Vector comprising gene associated with DNA repair deficiency and uses thereof

Abstract

The present disclosure relates, in general, to expression vectors comprising genes associated with DNA repair deficiencies, such as Fanconi anemia (FA), for use in gene therapy to treat DNA repair deficiency disorders. The expression vectors can be introduced directly into the patients or used ex vivo in a method for genome engineering a hematopoietic stem and progenitor cell (HSPC) to express a gene of interest and reintroduced into a patient.

Description

[0001] Docket No. 32757 / 70065

[0002] VECTOR COMPRISING GENE ASSOCIATED WITH DNA REPAIR DEFICIENCY AND USES THEREOF

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004]

[0001] The present application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 679,675, filed August 6, 2024, herein incorporated by reference in its entirety.

[0005] FIELD

[0006]

[0002] The present disclosure is directed to expression vectors comprising DNA repair genes, such as Fanconi anemia (FA) genes, for use in gene therapy to treat DNA deficiency repair disorders, such as FA and FA-associated cancers and other cancers.

[0007] BACKGROUND

[0008]

[0003] DNA repair deficiency disorders result from reduced function of genes or proteins involved in DNA repair mechanisms, including single stranded break repair, double strand break repair, Nucleotide excision repair (NER), mis-match repair and non-homologous end joining. DNA repair dysfunction can lead to conditions associated with developmental issues, skin disorders, premature aging, neurological syndromes and certain cancers.

[0009]

[0004] Fanconi anemia (FA) is a rare genetic disease characterized by congenital malformations, progressive bone marrow (BM) depletion, and an increased risk of developing solid and hematological tumors (Moreno et al., Biomed Rep 15, 74 (2021)). Genetically, FA is caused by mutations in one of the 23 known FA genes (Knies, J. Clin. Invest.127, 3013-3027 (2017)); Wegman-Ostrosky et al., Br. J. Haematol. 177, 526-542 (2017)) and is characterized mostly as an autosomal recessive genetic disorder (biallelic pathogenic mutations in FANC-A, -C, -D1 , -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -S, -T, -U, -V, -W, or -Y), rarely as an X-linked recessive (FANCB variants) or an autosomal dominant (FANCR variants) disorder. FA gene products comprise the FA pathway, which has numerous roles in maintaining genomic integrity. Most notably, the FA pathway is critical for the repair of interstrand crosslinks (ICLs), DNA adducts that covalently link two bases on complementary strands of a DNA duplex (Ceccaldi et al., Nat. Rev. Mol. Cell Biol. 17, 337- 349 (2016). These adducts can be caused by endogenously-produced metabolites, such as reactive aldehydes, and by exposure to cancer chemotherapeutics, such as Mitomycin C (MMC) (Clauson et al., Cold Spring Harb. Perspect. Biol. 5, a012732 (2013)). In the FA pathway for ICL repair, there are four main phases: (i) ICL recognition, (ii) enzymatic ICL unhooking, (iii) single-stranded (ss) DNA gap-filling via translesion synthesis (TLS), and (iv) Docket No. 32757 / 70065 homology directed repair (HDR)-mediated double stranded break (DSB) repair. Importantly, loss of any FA protein leads to genomic instability and exhaustion of hematopoietic stem and progenitor cells (HSPCs), contributing to cellular transformation (Garaycoechea, J. I. & Patel, K. J. Blood 123, 26-34 (2014); Ceccaldi et al., Cell Stem Cell 11 , 36-49 (2012); Nalepa et al., Nat. Rev. Cancer 18,168-185 (2018)). Furthermore, FA cells are hypersensitive to ICL- inducing agents and deficient in HDR-mediated DSB repair (Michl et al., EMBO J. 35, 909- 923 (2016)). These characteristics make treatment of FA and FA-associated cancers a challenge, as classical gene editing, and chemotherapeutics generate DSBs and are toxic to FA cells.

[0010]

[0005] Treatment by allogeneic hematopoietic stem cell transplant (HSCT) can be curative for the lethal BM manifestations of FA (aplastic anemia, myelodysplasia, or leukemia); however, the therapeutic benefits do not extend to solid tumors, such as those affecting the squamous epithelia of the upper aeroesophageal tract, vulvo-vaginal-cervical region and skin.

[0011]

[0006] Furthermore, allogeneic HSCT in FA patients is associated with significant risks of debilitating morbidities and mortality due to the pre- and post-transplant treatment procedures (MacMillan et al., Biol. Blood Marrow Transplant. 17, S109-14 (2011)), including the magnification of cancer risk. A Phase l / ll) gene therapy trial using complemented autologous HSPCs has been implemented (Rio et al., Blood 130, 1535-1542 (2017)). However, low numbers of HSPCs available in FA patients for genetic modification, risks of cellular toxicities and insertional mutagenesis with viral vector approaches, and an intolerance to preconditioning that typically provides a competitive advantage for the corrected repopulating cells limit this approach in FA.

[0012] SUMMARY

[0013]

[0007] The present disclosure provides gene editing systems for targeted insertion of a gene involved in DNA repair deficiency, such as a Fanconi anemia gene, in pluripotent stem cells or other cell types, including somatic cells, to correct a defective gene in a subject. The disclosure contemplates that the cell may be corrected in vivo, or ex vivo by use of the engineered cells as a cell-based gene therapy to treat a DNA repair deficiency, such as Fanconi anemia (FA). The present disclosure also shows that unexpectedly, HMEJ is a useful method to perform gene editing in non-cycling cells and treatments for gene therapy in these cell types.

[0014]

[0008] Provided herein is a polynucleotide expression cassette comprising a homology arm (HA), a polynucleotide encoding a gene associated with a DNA repair deficiency, a splice Docket No. 32757 / 70065 acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is capable of insertion at a locus targeted by the targeting molecule.

[0015]

[0009] Also provided is a polynucleotide expression cassette comprising homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is capable of insertion upstream of a gene associated with a DNA repair deficiency.

[0016]

[0010] The disclosure also provides a polynucleotide expression cassette comprising homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site for transposon delivery of a gene associated with a DNA repair deficiency, wherein the expression cassette is capable of insertion at a locus targeted by the transposon delivery site and / or is capable of insertion upstream of a a DNA repair deficiency.

[0017]

[0011] Further provided herein is a polynucleotide cassette comprising left and right homology arms, a polynucleotide encoding a gene associated with a DNA repair deficiency, and single-guide (sg) RNA cut sites to linearize donor DNA to be integrated into a locus in a cell genome.

[0018]

[0012] Also contemplated is a method using a dual nicking approach for linearization for HMEJ in which juxtaposed sgRNAs and a Cas nickase are useful to generate staggered double stranded breaks.

[0019]

[0013] The disclosure also provides a polynucleotide expression cassette comprising a homology arm (HA), a polynucleotide encoding a Fanconi anemia (FA) gene, a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is capable of insertion at a locus targeted by the targeting molecule.

[0020]

[0014] The disclosure also provides a polynucleotide cassette comprising left and right homology arms, a polynucleotide encoding a FA gene, and single-guide (sg) RNA cut sites to linearize the donor DNA to be integrated into a locus in a cell genome.

[0021]

[0015] Also provided is a polynucleotide expression cassette comprising homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is capable of insertion upstream of a gene associated with FA.

[0022]

[0016] In various embodiments, provided is a polynucleotide expression cassette comprising homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site for transposon delivery of a Fanconi anemia gene, wherein the expression cassette is capable of insertion Docket No. 32757 / 70065 at a locus targeted by the transposon delivery site and / or is capable of insertion upstream of a FA gene.

[0023]

[0017] In various embodiments, the homology arms are between 35 and 1000 nucleotides. In various embodiments, the homology arms are from 50-900 nucleotides, 50-750 nucleotides, 100-600 nucleotides, 100-500 nucleotides, or 200-400 nucleotides. In various embodiments, the homology arms are 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 nucleotides.

[0024]

[0018] In various embodiments, the nuclease dependent cleavage system comprises a CRISPR / Cas system, a Cas-CLOVER system, a zinc-finger nuclease (ZFN) system, a transcription activator like effector nuclease (TALEN) system, or a meganuclease system. In various embodiments, the CRISPR / Cas system comprises Cas9, Cas12a, Cas13a, or Cas13b. In some embodiments, the nuclease dependent cleavage system is a CRISPR / Cas system and the targeting molecule is a single guide RNA.

[0025]

[0019] In various embodiments, the locus targeted is an AAVS1 (PPP1 R12C) or FA gene locus. In various embodiments, the locus targeted is an AAVS1 (PPP1R12C) or gene locus for a DNA repair gene of interest.

[0026]

[0020] In various embodiments, the expression cassette comprises the promoter next to or near the gene associated with a DNA repair deficiency.

[0027]

[0021] In various embodiments, the expression cassette comprises the promoter next to or near the FA gene.

[0028]

[0022] In some embodiments, the promoter is an MND promoter, a CMV promoter, a CAG promoter, a PGK promoter, a EF1 A promoter, an AAV promoter, or an endogenous cell promoter, e.g., when the vector or cassette is introduced in gene therapy.

[0029]

[0023] In various embodiments, the FA gene is a donor polynucleotide that corrects a mutated genotype in a subject. In various embodiments, the mutated genotype is one of FANC-A, -B, -C, -D1, -D2, -E, -F, -G, -H, -I, -J, -K, -L, -M, -N, -O, -P, -Q, -S, -T, -U, -V, -W, or -Y.

[0030]

[0024] In various embodiments, the transgene comprises all or part of one or more of FANC-A, -B, -C, -D1, -D2, -E, -F, -G, -H, -I, -J, -K, -L, -M, -N, -O, -P, -Q, -S, -T, -U, -V, -W, or -Y.

[0031]

[0025] Also provided is a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette as described herein. Docket No. 32757 / 70065

[0032]

[0026] In various embodiments, the viral vector is a lentiviral vector, adenoviral vector, or AAV vector. In various embodiments, the viral vector is selected from the group consisting of a AAV6 vector, AAV1 vector, AAV-DJ vector, or VSVg-pseudotype lentiviral vector.

[0033]

[0027] In various embodiments, the vector comprises a FA donor polynucleotide in an empty vector backbone.

[0034]

[0028] In various embodiments, the vector comprises a gene associated with DNA repair deficiency donor polynucleotide in an empty vector backbone.

[0035]

[0029] Further disclosed is a method for genome engineering a hematopoietic stem and progenitor cell (HSPC) or a population of HSPC to express cDNA of a gene associated with a DNA repair deficiency from the endogenous locus comprising introducing into the HSPC cell or population of HSPC a viral vector, plasmid, nanoplasmid, lipid nanoparticle, minicircle, or GenCircle comprising an expression cassette comprising a homology arm (HA), a polynucleotide encoding a gene associated with a DNA repair deficiency, a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted at a locus targeted by the targeting molecule.

[0036]

[0030] In various embodiments, the plasmid comprises inverted terminal repeats flanking the polynucleotide encoding a gene associated with DNA repair deficiency for transposon delivery. In various embodiments, the plasmid for transposon delivery comprises a promoter, a gene associated with DNA repair deficiency, a biomarker, a regulatory element, and optionally a chimeric intron.

[0037]

[0031] Also provided is a method for genome engineering a hematopoietic stem and progenitor cell (HSPC) or a population of HSPC to express FA gene cDNA from the endogenous FA gene locus comprising introducing into the HSPC cell or population of HSPC using a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm (HA), a polynucleotide encoding a FA gene, a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted at a locus targeted by the targeting molecule.

[0038]

[0032] The disclosure provides a method for genome engineering a hematopoietic stem and progenitor cells HSPC or a population of HSPC to overexpress an endogenous FA gene comprising introducing into the HSPC or population of HSPC using a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site Docket No. 32757 / 70065 for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted upstream of a FA gene to be overexpressed.

[0039]

[0033] In various embodiments, the method further comprises transfecting the HSPC or population of HSPC with a Cas protein or polynucleotide encoding a Cas protein and guide RNA molecules that direct integration of the expression cassette to a target locus in the HSPC genome. In various embodiments, the target locus is AAVS1 (PPP1R12C) or FA gene loci, such as FANC-A. In various embodiments, the target locus is AAVS1 (PPP1R12C) or a loci for a gene associated with DNA repair deficiency.

[0040]

[0034] In various embodiments, the plasmid comprises inverted terminal repeats flanking the polynucleotide encoding a FA gene for transposon delivery. In various embodiments, the plasmid for transposon delivery comprises a promoter, a FA gene, a biomarker, a regulatory element, and optionally a chimeric intron.

[0041]

[0035] In various embodiments, the viral vector is an AAV vector, lentiviral vector, or adenoviral vector. In various embodiments, the AAV vector is an AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, or AAV-DJ vector. In various embodiments, the viral vector is selected from the group consisting of an AAV6 vector, AAV1 vector, AAV-DJ vector, VSVg-pseudotype, or lentiviral vector. In various embodiments, the FA donor polynucleotide is in an empty vector backbone. In various embodiments, the DNA repair gene donor polynucleotide is in an empty vector backbone.

[0042]

[0036] In various embodiments, the FA gene integrates into the HSPC genome via homology directed repair (HDR), homology-mediated end joining (HMEJ), or a combination of HDR / HMEJ.

[0043]

[0037] In various embodiments, the gene associated with DNA repair deficiency integrates into the HSPC genome via homology directed repair (HDR), homology-mediated end joining (HMEJ), or a combination of HDR / HMEJ.

[0044]

[0038] In various embodiments, the introduction of the plasmid, nanonplasmid, lipid nanoparticle, mini-circle, or GenCircle is by transfection or electroporation. In certain embodiments, the introduction of the viral vector is by electroporation. In various embodiments, the viral vector is introduced at a multiplicity of infection of 3 x 105- 1 x 107.

[0045]

[0039] In various embodiments, a plasmid for transposon delivery is electroporated with transposase mRNA and an expression cassette expressing the FA gene. In various embodiments, a plasmid for transposon delivery is electroporated with transposase mRNA and an expression cassette expressing the gene associated with DNA repair deficiency. Docket No. 32757 / 70065

[0046]

[0040] In various embodiments, efficiency of introduction is greater than 5%. In various embodiments, the HSPC cell population has a viability of greater than 50% after 3 days.

[0047]

[0041] In various embodiments, the HSPC cell or population of HSPC cells is CD34+.

[0048]

[0042] In various embodiments, the viral vector, plasmid nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprises the promoter next to or near the FA gene. In various embodiments, the viral vector, plasmid nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprises the promoter next to or near the gene associated with a DNA repair deficiency. In various embodiments, the promoter is an MND promoter, a CMV promoter, a CAG promoter, a PGK promoter, a EF1 A promoter, an AAV promoter, or an endogenous cell promoter. In various embodiments, the FA gene is a donor polynucleotide that corrects a mutated genotype in a subject. In various embodiments, the FA gene comprises all or part of one or more of FANC-A, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -S, -T, -U, -V, -W, or -Y.

[0049]

[0043] In another embodiment, the disclosure provides a method of making a gene edited hematopoietic stem and progenitor cell (HSPC) or population of HSPC, comprising:

[0050]

[0044] i) contacting a HSPC or population of HSPC with a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, and optionally comprising a polynucleotide encoding a gene associated with a DNA repair deficiency;

[0051]

[0045] ii) culturing the HSPC or population of HSPC of i) in a media that promotes expansion of HSPC cells;

[0052]

[0046] iii) isolating the HSPC or population of HSPC of ii) based on identification of a marker expressed only on a HSPC or population of HSPC carrying the viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle;

[0053]

[0047] iv) culturing the isolated cells of iii) in a culture medium to expand the isolated cells expressing the gene associated with a DNA repair deficiency.

[0054]

[0048] The disclosure also provides a method of making a gene edited hematopoietic stem and progenitor cell (HSPC) or population of HSPC, comprising:

[0055]

[0049] i) contacting a HSPC or population of HSPC with a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, and optionally comprising a polynucleotide encoding a FA associated gene; Docket No. 32757 / 70065

[0056]

[0050] ii) culturing the HSPC or population of HSPC of i) in a media that promotes expansion of HSPC cells;

[0057]

[0051] iii) isolating the HSPC or population of HSPC of ii) based on identification of a marker expressed only on a HSPC or population of HSPC carrying the viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle;

[0058]

[0052] iv) culturing the isolated cells of iii) in a culture medium to expand the isolated cells expressing the FA gene.

[0059]

[0053] In various embodiments, the method further comprises a step of stimulating, proliferating or activating the HSPC or population of HSPC prior to the contacting step. In various embodiments, the step of stimulating, proliferating or activating the HSPC or population of HSPC comprises contacting the cell(s) with one or more of IL-3, IL-6, SCF, Flt3L, TPO, GM-CSF, G-CSF and prostaglandin E2.

[0060]

[0054] In various embodiments, the method produces gene edited HSPC with an efficiency of greater than 5%. In various embodiments, the method maintains 50% viability of cells in culture after 3 days.

[0061]

[0055] Also provided is a gene edited HSPC or population of HSPC made by a method as described herein.

[0062]

[0056] The disclosure provides a gene edited hematopoietic stem and progenitor cell (HSPC) comprising, a heterologous polynucleotide sequence encoding all or part of a gene associated with DNA repair deficiency at a target location mediated by a nuclease dependent cleavage system, wherein the heterologous polynucleotide sequence is also flanked by portions of a homology arm and expressed via an endogenous promoter.

[0063]

[0057] Further provided is a gene edited hematopoietic stem and progenitor cell (HSPC) comprising, a heterologous polynucleotide sequence encoding all or part of a FA gene at a target location mediated by a nuclease dependent cleavage system, wherein the heterologous polynucleotide sequence is also flanked by portions of a homology arm and expressed via an endogenous promoter. In various embodiments, the FA gene is a donor polynucleotide that corrects a mutated genotype in a subject. In various embodiments, the gene associated with a DNA repair deficiency is a donor polynucleotide that corrects a mutated genotype in a subject.

[0064]

[0058] In another aspect, the disclosure provides a method of treating Fanconi Anemia in a subject in need thereof comprising administering to the subject a polynucleotide cassette or expression cassette as described herein. Docket No. 32757 / 70065

[0065]

[0059] Also provided is a method of treating Fanconi Anemia in a subject in need thereof comprising administering to the subject vector comprising a polynucleotide cassette or expression cassette or a vector as described herein.

[0066]

[0060] The disclosure further provides a method of treating Fanconi Anemia (FA) in a subject in need thereof comprising administering to the subject a gene edited HSPC or population of HSPC as described herein.

[0067]

[0061] In various embodiments, the administration alleviates one or more symptoms of FA selected from the group consisting of bone marrow failure, increased risk for malignancy, short stature, abnormal skin pigmentation, skeletal malformations of the upper and / or lower limbs, microcephaly, ophthalmic and genitourinary tract anomalies, pancytopenia, thrombocytopenia, or leukopenia.

[0068]

[0062] In various embodiments, the subject is receiving a second agent or standard of care therapy. In various embodiments, the second agent or standard of care therapy is oral androgens, granulocyte colony-stimulating factor, hematopoietic stem cell transplantation (HSCT), treatment of growth deficiency, treatment of limb anomalies, treatment of ocular anomalies, treatment of renal malformations, treatment of genital anomalies, treatment of hypothyroidism, treatment of cardiac anomalies, treatment of dermatologic manifestations, or vitamin D supplementation.

[0069]

[0063] In various embodiments, the methods and compositions described herein are useful in DNA repair deficiencies caused by mutations in DNA repair genes, e.g., other than in the FA gene. It is contemplated that a nucleotide expression construct, vector or cell as described herein can be modified to correct a genetic mutation in a DNA repair deficiency.

[0070]

[0064] In various embodiments, the disclosure provides a method of treating a DNA repair deficiency in a subject in need thereof comprising administering to the subject a polynucleotide cassette or an expression cassette, a vector, or a gene edited HSPC or population of HSPC described herein comprising a gene associated with DNA repair deficiency as described herein.

[0071]

[0065] In various embodiments, the DNA repair deficiency is xeroderma pigmentosum, ataxia telangiectasia, Nijmegen Breakage Syndrome, Bloom Syndrome, Werner Syndrome, Rothmund-Thompson Syndrome, DNA Ligase IV Deficiency Syndrome (LIG4 syndrome), trichothiodystrophy, progeria (Hutchinson-Gilford progeria syndrome) and Cockayne syndrome.

[0072]

[0066] In various embodiments, gene associated with a DNA repair deficiency is associated with xeroderma pigmentosum, ataxia telangiectasia, Nijmegen Breakage Syndrome, Bloom Docket No. 32757 / 70065

[0073] Syndrome, Werner Syndrome, Rothmund-Thompson Syndrome, DNA Ligase IV Deficiency Syndrome (LIG4 syndrome), trichothiodystrophy, progeria (Hutchinson-Gilford progeria syndrome) and Cockayne syndrome.

[0074]

[0067] In various embodiments, the gene associated with DNA repair deficiency for use in the compositions or methods is ATM, ATR, DNA-PKs, ERCC1 , ERCC2, ERCC4, ERCC5, ERCC6, ERCC8, GTF2H5, Ku70, Ku80, Lamin A, NRMT1 , RECQL4, SIRT6, SIRT7, Werner syndrome helicase, ZMPSTE24, Xeroderma pigmentosum, complementation group C (XPC), XPB (xeroderma pigmentosum type B), XPD, XPF, XPG, XPV, Endonuclease Ill-like protein 1 (NTHL1 ), MutS, BRCA1 / 2, LIG4, or a Rad gene.

[0075]

[0068] It is understood that each feature or embodiment, or combination, described herein is a non-limiting, illustrative example of any of the aspects of the invention and, as such, is meant to be combinable with any other feature or embodiment, or combination, described herein. For example, where features are described with language such as “one embodiment”, “various embodiments”, “some embodiments”, “certain embodiments”, “further embodiment”, “specific exemplary embodiments”, and / or “another embodiment”, each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature, or combination of features, described herein without having to list every possible combination. Such features or combinations of features apply to any of the aspects of the invention. Where examples of values falling within ranges are disclosed, any of these examples are contemplated as possible endpoints of a range, any and all numeric values between such endpoints are contemplated, and any and all combinations of upper and lower endpoints are envisioned.

[0076]

[0069] The headings herein are for the convenience of the reader and not intended to be limiting. Additional aspects, embodiments, and variations of the invention will be apparent from the Detailed Description and / or Drawings and / or claims.

[0077] BRIEF DESCRIPTION OF THE DRAWINGS

[0078]

[0070] Figure 1 A is a schematic of HMEJ-mediated FANCA complementation at the human FANCA locus. Bent arrow: translation start site. Stop sign: translation stop site. Figure 1 B shows % HMEJ repair in wild type of FANCA deficient cells.

[0079]

[0071] Figure 2 shows quantification of HDR-and HMEJ-mediated integration of splice acceptor (SA)-EGFP at the PPP1R12C locus in HCT116 WT, FANCA- / - and FANCD2- / - cells. Docket No. 32757 / 70065

[0080]

[0072] Figures 3A-3D show HMEJ in FA-proficient and -deficient patient-derived fibroblasts. (Fig. 3A) schematic of dual nicking HMEJ approach. Average percentage of HMEJ repair using a Cas9 nuclease or Cas9 nickase dual nicking approach in FA-proficient and -deficient patient-derived fibroblasts (Fig. 3B), HCT116 cells (Fig. 3C), and patient-derived lymphoblastoid cell lines (LCLs) (Fig. 3D).

[0081]

[0073] Figure 4 is a schematic of FANCA complementation using a rAAV HMEJ Nuclease (right) or HMEJ Dual Nickase approach (left).

[0082]

[0074] Figures 5A-5C show (A) Average percentage hFANCA KI in FANCA- / - LCLs. Unselected cells or cells selected with continuous 50 nM MMC treatment were collected for ddPCR analysis at several time points post transductions with rAAV-hFANCA-HMEJ- Nuclease (MOI: 105). (Fig. 5B) LCL viability assay in the absence of presence of several concentrations of MMC. (Fig. 5C) Average percentage hFANCA KI in FANCA- / - LCLs post transductions with rAAV-hFANCA-HMEJ-Nuclease or rAAV-hFANCA-HMEJ-Dual Nicking (MOI: 106).

[0083]

[0075] Figures 6A-6C show (Fig. 6A) flow analysis and bar graph (Fig. 6B) demonstrating that culturing RPE1 +hTERT cells at high confluency for 72 hours leads to a G0 / G1 arrest in day 4 and day 6 cultured cells. (Fig 6C) Quantitation of HMEJ-mediated gene targeting activity.

[0084]

[0076] Figures 7A-7B depict the Cas9 constructs for cell cycle arrest proteins (Fig. 7A) and the % HMEJ repair in cells expressing the construct (Fig. 7B).

[0085]

[0077] Figures 8A-8C show ABE mediated conservative substitution of the Spanish founder mutation reverts the FA phenotypes. Fig. 8A. Percentage of ABE mediated FANCA correction identified by Sanger sequencing five days post-transfection in patient-derived LCLs. MMC hypersensitivity in edited and unedited FA LCL showing cell viability (Fig. 8B) and total cells (Fig. 8C).

[0086] DETAILED DESCRIPTION

[0087]

[0078] Precision genetic modification of primary human stem cells has multiple applications in the field of gene therapy for genetic disorders. Precise genome modifications can be achieved by the introduction of the CRISPR / Cas9 system to induce a targeted DSB along with a DNA template homology-mediated end-joining (HMEJ), thereby integrating the DNA template into the host genome. Although FA cells are inherently deficient in classical homology directed repair (HDR) mechanisms, the inventors discovered that FA cells are highly proficient in HMEJ. These results suggest that HMEJ is a viable method to perform Docket No. 32757 / 70065 gene editing in non-cycling cells, which is unexpected and can have far reaching implications for gene therapy to target non-cycling cell populations that has been difficult to achieve. A DNA template is provided to encode a gene associated with a DNA repair deficiency disorder, such as Fanconi anemia, that can be used as a cell-based gene therapy to correct genetic deficiencies in DNA repair mechanism, e.g., those that cause FA.

[0088] Definitions

[0089]

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (3d ed. 2006); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1990); THE GLOSSARY OF GENETICS, 5TH ED., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THE COLLINS DICTIONARY OF BIOLOGY (3d, 2005).

[0090]

[0080] Each publication, patent application, patent, and other references cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure.

[0091]

[0081] It is noted here that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

[0092]

[0082] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0093]

[0083] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0094]

[0084] As used herein, the terms “about” and “approximately” or "consisting essentially of” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of” can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" or "consisting essentially of” can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the Docket No. 32757 / 70065 application and claims, unless otherwise stated, the meaning of "about" or "consisting essentially of” should be assumed to be within an acceptable error range for that particular value or composition.

[0095]

[0085] "Amplification" refers to any means by which a polynucleotide sequence is copied and thus expanded into a larger number of polynucleotide molecules, e.g., by reverse transcription, polymerase chain reaction, and ligase chain reaction.

[0096]

[0086] "cDNA" refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

[0097]

[0087] Conventional notation is used herein to describe polynucleotide sequences: the lefthand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as "upstream sequences"; sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

[0098]

[0088] "Complementary" refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide. Thus, the polynucleotide whose sequence 5'-TATAC-3' is complementary to a polynucleotide whose sequence is 5'- GTATA-3'. A nucleotide sequence is "substantially complementary" to a reference nucleotide sequence if the sequence complementary to the subject nucleotide sequence is substantially identical to the reference nucleotide sequence.

[0099]

[0089] "Conservative substitution" refers to the substitution in a polypeptide of an amino acid with a functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another:

[0100] 1) Alanine (A), Serine (S), Threonine (T);

[0101] 2) Aspartic acid (D), Glutamic acid (E);

[0102] 3) Asparagine (N), Glutamine (Q);

[0103] 4) Arginine (R), Lysine (K) ; Docket No. 32757 / 70065

[0104] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0105] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0106]

[0090] The term “fragment” when used in reference to polypeptides refers to polypeptides that are shorter than the full-length polypeptide by virtue of truncation at either the N- terminus or C-terminus of the protein or both, and / or by deletion of an internal portion or region of the protein. Fragments of a polypeptide can be generated by methods known in the art.

[0107]

[0091] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides ( / .e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and noncoding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0108]

[0092] "Expression control sequence" refers to a nucleotide sequence in a polynucleotide that regulates the expression (transcription and / or translation) of a nucleotide sequence operatively linked thereto. "Operatively linked" refers to a functional relationship between two parts in which the activity of one part (e.g., the ability to regulate transcription) results in an action on the other part e.g., transcription of the sequence). Expression control sequences can include, for example and without limitation, sequences of promoters e.g., inducible or constitutive), enhancers, transcription terminators, a start codon ( / .e., ATG), splicing signals for introns, and stop codons.

[0109]

[0093] The term “FA associated gene” or “gene associated with Fanconi anemia (FA)” as used herein refers genes that have been identified as a cause or contributing factor to one or more symptoms of FA. FA genes include FANC-A, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -S, -T, -U, -V, -W, or -Y. FA can be identified as an autosomal recessive genetic disorder (biallelic pathogenic mutations in FANC-A, -C, -D1 , -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -S, -T, -U, -V, -W, or -Y), as an X-linked recessive {FANCB variants) disorder or an autosomal dominant {FANCR variants) disorder. Docket No. 32757 / 70065

[0110]

[0094] The term "promoter" as used herein refers to a region of DNA that functions to control the transcription of one or more DNA sequences, and that is structurally identified by the presence of a binding site for DNA-dependent RNA-polymerase and of other DNA sequences, which interact to regulate promoter function. A functional expression promoting fragment of a promoter is a shortened or truncated promoter sequence retaining the activity as a promoter. Promoter activity may be measured in any of the assays known in the art e.g. in a reporter assay using Luciferase as reporter gene (Wood, 1991 ; de Wet et al. (1985), or commercially available.

[0111]

[0095] The term "vector" refers to any carrier of exogenous DNA or RNA that is useful for transferring exogenous DNA to a host cell for replication and / or appropriate expression of the exogenous DNA by the host cell. "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in vitro expression system. Expression vectors include all those known in the art, such as viral vectors, cosmids, plasmids e.g., naked or contained in liposomes), nanoplasmids, lipid nanoparticles, mini-circles, or GenCircles that incorporate the recombinant polynucleotide. A vector may also include an empty vector backbone.

[0112]

[0096] “Nanoplasmid™” refers to a 500 base-pair circular plasmid lacking traditional bacterial genes or antibiotic resistant genes, which in turn reduces cellular toxicity and inflammation of the plasmid-transfected cells, when compared to traditional plasmid. Minicircles similarly lack bacterial genes.

[0113]

[0097] “ Mini-circle” refers to a small circular plasmid derivative of approximately 4kb that lacks bacterial polynucleotide sequences. Mini-circles can be non-replicating and lack an origin of replication, or can be modified to comprise a non-bacterial replication element (e.g., a S / MAR element).

[0114]

[0098] “GenCicle™” (GenScript) refers to a novel small circular double stranded DNA vector with a 429bp vector backbone for use as a knock-in template, viral packaging plasmid, or non-viral vector. An antibiotic resistance gene and bacterial origin sequence have been removed from this vector.

[0115]

[0099] “Viral vector” refers to a vector that uses a viral backbone for carrying a polynucleotide expression cassette. Viral vectors include lentiviral vectors, adenoviral vectors or adeno-associated vectors (AAV).

[0116]

[0100] “Expression cassette” or “cassette” refers to a component of vector or plasmid DNA that controls expression of a gene or protein, and may be interchangeable and easily Docket No. 32757 / 70065 inserted or removed from a vector. Expression cassettes often comprise a promoter sequence, an open reading frame, and a 3' untranslated region that contains a polyadenylation site. A “therapeutic expression cassette” or “therapeutic cassette” refers to an expression cassette expressing a therapeutic protein for use in treating disease.

[0117]

[0101] A “polynucleotide cassette” refers to a polynucleotide comprising left and right homology arms, a polynucleotide encoding a FA gene, and single-guide (sg) RNA cut sites to linearize the donor DNA to be integrated into a genome.

[0118]

[0102] An "enhancer region" refers to a region of DNA that functions to increase the transcription of one or more genes. More specifically, the term "enhancer", as used herein, is a DNA regulatory element that enhances, augments, improves, or ameliorates expression of a gene irrespective of its location and orientation. It is contemplated that an enhancer may enhance expression of more than one promoter.

[0119]

[0103] A “homology arm” refers to a polynucleotide sequence at the 5' region and at the 3’ region immediately flanking, or within 3 or 4 nucleotides of, a DNA sequence of interest in an expression cassette that possesses homology to a selected insertion site in genomic DNA / RNA for the purpose of carrying out homologous recombination.

[0120]

[0104] “Transposon delivery” refers to use of transposon sequences and transposase enzyme for site-specific delivery of DNA to a cell genome. A DNA transposase makes a staggered cut at the target site producing sticky ends, cuts out the DNA transposon and ligates it into the target site. Exemplary transposons include, but are not limited to, Tn5, Tn3, Tn10, Sleeping Beauty, piggyBac, and Tol2.

[0121]

[0105] "Polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA"), including cDNA, and ribonucleic acid ("RNA") as well as nucleic acid analogs. Nucleic acid analogs include those which include non-naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptidenucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide Docket No. 32757 / 70065 sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."

[0122]

[0106] "Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides. Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.

[0123]

[0107] "Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell. A host cell that comprises the recombinant polynucleotide is referred to as a "recombinant host cell." The gene is then expressed in the recombinant host cell to produce, e.g., a "recombinant polypeptide." A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well. Recombinant protein refers to a protein encoded by a recombinant polynucleotide.

[0124]

[0108] "Substantially pure" or "isolated" means an object species is the predominant species present (i.e., on a molar basis, more abundant than any other individual macromolecular species in the composition), and a substantially purified fraction is a composition wherein the object species comprises at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition means that about 80% to 90% or more of the macromolecular species present in the composition is the purified species of interest. The object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) if the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species for purposes of this definition. In various embodiments, recombinant proteins of the disclosure are substantially pure or isolated with respect to the macromolecular starting materials used in their synthesis. In various embodiments, the pharmaceutical composition of the disclosure comprises a substantially purified or isolated therapeutic protein admixed with one or more pharmaceutically acceptable carriers, diluents or excipients. In various embodiments, a pharmaceutical composition of the disclosure Docket No. 32757 / 70065 comprises a polynucleotide cassette, expression cassette, vector or cell as described herein admixed with one or more pharmaceutically acceptable carriers, diluents or excipients.

[0125]

[0109] The term “endogenous” refers to a protein, polynucleotide, or other molecule that is naturally found in, or expressed by, a subject, e.g., a cell, organ, or tissue. The term “exogenous” refers to a protein, polynucleotide, or other molecule that is not naturally found in a subject, e.g., a cell, organ, or tissue.

[0126]

[0110] The term “genetically engineered” as used herein refers to a polynucleotide or polypeptide sequence that has been modified from its naturally-occurring sequence, e.g., by insertion, deletion or polynucleotide or amino acid substitution / modification, using recombinant DNA expression techniques to produce a polypeptide or polynucleotide sequence that differs from the previously unmodified sequence.

[0127]

[0111] The term “nuclease dependent cleavage system” as used herein refers to gene editing techniques that employ DNA or RNA dependent nucleases to cleave target DNA or RNA, respectively, and molecules or guides that direct the nuclease to the target DNA / RNA to be cleaved. Examples of nuclease dependent cleavage systems include CRISPR / Cas systems, Cas-CLOVER systems, zinc-finger nuclease (ZFN) systems, transcription activator like effector nuclease (TALEN) systems, or meganuclease systems.

[0128]

[0112] “Homozygous” for the donor polynucleotide as used herein refers to the result of the genetic modification in which both alleles of the modified gene express the donor polynucleotide. “Heterozygous” for the donor polynucleotide as used herein refers to the result of the genetic modification in which only one of the alleles of the gene express the donor polynucleotide.

[0129]

[0113] As used herein, the phrase “population of cells” or “population of gene -edited cells” refers to a number of cells that share common traits. In general, populations generally range from 1 x 106to 1 x 1010in number.

[0130]

[0114] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.

[0131] Tissues, cells and their progeny of a biological entity obtained in vivo, or cultured ex vivo or in vitro are also encompassed. The term "subject" may also refer to a human who has a tumor into which a population of lymphocytes that have left the human being's bloodstream have migrated and transformed into tumor infiltrating lymphocytes (TILs). In some embodiments, this human may be a patient in need of immunotherapy involving an expanded population of their own TILs (autologous TILs). In other embodiments, the human Docket No. 32757 / 70065 may be a patient in need of immunotherapy involving an expanded TILs from a person other than the patient (allogeneic TILs).

[0132]

[0115] The term “in vivo” refers to an event that takes place in a subject's body.

[0133]

[0116] The term “in vitro” refers to an event that takes places outside of a subject's body. In vitro assays encompass cell-based assays in which cells, alive or dead, are employed and may also encompass a cell-free assay in which no intact cells are employed.

[0134]

[0117] The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and / or organ which has been removed from a subject's body. The cell, tissue and / or organ may be returned to the subject's body in a method of surgery or treatment.

[0135]

[0118] The term “gene associated with a DNA repair deficiency” refers to a genetic mutation in a gene involved in a DNA repair mechanism which contributes to or is the cause of a disorder or condition in a subject resulting from a deficiency in a DNA repair mechanism.

[0136] The genetic mutation may be on a single allele or biallelic. “DNA repair deficiency” refers to a disease or condition that results from reduced function of genes or proteins involved in DNA repair mechanisms.

[0137] DNA Repair Deficiencies

[0138]

[0119] DNA repair deficiency disorders result from reduced function of genes or proteins involved in DNA repair mechanisms, including single stranded break repair, double strand break repair, Nucleotide excision repair (NER), mis-match repair and non-homologous end joining.

[0139]

[0120] It is contemplated that the methods and compositions described herein designed to mediate HMEJ-mediated gene complementation are applicable to addressing mutations in DNA repair deficient genetic disorders. Such disorders include xeroderma pigmentosum, ataxia telangiectasia, Nijmegen Breakage Syndrome, Bloom Syndrome, Werner Syndrome, Rothmund-Thompson Syndrome, DNA Ligase IV Deficiency Syndrome (LIG4 syndrome), trichothiodystrophy, progeria (Hutchinson-Gilford progeria syndrome) and Cockayne syndrome.

[0140]

[0121] Genetic mutations resulting in these DNA repair deficiencies, e.g., the ataxia telangiectasia mutated gene (ATM) in ataxia telangiectasia or Bloom Syndrome caused by BLM gene or RecQ Helicase mutations, can be corrected using a construct as described herein comprising elements to correct the desired DNA repair mutation. Nijmegen Breakage Syndrome is caused by mutations in the NBN gene which makes nibrin protein. Werner Docket No. 32757 / 70065

[0141] Syndrome results from mutations in the WRN gene that makes Werner protein. Rothmund- Thompson Syndrome is caused by mutations in the RECQL4 gene or ANAPC1 gene.

[0142]

[0122] DNA repair genes that contribute to DNA repair deficiency or cancer include, but are not limited to, FANCA genes, ATM, ATR, DNA-PKs, ERCC1 , ERCC2, ERCC4, ERCC5, ERCC6, ERCC8, GTF2H5, Ku70, Ku80, Lamin A, NRMT1 , RECQL4, SIRT6, SIRT7, Werner syndrome helicase, ZMPSTE24, Xeroderma pigmentosum, complementation group C (XPC), XPB (xeroderma pigmentosum type B), XPD, XPF, XPG, XPV, Endonuclease Ill-like protein 1 (NTHL1 ), MutS, BRCA1 / 2, LIG4 or Rad genes. See e.g., Thompson and Schil Mutation Research 509:49-78, 2002; Menck and Munford, Genet Mol Biol. 37(1 Suppl):220- 233, 2013 and Sharma et al., Front. Pediatr., 22 October 2020, herein incorporated by reference.

[0143]

[0123] It is contemplated that the methods and compositions herein alleviate one or more symptoms associated with a DNA repair deficiency including developmental abnormalities, blood abnormalities, skin abnormalities, premature aging, neurological symptoms and symptoms associated with cancer such as tumor volume, tumor burden or metastasis.

[0144] Fanconi Anemia (FA)

[0145]

[0124] Fanconi anemia (FA) is characterized by physical abnormalities, bone marrow failure, and increased risk for malignancy (e.g., AML or solid tumors). Physical abnormalities are present in approximately 75% of FA patients, and include short stature, abnormal skin pigmentation, skeletal malformations of the upper and / or lower limbs, microcephaly, ophthalmic abnormalities and genitourinary tract anomalies. Progressive bone marrow failure with pancytopenia often occurs in the first decade and may initially be present as thrombocytopenia or leukopenia. Solid tumors, including head and neck, skin, and genitourinary tract are more common in individuals with FA.

[0146]

[0125] There are several standard of care treatments for FA patients. Administration of oral androgens (e.g., oxymethoIone) improves blood counts (red cell and platelets) in approximately 50% of individuals with FA; granulocyte colony-stimulating factor improves the neutrophil count in some individuals; hematopoietic stem cell transplantation (HSCT) is the only curative therapy for the hematologic manifestations of FA, but the high risk for solid tumors remains and may even be increased in those undergoing HSCT. Treatment of growth deficiency, limb anomalies, ocular anomalies, renal malformations, genital anomalies, hypothyroidism, cardiac anomalies, and dermatologic manifestations as recommended by the subspecialty care provider are available to FA patients.

[0147]

[0126] Exemplary second agent or standard of care therapy include oral androgens, granulocyte colony-stimulating factor, hematopoietic stem cell transplantation (HSCT), Docket No. 32757 / 70065 treatment of growth deficiency, treatment of limb anomalies, treatment of ocular anomalies, treatment of renal malformations, treatment of genital anomalies, treatment of hypothyroidism, treatment of cardiac anomalies, treatment of dermatologic manifestations, or vitamin D supplementation.

[0148]

[0127] Although the use of ex vivo complemented autologous HSPCs overcomes many of the disadvantages of allogeneic HSCT treatments, there are also several detriments to the ex vivo manipulation and expansion of HSPCs prior to HSCT. For instance, ex vivo culture of HSPCs with cytokines to promote their expansion and for delivery of gene therapy negatively impacts their capacity for long-term repopulation (Charlesworth et al., Gene Targeting. (2018); Peterson et al., Blood 127, 2416-2426 (2016)). On top of these biological impediments there are also regulatory challenges as the ex vivo manipulation of HSPCs must be carried out at an accredited facility under good manufacturing practice conditions, substantially increasing costs. Moreover, the requirement for myeloablative preconditioning in patients prior to HSCT presents a critical risk factor and results in considerable morbidity.

[0149]

[0128] Thus, a desirable FA therapy would be to replace FA pathogenic alleles with wild type, functional alleles (i.e. gene complementation) using CRISPR / Cas9-mediated gene editing in vivo. Thus far, however, FA gene complementation has been limited by the HDR editing approaches used and the low target HSPC counts in most FA patients. For example, several preclinical studies attempted to complement FA patient primary cells with functional copies of FA genes using HDR. Unfortunately, the complementation was insufficient (<1 to 5%) due to the low HDR activity in FA cells (Osborn et al., Hum. Gene Ther. 26,114-126 (2015); Skvarova Kramarzova et al., Int. J. Mol. Sci. 18, (2017); Diez et al., EMBO Mol. Med. 9, 1574-1588 (2017)). Furthermore, correction of FA mutations by NHEJ-mediated restorative indels occurred at insufficient frequencies (Roman-Rodnguez, et al., Cell Stem Cell 25, 607-621 ,e7 (2019)) and cannot be used for many FA mutations (Dong et al., Hum. Genomics 9, 32 (2015)). However, it is shown herein that HMEJ is robust in FA cells, allowing for complementation approaches using genome editing. It is hypothesized that in vivo HMEJ-mediated complementation of FA genes in HSPCs is a sound therapeutic approach for the treatment of FA.

[0150] Homology-Mediated End Joining (HMEJ)

[0151]

[0129] Previous studies on introducing double stranded breaks (DSB) into a chromosome of interest and in donor plasmids carrying homologous sequence of the gene of interest, e.g., a FA gene, demonstrated that integration of the donor polynucleotide took place at a surprisingly high frequency and with great precision (i.e., very few indels), at least at the end harboring the homology (Nakade et al., Nat. Common. 5, 5560 (2014); Hisano et al., Sci. Docket No. 32757 / 70065

[0152] Rep. 5, 8841 (2015); Sakuma et al., Int. J. Mol. Sci. 16, 23849-23866 (2015); Sakuma et al., Nat. Protoc. 11 , 118-133 (2016); Aida et al. Genomics 17, 979 (2016)). Additional work demonstrated that introducing two DSBs into the vector to liberate a linear dsDNA donor with short homology at both ends, increased the frequency of integration and the precision (now at both ends) (Webber et al. Nat Biomed Eng (2023); Nakade et al., supra). The linear nature of the donor DNA proved important as the PCR generation of linear dsDNA donors with only 33 bp of homology also worked efficiently (Paix. et al. Proc. Natl. Acad. Sci. U. S. A. 114, E10745-E10754 (2017)). Other investigators repeated these studies and because they found a requirement for more homology (-300 to 600 bp) for high frequency integration they dubbed the process more generically as HMEJ (Yao et al., Cell Res.27, 801-814 (2017); Zhang et al., Genome Biol. 18, 35 (2017)).

[0153]

[0130] Recent studies have demonstrated that the HMEJ process can be adapted using homology arms as short as 24 to 48 bp, resulting in high frequency (20% to 100%) gene targeting at 8 different chromosomal loci in Zebrafish (Wierson et al. Elife 9:e53968, (2020)). It has also been demonstrated with homology arms of 48 bp that high frequency (generally 30+%) integration of donor DNA occurred nearly seamlessly (a remarkable near background 0.5% to 1 .5% indel / mutation frequency at the sites of integration) into the safe-harbor adeno- associated viral integration site 1 (AAVS1 ) locus in activated, primary human T-cells (Webber et al., Nat Biomed Eng (2023) doi:10.1038 / s41551 -023-01157-4.). For example, a donor DNA minicircle carrying 1 kb (HDR) or only 48 bp (HMEJ) homology arms was used for targeted integration at the AAVS1 (PPP1 R12C) locus, without, or with linearization, respectively. A successful outcome in either case required introducing a DSB at the AAVS1 locus. Importantly, HMEJ worked as well, or better, than HDR and at high frequency.

[0154] Moreover, it was found that HMEJ was well suited for large genetic cargo integration (2.6 kb at >30% and 6.3 kb >20%), which is significant since advanced genome engineering for immunotherapies requires increasingly more genetic cargo to be effective (Webber et al., supra).

[0155] Vectors

[0156]

[0131] Exemplary expression vectors include, but are not limited to, viral vectors, plasmids, nanoplasmids, lipid nanoparticles, mini-circles, or GenCircles™.

[0157]

[0132] Viral vectors include, but are not limited to the following: viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549 (1994); Borras et al., Gene Ther 6:515 524 (1999); Li and Davidson, PNAS 92:7700- 7704 (1995); Sakamoto et al., H Gene Ther 5:1088-1097 (1999); WO 94 / 12649, WO 93 / 03769; WO 93 / 19191 ; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno- Docket No. 32757 / 70065 associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86 (1998), Flanner et al., PNAS 94:6916-6921 (1997); Bennett et al., Invest Opthalmol Vis Sci 38:2857-2863 (1997); Jomary et al., Gene Ther 4:683-690 (1997), Rolling et al., Hum Gene Ther 10:641-648, (1999); Ali et al., Hum Mol Genet 5:591-594 (1996); Srivastava in WO 93 / 09239, Samulski et al., J. Vir. 63:3822-3828 (1989); Mendelson et al., Virol. 166:154-165 (1988); and Flotte et al., PNAS 90:10613-10617 (1993)); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319-23 (1997); Takahashi et al., J Virol 73:7812-7816 (1999)); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0158]

[0133] Recombinant adeno-associated viral vectors (rAAV) are small DNA viruses, with a packaging capacity of 4.7 kilobase (Kb), that have been used extensively as a vehicle for a DNA template delivery for CRISPR / Cas9 mediated transgene insertion in immune cells, including T cells1 2. The method for genome editing described here can be used to insert a promoter or splice acceptor to drive expression of a transgene encoding a secreted protein or enzyme using either a rAAV or a plasmid as an HDR or HMEJ DNA donor template. In various embodiments, the AAV vector is an AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, or AAV-DJ vector.

[0159]

[0134] Adenoviral vectors are non-enveloped double-stranded DNA vectors that can be rendered replication-deficient by deletion of the E1 region of the viral genome. Typical adenoviral vectors have a packaging capacity of up to 7.5 kbp. Adenoviral vectors are commonly used in gene therapy clinical trials. Useful adenoviral vectors are described in Wold et al., (Curr Gene Ther. 2013 Dec; 13(6): 421-433), hereby incorporated by reference.

[0160]

[0135] Lentiviral vectors are enveloped retroviruses with single stranded RNA genomes. Lentivirus is capable of infecting dividing and non-dividing cells. Lentivirus has a genome capacity of approximately 10 kb.

[0161]

[0136] Plasmids contemplated herein include, e.g., naked plasmids or contained in liposomes or another delivery vehicle. A nanoplasmid™” refers to a 500 base-pair circular plasmid lacking traditional bacterial genes or antibiotic resistant genes. The nanoplasmid construct reduces cellular toxicity and inflammation of the plasmid-transfected cells, when compared to traditional plasmid.

[0162]

[0137] Mini-circles similarly lack bacterial genes and are small circular plasmid derivatives of approximately 4kb that lack bacterial polynucleotide sequences. Mini-circles can be non Docket No. 32757 / 70065 replicating and lack an origin of replication, or can be modified to comprise a non-bacterial replication element (e.g., a S / MAR element).

[0163]

[0138] Vectors for transposon delivery of genetic information are also contemplated. The vectors comprise inverted terminal repeats flanking the polynucleotide encoding a gene of interest, e.g., a FA gene, for transposon delivery. The vector for transposon delivery comprises a promoter, a gene of interest, e.g., a FA gene, a biomarker, a regulatory element and optionally a chimeric intron. In various embodiments, the transposon is selected from the group consisting of Tn5, Tn3, Tn10, Sleeping Beauty, piggyBac, and Tol2.

[0164]

[0139] Lipid nanoparticles are an attractive delivery platform due to their low immunogenicity and ability to target specific tissues in vivo, including HSPCs. Lipid nanoparticles have been approved for use in delivering mRNA vaccines, and methods of making LNPs are known in the art. See e.g., Mehta et al., ACS Materials Au 3:600-619, 2023 and Cullis et al., Nature Reviews Drug Discovery 23:709-722, 2024. Micelle based nanoparticles are also contemplated for use herein.

[0165] Genome Engineering

[0166]

[0140] CRISPR / Cas and other nuclease-based gene editing systems open a new avenue to altering a gene of interest by creating double stranded breaks (DSB), leading to formation of small insertions or deletions created by semi-random repair via the Non-Homologous End Joining (NHEJ) pathway. Alternatively, precise genome modifications can be achieved by the introduction of CRISPR / Cas9 to induce a DSB along with a DNA template for Homology directed repair (HDR). A DNA template can be designed to encode a transgene of interest such as for a FA gene or encode a therapeutic protein / enzyme that can be used for cancer immunotherapy or protein / enzyme deficiency, respectively.

[0167]

[0141] In various embodiments, the method includes a technique to introduce a protein or nucleic acid into a target cell or population of target cells. Any suitable method of introducing a protein or nucleic acid may be used. In various embodiments, the method includes electroporation of a target cell or population of target cells to introduce genetic material including, for example, DNA, RNA, and / or mRNA. As used herein, electroporation may include nucleofection. Because plasmid DNA can be toxic to some cells, in some embodiments, mRNA or protein based approaches of genome editing are used. In various embodiments, a technique to introduce a protein or nucleic acid can include introducing a protein or nucleic acid via electroporation; microinjection; exosomes; liposomes; biolistics; jet injection; hydrodynamic injection; ultrasound; magnetic field-mediated gene transfer; electric pulse-mediated gene transfer; use of nanoparticles including, for example, lipid-based nanoparticles; incubation with an endosomolytic agent; use of cell-penetrating peptides; etc. Docket No. 32757 / 70065

[0168] In various embodiments, the method includes electroporation of a cell using a NEON transfection system, Lonza transfection system, or MaxCyte transfection system.

[0169]

[0142] In various embodiments, the method includes editing a gene, e.g., a FA gene. In various embodiments, the method includes editing a gene associated with a DNA repair deficiency. Editing a gene can include introducing one or more copies of the gene, altering the gene, deleting the gene, upregulating expression of the gene, downregulating expression of the gene, mutating the gene, methylating the gene, demethylating the gene, acetylating the gene, and / or deacetylating the gene. Mutating the gene can include introducing activating mutations, introducing inactivating and / or inhibitory mutations, and / or introducing point mutations.

[0170]

[0143] In various embodiments, the method includes inducing double stranded breaks in the genome of the method for genome engineering a hematopoietic stem and progenitor cell (HSPC). Double stranded breaks may be introduced using a nuclease dependent cleavage system including, for example, a transcription activator-like effector nucleases (TALEN), a zinc finger nuclease (ZFN), a CRISPR-associated nuclease, etc.

[0171]

[0144] Double stranded breaks may also be introduced using a transposase delivery system, or a nuclease dependent cleavage system including, for example, a transcription activator-like effector nucleases (TALEN), a zinc finger nuclease (ZFN), a CRISPR- associated nuclease, etc.

[0172]

[0145] If a CRISPR / Cas system is used, it includes use of a guide RNA (gRNA) or DNA (gDNA) targeting molecule. The gRNA target or gDNA target can include any suitable target. In various embodiments, the target includes a portion of the HSPC genome including, for example, a FA gene or a portion of a FA gene. In various embodiments, the target includes a portion of the HSPC genome including, for example, a gene associated with a DNA repair deficiency or a portion of said gene.

[0173]

[0146] Proposed herein is use of clustered regularly interspaced short palindromic repeats (CRISPR) to produce three DSBs to generate a chromosome with a DSB at the site of interest and a linear dsDNA donor with homology arms between 35 and 1000 nucleotides. In various embodiments, the homology arms are 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 nucleotides, or any value within these ranges. In various embodiments, the homology arms are approximately 40 bp homology arms. The relevant four ends then undergo 5’ > 3’ resection and reveal the regions of homology.

[0174]

[0147] The disclosure herein provides a method for genome engineering a target cell or a population of target cells to express a FA gene comprising introducing into the target cell or Docket No. 32757 / 70065 cell population a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle, comprising an expression cassette comprising a homology arm (HA), a polynucleotide encoding a FA gene, a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted at a locus targeted by the targeting molecule.

[0175]

[0148] The disclosure herein provides a method for genome engineering a target cell or a population of target cells to express a gene associated with a DNA repair deficiency comprising introducing into the target cell or cell population a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle, comprising an expression cassette comprising a homology arm (HA), a polynucleotide encoding a gene associated with a DNA repair deficiency, a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted at a locus targeted by the targeting molecule.

[0176]

[0149] In various embodiments, the target cell is a pluripotent stem cell, a HSPC, other stem cell, an epithelial cell, a fibroblast. In gene therapy for FA, it is contemplated that the target cell includes all cell types in the subject in order to correct a genetic deficiency. Such outcome is also contemplated when a gene associated with a DNA repair deficiency is used in a gene therapy method as described herein.

[0177]

[0150] The disclosure herein provides a method for genome engineering a target cell or a population of target cells to express a FA gene comprising introducing into the target cell or target cell population a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm (HA), a polynucleotide encoding a FA gene, a splice acceptor site, a promoter and a targeting site for transposon delivery of the FA gene, wherein the expression cassette is inserted at a locus targeted by the targeting molecule.

[0178]

[0151] The disclosure herein provides a method for genome engineering a target cell or a population of target cells to express a gene associated with a DNA repair deficiency comprising introducing into the target cell or target cell population a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle, comprising an expression cassette comprising a homology arm (HA), a polynucleotide encoding a gene associated with a DNA repair deficiency, a splice acceptor site, a promoter and a targeting site for transposon delivery of the gene associated with a DNA repair deficiency, wherein the expression cassette is inserted at a locus targeted by the targeting molecule.

[0179]

[0152] Also provided is a method for genome engineering a target cell or a population of target cells to express a FA gene comprising introducing into the target cell or target cell Docket No. 32757 / 70065 population a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle, comprising an expression cassette comprising a homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted upstream of a FA gene to be expressed.

[0180]

[0153] Also provided is a method for genome engineering a target cell or a population of target cells to express a gene associated with a DNA repair deficiency comprising introducing into the target cell or target cell population a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle, comprising an expression cassette comprising a homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule or a targeting site for transposon delivery, wherein the expression cassette is inserted upstream of a gene associated with a DNA repair deficiency to be expressed.

[0181]

[0154] Also provided is a method for genome engineering a target cell or a population of target cells, e.g., a HSPC, to express a FA gene comprising introducing into the target cell or target cell population a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle, comprising an expression cassette comprising a homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site for transposon delivery of a FA gene, wherein the expression cassette is inserted upstream of a FA gene to be expressed.

[0182]

[0155] In some embodiments, where transfection may be used to deliver a CRISPR / Cas9 system, the gRNA may preferably include a chemically modified gRNA. In some embodiments, the chemical modification to the gRNA preferably decreases a cell’s ability to degrade the RNA. In some embodiments, a chemically modified gRNA includes one or more of the following modifications: 2'-fluoro (2'-F), 2'-O-methyl (2'-0-Me), S-constrained ethyl (cEt), 2'-O-methyl (M), 2'-0-methyl-3'-phosphorothioate (MS), and / or 2'-O-methyl-3'- thiophosphonoacetate (MSP). In some embodiments, the chemically modified gRNA can include a gRNA and / or a chemical modification described in Hendel et al, Nature Biotechnology, 2015, 33(9):985-989 or Rahdar et al., PNAS, 2015, 112(51 ):E7110-7.

[0183]

[0156] Introduction of the plasmid, nanonplasmid, lipid nanoparticle, mini-circle, or GenCircle into a cell can be performed by transfection or electroporation.

[0184]

[0157] Introduction of a viral vector is by electroporation. In various embodiments, the viral vector is introduced at a multiplicity of infection (MOI) of 3 x 105- 1 x 107. In various embodiments, the MOI is between 5 x 105- 1 x 107, between 5 x 105- 5 x 106, between 5 x

[0185] 105- 1 x 106or between 3 x 105- 1 x 106. Docket No. 32757 / 70065

[0186]

[0158] A vector for transposon delivery is electroporated with transposase mRNA and an expression cassette for delivery of a transposon expressing the FA gene. A vector for transposon delivery is electroporated with transposase mRNA and an expression cassette for delivery of a transposon expressing a gene associated with a DNA repair deficiency.

[0187]

[0159] It is contemplated that the present method provides efficient transfer of the FA gene and provides improved viability of the cells after genome modification. For example, the efficiency of transfer of the FA gene, or overexpression of an endogenous polynucleotide is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more. Such outcome is also contemplated when a gene associated with a DNA repair deficiency is used in a gene therapy method as described herein.

[0188]

[0160] In various embodiments, the method includes selecting a gene edited cell. In various embodiments, the selection is performed after editing a gene. A gene edited cell can, in various embodiments, be selected using one or more of the following methods: flow sorting (including, for example, for cell surface marker expression); magnetic bead separation (including, for example, targeting a cell-surface marker); transient drug resistance gene expression (including, for example, antibiotic resistance).

[0189]

[0161] In various embodiments, the method includes expanding a population of gene edited cells in culture. In various embodiments, the expansion can be performed after selecting the gene edited cell. In various embodiments, a gene edited cell can be expanded (e.g., stimulating, proliferating or activating) by co-incubation with a cytokine or ligand including cell activation. For example, stimulating, proliferating or activating an HSPC or population of HSPC comprises contacting the cell(s) with one or more of IL-3, IL-6, SCF, Flt3L, TPO, GM- CSF, G-CSF and prostaglandin E2.

[0190] Nuclease dependent cleavage systems

[0191]

[0162] CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) is an RNA-mediated adaptive immune system found in bacteria and archaea, which provides adaptive immunity against foreign nucleic acids (Wiedenheft et al., Nature (2012) 482:331-8; Jinek et al., Science (2012) 337:816-21). Recent studies have shown that the biological components of this system can be used to modify the genome of mammalian cells. CRISPR-Cas systems are generally defined by a genomic locus called the CRISPR array, a series of 20-50 base-pair (bp) direct repeats separated by unique “spacers” of similar length and preceded by an AT-rich “leader” sequence (Wright et al., Cell (2016) 164:29-44).

[0192]

[0163] Three types of CRISPR / Cas systems exist, type I, II and III. The Type II CRISPR-Cas systems require a single protein, e.g., Cas9, to catalyze DNA cleavage (Sapranauskas et al., Docket No. 32757 / 70065

[0193] Nucleic Acids Res. (2011 ) 39(21): 9275-9282). Cas9 serves as an RNA-guided DNA endonuclease. Cas9 generates blunt double-strand breaks (DSBs) at sites defined by a 20- nucleotide guide sequence contained within an associated CRISPR RNA (crRNA) transcript. Cas9 requires both the guide crRNA and a trans-activating crRNA (tracrRNA) that is partially complementary to the crRNA for site-specific DNA recognition and cleavage (Deltcheva et al., Nature (2011 )4 71 (7340):602-7; Jinek et al., Science (2012) 337:816-21 ).

[0194]

[0164] The crRNA:tracrRNA complex can be synthesized as two separate molecules or as a single transcript (single-guide RNA or sgRNA) encompassing the features required for both Cas9 binding and DNA target site recognition. Using sgRNA, Cas from bacterial species, such as S. pyogenes, can be programmed to cleave double-stranded DNA at any site defined by the guide RNA sequence and including a protospacer-adjacent (PAM) motif (Sapranauskas et al., Nucleic Acids Res. (2011) 39(21): 9275-9282; Jinek et al., Science (2012) 337:816-21). The DSBs result in either non-homologous end-joining (NHEJ), which is error-prone and conducive to frameshift mutations that knock out gene alleles, or homology- directed repair (HDR), which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome. Therefore, in the presence of a homologous repair donor, the CRISPR / Cas9 system may be used to generate precise and defined modifications and insertions at a targeted locus through the HDR process. In the absence of a homologous repair donor, single DSBs generated by CRISPR / Cas9 are repaired through the error-prone NHEJ, which results in insertion or deletion (indel) mutations.

[0195]

[0165] Other publications describing the CRISPR systems and Cas9, include the following Cong et al. Science (2013) 339:819-23; Jinek et al., eLife 2013;2:e00471 . (2013) 2:e00471 ; Lei et al. Cell (2013) 152: 1173-1183; Gilbert et al. Cell (2013) 154:442-51 ; Lei et al. eLife (2014) 3:e04766; Perez-Pinela et al. Nat Methods (2013) 10: 973-976; Maider et al. Nature Methods (2013) 10, 977-979 which are incorporated by reference. The following U.S. and international patents and patent applications describe the methods of use of CRISPR, 8,697,359; 8,771 ,945; 8,795,965; 8,865,406; 8,871 ,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814; 8,945,839; 8,993,233; 8,999,641 ; 2014 / 0068797; and WO 2014 / 197568, each of which is incorporated by reference in their entirety.

[0196]

[0166] The CRISPR related protein, Cas9, can be from any number of species including, but not limited to, Streptococcus pyogenes, Staphylococcus aureus, Listeria innocua, and Streptococcus thermophilus. Docket No. 32757 / 70065

[0197]

[0167] Additional Cas proteins known in the art are contemplated for use in the methods, including Cas12a (Cpf 1 ) and Cas 13a / Cas13b (56). See also Yan et al., Cell Biology and Toxicology 35:489-492 (2019).

[0198]

[0168] In various embodiments, the nuclease dependent cleavage system comprises a CRISPR / Cas system, a Cas-CLOVER system, a zinc-finger nuclease (ZFN) system, a transcription activator like effector nuclease (TALEN) system, or a meganuclease system. In various embodiments, the CRISPR / Cas system comprises Cas9, Cas12a, Cas13a, or Cas13b.

[0199]

[0169] Cas-CLOVER™ systems are recently designed gene editing systems that utilize the Clo51 nuclease instead of the CRISPR protein. Cas-CLOVER™ comprises a nuclease- inactivated Cas9 protein fused to the Clo51 endonuclease. Cas-CLOVER uses two guide RNAs as well as a nuclease activity that requires dimerization of subunits associated with each guide RNA to provide target specificity.

[0200]

[0170] In one embodiment, the methods use a CRISPR-Cas system and one or more guide RNAs, repair templates and HDR to insert nucleotide bases into the genome at a FA cell locus. In various embodiments, the locus is the AAVS1 (PPP1 R12C) or FA gene locus. In various embodiments, the locus is the AAVS1 (PPP1R12C) or locus for a gene associated with a DNA repair deficiency.

[0201] Gene Therapy

[0202]

[0171] One approach for gene therapy in DNA repair deficiencies, such as FA, would be to circumvent the ex vivo manipulation of HSPCs and HSCT by executing the genetic complementation of HSPCs in vivo. Recent reports have demonstrated the promise of in vivo delivery of genome editing reagents through the use of recombinant adeno-associated virus (rAAV). A number of studies have delivered Cas9 nuclease and associated enzymes to HSPCs in vivo using rAAV48. In a mouse model of Hutchinson-Gilford progeroid syndrome - in which a dominant negative C*G-to-T*A mutation (c.1824 C>T; p.G608G) in Lamin A (Lmna) leads to the production of a toxic protein that induces rapid aging - a single injection of rAAV, encoding an adenosine base editor (ABE) resulted in a significant and long-lasting correction of the mutation, reduced levels of progeria, and rescued vascular pathology (Koblan et al., Nature 589, 608-614 (2021)). Furthermore, a single ABE injection at postnatal day 14 increased the lifespan of the mice from an average of 215 to 510 days (Koblan, supra).

[0203]

[0172] Provided herein is a therapy to replace DNA repair deficiency pathogenic alleles with wild type, functional alleles (i.e. gene complementation) using CRISPR / Cas9-mediated gene editing in vivo. Provided herein is a method for genome engineering a hematopoietic stem Docket No. 32757 / 70065 and progenitor cell (HSPC) or a population of HSPC to express cDNA of a gene associated with DNA repair from an endogenous locus comprising introducing into the HSPC cell or population of HSPC a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm (HA), a polynucleotide encoding a gene associated with a DNA repair deficiency, a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted at a locus targeted by the targeting molecule.

[0204]

[0173] Also provided is a method for genome engineering a hematopoietic stem and progenitor cells (HSPC) or a population of HSPC to overexpress an endogenous DNA repair gene comprising introducing into the HSPC or population of HSPC a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted upstream of a DNA repair gene to be overexpressed.

[0205]

[0174] Provided herein is an FA therapy to replace FA pathogenic alleles with wild type, functional alleles (i.e. gene complementation) using CRISPR / Cas9-mediated gene editing in vivo.

[0206]

[0175] Provided herein is a method for genome engineering a hematopoietic stem and progenitor cell (HSPC) or a population of HSPC to express FA gene cDNA from an endogenous FA locus comprising introducing into the HSPC cell or population of HSPC a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm (HA), a polynucleotide encoding a FA gene, a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted at a locus targeted by the targeting molecule.

[0207]

[0176] Also provided is a method for genome engineering a hematopoietic stem and progenitor cells (HSPC) or a population of HSPC to overexpress an endogenous FA gene comprising introducing into the HSPC or population of HSPC a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted upstream of a FA gene to be overexpressed.

[0208]

[0177] In various embodiments, the homology arms are between 35 and 1000 nucleotides. In various embodiments, the homology arms are from 50-900 nucleotides, 50-750 Docket No. 32757 / 70065 nucleotides, 100-600 nucleotides, 100-500 nucleotides, or 200-400 nucleotides. In various embodiments, the homology arms are 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 nucleotides.

[0209]

[0178] In various embodiments, the method further comprises transfecting the HSPC or population of HSPC with a Cas protein or polynucleotide encoding a Cas protein and guide RNA molecules that direct integration of the expression cassette to a target locus in the HSPC genome.

[0210]

[0179] The disclosure provides a method of making a gene edited hematopoietic stem and progenitor cell (HSPC) or population of HSPC, comprising:

[0211]

[0180] i) contacting a HSPC or population of HSPC with a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, and optionally comprising a polynucleotide encoding a gene associated with FA;

[0212]

[0181] ii) culturing the HSPC or population of HSPC of i) in a media that promotes expansion of HSPC cells;

[0213]

[0182] ill) isolating the HSPC or population of HSPC of ii) based on identification of a marker expressed only on a HSPC or population of HSPC carrying the viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle;

[0214]

[0183] iv) culturing the isolated cells of iii) in a culture medium to expand the isolated cells expressing the FA gene.

[0215]

[0184] Methods described herein for making an HSPC or population of HSPC comprising a FA gene are adapted to provide a method of making an HSPC or population of HSPC comprising a gene associated with a DNA repair deficiency, e.g., for cellular or gene therapy to treat a DNA repair deficiency.

[0216]

[0185] The disclosure provides a method of treating Fanconi Anemia in a subject in need thereof comprising administering to the subject an expression cassette as described herein or a vector comprising an expression cassette as described herein.

[0217]

[0186] Also provided is a method of treating Fanconi Anemia (FA) in a subject in need thereof comprising administering to the subject a gene edited HSPC or population of HSPC as described herein. Docket No. 32757 / 70065

[0218]

[0187] In various embodiments, the administration alleviates one or more symptoms of FA selected from the group consisting of bone marrow failure, increased risk for malignancy, short stature, abnormal skin pigmentation, skeletal malformations of the upper and / or lower limbs, microcephaly, ophthalmic and genitourinary tract anomalies, pancytopenia, thrombocytopenia, or leukopenia. In various embodiments, the treatment described herein increases red blood cell counts in a subject receiving treatment.

[0219]

[0188] Also provided is a method for treating a DNA repair deficiency, such as xeroderma pigmentosum, ataxia telangiectasia, Nijmegen Breakage Syndrome, Bloom Syndrome, Werner Syndrome, Rothmund-Thompson Syndrome, DNA Ligase IV Deficiency Syndrome (LIG4 syndrome), trichothiodystrophy, progeria (Hutchinson-Gilford progeria syndrome) and Cockayne syndrome, comprising administering to the subject a gene edited HSPC or population of HSPC comprising a gene associated with a DNA repair deficiency as described herein.

[0220] Kits

[0221]

[0189] The disclosure also provides kits which comprise one or more compounds or compositions packaged in a manner which facilitates their use to practice methods of the disclosure. In one embodiment, such a kit includes a compound or composition described herein (e.g., a composition comprising expression vector, viral vector or population of cells comprising an expression cassette comprising a gene associated with FA as described herein, or in combination with another agent), packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition in practicing the method. The kit may also comprise a compound or composition described herein (e.g., a composition comprising expression vector, viral vector or population of cells comprising an expression cassette comprising a gene associated with gene associated with a DNA repair deficiency as described herein.

[0222]

[0190] Preferably, the compound or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition according to a specific route of administration or for practicing a screening assay. Preferably, the kit contains a label that describes use of the particle compositions.

[0223]

[0191] Additional aspects and details of the disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting.

[0224] EXAMPLES Docket No. 32757 / 70065

[0225] Example 1 -Preparing Vectors Comprising a Gene Associated with Fanconi Anemia

[0226]

[0192] The use of ex vivo genetically complemented autologous HSPCs for hematopoietic stem cell transplantation (HSCT) has shown some success in the treatment of FA, however, there is substantial cost, risk, and mortality associated with the process. The ability to directly complement FA HSPCs in vivo could produce cures while circumventing the risks and costs associated with HSCT and offer an opportunity to address the non-hematopoietic manifestations of FA.

[0227]

[0193] FANC-A is the most commonly mutated gene in FA patients, accounting for approximately two-thirds of FA patient mutations (Moreno, Biomed Rep 15, 74 (2021)). To develop a vector to correct the FANCA mutations, rAAV (serotype 6) delivery of a HMEJ Fanca donor, Cas9, and gRNA to insert the corrected Fanca cDNA into the endogenous Fanca locus is generated, co-opting endogenous transcriptional activity. rAAV is a single stranded DNA virus that rapidly becomes double stranded upon entry to the nucleus and is therefore an ideal substrate for HMEJ (Weitzman, et al., Methods Mol. Biol. 807, 1-23 (2011); Wang et al. Proc. Natl. Acad. Sci. U. S. A. 104, 13104-13109 (2007); Ye et al. Nat Biomed Eng (2023) doi:10.1038 / s41551 -023-01058-6). The method utilizes (i) a rAAV expressing a wild-type Fanca cDNA (e.g., 4.2 kb) between HMEJ homology arms (48 bp) and (ii) a rAAV expressing a gRNA that facilitates Cas9 cleavage at Fanca exon 1 and that simultaneously cuts the HMEJ template out of the donor vector using the same gRNA. This rAAV will also express Staphylococcus aureus Cas9 (SaCas9), an engineered Cas9 homolog that is only 1053 amino acids (aa) long (compared to the full-length Streptococcus pyogenes Cas9 (SpCas9), which is 1368 aa) and which is effective at in vivo gene editing (Ran et al., Nature 520, 186-191 (2015)). Both vectors are within the size limitations of rAAV (-4.9 kb) (Samulski et al. AAV-Annu Rev Virol 1 , 427-451 (2014)). A control HDR-based rAAV vector with -300 bp homology arms, which will reach the cargo capacity limit of rAAV. gRNAs will be used (Webber et al., supra; Laoharawee et al. Int. J. Mol. Sci. 23, (2022);

[0228] Rollins et al., Nat. Commun. 14, 528 (2023); Kluesner et al., Nat. Commun. 12, 2437 (2021); Webber et al., Nat. Commun. 10, 5222 (2019); Pomeroy et al. Mol. Ther. 28, 52-63 (2020); Johnson et al., Sci. Rep. 8, 12144 (2018)). An optimal Fanca exon 1 gRNA is utilized for DSB induction at the Fanca exon 1 and to liberate the HMEJ template through incorporation of the gRNA target sequencing in the HMEJ vector (Figure 1).

[0229]

[0194] 72 hrs post-transduction, the percentage of HMEJ-mediated Fanca complementation in wild type or HCT116 Fanca- / - cells is measured by droplet digital PCR (ddPCR) (Webber et al., Nat. Commun. 10, 5222 (2019)). In addition to studying Fanca complementation in HCT116, HMEJ studies in FA patient derived lymphoblastoid cells are also carried out (Sipe Docket No. 32757 / 70065 et al., Int. J. Mol.Sci. 23, (2022)). Control groups include sham infections and infections with the HMEJ-donor or Cas9 / gRNA rAAVs alone. As a positive control, HMEJ-donor and Cas9 / gRNA rAAV plasmids are transfected via electroporation to confirm the infection results.

[0230]

[0195] Quantification of HDR- and HMEJ-mediated integration of EGFP at the PPP1 R12C locus in HCT116 WT, FANCA- / -, and FANCD2- / - cells was carried out. Results are shown in Figure 2. It is demonstrated herein for the first time that HMEJ-mediated targeted integration is effective in Fanconi anemia (FA)-deficient cells as evidenced by detectable expression in Fane deficient cells.

[0231]

[0196] Statistical considerations: In Vitro Studies: Two-tailed Student’s t-tests are used to analyze the differences between frequency of average CFU formation, percentage LSK, etc. in FA-proficient and -deficient cells. If more than two groups are being compared, a one-way ANOVA test is used. Experiments are performed in triplicate and three or more independent replicates will be used for analyses per genotype. The average percentage of radials per metaphase spread (n > 50 / genotype) is analyzed by a two-tailed Mann-Whitney test and the MMC-sensitivity assay (three independent experiments) will be analyzed by a two-way ANOVA. In Vivo Studies: For the rAAV treatment and competitive repopulation studies, the sample size will be 12 donor mice per group (untreated or treated). This sample size is based on the following parameters calculated using G*Power 3.1 software (Kang, H. J.

[0232] Educ. Eval. Health Prof. 18, 17 (2021); Faul et al. Behav. Res. Methods 39, 175-191 (2007)): effect size = 1 .2, a = 0.05, and power = 0.8. A two-tailed Student’s t-test is used to analyze the difference between the average percentage Y chromosome DNA in peripheral blood from recipients of BM HSPCs isolated from untreated or treated Adh5- / -:Fanca- / - mice.

[0233]

[0197] In an additional reporter assay, it was demonstrated that HMEJ-mediated integration of the whole FANCA cDNA into FANCA exon 1 of HCT 116 FANCA- / - cells (Figure 2). This is unexpected since hFANCA cDNA is very large (~4.3 kb) yet 5-7% knock-in efficiencies were still observed. This also demonstrates in vitro that HMEJ is useful for gene complementation.

[0234]

[0198] It is hypothesized that HMEJ-mediated FANCA / Fanca complementation will functionally rescue FA cellular phenotypes. To test this, in parallel to collecting cells for genomic analyses 72 hrs post-transduction, unedited control and rAAV-transduced cell populations are treated with mitomycin C (MMC), an ICL-producing chemotherapeutic drug, and (i) measure MMC-induced FANCD2 mono-ubiquitination via Western blot analysis, an indication of FA pathway activation, (ii) perform cell viability assays using Promega’s Cell Titer Gio 2 Kit, and (iii) score metaphase spreads for FA-associated chromosomal radial Docket No. 32757 / 70065 formations (“radials”) using confocal microscopy (Rogers et al., Cell Rep. 42, 112428 (2023); Webber et al., Nat. Commun. 10, 5222 (2019)).

[0235]

[0199] Cas9 mediated HMEJ

[0236]

[0200] Generation of Cas9-induced DSBs is suboptimal for gene therapy as it can lead to undesired on- and off-target mutagenesis and translocations. Since FA cells are inherently chromosomally unstable, a modified HMEJ approach compatible with Cas9 nickase was developed. Using two juxtaposed sgRNAs and Cas9 nickase has been shown to significantly reduce off-target mutagenesis and translocations compared to Cas9 nuclease. To assess the feasibility of this approach for HMEJ in FA-deficient cells, Cas9 nuclease / single sgRNA SA-EGFP HMEJ reporter assay as described above was compared to Cas9 nickase / dual sgRNA SA-EGFP HMEJ reporter assay (Figure 3A). These assays were performed in patient-derived E4 / VCD2-deficient PD20 fibroblasts (and a FANCD2 complemented control line), E4 / VCD2-proficient and -deficient HCT116 cells, and patient-derived lymphoblastoid cell lines (LCLs) with (UMN20 WT) or without (FA55 hMUT) FANCA expression. Surprisingly, it was found that Cas9 dual nickase-mediated HMEJ occurs at similar levels as Cas9 nuclease-mediated HMEJ in all three cell types (Figures 3B-D).

[0237]

[0201] AA V delivered FANCA genes

[0238]

[0202] Since HMEJ utilizes much shorter homology arms than HDR, it was postulated that the HMEJ FANCA cDNA donor could be delivered using a recombinant adeno-associated virus (rAAV) vector. FANCA cDNA is greater than 4.3 kb; therefore, with 48bp HMEJ homology arms (but not larger HDR homology arms), the donor fits within the size capacity of rAAV (i.e. -4.9 kb). To test this hypothesis, patient-derived FANCA ' LCLs were transfected with Cas9 nuclease mRNA and a sgRNA targeting FANCA exon 1 and transduced with rAAV-hFANCA-HMEJ-Nuclease donor (MOI 105; Figure 4, left panel). The percentage of hFANCA KI was measured at the endogenous FANCA locus by junction droplet digital PCR (ddPCR). While KI levels were low initially in LCLs, FANCA complemented cells were enriched for by culturing LCLs with medium containing MMC (Figure 5A). Phenotypic rescue was also measured by quantifying MMC sensitivity using a 72-hour ATPase-based cell viability assay (CellTiter-Glo) and by assessing MMC-induced FANCD2 monoubiquitination. It was found that a bulk population of FANCA complemented cells (-20% EA / VCA-complemented) exhibited (i) partial rescue of the MMC-hypersensitivity exhibited by the control FANCA ' LCLs (Figure 5B), (ii) expression of full-length FANCA protein, and (iii) MMC-induced FANCD2 monoubiquitination. Next, patient-derived FANCA7' LCLs were transfected with Cas9 nickase mRNA and two sgRNA targeting FANCA exon 1 and transduced with rAAV-hFANCA-HMEJ-Dual Nicking donor (MOI 106; Figure 4, right Docket No. 32757 / 70065 panel). Again, while initial FANCA KI is low in LCLs, as determined by junction ddPCR, FANCA complemented cells were enriched for by culturing in 50 nM MMC (Figure 5C).

[0239]

[0203] Taken together, the data supports the use of HMEJ for FANCA complementation using rAAV delivery. This approach has never been investigated for the treatment of patients with FA. Moreover, it was demonstrated that dual nicking is amenable to HMEJ-mediated KI.

[0240] Example 2 - In Vivo Analysis of HMEJ Effect of FA Cells

[0241]

[0204] After validating HMEJ-mediated Fanca complementation in human cells, similar rAAVs that contain a mouse Fanca cDNA are generated and targeted to the Fanca locus and perform HMEJ complementation assays in mouse embryonic fibroblasts (MEFs) derived from a Fanca- / - mice.

[0242]

[0205] To evaluate the efficacy of in vivo HMEJ-mediated Fanca complementation and phenotypic rescue, 129 S / v Adh5- / -:Fanca- / - mice are used. Alcohol dehydrogenase 5 (ADH5) catabolizes formaldehydes and prevents the accumulation of ICL-inducing reactive aldehydes, currently considered one of the main sources of endogenous DNA damage leading to HSPC attrition in the BM of FA patients (Pontel et al., Mol. Cell 60, 177-188 (2015)). Adh5- / -:Fanca- / - mice bred from Adh5- / - Fanca+ / - pairings are born at a Mendelian ratio (i.e. 25% liveborn double mutants). However, Ad h5- / - Fanca- / - mice spontaneously develop bone marrow failure (BMF) early in life and die by approximately 2-3 months of age from pancytopenia. This novel FA BMF mouse model is used to study in vivo HMEJ complementation.

[0243]

[0206] Murine specific rAAV described above are delivered intravenously at weaning using doses based on reports of in vivo editing in mouse models of human disease using rAAV (Koblan et al., Nature 589, 608-614 (2021); Chemello et al. Sci Adv 7, (2021); Yeh et al. Sci. Transl. Med. 12, (2020); Villiger et al. Nat. Med. 24, 1519-1525 (2018)) (i.e. 1 x 1011viral genomes per mouse). rAAV serotype 6 is used as the vector, as this vector has tropism to the BM niche and HSPCs (Fananas-Baquero et al. Mol Ther Methods Clin Dev 22, 237-248 (2021); Pandya et al., Cell Biol. 92, 116-123 (2014); Song et al. Cytotherapy 15, 986-998 (2013)). Equal numbers of female and male mice are used (12 animals per group). Control groups will include sham-treated and treatment with the HMEJ-donor or Cas9 / gRNA rAAVs alone. To determine the rate of correction in vivo, a separate cohort of mice 4-, 8-, or 16- weeks are euthanized post-injection, bone marrow processed into single-cell suspensions, and HSPCs (Lineage- Sca1 + c-Kit+; “LSK”) isolated via flow cytometry. NGS of the Fanca locus in multipotent HSPCs is carried out using MiSeq and analyze the percentage of BM Docket No. 32757 / 70065

[0244] HSPCs with Fanca complementation. DNA is collected from various tissues (e.g. liver, lung, kidney, spleen, heart, muscle, brain) to assess correction of somatic cells using the same approach. Off-target editing in these samples is assessed by NGS at the top 30 computationally predicted off-target sites, using Guide-seq, and TLA analysis.

[0245]

[0207] In addition, BM samples from Adh5- / -:Fanca- / - mice 4-, 8-, or 16-weeks posttreatment with rAAV are cultured and assessed for MMC-induced genomic instability phenotypes compared with animals treated without HMEJ donor rAAV controls as well as Adh5- / -:Fanca+ / + control mice. These phenotypes include MMC-induced FANCD2 mono- ubiquitination, cellular sensitivity to MMC treatment, and MMC-induced radial formation. Post-editing, BM samples will also be used to assess the HSPC population size (by flow cytometry) and function (colony forming unit (CFU) assays). To further demonstrate that gene corrected HSPCs are functional, a competitive repopulation assay is performed. Briefly, 2 x 105male BM cells from Adh5- / -: Fanca- / - mice (untreated or treated with the rAAV-HMEJ-donor alone) will be mixed with 2 x 105female BM cells from Adh5- / -:Fanca+ / + mice and injected intravenously into sub-lethally irradiated (500 cGy) female Adh5- / - :Fanca+ / + mice, as described (Xiao et al. J. Immunol. 199, 2701-2712 (2017); Tomaszowski et al. Nat. Common. 14, 1333 (2023)). Three experiments, each involving transplantation of BM cells from independent Adh5- deficient Fanca+ / +, Fanca- / - (unedited control), and Fanca- / - (rAAV-HMEJ-complemented) mice into four recipients, are performed. To control for the contribution of endogenous hematopoiesis in the recipient mice, 2 x 105female BM cells from Adh5- / -:Fanca+ / + mice are transplanted into male Adh5- / -:Fanca+ / + mice. Sixteen weeks post-transplant, the recipient mice are euthanized, and genomic DNA collected from peripheral blood to quantify the relative contribution of the Fanca mutant and wild type BM to peripheral blood chimerism using qPCR for the presence of the Y chromosome, as described (Tomaszowski et al. Nat. Common. 14, 1333 (2023); Kimura et al., Proc. Natl. Acad. Sci. U. S. A. 95, 1195-1200 (1998)). It is expected that the frequency of Y chromosome DNA in blood cells post-transplantation is significantly increased in recipients of rAAV-HMEJ-complemented Adh5- / -:Fanca- / - mice compared to recipients of unedited control Adh5- / -:Fanca- / - mice.

[0246]

[0208] It is hypothesized that the present HMEJ-mediated knock-in strategy will be similarly efficient in WT and FA-deficient cells in vitro and in vivo, representing a highly desired FA gene complementation approach. Notably, case reports from patients with (rarely occurring) somatic reversion of their FA mutation suggest that one or a few functionally corrected HSPCs may be sufficient to rescue the hematologic manifestations of FA (Mankad et al., Blood 107, 3084-3090 (2006)). It is also likely that complementation in somatic cells from various tissues will occur, particularly the liver where rAAV typically accumulate (Zhang et Docket No. 32757 / 70065 al., J. Control. Release 240, 332-348 (2016); Pupo et al. Mol. Ther. 30, 3515-3541 (2022)). If significant Fanca complementation in HSPCs is not observed or observe a lack of phenotypic rescue, elevated doses of rAAV or repeated doses to enhance the efficacy of rAAV delivery to the BM compartment are tested. As an alternative approach, mobilization of HSPCs prior to infusion of rAAV is tested, which was demonstrated to enhance viral transduction in vivo (Broxmeyer et al. J. Exp. Med. 201 , 1307-1318 (2005); Li et al. Blood 131 , 2915-2928 (2018)).

[0247]

[0209] It is hypothesized that HMEJ facilitates in vivo FA gene complementation and is capable of rescuing the effects of the pathogenic mutations, including BMF. The ability to directly complement FA HSPCs in vivo could produce cures while circumventing the risks and costs associated with HSCT and offer an opportunity to address the nonhematopoietic manifestations of FA and treat other systemic genetic disorders.

[0248]

[0210] These studies will demonstrate HMEJ-mediated FA gene complementation using rAAV delivery in vivo. Moreover, in addition to delivering HMEJ components to the BM HSPC compartment, it is hypothesized that the rAAVs herein will facilitate Fanca complementation in other stem and progenitor populations and somatic cells throughout the body.

[0249]

[0211] Further experimentation can be carried out by investigating in vivo delivery of HMEJ CRISPR / Cas9 reagents by lipid nanoparticles (LNPs). LNPs are an attractive alternative delivery platform due to their low immunogenicity and ability to target specific tissues in vivo, including HSPCs. The ability to directly complement FA HSPCs in vivo could produce cures while completely circumventing the risks and costs associated with HSCT and offer an opportunity to address the nonhematopoietic manifestations of FA and treat other systemic genetic disorders (e.g. Ataxia Telangiectasia, Nijmegen Breakage Syndrome, Bloom Syndrome, Werner Syndrome, and Rothmund-Thompson Syndrome).

[0250] Example 3-HMEJ Activity in Non-Cycling Cells

[0251]

[0212] To assess the cell cycle dependence of HMEJ, the G1- or S / G2-specific expression of a chimeric Cas9149, which is fused to either a portion of Cdc10-dependent transcript 1 (CDT1 ) (for G1 specific) or GEMININ (for S / G2-specific) expression will be determined.

[0252] CDT1 and GEMININ are both important DNA replication regulators. CDT1 is phosphorylated at the beginning of S phase by a cyclin-dependent kinase and this promotes its ubiquitin- mediated proteosomal degradation. Thus, the Cas9:CDT1 fusion protein is also degraded in early S phase and this restricts Cas9 activity to the G1 phase. In contrast, GEMININ is absent in G1 and then accumulates during S and G2 phases, before it is degraded in an Docket No. 32757 / 70065 anaphase-promoting complex dependent manner. Accordingly, the Cas9:GEMININ fusion protein is absent during G1 and this restricts Cas9 activity to the S / G2 phases of the cell cyclel 01 . Consequently, either Cas9:CDT 1 or Cas9:GEMININ is used in asynchronously growing RPE1 +hTERT cells and HMEJ targeting assay is performed. The correct cell cycle expression of these fusion proteins is confirmed by immunoblotting after cell sorting. Two biological replicates of this experiment have already been performed and each construct behaved as anticipated (data not shown). For additional controls, NHEJ71 and HDR66 extrachromosomal reporters can also be utilized with these fusion proteins to confirm that they show, as expected, more activity in G1 and S / G2 phases, respectively.

[0253]

[0213] It can be difficult to demonstrate that the actual repair is also occurring at that time as opposed to occurring later. To confirm, inhibitors to arrest cells at a particular cell cycle phase are used and then the HMEJ assay begun while the bulk of the cells are locked in that phase. To achieve a G1 arrest UCN-01 is used, which is a cyclin-dependent kinase inhibitor that causes a significant, reversible G1 phase arrest in cells (like HCT116 and RPE1+hTERT) with intact cell cycle checkpoints. Alternatively, cells will be synchronized at the G1 / S border using a double thymidine block, or in S phase using hydroxyurea, an inhibitor of ribonucleotide biosynthesis, or in G2 with nocodozole a microtubule inhibitor. All of these inhibitor treatments can be accompanied by cellular toxicity and rarely achieve perfect synchronization. Nonetheless, it is believed that they will be useful as independent confirmation of the experiments proposed above. Lastly, if indicated, cell sorting is used to obtain relatively pure populations of cells that are localized to specific phases of the cell cycle. If HMEJ is regulated like SSA then it may indeed be active in all phases of the cell cycle, which would open the door to optimizing its expression at the expense of most other DSB pathways.

[0254]

[0214] Initially duplicate plates of cells at either low confluency or at very high confluency were cultured and then let the cells grow for an additional 3 days or 6 days. At that time, one of the plates was assessed for their cell cycle status by flow cytometry. The day 3 low confluency cells showed the expected profile of -60% of the cells in G0 / G1 , -30% in S phase and -10% in G2 phase (Fig. 6A). In contrast, 98% of the high confluency cells were observed in G0 / G1 and only -1% were in either S or G2 phases (Fig. 6A, 6B). By day 6, low confluency cells were 53.6% in G0 / G1 while the high confluency cells 76% were in G0 / G1 . At this juncture the duplicate plates of the low and high confluency cells were subjected to a HMEJ assay. Several days later the correct integration of EGFP at the AAVS1 was assessed. As expected, the low confluency cells showed a -normal 7% HMEJ activity (Fig. 6C). Quite unexpectedly and remarkably, the high confluency cells showed nearly the same activity (Fig. 6C). While it cannot rule out that these cells were mostly in G1 versus GO, this Docket No. 32757 / 70065 data nonetheless strongly suggests that HMEJ is active in non-cycling cells. Therapeutically, this would be extremely important since many tissues / organs such as the liver or the brain, that are important therapeutic targets, consist mainly of non-dividing GO cells and are resistant to classical HDR gene targeting approaches.

[0255]

[0215] Different Cas protein constructs comprising either Geminin or Cdt1 were tested for their ability to modulate cell cycle arrest and HMEJ-mediated double strand break repair. Figure 7 shows that there is not a significant difference in % HMEJ as a result of the different constructs and their influence on cell cycle control. A chromatin flow assay (see e.g., Forment et al., 2015 Nature Protocols 10(9) : 1297-1307) is also used to assess the HMEJ in UCB-CD34 cells during the cell cycle. A rAAV6 vector expressing hFANCAexI for HMEJ is transfected into CD34 cells with an MOI of 105in either BNN (Bret’s CD34 medium) or XRC (X-vivo15+rapamycin+CHIR99021 ) media. Results show that there is approximately 16.86% integration of the construct in the BNN media while there is approximately 1 1 .37% integration in XRC media.

[0256]

[0216] These results show that HMEJ appears to work in non-cycling cells, unlike HDR. This is very unexpected and could have far reaching implications for gene therapy targeting noncycling cell populations.

[0257]

[0217] Existing mouse models lacking the Fanca gene have limitations in mimicking the full spectrum of FA manifestations observed in humans. Additionally, the development of a mouse model containing human DNA sequences will enable direct translation of CRISPR- based in vivo gene therapy approaches without the need for re-engineering gene editing reagents. To create the mouse model as described above in Example 2, murine Fanca exon 4 DNA sequences were replaced with human FANCA exon 4 DNA sequences harboring the most prevalent FA mutation (FANCA c.295C>T, i.e. Spanish founder mutation), termed FANCAhMUT / hMUT. Furthermore, to better recapitulate the human disease, KO mutations for Aldh2 and Adh5 were introduced, which are involved in the resolution of DNA ICLs, onto the FANCAhMUT / hMUTbackground. This is expected to lead to the accumulation of ICLs and exacerbate BMF and cancer development.

[0258]

[0218] The hypothesis is that the combination of FANCAhMUT / hMUTmice with predisposing Aldh2 or Adh5 KO background will yield animals exhibiting enhanced human FA phenotypes. Moreover, the utilization of human DNA sequences in FANCAhMUT / hMUTmice will facilitate preclinical testing of in vivo gene therapy with rapid translation.

[0259]

[0219] The impact of this research lies in the potential to advance the treatment of FA through in vivo correction of genetically humanized HSPCs and somatic cells. Traditional ex vivo approaches for HSCT have shown variable success and are associated with significant Docket No. 32757 / 70065 costs, risks, mortality, and accessibility inequalities. By directly correcting FA cells in vivo, the need for HSCT can be circumvented, offering a potentially curative approach with reduced risks and costs. Additionally, the use of a genetically humanized FA mouse model allows for direct translation of genome engineering techniques without the need for reoptimization for human DNA sequences.

[0260]

[0220] The most common FA founder mutation worldwide, the FANCA exon 4 c.295 C>T mutation (p.Q99X) found in those of Spanish Romani ancestry (carrier frequency of 1 / 64- 1 / 70) (“Spanish founder mutation”) was engineered into mice. This C>T substitution leads to a premature stop codon and truncated, non-functional FANCA protein. It has been confirmed that humanized mutant FANCA exon 4 (“FANCAhMUT / hMUT”) mice do not express Fanca protein or mRNA and their primary T cells are hypersensitive to MMC treatment (Figure 5C) compared to controls. Given that cells isolated from FANCAhMUT / hMUTmice are hypersensitive to MMC, a hallmark of FA patient cells, it was next assessed whether these mice developed BMF.

[0261]

[0221] To this end, femurs from age-matched FANCAhMUT / hMUTand control mice were isolated and bone marrow cells were stained for HSPC (Lin-, c-Kit+, Sca1 +; “LSK”) and analyzed by flow cytometry. Quantification of the HSPC population showed no changes in the percentage of LSK cells in FANCAhMUT / hMUTmice compared to controls. This observation is expected given that spontaneous BMF has not been observed in any previous FA mouse model, possibly due to the lack of exposure of lab mice to genotoxic environmental agents. Next, the functionality of the BM HSPCs in FANCAhMUT / hMUTand control mice was assessed. HSPCs from FANCAhMUT / hMUTmice exhibited decreased clonogenic potential compared to controls in MMC-treated colony-forming unit (CFU) assays. These data demonstrate that the genetically humanized mice are devoid of Fanca protein and mRNA, are hypersensitive to DNA cross linking agents, and do not have inherent BMF, as expected.

[0262]

[0222] Generation ofAldh2 andAdh5 KO mice. gRNAs were designed targeting Aldh2 exon 2 and Adh5 exon 2. gRNAs and Cas9 mRNA were electroporated into fertilized C57BL / 6 embryos and transferred to pseudo-pregnant mice. Numerous Aldh2 and Adh5 founders harboring frameshift mutations were obtained and bred them. to homozygosity. To further validate these models, RT-PCR and western blot analysis was performed, which confirmed a lack of mRNA transcript for both genes and lack of Aldh2 protein, respectively (Figure 6A, B). These data demonstrate generation of Aldh2 and Adh5 KO mice harboring frameshift mutations that lead to loss of Aldh2 protein and Aldh2 / Adh5 mRNA expression.

[0263]

[0223] In the case of correcting the FANCA Spanish founder mutation, the FANCA exon 4 C.295T premature stop codon cannot be directly converted back to the WT glutamine codon Docket No. 32757 / 70065

[0264] (CAG) using BE technology; however, Adenosine Base Editing (ABE) can be used to target the adenine of the stop codon (TAG) for conversion to guanine (TGG), resulting in a conservative amino acid substitution to tryptophan. Notably, in alignment of 248 metazoan FANCA sequences from Ensembl88, there is a diversity of amino acids, including tryptophan, present at the position in question, suggesting that a tryptophan at this position is compatible in mammals. Incredibly, it was found that targeting FANCA C.296A with a gRNA combined with advanced ABE (ABE8e) produced editing rates of >70% (Figure 8A), which restored FANCA expression and corrected FA phenotypes, namely MMC-sensitivity and MMC-induced FANCD2 mono-ubiquitination (Figure 8B-C). These data demonstrate that BEs are highly functional in FA cells, and ABE can be utilized to restore the FA pathway in FANCA- / - cells.

[0265]

[0224] rAAV delivery in BM cells from DH-deficient FANCAhMUT / hMUTmice is tested. Briefly, BM from 6- to 12-week-old DH-deficient FANCAhMUT / hMUTmice will be processed into a single cell suspension. Differentiated hematopoietic lineage cells (i.e., T lymphocytes, B lymphocytes, monocytes / macrophages, granulocytes, and erythrocytes) will be removed with the Mouse Hematopoietic Progenitor Stem Cell Enrichment kit (BD Biosciences), by way of immunomagnetic depletion. Cells are then stained for positive HSC markers c-Kit and Seal . A MACSQuant Tyto closed-system cell sorter is used to isolate the lineage- c-Kit+ Sca1 + CD150+ CD48low population. These cells are cultured in 5% oxygen at 37°C on fibronectin coated plates in serum-free media, as described. rAAV is overlaid on cells at -70% confluency. Three days post-treatment, cells treated with rAAV are collected and base editing at the target site analyzed by EditR software and high-throughput sequencing using the MiSeq System (Illumina). It is hypothesized that upwards of 70% FANCA gene correction in vitro is observed. Control cells receive rAAV encoding ABE without gRNA.

[0266]

[0225] In parallel with the experiments described herein, BM samples from DH-deficient FANCAhMUT / hMUTmice 4-, 8-, or 16-weeks post-treatment with rAAV are cultured and assessed for MMC-induced genomic instability phenotypes compared with untreated and DH-deficient FANCAhWT / hWTmice. These phenotypes include MMC-induced FANCD2 monoubiquitination, cellular sensitivity to MMC treatment, and MMC-induced radial formation. Post-editing, BM samples are also used to assess the HSPC population size (by flow cytometry) and function (CFU assays). To further demonstrate that gene-corrected HSPCs are functional, HSPCs are isolated from DH-deficient FANCAhMUT / hMUTmice 16- weeks post-injection with rAAV and HSPCs transplanted intravenously into sub-lethally irradiated DH-deficient FANCAhMUT / hMUTmice. Sixteen weeks post-transplant, peripheral blood is isolated and cellularity assessed by flow cytometry. It is expected final peripheral blood counts to be similar to DH-deficient FANCAhWT / hWTmice, indicating that HSPC Docket No. 32757 / 70065 engraftment was satisfactory. In addition, genomic DNA from all major organs (i.e. liver, heart, spleen, brain, muscle, kidney, etc.) is assessed for genetic correction of somatic cells via PCR / NGS.

[0267]

[0226] As a result of the anticipated in vivo FANCA correction in BM HSPCs, it is expected that BM cells from targeted DH-deficient FANCAhMUT / hMUTmice will exhibit (i) increased MMC-induced FANCD2 monoubiquitination, (ii) reduced sensitivity to MMC, and (iii) reduced radial formation compared to BM cells from non-targeted DH-deficient FANCAhMUT / hMUTmice, at similar levels to DH-deficient FANCAhWT / hWTmice. Furthermore, it is expected that the functional HSPC pool in the BM of targeted DH-deficient FANCAhMUT / hMUTmice to be larger in number than non-targeted DH-deficient FANCAhMUT / hMUTmice 16 weeks post-treatment with rAAV, at similar levels to that of DH-deficient FANCAhWT / hWTmice. Finally, it is hypothesized that transplant of HSPCs collected from DH-deficient FANCAhMUT / hMUTmice 16 weeks posttreatment into irradiated DH-deficient FANCAhMUT / hMUTwill rescue the low blood cellularity phenotype associated with FA-deficiency. It is also likely that base editing in somatic cells from various tissues is observed particularly the liver where rAAV tend to accumulate.

[0268]

[0227] It is understood that every embodiment of the disclosure described herein may optionally be combined with any one or more of the other embodiments described herein. Every patent literature and every non-patent literature cited herein are incorporated herein by reference in their entirety.

[0269]

[0228] It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description, and / or shown in the attached drawings. Consequently, only such limitations as appear in the appended claims should be placed on the disclosure.

Claims

1. Docket No. 32757 / 70065What is claimed is:1 . A polynucleotide expression cassette comprising a homology arm (HA), a polynucleotide encoding a gene associated with a DNA repair deficiency, a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is capable of insertion at a locus targeted by the targeting molecule.

2. A polynucleotide expression cassette comprising homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is capable of insertion upstream of a gene associated with a DNA repair deficiency.

3. A polynucleotide cassette comprising left and right homology arms, a polynucleotide encoding a gene associated with a DNA repair deficiency, and single-guide (sg) RNA cut sites to linearize donor DNA to be integrated into a locus in a cell genome.

4. A polynucleotide expression cassette comprising a homology arm (HA), a polynucleotide encoding a gene associated with Fanconi anemia (FA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is capable of insertion at a locus targeted by the targeting molecule.

5. A polynucleotide expression cassette comprising homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is capable of insertion upstream of a gene associated with Fanconi anemia (FA).

6. A polynucleotide cassette comprising left and right homology arms, a polynucleotide encoding a FA gene, and single-guide (sg) RNA cut sites to linearize donor DNA to be integrated into a locus in a cell genome.

7. The polynucleotide or expression cassette of any one of claims 1-6, wherein the homology arms are between 35 and 1000 nucleotides.

8. The polynucleotide or expression cassette of any one of claims 1 -2, 4-5 and 7, wherein the nuclease dependent cleavage system comprises a CRISPR / Cas system, a Cas-CLOVER system, a zinc-finger nuclease (ZFN) system, a transcription activator like effector nuclease (TALEN) system, or a meganuclease system.

9. The polynucleotide or expression cassette of any one of claims 1 -2, 4-5 and 7-8, wherein the nuclease dependent cleavage system is a CRISPR / Cas system and the targeting molecule is a single guide RNA.Docket No. 32757 / 7006510. The polynucleotide or expression cassette of any one of claims 1 -9 wherein the locus targeted is an AAVS1 (PPP1R12C) or FA gene locus.11 . The expression cassette of any one of claims 4-6 or 8-10, wherein the expression cassette comprises the promoter next to or near the FA gene.12 The expression cassette of any one of claims 1 -2, 4-5 or 9-10, wherein the promoter is an MND promoter, a CMV promoter, a CAG promoter, a PGK promoter, a EF1 A promoter, an AAV promoter, or a endogenous cell promoter.

13. The polynucleotide or expression cassette of any one of claims 4-12, wherein the FA gene is a donor polynucleotide that corrects a mutated genotype in a subject.

14. The polynucleotide or expression cassette of any one of claims 4-13, wherein the transgene comprises all or part of one or more of FANC-A, -B, -C, -D1, -D2, -E, -F, -G, - H, -I, -J, -K, -L, -M, -N, -O, -P, -Q, -S, -T, -U, - V, -W, or -Y.

15. A viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising a polynucleotide or expression cassette of any one of claims 1-14.

16. The viral vector of claim 15, wherein the viral vector is a lentiviral vector, adenoviral vector, or AAV vector.

17. The viral vector of claim 16, wherein the viral vector is selected from the group consisting of a AAV6 vector, AAV1 vector, AAV-DJ vector, or VSVg-pseudotype lentiviral vector.

18. A method for genome engineering a hematopoietic stem and progenitor cell (HSPC) or a population of HSPC to express cDNA of a gene associated with a DNA repair deficiency from the endogenous locus comprising introducing into the HSPC cell or population of HSPC a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm (HA), a polynucleotide encoding a gene associated with a DNA repair deficiency, a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted at a locus targeted by the targeting molecule.

19. A method for genome engineering a hematopoietic stem and progenitor cell (HSPC) or a population of HSPC to express FA gene cDNA from the endogenous FA locus comprising introducing into the HSPC cell or population of HSPC a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassetteDocket No. 32757 / 70065 comprising a homology arm (HA), a polynucleotide encoding a FA gene, a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted at a locus targeted by the targeting molecule.

20. A method for genome engineering a hematopoietic stem and progenitor cells HSPC or a population of HSPC to overexpress an endogenous FA gene comprising introducing into the HSPC or population of HSPC a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm(s) (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, wherein the expression cassette is inserted upstream of a FA gene to be expressed.21 . The method of any one of claims 18-20, wherein the homology arms are between 35 and 1000 nucleotides.

22. The method of any one of claims 18-21 , wherein the nuclease dependent cleavage system comprises a CRISPR / Cas system, a Cas-CLOVER system, a zinc-finger nuclease (ZFN) system, a transcription activator like effector nuclease (TALEN) system, or a meganuclease system.

23. The method of claim 22, wherein the CRISPR / Cas system comprises Cas9, Cas12a, Cas13a, or Cas13b.

24. The method of any one of claims 18-23, wherein the nuclease dependent cleavage system is a CRISPR / Cas system and the targeting molecule is a guide RNA.

25. The method of claim 24, wherein the method further comprises transfecting the HSPC or population of HSPC with a Cas protein or polynucleotide encoding a Cas protein and guide RNA molecules that direct integration of the expression cassette to a target locus in the HSPC genome.

26. The method of claim 25, wherein the target locus is AAVS1 (PPP1 R12C) or FA gene locus.

27. The method of any one of claims 18-26, wherein the viral vector is an AAV vector, lentiviral vector, or adenoviral vector.

28. The method of claim 27, wherein the viral vector is selected from the group consisting of an AAV6 vector, AAV1 vector, AAV-DJ vector, VSVg-pseudotype, or lentiviral vector.Docket No. 32757 / 7006529. The method of any one of claims 19-28, wherein the FA gene integrates into the HSPC genome via homology directed repair (HDR), homology-mediated end joining (HMEJ), or a combination of HDR / HMEJ.

30. The method of any one of claims 18-29, wherein the introduction of the plasmid, nanonplasmid, lipid nanoparticle, mini-circle, or GenCircle is by transfection or electroporation.31 . The method of any one of claims 18-30, wherein the introduction of the viral vector is by electroporation.

32. The method of claim 30 or 31 , wherein the viral vector is introduced at a multiplicity of infection of 3 x 105- 1 x 107.

33. The method of any one of claims 18-32, wherein efficiency of introduction is greater than 5%.

34. The method of any one of claims 18-33, wherein the HSPC cell population has a viability of greater than 50% after 3 days.

35. The method of any one of claims 18-34, wherein the HSPC cell or population of HSPC cells is CD34+.

36. The method of any one of claims 18-35, wherein the viral vector, plasmid nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprises the promoter next to or near the FA gene.

37. The method of any one of claims 18-36, wherein the promoter is an MND promoter, a CMV promoter, a CAG promoter, a PGK promoter, a EF1 A promoter, an AAV promoter, or an endogenous cell promoter.

38. The method of any one of claims 19-37, wherein the FA gene is a donor polynucleotide that corrects a mutated genotype in a subject.

39. The method of any one of claims 19-38, wherein the FA gene comprises all or part of one or more of FANC-A, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -S, - T, -U, -V, -W, or -Y.

40. A method of making a gene edited hematopoietic stem and progenitor cell (HSPC) or population of HSPC, comprising: i) contacting a HSPC or population of HSPC with a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm (HA), a splice acceptor site, a promoter and a targeting site for aDocket No. 32757 / 70065 nuclease dependent cleavage system targeting molecule, and optionally comprising a polynucleotide encoding a gene associated with a DNA repair deficiency; ii) culturing the HSPC or population of HSPC of i) in a media that promotes expansion of HSPC cells; iii) isolating the HSPC or population of HSPC of ii) based on identification of a marker expressed only on a HSPC or population of HSPC carrying the viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle; iv) culturing the isolated cells of iii) in a culture medium to expand the isolated cells expressing the gene associated with a DNA repair deficiency.41 . A method of making a gene edited hematopoietic stem and progenitor cell (HSPC) or population of HSPC, comprising: i) contacting a HSPC or population of HSPC with a viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle comprising an expression cassette comprising a homology arm (HA), a splice acceptor site, a promoter and a targeting site for a nuclease dependent cleavage system targeting molecule, and optionally comprising a polynucleotide encoding a FA gene associated with Fanconi anemia; ii) culturing the HSPC or population of HSPC of i) in a media that promotes expansion of HSPC cells; iii) isolating the HSPC or population of HSPC of ii) based on identification of a marker expressed only on a HSPC or population of HSPC carrying the viral vector, plasmid, nanoplasmid, lipid nanoparticle, mini-circle, or GenCircle; iv) culturing the isolated cells of iii) in a culture medium to expand the isolated cells expressing the FA gene.

42. The method of claim 40 or 41 , further comprising a step of stimulating, proliferating or activating the HSPC or population of HSPC prior to the contacting step.

43. The method of claim 42, wherein the step of stimulating, proliferating or activating the HSPC or population of HSPC comprises contacting the cell(s) with one or more of IL-3, IL-6, SCF, Flt3L, TPO, GM-CSF, G-CSF and prostaglandin E244. The method of any one of claims 40-43, wherein the method produces gene edited HSPC with an efficiency of greater than 5%.

45. The method of any one of claims 40-44, wherein the method maintains 50% viability of cells in culture after 3 days.Docket No. 32757 / 7006546. A gene edited HSPC or population of HSPC made by the method of any one of claims 18-45.

47. A gene edited hematopoietic stem and progenitor cell (HSPC) comprising, a heterologous polynucleotide sequence encoding all or part of a gene associated with DNA repair deficiency at a target location mediated by a nuclease dependent cleavage system, wherein the heterologous polynucleotide sequence is also flanked by portions of a homology arm and expressed via an endogenous promoter.

48. A gene edited hematopoietic stem and progenitor cell (HSPC) comprising, a heterologous polynucleotide sequence encoding all or part of a FA gene associated with Fanconi Anemia at a target location mediated by a nuclease dependent cleavage system, wherein the heterologous polynucleotide sequence is also flanked by portions of a homology arm and expressed via an endogenous promoter.

49. The gene edited HSPC of claim 48, wherein the FA gene is a donor polynucleotide that corrects a mutated genotype in a subject.

50. A method of treating a DNA repair deficiency in a subject in need thereof comprising administering to the subject an expression cassette of any one of claims 1 -3 or 7- 12, a vector of any one of claims 17-19 or a gene edited HSPC or population of HSPC of claim 47.51 . The method of claim 50, wherein the DNA repair deficiency is xeroderma pigmentosum, ataxia telangiectasia, Nijmegen Breakage Syndrome, Bloom Syndrome, Werner Syndrome, Rothmund-Thompson Syndrome, DNA Ligase IV Deficiency Syndrome (LIG4 syndrome), trichothiodystrophy, progeria (Hutchinson-Gilford progeria syndrome) and Cockayne syndrome.

52. A method of treating Fanconi anemia (FA) in a subject in need thereof comprising administering to the subject a polynucleotide or expression cassette of any one of claims 4-14.

53. A method of treating Fanconi anemia (FA) in a subject in need thereof comprising administering to the subject vector comprising an expression cassette of any one of claims 4-14 or a vector of any one of claims 15-17.

54. A method of treating Fanconi anemia (FA) in a subject in need thereof comprising administering to the subject a gene edited HSPC or population of HSPC of any one of claims 48-49.

55. The method of any one of claims 52-54, wherein the administration alleviates one or more symptoms of FA selected from the group consisting of bone marrow failure,Docket No. 32757 / 70065 increased risk for malignancy, short stature, abnormal skin pigmentation, skeletal malformations of the upper and / or lower limbs, microcephaly, ophthalmic and genitourinary tract anomalies, pancytopenia, thrombocytopenia, or leukopenia.

56. The method of any one of claims 52-55, wherein the subject is receiving a second agent or standard of care therapy.

57. The method of claim 56, wherein the second agent or standard of care therapy is oral androgens, granulocyte colony-stimulating factor, hematopoietic stem cell transplantation (HSCT), treatment of growth deficiency, treatment of limb anomalies, treatment of ocular anomalies, treatment of renal malformations, treatment of genital anomalies, treatment of hypothyroidism, treatment of cardiac anomalies, treatment of dermatologic manifestations, or vitamin D supplementation.

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