WO2017087961A1 - Hematopoietic cell gene editing - Google Patents

Abstract

The present invention provides methods for gene editing in cells using adeno-associated virus (AAV) particles and vectors. The AAV particles used in the methods of the invention are capable of targeting a specific genetic modification to a preselected genomic target region in a cellular genome by homologous pairing in the absence of site-specific nucleases.

Description

HEMATOPOIETIC CELL GENE EDITING

CROSS REFERENCE

[0001] This application is related to U.S. provisional patent application, Serial No. 62/257,601, filed November 19, 2015, the disclosure of which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No.

DK055759, awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] The sequence listing submitted herewith, entitled "16-1500- PCT_SequenceListing_ST25.txt" and 166kb in size, is incorporated by reference in its entirety.

BACKGROUND

[0004] Gene editing is a powerful technique for introducing site-specific sequence modifications into the human genome. Viral vectors based on the parvovirus adeno- associated virus (AAV) have been shown to carry out site-specific modifications in some types of human cells. The single-stranded AAV vector genome recombines with the homologous chromosomal locus to change the sequence of the target locus. However, in the case of hematopoietic cells, AAV vectors have never been shown to edit genes without the addition of site-specific nucleases to stimulate homologous recombination (Genovese et al., Nature 510:235-40 (2014); Wang et al, Nature Biotech 33(12): 1256-65 (2015)). Unfortunately, the introduction of a nuclease into cells can have unwanted side-effects, including off-target cutting, unpredictable repair of double-strand breaks (DSBs) by nonhomologous end joining (NHEJ), and the development of an immune response to the foreign nuclease protein. The discovery of a nuclease-free gene editing method for hematopoietic cells would be a major advance with significant implications for gene therapy. SUMMARY OF THE INVENTION

[0005] In a first aspect, the present invention provides a method for gene editing in a hematopoietic cell, comprising transducing an effective amount of a recombinant adeno- associated virus (AAV) particle into the hematopoietic cell, wherein the recombinant AAV particle comprises a targeting construct comprising: (a) an insert region comprising a DNA sequence which is substantially identical to a genomic target region except for a modification to be introduced at the genomic target region; (b) regions flanking the insert region, wherein the flanking regions facilitate homologous pairing between the insert region and the genomic target region resulting in the modification being introduced into the genomic target region; (c) a 5' inverted terminal repeat (ITR) sequence at the 5' end of the targeting construct; and (d) a 3' inverted terminal repeat (ITR) sequence at the 3' end of the targeting construct; wherein the recombinant AAV particle comprises an AAV serotype 6 capsid and is transduced into the hematopoietic cell in the absence of a site- specific nuclease, and wherein transduction results in gene editing through integration of the insert region into the genomic target region of the hematopoietic cell.

[0006] Hematopoietic cells are an especially important target cell for gene therapy, and clinical trials have been performed for several diseases based on the ex vivo delivery of genes by retroviral or lentiviral vectors. A major complication of this approach has been the development of malignancies due to the integration of vector-encoded promoter elements near cellular proto-oncogenes and/or aberrant expression of the vector-encoded transgene. For example, X-linked severe combined immunodeficiency (X-SCID) is caused by mutations in the IL2RG gene, which results in a lack of functional T-cells, B- cells and NK cells. X-SCID patients that underwent ex vivo delivery of a retroviral vector encoding IL2RG to their hematopoietic cells were able to express IL2RG and produce functional lymphocytes (Cavazzana-Calvo et al, Science 288:669-72 (2000)). However, many of these patients subsequently developed leukemia due to integration of the vector and activation of nearby proto-oncogenes such as LM02 (Hacein-Bey-Abina, et al, N Engl J Med 348:255-56 (2003)). In addition, the IL2RG gene itself may play a role in malignant transformation, since it is a component of several growth factor receptors and its aberrant expression has been linked to the development of lymphomas. There is a need for a gene editing tool that can be achieved in hematopoietic cells by AAV vectors, that solves the many problems encountered in gene therapy for hematopoietic diseases. The present disclosure demonstrates that gene editing can be achieved in hematopoietic cells by AAV vectors in the absence of site-specific nucleases.

[0007] In certain embodiments, the incidence of random integration of the insert region at the genomic target region is less than one percent. In some embodiments, the hematopoietic cell is transduced ex vivo and the genetic correction provides a selective survival advantage. In some embodiments, non-homologous end joining (NHEJ) does not occur at the genomic target region in the infected cells.

[0008] In a second aspect, the present invention provides a method of treating a subject in need thereof, wherein the method comprises: (a) obtaining a population of hematopoietic cells from the subject; (b) transducing the population of hematopoietic cells with a recombinant AAV particle comprising an AAV6 capsid, wherein the recombinant AAV particle comprises: (i) an insert region comprising a DNA sequence which is substantially identical to a genomic target region except for a modification to be introduced at the genomic target sequence; (ii) regions flanking the insert region, wherein the flanking regions facilitate homologous pairing between the insert region and the genomic target region resulting in the modification being introduced into the genomic target region; (iii) a 5' inverted terminal repeat (ITR) sequence at the 5' end of the targeting construct; and (iv) a 3' inverted terminal repeat (ITR) sequence at the 3' end of the targeting construct; wherein the hematopoietic cell is transduced by the recombinant AAV vector in the absence of a site-specific nuclease, and wherein transduction results in gene editing through integration of the insert region into the genomic target region of the hematopoietic cell; and (c) administering the transduced population of hematopoietic cell to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The disclosed exemplary aspects have other advantages and features which will be more readily apparent from the detailed description, the appended claims, and the accompanying figures. A brief description of the drawings is below.

[0010] FIG.1A shows wild-type and knockout I12rg loci with AAV vector maps.

[0011] FIG.1B shows that the wild-type (WT) I12rg allele transcribes mRNA from exon 1 to 8, and AAVmI12rg:ex3-8pA targeted allele produces a fusion I12rg transcript consisting exon 1, 2 and vector derived I12rg CDS. mPgk: murine pgk promotor, Neo: neomycin resistance gene, UTR: untranslated region.

[0012] FIG.1C shows AAV vector transfer to murine whole bone marrow (BM) cells. AAV vector (AAVscMSCVeGFPpA) was transduced into 5-FU treated BM cells at MOI of 0, 1,000 and 10,000. Three days after transducing, EGFP expression in cultured BM cells was analyzed by flow cytometry. These figures show EGFP positive populations in 7AAD negative cells.

[0013] FIG.1D shows GFP and Sca-1 expression in vitro in AAV-scMSCV-GFP- transduced BM cells.

[0014] FIG.1E shows representative CD4⁺ and CD8⁺ populations in CD3⁺ cells from mice treated with the indicated vectors.

[0015] FIG.1F shows CD4⁺ and CD8⁺ cell counts over time in the peripheral blood of mice treated with AAV-I12rg3-8 (squares), AAV-MSCV-GFP (diamonds), or no vector (black lines). *p < 0.05 (two-way ANOVA).

[0016] FIG.1G shows representative I12rg expression (red lines) and isotype (black lines) in CD4⁺ and CD8⁺ from treated mice.

[0017] FIG.1H shows mean fluorescent intensity (MFI) of I12rg in CD4⁺ and CD8⁺ cells. *p < 0.05., n.s. = no significant difference (one-way ANOVA). Each symbol represents a distinct mouse. Data from FIG.1 A through FIG. IF demonstrate that AAV- mediated gene editing restores T-cell I12rg expression.

[0018] FIG.2A shows representative flow cytometry of granulocyte (CD1 lb⁺, Gr-1⁺) and monocyte (CDl lb⁺, Gr-l^ne ) populations in peripheral blood of treated mice 28 weeks after transplant.

[0019] FIG.2B shows representative flow cytometry of NK cells (NK1.1⁺) and B- cells (B220⁺) in peripheral blood.

[0020] FIG.2C shows the number of each cell type present in peripheral blood. *p < 0.05 (one-way ANOVA). Data from FIG.2A through FIG.2C demonstrate the low marking rates in non-T-cell types in peripheral blood.

[0021] FIG.3A shows representative flow cytometry of T-cells (CD3⁺, NK1. l^ne ) in the spleens of treated mice 32 weeks after transplant.

[0022] FIG.3B shows representative flow cytometry of CD4⁺ and CD8⁺ populations in CD3⁺ splenocytes.

[0023] FIG.3C shows the number of CD3⁺, CD4⁺, and CD8⁺ cell counts per spleen. *p < 0.01, **p < 0.05 (one-way ANOVA).

[0024] FIG.3D shows representative flow cytometry showing the lack of B-cells and NK cells in the spleen. FIG.3A through FIG.3D demonstrate T-cell recovery in the spleens of transplant recipients.

[0025] FIG.4A shows the qPCR products used to detect donor cells (blue bar), random integrants (green bar), and internal vector sequences (edited alleles or random integrants; red bar) are shown.

[0026] FIG.4B shows the frequency of donor cells, cells with random integrants, and edited cells (internal vector sequence frequency minus random integrant frequency) as determined by qPCR of DNA from peripheral blood WBCs, bone marrow cells, CD3⁺ splenocytes, and CD3^ne splenocytes of mice treated with AAV-I12rg3-8 (black columns) or AAV-MSCV-GFP (white columns) 32 weeks after transplant. Total DNA amounts were determined by amplifying the Bcl2 gene (primers not shown) and used to calculate frequencies. *p < 0.05., n.s. = no significant difference (student's /-test).

[0027] FIG.4C shows representative GFP expression in peripheral blood WBCs at 28 weeks post-transplant. FIG.4A through FIG.4C demonstrate the detection of vector sequences in the genomic DNA of treated mice.

[0028] FIG.5A shows the locations of PCR primers used to specifically amplify edited Il2rg alleles from the genomic DNA of CD3⁺ splenocytes from an AAV-I12rg3-8- treated mouse are shown, along with the sequence (SEQ ID NO: 58) of the PCR product showing sequences uniquely found at the Il2rg target locus as nucleotides 1-46 of SEQ ID NO:58, sequences common to the target locus and vector are nucleotides 47-838 of SEQ ID NO:58, and sequences uniquely found in the vector are nucleotides 839-886 of SEQ ID NO:58.

[0029] FIG.5B shows Il2rg RT-PCR primers used to amplify the cDNA sequence (SEQ ID NO:59) derived from the mRNA of CD3⁺ splenocytes from an AAV-I12rg3-8- treated mouse, with alignment to the wild-type (WT) Il2rg cDNA sequence (SEQ ID NO:60). Sequences uniquely found at the Il2rg target locus are nucleotides 1-66 of SEQ ID NO:59, sequences common to the target locus and vector are nucleotides 67-429 of SEQ ID NO:59, and sequences uniquely found in the vector are nucleotides 430-1173 of SEQ ID NO:59.FIG.5A and FIG.5B demonstrate sequence confirmation of accurate Il2rg editing.

[0030] FIG.6A shows a time course of CD3⁺, CD4⁺, and CD8⁺ cells in the peripheral blood of secondary transplant recipients that received bone marrow cells from mice treated with AAV-I12rg3-8 (squares) or AAV-MSCV-GFP (circles). *p < 0.05 (two-way

ANOVA). [0031] FIG.6B shows the total number of CD3 , CD4 , and CD8 cells in the spleens of secondary recipients. *p < 0.05 (student's /-test).

[0032] FIG.6C shows a representative flow cytometry showing B-cell and NK cell populations in the spleens.

[0033] FIG.6D shows the total number of B-cells in these spleens. **p < 0.01 (student's /-test).

[0034] FIG.6E shows a representative flow cytometry analysis showing mature B- cells (IgD⁺, B220⁺) in the splenocytes of an AAV-I12rg3-8-treated secondary recipient. FIG.6A through FIG.6E demonstrate edited lymphocytes increase in secondary transplant recipients.

[0035] FIG.7A shows a representative CD3 expression levels in peripheral blood WBCs 8 and 16 weeks after secondary transplantation.

[0036] FIG.7B shows a representative flow cytometry of CD4⁺ and CD8⁺ populations in CD3⁺ blood cells.

[0037] FIG.7C shows the number of granulocytes and monocytes in peripheral blood at 16 weeks. *p < 0.05 (student's /-test).

[0038] FIG.7D shows a representative flow cytometry in the peripheral blood 16 weeks after secondary transplantation showing a lack of B (B220⁺) or NK (NKl. l ⁺) cells. FIG.7 A through FIG.7D demonstrate T-cell and B-cell populations in secondary transplant recipients.

[0039] FIG.8A shows an analysis of TCR νβ repertoire in the CD3⁺ splenocytes of untreated wild type mice and Il2rg-/- knockout mice treated with AAV-I12rg3-8 or AAV- MSCV-GFP.

[0040] FIG.8B shows a representative flow cytometry of naive CD4⁺ splenocytes (CD62L⁺, CD4⁺) in mice treated with AAV-I12rge3-8, AAV-MSCV-GFP or no AAV. *p < 0.05 (one-way ANOVA).

[0041] FIG.8C shows total number of naive CD4⁺ splenocytes (CD62L⁺, CD4⁺) in mice treated with AAV-I12rge3-8, AAV-MSCV-GFP or no AAV. *p < 0.05 (one-way ANOVA).

[0042] FIG.8D shows a representative flow cytometry of naive CD8⁺ splenocytes (CD62L⁺, CD8⁺) population and isotype control in AAV-I12rg3-8-treated mice (note that AAV-MSCV-GFP-treated mice have no CD8+ cells). FIG.8A through FIG.8D demonstrate edited T-cell characterization. [0043] FIG.9A shows the total white blood cell numbers from recipient mice at each time points (4, 8, and 12 weeks) when AAVmI12rg:ex3-8pA was transduced and control BM cells were transplanted into irradiated mice. The circles and line: recipients of AAVmI12rg:ex3-8pA transduced BM cells; the squares and line: recipients of

AAVscMSCVeGFPpA transduced BM cells; the rhombus and line: recipients of vector free BM cells. Data show mean ± standard error of mean (SEM). (*p < 0.05).

[0044] FIG.9B shows flow cytometric profiles from typical experimental and control mice at 8 weeks after transplanting. Upper panels show CD3+ cell populations in 7AAD negative (live) lymphocytes and down panels show CD4+ and CD8+ cell populations in CD3+ cells (left: recipient of AAVmI12rg:ex3-8pA transduced BM cells (I12rg); middle: recipient AAVscMSCVeGFPpA transduced BM cells (EGFP); right: recipient of vector free BM cells (VF)).

[0045] FIG.9C shows the number of CD3+, CD4+, and CD8+ cells at 8 and 12 weeks after transplanting. Data points represent individual mice; horizontal bars represent mean values and vertical bars represent standard deviation (SD). (*p < 0.05).

[0046] FIG.9D shows flow cytometric profiles from typical recipients of

AAVscMSCVeGFPpA transduced or vector free BM cells at 8 weeks after transplanting. These figures show low or undetectable levels of EGFP positive peripheral blood derived mononuclear cells. FIG.9A through FIG.9D demonstrate reconstitution in recipient mice.

DETAILED DESCRIPTION OF THE INVENTION

[0047] All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al, 1989, Cold Spring Harbor Laboratory Press), Gene Expression

Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX).

[0048] Terms used in the claims and specification are defined as set forth below unless otherwise specified. In the case of direct conflict with a term used in a parent provisional patent application, the term used in the instant specification shall control.

[0049] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0050] The following definitions and explanations are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

[0051] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "And" as used herein is interchangeably used with "or" unless expressly stated otherwise.

[0052] The terms "nucleic acid," "polynucleotide" and "oligonucleotide" are used interchangeably and refer to deoxyribonucleotides or ribonucleotides or modified forms of either type of nucleotides, and polymers thereof in either single- or double-stranded form. The terms should be understood to include equivalents, analogs of either RNA or DNA made from nucleotide analogs and as applicable to the embodiment being described, single stranded or double stranded polynucleotides.

[0053] The term "homologous pairing," as used herein, refers to the pairing that can occur between two nucleic acid sequences or subsequences that are complementary, or substantially complementary, to each other. Two sequences are substantially

complementary to each other when one of the sequences is substantially identical to a nucleic acid that is complementary to the second sequence.

[0054] The term "identical" in the context of two nucleic acid or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection. An indication that two nucleic acid sequences are "substantially identical" is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. Another indication that two nucleic acid sequences are substantially identical is that the two molecules and/or their complementary strands hybridize to each other under stringent conditions.

[0055] The term "operably linked" refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0056] The term "AAV" refers to an adeno-associated virus. The term may be used to refer to the virus or derivatives thereof, virus subtypes, and naturally occurring and recombinant forms, unless otherwise indicated. AAV has over 100 different subtypes, which are referred to as AAV1, AAV2, AAV3, AAV4, AAV6, AAV6 etc., and includes both human and non-human derived AAV. There are about a dozen AAV serotypes. The various subtypes of AAVs can be used as recombinant gene transfer viruses to transduce many different cell types.

[0057] The term "recombinant parvoviral vector genome" refers to a vector genome derived from a parvovirus that carries non-parvoviral DNA in addition to parvoviral viral DNA. The recombinant vector genome will typically include at least one targeting construct.

[0058] The term "genomic target region," as used herein, refers to a region of a cellular genome at which a genetic modification is desired. The target region typically includes the specific nucleotides to be modified, as well as additional nucleotides on one or both sides of the modification sites.

[0059] The term "insert region," as used herein, refers to a DNA sequence that is substantially identical to the genomic target region, except for the modification(s) to be introduced into the host cell genome at the genomic target region.

[0060] The term "targeting construct" refers to a DNA molecule that is present in the recombinant AAV particles used in the methods of the invention and includes a region that is identical to, or substantially identical to, a part of the genomic target region, except for the modification or modifications that are to be introduced into the host cell genome at the genomic target region. The modification can be at either end of the targeting construct, or can be internal to the targeting construct. The modification can be one or more deletions, point mutations, and/or insertions, or combinations thereof, compared to DNA in the wild- type target cell.

[0061] The term "transduction" refers to the transfer of genetic material by infection of a recipient cell by a recombinant viral particle or vector. A cell that has received recombinant AAV particle or vector DNA, thereby undergoing genetic modification, is referred to herein as a "transduced cell," a "transfected cell," a "modified cell," or a "recombinant cell," as are progeny and other descendants of such cells.

[0062] The term "transgenic cell" refers to a cell that includes a specific modification of the cell's chromosomal or other nucleic acids, which specific modification was introduced into the cell, or an ancestor of the cell. Such modifications can include one or more point mutations, deletions, insertions, or combinations thereof. When referring to an animal, the term "transgenic" means that the animal includes cells that are transgenic. An animal that is composed of both transgenic cells and non-transgenic cells is referred to herein as a "chimeric" animal.

[0063] The term "vector" refers to an agent for transferring a nucleic acid (or nucleic acids) to a host cell. A vector comprises a nucleic acid that includes the nucleic acid fragment to be transferred, and optionally comprises a viral capsid or other materials for facilitating entry of the nucleic acid into the host cell and/or replication of the vector in the host cell (e.g., reverse transcriptase or other enzymes which are packaged within the capsid, or as part of the capsid).

[0064] The term "viral particle" refers to a particle that comprises a viral nucleic acid or viral vector and can also include a viral capsid.

[0065] All embodiments disclosed herein can be used in combination unless the context clearly dictates otherwise.

[0066] In a first aspect, the present invention provides a method for gene editing in a hematopoietic cell, comprising transducing an effective amount of a recombinant adeno- associated virus (AAV) particle into the hematopoietic cell, wherein the recombinant AAV particle comprises a targeting construct comprising: (a) an insert region comprising a DNA sequence which is substantially identical to a genomic target region except for a modification to be introduced at the genomic target region; (b) regions flanking the insert region, wherein the flanking regions facilitate homologous pairing between the insert region and the genomic target region resulting in the modification being introduced into the genomic target region; (c) a 5' inverted terminal repeat (ITR) sequence at the 5' end of the targeting construct; and (d) a 3' inverted terminal repeat (ITR) sequence at the 3' end of the targeting construct; wherein the recombinant AAV particle comprises an AAV serotype 6 capsid and is transduced into the hematopoietic cell in the absence of a site- specific nuclease, and wherein transduction results in gene editing through integration of the insert region into the genomic target region of the hematopoietic cell.

[0067] In some embodiments, the methods of the invention result in an incidence of random integration (i.e., integration from non-homologous pairing) of the insert region at the genomic target region of less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, or less than 0.04%. In other embodiments, the recombinant AAV particle transduces the desired specific genetic modification at a much higher frequency than previously possible with other methods of site-specific modification of DNA in hematopoietic cells without the use of site-specific nucleases. In certain embodiments, the desired modification frequencies can be greater than 0.01%, or greater than 0.1%, or greater than 1%, or greater than 2%, or greater than 3%, or greater than 4%, or greater than 5%, greater than 6%, or greater than 7% or greater than 8% or greater than 9% or greater than 10% or more can be obtained in hematopoietic cells using the methods described herein. In some embodiments, the insert region of the targeting construct can be integrated into the genomic target region of the hematopoietic cell's chromosome in at least 1%, 5%, 10%, 20%, 40%, or 50% or more of the hematopoietic cells. The efficiency of integration can depend in part on the multiplicity of infection (MOI; defined herein in units of vector particles per cell) used for the transduction, as well as the type of cell being transduced. In some embodiments, a MOI of about 1 to 10¹² vector particles per cell is used to transduce a hematopoietic cell. For example, a MOI of about 10² vector particles per cell, a MOI of about 10³ vector particles per cell, a MOI of about 10⁴ vector particles per cell, a MOI of about 10 vector particles per cell, a MOI of about 10 vector particles per cell, a MOI of about 10⁷ vector particles per cell, a MOI of about 10^s vector particles per cell, a MOI of about 10⁹ vector particles per cell, a MOI of about 10¹⁰ vector particles per cell, a MOI of about 10¹¹ vector particles per cell, or a MOI of about 10¹² or more vector particles per cell can be used to transduce the hematopoietic cells.

[0068] In another embodiment, wherein the hematopoietic cell is transduced ex vivo and the genetic correction provides a selective survival advantage. In certain

embodiments, the correct integration of the insert region confers a proliferation and survival advantage to the transduced cell population. In some embodiments, a subject is infused with a population of hematopoietic cells transduced using the methods described and correct insertion of the insert region in the hematopoietic cells results in a therapeutic response conferring a proliferation and survival advantage to the transduced cell population in the subject. In a number of genetic diseases, the genetic defect imparts a survival disadvantage to the affected cell population. In such diseases, a corrected cell population would undergo spontaneous in vivo selection in the absence of any

exogenously applied selective pressure. For example, in X-linked SCID, the introduction of a therapeutic insert region confers a proliferation and survival advantage to the transduced cell population in the subject. Non-limiting examples of genetic diseases amenable to gene editing using the methods provided herein include, but are not limited to, severe combined immunodeficiency syndrome (SCID), adenosine deaminase (ADA) deficiency, Wiskott-Aldrich syndrome (WAS), Fanconi anemia, thalassemia, and sickle cell disease. Non-limiting examples of genes that could be targeted by the methods described herein, include but are not limited to IL2RG gene (interleukin 2 receptor subunit gamma; also known as: P64, CIDX, IMD4, CD132, SCIDX, IL-2RG, SCIDX1; H.

sapiens nucleotide sequence NCBI RefSeq No. : NM_000206.2 (SEQ ID NO:01); H. sapiens amino acid sequence NCBI RefSeq No. : NP_000197.1 (SEQ ID NO:02)), ADA gene (adenosine deaminase; also known as: ADA, ADAl; H. sapiens nucleotide sequence NCBI RefSeq No. : NM_000022.3 (SEQ ID NO:03); H. sapiens amino acid sequence NCBI RefSeq No.: NP_000013.2 (SEQ ID NO: 04)), IL7R gene (interleukin 7 receptor; also known as: ILRA, CD127, IL7RA, CDW127, IL-7R-alpha; H. sapiens nucleotide sequence NCBI RefSeq No. : NM_002185.3 (SEQ ID NO:05); H. sapiens amino acid sequence NCBI RefSeq No. : NP_002176.2 (SEQ ID NO:06)), RAGl gene (recombination activating 1, V(D)J recombination-activating protein 1; also known as: RAG-1, RNF74; H. sapiens nucleotide sequence NCBI RefSeq No. : NM_000448.2 (SEQ ID NO:07); H. sapiens amino acid sequence NCBI RefSeq No. : NP_000439.1 (SEQ ID NO:08)), RAG2 gene (recombination activating 2, V(D)J recombination-activating protein 2; also known as: RAG-2; H. sapiens nucleotide sequence NCBI RefSeq No. : NM_000536.3 (SEQ ID NO:09); H. sapiens amino acid sequence NCBI RefSeq No. : NP_000527.2 (SEQ ID NO: 10)), ZAP70 gene (zeta chain of T cell receptor associated protein kinase 70; also known as: SRK; STD; TZK; STCD; IMD48; ADMI02; ZAP-70; H. sapiens nucleotide sequence NCBI RefSeq No. : NM_001079.3 (SEQ ID NO: l 1); H. sapiens amino acid sequence NCBI RefSeq No. : NP_001070.2 (SEQ ID NO: 12)), JAK3 gene (Janus kinase 3; also known as: JAKL, LJAK, JAK-3, L-JAK, JAK3; H. sapiens nucleotide sequence NCBI RefSeq No. : NM_000215.3 (SEQ ID NO: 13); H. sapiens amino acid sequence NCBI RefSeq No.: NP_000206.2 (SEQ ID NO: 14)), BTK gene (Bruton tyrosine kinase; also known as: AT, ATK, BPK, XLA, IMD1, AGMX1, PSCTK1; H. sapiens nucleotide sequence NCBI RefSeq No. : NM_000061.2 (SEQ ID NO: 15); H. sapiens amino acid sequence NCBI RefSeq No. : NP_000052.1 (SEQ ID NO: 16)), WAS gene (Wiskott- Aldrich syndrome; also known as: THC, IMD2, SCNX, THC1, WASP, WASPA; H. sapiens nucleotide sequence NCBI RefSeq No. : NM_000377.2 (SEQ ID NO: 17); H. sapiens amino acid sequence NCBI RefSeq No. : NP_000368.1 (SEQ ID NO: 18)), LAD-1 gene (ladinin 1; also known as: LadA, LAD-1; H. sapiens nucleotide sequence NCBI RefSeq No.: NM_005558.3 (SEQ ID NO: 19); H. sapiens amino acid sequence NCBI RefSeq No. : NP_005549.2 (SEQ ID NO:20)), or Fanconi's anemia (FANC) gene (e.g., FANCA (Fanconi anemia complementation group A; also known as: FA, FA1, FAA, FAH, FA-H, FACA, FANCH; H. sapiens nucleotide sequence NCBI RefSeq No. :

NM_000135.2 (SEQ ID NO:21); H. sapiens amino acid sequence NCBI RefSeq No.: NP_000126.2 (SEQ ID NO:22)); FANCC (Fanconi anemia complementation group C; also known as: FA3, FAC, FACC; H. sapiens nucleotide sequence NCBI RefSeq No. : NM_000136.2 (SEQ ID NO:23); H. sapiens amino acid sequence NCBI RefSeq No.: NP_000127.2 (SEQ ID NO:24)); FANCG (Fanconi anemia complementation group G; also known as: FAG, XRCC9; H. sapiens nucleotide sequence NCBI RefSeq No. :

NM_004629.1 (SEQ ID NO:25); H. sapiens amino acid sequence NCBI RefSeq No.: NP 004620.1 (SEQ ID NO:26)); or FANCB, FANCD1/BCRA2, FANCD2, FANCE, FANCF, FANCJ, FANCL, FANCM).

[0069] In certain embodiments, the insert region can be integrated into the genomic target region through homologous pairing without the need for DNA cleavage prior to integration. In some embodiments, the insert region can be integrated into the genome at the genomic target region through homologous pairing without the need for the addition of exogenous site-specific nucleases such as a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or an RNA guided nuclease (CRISPR/Cas).

[0070] In yet another embodiment, non-homologous end joining (NHEJ) does not occur at the genomic target region in the infected cells.

[0071] In some embodiments, the hematopoietic cell is a hematopoietic stem cell. Hematopoietic stem cells (HSCs) are the stem cells that give rise to all the other blood cells through the process of hematopoiesis and encompass long-term repopulating cells. HSCs are derived from mesoderm and located in the red bone marrow, which is contained in the core of most bones (e.g., pelvis, femur and sternum), and they give rise to both the myeloid (e.g., monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, and megakaryocytes or platelets) and lymphoid (e.g., T-cells, B-cells, and natural killer (NK) cells lineages of blood cells). Sources of hematopoietic stem cells include, but are not limited to, whole bone marrow, mobilized peripheral blood, or fetal cord blood. In some embodiments, HSCs and progenitor cells can be taken from bone marrow of the pelvis or femur using a needle and syringe. In other embodiments, HSCs can be harvested from circulating peripheral blood. In such an embodiment, the blood donor can be injected with a cytokine (e.g., granulocyte-colony stimulating factor (G- CSF)), that induces cells to leave the bone marrow and circulate in the blood vessels.

[0072] In another embodiment, the hematopoietic cell can be a hematopoietic progenitor cell. A hematopoietic progenitor cell is an oligopotent cell that can

differentiate into a number of different specific types of cells, but can be pushed to differentiate into a specific type of cell. Hematopoietic progenitor cells typically have a limited capacity for self-renewal (i.e., a limited number divisions), while hematopoietic stem cells typically can replicate indefinitely. Examples of hematopoietic progenitor cell sources include, but are not limited to, lymphoid progenitor cells, whole bone marrow, mobilized peripheral blood, or fetal cord blood.

[0073] In other embodiments, the hematopoietic cell can be a T-lymphocyte, B- lymphocyte, monocyte, macrophage, natural killer cell, natural killer T-cell,

megakaryocyte, erythroid cell, or granulocyte.

[0074] In an embodiment, the recombinant AAV genomes of the AAV particles used in the methods for gene editing in a hematopoietic cell include a targeting construct comprising an insert region (which is substantially identical to a genomic target region except for a modification to be introduced at the genomic target sequence), regions flanking the insert region (the flanking regions facilitate homologous pairing between the insert region and the genomic target region resulting in the modification being introduced into the genomic target region), a 5' ITR and a 3' ITR. The targeting construct generally includes at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, at least about 1,000 nucleotides, at least about, at least about 2,000 nucleotides, at least about 3,000 nucleotides, at least about 4,000 nucleotides, or at least about 5,000 nucleotides or more.

[0075] In certain embodiments, the adenovirus-associated virus ("adeno-associated viruses" or "AAV") particles can include particles from AAV isolates or serotypes designated AAV2, AAV3 or AAV6. In some embodiments, the AAV serotype is AAV6. As used herein, the term "adeno-associated virus" or "AAV" means a member of

Dependovirus genus of the Parvidoviridae family of viruses. This genus of parvoviruses is characterized by a dependence on a helper virus for complete AAV replication and by the ability to stably integrate into a host cell chromosome in the absence of helper virus. In some embodiments, to obtain integration of an AAV genome into a mammalian cell, the cell is infected with the AAV in the absence of a helper virus. The term "helper virus" for AAV as used herein refers to a virus that allows AAV to be replicated and packaged by a mammalian cell. Helper viruses for AAV are known in the art, and include, for example, adenoviruses (such as Adenovirus type 5 of subgroup C), herpes viruses (such as herpes simplex viruses, Epstein-Bar viruses, and cytomegaloviruses) and poxviruses. As used herein, the term "serotype," when used in reference to AAV, means a subdivision of AAV that is identifiable by serologic or DNA sequencing methods and can be distinguishable by its antigenic character. In addition, the term "isolate," when used in reference to AAV, means a particular AAV serotype obtained from a specific source. The skilled artisan readily will recognize the difference between an "isolated AAV," which refers to the relative purity of an AAV sample, and an "AAV isolate," which refers to a clonally derived preparation of a particular AAV serotype, based on the context in which the term is used. In the absence of a helper virus, AAV produces no progeny virus but, instead, can integrate into a host chromosome. The viral genome of AAV6 (SEQ ID NO: 27) can useful for constructing AAV particles, AAV viral vectors and AAV vector plasmids, which can be used to introduce a heterologous nucleic acid sequence (i.e., the insert region) into a selected population of hematopoietic cells. As used herein, the term "viral genome," when used in reference to AAV, means the full length nucleic acid molecule found in the wild-type virus. It is recognized that an AAV viral genome is a single stranded nucleic acid molecule, which can be the plus strand or the minus strand, but that the combination of plus and minus strands in solution results in hybridization of the complementary strands to produce a double stranded form of the AAV viral genome. In addition, a viral genome can be in a double stranded form, which can be contained in plasmid. In addition, the term "vector genome" is used herein in reference to the single stranded nucleic acid molecule that is derived from an AAV viral genome and comprises at least a functional portion of the AAV viral genome. It is recognized, however, that a vector genome, such as an AAV6 vector genome, for example, also can contain portions of a viral genome from an AAV isolate other than AAV6, thus producing a hybrid vector genome, and that an AAV viral vector containing such an AAV6 vector genome can contain AAV6 viral proteins or can contain viral proteins of a different AAV isolate or serotype, thus producing a hybrid AAV viral vector. A vector genome can be in a double stranded form, which can be contained in a plasmid.

[0076] As used herein, the term "recombinant AAV particle" means an AAV viral particle containing an AAV vector genome with the targeting construct. If desired, the polypeptide components of the AAV particle can be from the same AAV serotype as the vector genome or can be from a different serotype, thus producing a hybrid AAV viral vector. Furthermore, a polypeptide component of a hybrid AAV viral particle can be hybrid viral protein, for example, a hybrid capsid protein, which can consist of a portion of a capsid protein of a first AAV serotype and a portion of a capsid protein of a second, different AAV serotype. In particular, a hybrid capsid can be constructed so as to lack one or more epitopes recognized by neutralizing antibodies, thereby allowing administration of an AAV particle comprising the hybrid capsid protein to an individual having neutralizing antibodies to a particular AAV serotype. A non-limiting example of a hybrid capsid can be AAV particles comprising at least a portion of an AAV3B capsid and an AAV6 capsid.

[0077] In some embodiments, the AAV particles comprise an AAV6 capsid protein (SEQ ID NO:28).

SEQ ID NO:28:

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKG EPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKR VLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQ PLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWA LPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPK RLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFM IPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRL MNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDN NNSNFTWTGASKYNLNGRESI INPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNV MITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWA KIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVE IEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL

[0078] The AAV6 sequence was first published by Rutledge et al, (1998, J. Virol. V72 p309). In certain embodiments, an AAV6 capsid comprises a protein that has at least 80%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 1 to 736 of SEQ ID NO:28.

[0079] In certain embodiments, the targeting construct lacks a promoter sequence or an enhancer sequence operatively linked to the insert region. In such an embodiment, the DNA sequence of the insert region will only be expressed if it correctly integrates at the genomic target region and thus uses the endogenous promoter/regulatory elements at the genomic target region. For example, if the insert region (or targeting cassette) does not contain a promoter to drive expression of the nucleotide sequence, then expression of the promoter-less nucleotide sequence demonstrates that the nucleotide sequence was correctly integrated into the hematopoietic cell. Thus, if the insert region does not contain a promoter, then the expression of the DNA sequence after integration into the genomic target region may be controlled by one or more regulatory elements of the hematopoietic cell. In certain embodiments, expression of the promoter-less DNA sequence

demonstrates that the DNA sequence was correctly integrated into the cell.

[0080] The targeting construct includes an insert region with the genetic modification or modifications that are to be introduced into the genomic target region. The

modifications can include one or more insertions, deletions, or point mutations, or combinations thereof, relative to the DNA sequence of the genomic target region. For example, to modify a genomic target region by introducing a point mutation, the insert region will include a DNA sequence that is at least substantially identical to the genomic target region except for the specific point mutation to be introduced. Upon introduction of the recombinant AAV genome into the cell, homologous pairing occurs between the portions of the targeting construct (i.e., flanking regions) that are substantially identical to the corresponding regions of the genomic target region, after which the DNA sequence of the mutation to be introduced that is present in the insert region of the targeting construct replaces that of the genomic target region (e.g., correction of genomic target region containing a mutation by replacement with a wild-type sequence). In certain

embodiments, the insert region can be of any length, but is typically between 10 and 4,500 nucleotides or longer. For example, the insert region can be from 100 to about 3,000 nucleotides, from 2,000 to about 4,000 nucleotides, from 3,000 to about 5,000 nucleotides, from 400 to about 4,000 nucleotides, or more than about 4,500 nucleotides long.

Accordingly, insert regions of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 3500, 4000, 4500 or more nucleotides in length are contemplated. In some embodiments, the insert region generally includes at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, at least about 1,000 nucleotides, at least about, at least about 2,000 nucleotides, at least about 3,000 nucleotides, at least about 4,000 nucleotides, or at least about 5,000 nucleotides or more that are identical to, or substantially identical to, the nucleotide sequence of a corresponding region of the genomic target region. By

"substantially identical" is meant that this portion of the targeting construct is at least about 80% identical, at least about 90% identical, or at least about 99% identical to the corresponding region of the genomic target region. The insert region can have the genetic modifications at either end of, or within the region of the insert that is identical to, or substantially identical to, the genomic target region. To delete a portion of a genomic target region, for example, the genetic modification will generally be within the insert region, being flanked by two regions of substantial identity to the target locus.

Homologous pairing between the two flanking regions of substantial identity and their corresponding regions of the genomic target region result in a portion of the sequence of the insert region, including the deletion, becoming incorporated into the genomic target region. Deletions can be precisely targeted to a desired location by this method. Similarly, genetic modifications that involve site-specific insertion of DNA sequences into a genomic target region can be made by use of a targeting construct with an insert region that has the DNA sequence to be inserted flanked by or next to regions of substantial identity to the genomic target region. Homologous pairing between the targeting construct and the corresponding regions of the genomic target locus is followed by incorporation of the insertion sequence into the genomic target region.

[0081] In an embodiment, the genomic target region refers to a region of a cellular genome at which a genetic modification is desired. In certain embodiments, the genomic target region of the cell genome may be any region of the genome where it is desired that the gene editing of the cell genome occur. In some embodiments, the genomic target region is a locus associated with a disease state as described herein. In certain

embodiments, the genomic target region can be of any length, but is typically between 10 and 4,500 nucleotides or longer. For example, the genomic target region can be from 100 to about 3,000 nucleotides, from 2,000 to about 4,000 nucleotides, from 3,000 to about 5,000 nucleotides, from 400 to about 4,000 nucleotides, or more than about 4,500 nucleotides long. Accordingly, genomic target regions of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 3500, 4000, 4500 or more nucleotides in length are contemplated. In some embodiments, the genomic target region generally includes at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, at least about 1 ,000 nucleotides, at least about, at least about 2,000 nucleotides, at least about 3,000 nucleotides, at least about 4,000 nucleotides, or at least about 5,000 nucleotides.

[0082] The genomic target region typically includes the specific nucleotides to be modified, as well as additional nucleotides on one or both sides of the modification sites. The genomic target region can be an exon, an intron, a promoter, a splice donor site, a splice acceptor site, a sequence encoding mRNA, a sequence encoding a non-coding RNA, a gene, a cDNA sequence, a partial cDNA sequence or combination thereof. A genomic target region can comprise a mutant target sequence comprising one or more mutant nucleotides, relative to a corresponding wild type target sequence. In some embodiments, the mutant target sequence is known to cause a genetic disorder in a subject. Examples of genetic disorders include, but are not limited to: achondroplasia, adenosine deaminase (ADA) deficiency, alpha- 1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, breast cancer, Charcot-Marie-Tooth, colon cancer, Cri du chat, Crohn's disease, cystic fibrosis, Dercum disease, down syndrome, Duane syndrome, Duchenne muscular dystrophy, Factor V Leiden thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, Fanconi anemia, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, prostate cancer, retinitis pigmentosa, severe combined immunodeficiency (SCID), sickle cell disease, spinal muscular atrophy, Tay-Sachs, thalassemia, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, WAGR syndrome, Wilson disease, and Wiskott-Aldrich syndrome (WAS).

[0083] In an embodiment, the mutant target sequence comprises one or more of a point mutation, a missense mutation, a nonsense mutation, an insertion of one or more nucleotides, a deletion of one or more nucleotides, or combinations thereof. In some embodiments, the genomic target region comprises the IL2RG gene, ADA gene, IL7R gene, RAGl gene, RAG2 gene, ZAP70 gene, JAK3 gene, BTK gene, WASP gene, LAD-1 gene, or Fanconi 's anemia (FANC) gene.

[0084] In certain embodiments, the targeting construct includes flanking regions on both sides of the insert region. The flanking regions facilitate homologous pairing between the insert region and the genomic target region resulting in the modification being introduced into the genomic target region. As used herein, "homologous pairing" refers to the pairing that can occur between two nucleic acid sequences or subsequences that are complementary, or substantially complementary, to each other. In certain embodiments, the insert region can be integrated into the genomic target region through homologous pairing (i.e., homologous recombination) facilitated by the flanking regions without the need for DNA cleavage prior to integration. In some embodiments, integration of the insert region at the genomic target region occurs through homologous pairing (i.e., homologous recombination) without the need for the addition of exogenous site-specific nucleases. In an embodiment, one of the flanking regions is 5' to the insert region and comprises a DNA sequence homologous to the DNA sequence that is upstream of the genomic target region (insert region). In another embodiment, the other flanking region is 3' to the insert region and comprises a DNA sequence homologous to the DNA sequence that is downstream of the genomic target region (insert region). In some embodiments, the flanking regions have substantially equal nucleotide lengths. In other embodiments, the flanking regions have asymmetrical nucleotide lengths. In some embodiments, the flanking region 5' to the insert region has at least about 90% (e.g., at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5%) nucleotide sequence identity to the region 5' to the genomic target region. In some embodiments, the flanking region 3' to the insert region has at least about 90% (e.g., at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5%) nucleotide sequence identity to the region 3' to the genomic target region. In some embodiments, the flanking region 5' to the insert region has 100% sequence identity to the region 5' to the genomic target region, and the flanking region 3' to the insert region has 100% sequence identity to the region 3' to the genomic target region. In some embodiments, each of the flanking regions independently is between about 100 to 4,500 nucleotides. For example, the flanking regions can be at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, at least about 1,000 nucleotides, at least about, at least about 2,000 nucleotides, at least about 3,000 nucleotides, at least about 4,000 nucleotides, or at least about 4,500 nucleotides or more and are identical to, or substantially identical to, the nucleotide sequence of a corresponding region 5' and 3' of the genomic target region (insert region).

[0085] In certain embodiments, the targeting constructs have all or a portion of at least one of the inverted terminal repeat sequence (ITR) or a functional equivalent inverted terminal repeat sequence at each end (i.e., a 5' ITR and a 3' ITR). ITR sequences or a functional equivalent, are generally required for the AAV vectors to replicate and be packaged into parvovirus particles. A functional equivalent of an ITR is typically an inverted repeat which can form a hairpin structure. Both a 5' ITR and a 3' ITR are typically present in the recombinant AAV vector DNAs used in the methods. In certain embodiments, a 5' ITR nucleotide sequence is 5' to the 5' flanking region, and a 3' ITR nucleotide sequence is 3' to the 3' flanking region.

[0086] In some embodiments, each the 5' ITR and 3' ITR sequences independently is between about 100 to 200 nucleotides. In another embodiment, the 5' ITR sequence and the 3' ITR sequence are substantially identical (e.g., at least 90%, at least 95%, at least 98%, at least 99% identical or 100% identical) to an AAV2 virus 5' ITR and an AAV2 virus 3' ITR, respectively. In some embodiments, the 5' ITR sequence has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:29, and the 3' ITR sequence has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:30. In some embodiments, the 5' ITR sequence and the 3' ITR sequence are substantially inverted repeats of each other (e.g., are inverted repeats of each other except for at 1, 2, 3, 4 or 5 nucleotide positions in the 5' or 3' ITR).

[0087] Exemplary AAV2 5' ITR (SEQ ID NO:29):

TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGC CCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATC ACTAGGGGTTCCT

[0088] Exemplary AAV2 3' ITR (SEQ ID NO: 30):

CTCTCCCCCCTGTCGCGTTCGCTCGCTCGCTGGCTCGTTTGGGGGGGTGGCAGCTCAAAGAGCTGC CAGACGACGGCCCTCTGGCCGTCGCCCCCCCAAACGAGCCAGCGAGCGAGCGAACGCGACAGGGGG GAGAGTGCCACACTCTCAAGCAAGGGGGTTTTGTA

[0089] In certain embodiments, the insert region to be integrated into the genomic target region of the hematopoietic cell genome may be a therapeutic nucleotide sequence. The term "therapeutic" as used herein refers to a substance or process that results in the treatment of a disease or disorder. Thus, a therapeutic nucleotide sequence is a nucleotide sequence that provides a therapeutic effect. The therapeutic effect can be direct (e.g., substitution of a nucleic acid of a gene expressed as a protein, or insertion of a cDNA into an intron for expression) or indirect (e.g., correction of a regulatory element such as a promoter). In certain embodiments, the therapeutic insert region may be a gene, a cDNA, a variant, fragment, or mutant thereof. In certain embodiments, when gene editing or gene therapy is desired, the therapeutic insert region may be any nucleotide sequence that encodes a protein that is therapeutically effective. The AAV particles comprising the therapeutic insert region nucleotide sequences are preferably administered in a therapeutically effective amount via a suitable route of administration, such as injection, inhalation, absorption, ingestion or other methods. Preparing Vectors

[0090] In certain embodiments, the methods of the invention involve the construction of recombinant AAV vector genomes and, the packaging of these viral genomes into AAV particles. Methods for achieving these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant viral genomes are well-known to persons of skill. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, Calif; Sambrook et al. (1989) Molecular Cloning— A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY; Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement); U.S. Pat. No. 5,017,478 and European Patent No. 0 246 864.

[0091] Typically, the recombinant viral genomes are initially constructed as plasmids using standard cloning techniques. The targeting constructs are inserted into the viral vectors, which include at least one of the two inverted terminal repeats or their functional equivalent. In some embodiments, the viral vector DNA is packaged into complete virus particles (i.e. , virions) that consist of an RNA or DNA core with a protein coat for use to infect the target cells. Viral vectors to be packaged can include in the viral genome DNA sequences necessary for replication and packaging of the recombinant viral genome into particles. In most embodiments, however, one or more of the replication and/or packaging polypeptides is provided by a producer cell line and/or a helper virus (e.g., adenovirus or herpesvirus). In certain embodiments, the AAV virus is dependent on a helper virus for complete AAV replication. An AAV virus has the ability to stably integrate into a host cell chromosome in the absence of helper virus. In some

embodiments, to obtain integration of an AAV genome into a mammalian cell, a cell is infected with the AAV in the absence of a helper virus. Helper virus functions include, for example, the Rep expression products, which are required for replicating the AAV genome (see, e.g., Muzyczka, (1992) Current Topics in Microbiol, and Immunol. 158: 97- 129 and Kotin, (1994) Human Gene Therapy 5:793-801). The human herpesvirus 6 (HHV-6) rep gene can serve as a substitute for an AAV rep gene (Thomson et al. (1994) Virology 204: 304-311). The cap region, which encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof, is also typically provided by a helper virus or producer cell line.

[0092] In certain embodiments, the recombinant viral genomes are grown as a plasmid and packaged into AAV particles (i.e., virions) by standard methods. See, e.g., Muzyczka, supra., Russell et al. (1994) Proc. Natl Acad. Sci. USA 91 : 8915-8919, Alexander et al. (1996) Human Gene Ther. 7: 841-850; Koeberl et al. (1997) Proc. Natl Acad. Sci. USA 94: 1426-1431; Samulski et al. (1989) J. Virol. 63: 3822-3828; Tratschin et al. (1985) o/. Cell. Biol. 5: 3251-3260; and Hermonat and Muzyczka (1984) Proc. Natl Acad. Sci. USA 81 : 6466-6470.

[0093] Transducing cells with viral vectors can involve, for example, incubating vectors with cells within the viral host range under conditions and concentrations necessary to cause transduction. See, e.g., Methods in Enzymology, vol. 185, Academic Press, Inc., San Diego, Calif. (D. V. Goeddel, ed.) (1990) or M. Krieger, Gene Transfer and Expression~A Laboratory Manual, Stockton Press, New York, N.Y.; and Muzyczka (1992) Curr. Top. Microbiol. Immunol. 158: 97-129, and references cited in each. The culture of cells, including cell lines and cultured cells from tissue samples is well known in the art. Freshney (Culture of Animal Cells, a Manual of Basic Technique, Third edition Wiley-Liss, New York (1994)) provides a general guide to the culture of cells.

[0094] The recombinant AAV genomes can be introduced into target cells by any of several methods. In certain embodiments, the AAV genomes into AAV particles which are then used to infect the target cells. Alternatively, the recombinant viral genomes can be introduced into cells in an unpackaged form. For example, standard methods for introducing DNA into cells can be employed to introduce the viral genomes, such as by microinjection, transfection, electroporation, lipofection, lipid encapsulation, biolistics, and the like. The recombinant viral genomes can be incorporated into viruses other than parvoviruses (e.g., an inactivated adenovirus), or can be conjugated to other moieties for which a target cell has a receptor and/or a mechanism for cellular uptake (see, e.g. , Gao et al. (1993) Hum. Gene Ther. 4: 17-24). The recombinant viral genomes can be introduced into either the nucleus or the cytoplasm of the target cells. Methods of transfecting and expressing genes in vertebrate cells are known in the art.

[0095] In certain embodiments, targeting enhancers can be included, such as recombinogenic proteins. See, e.g., Pati et al. (1996) Molecular Biol, of Cancer 1 : 1; Sena and Zarling (1996) Nature Genet. 3: 365; Revet et al. (1993) J. Mol. Biol. 232: 779-791; Kowalczkowski & Zarling in Gene Targeting (CRC 1995, Ch. 7). The parvoviral vector nucleic acids can be associated with the recombinogenic proteins prior to being introduced into the cells, or the recombinogenic proteins can be introduced into the cells

independently of the parvoviral vectors. In an embodiment, the AAV vector can be packaged in the presence of the recombinogenic protein, resulting in recombinogenic protein becoming packaged into the viral particles. An example of a recombinogenic protein is recA from E. coli and is available from Pharmacia (Piscataway N. J.). In addition to the wild-type protein, a number of mutant recA-like proteins have been identified (e.g., recA803). In some embodiments, the efficiency of gene editing can also be improved by treating the host cell in conjunction with the introduction of the recombinant viral genome. For example, one can administer to the target cells an agent that affects the cell cycle. These agents include, for example, DNA synthesis inhibitors (e.g., hydroxyurea, aphidicolin), microtubule inhibitors (e.g., vincristine), and genotoxic agents (e.g., radiation, alkylators).

[0096] In certain embodiments, other agents that can improve the efficiency of gene targeting include those that affect DNA repair, DNA recombination, DNA synthesis, protein synthesis, and levels of receptors for AAV. Also of interest are agents that affect, chromatin packaging, gene silencing, DNA methylation, and the like, as less condensed DNA is more likely to be accessible for gene targeting. These agents include, for example, topoisomerase inhibitors such as etoposide and camptothecin, and histone deacetylase inhibitors such as sodium butyrate and trichostatin A. Agents that inhibit apoptosis can also increase gene targeting by virtue of their ability to reduce the tendency of high concentrations of AAV to induce apoptosis. Suitable agents for these applications are described in, for example, U.S. Pat. No. 5,604,090, Russell et al. (1995) Proc. Nat'l. Acad. Sci. USA 92: 5719; Chen et al. (1997) Proc. Nat'l. Acad. Sci. USA 94: 5798;

Alexander et al. (1994) J. Virol. 68: 8282; and Ferrari et al. 41995) J. Neurosci. 15: 2857- 66, (1998) Mol. Cell. Biol. 18: 6482-92, (1994) EMBO J. 13: 5922-8 (70:3227)).

[0097] The parvoviral AAV particles can also be targeted to a particular cell or tissue type by use of a capsid or virion or delivery vehicle that displays a molecule that binds to a moiety that is specific for a desired target cell (i.e., hematopoietic cell). In some embodiments, polynucleotides that encode a polypeptide which can specifically bind to the target cell are incorporated into a gene that encodes a parvoviral capsid protein. Upon packaging of the parvoviral vector genome, the modified capsid (i.e. , an AAV6 serotype 6 capsid) is displayed on the surface of the particle, thus allowing the particles to preferentially deliver the nucleic acid to the desired target cell (i.e., a hematopoietic cell). Modification of parvoviral AAV capsid proteins for other purposes, as well as cell lines useful for expressing the genes that encode the modified proteins and methods of in vitro packaging using the modified capsid proteins, are described, for example, in U.S. Patent No. 5,863,541.

[0098] In a second aspect, the invention provides a method of treating a subject in need thereof, wherein the method comprises: (a) obtaining a population of hematopoietic cells from the subject; (b) transducing the population of hematopoietic cells with a recombinant AAV particle comprising an AAV6 capsid, wherein the recombinant AAV particle comprises: (i) an insert region comprising a DNA sequence which is substantially identical to a genomic target region except for a modification to be introduced at the genomic target sequence; (ii) regions flanking the insert region, wherein the flanking regions facilitate homologous pairing between the insert region and the genomic target region resulting in the modification being introduced into the genomic target region; (iii) a 5' inverted terminal repeat (ITR) sequence at the 5' end of the targeting construct; and (iv) a 3' inverted terminal repeat (ITR) sequence at the 3' end of the targeting construct;

wherein the hematopoietic cell is transduced by the recombinant AAV vector in the absence of a site-specific nuclease, and wherein transduction results in gene editing through integration of the insert region into the genomic target region of the hematopoietic cell; and (c) administering the transduced population of hematopoietic cell to the subject.

[0099] In certain embodiments, the population of hematopoietic cells is autologous to the subject. In another aspect the hematopoietic cell population is allogeneic or xenogeneic to the subject.

Ex Vivo Applications

[00100] In certain embodiments, the methods provided are useful for ex vivo applications, in which cells are harvested from a subject organism, genetically modified (i.e., transduced) using the methods provided herein, and reintroduced (i.e., administered) or infused into the subject. In some embodiments, harvested cells that are transduced using the methods provided are cultured ex vivo for a time in order to expand the cell population before being infused into the subject organism. In some embodiments, the harvested cells are cultured (i.e., expanded) ex vivo for a specified time before being genetically modified (i.e., transduced) and then the transduced cells are administered to the subject. In certain embodiments, the harvested cells are cultured ex vivo before being transduced, then the transduced cells are expanded for a time before being infused into the subject. The genetically modified (i. e., transduced) cells can be introduced into the same subject (i.e., autologous or syngeneic) from which the cells were originally obtained, or can be introduced into a different subject (i. e., allogeneic), or can be introduced into a different species (i. e., xenogeneic). Ex vivo therapy is useful, for example, in treating genetic diseases such as severe combined immunodeficiency, adenosine deaminase deficiency, hemophilia and certain types of thalassemia, as well as other diseases that are characterized by a defect in a cell that can be removed from the animal, modified using the methods of the invention, and reintroduced into the organism. The cells can be, for example, hematopoietic cells, hematopoietic stem cells or hematopoietic progenitor cells which can be derived from whole bone marrow, peripheral blood stem cell (PBSC) or fetal cord blood, T-lymphocytes, B-lymphocytes, monocytes, liver cells, muscle cells, fibroblasts, stromal cells, skin cells, or stem cells. The cells can be cultured from a subject or patient, or can be those stored in a cell bank (e.g., a blood bank). These methods are useful for treating humans, and also for veterinary purposes.

[00101] In some embodiments, the transduced cells are administered to the subject or patient at a rate determined by the LD₅₀ of transduced cell type, and the side-effects of cell type at various concentrations, as applied to the mass and overall health of the patient. For example, the transduced population of cells to be administered to the subject can be between 1 cell and 5x10⁹ cells per kilogram of body weight of the subject. Administration can be accomplished via single or divided doses. In certain embodiments, the transduced cells can be administered in a therapeutically effective amount via a suitable route of administration, such as injection, inhalation, absorption, ingestion or other methods.

[00102] Animal models and clinical protocols for ex vivo gene therapy have been established for hematopoietic cells (Blaese et al. (1995) Science 270: 475-480; Kohn et al. (1995) Nature Med. 1 : 1017-1023), liver cells (Grossman et al. (1994) Nature Genet. 6: 335-341), muscle cells (Bonham et al. (1996) Human Gene Ther. 7: 1423-1429), skin cells (Choate ei a/. (1996) Nature Med. 2: 1263-1267) and fibroblasts (Palmer et al. (1989) Blood 73: 438-445).

In Vivo Therapy

[00103] In some embodiments, the methods provided can be useful for correcting genetic defects in vivo. Muscular dystrophy is just one example of a genetic disease that is often the result of one or a few mutations that result in an abnormal polypeptide being expressed that is unable to carry out its function properly. The precise mutations for many variants of these and other genetic diseases are known to those of skill in the art, as are methods for identifying undesirable genetic mutations. Examples include, but are not limited to, Charcot-Marie-tooth disease, Coffin-Lowry syndrome, cystic fibrosis, fragile x syndrome, hemophilia, hereditary thrombotic predisposition (Factor V mutation)

Huntington's disease, medium-chain acyl-coemzyme a dehydrogenase deficiency (mead), myotonic dystrophy, neurofibromatosis (nfl), sickle cell disease and globin chain variations, spinal muscular atrophy, spinocerebellar ataxia, alpha and beta thalassemia, von Hippel-Lindau disease, and the like. Genetic diseases are reviewed in, for example, Shaw, D J (Ed.), Molecular Genetics of Human Inherited Disease, John Wiley & Sons, 1995; Davies and Read, Molecular Basis of Inherited Disease, 2^nd Edition, IRL Press, 1992. Human genetic diseases are treatable using the methods of the invention, as are those of other vertebrates. Non-genetic diseases can also be treated by manipulating genes. For example, one can modify a co-receptor for HIV so that the receptor is no longer able to bind to HIV particles.

[00104] In another embodiment, the AAV particles containing recombinant parvoviral genomes can be administered directly to the subject for modification of cells in vivo. Administration can be by any of the routes normally used for introducing viral vectors into ultimate contact with blood or tissue cells. The viral vectors used in the present inventive method are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such viral vectors in the context of the present invention to a patient are available, and, although more than one route can be used to administer a particular viral vector, a particular route can often provide a more immediate and more effective reaction than another route.

[00105] Pharmaceutically acceptable carriers are determined in part by the particular viral vector being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical compositions of the present invention. In some embodiments, formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the vector dissolved in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, com starch, potato starch, tragacanth,

microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the viral vector, carriers known in the art.

[00106] The AAV vector, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. Because the bronchial passageways are the usual route of choice for certain viruses, corresponding vectors are appropriately administered by this method. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[00107] Suitable formulations for rectal administration include, for example, suppositories, which consist of the active viral vector with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the viral vector with a base, including, for example, liquid triglyercides, polyethylene glycols, and paraffin hydrocarbons.

[00108] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, intrathecal (in the cerebrospinal fluid), and subcutaneous routes, include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and in some embodiments, can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[00109] The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time. The dose will be determined by the efficacy of the particular viral vector employed and the condition of the patient or animal, as well as the body weight or surface area of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector or modified cell type in a particular subject or patient.

[00110] In determining the effective amount of the viral vector to be administered in the treatment or prophylaxis of a particular disease, the physician or veterinarian needs to evaluate circulating plasma levels, vector toxicities, and progression of the disease.

[00111] In some embodiments, for the practice of this invention, the parvoviral vectors can be administered, for example, by aerosolization and inhalation, intravenous infusion, orally, topically, intramuscularly, intraperitoneally, intravesically or intrathecally. In certain embodiments, the method of administration will often be intravenous or by inhalation, but the parvoviral vectors can be applied in a suitable vehicle for the local and topical treatment of virally-mediated conditions.

[00112] For administration, parvoviral vectors and genetically modified cell types of the present invention can be administered at a rate determined by the LD₅₀ of the parvoviral vectors, and the side-effects of the parvoviral vector or cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses.

[00113] Protocols for in vivo gene therapy using adeno-associated viral vectors have been described for the brain (Alexander et al. (1996) Human Gene Ther. 7: 841-850), liver (Koeberl et al. (1997) Proc. Nat'l. Acad. Sci. USA 94: 1426-1431), lung (Flotte et al. (1993) Proc. Nat'l. Acad. Sci. USA 90: 10613-10617), and muscle (Xiao et al. (1996) J Virol. 70: 8098-8108). These methods can be adapted to other target organs by those of skill in the art.

[00114] One of skill in art would appreciate that the methods described herein are useful for nuclease-free AAV-mediated gene editing in hematopoietic cells. Several other groups reported that site-specific nucleases were required for editing and they did not observe editing without a nuclease (Wang et al., (2015) Nature Biotechnology 33(12): 1256-65). As described herein and demonstrated in the examples below, these methods provide for nuclease-free AAV-mediated gene editing in hematopoietic cells. A therapeutic level of targeting was obtained without a nuclease, based on the development of circulating mature CD8 cells. Furthermore, nuclease-free edited cells had an in vivo selective advantage and increased in number after their infusion into recipients, and a cDNA molecule that was integrated in the host cell genome and was appropriately regulated (and non-functional unless integrated appropriately). Furthermore, random integration of the AAV targeting construct' s insert region was not significant, and NHEJ did not occur at the target locus in the infected cells.

EXEMPLARY ASPECTS

[00115] Below are examples of specific aspects for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g. , amounts, temperatures, and the like), but some experimental error and deviation should, of course, be allowed for.

MATERIALS AND METHODS

Mice

[00116] I12rg-mutant mice (B6.129S4-I12rgtml Wjl/J), originally established by Cao et al., ("Defective lymphoid development in mice lacking expression of the common cytokine receptor gamma chain. Immunity 2:223-38 (1995)), and wild type C57BL/6 mice were obtained from The Jackson Laboratory. Mice were housed in pathogen-free conditions with unrestricted access to sterilized food and drinking water. All experiments were performed in compliance with the University of Washington Institutional Animal Care and Use Committee.

AA V construction

[00117] The targeting vector plasmids were constructed using routine molecular cloning methods and confirmed by DNA sequencing. The sequence of the I12rg knockout chromosomal locus was determined by sequencing PCR-amplified genomic DNA from an I12rg-/- mouse. The I12rg coding sequence was amplified from complementary DNA (cDNA) synthesized from total RNA harvested from wild type mouse peripheral blood. These sequences were assembled in an AAV vector backbone to produce pAAV-I12rge3- 8. The control vector plasmids pAAV-scMSCV-GFP and pAAV-MSCV-GFP contain a green fluorescent protein (GFP) gene driven by a murine stem cell virus (MSCV) promoter and a human growth hormone polyadenylation signal. The pAAV-MSCV-GFP vector plasmid contains additional stuffer DNA to expand the genome size beyond 2.5 kb and force packaging of non-self-complementary vectors.

AAV production and quantification

[00118] AAV vectors of serotype 6 (AAVmI12rg:ex3-8pA or AAVscMSCVeGFPpA) were produced by co-transfection of HEK 293T cells with pDGM6 (Gregorevic et al. "Systemic delivery of genes to striated muscles using adeno-associated viral vectors." Nature medicine 10:828-834, (2004)), and AAV plasmids using Calcium Phosphate precipitation method. Crude AAV particles were purified through iodixanol step gradient (Sigma- Aldrich, St. Louis, MO). Following heparin column binding and salt exchange (GE Life Sciences, Fairfield, CT), purified AAV particles were obtained and quantified by quantitative Southern blots as described {see Khan et al, Nature Protocols 6:482-501 (2011)). .

AA V vector transduced bone marrow cell preparation and transplantation

[00119] For preparation of bone marrow cells, 6-week-old male I12rg-mutant mice were treated with 5-fluorouracil (5-FU) (150 mg / kg per mouse) (APP Pharmaceuticals, Schaumburg, IL). Four days later, whole bone marrow (BM) cells from the femurs and tibias of donors were harvested and pooled. After lysing red blood cells using ACK lysis buffer (containing 155 mM NH₄C1, 10 mM KHC0₃ and 0.1 mM EDTA · 2Na (All reagents were purchased from ThermoFisher Scientific, Waltham, MA)), AAVmI12rg:ex3- 8pA or AAVscMSCVeGFPpA were transduced overnight at a multiplicity of infection of 10,000 genome-containing parti cles/cell (except where indicated otherwise in FIG. ID) in 6 well plates (Corning, Corning, New York, USA) seeded with 2xl0⁶ cells per well and cultured in RPMI 1640 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) containing 10% HyClone fetal bovine serum (Thermo Fisher Scientific), 1% GlutaMax (Thermo Fisher Scientific), 100 ng/mL recombinant mouse stem cell factor (PeproTech, Rocky Hill, New Jersey, USA), 100 ng/mL recombinant mouse FMS-related tyrosine kinase 3 ligand (Affymetrix ebioscience, San Diego, California, USA), and 10 ng/mL recombinant mouse thrombopoietin (PeproTech). The media was changed the next day. GFP expression after transducing with AAV-scMSCV-GFP was analyzed 3 days after infection by flow cytometry. AAV vector free BM cells were prepared by same methods. The next day, AAV -transduced BM cells were collected and washed by calcium/magnesium free phosphate buffered saline (PBS) (ThermoFisher Scientific).

[00120] For primary transplantation, BM cells from 6 week old male I12rg^_/" mice were transduced overnight as described above with AAV-I12rg3-8 or AAV-MSCV-GFP and 3x10⁶ BM cells were injected intravenously the next day into 6 week old irradiated (800 cGy) female I12rg^_/" recipients. For secondary transplantation, 2x10⁷ whole BM cells from primary recipients 20 weeks post-transplant were injected intravenously into irradiated 6 week old female mice. All transplant recipients were given Enrofloxacin (Bayer, Leverkusen, North Rhine-Westphalia, Germany) in the drinking water for 4 weeks after transplant.

[00121] For transducing efficiency analysis of AAVscMSCVeGFPpA, transduced BM cells were cultured in fresh DMEM based medium in 37°C 5% C02 incubator for 3 days.

[00122] For BM transplantation, 3 million BM cells were resuspended in 100 uL PBS and injected into an irradiated (800 cGy) age-matched female I12rg-mutant mouse (recipient mice) by retro-orbital injection. After injection, recipient mice were nurtured with sterile water with BAYTRIL® (1.7 mL in 1.7 OZ water) (Bayer, Shawnee Mission, KS) for 4 weeks.

Analysis of recipient peripheral blood

[00123] More than 100 uL peripheral blood (PB) was harvested from each recipient mouse, untreated I12rg-mutant mice, and C57BL/6 mice. Each lOOuL PB was treated by ACK lysis buffer, stained with Turk's solution, and counted. Then, the cells were incubated with anti-CD 16/32 antibodies (BD biosciences, Franklin Lakes, NJ) for 5 minutes on ice. After washing by PBS, the cells were stained by Allophycocyanin (APC) conjugated rat anti-mouse CD3 (Clone: 17A2) (BioLegend, SanDiego, CA), APC-cyanin 7 (Cy7) conjugated rat anti-mouse CD4 (Clone: GK1.5) (BD biosciences), and Brilliant Violet (BV) 510 conjugated rat anti-mouse CD8 (Clone: 53-6.7) (BioLegend) for 30 minutes on ice. For isotype control, about 1 x lO⁵ cells from C57BL/6 mouse were stained by APC conjugated rat IgG2b, APC-Cy7 conjugated rat IgG2b, and BV 510 conjugated rat IgG2a (all isotype antibodies were purchased from BioLegend). After staining, cells were washed by PBS, resuspended by 100 uL PBS with 2 uL 7AAD (BD biosciences), and analyzed using BD FACSCanto™ II Cell Analyzer (BD biosciences).

[00124] 100 uL of peripheral blood was collected monthly by retro-orbital bleeding from transplant recipients. Red blood cells were lysed by ACK solution (150 mM NH4C1, 10 mM KHC03, 1 mM ethylenediamine tetra-acetic acid) and the white blood cells (WBCs) were counted after staining with Turk's solution (Sigma- Aldrich, St. Louis, Missouri, USA). For flow cytometry, 10⁵ cells were suspended in PBS with 0.5 % bovine serum albumin and 2 mM EDTA, and incubated with each antibody for 30 minutes at 4°C. The antibody concentrations were established by staining wild type WBCs before each experiment. BM cells and splenocytes were stained similarly. The antibodies used were Allophycocyanin (APC) conjugated anti-CD3 (17A2, BioLegend, San Diego, California, USA), APC-cyanin 7 (Cy7) conjugated anti-CD4 (L3T4, BD Pharmingen, Franklin Lakes, New Jersey, USA), Brilliant Violet 510 conjugated anti-CD8a (53-6.7, BioLegend), APC conjugated anti-CDl lb (Ml/70, BioLegend), Phycoerythrin (PE) conjugated anti-CD45.2 (104, Affymetrix ebioscience), PE-Cy7 conjugated anti-CD45.2 (104-2, Miltenyi Biotec, San Diego, California, USA), PE conjugated anti-CD132 (TUGm2, BioLegend), PE conjugated anti-CD62L (MEL-14, BioLegend), fluorescein conjugated anti-IgD (l l-26c, BD Pharmingen), PE conjugated anti-Seal (D7, Miltenyi Biotec), PE conjugated anti- NK1.1 (PK136, BioLegend), Brilliant Violet 421 conjugated anti-B220 (RA3-6B2, BioLegend) and fluorescence-conjugated isotype controls (all BioLgend). Live, stained cells were analyzed by using the BD Cell Viability kit (BD pharmingen) on a

FACSCanto™ II flow cytometer (BD Pharmingen).

Quantitative PCR and sequencing

[00125] Genomic DNA was harvested from BM cells, CD3⁺ and CD3^" splenocytes, and peripheral blood WBCs by using the QIAamp DNA Micro Kit (Qiagen, Hilden, Germany). The CD3⁺ and CD3^" splenocytes were collected by staining with APC conjugated anti-CD3 body and PE conjugated anti-CD45.2 and sorting on a BD

FACSAria™ III (BD Pharmingen). Quantitative PCR was performed with the GoTaq Probe qPCR Master Mix (Promega) on a StepOnePlus real-time PCR system (Thermo Fisher Scientific). To confirm that editing occurred, an edited allele-specific product was amplified from the genomic DNA of CD3⁺ splenocytes isolated from AAV-I12rg3-8- treated mice with PHUSION® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, Massachusetts, USA) on a C 1000 Touch Thermal Cycler (BIO-RAD, Hercules, California, USA), then cloned and sequenced the gel-isolated product. Total RNA was prepared from the CD3⁺ splenocytes of AAV-I12rg3-8-treated mice with the RNeasy Mini Kit (Qiagen), synthesized cDNA with the SUPERSCRIPT® III First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific), amplified an edited allele-specific cDNA and sequenced the product. The primers, probes and conditions used are shown in Table 1. Table 1. PCR primers and probes.

(SEQ ID NO:52) genomic DNA levels

ITR qPCR FW GGAACCCCTAGTGATGGAGTT (SEQ ID NO:53) qPCR of vector ITR sequences

ITR qPCR RV CGGCCTCAGTGAGCG (SEQ ID NO:54) qPCR of vector ITR sequences

ITR qPCR probe (5'6-FAM)-CACTCCCTCTCTGCGCGCTCG-(3IABkFQ) qPCR of vector ITR

(SEQ ID NO:55) sequences

MuTCB3C GCCAGAAGGTAGCAGAGACCC (SEQ ID NO:56) Common primer for

TCR repertoire analysis

MuTCB for 2^nd primer (5'6-FAM)-TTGGGTGGAGTCACATTTCTC (SEQ ID Analyzing TCR

NO:57) fragment length

T cell receptor (TCR) repertoire analysis

[00126] Variable (V) β chain repertoires in complementary determining region 3 were analyzed by preparing total R A from CD3⁺ splenocytes with the RNeasy Mini Kit and synthesizing cDNA with the iSCRIPT® Select cDNA Synthesis Kit (BIO-RAD). A first step PCR was performed using a specific primer for the constant region of the TCR β chain (C β) in combination with 21 V gene segment-specific primers and the products were underwent a second PCR using an internal FAM-labeled C β specific primer. Primer sequences and PCR conditions were based on a previous report (Pannetier et al, (1993), Proc Natl Acad Sci U.S.A. 90(9);4319-23). FAM-labeled products were analyzed on a DNA sequencer by the Genomics Facility of the Fred Hutchinson Cancer Research Center (Seattle, WA, USA) and the data were analyzed using Peak Scanner Software (Thermo Fisher Scientific).

Statistical analysis

[00127] Statistical analysis was performed using Graphpad Prism 5 (GraphPad

Software, San Diego, CA). Values are presented as the mean ± standard deviation and P < 0.05 was considered significant. Statistical analyses were performed by the unpaired two- tailed Student's t-test for pairwise comparisons or one-way or two-way analysis of variance (ANOVA) with Bonferroni's multiple comparison post-test for more than three groups using Prism software (Graph Pad Software, San Diego, CA. USA).

Example 1. Generation of AAV vector for gene editing murine I12rg locus

[00128] In the chromosomes of I12rg-mutant mouse, a neomycin resistance cassette replaced part of exon 3 and all of exons 4-8 of chromosomal I12rg locus, resulting in the loss of most of the extracellular domain and all of the transmembrane and cytoplasmic domains of the protein. The AAV targeting vector AAVmI12rg:ex3-8pA (SEQ ID NO: 61) was designed to insert the cDNA sequence from exon 3 to exon 8 of the I12rg gene into the mutant locus upstream of the neomycin cassette (see Figure 1). Homology arms flank this partial cDNA cassette. Because the AAV vector doesn't contain promoter sequences, a transcription start site or a translation start site, random integrants of the AAV targeting vector will not produce functional I12rg mRNA or protein.

[00129] An AAV vector homologous to the deleted I12rg locus in X-SCID mice 4 was constructed, but containing a partial I12rg cDNA at exon 3 (see FIG.1A). AAV-I12rg3-8 does not include the I12rg promoter or initiation codon, so random integration events will not lead to I12rg expression, but homologous recombination at the endogenous locus creates a complete I12rg reading frame expressed from the I12rg promoter. AAV- scMSCV-GFP is a self-complementary control vector that does not require second strand synthesis or annealing to express GFP from its murine stem cell virus (MSCV) promoter. AAV-scMSCV-GFP packaged in serotype 6 capsids (Rutledge et al, J Virol 72:309-19 (1998).) transduced over 90% of mouse bone marrow cells at an MOI of 20,000, including primitive Sca-1+ cells (FIG. ID), confirming that serotype 6 vectors are able to efficiently enter hematopoietic cells (Wang et al, Nature Biotech 33(12): 1256-65 (2015)). The full- length control vector AAV-MSCV-GFP was used in transplantation experiments to more accurately model the similarly sized AAV-I12rg3-8 vector.

[00130] X-SCID mouse bone marrow cells were infected overnight with AAV vectors, and then delivered by intravenous injection into irradiated X-SCID recipients. AAV- I12rg3-8-treated mice developed circulating CD4⁺ and CD8⁺ T-cells that increased in number over time, which did not occur in mice that received AAV-MSCV-GFP-infected or uninfected cells (FIG. IE, FIG. IF). The circulating T-cells expressed I12rg on their cell surface (FIG. IG), and in CD8+ cells the level was statistically indistinguishable from that of wild type cells, demonstrating properly regulated expression of the I12rg transcript at the edited locus. Cell surface expression of I12rg in CD4⁺ cells was slightly below wild type levels (FIG.1G), as expected for a mixed population of edited and unedited cells. The levels of other peripheral blood cell types in AAV-I12rg3-8-treated mice were unchanged from controls, except for a modest increase in circulating monocytes (FIG.2A, FIG.2C). These results are consistent with in vivo selection and expansion of edited T-cells, but not of edited myeloid cells, B-cells or their precursors. Natural Killer (NK) cell numbers also were not significantly changed in treated mice (FIG.2A-2C), although they require I12rg- mediated signaling for their formation (Rochman et al., Nature reviews. Immunology 9:480-90 (2009). Selective expansion of T-cells was also observed in the spleens of AAV- I12rg3-8-treated mice (FIG.3A-3D).

Example 2. AAV serotype 6 transduces BM cells in vitro

[00131] Whole BM cells from I12rg-mutant mice were transduced with

AAVscMSCVeGFPpA and analyzed GFP expression by flow cytometry 3 days after. About 6 % and 50 % of BM cells transiently expressed enhanced GFP (EGFP) with AAV transduction at multiplicity of infection (MOI) of 1,000 and 10,000 vector particles per cell respectively (FIG. IC). These data showed that AAV serotype 6 at MOI of 10,000 can efficiently transduce hematopoietic stem/progenitor cells.

[00132] The frequency of transduced cells was measured in the transplanted mice by quantitative PCR (qPCR) with primers designed to detect either donor cells (Y

chromosome sequences), cells containing randomly integrated vector genomes (AAV vector sequences outside of the homology arms), or all transduced cells (I12rg exon 5 sequences only found in the AAV-I12rg3-8 vector genome) (FIG.4A). The frequency of edited cells was then calculated by subtracting the random integration frequency from the frequency of all transduced cells. While random AAV vector integrants may have terminal deletions of varying sizes, the vast majority of integrated genomes are expected to contain the sequences amplified in this assay, based on large-scale studies of vector integration in human and mouse cells (Miller et al. Journal of virology 79: 11434-42 (2005); Nakai et al, Journal of virology 79:3606-14 (2005). qPCR analysis was performed on peripheral blood white blood cells (WBCs), bone marrow (BM) cells, and CD3⁺ or CD3^" splenocytes 32 weeks after transplantation (FIG.4B). Substantial donor cell marking (32-62%) was found in all samples, regardless of the vector used. Edited cells constituted -10% of the peripheral blood WBCS of AAV-I12rg3-8-treated mice, and a higher proportion of their CD3⁺ splenocytes (> 58% of donor cells), whereas edited BM cells and CD3^" splenocytes were significantly lower (< 1%). In general, the qPCR-based editing frequencies correlated with the frequency of CD3⁺ cells in each sample, confirming that the increase in T-cell numbers represented edited cells. Although random integration frequencies were detectable, they were extremely rare (< 0.4%) in all samples, despite the fact that most of the cells presumably contained biologically active vector genomes soon after infection (see FIG. ID). This was confirmed by measuring GFP expression in AAV-MSCV-GFP-treated mice, which showed that 0.04 % of peripheral blood WBCs were GFP⁺ (FIG.4C).

[00133] An edited allele-specific PCR product was amplified from the CD3⁺ cells of AAV-I12rg3-8-treated mice by long-range PCR with primers inside the vector and outside the homology arms. This PCR product had unique chromosomal and vector sequences that could only be due to I12rg gene editing (FIG.5 A). Similarly, the sequence of an mRNA amplified by RT-PCR from the CD3⁺ splenocytes of treated mice showed chromosomal-specific and vector-specific sequences (FIG.5B), confirming that accurate I12rg gene editing had occurred. The entire I12rg coding region of this mRNA was identical to the wild-type transcript.

Example 3. Reconstitution of donor cells in recipient mice

[00134] Oligonucleotide AAVmI12rg:ex3-8pA transduced (MOI = 10,000) male I12rg- mutant BM cells were transplanted into irradiated female I12rg-mutant mice (experimental mice). As a control, AAVscMSCVeGFPpA transduced (MOI=10,000) or AAV vector free BM cells were injected into recipient mice by the same methods as experimental mice. After transplanting, peripheral blood from recipients, untreated wild type C57BL/6 mice, and untreated I12rg-mutant mice was harvested every 4 weeks. Peripheral blood cells from each mouse were analyzed for T-cell populations using flow cytometer and cell numbers of total white blood cells (WBCs), CD4⁺ T cells, and CD8⁺ T-cells were measured.

[00135] After transplanting, WBC numbers from experimental mice had increased over 12 weeks whereas that from controls reached plateau at 8 weeks (FIG.9A). CD8⁺ T- cells first appeared from experimental mice at 8 weeks (FIG.9B) and both CD4⁺ and CD8⁺ T-cells further increased over 12 weeks (FIG.9C). These data showed that AAV targeting vector treated BM reconstituted T cells in I12rg-mutant mice. As with X-SCID patients, I12rg-mutant mice do not develop CD3⁺ T-cells, including both mature CD4⁺ and mature CD8 T-cells. The lack of T-cells causes life-threatening immunodeficiency. The appearance of these cells in vector-treated mice demonstrates that the chromosomal I12rg gene was successfully edited, and that it was expressed correctly in the proper cell lineages. This requires that gene editing occurred in the T-cell precursor or earlier stage. These findings demonstrate that a therapeutic effect can be obtained by nuclease-free hematopoietic cell gene editing.

[00136] At 8 weeks, very low levels of EGFP⁺ cells were detected in the peripheral blood of AAVscMSCVeGFPpA transduced controls (FIG.9D), and this may not have been statistically different from non-specific background EGFP readings. This indicated that if AAV vectors integrate non-homologously (i.e., randomly) into host genomes, it occurs at a very low frequency (less than 0.2% of total PB).

[00137] Bone marrow cells were isolated from treated mice 20 to 32 weeks after transplantation and used to transplant additional I12rg-/- mice. Secondary recipients of AAV-I12rg3-8-treated BM cells had increases in peripheral blood lymphocytes over time, including CD3⁺, CD4⁺ and CD8⁺ cells that by 16 weeks reached levels slightly below those of primary recipients (FIG.6A, FIG.7A and FIG.7B). These secondary recipients also had a modest increase in peripheral blood monocytes, and no increase in

granulocytes, NK cells or B cells as compared to AAV-MSCV-GFP-treated controls (FIG.6C, FIG.6D), suggesting that the edited T-cells were selectively expanding in vivo. The spleens of secondary AAV-I12rg3-8-treated recipients also contained T lymphocytes, with CD3⁺ and CD4⁺ cells present at higher levels than in primary recipients (FIG.6B). These combined results show that long-term repopulating cells were edited and that their progeny continued to increase in number in secondary recipients.

[00138] These long-lived repopulating cells could be true hematopoietic stem cells, or possibly more committed lymphoid progenitors that retained proliferative potential. In contrast to primary transplant recipients, the spleens of AAV-I12rg3-8-treated secondary- recipients contained ~6 % B220⁺ B lymphocytes and a total of ~2xl0⁵ B cells per spleen (FIG.6C, FIG.6D). Approximately one-third of these B-cells were IgD⁺ (FIG.6E), demonstrating that antibody gene rearrangement had occurred (Geisberger et al.,

Immunology 118:429-37 (2006).

[00139] A key indicator of successful X-SCID gene therapy is the development of a diverse T cell receptor (TCR) repertoire. The TCR νβ genes of CD3 splenocytes from primary transplant recipients were examined by PCR amplification of CDR3 variant beta chain regions. The repertoire of AAV-I12rg3-8-treated mice was similar to that of wild- type mice, and significantly more diverse than that of AAV-MSCV-GFP-treated mice (FIG.7A). Naive CD4⁺ and CD8⁺ T cells were both increased by AAV-I12rg3-8 treatment (FIG.7B and FIG.7C), suggesting that gene editing can help establish the complex regulatory networks required for effective immunity.

[00140] The results here demonstrate that the nuclease-free AAV-mediated gene editing of I12rg^_/" mouse BM cells can restore expression of I12rg, which is the common subunit of the receptors for interleukins 2, 4, 7, 9, 15, and 21. These cytokines are required for normal T cell development and homeostasis, which allowed edited, I12rg⁺ T cells to selectively proliferate in vivo, undergo TCR rearrangements, and produce naive CD4⁺ and CD8⁺ cells. Edited T cells persisted for more than 8 months in primary recipients, and at least 4 more months in secondary recipients, demonstrating that editing had occurred in either hematopoietic stem cells or long-lived lymphoid progenitors.

[00141] The examples demonstrate site-directed gene editing for long term

repopulating hematopoietic stem cells that are free from any side effects, including malignancy. Unlike nuclease-mediated gene editing, which has the possibility of unwanted side-effects (including off-target cutting and a high frequency of unpredictable nucleotide insertion or deletion by the double strand brakes (DSBs) repair due to nonhomologous end joining (NHEJ)), the methods of AAV mediated recombination provided here do not result in DSBs nor NHEJ. The results demonstrate that nuclease-free, AAV- mediated gene editing of hematopoietic cells could be used to treat humans. For example, patients with X-SCID, ADA SCID, RAGl/2 deficiency, Wiskott Aldrich Syndrome, and Fanconi's anemia, could be provided therapeutic effects using the methods described herein.

[00142] Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words "herein," "above," and "below" and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

[00143] While the invention has been particularly shown and described with reference to an aspect and various alternate aspects, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize

[00144] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes. Aspects of the disclosure can be modified, if necessary, to employ the systems, functions, and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description.

Claims

Claims

1. A method for gene editing in a hematopoietic cell, comprising transducing an effective amount of a recombinant adeno-associated virus (AAV) particle into the hematopoietic cell, wherein the recombinant AAV particle comprises a targeting construct comprising:

(a) an insert region comprising a DNA sequence which is substantially identical to a genomic target region except for a modification to be introduced at the genomic target sequence;

(b) regions flanking the insert region, wherein the flanking regions facilitate homologous pairing between the insert region and the genomic target region resulting in the modification being introduced into the genomic target region;

(c) a 5' inverted terminal repeat (ITR) sequence at the 5' end of the targeting construct; and

(d) a 3' inverted terminal repeat (ITR) sequence at the 3' end of the targeting construct;

wherein the recombinant AAV particle comprises an AAV serotype 6 capsid and is transduced into the hematopoietic cell in the absence of a site-specific nuclease, and wherein transduction results in gene editing through integration of the insert region into the genomic target region of the hematopoietic cell.

2. The method of claim 1, wherein incidence of random integration is less than one percent.

3. The method of any one of claims 1 to 2, wherein the hematopoietic cell is transduced ex vivo.

4. The method of any one of claims 1 to 3, wherein the genetic correction provides a selective survival advantage.

5. The method of any one of claims 1 to 4, wherein non-homologous end joining ( HEJ) does not occur at the genomic target region in the transduced cells.

6. The method of any one of claims 1 to 5, wherein the AAV6 capsid comprises an amino acid sequence that has at least 80%, at least 85%>, at least 86%>, at least 88%>, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid sequence identity to SEQ ID NO:28.

7. The method of any one of claims 1 to 6, wherein the AAV serotype 6 capsid comprises the amino acid sequence of SEQ ID NO:28.

8. The method of any one of claims 1 to 7, wherein the hematopoietic cell is a hematopoietic stem cell.

9. The method of claim 8, wherein the hematopoietic stem cell is derived from whole bone marrow, mobilized peripheral blood, or fetal cord blood.

10. The method of claim 9, wherein the hematopoietic stem cell is derived from whole bone marrow.

11. The method of any one of claims 1 to 7, wherein the hematopoietic cell is a

hematopoietic progenitor cell.

12. The method of claim 11, wherein the hematopoietic progenitor cell is derived from lymphoid progenitor cells, whole bone marrow, mobilized peripheral blood, or fetal cord blood.

13. The method of claim 12, wherein the hematopoietic progenitor cell is derived from lymphoid progenitor cells.

14. The method of any one of claims 1 to 7, wherein the hematopoietic cell is a T- lymphocyte, B-lymphocyte, monocyte, macrophage, natural killer cell, natural killer T cell, megakaryocyte, erythroid cell, or granulocyte.

15. The method of any one of claims 1 to 14, wherein the genomic target region is an exon, an intron, a promoter, a splice donor site, a splice acceptor site, a sequence encoding mRNA, a sequence encoding a non-coding RNA, a gene, a cDNA sequence , a partial cDNA sequence or combination thereof.

16. The method of any one of claims 1 to 15, wherein the genomic target region comprises a mutant target sequence comprising one or more mutant nucleotides, relative to a

corresponding wild type target sequence.

17. The method of claim 16, wherein the mutant target sequence comprises one or more selected from a point mutation, a missense mutation, a nonsense mutation, an insertion of one or more nucleotides, a deletion of one or more nucleotides, or combinations thereof.

18. The method of any one of claims 1 to 17, wherein the genomic target region comprises the IL2RG gene, ADA gene, IL7R gene, RAG1 gene, RAG2 gene, ZAP70 gene, JAK3 gene, BTK gene, WASP gene, LAD-1 gene, or Fanconi's anemia (FANC) gene.

19. The method of any one of claims 1 to 18, wherein the targeting construct lacks a promoter sequence or an enhancer sequence operatively linked to the insert region.

20. The method of any one of claims 1 to 19, wherein each of the flanking regions independently is between about 100 to 4,500 nucleotides.

21. The method of any one of claims 1 to 20, wherein each the 5' ITR and the 3' ITR sequences independently is between about 100 to 200 nucleotides.

22. The method of any one of claims 1 to 21, wherein the 5' ITR comprises the amino acid sequence of SEQ ID NO: 29 and the 3' ITR comprises the amino acid sequence of SEQ ID NO:30.

23. A method of treating a subject in need thereof, wherein the method comprises:

(a) obtaining a population of hematopoietic cells from the subject;

(b) transducing the population of hematopoietic cells with a recombinant AAV particle comprising an AAV6 capsid, wherein the recombinant AAV particle comprises:

(i) an insert region comprising a DNA sequence which is substantially identical to a genomic target region except for a modification to be introduced at the genomic target sequence;

(ii) regions flanking the insert region, wherein the flanking regions facilitate homologous pairing between the insert region and the genomic target region resulting in the modification being introduced into the genomic target region;

(iii) a 5' inverted terminal repeat (ITR) sequence at the 5' end of the targeting construct; and

(iv) a 3' inverted terminal repeat (ITR) sequence at the 3' end of the targeting construct;

wherein the hematopoietic cell is transduced by the recombinant AAV vector in the absence of a site-specific nuclease, and wherein transduction results in gene editing through integration of the insert region into the genomic target region of the hematopoietic cell; and

(c) administering the transduced population of hematopoietic cell to the subject.

24. The method of claim 23, wherein the population of hematopoietic cells is autologous to the subject.

25. The method of claim 23 or claim 24, wherein the transduced population of hematopoietic cells administered to the subject is between 1 and 5xl0⁹ cells per kilogram of body weight of the subject.

26. The method of any one of claims 23 to 25, wherein the subject has a mutation in the IL2RG gene, ADA gene, IL7R gene, RAG1 gene, RAG2 gene, ZAP70 gene, JAK3 gene, BTK gene, WASP gene, LAD-1 gene, or Fanconi's anemia (FANC) gene.

27. The method of any one of claims 23 to 26, wherein the subject has a genetic disorder selected from achondroplasia, adenosine deaminase (ADA) deficiency, alpha- 1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, breast cancer, Charcot-Marie-Tooth, colon cancer, Cri du chat, Crohn's disease, cystic fibrosis, Dercum disease, down syndrome, Duane syndrome, Duchenne muscular dystrophy, Factor V Leiden thrombophilia, familial hypercholesterolemia, familial

Mediterranean fever, Fanconi anemia, fragile X syndrome, Gaucher disease,

hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, prostate cancer, retinitis pigmentosa, severe combined immunodeficiency (SCID), sickle cell disease, spinal muscular atrophy, Tay-Sachs, thalassemia, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, WAGR syndrome, Wilson disease, and Wiskott-Aldrich syndrome (WAS).

28. The method of any one of claims 23 to 27, wherein incidence of random integration is less than one percent.

29. The method of any one of claims 23 to 28, wherein the population of hematopoietic cells is transduced ex vivo.

30. The method of any one of claims 23 to 29, wherein the genetic correction provides a selective survival advantage.

31. The method of any one of claims 23 to 30, wherein non-homologous end joining (NFIEJ) does not occur at the genomic target region in the transduced cells.

32. The method of any one of claims 23 to 31, wherein the AAV6 capsid comprises an amino acid sequence that has at least 80%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid sequence identity to SEQ ID NO:28.

33. The method of any one of claims 23 to 32, wherein the AAV serotype 6 capsid comprises the amino acid sequence of SEQ ID NO:28.

34. The method of any one of claims 23 to 33, wherein the population of hematopoietic cells comprises hematopoietic stem cells.

35. The method of claim 34, wherein the population of hematopoietic stem cells are derived from whole bone marrow, mobilized peripheral blood, or fetal cord blood.

36. The method of claim 35, wherein the population of hematopoietic stem cells are derived from whole bone marrow.

37. The method of any one of claims 23 to 33, wherein the population of hematopoietic cells are hematopoietic progenitor cells.

38. The method of claim 37, wherein the population of hematopoietic progenitor cells are derived from lymphoid progenitor cells, whole bone marrow, mobilized peripheral blood, or fetal cord blood.

39. The method of claim 38, wherein the population of hematopoietic progenitor cells are derived from lymphoid progenitor cells.

40. The method of any one of claims 23 to 33, wherein the population of hematopoietic cells are T-lymphocytes, B-lymphocytes, monocytes, macrophages, natural killer cells, natural killer T cells, megakaryocytes, erythroid cells, or granulocytes.

41. The method of any one of claims 23 to 40, wherein the genomic target region is an exon, an intron, a promoter, a splice donor site, a splice acceptor site, a sequence encoding mRNA, a sequence encoding a non-coding RNA, a gene, a cDNA sequence , a partial cDNA sequence or combination thereof.

42. The method of any one of claims 23 to 41, wherein the genomic target region comprises a mutant target sequence comprising one or more mutant nucleotides, relative to a corresponding wild type target sequence.

43. The method of claim 42, wherein the mutant target sequence comprises one or more selected from a point mutation, a missense mutation, a nonsense mutation, an insertion of one or more nucleotides, a deletion of one or more nucleotides, or combinations thereof.

44. The method of any one of claims 23 to 43, wherein the genomic target region comprises the IL2RG gene, ADA gene, IL7R gene, RAG1 gene, RAG2 gene, ZAP70 gene, JAK3 gene, BTK gene, WASP gene, LAD-1 gene, or Fanconi's anemia (FANC) gene.

45. The method of any one of claims 23 to 44, wherein the targeting construct lacks a promoter sequence or an enhancer sequence operatively linked to the insert region.

46. The method of any one of claims 23 to 45, wherein each of the flanking regions independently is between about 100 to 4,500 nucleotides.

47. The method of any one of claims 23 to 46, wherein each the 5' ITR and the 3' ITR sequences independently is between about 100 to 200 nucleotides.

48. The method of any one of claims 23 to 47, wherein the 5' ITR comprises the amino acid sequence of SEQ ID NO: 29 and the 3' ITR comprises the amino acid sequence of SEQ ID NO:30.