In one embodiment, the HSCs or hematopoietic precursors are derived from a subject with reduced or no protein activity from one or more FANCA-encoded proteins. In one embodiment, the subject has FA-A. In one embodiment, the endogenous FANCA gene of the HSCs is deleted and / or mutated.

[0121] In one embodiment, transducing a cell with a gene therapy vector results in the integration into the cell genome of an expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding a therapeutic agent within the gene therapy vector, hi some embodiments, transducing a cell with a gene therapy vector results in the expression of a therapeutic agent, e.g., a biologically active FANCA protein.

[0122] For example, biologically active FANCA protein forms part of the FA core complex. In certain embodiments, the FANCA gene is delivered via a viral vector. In one embodiment, the FANCA gene is delivered via a lentiviral vector. In certain embodiments, the lentiviral vector is PGK-FANCA.WPRE*LV. It is contemplated that after transduction of bone marrow (BM) cells or stem or progenitor cells from FA-A patients with a FANCA lentiviral vector (LV), the therapeutic vector is integrated into the genome of the cells. Once integrated, the therapeutic protein (e.g., human FANCA protein) is expressed by the cells. The transduced FA cells are genetically corrected and thus can activate the FA pathway by monoubiquitination of FANCD2 and FANCI. These proteins can then migrate to areas of DNA damage and cooperate with other DNA repair proteins to facilitate the repair of DNA in these cells, as occurs in healthy cells.

[0123] As discussed herein, the subject method and composition finds use in expressing transgenes, such as FANCA, in animal cells.For example, the subject composition can be used in research, for example, to determine the effect of genes on cell viability and / or function.As another example, the subject composition can be used in medicine, for example, to treat disorders such as FA.

[0124] Thus, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising providing or administering to the subject a combination of a high stringency CD34 enriched cell population prepared as described herein, and a low stringency CD34 enriched cell population, wherein one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population is transduced with a recombinant gene therapy vector comprising a polynucleotide sequence encoding a therapeutic agent or comprising an expression cassette expressing a therapeutic agent, and wherein the therapeutic agent is effective to treat the disease or disorder. In certain embodiments, the disease is FA and the therapeutic agent is a Fanconi Anemia Complementation Group A (FANCA) polypeptide or a functional variant or fragment thereof.

[0125] In certain embodiments, the method comprises: (a) detecting CD34 under high stringency conditions; + (b) preparing a high stringency CD34 enriched cell population from a first biological sample obtained from the subject by selecting cells that are CD34 enriched under low stringency conditions; +The method includes preparing a low stringency CD34 enriched cell population from a second biological sample obtained from the subject by selecting cells; (c) transducing one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population with a recombinant gene therapy vector comprising a polynucleotide sequence encoding a therapeutic agent; and (d) providing or administering the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population to the subject, wherein one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population are transduced with the recombinant gene therapy vector, and the therapeutic agent is effective to treat a disease or disorder. In a specific embodiment, the disease is FA and the therapeutic agent is a Fanconi anemia complementation group A (FANCA) polypeptide or a functional variant or fragment thereof. In a specific embodiment, all of the cells obtained from each of the two cell populations are administered to the patient. The cell dose of each of the two cell populations can be similar or different, for example, more total cells and more total CD34+ cells are derived from the low stringency population. In certain embodiments, cells initially obtained from a patient are split into two approximately equal populations, one population treated under high stringency CD34+ selection conditions and one treated under low stringency CD34+ selection conditions, which can result in approximately 2x more CD34+ cells and 5-10x more total cells resulting from the low stringency selection process.

[0126] In certain embodiments of any of the treatment methods described herein, the first biological sample and the second biological sample are each independently peripheral blood or bone marrow.In certain embodiments, the first biological sample and the second biological sample are peripheral blood obtained after the subject is treated with G-CSF, plerixafor, or a combination of G-CSF and plerixafor.

[0127] In certain embodiments of the therapeutic methods described herein, CD34 + Selecting cells is done by using CD34+ The method includes applying a first biological sample to a column that binds the cells, washing the loaded column one or more times with a wash buffer, and eluting the high stringency CD34 enriched cell population from the column with an elution buffer. In one embodiment, the applying step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 20-25 mL / min, or 25-30 mL / min. In one embodiment, the elution step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 25-25 mL / min, or 25-30 mL / min. In one embodiment, the applying step is performed at 10-20 mL / min. In one embodiment, the elution step is performed at 20 mL / min.

[0128] In certain embodiments of the therapeutic methods described herein, CD34 + Selecting cells is done by using CD34 + The method includes loading a second biological sample onto a column that binds the cells, flowing the second biological sample through the column, and eluting the low stringency CD34 enriched cell population from the column using an elution buffer. In one embodiment, the application step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 20-25 mL / min, or 25-30 mL / min. In one embodiment, the elution step is performed at 5-10 mL / min, 5-15 mL / min, 15-20 mL / min, 25-25 mL / min, or 25-30 mL / min. In one embodiment, the application step is performed at 10-20 mL / min. In one embodiment, the elution step is performed at 20 mL / min.

[0129] In certain embodiments of the therapeutic methods described herein, a high stringency CD34 enriched cell population is transduced and / or a low stringency CD34 enriched cell population is transduced.

[0130] In some embodiments, the subject method provides a therapeutic benefit, such as preventing the onset of a disorder, halting the progression of a disorder, or reversing the progression of a disorder. For example, in one embodiment, the disorder is FA. In another embodiment, the disease or 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 subject method includes detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such measurements of therapeutic benefit are applicable to the particular disease being improved, and will recognize the appropriate detection methods to use to measure therapeutic efficacy.

[0131] As described in further detail in the Examples, clinical data on bone marrow cells mobilized into peripheral blood biosamples and administered according to the methods of the present invention unexpectedly demonstrated more rapid in vivo selective engraftment kinetics compared to other FA patients transplanted by existing technology methods. In certain embodiments, the methods and compositions of the present disclosure are used to treat FA.

[0132] Thus, the present invention provides a method for treating FA or one or more hematological manifestations of FA. In one embodiment, the hematological manifestations of FA are selected from one or more of bone marrow failure (BMF), thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia. In a particular embodiment, the hematological manifestation is BMF, which appears in the pediatric age of most FA 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.

[0133] It is also an object of the present invention to repair endogenous FANC genes (e.g., FANCA, FANCC, and / or FANCG) using, for example, a CRISPR / Cas gene editing system. Cas9-mediated repair of genes associated with Fanconi anemia has been demonstrated in vitro, for example, in Obsorn et al. Fanconi anemia gene editing by the CRISPR / Cas9 system. Hum Gene Ther. 2015 Feb;26(2):114-26. The present invention provides methods for treating Fanconi anemia that result in improved in vivo efficiency of repair and improved bone marrow reconstitution, as well as methods for preparing genetically modified cells. In some cases, the gene editing system repairs single nucleotide polymorphisms, deletions, insertions, indels, or another genetic defect. Repair is, in some cases, directed to the coding region of a gene, an intron, or an upstream or downstream region, for example, a gene regulatory region of the genome proximal or distal to a gene associated with a disease or disorder.

[0134] In one embodiment of the methods of the present disclosure, the present disclosure provides a method for treating Fanconi anemia in a subject in need thereof, comprising: + preparing a high stringency CD34 enriched cell population from a first biological sample obtained from a subject by selecting cells that are CD34 enriched under low stringency conditions; +The method includes preparing a low stringency CD34 enriched cell population from a second biological sample obtained from a subject by selecting cells, contacting one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population with a recombinant gene therapy vector for Fanconi anemia, and administering the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population to a subject, contacting one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population with a recombinant gene therapy vector, the gene therapy vector comprises a gene editing system capable of directed repair of endogenous genes, thereby treating Fanconi anemia.In some cases, the gene editing system is a CRISPR-Cas system (e.g., CRISPR-Cas9).

[0135] In some embodiments, the gene editing system comprises a nucleic acid encoding a Cas, Cas9, or spCas gene product (mRNA, cDNA, plasmid, etc.) operably linked to a promoter. In some embodiments, the gene editing system comprises a nucleic acid encoding a Cas, Cas9, or spCas protein. In some cases, the gene therapy vector is a liposome or lipid nanoparticle, or other lipid-based or lipid-like delivery system. In some cases, the gene therapy vector is a virus, such as a lentivirus, adenovirus, or adeno-associated virus. In some embodiments, the gene editing system comprises a guide RNA, e.g., a single guide RNA (sgRNA), and optionally a repair template. The repair template and the guide RNA are, in some cases, different molecules, or in some cases, the same molecule. The repair template and the guide RNA can be covalently or non-covalently linked. The guide RNA is, in some cases, preloaded with a Cas, Cas9, or spCas protein. The components of the gene editing system are, in some cases, delivered in a single gene therapy vector. In some cases, the gene editing system is delivered in a separate gene therapy vector. Multiple gene therapy vectors can be used. For example, but not limited to, multiple genetic lesions can be repaired with the same or different gene therapy vectors. In some cases, two or more FANC genes are repaired simultaneously. In some cases, one gene therapy vector is contacted with a high stringency CD34 enriched cell population, and another gene therapy vector is contacted with a low stringency CD34 enriched cell population. In some cases, the two gene therapy vectors are the same. In some cases, one gene therapy vector contains a transgene for a disorder associated with a given gene (e.g., FANCA), and the other gene therapy vector contains a gene editing system.

[0136] In some cases, gene therapy vector provides or repairs the transgene of a gene other than the gene associated with a disease or disorder.For example, but not limited to, gene therapy vector can up- or down-regulate immune effector genes, change cell surface markers, provide alternative MHC molecules, or code immunoglobulin genes.In some cases, gene therapy vector or vector is specifically intended to provide the use of allogeneic or mismatched donor transplantation by changing immune markers (e.g., HLA or MHC genes) or causing the expression of immune effector genes.

[0137] In some embodiments, a method of treating a disease or disorder in a subject in need thereof is provided, comprising providing a combination of a high stringency CD34 enriched cell population and a low stringency CD34 enriched cell population to the subject, both of which are prepared from one or more biological samples obtained from the subject, wherein the CD34 cells obtained from the subject contain one or more genetic mutations associated with or causing the disease or disorder, and one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population are subjected to gene editing to repair the one or more genetic mutations before providing the cell population to the subject. In certain embodiments, gene editing is performed by contacting one or both of the CD34 enriched cell populations with a Cas (e.g., Cas9 or spCas) protein, a guide RNA (e.g., a single guide RNA, sgRNA), and a repair template. In certain embodiments, the disease is FA, and the genetic mutation to be repaired is a mutation in the Fanconi anemia complementation group A (FANCA) gene.

[0138] In certain embodiments, the gene therapy vectors disclosed herein comprise a polynucleotide comprising a promoter operably linked to a sequence encoding a therapeutic agent, i.e., the coding sequence. As used herein, the term "operably linked" means that the promoter is capable of driving expression of the sequence encoding the therapeutic agent.

[0139] In some embodiments, polynucleotide comprises one or more enhancers.Enhancers are nucleic acid elements known in the art to enhance transcription, and can be located anywhere relative to the gene they regulate, for example, upstream, downstream, in intron, etc.Any enhancer element can be used in the polynucleotide cassette and gene therapy vector of the present disclosure, as long as it enhances the expression of gene when used in combination with promoter.

[0140] The coding sequence expressed in the cell can be any polynucleotide sequence, e.g., a gene or cDNA encoding a gene product, e.g., a polypeptide or an RNA-based therapeutic (e.g., siRNA, antisense, ribozyme, shRNA, etc.). The coding sequence can be heterologous to the promoter sequence to which it is operably linked, i.e., not naturally associated with it. Alternatively, the coding sequence can be endogenous to the promoter sequence to which it is operably linked, i.e., naturally associated with the promoter. The gene product can act endogenously in the mammalian cell or exogenously, e.g., it can be secreted. For example, when the transgene is a therapeutic gene, the coding sequence can be any gene encoding a desired gene product or a functional fragment or variant thereof that can be used as a therapeutic agent to treat a disease or disorder. In various embodiments, the transgene encodes human FANCA.

[0141] In one embodiment of the present invention, the transgene coding sequence is modified or "codon-optimized" to enhance expression by replacing low-occurrence codons with higher-occurrence codons. The coding sequence is the part of the mRNA sequence that codes for amino acids for translation. During translation, each of the 61 trinucleotide codons is translated into one of the 20 amino acids, resulting in degeneracy or redundancy in the genetic code. However, different cell types and different animal species utilize tRNAs (each with an anticodon) that code for the same amino acid at different frequencies. If a gene sequence contains a low-occurrence codon in the corresponding tRNA, the ribosomal translation machinery may slow down and prevent efficient translation. Expression can be improved by "codon optimization" for a particular species, where the coding sequence is modified to code for the same protein sequence but utilizes highly occurring codons and / or is utilized by highly expressed human proteins (Cid-Arregui et al., 2003; J. Virol. 77:4928). In one aspect of the present invention, the coding sequence of transgene is modified to replace the codons that are less frequently expressed in mammals or primates with the codons that are more frequently expressed in primates.For example, in some embodiments, the coding sequence encoded by transgene encodes a polypeptide that has at least 85% sequence identity, for example, at least 90% sequence identity, for example, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the polypeptide encoded by the sequence described above and disclosed herein, and at least one codon of the coding sequence has a higher tRNA frequency in humans than the corresponding codon of the sequence described above or disclosed herein.

[0142] In additional embodiments of the present invention, the transgene coding sequence is modified to enhance expression by terminating or removing open reading frames (ORFs) that do not code for the desired transgene. An open reading frame (ORF) is a nucleic acid sequence that follows a start codon and does not contain a stop codon. The ORF may be in forward or reverse orientation, and may 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 alongside the gene of interest, which may result in undesirable adverse effects. In one aspect of the present invention, the coding sequence of the transgene is modified to remove open reading frames by further altering the codon usage. This may be done by removing the start codon (ATG) and introducing a stop codon (TAG, TAA, or TGA) in the reverse or out-of-frame ORF, while conserving the amino acid sequence and maintaining the codons that are highly utilized in the gene of interest (i.e., avoiding codons with a frequency of less than 20%). In the present disclosure, the transgene coding sequence can be optimized using either or both of the techniques of codon optimization and removal of non-transgene ORFs.As will be apparent to those skilled in the art, it is preferable to remove or minimize non-transgene ORFs after codon optimization in order to remove the ORFs introduced during codon optimization.

[0143] The present disclosure includes plasmids comprising the expression or transfer cassettes described herein. In certain embodiments, the plasmid is pCCL-PGK-FANCA-WPRE* (SEQ ID NO:24) and / or the plasmid comprises the expression cassette from pCCL-PGK-FANCA-WPRE (SEQ ID NO:25).

[0144] In certain embodiments, the gene therapy vector or gene transfer cassette therein comprises one or more additional elements, such as one or more elements selected from 5'LTR, 3'LTR, cPPT, CTS, RRE, enhancer sequence, and packaging signal. In some embodiments, the gene therapy vector or gene transfer cassette is any vector or cassette disclosed in International Patent Publication No. WO 2018 / 049273 A1, the disclosure of which is incorporated herein in its entirety for all purposes. Polynucleotide sequences encoding Fanconi Anemia Complementation Group (FANC) polypeptides include those disclosed in U.S. Patent No. 5,952,190, the disclosure of which is incorporated herein in its entirety for all purposes.

[0145] The RRE sequence improves the efficiency of gene transfer. In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the RRE sequence comprises or consists of any of the following sequences, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequences (all displayed in the 5' to 3' direction): TIFF2025004171000002.tif98162 A sequence comprising or consisting of nucleotides 2649 to 2882 of SEQ ID NO: 24 or polynucleotides 1296 to 2153 of SEQ ID NO: 25.

[0146] The retroviral leader region contains the packaging signal (ψ) and is responsible for packaging the retroviral genome into the viral capsid. LV vectors were thought to require approximately 300 bp of the Gag gene in this region. Currently, this Gag sequence has been reduced to only 40 bp (Figure 65). In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the ψ sequence is the HIV-1 ψ sequence, or the ψ sequence comprises or consists of any of the following sequences, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequences: TIFF2025004171000003.tif25160 A sequence comprising or consisting of polynucleotides 2031 to 2156 of SEQ ID NO: 24 or polynucleotides 889 to 933 of SEQ ID NO: 25.

[0147] In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the truncated HIV-1 5'LTR comprises or consists of any of the following sequences, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of the following sequences: TIFF2025004171000004.tif44160 A sequence comprising or consisting of polynucleotides 1586 to 9495 of SEQ ID NO: 24 or polynucleotides 934 to 1295 of SEQ ID NO: 25.

[0148] In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the HIV-1 self-inactivating 3'LTR comprises or consists of any of the following sequences, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequences: TIFF2025004171000005.tif54160 A sequence comprising or consisting of polynucleotides 9262 to 9495 of SEQ ID NO: 24 or polynucleotides 8056 to 8289 of SEQ ID NO: 25.

[0149] In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the human cytomegalovirus (CMV) immediate early promoter comprises or consists of any of the following sequences, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of the following sequences: TIFF2025004171000006.tif70160 A sequence comprising or consisting of polynucleotides 1586 to 1789 of SEQ ID NO: 24 or polynucleotides 1 to 577 of SEQ ID NO: 25.

[0150] In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the Rous Sarcoma Virus (RSV) promoter comprises or consists of any of the following sequences, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of the following sequences: TIFF2025004171000007.tif25160

[0151] The cPPT, which promotes nuclear translocation of the preintegration complex, along with the CTS, which is involved in the detachment of reverse transcriptase, have been confirmed to improve viral titers (Zennou, et al. 2000; Follenzi et al. 2000). In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the central polypurine tract and central termination sequence of HIV-1 (cPPT / CTS) comprises or consists of any of the following sequences, functional fragments thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of the following sequences: TIFF2025004171000008.tif50160 A sequence comprising or consisting of nucleotides 3378 to 3495 of SEQ ID NO:24 or nucleotides 2176 to 2312 of SEQ ID NO:25.

[0152] In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the human phosphoglycerate kinase 1 (hPGK) promoter comprises or consists of any of the following sequences, functional fragments thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of the following sequences: TIFF2025004171000009.tif49159 A sequence comprising or consisting of nucleotides 3541 to 4051 of SEQ ID NO:24 or nucleotides 2335 to 2845 of SEQ ID NO:25.

[0153] In certain embodiments, the expression cassettes and gene therapy vectors disclosed herein further comprise one or more additional elements, e.g., a CMV promoter and / or enhancer, an SV 40 polyA sequence, an origin of replication, e.g., an SV40 ori sequence, or any of the elements disclosed herein.

[0154] In certain embodiments of the expression cassettes and gene therapy vectors described herein, the human CMV enhancer comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025004171000010.tif36160

[0155] In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the simian virus 40 (SV40) poly(A) signal comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025004171000011.tif13159 A sequence comprising or consisting of nucleotides 8361 to 8482 of SEQ ID NO: 25.

[0156] In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the SV40 origin of replication comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025004171000012.tif41160 A sequence comprising or consisting of nucleotides 8502 to 8657 of SEQ ID NO: 25.

[0157] In some embodiments of any of the expression cassettes and gene therapy vectors described herein, the dNEF signal present in any of the expression cassettes or gene therapy vectors described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025004171000013.tif12159

[0158] In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the KanR sequence present in any of the expression cassettes or gene therapy vectors described herein comprises or consists of the following sequence: TIFF2025004171000014.tif66160

[0159] In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the RNA-OUT sequence present in any of the expression cassettes or gene therapy vectors described herein comprises or consists of the following sequence: TIFF2025004171000015.tif19162 A sequence comprising or consisting of nucleotides 9731 to 9871 of SEQ ID NO:25.

[0160] In some embodiments of any of the expression cassettes and gene therapy vectors described herein, the rrnG terminator (the transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)) present in any of the expression cassettes or gene therapy vectors described herein comprises or consists of the following sequence: TIFF2025004171000016.tif12162

[0161] In some embodiments of any of the expression cassettes and gene therapy vectors described herein, the ori (high copy number ColE1 / pMB1 / pBR322 / pUC origin of replication) present in any of the expression cassettes or gene therapy vectors described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025004171000017.tif140160 A sequence comprising or consisting of nucleotides 9731 to 9871 of SEQ ID NO: 24 or nucleotides 8700 to 9714 of SEQ ID NO: 25.

[0162] In some embodiments of any of the expression cassettes and gene therapy vectors described herein, the CAP binding site present in any of the expression cassettes or gene therapy vectors described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025004171000018.tif6128

[0163] In some embodiments of any of the expression cassettes and gene therapy vectors described herein, the E. coli lac promoter present in any of the expression cassettes or gene therapy vectors described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025004171000019.tif6128

[0164] In some embodiments of any of the expression cassettes and gene therapy vectors described herein, the lac operator present in any of the expression cassettes or gene therapy vectors described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025004171000020.tif4128

[0165] In some embodiments of any of the expression cassettes and gene therapy vectors described herein, the T3 promoter (promoter of bacteriophage T3 RNA polymerase) present in any of the expression cassettes or gene therapy vectors described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025004171000021.tif6128

[0166] In some embodiments of any of the expression cassettes and gene therapy vectors described herein, the T7 promoter (promoter of bacteriophage T7 RNA polymerase) present in any of the expression cassettes or gene therapy vectors described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025004171000022.tif6128

[0167] In some embodiments of any of the expression cassettes and gene therapy vectors described herein, the f1 ori (f1 bacteriophage origin of replication) present in any of the expression cassettes or gene therapy vectors described herein comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025004171000023.tif36160

[0168] As discussed herein, the expression cassettes and gene therapy vectors of the present invention may include an RNA nuclear export signal. Exemplary RNA nuclear export sequences include, but are not limited to, WPRE. WPRE significantly increases transgene expression in target cells by increasing the stability of RNA in a transgene, promoter, and vector-independent manner (Zuffrey et al., 1999). However, it can express a truncated 60-amino acid protein derived from the WHV X gene, which is involved in liver cancer (Kingsman et al., 2005). Thus, most preclinical protocols and clinical trials include a mutated version of the WPRE element (Zanta-Boussif et al., 2009). Meanwhile, the use of two SV40-USE elements in SIN-LV vectors has been found to be more efficient than the WPRE sequence in suppressing transcriptional read-through (Schambach et al., 2007). More precisely, the WPRE disclosed herein is a chimeric wPRE carrying 589 nucleotides from the modified WPRE (WO 2008 / 136670 A2) performed by Axel Schambach (nucleotides 1-589) and 88 nucleotides from the previous WPRE (nucleotides 590-677) (Zuffrey et al, 1999). The data disclosed herein show that this chimeric WPRE functions better than the previous wPRE. The chimeric wPRE sequence includes the following sequences, functional fragments thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequences: TIFF2025004171000024.tif60151

[0169] In certain embodiments, the mutant WPRE sequence comprises or consists of WPRE* and corresponds to nucleotides 8502 to 9178 of SEQ ID NO:24 or nucleotides 7293 to 7888 of SEQ ID NO:25, 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 or SEQ ID NO:25.

[0170] Other combinations of both elements disclosed herein or known in the art will be readily apparent to those skilled in the art.

[0171] In addition, as will be recognized by those of skill in the art, expression cassettes and gene therapy vectors may optionally contain other elements, including, but not limited to, restriction sites to facilitate the cloning and regulatory elements of the particular gene therapy vector.

[0172] In some aspects of the invention, the subject polynucleotide cassettes are used to deliver genes to cells, e.g., to determine the effect of the gene on cell viability and / or function, to treat cellular disorders, etc. In various embodiments, delivery of the viral vector to cells by transduction can occur in vitro, ex vivo, or in vitro. Thus, in some aspects of the invention, the composition that provides expression of a transgene in a mammalian cell is a gene therapy vector, and the gene therapy vector comprises a polynucleotide cassette of the present disclosure, e.g., a gene transfer cassette.

[0173] Genetic correction of HSCs from FA patients followed by autologous transplantation of these cells (hematopoietic gene therapy) is a good alternative for FA patients, especially those who lack HLA-homogenous siblings. In one embodiment, hematopoietic gene therapy is the preferred treatment regimen for patients who lack HLA-homogenous siblings. In another embodiment, hematopoietic gene therapy is the treatment regimen for patients who have HLA-homogenous siblings.

[0174] In certain embodiments of any of the methods disclosed herein, a gene therapy vector comprises a polynucleotide sequence encoding a FAC protein, and cells transfected with the vector are provided to a subject to treat FA.

[0175] Because most FA patients belong to the FA-A complementation group (Casado et al., 2007, Levitus et al., 2004, Taniguchi et al., 2006), vectors carrying a functional FANCA gene have been developed that contain a mutated posttranscriptional regulatory element of the Woodchuck Hepatitis Virus (WPRE*) and lack any residual open reading frame (Schambach A et al. Gene Ther. 2006;13:641-645) to improve expression levels and stability of the therapeutic gene.

[0176] In some embodiments, the polynucleotide cassette comprises: (i) a phosphoglycate 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) Posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE) sequence Includes.

[0177] In some embodiments, the polynucleotide cassette comprises: (i) human phosphoglycerate kinase (PGK) promoter sequence, (ii) a sequence encoding the human FANCA protein, and (iii) Mutant WPRE sequence Includes.

[0178] In some embodiments, the polynucleotide cassette comprises: a) 5'LTR, optionally modified 5'LTR; b) cPPT sequence, c) a PGK promoter sequence, optionally a human PGK promoter sequence, and d) a sequence encoding the human FANCA protein, optionally a cDNA sequence or a codon-optimized sequence; e) a mutant WPRE sequence; f) 3'LTR, optionally modified 3'LTR Includes.

[0179] In one embodiment, the modified WPRE is referred to as WPRE*, which is a modified WPRE that lacks an open reading frame (see, e.g., Schambach et al, 2006 Gene Ther. 13:641-645).

[0180] Since most FA patients belong to the FA-A complementation group (Casado et al., 2007, Levitus et al., 2004, Taniguchi et al., 2006), in certain embodiments the encoded therapeutic gene product is FANCA, although the present disclosure contemplates that FA proteins of other complementation groups may also be delivered and thus encoded in the expression cassettes disclosed herein, for example, in place of FANCA.

[0181] In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the polynucleotide sequence encoding the codon-optimized FANCA is a human FANCA cDNA sequence that comprises or consists of the following sequence, or a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF2025004171000025.tif80160TIFF2025004171000026.tif236160TIFF2025004171000027.tif244160TIFF2025004171000028.tif144160

[0182] The present disclosure includes a plasmid comprising the expression cassette or transfer cassette described herein. In a specific embodiment, the plasmid is pCCL-PGK-FANCA-WPRE* or pCCL-PGK-FANCAW-82-RO (SEQ ID NO:25).

[0183] In some embodiments, the gene therapy vector is a self-limiting LV. In certain embodiments of any of the expression cassettes and gene therapy vectors described herein, the transfer cassette is the pCCL-SIN-cPPT / CTS-hPGK-hFANCA-WPRE of the present disclosure, which comprises or consists of the following sequence, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:24. SEQ ID NO:24 corresponds to the pCCL-PGK-FANCA-WPRE* plasmid. SEQ ID NO:25 corresponds to the pCCL-PGK-FANCAW-82-RO plasmid.

[0184] In one embodiment, the FANCA gene is delivered via lentiviral vector (LV). The FANCA LV described herein utilizes a self-inactivating lentiviral vector (LV). In one embodiment, the FANCA LV contains the promoter of the human phosphoglycerate (PGK) gene. The safety of this vector is significantly improved compared to the gamma retroviral vector already used in the clinic, which contains a strong viral promoter.

[0185] In a particular embodiment, the lentiviral vector is PGK-FANCA.WPRE*LV and comprises a gene transfer cassette comprising the sequence disclosed in SEQ ID NO:24. The PGK-FANCA-WPRE*LV gene expression cassette portion comprises the human PGK promoter, the coding sequence of FANCA cDNA, and WPRE* and corresponds to nucleotides 3541-9178 of SEQ ID NO:24. The PGK-FANCA-WPRE*LV transfer cassette portion comprises the sequence from about the 5'LTR (U5) to about the 3'LTR (U5). With respect to SEQ ID NO:24, nucleotides 1586-1789 of SEQ ID NO:24 comprise the human CMV immediate early promoter. Nucleotides 2031-2156 of SEQ ID NO:24 comprise the HIV1 psi packaging signal. Nucleotides 2649-2882 of SEQ ID NO:24 comprise the HIV1 RRE element. Nucleotides 3378-3495 of SEQ ID NO:24 comprise the HIV cPPT / CTS element. Nucleotides 3541-4051 of SEQ ID NO:24 contain the hPGK promoter. Nucleotides 4078-8445 of SEQ ID NO:24 contain the human FANCA-A cDNA. Nucleotides 8502-9178 of SEQ ID NO:24 contain a mutated WPRE element. Nucleotides 9262-9495 of SEQ ID NO:24 contain the HIV delta U3'LTR.

[0186] In a particular embodiment, the lentiviral vector is pCCL-PGK-FANCAW-82-RO and comprises a gene transfer cassette comprising the sequence disclosed in SEQ ID NO:25. The pCCL-PGK-FANCAW-82-RO gene expression cassette portion comprises the human PGK promoter, the coding sequence of FANCA cDNA, and WPRE*, and corresponds to nucleotides 2335-7888 of SEQ ID NO:25. The pCCL-PGK-FANCAW-82-RO transfer cassette portion comprises the sequence from about the 5'LTR (U5) to about the 3'LTR (U5). With respect to SEQ ID NO:25, nucleotides 1-577 of SEQ ID NO:25 comprise the human CMV immediate early promoter. Nucleotides 889-933 of SEQ ID NO:25 comprise the HIV1 psi packaging signal. Nucleotides 1296-253 of SEQ ID NO:25 comprise the HIV1 RRE element. Nucleotides 2176-2191 of SEQ ID NO:25 contain the HIV cPPT / CTS element. Nucleotides 2335-2845 of SEQ ID NO:25 contain the hPGK promoter. Nucleotides 2872-7242 of SEQ ID NO:25 contain the human FANCA-A cDNA. Nucleotides 7293-7888 of SEQ ID NO:25 contain a mutated WPRE element. Nucleotides 8056-8289 of SEQ ID NO:25 contain the HIV delta U3'LTR.

[0187] In yet another embodiment, the lentiviral vector comprises the following elements: (i) a lentiviral vector backbone derived from the earlier pCCLsin-cppt-hPGK-eGFP-WPRE (Dull et al, 1998; J. Virol 72 (11), 9873-9880). The pCCL backbone utilizes a heterologous CMV-HIV 5'LTR to obtain high levels of viral RNA transcription in producer cells. Such a heterologous LTR renders the construct independent of the need to use the HIV Tat protein for the generation of rHIV particles, which is characterized by its safety. The U3 region of the 3'LTR contains a 400 bp deletion as described in (Zufferey et al J Virol, 1998), which confers self-inactivating properties to the vector; (ii) a cDNA of the human condon-optimized FANCA gene (4368 bp GenBank Accession No: X_99226 or as disclosed herein) encoding the FANCA protein (1455AA) under the control of the human PGK promoter. The promoter is characterized by its stable activity in vivo and improved safety profile compared to other promoters already used in gene therapy, and (iii) a mutated version of the Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE) is deleted in the 3' region of the sequence coding for the X protein and any remaining ORF or WPRE* described by Schambach et al. (Gene therapy, 2006; 13, 641-645).

[0188] In certain embodiments, the FANCA LV described herein utilizes a self-inactivating lentiviral vector (LV). In one embodiment, the FANCA LV contains the promoter of the human phosphoglycerate (PGK) gene. The safety of this vector is significantly improved compared to the gamma retroviral vector already used in the clinic, which contains a strong viral promoter. In one embodiment, the FANCA gene is delivered via a lentiviral vector. In a specific embodiment, the lentiviral vector is PGK-FANCA.WPRE*LV.

[0189] Gene therapy vectors encapsulating the polynucleotide cassettes of the present disclosure may be produced using standard methods. For example, in the case of LV virions, an LV expression vector according to the present invention may be introduced into producer cells, followed by an LV helper construct, which contains an LV coding region capable of being expressed in the producer cells and complements LV helper functions in the absence of the LV vector. Following this, a helper virus and / or additional vectors are introduced into the producer cells, which provide accessory functions capable of supporting efficient LV virus production. The producer cells are then cultured to produce LV. These steps are performed using standard methods. In certain embodiments, the plasmids shown in Figures 38-41 are used to produce gene therapy vectors.

[0190] Any suitable method for producing viral vector particles for delivery of a polynucleotide cassette of interest can be used, including but not limited to those described in the Examples below. Any concentration of infectious viral vector particles suitable for efficiently transducing mammalian cells can be prepared for contact with mammalian cells in vitro or in vivo. Viral particles can be at least 10 per ml. 8 Infectious units or more, e.g., 5x10 per mL 8 Infectious units, 10 per mL 9 Infectious units, 5x10 per mL 9 Infectious units, 10 per mL 10 Infectious units, 5x10 per mL 10 Infectious units, 10 per mL 11 Infectious units, 5x10 per mL 11 Infectious units, 10 per mL 12 Infectious units, 5x10 per mL 12 Infectious units, 10 per mL 13 Infectious units, 1.5x10 per mL 13 Infectious units, 3x10 per mL 13Infectious units, 5x10 per mL 13 Infectious units, 7.5x10 per mL 13 Infectious units, 9x10 per mL 13 Infectious units, 1x10 per mL 14 Infectious units, 5x10 per mL 14 Infectious units or more, but typically 1x10 per mL 15 It may be formulated at a concentration of less than the infectious unit.

[0191] In preparing the subject LV gene therapy vector, any host cell for producing LV virions can be used, including, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell can also be a packaging cell in which the LV rep and cap genes are stably maintained in the host cell or producer cell 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. The LV vector is purified and formulated using standard techniques known in the art.

[0192] In certain embodiments, the invention includes cells comprising a gene expression cassette, gene transfer cassette, or gene therapy vector disclosed herein. In related embodiments, the cells are transduced with a gene therapy vector comprising an expression cassette disclosed herein, or have an expression cassette disclosed herein integrated into the genome of the cell. In certain embodiments, the cells are cells used to produce viral gene therapy vectors, e.g., packaging cells.

[0193] In other embodiments, the cells are cells that are delivered to a subject to provide the subject with a gene product encoded by the expression cassette. Thus, in certain embodiments, the cells are autologous to the subject to be treated or are obtained from the subject to be treated. In other embodiments, the cells are allogeneic to the subject to be treated or are obtained from a donor other than the subject to be treated. In certain embodiments, the cells are mammalian cells, e.g., human cells. In certain embodiments, the cells are blood cells, red blood cells, hematopoietic progenitor cells, bone marrow cells, e.g., lineage-depleted bone marrow cells, hematopoietic stem cells (e.g., CD34+), or committed hematopoietic erythroid progenitor cells. In certain embodiments, the cells are CD34+ cells obtained from a subject to be treated with the cells after the cells are transduced with a gene therapy vector disclosed herein. In certain embodiments, the cells are CD34+ FA cells obtained from a subject diagnosed with FA.

[0194] In some embodiments, the methods disclosed herein provide a therapeutic benefit, such as preventing the onset of a disorder, halting the progression of a disorder, or reversing the progression of a disorder. 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 subject method includes detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such measurements of therapeutic benefit are applicable to the particular disease to be improved, and will recognize the appropriate detection method to use to measure therapeutic efficacy.

[0195] In another embodiment, the present invention includes a method of treating a disease in a subject in need thereof, comprising providing an effective amount of either or both of a high stringency CD34 enriched cell population and a low stringency CD34 enriched cell population to the subject, and contacting either or both with a gene therapy vector, e.g., a viral vector, that expresses a therapeutic gene product in the cells. In certain embodiments, either or both of the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population are autologous to the subject. In certain embodiments, the cells are erythroid cells, e.g., hematopoietic stem cells or committed hematopoietic erythroid progenitor cells. In some embodiments, the cells are bone marrow cells, e.g., lineage-depleted bone marrow cells. In certain embodiments, the method is used to treat FA, and the viral vector is an expression construct disclosed herein that includes a human PGK promoter operably linked to a FANCA gene cDNA or coding sequence, and an LV that includes a mutant wPRE disclosed herein. In certain embodiments, the cells are provided to the subject parenterally, e.g., via intravenous injection.

[0196] In another embodiment, the present invention includes a method of treating FA in a subject in need thereof, comprising providing to the subject an effective amount of an autologous high stringency CD34 enriched and / or low stringency CD34 enriched cell population of stem cells transduced with an LV vector expressing FANCA cDNA in the cells, the LV vector comprising a human PGK promoter operably linked to the FANCA cDNA or coding sequence, and a mutant wPRE sequence as disclosed herein. In certain embodiments, the cells are hematopoietic stem cells or committed hematopoietic erythroid progenitor cells, e.g., bone marrow cells. In certain embodiments, the cells are provided to the subject parenterally, e.g., via intravenous injection.

[0197] The expression of the transgene using the subject transgene is expected to be robust. Thus, in some cases, the expression of the transgene, as detected, for example, by measuring the level of gene product, measuring therapeutic efficacy, etc., can be observed 2 months or less after administration of the subject composition, for example, 4, 3, or 2 weeks or less after administration, for example, 1 week after administration. The expression of the transgene is also expected to persist over time. Thus, in some cases, the expression of the transgene, as detected, for example, by measuring the level of gene product, measuring therapeutic efficacy, etc., can be observed 2 months or more after administration of the subject composition, for example, 4, 6, 8, or 10 months or more, in some cases, 1 year or more, for example, 2, 3, 4, or 5 years, and in certain cases, more than 5 years.

[0198] In certain embodiments, the method includes detecting expression of the transgene in a cell or subject, and expression is enhanced compared to expression from a polynucleotide cassette that does not include one or more of the improved elements of the present disclosure. Typically, expression will be enhanced by 2-fold or more, e.g., 3-fold, 4-fold, or 5-fold or more, and in some cases 10-fold, 20-fold, or 50-fold or more, e.g., 100-fold, compared to expression from a reference, i.e., control, polynucleotide cassette, as evidenced, for example, by earlier detection, higher levels of gene product, stronger functional effect on the cell, etc.

[0199] In some embodiments, the dose of cells a patient receives by infusion will be that resulting from the transduction process. In various preferred embodiments, the dose of cells received by a patient will be at least about 1×10 of the patient's body weight. 1 , 1x10 2 , 1x10 3 , 1x10 4 , 1x10 5 , 1x10 6 , 1x10 7 , 1x10 8 or more high stringency CD34 enriched cells / kg are infused into the patient. In various preferred embodiments, at least about 1x10 1, 1x10 2 , 1x10 3 , 1x10 4 , 1x10 5 , 1x10 6 , 1x10 7 , 1x10 8 In some embodiments, 1×10 low stringency CD34 enriched cells / kg or more are infused into the patient. 6 ~4×10 6 In another embodiment, 3×10 high stringency CD34 enriched cells / Kg of patient body weight are infused into the patient. 5 ~4×10 6 In some embodiments, 1×10 high stringency CD34 enriched cells / Kg of patient body weight are infused into the patient. 6 ~4×10 6 In another embodiment, 3×10 high stringency CD34 enriched cells / Kg of patient body weight are infused into the patient. 5 ~4×10 6 of high stringency CD34 enriched cells / Kg are infused into the patient. In some embodiments, the cells are infused into the patient in a single dose. In other embodiments, the cells are infused into the patient in multiple doses (e.g., high stringency and low stringency CD34 enriched cell populations are administered sequentially in one or more doses). The transduced cells can be infused immediately after the transduction process is completed. In certain embodiments, the transduced cells are stored or frozen prior to use, although in certain embodiments, they are provided to the subject immediately or shortly after transduction, e.g., within 1 hour, 2 hours, 4 hours, or 8 hours.

[0200] Once integrated, the therapeutic protein (e.g., human FANCA protein) is expressed by the cells. The transduced FA cells are genetically corrected and thus able to activate the FA pathway by monoubiquitination of FANCD2 and FANCI. These proteins migrate to areas of DNA damage and cooperate with other DNA repair proteins to facilitate the repair of DNA in these cells, as occurs in healthy cells.

[0201] As described in more detail in the Examples, preclinical in vitro data using BM samples from human FA patients has already demonstrated the efficacy of FANCA LV to modify the phenotype of these cells.

[0202] In one embodiment, at least 1-4 x 10 per kg of patient body weight 6 CD34 + Correction cells (e.g., FANCA transduced HSCs) are administered, e.g., to restore hematopoiesis in unconditioned FA patients. In some embodiments, the transduced cells are infused or administered to the patient immediately after transduction. In other embodiments, the transduced cells are frozen prior to infusion or administration to the patient.

[0203] Genetic correction of HSCs from FA patients followed by autologous transplantation of these cells (hematopoietic gene therapy) is a good alternative for FA patients, especially those who lack HLA-homogenous siblings. In one embodiment, hematopoietic gene therapy is the preferred treatment regimen for patients who lack HLA-homogenous siblings. In another embodiment, hematopoietic gene therapy is the preferred treatment regimen for patients who have HLA-homogenous siblings.

[0204] Compositions and Formulations Certain aspects of the present disclosure relate to systems or combinations of high stringency CD34 enriched cell populations and low stringency CD34 enriched cell populations, either or both of which are transduced with gene therapy vectors. Some embodiments include combinations of high stringency CD34 enriched and low stringency CD34 enriched cell populations, either or both of which are transduced with lentiviral vectors containing human FANC gene, e.g., FANCA, FANCC, or FANCG, or nucleic acid sequences encoding FANC protein, including functional variants and fragments thereof. Some embodiments include combinations of high stringency CD34 enriched and low stringency CD34 enriched cell populations, either or both of which have undergone gene editing or gene repair, such as CRISPR, TALEN, zinc finger, or meganuclease gene editing systems. Some embodiments include combinations of high stringency CD34 enriched and low stringency CD34 enriched cell populations, which are transduced with lentiviral (or other viral) vectors containing transgenes related to diseases or conditions, such as immune deficiency disorders.

[0205] In some aspects of the present disclosure, a formulation for the treatment of disease or condition is provided.The formulation may comprise high stringency CD34 enriched cell population(s), or low stringency CD34 enriched cell population(s), or both, together with physiologically acceptable carrier or pharmaceutically acceptable carrier as described herein.The formulation may comprise high stringency CD34 enriched cell population(s), or low stringency CD34 enriched cell population(s), or both, where one or both cell populations are transduced with gene therapy vector and express or can express therapeutic agent together with physiologically acceptable carrier or pharmaceutically acceptable carrier as described herein.

[0206] The present invention includes pharmaceutical compositions and formulations comprising either or both of the high stringency CD34 enriched cell population and low stringency CD34 enriched cell population gene therapy vectors described herein, and pharma- ceutical acceptable carriers, diluents, or excipients.The subject high stringency CD34 enriched cell population and / or low stringency CD34 enriched cell population can be combined with pharma-ceutical acceptable carriers, diluents, and reagents useful in the preparation of generally safe, non-toxic, and desirable formulations, including excipients that are acceptable for use in primates.Examples of such excipients, carriers, or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.Additionally, supplementary active compounds can be incorporated into the formulation. The solutions or suspensions used in the formulations can include sterile diluents such as water for injection, saline solution, dimethylsulfoxide (DMSO), fixed oils, polyethylene glycol, 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 acetate, citrate, or phosphate, detergents such as Tween 20 to prevent aggregation, and compounds for adjusting osmotic pressure such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. In certain embodiments, the formulations are sterile.

[0207] In some embodiments, the CD34 enriched cell population is manufactured according to current good manufacturing practice. Manufactured according to current good manufacturing practice means that the formulation prepared for administration is sufficiently safe to allow administration to human subjects under governing regulations and governmental authorization. Generally, governing regulations and authorizations stipulate that the formulation meets pre-approved acceptance criteria for identity, strength, quality, and purity. Acceptance criteria include numerical limits, ranges, or other appropriate measures of test results used to determine whether the formulation meets current good manufacturing practice. Specifications describe analytical procedures used to test compliance with the acceptance criteria. The formulation can be evaluated in batches. A batch is a specific amount of formulation that is tested to ensure compliance with the acceptance criteria.

[0208] The formulations may be included in a container, pack, or dispenser, eg, a syringe, eg, a prefilled syringe, together with instructions for administration.

[0209] Where necessary or beneficial, the formulation can include a local anesthetic such as lidocaine to ease pain at the site of the injection.

[0210] The therapeutically effective amount of cells in the formulation is 10 2 More than 10 cells 3 More than 10 cells 4 More than 10 cells 5 More than 10 cells 6 More than 10 cells 7 More than 10 cells 8 More than 10 cells 9 More than 10 cells 10 More than 10 cells 11 It can exceed.

[0211] In the formulations disclosed herein, the cells are generally in a volume of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Thus, the density of cells administered is typically less than 10 4 cells / ml, 107 cells / ml, or 10 8 More than cells / ml.

[0212] The formulations disclosed herein can be prepared for administration, for example, by injection, infusion, perfusion, or lavage. The therapeutically effective amount administered is within the range of 10 2 More than 10 cells 3 More than 10 cells 4 More than 10 cells 5 More than 10 cells 6 More than 10 cells 7 More than 10 cells 8 More than 10 cells 9 More than 10 cells 10 More than 10 cells 11 In certain embodiments, the minimum dose is greater than 2×10 6 Cells / kg subject weight.

[0213] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of either or both of the high stringency CD34 enriched cell populations and low stringency CD34 enriched cell populations disclosed herein in admixture with pharma- ceutically acceptable carriers and / or excipients, such as saline, phosphate buffered saline, phosphate, and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, sugars, etc. are octylphenoxypolyethoxyethanol compounds, 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, citric acid, acetic acid, Ringer's solution and Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol.Preferably, the formulation is stable at 4°C for at least 6 months.

[0214] In some embodiments, the pharmaceutical compositions provided herein contain 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 in Good et al. (1966) Biochemistry 5: 467. The pH of the buffer in which the pharmaceutical composition containing the tumor suppressor gene contained in the adenoviral vector delivery system is contained may range from 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0215] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication in the event of a conflict.

[0216] It is further noted that the claims may be drafted to exclude any optional element, and as such, this statement is intended to serve as a predicate to the use of exclusive terminology such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0217] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0218] 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, although other FANCA genes can be utilized to treat other complementation groups as well.

[0219] The present disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims. EXAMPLES

[0220] Example 1 Treatment of patients with high and low stringency CD34 enriched cell populations Patient 1 presented with Fanconi anemia. Patient 1 was then mobilized with G-CSF and Plerixan, followed by two apheresis collections on consecutive days. Compared to other FA patients in the same clinical trial, patient 1 had modest mobilization of CD34 cells as shown by peripheral blood analysis of circulating CD34 cells, with mobilization kinetics similar to other patients. After the two apheresis collections, the collected HSPCs were divided into two peripheral blood biosamples.

[0221] High stringency CD34 selection: The first biological sample was enriched by selecting for CD34+ cells under high stringency conditions using a Miltenyi Biotec CliniMACS® system in enrichment mode with Miltenyi Biotec CD34 reagent for enrichment as described in U.S. Pat. No. 8,727,132. High stringency CD34 selection is performed by selecting for CD34+ cells under high stringency conditions using a Miltenyi Biotec CliniMACS® system in enrichment mode with Miltenyi Biotec CD34 reagent for enrichment as described in U.S. Pat. No. 8,727,132. + This resulted in a cell yield of 29.0% and a relative purity of 36%.

[0222] Low stringency CD34 selection: The second biological sample was enriched by selecting for CD34+ cells under low stringency conditions using a modification of the depletion mode of the Miltenyi Biotec CliniMACS® system. Briefly, the second biological sample was labeled using Miltenyi Biotec CD34 reagent. The sample was then loaded onto the column using the depletion mode program of the instrument. After loading the sample in volume with the magnet "ON", the cell collection bag (used in the normal operation of the instrument to collect target cells) was removed and not used for stem cell transplantation. The "non-target cell bag" was attached, then the magnet was turned "OFF", then elution buffer was applied to the instrument, and CD34 + The cells were eluted into a non-target cell bag. The cell population collected in the non-target cell bag was saved and designated as the low stringency CD34 enriched cell population. Low stringency CD34 selection resulted in CD34 enrichment with a relative purity of 5.8%. + The low purity was due to the CD34 + The results are summarized in Tables 1A and 1B.

[0223] (Table 1A) Before CD34 enrichment TIFF2025004171000029.tif22128 (Table 1B) After CD34 enrichment TIFF2025004171000030.tif26148 Starting cell numbers and CD34+ cell purity from Fanconi anemia-A patients, and resulting cell populations Aph: Apheresis TNC: Total nucleated cell count Low: Modified CliniMACS depletion program High: Standard CliniMACS CD34 Enrichment Program

[0224] Both the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population were then each individually transduced with a recombinant gene therapy vector encoding the FANCA gene product (PGK-FANCA-WPRE*) (SEQ ID NO:25), and the resulting genetically modified cells were designated Product 1.1 and Product 1.2, respectively.

[0225] Product 1.1 and Product 1.2 were mixed together and then administered to Patient 1 by continuous intravenous infusion over a period of 10-30 minutes while monitoring clinical endpoints.

[0226] Patient 1 shows the fastest or nearly fastest in vivo selected engraftment kinetics among all FA patients transplanted at the participating institutions. The early trend toward stabilization of hematopoietic lineages, which were depressed in this FA patient before transplant, is a compelling endpoint for the clinical success unexpectedly achieved by this treatment. Five other FA patients have been transplanted with a standard CD34 selection approach, and none have shown engraftment kinetics comparable to those of patient 1.

[0227] Without being bound by theory, it is believed that the mixture of high stringency (Product 1.1) and low stringency (Product 1.2) CD34 enriched cell populations confers an in vivo selective advantage to the genetically modified FA hematopoietic cells, resulting in a progressive expansion of genetically modified cells over time. Product 1.1 (high stringency CD34 enriched cell population) is believed to provide the majority of genetically modified cells contributing to hematopoiesis, and Product 1.2 (low stringency CD34 enriched cell population) is believed to have promoted robust engraftment and hematopoietic repopulation by Product 1.1.

[0228] Example 2 Comparative efficacy of treatments with lineage-depleted cell populations Patient 2 presented with Fanconi anemia. A peripheral blood biosample was obtained from Patient 2 by mobilized apheresis using a procedure equivalent to Example 1. A lineage-depleted cell population was prepared by labeling the biosample with CD3 / CD14 / CD16 / CD19 reagents and depleting the sample of labeled cells using the Miltenyi Biotec CliniMACS® system in depletion mode. Lineage-depleted CD34 selection yielded CD34 with a relative purity of 1.6%. + The low purity was due to the CD34 + This is due to other hematopoietic cell types that were not purified from the cells. The yield and purity of CD34 cells was equivalent to that achieved in patient 1 in the low stringency CD34 enriched cell population. A high stringency CD34 enriched cell population was not prepared. The lineage-depleted cell population was transduced with a recombinant gene therapy vector encoding the FANCA gene product and designated as product 2.1.

[0229] Product 2.1 was administered to patient 2 by continuous intravenous infusion over 10-30 minutes with monitoring of clinical indicators of cell numbers comparable to those used in patient 1. In contrast to patient 1, no genetically modified cells were detectable in the blood of patient 2 6 months after transplantation. Product 2.1, which is therefore considered similar to product 1.2 alone, does not result in detectable hematopoietic recovery.

[0230] Example 3 Modification of low stringency conditions for higher yields and treatment with the resulting cell population Patient 3 presents with Fanconi anemia. A peripheral blood biosample is obtained from Patient 3 by mobilization apheresis using a procedure equivalent to Example 1. After apheresis collection, the collected HSPCs are split into two peripheral blood biosamples.

[0231] High stringency CD34 selection: The first biological sample was enriched by selecting for CD34+ cells under high stringency conditions using a Miltenyi Biotec CliniMACS® system in enrichment mode with Miltenyi Biotec CD34 reagent for enrichment as described in Example 1. High stringency CD34 selection was performed by selecting for CD34+ cells under high stringency conditions using a Miltenyi Biotec CliniMACS® system in enrichment mode with Miltenyi Biotec CD34 reagent for enrichment as described in Example 1. + This results in a yield of cells greater than 20% and a relative purity greater than 20%. The CD34 selection program on the CliniMACS® system was run using samples as described above with parameters as shown in Table 2. The process lasts approximately 20-30 minutes. The "target cell bag" of the CliniMACS® system contains the high stringency CD34 enriched cell population.

[0232] (Table 2) TIFF2025004171000031.tif225153

[0233] Low stringency CD34 selection: the second biological sample was enriched for CD34 compared to the low stringency enrichment applied to patient 1. + Cells were enriched by selecting for CD34+ cells under low stringency conditions using a modification of the Miltenyi Biotec CliniMACS® system using depletion mode as described in Example 1, with modifications intended to provide a higher yield of cells.

[0234] The apheresis product derived from peripheral blood anticoagulated with anticoagulant acid citrate dextrose solution (ACD-A) was 60 × 10 9 ~6×10 8 of total cells (if using one vial of CD34 labeling reagent) or 120 x 10 9 ~12×10 8The apheresis product is delivered either in 100 mL of total cells (if two vials of CD34 labeling reagent are used). In a laminar flow hood under sterile conditions, cell count, cell viability, and number of CD3+, CD19+, or CD34+ cell subpopulations are measured. The apheresis product is delivered to an introduction bag filled to a total volume of 600 mL with phosphate-buffered saline (PBS). The contents are centrifuged at 230 G for 15 minutes, the supernatant is removed, leaving 90 mL, 5 mL of immunoglobulin is added, and then mixed at room temperature for 10 minutes. One or two 7.5 ml vials of CliniMACS® CD34 reagent are injected into the bag, which is mixed and incubated under agitation at room temperature for 30 minutes. The cells are centrifuged at 230 G for 15 minutes and resuspended in 150 mL of PBS.

[0235] The CD34 selection program on the CliniMACS® system was run using samples as described above with parameters as shown in Table 2. The process lasts approximately 20-30 minutes. The program is stopped before any of the wash steps are performed and the CD34 enriched cells and other non-specific cells are flushed into the "target cell bag". The "target cell bag" of the CliniMACS® system contains the low stringency CD34 enriched cell population. The "non-target cell bag" of the CliniMACS® system contains the non-target cells.

[0236] Low stringency CD34 selection is + Results in a yield of approximately 35%-60% of cells, and a relative purity of approximately 10%-30%.

[0237] Both the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population were then transduced, each individually or in combination, with a recombinant gene therapy vector encoding the FANCA gene product as described in Example 1, and the resulting genetically modified cells were designated Product 3.1 and Product 3.2, respectively.

[0238] Individually transfected product 3.1 and product 3.2 were mixed together and then administered to patient 3 by intravenous bolus.

[0239] Patient 3 shows rapid in vivo selective engraftment kinetics. This treatment protocol results in multilineage hematopoiesis, neutrophils, erythrocytes, and platelets. Stabilization of the hematopoietic lineages, which were depressed in patient 3 prior to transplant, is a compelling endpoint for clinical success.

[0240] Example 4 Gene therapy using CRISPR-CAS in high- and low-stringency CD34-enriched cell populations Biological samples are taken from additional patients and high and low stringency CD34 enriched cell populations are prepared as described in Example 3. A recombinant gene therapy is created, which is designed to provide a gene editing system capable of directed repair of the endogenous FANC gene. The gene editing system includes a Cas protein or a polynucleotide encoding a Cas protein, a gRNA, and a repair template. The repair template includes a sequence fragment that overlaps with a known mutation(s) for the FANC gene (e.g., FANCA) in the additional patient. One or both of the high and low stringency CD34 enriched populations are contacted with a recombinant gene therapy vector, and then a mixture of the two CD34 enriched cell populations is autologously transplanted into each of the patients. The patients show rapid in vivo selection engraftment kinetics. This treatment protocol results in a multilineage increase in hematopoiesis, neutrophils, red blood cells, and platelets. Restoration of hematopoietic lineages that were reduced in the patient before transplantation is a compelling endpoint for clinical success.

[0241] Sequence information SEQUENCE LISTING <110> Spacecraft Seven, LLC Fundacion para la Investigacion Biomedica del Hospital Infantil Universitario Nino Jesus <120> COMPOSITIONS AND METHODS FOR STEM CELL TRANSPLANT <150> US 62 / 656,292 <151> 2018-04-11 <160> 37 <170> PatentIn version 3.5 <210> 1 <211> 234 <212> DNA <213> Artificial Sequence <220> <223> Recombinant Rev response element <400> 1 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat 60 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 120 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca 180 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcct 234 <210> 2 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> Recombinant packaging signal sequence <400> 2 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga 60 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag 120 agcgtc 126 <210> 3 <211> 181 <212> DNA <213> Artificial Sequence <220> <223> Recombinant truncated HIV-1 5 prime LTR sequence <400> 3 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 120 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 180 a 181 <210> 4 <211> 234 <212> DNA <213> Artificial Sequence <220> <223> Recombinant HIV-1 self-inactivating 3 prime LTR sequence <400> 4 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc 60 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 120 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 180 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agca 234 <210> 5 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> Recombinant human cytomegalovirus immediate early promoter <400> 5 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 60 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 120 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 180 tgggaggtct atataagcag agct 204 <210> 6 <211> 118 <212> DNA <213> Artificial Sequence <220> <223> Recombinant HIV-1 central polypurine tract and central termination sequence <400> 6 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 60 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaatttt 118 <210> 7 <211> 511 <212> DNA <213> Artificial Sequence <220> <223> Recombinant human phosphoglycerate kinase 1 promoter sequence <400> 7 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc 60 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc 120 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 180 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 240 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 300 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag 360 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 420 gcccgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 480 cgttgaccga atcaccgacc tctctcccca g 511 <210> 8 <211> 4368 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized FANCA sequence <400> 8 atgtccgact cgtgggtccc gaactccgcc tcgggccagg acccaggggg ccgccggagg 60 gcctgggccg agctgctggc gggaagggtc aagaggggaaa aataatcc tgaaagggca 120 cagaaattaa aggaatcagc tgtgcgcctc ctgcgaagcc atcaggacct gaatgccctt 180 ttgcttgagg tagaaggtcc actgtgtaaa aaattgtctc tcagcaaagt gattgactgt 240 gacagttctg aggcctatgc taatcattct agttcattta taggctctgc tttgcaggat 300 caagcctcaa ggctgggggt tcccgtgggt attctctcag ccgggatggt tgcctctagc 360 gtgggacaga tctgcacggc tccagcggag accagtcacc ctgtgctgct gactgtggag 420 cagagaaaga agctgtcttc cctgttagag tttgctcagt atttattggc acacagtatg 480 ttctcccgtc tttccttctg tcaagaatta tggaaaatac agagttcttt gttgcttgaa 540 gcggtgtggc atcttcacgt acaaggcatt gtgagcctgc aagagctgct ggaaagccat 600 cccgacatgc atgctgtggg atcgtggctc ttcaggaatc tgtgctgcct ttgtgaacag 660 atggaagcat cctgccagca tgctgacgtc gccagggcca tgctttctga ttttgttcaa 720 atgtttgttt tgaggggatt tcagaaaaac tcagatctga gaagaactgt ggagcctgaa 780 aaaatgccgc aggtcacggt tgatgtactg cagagaatgc tgatttttgc acttgacgct 840 ttggctgctg gagtacagga ggagtcctcc actcacaaga tcgtgaggtg ctggttcgga 900 gtgttcagtg gacacacgct tggcagtgta atttccacag atcctctgaa gaggttcttc 960 agtcataccc tgactcagat actcactcac agccctgtgc tgaaagcatc tgatgctgtt 1020 cagatgcaga gagagtggag ctttgcgcgg acacaccctc tgctcacctc actgtaccgc 1080 aggctctttg tgatgctgag tgcagaggag ttggttggcc atttgcaaga agttctggaa 1140 acgcaggagg ttcactggca gagagtgctc tcctttgtgt ctgccctggt tgtctgcttt 1200 ccagaagcgc agcagctgct tgaagactgg gtggcgcgtt tgatggccca ggcattcgag 1260 agctgccagc tggacagcat ggtcactgcg ttcctggttg tgcgccaggc agcactggag 1320 ggcccctctg cgttcctgtc atatgcagac tggttcaagg cctcctttgg gagcacacga 1380 ggctaccatg gctgcagcaa gaaggccctg gtcttcctgt ttacgttctt gtcagaactc 1440 gtgccttttg agtctccccg gtacctgcag gtgcacattc tccacccacc cctggttccc 1500 agcaagtacc gctccctcct cacagactac atctcattgg ccaagacacg gctggccgac 1560 ctcaaggttt ctatagaaaa catgggactc tacgaggatt tgtcatcagc tggggacatt 1620 actgagcccc acagccaagc tcttcaggat gttgaaaagg ccatcatggt gtttgagcat 1680 acggggaaca tcccagtcac cgtcatggag gccagcatat tcaggaggcc ttactacgtg 1740 tcccacttcc tccccgccct gctcacacct cgagtgctcc ccaaagtccc tgactcccgt 1800 gtggcgttta tagagtctct gaagagagca gataaaatcc ccccatctct gtactccacc 1860 tactgccagg cctgctctgc tgctgaagag aagccagaag atgcagccct gggagtgagg 1920 gcagaaccca actctgctga ggagcccctg ggacagctca cagctgcact gggagagctg 1980 agagcctcca tgacagaccc cagccagcgt gatgttatat cggcacaggt ggcagtgatt 2040 tctgaaagac tgagggctgt cctgggccac aatgaggatg acagcagcgt tgagatatca 2100 aagattcagc tcagcatcaa cacgccgaga ctggagccac gggaacacat tgctgtggac 2160 ctcctgctga cgtctttctg tcagaacctg atggctgcct ccagtgtcgc tcccccggag 2220 aggcagggtc cctgggctgc cctcttcgtg aggaccatgt gtggacgtgt gctccctgca 2280 gtgctcaccc ggctctgcca gctgctccgt caccagggcc cgagcctgag tgccccacat 2340 gtgctggggt tggctgccct ggccgtgcac ctgggtgagt ccaggtctgc gctcccagag 2400 gtggatgtgg gtcctcctgc acctggtgct ggccttcctg tccctgcgct ctttgacagc 2460 ctcctgacct gtaggacgag ggattccttg ttcttctgcc tgaaattttg tacagcagca 2520 atttcttact ctctctgcaa gttttcttcc cagtcacgag atactttgtg cagctgctta 2580 tctccaggcc ttattaaaaa gtttcagttc ctcatgttca gattgttctc agaggcccga 2640 cagcctcttt ctgaggagga cgtagccagc ctttcctgga gacccttgca ccttccttct 2700 gcagactggc agagagctgc cctctctctc tggacacaca gaaccttccg agaggtgttg 2760 aaagaggaag atgttcactt aacttaccaa gactggttac acctggagct ggaaattcaa 2820 cctgaagctg atgctctttc agatactgaa cggcaggact tccaccagtg ggcgatccat 2880 gagcactttc tccctgagtc ctcggcttca gggggctgtg acggagacct gcaggctgcg 2940 tgtaccattc ttgtcaacgc actgatggat ttccaccaaa gctcaaggag ttatgaccac 3000 tcagaaaatt ctgatttggt ctttggtggc cgcacaggaa atgaggatat tatttccaga 3060 ttgcaggaga tggtagctga cctggagctg cagcaagacc tcatagtgcc tctcggccac 3120 accccttccc aggagcactt cctctttgag attttccgca gacggctcca ggctctgaca 3180 agcgggtgga gcgtggctgc cagccttcag agacagaggg agctgctaat gtacaaacgg 3240 atcctcctcc gcctgccttc gtctgtcctc tgcggcagca gcttccaggc agaacagccc 3300 atcactgcca gatgcgagca gttcttccac ttggtcaact ctgagatgag aaacttctgc 3360 tcccacggag gtgccctgac acaggacatc actgcccact tcttcagggg cctcctgaac 3420 gcctgtctgc ggagcagaga cccctccctg atggtcgact tcatactggc caagtgccag 3480 acgaaatgcc ccttaatttt gacctctgct ctggtgtggt ggccgagcct ggagcctgtg 3540 ctgctctgcc ggtggaggag acactgccag agcccgctgc cccgggaact gcagaagcta 3600 caagaaggcc ggcagtttgc cagcgatttc ctctcccctg aggctgcctc cccagcaccc 3660 aacccggact ggctctcagc tgctgcactg cactttgcga ttcaacaagt cagggaagaa 3720 aacatcagga agcagctaaa gaagctggac tgcgagagag aggagctatt ggttttcctt 3780 ttcttcttct ccttgatggg cctgctgtcg tcacatctga cctcaaatag caccacagac 3840 ctgccaaagg ctttccacgt ttgtgcagca atcctcgagt gtttagagaa gaggaagata 3900 tcctggctgg cactctttca gttgacagag agtgacctca ggctggggcg gctcctcctc 3960 cgtgtggccc cggatcagca caccaggctg ctgcctttcg ctttttacag tcttctctcc 4020 tacttccatg aagacgcggc catcagggaa gaggccttcc tgcatgttgc tgtggacatg 4080 tacttgaagc tggtccagct cttcgtggct ggggatacaa gcacagtttc acctccagct 4140 ggcaggagcc tggagctcaa gggtcagggc aaccccgtgg aactgataac aaaagctcgt 4200 ctttttctgc tgcagttaat acctcggtgc ccgaaaaaga gcttctcaca cgtggcagag 4260 ctgctggctg atcgtgggga ctgcgaccca gaggtgagcg ccgccctcca gagcagacag 4320 caggctgccc ctgacgctga cctgtcccag gagcctcatc tcttctga 4368 <210> 9 <211> 380 <212> DNA <213> Artificial Sequence <220> <223> Recombinant human CMV enhancer sequence <400> 9 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360 tagtcatcgc tattaccatg 380 <210> 10 <211> 122 <212> DNA <213> Artificial Sequence <220> <223> Recombinant Simian virus 40 polyA signal sequence <400> 10 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120 ta 122 <210> 11 <211> 136 <212> DNA <213> Artificial Sequence <220> <223> Recombinant Simian virus 40 origin of replication sequence <400> 11 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 60 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga 120 ggcttttttg gaggcc 136 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Recombinant HIV-1 central polypurine tract and central termination sequence <400> 12 tttaaaagaa aaggggggat tggggggt 28 <210> 13 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> Recombinant dNEF signal sequence <400> 13 gaattcgagc tcggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca 60 ctttttaaaa gaaaaggggg gac 83 <210> 14 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> Recombinant KanR sequence <400> 14 atgattgaac aagatggatt gcacgcaggt tctccggcgg cttgggtgga gaggctattc 60 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120 gcgcaggggc gtccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180 caagacgagg cagcgcggct atcgtggctg gcgacgacgg gcgttccttg cgcggctgtg 240 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgtc tatgcccgac 540 ggcgaggatc tcgtcgtgac ccacggcgat gcctgcttgc cgaatatcat ggtggaaaat 600 ggccgctttt ctggattcat cgactgtggc cgtctgggtg tggcggaccg ctatcaggac 660 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720 cttgtgcttt acggtatcgc cgcgcccgat tcgcagcgca tcgccttcta tcgccttctt 780 gacgagttct tctga 795 <210> 15 <211> 137 <212> DNA <213> Artificial Sequence <220> <223> Recombinant rrnG terminator sequence <400> 15 gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc 60 cagattcagc aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac 120 cagaaattta tccttaa 137 <210> 16 <211> 589 <212> DNA <213> Artificial Sequence <220> <223> Recombinant high-copy-number ColE1, pMB1, pBR322, pUC origin of replication <400> 16 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 60 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 120 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 180 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 240 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 300 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 360 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 420 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 480 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 540 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaa 589 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Recombinant CAP binding site <400> 17 taatgtgagt tagctcactc at 22 <210> 18 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Recombinant lac promoter sequence <400> 18 tttacacttt atgcttccgg ctcgtatgtt g 31 <210> 19 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Recombinant lac operator sequence <400> 19 ttgtgagcgg ataacaa 17 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Recombinant T3 promoter sequence <400> 20 aattaaccct cactaaagg 19 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Recombinant T7 promoter sequence <400> 21 cctatagtga gtcgtatta 19 <210> 22 <211> 429 <212> DNA <213> Artificial Sequence <220> <223> Recombinant f1 bacteriophage origin of replication sequence <400> 22 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 60 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 120 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 180 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 240 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 300 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 360 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 420 acgcgaatt 429 <210> 23 <211> 677 <212> DNA <213> Artificial Sequence <220> <223> Chimeric wPRE sequence <400> 23 cgagcatctt accgccattt attcccatat ttgttctgtt tttcttgatt tgggtataca 60 tttaaatgtt aataaaacaa aatggtgggg caatcattta catttttagg gatatgtaat 120 tactagttca ggtgtattgc cacaagacaa acatgttaag aaactttccc gttatttacg 180 ctctgttcct gttaatcaac ctctggatta caaaatttgt gaaagattga ctgatattct 240 taactatgtt gctcctttta cgctgtgtgg atatgctgct ttaatgcctc tgtatcatgc 300 tattgcttcc cgtacggctt tcgttttctc ctccttgtat aaatcctggt tgctgtctct 360 tttgaggag ttgtggcccg ttgtccgtca acgtggcgtg gtgtgctctg tgtttgctga 420 cgcaaccccc actggctggg gcattgccac cacctgtcaa ctcctttctg ggactttcgc 480 tttccccctc ccgatcgcca cggcagaact catcgccgcc tgccttgccc gctgctggac 540 aggggctagg ttgctgggca ctgaaattc cgtggtgttg tcggggaagg gcctgctgcc 600 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg 660 ggccgcctcc ccgcctg 677 <210> 24 <211> 11433 <212> DNA <213> Artificial Sequence <220> <223> pCCL-PGK-FANCA-WPRE or pCCL-SIN-cPPT CTS-hPGK-hFANCA-WPRE <400> 24 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 60 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 120 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 180 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 240 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 300 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 360 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 420 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 480 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 540 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 600 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 660 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 720 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 780 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 840 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 900 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag 960 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 1020 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa 1080 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt ggccattgca 1140 tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa cattaccgcc 1200 atgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca 1260 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 1320 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 1380 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 1440 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 1560 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 1680. gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccggggtctc tctggttaga ccgatctga gcctgggagc tctctggcta actagggac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagaga tgggtgcgag agcgtcagta 2160 ttaagcgggg gagaattaga tcgcgatgggg aaaaatttcg gttaaggcca gggggaaga 2220 aaaataata attaaaacat atagtaggg caagcaggga gctagaacga ttcgcagtta 2280 atcctggcct gttagaaca tcagaggct gtagacaat actgggacag ctacaaccat 2340 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt 2400 gtgtgcatca aaggatagag aaaagaca ccaaggaagc tttagacaag atagaggag 2460 agcaaaaaaa agtaagacc accgcacagc aagcggccgc tgatctcag acctgagga 2520 ggagaattga gggacaattg gagagtgaa ttataat aataagtagt aaaattgaa 2580 ccattaggag tagcaccac ccaggchaag agagagtgg tgcagagaga aaaagagca 2640 gtgggaatag gagctttgtt ccttggggttc ttgggagcag caggaagcac tatgggcgca 2700 gcgtcaatga cgctgacggt acaggccaga cattattgt ctggtatagt gcagcagcag 2760 aacaatttgc tgaggctat tgaggcgca cagcatctgt tgcactcac agtctggggc 2820 atcaagcagc tccaggcaag aatcctggct gtggaagat acctaagga tcacagctc 2880 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct 2940 agttggagta ataatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac 3000 agagaaatta acattacac aagcttaata cactccttaa ttgaagaatc gcaaaccag 3060 caghaaaga atgaaga attattgaa ttagataat gggcaagtttt gtggaatttgg 3120 tttaacataa caattggct gtgtatata aattattca taatgatagt aggaggctg 3180 gtaggtttaa gatagtttt tgctgtactt tctatagtga atagagttag gcaggtat 3240 tcaccattat cgtttcagac ccaccccca accccgaggg gaccgacag gcccgaagga 3300 atagagaag aggtggaga gagagacaga gatacca ttcgattagt gaacggatct 3360 cgacggtatc ggttaacttt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 3420 agaatagtag acatatagc aagacata aactaag attaxaaaaaattaca 3480 aaaattcaaa attttatcga tcacgagact agcctcgaga agcttgatat cgaattccac 3540 ggggttgggg tgcgccttt tccaggcag ccctggttt gcgcagggac gcggctgctc 3600 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc 3660 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 3720 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 3780 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 3840 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag 3900 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 3960 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 4020 cgttgaccga atcaccgacc tctctcccca gggggatccc ccgggctgca ggaattcatg 4080 tccgactcgt gggtcccgaa ctccgcctcg ggccaggacc cagggggccg ccggagggcc 4140 tgggccgagc tgctggcggg aagggtcaag agggaaaaat ataatcctga aagggcacag 4200 aaattaaagg aatcagctgt gcgcctcctg cgaagccatc aggacctgaa tgcccttttg 4260 cttgaggtag aaggtccact gtgtaaaaaa ttgtctctca gcaaagtgat tgactgtgac 4320 agttctgagg cctatgctaa tcattctagt tcatttatag gctctgcttt gcaggatcaa 4380 gcctcaaggc tgggggttcc cgtgggtatt ctctcagccg ggatggttgc ctctagcgtg 4440 ggacagatct gcacggctcc agcggagacc agtcaccctg tgctgctgac tgtggagcag 4500 agaaagaagc tgtcttccct gttagagttt gctcagtatt tattggcaca cagtatgttc 4560 tcccgtcttt ccttctgtca agaattatgg aaaatacaga gttctttgtt gcttgaagcg 4620 gtgtggcatc ttcacgtaca aggcattgtg agcctgcaag agctgctgga aagccatccc 4680 gacatgcatg ctgtgggatc gtggctcttc aggaatctgt gctgcctttg tgaacagatg 4740 gaagcatcct gccagcatgc tgacgtcgcc agggccatgc tttctgattt tgttcaaatg 4800 tttgttttga ggggatttca gaaaaactca gatctgagaa gaactgtgga gcctgaaaaa 4860 atgccgcagg tcacggttga tgtactgcag agaatgctga tttttgcact tgacgctttg 4920 gctgctggag tacaggagga gtcctccact cacaagatcg tgaggtgctg gttcggagtg 4980 ttcagtggac acacgcttgg cagtgtaatt tccacagatc ctctgaagag gttcttcagt 5040 cataccctga ctcagatact cactcacagc cctgtgctga aagcatctga tgctgttcag 5100 atgcagagag agtggagctt tgcgcggaca caccctctgc tcacctcact gtaccgcagg 5160 ctctttgtga tgctgagtgc agaggagttg gttggccatt tgcaagaagt tctggaaacg 5220 caggaggttc actggcagag agtgctctcc tttgtgtctg ccctggttgt ctgctttcca 5280 gaagcgcagc agctgcttga agactgggtg gcgcgtttga tggcccaggc attcgagagc 5340 tgccagctgg acagcatggt cactgcgttc ctggttgtgc gccaggcagc actggagggc 5400 ccctctgcgt tcctgtcata tgcagactgg ttcaaggcct cctttgggag cacacgaggc 5460 taccatggct gcagcaagaa ggccctggtc ttcctgttta cgttcttgtc agaactcgtg 5520 ccttttgagt ctccccggta cctgcaggtg cacattctcc acccacccct ggttcccagc 5580 aagtaccgct ccctcctcac agactacatc tcattggcca agacacggct ggccgacctc 5640 aaggtttcta tagaaaacat gggactctac gaggatttgt catcagctgg ggacattact 5700 gagccccaca gccaagctct tcaggatgtt gaaaaggcca tcatggtgtt tgagcatacg 5760 gggaacatcc cagtcaccgt catggaggcc agcatattca ggaggcctta ctacgtgtcc 5820 cacttcctcc ccgccctgct cacacctcga gtgctcccca aagtccctga ctcccgtgtg 5880 gcgtttatag agtctctgaa gagagcagat aaaatccccc catctctgta ctccacctac 5940 tgccaggcct gctctgctgc tgaagagaag ccagaagatg cagccctggg agtgagggca 6000 gaacccaact ctgctgagga gcccctggga cagctcacag ctgcactggg agagctgaga 6060 gcctccatga cagaccccag ccagcgtgat gttatatcgg cacaggtggc agtgatttct 6120 gaaagactga gggctgtcct gggccacaat gaggatgaca gcagcgttga gatatcaaag 6180 attcagctca gcatcaacac gccgagactg gagccacggg aacacattgc tgtggacctc 6240 ctgctgacgt ctttctgtca gaacctgatg gctgcctcca gtgtcgctcc cccggagagg 6300 cagggtccct gggctgccct cttcgtgagg accatgtgtg gacgtgtgct ccctgcagtg 6360 ctcacccggc tctgccagct gctccgtcac cagggcccga gcctgagtgc cccacatgtg 6420 ctggggttgg ctgccctggc cgtgcacctg ggtgagtcca ggtctgcgct cccagaggtg 6480 gatgtgggtc ctcctgcacc tggtgctggc cttcctgtcc ctgcgctctt tgacagcctc 6540 ctgacctgta ggacgaggga ttccttgttc ttctgcctga aattttgtac agcagcaatt 6600 tcttactctc tctgcaagtt ttcttcccag tcacgagata ctttgtgcag ctgcttatct 6660 ccaggcctta ttaaaaagtt tcagttcctc atgttcagat tgttctcaga ggcccgacag 6720 cctctttctg aggaggacgt agccagcctt tcctggagac ccttgcacct tccttctgca 6780 gactggcaga gagctgccct ctctctctgg acacacagaa ccttccgaga ggtgttgaaa 6840 gaggaagatg ttcacttaac ttaccaagac tggttacacc tggagctgga aattcaacct 6900 gaagctgatg ctctttcaga tactgaacgg caggacttcc accagtgggc gatccatgag 6960 cactttctcc ctgagtcctc ggcttcaggg ggctgtgacg gagacctgca ggctgcgtgt 7020 accattcttg tcaacgcact gatggatttc caccaaagct caaggagtta tgaccactca 7080 gaaaattctg atttggtctt tggtggccgc acaggaaatg aggatattat ttccagattg 7140 caggagatgg tagctgacct ggagctgcag caagacctca tagtgcctct cggccacacc 7200 ccttcccagg agcactcct ctttgagat ttcgcagac ggctccaggc tctgacaagc 7260 gggtggagcg tggctgccag ccttcagaga cagagggagc tgctaatgta caaacggatc 7320 ctcctccgcc tgccttcgtc tgtcctctgc ggcagcagct tccaggcaga acagcccatc 7380 actgccagat gcgagcagtt cttccacttg gtcactctg agatgagaa cttctgctcc 7440 cacggaggtg cctgacaca ggacacact gcccactct tcaggggcct cctgaacgcc 7500 tgtctgcgga gcagagaccc ctccctgatg gtcgactca tactggccaa gtgccagacg 7560 aaatgcccct taattttgac ctctgctctg gtgtgtgc cgagcctgga gcctgtgctg 7620 ctctgccggt ggaggagaca ctgccagagc ccgctgcccc gggaactgca gaagctacaa 7680 gaaggccggc agtttgccag cgatttcctc tccctgagg ctgcctcccc agcacccac 7740 ccggactggc tctcagctgc tgcactgcac tttgcgattc aacaagtcag ggaagaaaac 7800 atcaggaagc agctaagaa gctggactgc gagagagagg agctattggt ttccttttc 7860 ttctctcct tgatgggcct gctgtcgtca catctgacct caatagcac cacagacctg 7920 ccaaaggctt tccacgtttg tgcagcaatc ctcgagtgtt tagagaagag gaagatatcc 7980 tggctggcac tctttcagtt gacagagagt gacctcaggc tggggcggct cctcctccgt 8040 gtggccccgg atcagcacac caggctgctg cctttcgctt tttacagtct tctctcctac 8100 ttccatgaag acgcggccat cagggaagag gccttcctgc atgttgctgt ggacatgtac 8160 ttgaagctgg tccagctctt cgtggctggg gatacaagca cagtttcacc tccagctggc 8220 aggagcctgg agctcaaggg tcagggcaac cccgtggaac tgataacaaa agctcgtctt 8280 tttctgctgc agttaatacc tcggtgcccg aaaaagagct tctcacacgt ggcagagctg 8340 ctggctgatc gtggggactg cgacccagag gtgagcgccg ccctccagag cagacagcag 8400 gctgcccctg acgctgacct gtcccaggag cctcatctct tctgatgaga attcgatatc 8460 aagcttatcg ataccgtcga atcccccggg ctgcaggaat tcgagcatct taccgccatt 8520 tattcccata tttgttctgt ttttcttgat ttgggtatac atttaaatgt taataaaaca 8580 aaatggtggg gcaatcattt acatttttag ggatatgtaa ttactagttc aggtgtattg 8640 ccacaagaca aacatgttaa gaaactttcc cgttatttac gctctgttcc tgttaatcaa 8700 cctctggatt acaaaatttg tgaaagattg actgatattc ttaactatgt tgctcctttt 8760 acgctgtgtg gatatgctgc tttaatgcct ctgtatcatg ctattgcttc ccgtacggct 8820 ttcgttttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc 8880 gttgtccgtc aacgtggcgt ggtgtgctct gtgtttgctg acgcaacccc cactggctgg 8940 ggcattgcca ccacctgtca actcctttct gggactttcg ctttccccct cccgatcgcc 9000 acggcagaac tcatcgccgc ctgccttgcc cgctgctgga caggggctag gttgctgggc 9060 actgataatt ccgtggtgtt gtcggggaag ggcctgctgc cggctctgcg gcctcttccg 9120 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgga 9180 attcgagctc ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact 9240 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc 9300 tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct 9360 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt 9420 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt 9480 ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta taacttgcaa 9540 agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat 9600 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 9660 gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac 9720 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 9780 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta 9840 gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat 9900 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 9960 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 10020 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt 10080 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 10140 agcgccctag cgcccgctcc ttcgctttc ttcccttcct ttctcgccac gttcgccggc 10200 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 10260 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 10320 tagacggtt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 10380 caaactggaa caacactcaa ccctatctcg gtctattcttt ttgatttata agggattttg 10440 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattttt 10500 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc 10560 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 10620 gataaatgct tcaataatag cacctagatc aagagacagg atgaggatcg tttcgcatga 10680 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 10740 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 10800 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag 10860 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 10920 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 10980 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 11040 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 11100 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 11160 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg 11220 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 11280 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 11340 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 11400 tgctttacgg tatcgccgct cccgattcgc agc 11433 <210> 25 <211> 5554 <212> DNA <213> Artificial Sequence <220> <223> pCCL-PGK-FANCAW-82-RO <400> 25 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc 60 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc 120 cgtcgcagc gtcacccgga tctcgcgc tacccttgtg ggccccccgg cgacgcttcc 180 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 240 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 300 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag 360 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 420 gcccgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 480 cgttgaccga atcaccgacc tctctcccca gggggatccc ccgggctgca ggaattcatg 540 tccgactcgt gggtcccgaa ctccgcctcg ggccaggacc cagggggccg ccggagggcc 600 tgggccgagc tgctggcggg aagggtcaag agggaaaaat ataatcctga aagggcacag 660 aaattaaagg aatcagctgt gcgcctcctg cgaagccatc aggacctgaa tgcccttttg 720 cttgaggtag aaggtccact gtgtaaaaaa ttgtctctca gcaaagtgat tgactgtgac 780 agttctgagg cctatgctaa tcattctagt tcatttatag gctctgcttt gcaggatcaa 840 gcctcaaggc tgggggttcc cgtgggtatt ctctcagccg ggatggttgc ctctagcgtg 900 ggacagatct gcacggctcc agcggagacc agtcaccctg tgctgctgac tgtggagcag 960 agaaagaagc tgtcttccct gttagagttt gctcagtatt tattggcaca cagtatgttc 1020 tcccgtcttt ccttctgtca agaattatgg aaaatacaga gttctttgtt gcttgaagcg 1080 gtgtggcatc ttcacgtaca aggcattgtg agcctgcaag agctgctgga aagccatccc 1140 gacatgcatg ctgtgggatc gtggctcttc aggaatctgt gctgcctttg tgaacagatg 1200 gaagcatcct gccagcatgc tgacgtcgcc agggccatgc tttctgattt tgttcaaatg 1260 tttgttttga ggggatttca gaaaaactca gatctgagaa gaactgtgga gcctgaaaaa 1320 atgccgcagg tcacggttga tgtactgcag agaatgctga tttttgcact tgacgctttg 1380 gctgctggag tacaggagga gtcctccact cacaagatcg tgaggtgctg gttcggagtg 1440 ttcagtggac acacgcttgg cagtgtaatt tccacagatc ctctgaagag gttcttcagt 1500 cataccctga ctcagatact cactcacagc cctgtgctga aagcatctga tgctgttcag 1560 atgcagagag agtggagctt tgcgcggaca caccctctgc tcacctcact gtaccgcagg 1620 ctctttgtga tgctgagtgc agaggagttg gttggccatt tgcaagaagt tctggaaacg 1680 caggaggttc actggcagag agtgctctcc tttgtgtctg ccctggttgt ctgctttcca 1740 gaagcgcagc agctgcttga agactgggtg gcgcgtttga tggcccaggc attcgagagc 1800 tgccagctgg acagcatggt cactgcgttc ctggttgtgc gccaggcagc actggagggc 1860 ccctctgcgt tcctgtcata tgcagactgg ttcaaggcct cctttgggag cacacgaggc 1920 taccatggct gcagcaagaa ggccctggtc ttcctgttta cgttcttgtc agaactcgtg 1980 ccttttgagt ctccccggta cctgcaggtg cacattctcc acccacccct ggttcccagc 2040 aagtaccgct ccctcctcac agactacatc tcattggcca agacacggct ggccgacctc 2100 aaggtttcta tagaaaacat gggactctac gaggatttgt catcagctgg ggacattact 2160 gagccccaca gccaagctct tcaggatgtt gaaaaggcca tcatggtgtt tgagcatacg 2220 gggaacatcc cagtcaccgt catggaggcc agcatattca ggaggcctta ctacgtgtcc 2280 cacttcctcc ccgccctgct cacacctcga gtgctcccca aagtccctga ctcccgtgtg 2340 gcgtttatag agtctctgaa gagagcagat aaaatccccc catctctgta ctccacctac 2400 tgccaggcct gctctgctgc tgaagagaag ccagaagatg cagccctggg agtgagggca 2460 gaacccaact ctgctgagga gcccctggga cagctcacag ctgcactggg agagctgaga 2520 gcctccatga cagaccccag ccagcgtgat gttatatcgg cacaggtggc agtgatttct 2580 gaaagactga gggctgtcct gggccacaat gaggatgaca gcagcgttga gatatcaaag 2640 attcagctca gcatcaacac gccgagactg gagccacggg aacacattgc tgtggacctc 2700 ctgctgacgt ctttctgtca gaacctgatg gctgcctcca gtgtcgctcc cccggagagg 2760 cagggtccct gggctgccct cttcgtgagg accatgtgtg gacgtgtgct ccctgcagtg 2820 ctcacccggc tctgccagct gctccgtcac cagggcccga gcctgagtgc cccacatgtg 2880 ctggggttgg ctgccctggc cgtgcacctg ggtgagtcca ggtctgcgct cccagaggtg 2940 gatgtgggtc ctcctgcacc tggtgctggc cttcctgtcc ctgcgctctt tgacagcctc 3000 ctgacctgta ggacgaggga ttccttgttc ttctgcctga aattttgtac agcagcaatt 3060 tcttactctc tctgcaagtt ttcttcccag tcacgagata ctttgtgcag ctgcttatct 3120 ccaggcctta ttaaaaagtt tcagttcctc atgttcagat tgttctcaga ggcccgacag 3180 cctctttctg aggaggacgt agccagcctt tcctggagac ccttgcacct tccttctgca 3240 gactggcaga gagctgccct ctctctctgg acacacagaa ccttccgaga ggtgttgaaa 3300 gaggaagatg ttcacttaac ttaccaagac tggttacacc tggagctgga aattcaacct 3360 gaagctgatg ctctttcaga tactgaacgg caggacttcc accagtgggc gatccatgag 3420 cactttctcc ctgagtcctc ggcttcaggg ggctgtgacg gagacctgca ggctgcgtgt 3480 accattcttg caacgcact gatggattc caccaagct caggagtta tgaccactca 3540 gaaaattctg atttggtctt tggtggccgc acaggaatg aggatattat ttccattg 3600 caggagatgg tagctgacct ggagctgcag caggacctca tagtgccct cggccacacc 3660 ccttcccagg agcactctcct ctttgagat ttcgcagac ggctccaggc tctgacaagc 3720 gggtggagcg tggctgccag ccttcagaga cagagggagc tgctaatgta caaacggatc 3780 ctcctccgcc tgccttcgtc tgtcctctgc ggcagcagct tccaggcaga acagcccatc 3840 actgccagat gcgagcagtt cttccacttg gtcactg agatgagaa cttctgctcc 3900 cacggaggtg cctgacaca ggacacact gcccactct tcaggggct cctgaacgcc 3960 tgtctgcgga gcagagaccc ctccctgatg gtcgacttca tactggccaa gtgccagacg 4020 aaatgcccct taattttgac ctctgctctg gtgtggtggc cgagcctgga gcctgtgctg 4080 ctctgccggt ggaggagaca ctgccagagc ccgctgcccc gggaactgca gaagctacaa 4140 gaaggccggc agtttgccag cgatttcctc tccctgagg ctgcctcccc agcacccac 4200 ccggactggc tctcagctgc tgcactgcac tttgcgattc aacaagtcag ggaagaaaac 4260 atcaggaagc agctaaagaa gctggactgc gagagagagg agctattggt tttccttttc 4320 ttcttctcct tgatgggcct gctgtcgtca catctgacct caaatagcac cacagacctg 4380 ccaaaggctt tccacgtttg tgcagcaatc ctcgagtgtt tagagaagag gaagatatcc 4440 tggctggcac tctttcagtt gacagagagt gacctcaggc tggggcggct cctcctccgt 4500 gtggccccgg atcagcacac caggctgctg cctttcgctt tttacagtct tctctcctac 4560 ttccatgaag acgcggccat cagggaagag gccttcctgc atgttgctgt ggacatgtac 4620 ttgaagctgg tccagctctt cgtggctggg gatacaagca cagtttcacc tccagctggc 4680 aggagcctgg agctcaaggg tcagggcaac cccgtggaac tgataacaaa agctcgtctt 4740 tttctgctgc agttaatacc tcggtgcccg aaaaagagct tctcacacgt ggcagagctg 4800 ctggctgatc gtggggactg cgacccagag gtgagcgccg ccctccagag cagacagcag 4860 gctgcccctg acgctgacct gtcccaggag cctcatctct tctgatgaga attcgatatc 4920 aagcttatcg ataccgtcga atcccccggg ctgcaggaat tcgagcatct taccgccatt 4980 tattcccata tttgttctgt ttttcttgat ttgggtatac atttaaatgt taataaaaca 5040 aaatggtggg gcaatcattt acatttttag ggatatgtaa ttactagttc aggtgtattg 5100 ccacaagaca aacatgttaa gaaactttcc cgttatttac gctctgttcc tgttaatcaa 5160 cctctggatt acaaaatttg tgaaagattg actgatattc ttaactatgt tgctcctttt 5220 acgctgtgtg gatatgctgc tttaatgcct ctgtatcatg ctattgcttc ccgtacggct 5280 ttcgttttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc 5340 gttgtccgtc aacgtggcgt ggtgtgctct gtgtttgctg acgcaacccc cactggctgg 5400 ggcattgcca ccacctgtca actcctttct gggactttcg ctttccccct cccgatcgcc 5460 acggcagaac tcatcgccgc ctgccttgcc cgctgctgga caggggctag gttgctgggc 5520 actgataatt ccgtggtgtt gtcggggaag ggcc 5554 <210> 26 <211> 858 <212> DNA <213> Artificial Sequence <220> <223> Recombinant Rev response item <400> 26 gatcttcaga cctggaggag gagatatgag ggacaatttgg agagtgaat tataata 60 taaagtagta aaattgaac cattaggagt agcacccacc aaggxaag gagagtggt 120 gcagagagaa aaagagcag tgggaatagg agctttgttc cttggttct tgggagcagc 180 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc 240 tggtatagtg cagcagcaga acaatttgct gaggctatt gaggcgcaac agcatctgtt 300 gcaaccaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaagata 360 cctaaaggat cacagctcc tgggatttg gggttgctct ggaaaactca tttgcaccac 420 tgctgtgcct tggaatgcta gttggagtaa taatctctg gaacagattt ggaatcacac 480 gacctggatg gagtgggaca gagaattta cattacaca agcttaatac actccttaat 540 tgaagaatcg haaaaccagc aaaaaagaa tgaacaa ttattggaat tegataatg 600 ggcaagtttg tggaatttggt ttacataac aaattggctg tggtatataa aattattcat 660 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa 720 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg 780 acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat 840 tcgattagtg aacggatc 858 <210> 27 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Recombinant packaging signal sequence <400> 27 tgagtacgcc aaaaattttg actagcggag gctagaagga gaga 44 <210> 28 <211> 188 <212> DNA <213> Artificial Sequence <220> <223> Recombinant truncated HIV-1 5 prime LTR sequence <400> 28 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact 60 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg 120 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag 180 tggcgccc 188 <210> 29 <211> 234 <212> DNA <213> Artificial Sequence <220> <223> Recombinant HIV-1 self-inactivating 3 prime LTR sequence <400> 29 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc 60 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 120 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 180 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agca 234 <210> 30 <211> 577 <212> DNA <213> Artificial Sequence <220> <223> Recombinant human cytomegalovirus immediate early promoter sequence <400> 30 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 60 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 120 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 180 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 240 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 300 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 360 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 420 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 480 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 540 gggcggtagg cgtgtacggt gggaggtcta tataagc 577 <210> 31 <211> 233 <212> DNA <213> Artificial Sequence <220> <223> Recombinant Rous sarcoma virus promoter sequence <400> 31 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa 60 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac 120 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa 180 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacaataa acg 233 <210> 32 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Recombinant HIV-1 central polypurine tract and central termination sequence <400> 32 aaaagaaaag ggggga 16 <210> 33 <211> 121 <212> DNA <213> Artificial Sequence <220> <223> Recombinant HIV-1 central polypurine tract and central termination sequence <400> 33 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta 60 aagaattaca aaaacaaatt acaaaaattc aaaattttat cgatcacgag actagcctcg 120 a 121 <210> 34 <211> 156 <212> DNA <213> Artificial Sequence <220> <223> Recombinant Simian virus 40 origin of replication sequence <400> 34 ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggccgag gcggcctcgg 60 cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg 120 gagttagggg cgggatgggc ggagttaggg gcggga 156 <210> 35 <211> 141 <212> DNA <213> Artificial Sequence <220> <223> Recombinant RNA-OUT sequence <400> 35 gtagaattgg taaagagagt cgtgtaaaat atcgagttcg cacatcttgt tgtctgatta 60 ttgatttttg gcgaaaccat ttgatcatat gacaagatgt gtatctacct taacttaatg 120 attttgataa aaatcattag g 141 <210> 36 <211> 4371 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized FANCA sequence <400> 36 atgtccgact cgtgggtccc gaactccgcc tcgggccagg acccaggggg ccgccggagg 60 gcctgggccg agctgctggc gggaagggtc aagaggggaaa aataatcc tgaaagggca 120 cagaaattaa aggaatcagc tgtgcgcctc ctgcgaagcc atcaggacct gaatgccctt 180 ttgcttgagg tagaaggtcc actgtgtaaa aaattgtctc tcagcaaagt gattgactgt 240 gacagttctg aggcctatgc taatcattct agttcattta taggctctgc tttgcaggat 300 caagcctcaa ggctgggggt tcccgtgggt attctctcag ccgggatggt tgcctctagc 360 gtgggacaga tctgcacggc tccagcggag accagtcacc ctgtgctgct gactgtggag 420 cagagaaaga agctgtcttc cctgttagag tttgctcagt atttattggc acacagtatg 480 ttctccccgtc tttccttctg tcaagaatta tggaaaatac agagttcttt gttgcttgaa 540 gcggtgtggc atcttcacgt acaaggcatt gtgagcctgc aagagctgct ggaaagccat 600 cccgacatgc atgctgtggg atcgtggctc ttcaggaatc tgtgctgcct ttgtgaacag 660 atggaagcat cctgccagca tgctgacgtc gccagggcca tgctttctga ttttgttcaa 720 atgtttgttt tgaggggatt tcagaaaaac tcagatctga gaagaactgt ggagcctgaa 780 aaaatgccgc aggtcacggt tgatgtactg cagagaatgc tgatttttgc acttgacgct 840 ttggctgctg gagtacagga ggagtcctcc actcacaaga tcgtgaggtg ctggttcgga 900 gtgttcagtg gacacacgct tggcagtgta atttccacag atcctctgaa gaggttcttc 960 agtcataccc tgactcagat actcactcac agccctgtgc tgaaagcatc tgatgctgtt 1020 cagatgcaga gagagtggag ctttgcgcgg acacaccctc tgctcacctc actgtaccgc 1080 aggctctttg tgatgctgag tgcagaggag ttggttggcc atttgcaaga agttctggaa 1140 acgcaggagg ttcactggca gagagtgctc tcctttgtgt ctgccctggt tgtctgcttt 1200 ccagaagcgc agcagctgct tgaagactgg gtggcgcgtt tgatggccca ggcattcgag 1260 agctgccagc tggacagcat ggtcactgcg ttcctggttg tgcgccaggc agcactggag 1320 ggcccctctg cgttcctgtc atatgcagac tggttcaagg cctcctttgg gagcacacga 1380 ggctaccatg gctgcagcaa gaaggccctg gtcttcctgt ttacgttctt gtcagaactc 1440 gtgccttttg agtctccccg gtacctgcag gtgcacattc tccacccacc cctggttccc 1500 agcaagtacc gctccctcct cacagactac atctcattgg ccaagacacg gctggccgac 1560 ctcaaggttt ctatagaaaa catgggactc tacgaggatt tgtcatcagc tggggacatt 1620 actgagcccc acagccaagc tcttcaggat gttgaaaagg ccatcatggt gtttgagcat 1680 acggggaaca tcccagtcac cgtcatggag gccagcatat tcaggaggcc ttactacgtg 1740 tcccacttcc tccccgccct gctcacacct cgagtgctcc ccaaagtccc tgactcccgt 1800 gtggcgttta tagagtctct gaagagagca gataaaatcc ccccatctct gtactccacc 1860 tactgccagg cctgctctgc tgctgaagag aagccagaag atgcagccct gggagtgagg 1920 gcagaaccca actctgctga ggagcccctg ggacagctca cagctgcact gggagagctg 1980 agagcctcca tgacagaccc cagccagcgt gatgttatat cggcacaggt ggcagtgatt 2040 tctgaaagac tgagggctgt cctgggccac aatgaggatg acagcagcgt tgagatatca 2100 aagattcagc tcagcatcaa cacgccgaga ctggagccac gggaacacat tgctgtggac 2160 ctcctgctga cgtctttctg tcagaacctg atggctgcct ccagtgtcgc tcccccggag 2220 aggcagggtc cctgggctgc cctcttcgtg aggaccatgt gtggacgtgt gctccctgca 2280 gtgctcaccc ggctctgcca gctgctccgt caccagggcc cgagcctgag tgccccacat 2340 gtgctggggt tggctgccct ggccgtgcac ctgggtgagt ccaggtctgc gctcccagag 2400 gtggatgtgg gtcctcctgc acctggtgct ggccttcctg tccctgcgct ctttgacagc 2460 ctcctgacct gtaggacgag ggattccttg ttcttctgcc tgaaattttg tacagcagca 2520 atttcttact ctctctgcaa gttttcttcc cagtcacgag atactttgtg cagctgctta 2580 tctccaggcc ttattaaaaa gtttcagttc ctcatgttca gattgttctc agaggcccga 2640 cagcctcttt ctgaggagga cgtagccagc ctttcctgga gacccttgca ccttccttct 2700 gcagactggc agagagctgc cctctctctc tggacacaca gaaccttccg agaggtgttg 2760 aaagaggaag atgttcactt aacttaccaa gactggttac acctggagct ggaaattcaa 2820 cctgaagctg atgctctttc agatactgaa cggcaggact tccaccagtg ggcgatccat 2880 gagcactttc tccctgagtc ctcggcttca gggggctgtg acggagacct gcaggctgcg 2940 tgtaccattc ttgtcaacgc actgatggat ttccaccaaa gctcaaggag ttatgaccac 3000 tcagaaaatt ctgatttggt ctttggtggc cgcacaggaa atgaggatat tatttccaga 3060 ttgcaggaga tggtagctga cctggagctg cagcaagacc tcatagtgcc tctcggccac 3120 accccttccc aggagcactt cctctttgag attttccgca gacggctcca ggctctgaca 3180 agcgggtgga gcgtggctgc cagccttcag agacagaggg agctgctaat gtacaaacgg 3240 atcctcctcc gcctgccttc gtctgtcctc tgcggcagca gcttccaggc agaacagccc 3300 atcactgcca gatgcgagca gttcttccac ttggtcaact ctgagatgag aaacttctgc 3360 tcccacggag gtgccctgac acaggacatc actgcccact tcttcagggg cctcctgaac 3420 gcctgtctgc ggagcagaga cccctccctg atggtcgact tcatactggc caagtgccag 3480 acgaaatgcc ccttaatttt gacctctgct ctggtgtggt ggccgagcct ggagcctgtg 3540 ctgctctgcc ggtggaggag acactgccag agcccgctgc cccgggaact gcagaagcta 3600 caagaaggcc ggcagtttgc cagcgatttc ctctcccctg aggctgcctc cccagcaccc 3660 aacccggact ggctctcagc tgctgcactg cactttgcga ttcaacaagt cagggaagaa 3720 aacatcagga agcagctaaa gaagctggac tgcgagagag aggagctatt ggttttcctt 3780 ttcttcttct ccttgatggg cctgctgtcg tcacatctga cctcaaatag caccacagac 3840 ctgccaaagg ctttccacgt ttgtgcagca atcctcgagt gtttagagaa gaggaagata 3900 tcctggctgg cactctttca gttgacagag agtgacctca ggctggggcg gctcctcctc 3960 cgtgtggccc cggatcagca caccaggctg ctgcctttcg ctttttacag tcttctctcc 4020 tacttccatg aagacgcggc catcagggaa gaggccttcc tgcatgttgc tgtggacatg 4080 tacttgaagc tggtccagct cttcgtggct ggggatacaa gcacagtttc acctccagct 4140 ggcaggagcc tggagctcaa gggtcagggc aaccccgtgg aactgataac aaaagctcgt 4200 ctttttctgc tgcagttaat acctcggtgc ccgaaaaaga gcttctcaca cgtggcagag 4260 ctgctggctg atcgtgggga ctgcgaccca gaggtgagcg ccgccctcca gagcagacag 4320 caggctgccc ctgacgctga cctgtcccag gagcctcatc tcttctgatg a 4371 <210> 37 <211> 1015 <212> DNA <213> Artificial Sequence <220> <223> Recombinant high copy number ColE1, pMB1, pBR322, pUC origin of replication sequence <400> 37 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 60 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 120 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 180 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 240 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 300 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 360 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 420 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 480 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 540 ataaggcgca gcggtcgggc tgaacgggg gttcgtgcac acagcccagc ttggagcgaa 600 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 660 aaggggaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 720 gggagcttcc aggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 780 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 840 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 900 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 960 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaaga 1015

Claims

1. 1. A method of treating Fanconi anemia in a subject in need thereof, comprising: (i) detecting CD34 under high stringency conditions from a first biological sample obtained from the subject; ＋ A high stringency CD34 enriched cell population prepared by cell selection, and (ii) isolating CD34 under low stringency conditions from a second biological sample obtained from the subject. ＋ Low stringency CD34 enriched cell population prepared by cell selection providing to said subject one or both of the high stringency CD34 enriched cell population and / or the low stringency CD34 enriched cell population (i) a recombinant gene therapy vector comprising a polynucleotide sequence encoding a Fanconi Anemia Complementation Group (FANC) polypeptide or a functional variant or fragment thereof; or (ii) A gene editing system that targets the direct repair of endogenous mutant FANC genes. genetically modified by, and The method, wherein the first biological sample and the second biological sample are optionally the same biological sample.

2. (a) CD34 under high stringency conditions ＋ preparing a high stringency CD34 enriched cell population from a first biological sample obtained from said subject by selecting cells; (b) CD34 under low stringency conditions ＋ preparing a low stringency CD34 enriched cell population from a second biological sample obtained from said subject by selecting cells; (c) subjecting one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population to (i) a recombinant gene therapy vector comprising a polynucleotide sequence encoding a Fanconi anemia complementation group A (FANCA) polypeptide or a functional variant or fragment thereof; or (ii) the gene editing system that targets direct repair of the endogenous mutant FANC gene. and genetically modifying the (d) providing said high stringency CD34 enriched cell population and said low stringency CD34 enriched cell population to said subject. Further comprising: The method of claim 1 , thereby treating Fanconi anemia.

3. 3. The method of claim 1 or 2, wherein the first biological sample and the second biological sample are each independently peripheral blood or bone marrow.

4. 3. The method of claim 1 or 2, wherein the first biological sample and the second biological sample are peripheral blood obtained after the subject has been treated with G-CSF, plerixafor, or a combination of G-CSF and plerixafor.

5. Under the high stringency conditions, CD34 ＋ Selecting the cells includes selecting the cells from ＋ 3. The method of claim 2, comprising applying the first biological sample to a capture matrix that binds cells, washing the capture matrix one or more times using a wash buffer, and eluting the high stringency CD34 enriched cell population from the capture matrix using an elution buffer.

6. Under the low stringency conditions, CD34 ＋ Selecting the cells includes selecting the cells from ＋ 3. The method of claim 2, comprising applying the second biological sample to a capture matrix that binds cells, allowing an unbound fraction of the second biological sample to flow through the capture matrix, and eluting the low stringency CD34 enriched cell population from the capture matrix using an elution buffer.

7. The method of claim 1 or 2, wherein the high stringency CD34 enriched cell population is genetically modified.

8. The method of claim 1, 2, 6, or 7, wherein the low stringency CD34 enriched cell population is genetically modified.

9. The method according to any one of claims 1 to 8, wherein the percentage of CD34+ cells in the high stringency CD34 enriched cell population is 2 to 4 times greater than the percentage of CD34+ cells in the low stringency CD34 enriched cell population.

10. The method of any one of claims 1 to 9, wherein the high stringency CD34 enriched cell population comprises CD34+ cells at a purity of more than 20% or more than 30%.

11. The method of any one of claims 1 to 10, wherein the low stringency CD34 enriched cell population comprises CD34+ cells at a purity of less than 30%.

12. The method of any one of claims 1 to 11, wherein the high stringency CD34 enriched cell population comprises CD34+ cells at a yield of more than 20%.

13. The method of any one of claims 1 to 12, wherein the low stringency CD34 enriched cell population comprises CD34+ cells at a yield of more than 35%.

14. The recombinant gene therapy vector comprises: (a) a promoter sequence active in eukaryotes, and (b) a sequence encoding a human FANC gene polypeptide or a functional fragment or variant thereof In the 5' to 3' order, The method of any one of claims 1 to 3, wherein the sequence encoding the human FANC gene polypeptide or a functional fragment or variant thereof is operably linked to a promoter sequence active in the eukaryote, and the FANC gene is selected from FANCA, FANCC, and FANCG.

15. The gene editing system comprises: (a) a Cas protein or a polynucleotide encoding a Cas protein; (b) a gRNA, and (c) a repair template comprising a sequence comprising the endogenous FANC gene or a fragment thereof that overlaps with one or more mutations in the FANC gene. Including, 2. The method of claim 1, wherein an sgRNA is configured to direct the repair template to the FANC gene, the FANC gene being selected from FANCA, FANCC, and FANCG, and the gene editing system is capable of direct repair of an endogenous FANC gene.

16. 16. The method of any one of claims 1 to 15, wherein the hematological manifestations of Fanconi anemia are optionally selected from one or more of bone marrow failure, thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia, wherein the hematological manifestations of Fanconi anemia are inhibited from occurring, halted from progressing, and / or reversed from progressing in the subject.

17. The method of claim 2 , wherein the selection is performed by bead-based magnetic selection.

18. 5. The method of claim 4, further comprising performing one or more apheresis on the peripheral blood.

19. 19. The method of any one of claims 1 to 18, which results in a progressive increase in genetically modified Franconia anemia cells over time.

20. 20. The method of any one of claims 1 to 19, which results in restoration of one or more hematopoietic lineages that were depleted in the subject prior to administering to the subject the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population.

21. 21. The method of claim 20, wherein the one or more hematopoietic lineages include one or more of lymphocytes, eosinophils, neutrophils, erythrocytes, and platelets.

22. 20. The method of any one of claims 1 to 19, which results in restoration of one or more hematological parameters that were decreased in the subject prior to administering to the subject the high stringency CD34 enriched cell population and the low stringency CD34 enriched cell population.

23. 23. The method of claim 22, wherein the hematological parameter is hemoglobin.

24. 1. A method for preparing a genetically modified cell for the treatment of Fanconi anemia, comprising: (a) CD34 under high stringency conditions ＋ preparing a high stringency CD34 enriched cell population from a first biological sample obtained from a subject by selecting cells; (b) preparing a low stringency CD34 enriched cell population from a second biological sample obtained from the subject by selecting CD34+ cells under low stringency conditions; and (c) genetically modifying one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population with a recombinant gene therapy vector for Fanconi anemia, wherein the gene therapy vector optionally comprises a polynucleotide encoding a Fanconi anemia complementation group (FANC) polypeptide or a functional variant or fragment thereof. The method comprising:

25. 25. The method of claim 24, wherein the first biological sample and the second biological sample are each independently peripheral blood or bone marrow.

26. 26. The method of claim 25, wherein the first biological sample and the second biological sample are peripheral blood obtained after the subject has been treated with G-CSF, plerixafor, or a combination of G-CSF and plerixafor.

27. Under the high stringency conditions, CD34 ＋ Selecting the cells includes selecting the cells from ＋ 25. The method of claim 24, comprising applying the first biological sample to a capture matrix that binds cells, washing the capture matrix one or more times using a wash buffer, and eluting the high stringency CD34 enriched cell population from the capture matrix using an elution buffer.

28. Under the low stringency conditions, CD34 ＋ Selecting the cells includes selecting the cells from ＋ 25. The method of claim 24, comprising applying the second biological sample to a capture matrix that binds cells, allowing an unbound fraction of the second biological sample to flow through the capture matrix, and eluting the low stringency CD34 enriched cell population from the capture matrix using an elution buffer.

29. 25. The method of claim 24, wherein the high stringency CD34 enriched cell population is contacted with the recombinant gene therapy vector.

30. The method of any one of claims 24 to 29, wherein the low stringency CD34 enriched cell population is contacted with the recombinant gene therapy vector.

31. The method of any one of claims 240 to 30, wherein the percentage of CD34+ cells in the high stringency CD34 enriched cell population is 2 to 4 times greater than the percentage of CD34+ cells in the low stringency CD34 enriched cell population.

32. The method of any one of claims 24 to 31, wherein the high stringency CD34 enriched cell population comprises CD34+ cells at a purity of more than 20% or more than 30%.

33. The method of any one of claims 24 to 32, wherein the low stringency CD34 enriched cell population comprises CD34+ cells at a purity of less than 30%.

34. The method of any one of claims 24 to 33, wherein the high stringency CD34 enriched cell population comprises CD34+ cells at a yield of more than 20%.

35. The method of any one of claims 24 to 34, wherein the low stringency CD34 enriched cell population comprises CD34+ cells at a yield of greater than 35%.

36. The recombinant gene therapy vector for Fanconi anemia comprises (a) a promoter sequence active in eukaryotes, and (b) a sequence encoding a human FANC gene polypeptide or a functional fragment or variant thereof In the 5' to 3' order, The method of any one of claims 24 to 35, wherein the sequence encoding the human FANC gene polypeptide or a functional fragment or variant thereof is operably linked to a promoter sequence active in the eukaryote, and the FANC gene is selected from FANCA, FANCC, and FANCG.

37. The recombinant gene therapy vector for Fanconi anemia comprises a gene editing system capable of directly repairing an endogenous FANC gene, the gene editing system comprising: (a) a Cas protein or a polynucleotide encoding a Cas protein; (b) a gRNA, and (c) a repair template comprising a sequence comprising the FANC gene or a fragment thereof that overlaps with one or more mutations in the endogenous FANC gene. Including, The method of any one of claims 24 to 35, wherein the sgRNA is configured to guide the repair template to the FANC gene, the FANC gene being selected from FANCA, FANCC, and FANCG.

38. 25. The method of claim 24, wherein the selection of (a) and / or (b) is carried out by bead-based magnetic selection.

39. The method of claim 24 or 38, wherein the selection of (a) and / or (b) is carried out using an antibody or a functional fragment thereof that specifically binds to CD34.

40. The method of claim 24 or 38, wherein the selecting of (a) and / or (b) is carried out using a flow rate of 10 to 20 mL / min.

41. (a) isolating CD34 from a first biological sample under high stringency conditions; ＋ A high stringency CD34 enriched cell population prepared by cell selection, and (b) a low stringency CD34 enriched cell population prepared by selecting CD34+ cells under low stringency conditions from the second biological sample. Including, The composition, wherein one or both of the high stringency CD34 enriched cell population or the low stringency CD34 enriched cell population are contacted with a recombinant gene therapy vector for Fanconi anemia.

42. 42. The composition of claim 41, wherein the first biological sample and the second biological sample are each independently peripheral blood or bone marrow.

43. 42. The composition of claim 41, wherein the first biological sample and the second biological sample are peripheral blood obtained from the subject after the subject has been treated with G-CSF, plerixafor, or a composition of G-CSF and plerixafor.

44. The composition of any one of claims 41 to 43, wherein the percentage of CD34+ cells in the high stringency CD34 enriched cell population is 2 to 4 times greater than the percentage of CD34+ cells in the low stringency CD34 enriched cell population.

45. The composition of any one of claims 41 to 44, wherein the high stringency CD34 enriched cell population comprises CD34+ cells at a purity of greater than 20% or greater than 30%.

46. The composition of any one of claims 41 to 45, wherein the low stringency CD34 enriched cell population comprises CD34+ cells at a purity of less than 30%.

47. The composition of any one of claims 41 to 46, wherein the high stringency CD34 enriched cell population comprises CD34+ cells at a yield of greater than 20%.

48. 48. The composition of any one of claims 41 to 47, wherein the low stringency CD34 enriched cell population comprises CD34+ cells at a yield of greater than 35%.

49. The composition of any one of claims 41 to 48, wherein the high stringency CD34 enriched cell population is contacted with the recombinant gene therapy vector.

50. 50. The composition of any one of claims 41 to 49, wherein the low stringency CD34 enriched cell population is contacted with the recombinant gene therapy vector.

51. The recombinant gene delivery vector comprises: (a) a promoter sequence active in eukaryotes, and (b) a sequence encoding a human FANCA polypeptide or a functional fragment or variant thereof. In the 5' to 3' order, The composition of any one of claims 41 to 50, wherein the sequence encoding the human FANC polypeptide or a functional fragment or variant thereof is operably linked to a promoter sequence active in the eukaryote, and the FANC gene is selected from FANCA, FANCC, and FANCG.

52. A pharmaceutical composition comprising the composition of any one of claims 41 to 51 and a pharma- ceutically acceptable carrier.

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