WO2022232839A1 - Methods for improved production of primary cd34+ cells - Google Patents

Abstract

The present disclosure describes methods to produce genetically modified cells from human stem cells derived from a subject. In addition, the present disclosure describes correction of a gene through homology-directed repair in order to correct aberrant expression that results in a disease.

Description

METHODS FOR IMPROVED PRODUCTION OF PRIMARY CD34+ CELLS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to U.S. Provisional Application No.63/182,042 filed April 30, 2021, the disclosure of which is incorporated by reference in its entirety for all purposes. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH [0002] This invention was made with Government support under contract R01HL135607 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND [0003] Genome engineering is rapidly becoming an attractive avenue to develop therapeutics and tools that address many unmet needs related to curing human disease. Many diseases are caused by aberrant expression of a gene, thereby causing the manifestation of a disease. For example, some disease may occur due to inadequate expression of a disease. In these cases, approaches such as enzyme replacement therapy (ERT) may be a possible approach; however, the patient may require continual administration throughout his or her life in order to keep the disease at bay. A more permanent solution is more attractive. [0004] Hematopoietic stem and progenitor cells (HSPCs) are stem cells that can give rise to all blood cell types such as the white blood cells of the immune system (e.g., virus-fighting T cells and antibody-producing B cells) and red blood cells. The therapeutic administration of HSPCs can be used to treat a variety of adverse conditions including immune deficiency diseases, blood disorders, malignant cancers, infections, and radiation exposure (e.g., cancer treatment, accidental, or attack-based). HSPCs can be distinguished by their expression of the CD34 cell surface protein and can be referred to as “CD34+ stem cells.” Isolation of CD34+ stem cells and subsequent manipulation of said cells can be useful alternatives to ERTs, which require continuous and lifelong administration of the replacement enzyme. BRIEF SUMMARY [0005] The present disclosure provides a large scale ex vivo method of generating a population of genetically modified stem cells from a population of stem cells, the method comprising: 1) obtaining the population of stem cells from a subject, 2) culturing the population of stem cells, 3) introducing into the population of stem cells a CRISPR-associated Cas9 nuclease and a guide polynucleotide sequence that hybridizes to a target sequence within a target gene; and 4) contacting the population of stem cells with an AAV vector comprising a donor polynucleotide, thereby generating the population of genetically modified stem cells. In some embodiments, the population of stem cells obtained in step 1) is frozen. In some embodiments, the population of stem cells obtained in step 1) is an apheresis product. In some embodiments, the population of stem cells cultured in step 2) is enriched from the apheresis product. In some embodiments, step 2) can further comprise cryopreservation of the population of stem cells. In some embodiments, the cryopreservation is performed in cryopreservation media comprising heparin and pulmozyme. In some embodiments, the cryopreservation media further comprises human serum albumin. [0006] In some embodiments, the CRISPR-associated Cas9 nuclease is a high-fidelity CRISPR- associated Cas9 nuclease. [0007] In some embodiments, the CRISPR-associated Cas9 nuclease comprises a mutation at position R691. In some embodiments, the mutation at position R691 is an alanine. In some embodiments, the CRISPR-associated Cas9 nuclease introduces a double stranded break. [0008] In some embodiments, the population of stem cells is cultured in cytokine-rich cell culture media. In some embodiments, the cytokine-rich media comprises human SCF, human TPO, human Flt3 ligand, human IL-6, or a combination thereof. [0009] In some embodiments, the culturing is performed in the presence of UM171. [0010] In some embodiments, the culturing is performed at a low-cell density. In some embodiments, the low-cell density comprises 2.5-5.0 x 10⁵ cells/ml. [0011] In some embodiments, the AAV vector is AAV6. [0012] In some embodiments, the culturing is performed at low oxygen conditions. In some embodiments, the low oxygen conditions comprise 5% O2. In some embodiments, the low oxygen conditions further comprise 5% CO2. [0013] In some embodiments, step 4) occurs within 20 minutes of step 3). In some embodiments, step 4) occurs within 15 minutes of step 3). In some embodiments, step 4) occurs within 6 minutes of step 3). [0014] In some embodiments, the culturing of step 2) results in reduced production of pro- inflammatory cytokines. In some embodiments, the culturing of step 2) results in at least 50% reduced production of pro-inflammatory cytokines. [0015] In some embodiments, the population of stem cells comprises a population of human stem and progenitor cells (HSPCs). In some embodiments, the population of stem cells is mobilized by plerixafor. [0016] In some embodiments, the method results in at least 20% gene edited (i.e. genetically modified) stem cells within the population of stem cells. In some embodiments, the method generates a yield of at least 10%- 60% genetically modified stem cells within the population of stem cells. In some embodiments, the method generates at least 50-150 x 10⁶ genetically modified stem cells. [0017] In some embodiments, the method generates the genetically modified stem cells in at least 5L, 10L, 50L, 100L, 300L, or 500L. [0018] In some embodiments, the population of stem cells comprises at least one mutation in the HBB gene. In some embodiments the target gene is the HBB gene. In some embodiments, the donor polynucleotide comprises a sequence comprising SEQ ID NO: 5 flanked by a 5’ homology arm and 3’ homology arm. In some embodiments, the 5’ homology arm and 3’ homology arm comprises a nucleic acid sequence having at least 75% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4, respectively. In some embodiments, the target sequence comprises SEQ ID NO: 1. [0019] In some embodiments, the genetically modified stem cell comprises at least 10%, 20%, 30%, 40%, 50% greater expression of wild-type beta globin compared to a cell that was not genetically modified. In some embodiments, the genetically modified stem cell comprises at least 10%, 20%, 30%, 40%, 50% less expression of a mutant form of beta globin compared to a cell that was not genetically modified. [0020] In some embodiments, the donor polynucleotide sequence comprises SEQ ID NO: 2. [0021] The present disclosure also provides a large scale ex vivo method of generating a population of genetically modified stem cells from a population of stem cells, wherein the population of stem cells comprises at least one mutation in the HBB gene, the method comprising: 1) obtaining the population of stem cells from a subject, 2) culturing the population of stem cells, 3) introducing into the population of stem cells a CRISPR-associated Cas9 nuclease and a guide polynucleotide sequence that hybridizes to a target sequence in the HBB gene and wherein the target sequence comprises SEQ ID NO 1, and contacting the population of stem cells with an AAV vector comprising a donor polynucleotide sequence comprising SEQ ID NO: 5 flanked by a 5’ homology and 3’ homology arm having at least 75% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4, respectively, thereby generating the population of genetically modified stem cells. [0022] In some embodiments, the population of stem cells obtained in step 1) is frozen. In some embodiments, the population of stem cells obtained in step 1) is an apheresis product. In some embodiments, the population of stem cells cultured in step 2) is enriched from the apheresis product. In some embodiments, step 2) can further comprise cryopreservation of the population of stem cells. [0023] In some embodiments, the cryopreservation is performed in cryopreservation media comprising heparin and pulmozyme. In some embodiments, the cryopreservation media further comprises human serum albumin. In some embodiments, the method results in at least 20% of gene edited cells. In some embodiments, the CRISPR-associated Cas9 nuclease introduces a double stranded break. [0024] In some embodiments, the CRISPR-associated Cas9 nuclease is a high-fidelity Cas9 nuclease. In some embodiments, the CRISPR-associated Cas9 nuclease comprises a mutation at position R691. In some embodiments, the mutation at position R691 is an alanine. [0025] In some embodiments, the population of stem cells is cultured in cytokine-rich cell culture media. In some embodiments, the cytokine-rich media comprises human SCF, human TPO, human Flt3 ligand, human IL-6, or a combination thereof. [0026] In some embodiments, the culturing is performed in the presence of UM171. [0027] In some embodiments, the culturing is performed at a low-cell density. In some embodiments, the low-cell density comprises 2.5-5.0 x 10⁵ cells/ml. [0028] In some embodiments, the genetically modified stem cell comprises at least 10%, 20%, 30%, 40%, 50% greater expression of wild-type beta globin compared to a cell that was not genetically modified. In some embodiments, the genetically modified stem cell comprises at least 10%, 20%, 30%, 40%, 50% less expression of a mutant form of beta globin compared to a cell that was not genetically modified. [0029] In some embodiments, the AAV vector is AAV6. [0030] In some embodiments, the culturing is performed at low oxygen conditions. [0031] In some embodiments, the low oxygen conditions comprise 5% O2. [0032] In some embodiments, the low oxygen conditions further comprise 5% CO2. [0033] In some embodiments, step 4) occurs within 20 minutes of step 3). In some embodiments, step 4) occurs within 15 minutes of step 3). In some embodiments, step 4) occurs within 6 minutes of step 3). In some embodiments, the culturing of step 2) results in reduced production of pro-inflammatory cytokines. In some embodiments, the culturing of step 2) results in at least 50% reduced production of pro-inflammatory cytokines. [0034] In some embodiments, the population of stem cells comprises a population of human stem and progenitor cells (HSPCs). In some embodiments, the population of stem cells is mobilized by plerixafor. In some embodiments, the method generates a yield of at least 10%-60% genetically modified stem cells. In some embodiments, the method generates at least 50-150 x 10⁶ genetically modified stem cells. [0035] In some embodiments, the method generates the genetically modified stem cells in at least 5L, 10L, 50L, 100L, 300L, or 500L. [0036] In some embodiments, the donor polynucleotide sequence comprises SEQ ID NO: 2. [0037] Provided in the present disclosure is also a method of treating a hematological disease in a subject in need thereof, the method comprising: 1) generating a population of genetically modified stem cells according to present disclosure; and 2) administering the population of genetically modified stem cells to the subject in need thereof. In some embodiments, the population of genetically modified cells administered in step 2) comprises 50-150 x 10⁶ genetically modified stem cells. [0038] In some embodiments, the subject has sickle cell disease (SCD). [0039] In some embodiments, the administering is performed intravenously. [0040] The present disclosure also provides a large scale ex vivo method for generating genetically modified stem cells at a clinical scale for use in treating sickle cell disease in a subject in need thereof, the method comprising: 1) obtaining a population of cells from the subject, 2) isolating a population of hematopoietic stem cells from the population of cells, 3) culturing the population of stem cells, 4) introducing into the population of stem cells a CRISPR- associated Cas9 nuclease and b) a guide polynucleotide sequence that hybridizes to a target sequence comprising SEQ ID NO: 1, and 5) contacting the population of stem cells with an AAV vector comprising a donor polynucleotide sequence comprising SEQ ID NO 2, thereby generating the genetically modified stem cells. INCORPORATION BY REFERENCE [0041] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS [0042] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [0043] FIGS.1A-1H show gene correction in healthy donor-derived HSPCs. FIG.1A shows schematics of cell manufacturing protocol, in vitro readouts and experimental design of in vivo NSG mouse studies. FIG.1B shows the percentage of in vitro gene-corrected alleles (gcHBB) (left), viability and CD34 expression (right) in up to 13 cell donors; black lines indicate mean values. FIG.1C shows human chimerism in bone marrow of NSG mice at week 16 post- injection. Control cells were i. electroporated only (Mock), ii. coupled with either RNP (Cas9 only) or iii. AAV6 (AAV6 only). One-way ANOVA Kruskal-Wallis test plus Dunn’s multiple comparisons test; ns, not significant. FIG.1D shows the percent distribution of human hematopoietic lineages within the human cell population. FIG.1E shows the percent gcHBB alleles in the human cell population (bulk) and in the respective hematopoietic lineages. FIG.1F shows the percent gcHBB alleles in the bulk population and in a mouse-by-mouse analysis of human lineages in bone marrow samples (data points from the same mouse are connected), mice showing good gcHBB allele representation in all lineages (left) or mostly in the myeloid compartment (right). FIG.1G shows the quantification of HBB allele distribution in single CFU colonies derived from human HSPCs extracted at terminal analysis from bone marrow of engrafted mice. WT: wild type, INDEL: insertion/deletions, HR: homologous recombination. Data point colors indicate cellular outcome with respect to HBB expression: white “neutral”, red “deficient”, green “corrective”. Brackets group genotypes per cellular outcomes; black bars indicate mean values. FIG.1H shows the percent of gcHBB alleles in vivo (bone marrow) linked to genotype of single sorted HSPC-derived CFUs from the same mouse. Pearson r test, p<0.001. Black bars and lines indicate median values if not differently specified. [0044] FIGS.2A-2G show gene correction in SCD patient derived HSPCs. FIG.2A shows the percent allele modification in six gcHBB-SCD biological replicates (Exp 1-6). FIG.2B shows the viability (n=5) and CD34 (n=3) expression in vitro. FIG.2C shows the percent of in vitro differentiated erythroid cells displaying CD71+ CD235a+ markers (n=3). FIG.2D shows the quantification of hemoglobin tetramers (HPLC) on erythroid differentiated populations (n=3). FIG.2E shows human chimerism in the bone marrow of NSG mice analyzed at week-16 post- injection (n=7). Mann-Whitney test; *, p<0.01. Black bars indicate median values. FIG.2F shows the percent distribution of human hematopoietic lineages in the NSG bone marrow. FIG. 2G shows the percent of gcHBB alleles in the human cell population (bulk) in vitro (n=3) and in vivo mouse-by-mouse (n=7), along with the respective human hematopoietic lineages collected from NSG bone marrow. Mann-Whitney test; *, p<0.05. Black bars and lines indicate mean (± SD) if not differently specified. [0045] FIGS.3A-3D show the scale up manufacturing of CD34+ cells. FIG.3A shows the percent of allele modification in six medium-scale cell manufacturing runs (Run 1-6). FIG.3B shows the CFU frequency for gcHBB-SCD cell products and untreated cell counterpart. Paired t test. *, p<0.05. FIG.3C shows a scatter plot linking gene correction of HBB alleles in the bulk cell population to single genotyped colonies. Pearson r test *, p<0.05. FIG.3D shows quantification of HBB allele distribution in single CFU colonies (genotype). WT: wild type, INDEL: insertion/deletions, HR: homologous recombination. Data point colors indicate cellular outcome with respect to HBB expression: white “neutral’, red ‘deficient’, green ‘corrective’. Brackets group genotypes per cellular outcome. [0046] FIGS.4A-4E show the effects of UM171 on gene correction of CD34+ cells as a comparison of the two cell culture conditions, with (+) and without (-) UM171. FIG.4A shows gcHBB allele frequencies in vitro. FIG.4B shows human chimerism in bone marrow of transplanted NSG mice (week 16). FIG.4C shows In vivo retained gcHBB alleles (bone marrow) expressed as a ratio to the injected in vitro cell population; median with interquartile range. FIG.4D shows quantification of total gcHBB-SCD cell number in the bone marrow at week 16. FIG.4E shows the percent of gcHBB alleles in the human graft in bone marrow of primary and secondary recipients. Black lines show median values. [0047] FIGS.5A-5E show different protocol optimization of the gene correction of CD34+ cells. FIG.5A shows cell growth in vitro (day-2, pre-stimulation phase). Values are expressed as fold- change with respect to the initial culture cell number (day 0). FIG.5B shows HBB gene targeting frequencies with an AAV6.GFP donor vector. Percentage of GFP positive cells as a function of cell culture concentration. FIG.5C shows a heatmap representation of top-scoring pro- inflammatory cytokines that decrease as a function of cell concentration. Data are expressed as fold-change with respect to the high-density condition (1 x 10⁶ cells/mL; orange, 1.0). The cursor indicates beginning of statistical significance. Two-way ANOVA with Bonferroni post-test; p<0.001. FIG.5D shows the percent of gcHBB alleles in cells edited with WT and HiFi Cas9. FIG.5E shows the percent of INDELs at OT1 quantified in human cells engrafted in the NSG bone marrow. Black lines indicate mean values. [0048] FIG.6 shows a schematic of the manufacturing of CD34+ cells for HBB gene correction. [0049] FIGS.7A-7B show the efficiency of gene correction of HBB. FIG.7A shows the stacked distribution of the different expected outcomes of the gene correction protocol. FIG. 7B shows the percent of HBB allele grouped by the different expected outcomes. [0050] FIGS.8A-8B demonstrate the purity and viability of the generated gene corrected drug products comprising the genetically modified cells. FIG.8A shows the viability following acridine orange and propidium iodide to quantify the viability of the cells. FIG.8B shows the percent (%) of CD34+ cells in the product. [0051] FIGS.9A-9C show the engraftment of the drug product into NSG mice. FIG.9A shows the engraftment protocol of obtaining thawed gene corrected cells and dosing NSG mice, and quantifying the engraftment efficiency. FIG.9B shows the percent of cells taken from bone marrow. FIG.9C shows the percent of gene corrected HBB from different cellular population derived from the harvested bone marrow. These results were compared to the in vitro gene corrected frequency. [0052] FIG.10 shows the percent of alleles from gene corrected HBB from two different increased scale manufacturing runs. DETAILED DESCRIPTION Introduction [0053] The present disclosure provides a method of generating CD34+ stem cells from a subject and using said CD34+ stem cells for genetic manipulation to correct aberrant expression of a gene harboring one or more mutations that causes disease, for example, a mutated beta- hemoglobin gene. The method disclosed herein provides several advantages: 1) generation of genetically modified CD34+ stem cells at increased scale, yield and viability; and 2) use of a closed system to generate genetically modified CD34+ stem cells that is substantially free of contaminants. Manufacturing cells at a scale greater than what is achievable in a standard laboratory is not a trivial task and requires optimization and systems to enable production of cells in a greater number and is amenable as a therapeutic for human use. The present disclosure further describes the present method of manufacturing of said CD34+ stem cells for genetic manipulation for said human therapeutic use. [0054] The present disclosure also provides an ex vivo method of manufacturing and generating CD34+ stem cells that have been engineered to produce a therapeutic protein. In some cases, the subject has at least one mutation in beta-globin (HBB). In some cases, the subject who has the mutation has a hemoglobinopathy. In some cases, the hemoglobinopathy is sickle cell disease (SCD). Definitions [0055] As used herein, the following terms have the meanings ascribed to them unless specified otherwise. [0056] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth. [0057] The term “gene” refers to a combination of polynucleotide elements, that when operatively linked in either a native or recombinant manner, provide some product or function. The term “gene” is to be interpreted broadly, and can encompass mRNA, cDNA, cRNA and genomic DNA forms of a gene. [0058] The term “homology-directed repair” or “HDR” refers to a mechanism in cells to accurately and precisely repair double-strand DNA breaks using a homologous template to guide repair. The most common form of HDR is homologous recombination (HR). [0059] The term “homologous recombination” or “HR” refers to a genetic process in which nucleotide sequences are exchanged between two similar molecules of DNA. Homologous recombination (HR) is used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks or other breaks that generate overhanging sequences. [0060] The term “single guide RNA” or “sgRNA” refer to a DNA-targeting RNA containing a guide sequence that targets the Cas nuclease to the target genomic DNA and a scaffold sequence that interacts with the Cas nuclease (e.g., tracrRNA), and optionally, a donor repair template [0061] The term “Cas polypeptide” or “Cas nuclease” refers to a Clustered Regularly Interspaced Short Palindromic Repeats-associated polypeptide or nuclease that cleaves DNA to generate blunt ends at the double-strand break at sites specified by a 20-nucleotide guide sequence contained within a crRNA transcript. A Cas nuclease requires both a crRNA and a tracrRNA for site-specific DNA recognition and cleavage. The crRNA associates, through a region of partial complementarity, with the tracrRNA to guide the Cas nuclease to a region homologous to the crRNA in the target DNA called a “protospacer.” [0062] The term “ribonucleoprotein complex” or “RNP complex” refers to a complex comprising an sgRNA and a Cas polypeptide. [0063] The term “donor polynucleotide” refers to a polynucleotide sequence comprising an exogenous polynucleotide sequence that is flanked by 5’ and 3’ homology arms that are sufficiently complementary to the target region such that integration of the exogenous polynucleotide sequence occurs by homology directed repair, resulting in insertion of the exogenous polynucleotide sequence into the target region. This can also refer to a nucleic acid stand, e.g., DNA strand that is the recipient strand during homologous recombination strand invasion that is initiated by the damaged DNA, in some cases, resulting from a double-stranded break. The donor polynucleotide serves as template material to direct the repair of the damaged DNA region. [0064] The terms “sequence identity” or “percent identity” in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same (“identical”) or have a specified percentage of amino acid residues or nucleotides that are identical (“percent identity”) when compared and aligned for maximum correspondence with a second molecule, as measured using a sequence comparison algorithm (e.g., by a BLAST alignment, or any other algorithm known to persons of skill), or alternatively, by visual inspection. [0065] The term “homologous” refers to two or more amino acid sequences when they are derived, naturally or artificially, from a common ancestral protein or amino acid sequence. [0066] Similarly, nucleotide sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid. [0067] The term “primary cell” refers to a cell isolated directly from a multicellular organism. Primary cells typically have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous (tumor or artificially immortalized) cell lines. In some cases, primary cells are cells that have been isolated and then used immediately. In other cases, primary cells cannot divide indefinitely and thus cannot be cultured for long periods of time in vitro. [0068] The term “genetically modified cell” refers to a cell or a primary cell into which a heterologous nucleic acid has been introduced in some cases, into its endogenous genomic DNA. It generally refers to a cell that has undergone gene correction through the method disclosed herein. “Genetically modified cell” and “gene corrected cells” can be used interchangeably. The genetically modified cell has undergone a genetic alteration and may have a different expression profile than the parental cell. [0069] The term “primary immune cell” or “primary leukocyte” refers to a primary white blood cell including but not limited to a lymphocyte, granulocyte, monocyte, macrophage, natural killer cell, neutrophil, basophil, eosinophil, macrophage, stem cell thereof, or progenitor cell thereof. For instance, a primary immune cell can be a hematopoietic stem cell or a hematopoietic progenitor cell. A hematopoietic stem cell or a hematopoietic progenitor cell can give rise to blood cells, including but not limited to, red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, and all types thereof. [0070] The term “administering” or “administration” refers to the process by which agents, compositions, dosage forms and/or combinations disclosed herein are delivered to a subject for treatment or prophylactic purposes. Compositions, dosage forms and/or combinations disclosed herein are administered in accordance with good medical practices taking into account the subject's clinical condition, the site and method of administration, dosage, subject age, sex, body weight, and other factors known to the physician. For example, the terms “administering” or “administration” include providing, giving, dosing and/or prescribing agents, compositions, dosage forms and/or combinations disclosed herein by a clinician or other clinical professional. [0071] The term “treating” refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested. [0072] The terms “culture,” “culturing,” “grow,” “growing,” “maintain,” “maintaining,” “expand,” “expanding,” etc., when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that a cell (e.g., primary cell) is maintained outside its normal environment under controlled conditions, e.g., under conditions suitable for survival. Cultured cells are allowed to survive, and culturing can result in cell growth, stasis, differentiation or division. The term does not imply that all cells in the culture survive, grow, or divide, as some may naturally die or senesce. Cells are typically cultured in media, which can be changed during the course of the culture. [0073] The terms “subject,” “patient,” and “individual” are used herein interchangeably to include a human or animal. For example, the animal subject may be a mammal, a primate (e.g., a monkey), a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance. The subject can comprise at least one mutation in a gene, wherein the gene is replaced with a gene lacking the at least one mutation using the ex vivo method described in the present disclosure. [0074] The term “ex vivo” refers generally to activities that take place outside an organism, such as experimentation or measurements done in or on living tissue in an artificial environment outside the organism, preferably with minimum alteration of the natural conditions. In particular embodiments, “ex vivo” procedures involve living cells or tissues taken from an organism and cultured or modulated in a laboratory apparatus, usually under sterile conditions, and typically for a few hours or up to about 24 hours, but including up to 48 or 72 hours, depending on the circumstances. In certain embodiments, such tissues or cells can be collected and frozen, and later thawed for ex vivo treatment. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissue are typically considered to be “in vitro,” though in certain embodiments, this term can be used interchangeably with ex vivo. Methods of Generating Genetically Modified Stem Cells from a Subject [0075] Provided in the present disclosure are methods for manufacturing cells, for example, to generate populations of cells for use in adoptive cell therapy. The methods include those useful for gene editing applications utilizing site-specific nucleases for knock-out of targeted genomic sequences or knock-in of exogenous sequences, and for transferring exogenous sequences to the cells by viral transduction through the use of recombinant viral vectors. The exogenous sequences generally encode recombinant molecules to be expressed in the cells, e.g., for use in cell therapy. Processing steps of the methods can also or alternatively include all or a portion of cell washing, dilution, selection, isolation, separation, cultivation, stimulation, packaging, and/or formulation. The methods generally allow for the processing, e.g., selection or separation and/or transduction, of cells on a large scale (such as in compositions of volumes greater than or at about 50 mL). [0076] The provided methods offer various advantages compared with available methods for cell processing, including for transduction and selection, particularly those for large-scale cell processing. In some embodiments, the provided methods are suitable for large-scale and/or clinical-grade cell production, while still providing desirable features otherwise available only with small-scale production methods, and offering additional advantages not provided by available methods. For example, the methods for cell transduction and/or affinity-based selection offer advantages compared with available methods performed in flexible plastic bags or plastic multi-well plates. [0077] Currently, existing CD34+ stem cells manufacturing methods include various complex steps for the isolation, transduction and expansion of CD34+ stem cells. In contrast, the present disclosure provides use of a closed system for a genetically modified CD34+ cell manufacturing platform for a medium to large-scale clinical cGMP manufacturing process for engineered CD34+ HSPCs. [0078] The present disclosure provides methods to enrich CD34+ stem cells obtained from a subject and performing an ex vivo method to perform a targeted genetic manipulation to correct at least one mutation in a gene that results in aberrant expression or result in a disease in the subject. A non-limiting example can be, a population of cells (e.g., apheresis product) are obtained from a subject, in which the CD34+ stem cells are isolated using an enrichment method. A Cas9 nuclease and a guide polynucleotide specific for the target gene, as well as, a donor polynucleotide sequence are introduced into the enriched cell, thereby generating a population of genetically modified cells. The genetically modified cells are separated from the cells that have not been genetically modified to obtain a population that is substantially free of non-genetically modified cells. The population of genetically modified cells can be used to treat a subject having a hemoglobinopathy, such as SCD. Closed System Manufacturing [0079] In some embodiments, cells are manufactured using a closed processing system, or in combination with a closed cell processing system. Closed cell processing systems automate processes including treatment, centrifugation, incubation, media addition, cell selection, cell washing, and final fill and finish within “closed” or resealable vessels. Closed cell processing systems integrate and automate the processes and replicate many qualitatively controlled manual tasks to provide consistent and operator-independent quality. [0080] The benefits associated with an automated, closed-cell processing system would include significant reduction in the cost of therapies (typically 25-90%) and the number of operators required (typically >70%); lowered dependence on skilled labor; significant savings in capital investment through better facility use (typically 30-50%); improved quality and fewer quality events; and an ability to more rapidly scale up and scale out to match market demands. [0081] In various examples, a method for manufacturing therapeutic gene edited cell compositions comprises obtaining a population of cells comprising CD34+ cells or HSPCs using a closed system process. In some embodiments, the isolated population of cells is seeded at a particular density to initiate cultures. In particular examples, populations of stem cells comprising CD34+ cells that are transduced with a viral vector encoding an exogenous polynucleotide sequence. Manufactured CD34+ cells or HSPCs comprising the genetical modified CD34+ cells may then be used to treat subjects in need thereof or frozen for later use. [0082] A closed system may provide reduced contaminants that can be introduced into the cellular culture. Methods for Isolating and Purifying CD34+ Stem Cells from a Subject [0083] The present disclosure provides a method of obtaining a population of cells from a product that is collected from a subject, such as a patient or subject in need thereof. The product can be an apheresis product that has been obtained from a product and contains a heterogeneous mixture of cells that have been collected from the subject. Said heterogenous mixture of cells can contain primary cells as well as primary CD34+ cells and/or human stem and progenitor cells (HSPCs). The CD34+ cells and/or HSPCs can be isolated or separated from the other cells in order to obtain a population of stem cells. Following the separation of CD34+ HSPCs, the resulting population of stem cells are substantially free of non-CD34+ cells and are ready for subsequent genetic manipulation. [0084] In some embodiments, the CD34+ HSPCs are separated from the population of primary cells using flow cytometry. In some instances, the flow cytometry comprises fluorescence- activated cell sorting (FACS). In certain other embodiments, the CD34+ HSPCs are separated from the population of primary cells using magnetic bead separation. In some instances, the magnetic bead separation comprises magnetic-activated cell sorting (MACS). In certain other embodiments, the CD34+ HSPCs are separated using a device configured for CD34+ HSPC enrichment, such as the Miltenyi Biotec CliniMACS cell manufacturing platform. [0085] In some embodiments, the obtained population of stem cells comprise at least about 50 % viability to about 99 % viability. In some embodiments, the obtained population of stem cells comprise at least at least about 50 % viability. In some embodiments, the obtained population of stem cells comprise at least at most about 99 % viability. In some embodiments, the obtained population of stem cells comprise at least about 50 % viability to about 60 % viability, about 50 % viability to about 70 % viability, about 50 % viability to about 80 % viability, about 50 % viability to about 90 % viability, about 50 % viability to about 95 % viability, about 50 % viability to about 97 % viability, about 50 % viability to about 99 % viability, about 60 % viability to about 70 % viability, about 60 % viability to about 80 % viability, about 60 % viability to about 90 % viability, about 60 % viability to about 95 % viability, about 60 % viability to about 97 % viability, about 60 % viability to about 99 % viability, about 70 % viability to about 80 % viability, about 70 % viability to about 90 % viability, about 70 % viability to about 95 % viability, about 70 % viability to about 97 % viability, about 70 % viability to about 99 % viability, about 80 % viability to about 90 % viability, about 80 % viability to about 95 % viability, about 80 % viability to about 97 % viability, about 80 % viability to about 99 % viability, about 90 % viability to about 95 % viability, about 90 % viability to about 97 % viability, about 90 % viability to about 99 % viability, about 95 % viability to about 97 % viability, about 95 % viability to about 99 % viability, or about 97 % viability to about 99 % viability. [0086] Selectable markers, detectable markers, cell surface markers, and cell purification markers, alone or in combination, can be used to isolate and/or purify genetically modified primary cells, e.g., genetically modified human primary cells. Expression of a selectable marker gene encoding an antibiotic resistance factor can provide for preferential survival of genetically modified cells in the presence of the corresponding antibiotic, whereas other cells present in the culture will be selectively killed. Alternatively, expression of a fluorescent protein such as GFP or expression of a cell surface marker not normally expressed on the primary cells may permit genetically modified primary cells to be identified, purified, or isolated by fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), or analogous methods. Suitable cell surface markers include CD8, truncated CD8, CD19, truncated CD19, truncated nerve growth factor receptor (tNGFR), and truncated epidermal growth factor receptor (tEGFR), CD34, although other cell surface markers can also fulfill the same function. [0087] Methods for isolating or purifying the genetically modified primary cells are known in the art. In some embodiments, a population of genetically modified primary cells is isolated or purified (e.g., separated) from a population of unmodified primary cells in accordance with the enrichment scheme of the present disclosure. For example, FACS or MACS methods can be used to enrich genetically modified human primary cells expressing a fluorescent protein such as GFP or a cell surface marker such as CD34 as described herein. [0088] Methods for culturing or expanding the genetically modified cells are known in the art. Methods for culturing primary cells and their progeny are known, and suitable culture media, supplements, growth factors, and the like are both known and commercially available. Typically, human primary cells are maintained and expanded in serum-free conditions. Alternative media, supplements and growth factors and/or alternative concentrations can readily be determined by the skilled person and are extensively described in the literature. In some embodiments, the isolated or purified gene modified cells can be expanded in vitro according to standard methods known to those of ordinary skill in the art. Cryopreservation [0089] In particular embodiments, the CD34+ HSPC manufacturing methods contemplated herein are practiced using freshly isolated populations of cells comprising CD34+ HSPCs. In other particular embodiments, the methods contemplated herein are practiced using cryopreserved populations of cells comprising CD34+ HSPCs. Cells may be cryopreserved following harvest or isolation of CD34+ HSPCs, after culture initiation and activation, after transduction, or after expansion or after any process step. Manufactured CD34+ HSPCs may also be cryopreserved following the CD34+ HSPC manufacturing process. The freeze-thaw cycle may provide a more uniform CD34+ HSPC composition by removing the non-CD34+ HSPC population. [0090] Cell populations may be cryopreserved in a suitable cell culture vessel as contemplated herein, see supra, under PBMC isolation and elsewhere herein. The cell populations may be frozen in a suitable cell culture medium and/or freezing medium, e.g., 50% plasmalyte and 50% Cryostor 10; 50/40/10 (XVIVO/HABS/DMSO); Cryostor 10; PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A. Cells then are frozen to a temperature of about í80° C to about í135° C at a rate of 1° C per minute in a controlled rate freezer and stored in the vapor phase of a liquid nitrogen storage tank. Other illustrative methods of controlled freezing may be used as well as uncontrolled freezing immediately at í20° C or in liquid nitrogen. [0091] After cryopreservation, cells are thawed in a 37° C water bath and washed in a suitable cell culture media or buffer. In some embodiments, the thawing process occurs in the presence of pulmozyme and/or heparin. The thawed cells may be subsequently used, either for the manufacturing methods contemplated herein or for administration to a subject. Wash steps may be performed as contemplated elsewhere herein. [0092] In particular embodiments, CD34+ HSPCs are collected by apheresis, enriched from the apheresis product, then cryopreserved prior to performing any of the gene editing methods described herein (e.g., gene knock-out, gene knock-in, gene correction). As shown in the workflow depicted in FIG.6, cryopreservation may be introduced after mobilization (e.g. with plerixafor) and collection (e.g. by apheresis) of stem cells and selection for CD34+ HSPCs. Following cryopreservation, an assessment can be made on whether the threshold number of CD34+ HSPCs has been collected from the donor to proceed with the gene editing steps that follow. If a threshold number of cells has not been reached from a single round of mobilization, collection, selection and cryopreservation, subsequent rounds may be performed until the threshold number of cells has been reached. Threshold numbers of CD34+ HSPCs to be collected may vary depending on a number of factors, including but not limited to, the gene editing procedure performed (e.g., gene knock-out, gene knock-in, gene correction), the targeted gene to be edited, the mechanism by which the targeted gene is modified (e.g., homology dependent repair (HDR)), the efficiency of the editing procedure (e.g. HDR efficiency) and the therapeutic threshold for treatment of a specific disease. In some embodiments, the threshold number of CD34+ HSPCs to be collected from a donor prior to gene editing is about 1 x 10⁴ to 1 x 10⁵, 1 x 10⁵ to 1 x 10⁶, 1 x 10⁶ to 1 x 10⁷ cells/kg or more. In some embodiments, at least about 1 x 10⁵ to 1 x 10⁷ cells/kg are collected prior to gene editing. In some embodiments, at least about 1 x 10⁴, 2 x 10⁴, 3 x 10⁴, 4 x 10⁴, 5 x 10⁴, 6 x 10⁴, 7 x 10⁴, 8 x 10⁴, 9 x 10⁴, 1 x 10⁵, 2 x 10⁵, 3 x 10⁵, 4 x 10⁵, 5 x 10⁵, 6 x 10⁵, 7 x 10⁵, 8 x 10⁵, 9 x 10⁵, 1 x 10⁶, 2 x 10⁶, 3 x 10⁶, 4 x 10⁶, 5 x 10⁶, 6 x 10⁶, 7 x 10⁶, 8 x 10⁶, 9 x 10⁶, 1 x 10⁷, 2 x 10⁷, 3 x 10⁷, 4 x 10⁷, 5 x 10⁷, 6 x 10⁷, 7 x 10⁷, 8 x 10⁷, 9 x 10⁷, or about 1 x 10⁸ CD34+ HSPCs/kg are collected prior to proceeding with gene editing of the collected cells. Once the threshold number of CD34+ HSPCs are mobilized, collected, selected for, and cryopreserved, the cells can then proceed to thaw, culture and gene correction. [0093] Cryopreservation prior to gene editing confers several advantages to the manufacturing methods provided herein. First, cryopreservation provides the ability to generate a cell bank of a single donor’s CD34+ HSPCs having a sufficient number of starting cells for the gene editing procedure. This improves the likelihood that the editing procedure will yield, in a single round of editing, the minimum number of gene edited cells needed for an effective dose of the cell therapy. This also avoids having to generate multiple drug products from multiple apheresis products having varying critical quality attributes, such as varying HDR rates where HDR- mediated knock-in of exogenous DNA is desired. Manufacturing cost and efficiency is also increased by producing only a single drug product batch as opposed to multiple lots per batch of donor cells. Benefits to the donor include the ability to harvest donor cells over multiple apheresis cycles compared to a single, prolonged collection process, which allows for patient recovery between cycles. [0094] Accordingly, the present disclosure provides in further aspects an ex vivo method of generating a population of genetically modified CD34+ hematopoietic stem and progenitor cells (HSPCs) from a population of stem cells, the method comprising: (a) isolating the population of stem cells from a subject, wherein the isolating comprises one or more steps selected from mobilizing the stems cells in the subject and collecting the mobilized stem cells from the subject; (b) selecting for a CD34+ HSPC enriched stem cell population from the isolated stem cells; (c) cryopreserving the CD34+ HSPC-enriched stem cell population; (d) optionally, repeating steps (a) to (c) until a threshold number of CD34+ HSPCs required for gene editing has been isolated from the subject; (e) thawing the one or more cryopreserved CD34+ HSPC-enriched stem cell populations to obtain a thawed population of CD34+ HSPCs, wherein the thawed population of CD34+ HSPCs comprises the threshold number of CD34+ HSPCs of step (d); and (f) gene editing one or more target genes of the CD34+ HSPCs; thereby generating the population of genetically modified CD34+ HSPCs. [0095] In some embodiments, the gene editing comprises: (a) introducing into the thawed population of CD34+ HSPCs: (i) a CRISPR-associated Cas9 nuclease; and (ii) a guide polynucleotide sequence that hybridizes to a target sequence in the genome of the CD34+ HSPCs; and (b) contacting the thawed population of CD34+ HSPCs with an AAV vector comprising a donor polynucleotide sequence; whereupon induction of a double-strand break by the Cas9 nuclease and guide polynucleotide, the donor polynucleotide sequence is integrated into the genome of the cell by homology directed repair. [0096] In some embodiments, the ex vivo method generates the genetically modified stem cells in at least 5L, 10L, 50L, 100L, 300L, or 500L. In some embodiments, the percentage of CD34+ HSPCs recovered following gene editing relative to the threshold number of CD34+ HSPCs is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. Genetically Modifying Primary CD34+ Cells [0097] Provided in the present disclosure are methods to generate genetically modified CD34+ stem cells by introducing a guide polynucleotide, a CRISPR-associated Cas9 nuclease, and a donor polynucleotide sequence into a primary CD34+ stem cell. Through introduction of these components into a cell, a double stranded break can be introduced at a specific site as directed by the guide polynucleotide sequence and the CRISPR-associated Cas9 nuclease. A donor polynucleotide containing a sequence of interest can be further introduced into the cell and through homology directed recombination, the sequence of interest can be inserted into the cell. The transfer of the donor polynucleotide sequence can be carried out by transduction. The methods for viral transfer, e.g., transduction, generally involve at least initiation of transduction by incubating in a centrifugal chamber an input composition comprising the cells to be transduced and viral vector particles containing the vector, under conditions whereby cells are transduced or transduction is initiated in at least some of the cells in the input composition, wherein the method produces an output composition comprising the transduced cells. [0098] Methods for introducing polypeptides, nucleic acids, and viral vectors (e.g., viral particles) into a primary cell, target cell, or host cell are known in the art. Any known method can be used to introduce a polypeptide or a nucleic acid (e.g., a nucleotide sequence encoding the DNA nuclease or a modified sgRNA) into a primary cell, e.g., a human primary cell. Non- limiting examples of suitable methods include electroporation (e.g., nucleofection), viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like. [0099] Introduction of the Cas9 nuclease and a guide polynucleotide sequence can be introduced into a cell in order to provide a means of introducing a double stranded break at the site at which the guide polynucleotide guides the Cas9 nuclease. In some embodiments, the Cas9 nuclease and the guide polynucleotide sequence is introduced into the CD34+ cell through electroporation. [0100] In some embodiments the Cas9 nuclease can be in the form of a protein. In some embodiments, the Cas9 nuclease can be in the form of a plasmid, thereby allowing a cell that carries this expression construct to then express the Cas9 nuclease. [0101] In some embodiments, the Cas9 nuclease and the guide polynucleotide can be on the same or different polynucleotide sequence or plasmid. [0102] Introduction of the donor polynucleotide can occur through viral transduction using a delivery vector, such as adeno associated virus (AAV). In some embodiments, the viral transduction occurs within 30 minutes of the electroporation. In some embodiments, the viral transduction occurs simultaneously with the electroporation. In some embodiments, the viral transduction occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 minutes of the electroporation. Donor Polynucleotide [0103] The donor polynucleotide can comprise an exogenous polynucleotide sequence that replaces and endogenous sequence in a cell. To promote HDR of the exogenous polynucleotide through the 5’ and 3’ homology arms that are flanking the donor gene, the exogenous polynucleotide sequence may be altered in order to reduce the percent identity of the donor gene and the endogenous gene sequence to be replaced. The homology arms can be of varying lengths, affecting the size of the overall donor polynucleotide as well as affecting specificity during homology directed repair [0104] The homology arms can be of variable lengths. In some embodiments, the 5’ and 3’ homology arms can be identical in length. In some embodiments the 5’ and 3’ homology arms can be different lengths. [0105] In some embodiments, the 5' homology arm comprises about 50 base pairs to about 1,000 base pairs. In some embodiments, the 5' homology arm comprises at least about 50 base pairs. In some embodiments, the 5' homology arm comprises at most about 1,000 base pairs. In some embodiments, the 5' homology arm comprises about 50 base pairs to about 100 base pairs, about 50 base pairs to about 150 base pairs, about 50 base pairs to about 200 base pairs, about 50 base pairs to about 250 base pairs, about 50 base pairs to about 300 base pairs, about 50 base pairs to about 350 base pairs, about 50 base pairs to about 400 base pairs, about 50 base pairs to about 450 base pairs, about 50 base pairs to about 500 base pairs, about 50 base pairs to about 750 base pairs, about 50 base pairs to about 1,000 base pairs, about 100 base pairs to about 150 base pairs, about 100 base pairs to about 200 base pairs, about 100 base pairs to about 250 base pairs, about 100 base pairs to about 300 base pairs, about 100 base pairs to about 350 base pairs, about 100 base pairs to about 400 base pairs, about 100 base pairs to about 450 base pairs, about 100 base pairs to about 500 base pairs, about 100 base pairs to about 750 base pairs, about 100 base pairs to about 1,000 base pairs, about 150 base pairs to about 200 base pairs, about 150 base pairs to about 250 base pairs, about 150 base pairs to about 300 base pairs, about 150 base pairs to about 350 base pairs, about 150 base pairs to about 400 base pairs, about 150 base pairs to about 450 base pairs, about 150 base pairs to about 500 base pairs, about 150 base pairs to about 750 base pairs, about 150 base pairs to about 1,000 base pairs, about 200 base pairs to about 250 base pairs, about 200 base pairs to about 300 base pairs, about 200 base pairs to about 350 base pairs, about 200 base pairs to about 400 base pairs, about 200 base pairs to about 450 base pairs, about 200 base pairs to about 500 base pairs, about 200 base pairs to about 750 base pairs, about 200 base pairs to about 1,000 base pairs, about 250 base pairs to about 300 base pairs, about 250 base pairs to about 350 base pairs, about 250 base pairs to about 400 base pairs, about 250 base pairs to about 450 base pairs, about 250 base pairs to about 500 base pairs, about 250 base pairs to about 750 base pairs, about 250 base pairs to about 1,000 base pairs, about 300 base pairs to about 350 base pairs, about 300 base pairs to about 400 base pairs, about 300 base pairs to about 450 base pairs, about 300 base pairs to about 500 base pairs, about 300 base pairs to about 750 base pairs, about 300 base pairs to about 1,000 base pairs, about 350 base pairs to about 400 base pairs, about 350 base pairs to about 450 base pairs, about 350 base pairs to about 500 base pairs, about 350 base pairs to about 750 base pairs, about 350 base pairs to about 1,000 base pairs, about 400 base pairs to about 450 base pairs, about 400 base pairs to about 500 base pairs, about 400 base pairs to about 750 base pairs, about 400 base pairs to about 1,000 base pairs, about 450 base pairs to about 500 base pairs, about 450 base pairs to about 750 base pairs, about 450 base pairs to about 1,000 base pairs, about 500 base pairs to about 750 base pairs, about 500 base pairs to about 1,000 base pairs, or about 750 base pairs to about 1,000 base pairs. [0106] In some embodiments, the 3' homology arm comprises about 50 base pairs to about 1,000 base pairs. In some embodiments, the 3' homology arm comprises at least about 50 base pairs. In some embodiments, the 3' homology arm comprises at most about 1,000 base pairs. In some embodiments, the 3' homology arm comprises about 50 base pairs to about 100 base pairs, about 50 base pairs to about 150 base pairs, about 50 base pairs to about 200 base pairs, about 50 base pairs to about 250 base pairs, about 50 base pairs to about 300 base pairs, about 50 base pairs to about 350 base pairs, about 50 base pairs to about 400 base pairs, about 50 base pairs to about 450 base pairs, about 50 base pairs to about 500 base pairs, about 50 base pairs to about 750 base pairs, about 50 base pairs to about 1,000 base pairs, about 100 base pairs to about 150 base pairs, about 100 base pairs to about 200 base pairs, about 100 base pairs to about 250 base pairs, about 100 base pairs to about 300 base pairs, about 100 base pairs to about 350 base pairs, about 100 base pairs to about 400 base pairs, about 100 base pairs to about 450 base pairs, about 100 base pairs to about 500 base pairs, about 100 base pairs to about 750 base pairs, about 100 base pairs to about 1,000 base pairs, about 150 base pairs to about 200 base pairs, about 150 base pairs to about 250 base pairs, about 150 base pairs to about 300 base pairs, about 150 base pairs to about 350 base pairs, about 150 base pairs to about 400 base pairs, about 150 base pairs to about 450 base pairs, about 150 base pairs to about 500 base pairs, about 150 base pairs to about 750 base pairs, about 150 base pairs to about 1,000 base pairs, about 200 base pairs to about 250 base pairs, about 200 base pairs to about 300 base pairs, about 200 base pairs to about 350 base pairs, about 200 base pairs to about 400 base pairs, about 200 base pairs to about 450 base pairs, about 200 base pairs to about 500 base pairs, about 200 base pairs to about 750 base pairs, about 200 base pairs to about 1,000 base pairs, about 250 base pairs to about 300 base pairs, about 250 base pairs to about 350 base pairs, about 250 base pairs to about 400 base pairs, about 250 base pairs to about 450 base pairs, about 250 base pairs to about 500 base pairs, about 250 base pairs to about 750 base pairs, about 250 base pairs to about 1,000 base pairs, about 300 base pairs to about 350 base pairs, about 300 base pairs to about 400 base pairs, about 300 base pairs to about 450 base pairs, about 300 base pairs to about 500 base pairs, about 300 base pairs to about 750 base pairs, about 300 base pairs to about 1,000 base pairs, about 350 base pairs to about 400 base pairs, about 350 base pairs to about 450 base pairs, about 350 base pairs to about 500 base pairs, about 350 base pairs to about 750 base pairs, about 350 base pairs to about 1,000 base pairs, about 400 base pairs to about 450 base pairs, about 400 base pairs to about 500 base pairs, about 400 base pairs to about 750 base pairs, about 400 base pairs to about 1,000 base pairs, about 450 base pairs to about 500 base pairs, about 450 base pairs to about 750 base pairs, about 450 base pairs to about 1,000 base pairs, about 500 base pairs to about 750 base pairs, about 500 base pairs to about 1,000 base pairs, or about 750 base pairs to about 1,000 base pairs. [0107] In some embodiments, the 5’ homology arm comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In some embodiments, the 3’ homology arm comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. [0108] While the homology arms contain high sequence homology or sequence identity between the homology arms and the endogenous target site, the exogenous polynucleotide sequence between the homology arms may not be identical to the target gene. By having decreased homology compared to the homology arms, homology directed repair can be directed to improve homologous recombination rates utilize the homology arms rather than the exogenous polynucleotide sequence. [0109] In some embodiments, the percent identity between the exogenous polynucleotide sequence and endogenous polynucleotide sequence to be replaced comprises at least 60% sequence identity. In some embodiments, the percent identity between the exogenous polynucleotide sequence and endogenous polynucleotide sequence to be replaced is about 60% to about 99%. In some embodiments, the percent identity between the exogenous polynucleotide sequence and endogenous polynucleotide sequence to be replaced is at least about 60%. In some embodiments, the percent identity between the exogenous polynucleotide sequence and endogenous polynucleotide sequence to be replaced is at most about 99%. In some embodiments, the percent identity between the exogenous polynucleotide sequence and endogenous polynucleotide sequence to be replaced is about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 97%, about 60% to about 98%, about 60% to about 99%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%, about 65% to about 90%, about 65% to about 95%, about 65% to about 97%, about 65% to about 98%, about 65% to about 99%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 97%, about 70% to about 98%, about 70% to about 99%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 97%, about 75% to about 98%, about 75% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 97%, about 80% to about 98%, about 80% to about 99%, about 85% to about 90%, about 85% to about 95%, about 85% to about 97%, about 85% to about 98%, about 85% to about 99%, about 90% to about 95%, about 90% to about 97%, about 90% to about 98%, about 90% to about 99%, about 95% to about 97%, about 95% to about 98%, about 95% to about 99%, about 97% to about 98%, about 97% to about 99%, or about 98% to about 99%. Delivery Vector [0110] Provided herein are delivery vectors that will enable introduction of the compositions described herein into a cell. The delivery vector may include a surface modification that targets the vector to a cell of the subject, such as an antibody linked to an external surface of the viral delivery vector, wherein the antibody targets hematopoietic stem cells, or precursors thereof. The composition may include a particle (e.g., lipid nanoparticle or liposome) containing the globin gene and the gene editing reagents, or a plurality of lipid nanoparticles having the globin gene and the gene editing reagents comprised or embedded therein. For example, the plurality of lipid nanoparticles may include at least: a first solid lipid nanoparticle comprising a segment of DNA that includes the globin gene; a second solid lipid nanoparticle that includes at least one Cas endonuclease complexed with a guide RNA (gRNA) that targets the Cas endonuclease to a locus within an alpha-globin gene cluster in chromosome 16. The particle(s) may be provided as one or a plurality of liposomes enveloping one or more of the globin gene and the gene editing reagents. [0111] Donor polynucleotide sequences described herein may be incorporated within a wide variety of gene therapy constructs, e.g., to deliver a nucleic acid encoding a protein to a subject in need thereof. A vector construct refers to a polynucleotide molecule including all or a portion of a viral genome and an exogenous polynucleotide sequence. In some instances, gene transfer can be mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV). Other vectors useful in methods of gene therapy are known in the art. For example, a construct of the present disclosure can include an alphavirus, herpesvirus, retrovirus, lentivirus, or vaccinia virus. [0112] Adenoviruses are a relatively well characterized group of viruses, including over 50 serotypes. Adenoviruses are tractable through the application of techniques of molecular biology and may not require integration into the host cell genome. Recombinant Ad-derived vectors, including vectors that reduce the potential for recombination and generation of wild-type virus, have been constructed. Wild-type AAV has high infectivity and is capable of integrating into a host genome with a high degree of specificity. [0113] AAV of any serotype or pseudotype can be used. Certain AAV vectors are derived from single stranded (ss) DNA parvoviruses that are nonpathogenic for mammals. Briefly, rep and cap viral genes that can account for 96% of the archetypical wild-type AAV genome can be removed in the generation of certain AAV vectors, leaving flanking inverted terminal repeats (ITRs) that can be used to initiate viral DNA replication, packaging and integration. Wild type AAV integrates into the human host cell genome with preferential site specificity at chromosome 19q13.3. Alternatively, AAV can be maintained episomally. [0114] At least twelve human serotypes of AAV (AAV serotype 1 (AAV-1) to AAV-12) and more than 100 serotypes from nonhuman primates have been discovered to date. Any of these serotypes, as well as any combinations thereof, may be used within the scope of the present disclosure. [0115] A serotype of a viral vector used in certain embodiments described herein can be selected from the group consisting from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. Other serotypes are known in the art or described herein and are also applicable to the present disclosure. In particular instances, the present disclosure provides a AAV6 viral vector containing donor polynucleotide sequence. [0116] A vector of the present disclosure can be a pseudotyped vector. Pseudotyping provides a mechanism for modulating a vector's target cell population. For instance, pseudotyped AAV vectors can be utilized in various methods described herein. Pseudotyped vectors are those that contain the genome of one vector, e.g., the genome of one AAV serotype, in the capsid of a second vector, e.g., a second AAV serotype. Methods of pseudotyping are well known in the art. For instance, a vector may be pseudotyped with envelope glycoproteins derived from Rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Chandipura strains), rabies virus (e.g., various Evelyn-Rokitnicki-Abelseth ERA strains and challenge virus standard (CVS)), Lyssavirus Mokola virus, a rabies-related virus, vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), rabies virus glycoprotein (RV-G), glycoprotein B type (FuG-B), a variant of FuG-B (FuG-B2) or Moloney murine leukemia virus (MuLV). [0117] Without limitation, illustrative examples of pseudotyped vectors include recombinant AAV2/1, AAV2/2, AAV2/5, AAV2/6, AAV2/7, and AAV2/8 serotype vectors. It is known in the art that such vectors may be engineered to include a transgene encoding a human protein or other protein. In particular instances, the present disclosure includes a AAV6 vector for delivery. [0118] In some instances, a particular AAV serotype vector may be selected based upon the intended use, e.g., based upon the intended route of administration. For example, for direct injection into the brain, e.g., either into the striatum, an AAV2 serotype vector can be used. [0119] Various methods for application of AAV vector constructs in gene therapy are known in the art, including methods of modification, purification, and preparation for administration to human. CRISPR-associated Cas9 [0120] In some embodiments, the insertion is carried out using one or more DNA-binding nucleic acids, such as disruption via a nucleic acid-guided nuclease. For example, in some embodiments, the insertion is carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins via introduction of a double-stranded break in a DN sequence. [0121] In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide polynucleotide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus. [0122] In some embodiments, the CRISPR/Cas nuclease or CRISPR/Cas nuclease system includes a non-coding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains). [0123] In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes or Staphylococcus aureus. [0124] In some embodiments, a Cas nuclease and gRNA (including a fusion of crRNA specific for the target sequence and fixed tracrRNA) are introduced into the cell. In general, target sites at the 5ƍ end of the gRNA target the Cas nuclease to the target site, e.g., the gene, using complementary base pairing. In some embodiments, the target site is selected based on its location immediately 5ƍ of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG. In this respect, the gRNA is targeted to the desired sequence by modifying the first 20 nucleotides of the guide RNA to correspond to the target DNA sequence. [0125] In some embodiments, the CRISPR system induces DSBs at the target site, followed by disruptions as discussed herein. In other embodiments, Cas9 variants, deemed “nickases” are used to nick a single strand at the target site. In some embodiments, paired nickases are used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5ƍ overhang is introduced. [0126] In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. Typically, in the context of formation of a CRISPR complex, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. [0127] The target sequence may comprise any polynucleotide, such as DNA polynucleotides. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. In some embodiments, the target sequence may be within an organelle of the cell. Generally, a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “donor template” or “donor polynucleotide” or “donor sequence”. In some embodiments, an exogenous polynucleotide may be referred to as an donor template or donor polynucleotide. In some embodiments, the donor polynucleotide comprises an exogenous polynucleotide sequence. In some embodiments, the recombination is homologous recombination or homology-directed repair (HDR). [0128] Typically, in the context of an endogenous CRISPR system, formation of the CRISPR complex (comprising the guide sequence hybridized to the target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild- type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of the CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. In some embodiments, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of the CRISPR complex. [0129] As with the target sequence, in some embodiments, complete complementarity is not necessarily needed. In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, one or more vectors driving expression of one or more elements of the CRISPR system are introduced into the cell such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. In some embodiments, CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter. [0130] In some embodiments, the nucleic acid guide programmable nuclease can be a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments the CRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes, S. aureus or S. pneumoniae. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. [0131] In some embodiments, a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme. Non-limiting examples of mutations in a Cas9 protein are known in the art (see e.g., WO 2015/161276), any of which can be included in a CRISPR/Cas9 system in accord with the provided methods. In some embodiments, the CRISPR enzyme is mutated such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). In some embodiments, a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ. [0132] In further embodiments, the CRISPR-Cas9 comprises high-fidelity Cas9 variants having improved on-target specificity and reduced off-target activity. Examples of high-fidelity Cas9 variants include but are not limited to those described in PCT Publication Nos. WO/2018/068053 and WO/2019/074542, each of which is herein incorporated by reference in its entirety. In some embodiments, the CRISPR-associated Cas9 nuclease is a high-fidelity CRISPR-associated Cas9 nuclease comprising a mutation at position R691. In some embodiments, the mutation at position R691 is an alanine. [0133] In some embodiments, an enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding the CRISPR enzyme corresponds to the most frequently used codon for a particular amino acid. [0134] In general, a guide sequence includes a targeting domain comprising a polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. In some examples, the targeting domain of the gRNA is complementary, e.g., at least 80, 85, 90, 95, 98 or 99% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid. [0135] Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of the CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of the CRISPR system sufficient to form the CRISPR complex, including the guide sequence to be tested, may be provided to the cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of the CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. [0136] A guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. In some embodiments, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm. [0137] In general, a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences. [0138] Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%,

%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. In some aspects, loop forming sequences for use in hairpin structures are four nucleotides in length, and have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. In some embodiments, the sequences include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG. In some embodiments, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In some embodiments, the transcript has two, three, four or five hairpins. In a further embodiment, the transcript has at most five hairpins. In some embodiments, the single transcript further includes a transcription termination sequence, such as a polyT sequence, for example six T nucleotides. [0139] In some embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a CR ISPR enzyme are described in US20110059502, incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence. [0140] In some embodiments, a CRISPR enzyme in combination with (and optionally complexed with) a guide sequence is delivered to the cell. In some embodiments, methods for introducing a protein component into a cell according to the present disclosure (e.g., Cas9/gRNA RNPs) may be via physical delivery methods (e.g., electroporation, particle gun, Calcium Phosphate transfection, cell compression or squeezing), liposomes or nanoparticles. [0141] For example, CRISPR/Cas9 technology may be used to knock-down gene expression in the engineered cells. In an exemplary method, Cas9 nuclease (e.g., that encoded by mRNA from Staphylococcus aureus or from Stretpococcus pyogenes, e.g., pCW-Cas9, Addgene #50661, Wang et al. (2014) Science, 3:343-80-4; or nuclease or nickase lentiviral vectors available from Applied Biological Materials (ABM; Canada) as Cat. No. K002, K003, K005 or K006) and a guide RNA specific to the target gene are introduced into cells, for example, using lentiviral delivery vectors or any of a number of known delivery method or vehicle for transfer to cells, such as any of a number of known methods or vehicles for delivering Cas9 molecules and guide RNAs. Non-specific or empty vector control cells also are generated. Degree of Knockout of a gene (e.g., 24 to 72 hours after transfer) is assessed using any of a number of well-known assays for assessing gene disruption in cells. [0142] It is within the level of a skilled artisan to design or identify a gRNA sequence that is or comprises a sequence targeting a target gene of interest, such as any described herein, including the exon sequence and sequences of regulatory regions, including promoters and activators. A genome-wide gRNA database for CRISPR genome editing is publicly available, which contains exemplary single guide RNA (sgRNA) target sequences in constitutive exons of genes in the human genome or mouse genome (see e.g., genescript.com/gRNA-database.html; see also, Sanjana et al. (2014) Nat. Methods, 11:783-4; http://www.e-crisp.org/E-CRISP/; http://crispr.mit.edu/; https://www.dna20.com/eCommerce/cas9/input). In some embodiments, the gRNA sequence is or comprises a sequence with minimal off-target binding to a non-target gene. [0143] In some embodiments, design gRNA guide sequences and/or vectors for any gene are generated using any of a number of known methods, such as those for use in gene knockdown via CRISPR-mediated, TALEN-mediated and/or related methods. [0144] In some embodiments, target polynucleotides are modified in a eukaryotic cell. In some embodiments, the method comprises allowing the CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises the CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. [0145] Binding of the polynucleotide sequence recruits the Cas9 protein and facilitates a double- stranded break into the polynucleotide sequence by the Cas9 nuclease. In some embodiments, guide polynucleotide sequence binds to a region of a gene corresponding to the coding sequence. In some embodiments, the coding sequence is an exon. [0146] Guide polynucleotide sequences are specific to the target that they bind. In some embodiments, the guide polynucleotide sequence target is hemoglobin B (HBB). In some embodiments, the guide polynucleotide sequence binds to an exon of HBB. In some embodiments, the guide polynucleotides binds to exon 1, exon 2, or exon 3 of HBB. [0147] In some embodiments, the guide polynucleotide sequence comprises a modification. In some embodiments, the guide polynucleotide sequence comprises a 2^-O-methyl-3^- phosphorothioate modification. Methods of Culturing HSPCs [0148] Following introduction of the exogenous polynucleotide sequence into a primary CD34+ cell, thereby generating a population of genetically modified cells, the population of genetically modified cells are cultured in conditions that increase the viability of the genetically modified cells. Provided herein and further exemplified in the Examples are methods of culturing stem cells in order to provide a population genetically modified stem cells. [0149] In some embodiments, the method for manufacturing the HSPCS comprises harvesting cells from a subject and isolating a population of cells using a closed system process. In particular embodiments, cells may be isolated from any suitable fresh or frozen sources. In certain embodiments, the isolated population of cells comprises hematopoietic stem and progenitor cells (HSPCs). The isolated population of cells is seeded to initiate cultures and maintained in appropriate media to expand and culture stem cells. Manufactured gene edited cell compositions may then be used to treat subjects in need thereof or frozen for later uses. [0150] Adoptive cellular therapies manufactured using the methods contemplated herein are effective in producing cellular drug products with reproducible levels of expansion, cellular profiles, and that are effective in treating diseases caused by a mutation in a gene. [0151] Cells may be grown in low oxygen conditions, because it is thought that low oxygen conditions reduce the production of reactive oxygen species. In some embodiments, the low oxygen conditions can reduce the production and release of pro-inflammatory cytokines in the media. Proinflammatory cytokines can cause unnecessary stimulation to HSPCs can cause HSPCs to activate inappropriately. In some embodiments, the method of culturing results in a 10%, 20%< 30%, 40%< 50% reduction of pro inflammatory cytokines into the cell culture media. In some embodiments, the genetically modified cells are cultured in low O2 conditions. In some embodiments, the low O2 condition comprises 5% O2. [0152] In addition to low oxygen conditions, the CD34+ cells can be cultured in a low-density culture. A low-density culture can be employed to reduce the cell-to-cell contact between cells. In some embodiments, the CD34+ cells are cultured at a cell density comprising no more than 5 x 10⁵ cells / mL. In some embodiments, the CD34+ cells are cultured at a cell density comprising no more than 2.5 x 10⁵ cells / mL. In some embodiments, the CD34+ cells are cultured at a cell density comprising no more than 2.5 x 10⁵ - 5 x 10⁵ cells / mL. [0153] As used herein, “UM171” refers to a compound having the structure and biological activity of UM171 as described, e.g., in US 2015/0011543, the disclosure of which is incorporated herein by reference. UM171 has been previously shown to promote HSC cycling and self-renewal. [0154] In some embodiments, the population of stem cells are cultured in in the presence of UM171. Suitable amounts of UM171 for use in the present methods are from about 10 to about 100 nM, preferably from about 30 to about 100 nM, alternatively from about 35 nM to about 100 nM. [0155] Isolated populations of CD34+ stem cells or populations or populations of genetically modified stem cells can be frozen in freeze media and stored for long periods of time to provide a stopping point prior to subsequent genetic manipulation. This can provide a means of attaining the dose required for the therapeutic application of the subsequently generated genetically modified stem cells (FIG. 6). Freeze-thawing cycles must be limited to in order to maintain the efficacy and the viability stem cells. In order to mitigate any loss derived from thawing, cells can be thawed in the presence of pulmozyme and/or heparin. [0156] Cell culture media supplemented with cytokines can provide additional support to allow for optimized growth and maintenance of stem cells during the method disclosed herein. In some embodiments, the cell culture media can be further supplemented with at least one of human SCF, human TPO, human Flt3 ligand, or human IL-6. Manufacturing at a Large Scale at Clinical Requirements [0157] In particular embodiments, methods for manufacturing gene edited cells contemplated herein comprise a step of recovering the manufactured stem cells comprising harvesting and washing the expanded cells in using a semiautomated flow through centrifuge, e.g., the Cobe 2991 cell processor, the Cell Saver 5, the Baxter CytoMate, LOVO, or the like. [0158] Prior to harvesting the expanded cells, pre-harvest sample aliquots may be taken to establish cell counts, viability, cell characterization, e.g., FACs analysis, purity, and/or other general release criteria for the cells. In addition, post-harvest sample aliquots may be taken to establish cell counts and/or viability. [0159] The populations of genetically modified stem cells are then transferred to one or more IV bags or other suitable vessels, e.g., MACS® GMP Cell Expansion Bags, MACS® GMP Cell Differentiation Bags, EXP-Pak™ Cell Expansion Bio-Containers, VueLife™ bags, KryoSure™ bags, KryoVue™ bags, Lifecell® bags, PermaLife™ bags, X-Fold™ bags, Si-Culture™ bags, Origen biomedical cryobags and VectraCell™ bags and cryopreserved in a controlled rate freezer as discussed supra and elsewhere herein, until the cells are ready for use. In particular embodiments, cells are frozen in 50% plasmalyte and 50% Cryostor 10; 50/40/10 (XVIVO/HABS/DMSO);; Crytostor 5 or Cryostor 10. [0160] Bags (10 to 250 mL capacity) containing genetically modified stem cells are stored in blood bank conditions in a monitored í80° C to í135° C. Infusion bags are stored in the freezer until needed. [0161] The method disclosed herein can be used generate CD34+ HSPCs or CD34+ genetically modified cells at a total cell number that is appropriate for the treatment of a subject in need thereof. In some embodiments, the treatment is an autologous cell therapy. In some embodiments, the scale of said method can result in a production of from about 50 million CD34+ cells to about 200 million CD34+ cells. In some embodiments, the scale of said method can result in a production of at least about 50 million CD34+ cells. In some embodiments, the scale of said method can result in a production of at most about 200 million CD34+ cells. In some embodiments, the scale of said method can result in a production of from about 50 million CD34+ cells to about 60 million CD34+ cells, about 50 million CD34+ cells to about 70 million CD34+ cells, about 50 million CD34+ cells to about 80 million CD34+ cells, about 50 million CD34+ cells to about 90 million CD34+ cells, about 50 million CD34+ cells to about 100 million CD34+ cells, about 50 million CD34+ cells to about 125 million CD34+ cells, about 50 million CD34+ cells to about 150 million CD34+ cells, about 50 million CD34+ cells to about 175 million CD34+ cells, about 50 million CD34+ cells to about 200 million CD34+ cells, about 60 million CD34+ cells to about 70 million CD34+ cells, about 60 million CD34+ cells to about 80 million CD34+ cells, about 60 million CD34+ cells to about 90 million CD34+ cells, about 60 million CD34+ cells to about 100 million CD34+ cells, about 60 million CD34+ cells to about 125 million CD34+ cells, about 60 million CD34+ cells to about 150 million CD34+ cells, about 60 million CD34+ cells to about 175 million CD34+ cells, about 60 million CD34+ cells to about 200 million CD34+ cells, about 70 million CD34+ cells to about 80 million CD34+ cells, about 70 million CD34+ cells to about 90 million CD34+ cells, about 70 million CD34+ cells to about 100 million CD34+ cells, about 70 million CD34+ cells to about 125 million CD34+ cells, about 70 million CD34+ cells to about 150 million CD34+ cells, about 70 million CD34+ cells to about 175 million CD34+ cells, about 70 million CD34+ cells to about 200 million CD34+ cells, about 80 million CD34+ cells to about 90 million CD34+ cells, about 80 million CD34+ cells to about 100 million CD34+ cells, about 80 million CD34+ cells to about 125 million CD34+ cells, about 80 million CD34+ cells to about 150 million CD34+ cells, about 80 million CD34+ cells to about 175 million CD34+ cells, about 80 million CD34+ cells to about 200 million CD34+ cells, about 90 million CD34+ cells to about 100 million CD34+ cells, about 90 million CD34+ cells to about 125 million CD34+ cells, about 90 million CD34+ cells to about 150 million CD34+ cells, about 90 million CD34+ cells to about 175 million CD34+ cells, about 90 million CD34+ cells to about 200 million CD34+ cells, about 100 million CD34+ cells to about 125 million CD34+ cells, about 100 million CD34+ cells to about 150 million CD34+ cells, about 100 million CD34+ cells to about 175 million CD34+ cells, about 100 million CD34+ cells to about 200 million CD34+ cells, about 125 million CD34+ cells to about 150 million CD34+ cells, about 125 million CD34+ cells to about 175 million CD34+ cells, about 125 million CD34+ cells to about 200 million CD34+ cells, about 150 million CD34+ cells to about 175 million CD34+ cells, about 150 million CD34+ cells to about 200 million CD34+ cells, or about 175 million CD34+ cells to about 200 million CD34+ cells. [0162] Illustrative embodiments of cell culture bags include, but are not limited to MACS® GMP Cell Expansion Bags, MACS® GMP Cell Differentiation Bags, EXP-Pak™ Cell Expansion Bio-Containers, VueLife™ bags, KryoSure™ bags, KryoVue™ bags, Lifecell® bags, PermaLife™ bags, X-Fold™ bags, Si-Culture™ bags, Origen biomedical cryobags, and VectraCell™ bags. In particular embodiments, cell culture bags comprise one or more of the following characteristics: gas permeability (materials have suitable gas transfer rates for oxygen, carbon dioxide and nitrogen); negligible water loss rates (materials are practically impermeable to water); chemically and biologically inert (materials do not react with the vessel contents), and retention of flexibility and strength in various conditions (materials enable vessel to be microwaved, treated with UV irradiation, centrifuged, or used within a broad range of temperatures, e.g., from í100° C to +100° C). [0163] Exemplary large scale volumes of the cell culture vessel contemplated herein include, without limitation, volumes of about 10 mL, about 25 mL, about 50 mL, about 75 mL, about 100 mL, about 150 mL, about 250 mL, about 500 mL, about 750 mL, about 1000 mL, about 1250 mL, about 1500 mL, about 1750 mL, about 2000 mL, or more, including any intervening volume. For example, intervening volumes between 10 mL and 25 mL, include 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, and 24 mL. In one embodiment, the volume of the cell culture vessel is from about 100 mL to about 500 L. In some embodiments, the method of manufacturing is prepared in a volume of at least 10L – 500L. In some embodiments, the method is prepared in a volume of at least 10L, 20L, 50L, 100L, 250L, 500L. Methods of Treatment [0164] The present disclosure, provides in further aspects, a method of preventing or treating a disease in a subject in need thereof, the method comprising administering to the subject any of the genetically modified primary cells described herein, or any of the pharmaceutical compositions described herein, to prevent the disease or ameliorate one or more symptoms of the disease. In some embodiments, the genetically modified primary cells or pharmaceutical compositions thereof are autologous therapies. In some embodiments, the genetically modified primary cells or pharmaceutical compositions thereof are allogeneic therapies. [0165] In some embodiments, the step of administering comprises a delivery route selected from the group consisting of intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, intrathecal, intraosseous, or a combination thereof. [0166] The disease can be selected from the group consisting of a hemoglobinopathy, a viral infection, X-linked severe combined immune deficiency, Fanconi anemia, hemophilia, neoplasia, cancer, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, cystic fibrosis, blood diseases and disorders, inflammation, immune system diseases or disorders, metabolic diseases, liver diseases and disorders, kidney diseases and disorders, muscular diseases and disorders, bone or cartilage diseases and disorders, neurological and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and lysosomal storage disorders. In some instances, the hemoglobinopathy is sickle cell disease, Į- thalassemia, ȕ-thalassemia, or į-thalassemia. In other instances, the viral infection is selected from the group consisting of a hepatitis B virus infection, hepatitis C virus infection, human papilloma virus infection, human immunodeficiency virus (HIV) infection, human T- lymphotrophic virus (HTLV) infection, Epstein-Barr virus infection, herpes virus infection, cytomegalovirus infection, and any other chronic viral infection. In yet other instances, the muscular diseases and disorders are selected from the group consisting of Becker muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, any other muscular dystrophy, and muscular atrophy. [0167] In particular embodiments, the genetically modified primary cells or pharmaceutical compositions described herein are administered to the subject in a sufficient amount to correct a mutation in the target nucleic acid that is associated with the disease. In some instances, the mutation is corrected by replacing a mutant allele in the target nucleic acid with the wild-type allele. In other instances, the mutation is corrected by inserting an open reading frame (ORF) that corresponds to a wild-type cDNA of the target nucleic acid. As a non-limiting example, the method described herein can be used to prevent or treat sickle cell disease by administering genetically modified primary cells or pharmaceutical compositions thereof wherein the sickle cell disease-causing E6V mutation in the HBB gene has been corrected via editing the nucleotide mutation (e.g., by introducing a homologous donor AAV vector comprising the sickle cell disease nucleotide correction donor template set forth in SEQ ID NO: 2, wherein a “T” nucleotide at position 1194 has been changed to an “A” nucleotide to correct the E6V mutation) or knocking in a wild-type HBB cDNA. Examples Example 1: Isolation of CD34+ cells from an Apheresis Product from Human Donors [0168] Apheresis products are collected from donor subjects according to standard clinical procedures. Prior to isolation of CD34+ cells from the apheresis product, the white blood cell (WBC) count is assessed. The WBC concentration should be less than or equal to 2 x 10⁸ cells/mL. If the cell concentration is higher, dilution using a 2% human serum albumin in Normosol pH 7.4 or in Plasma-Lyte-A Bag is used to achieve the desired cell concentration of 2 x 10⁸ cells/mL. [0169] The number of CD34+ cells are counted from the diluted apheresis product. The maximum loading dose is 1.2 x 10⁹ total CD34+ cells or 120 x 10⁹ total WBC and the apheresis product will be further diluted if either value is greater than what is noted. [0170] For isolation of CD34+ cells, a CliniMACS ® CD34 Reagent System is used to isolation CD34+ hematopoietic stem cells from apheresis products. For preparation of cells, a 0.5% human serum albumin in CliniMACS ® Buffer is prepared. The product is washed to deplete platelets from the product by either centrifugation or by the LOVO Cell Processing System. [0171] For centrifugation, the product is prepared to a solution of 600 mL in phosphate buffered saline (PBS), EDTA, 0.5% HSA and centrifuged for 12 minutes at 5200 x g with the brake set at 2. A pellet will form, and the supernatant is discarded. The pellet is dispersed and resuspended with PBS, EDTA, 0.5% HSA and centrifuged again until a platelet depletion of 70% or greater is achieved. [0172] For LOVO, use standard manufacturing operating procedures. Use a 0.22 ^M or 0.45 ^M filter in the source line. Use the “Platelet Wash” program and enter in the Source Volume, %PCV-WBC concentration, % HCT and PLT concentration. Determine the % Platelet depletion. [0173] Optionally, a 10% IVIG solution (Gammagard) is incubated in the product for 5 minutes at 5-10 RPM prior to adding a CD34 reagent. If the WBC is less than 60 x 10⁹ or the CD34+ cells is less than 600 x 10⁹ than only 1 mL of 10% IVIG is added. If the total WBC is greater than 60 x 10⁹ or the total CD34+ cells is greater than 600 x 10⁹ than only 2 mL of 10% IVIG is added. [0174] The amount of CliniMACS CD34 Reagent to add to the product is dependent on the number of cells. If the WBC is less than 60 x 10⁹ or the CD34+ cells is less than 600 x 10⁹ than only 1 vial of CD34 reagent is added. If the total WBC is greater than 60 x 10⁹ or the total CD34+ cells is greater than 600 x 10⁹ than only 2 vials of CD34 reagent is added. An antibody wash is performed by either centrifugation twice at 350 x g for 15 minutes and discarding the supernatant or by utilizing the LOVO cell processing system. CD34 Cell Selection [0175] For selection of CD34+ cells from the product, the CliniMACS system is used to enrich for CD34+ cells. A standard operating procedure to identify and isolate the correct cells are assessed by an experience cellular technologist. After cell enrichment, cell counts are assessed using a hematology analyzer or the Cellometer Auto-2000. Cell viability, endotoxin (sterility) levels, number of TCR ^/^ cells, and number of CD34+ cells are assessed. [0176] After CD34+ cell enrichment, cells are frozen for long-term storage. Cryopreservation of CD34 cells for storage [0177] For cryopreservation, cells will be preserved at a concentration of 5 x 10⁶ cells/mL to 50 x 10⁶ cells/mL. Cells are frozen in Cryopreservation media (Cryostor) (CS-5 or CS-10) with 2% HSA. Example 2: Generating Genetically Modified CD34+ Stem Cells Preparation of SCGM Cytokine Rich Media Table 1: Cytokines added to SCGM Media

[0178] SCGM-CRM media is prepared according to Table 1, or in their respective ratios. [0179] Cytokine vials are resuspended in 1 mL of WFI water. In addition, 1 mL of 50 ^M of UM171 is added per 1 mL of media. Preparation of cells for Gene Modification [0180] Thaw media is prepared by preparing 2% HAS in Normosol or Plasma-Lyte-A. The thaw media is further supplemented with 2% pulmozyme and 3 % heparin. [0181] Frozen cells are transferred into a pre-warmed water bath set at 37° C until a small ice crystal remains (approximately 2-5 minutes). Thaw media is added at approximately 2 times the cell bag volume (i.e.200 mL thaw media in 100 mL frozen culture). After thawing, assess cells for endotoxin and gram stain testing. The resuspended, thawed cells are transferred to 250 mL conical tubes and centrifuged for 10 minutes at 400 x g with the brake set to 7. The supernatant is aspirated and the cell are resuspended in 50 mL of SCRM-CRM prepared as previously disclosed. Cell viability is assessed by AO/PI stain. Cells are resuspended to a cell density of 2.5 x 10⁵ to 5.0 x 10⁵ cells/mL. [0182] Resuspended cells are placed in a 5% CO₂ + 5% O₂ incubator for 68-72 hours. Electroporate and Transduction of CD34+ Cells [0183] Pre-warm cell media (SCGM-CRM) at room temperature. Wipe the surface of culture bags with a dry Kimwipe. DO NOT disinfect bags with IPA as it may damage the FEP material. Aseptically connect each individual culture bag to tubing on a 500mL closed centrifuge tube system and transfer the entire cell suspension to the tube. [0184] Each bag may be rinsed with an additional 20mL of HSPC-CRM as needed. Remove the dip-tube connected lid of the closed centrifuge bottles and replace with a compatible sterile screw top lid. Repeat the steps for each bag. Tubes are spun at 300 x g for 10 minutes. The supernatant is aspirated, and the pellets are pooled and resuspended in approximately 100 mL of SCGM- CRM. Cell viability is assessed using AO/PI staining. P3 Nucleofector Solution and Supplement 1 are warmed at room temperature for at least 30 minutes prior to use.5 mL of Supplement 1 is added to 1 vial containing 22.5 mL of P3 solution. Determine how many Nucleofector cartridges are required as each cassette has a maximum fill volume of 1 mL and must contain 75 x 10⁶ – 150 x 10⁶ cells/mL. Preparation of Cas9-sgRNA complex [0185] The Cas9-sgRNA complex is prepared by suspending 240 ^g of sgRNA with 450 ^g per cartridge used. [0186] The amount of AAV6 virus encoding the donor template. The virus is thawed at room temperature. Electroporation and Transduction [0187] For electroporation, a Lonza Nucleofector (LV unit) is used. From the drop-down menu, select the CD34, Primary Human Cells Program. For solution type, select ‘P3 Primary Cells.’ Type in with verification the ‘DZ-100’ program [0188] Prior to electroporation, centrifuge cells at 300 x g for 10 minutes. Aspirate the supernatant minimizing disturbance to the cell pellet. Aspirate as much as possible to ensure that the pellet is as dry as possible. Prepare an appropriate amount of AAV6 virus into a 18 mL of SCGM-CRM. Using the selected electroporate protocol, ensure that the volume to be electroporated is 1 mL. If less than 1 mL add P3 solution and supplement 1 to attain 1 mL total volume. Load to electroporation cartridge. After electroporation, transfer pooled electroporated cells into the cell-media containing the virus. The transduction should occur within 15 minutes of electroporation. After transduction, cells are incubated at 5% O₂ + 5% CO₂ for 16- 20 hours. [0189] After incubation, the cells are spun down at 300 x g for 10 minutes. The supernatant is aspirated and then the pellet is resuspended in SCGM-CRM. [0190] After cultivating cells, the genetically modified cells are frozen for subsequent steps prior to dosing. Example 3: Gene Correction of Donor-Derived Cells Healthy donor-derived cells [0191] An optimized manufacturing protocol using UM171, low density culture condition and HiFi Cas9 (FIG.1A) was used to increase the ~5% gcHBB allele frequency in long-term engrafting plxHSPCs. The gcHBB-SCD were manufactured using plxHSPC from healthy donors and resulted in an average of 65% gcHBB alleles, while consistently showing >80% viability and >90% CD34+ cellular content (FIG.1B). When studying the kinetics of rAAV6 transduction, an 8-hour minimum period of rAAV6 incubation was necessary for efficient allele correction frequency. NSG mice were transplanted with cells harvested 48 hours after RNP delivery and AAV transduction. gcHBB-SCD cells showed similar engraftment frequencies (28% median engraftment, n=15) to control cells, including electroporated only (mock, n=9), RNP-only (n=4) and AAV6-only (n=4) (FIG.1C). Engrafted human cells derived from transplanted gcHBB-SCD were sorted from bone marrow at week 16 using human lineage-specific markers. The human graft was multi-lineage and consisted mostly of CD19+ B-cells and CD33+ myeloid cells (FIG. 1D). Genomic DNA was extracted from the different cell types and gcHBB allele frequencies were analyzed in specific lineages. Across all the mice transplanted (n=15), there was a median gcHBB allele frequency of 21%, 20%, 31%, 27%, and 24% in the bulk human cell population, B- cells, myeloid cells, T/NK cells, and HSPCs, respectively (FIG.1E). A mouse-by-mouse analysis highlighted that the majority of NSG mice had several human hematopoietic lineages derived from transplanted gcHBB-SCD, with similar frequencies of gcHBB alleles among the different lineages (FIG.1F, left). A subset of mice was found to have reduced B cell gene correction frequencies but sustained higher correction frequencies in the myeloid lineage (FIG. 1F, right). To estimate the percent of HSPCs that had at least one gcHBB allele in vivo, CD34+ HSPCs were harvested from bone marrow of NSG mice (n=8) and single-cell sorted into methylcellulose and genotyped single CFU colonies. Positive colonies for the gcHBB allele(s) were an average of 30% frequency (up to 50% in some donors), with an average of 12% carrying a bi-allelic gcHBB (FIG.1G). Notably, these frequencies of gcHBB positive colonies were ~1.3 times more than the allele frequency in the bulk cell population from NSG bone marrow (FIG. 1H). These results indicate that gcHBB-SCD contains long-term repopulating HSCs with ~30% of cells carrying at least one HBB corrected allele. A 30% cell frequency of at least one corrected allele matches the 30% threshold for donor cell chimerism needed for hematologic cure following allo-HSCT. [0192] For CFU analysis, HSPCs were stained with CD34 APC (561; BioLegend, San Diego, CA, USA), Ghost Dye Red 780 (Tonbo Biosciences, San Diego, CA, USA) and live CD34+ were single cell sorted into 96-well plates containing MethoCult Optimum (STEMCELL Technologies, Vancouver, Canada). After 2 weeks, colonies were appropriately scored based on external appearance in a blinded manner. Subsequently, single cell colonies were individually harvested and genotyped as mentioned previously for targeted allelic analysis. SCD-Donor Derived Cells [0193] To understand if the optimized plxHSPC manufacturing from healthy donors would translate to sickle cell HSPCs, cryopreserved CD34+ plxHSPCs were obtained from two different SCD patients. gcHBB-SCD manufacturing reproducibility, and ability to differentiate into erythrocytes and express HgbA tetramers was assessed. In six independent experiments (3 technical replicates in each biological donor), >60% gcHBB alleles (FIG.2A) with minimal effect on cell viability (>80% viable cells) and expression level of CD34 (>90% CD34+ cells) (FIG.2B) was observed, showing feasibility of the gene correction protocol in SCD derived HSPCs. gcHBB-SCD and mock control were plated (electroporated HSPCs) into an erythroid differentiation medium and obtained >85% CD45-/CD34-CD71+/GPA+ red blood cells (FIG. 2C). gcHBB-SCD derived erythroblast in vitro restored output of ‘corrective’ hemoglobin (<10% HgbS, >25% HgbF, >65% HgbA) in contrast with the mock control derived erythroblasts (>85% HgbS, >10% HgbF, 0% HgbA) (FIG.2D). As expected, the level of HgbA restoration increased with the higher gcHBB allele frequencies (FIG.2A). Of note, a slight increase of HgbF levels could be observed in in vitro erythroid differentiated cells, albeit with no statistical significance between gcHBB-SCD- and control-derived erythroid cells. This can be attributed to stress erythropoiesis with concomitant upregulation of Ȗ-globin. These data indicate that high frequencies of gene correction (for the codon 6 HBB gene mutation) can be achieved in SCD patients’ HSPCs with restoration of non-pathologic HgbA expression in vitro. [0194] The ability of gcHBB-SCD manufactured from SCD patients to engraft long-term and reconstitute hematopoiesis was tested. gcHBB-SCD along with mock controls were injected IV into sub-lethally irradiated NSG mice. Bone marrow human chimerism as well as gcHBB allele frequencies within the human cell compartment were evaluated 16-20 weeks post- transplantation. Overall, SCD patient-derived plxHSPCs showed robust engraftment capacity with a median human chimerism of 80% and 42% for mock electroporated HSPCs and gcHBB- SCD, respectively (FIG.2E). The human graft was multi-lineage and consisted of mostly CD19+ B cells and CD33+ myeloid cells (FIG.2F). The bulk human cell population in the bone marrow of mice injected with gcHBB-SCD showed a robust percentage of gcHBB alleles (median 30%). Furthermore, B cells, myeloid cells, erythroid cells and HSPCs were isolated from the bulk human population and assessed similar gene correction frequencies in all the human hematopoietic lineages (FIG.2G). These findings demonstrate long-term reconstitution of HBB gene-corrected HSCs with multi-lineage potential. In the erythroid compartment (GPA+) gene correction frequencies were higher with a median of 60% gcHBB alleles (FIG.2G). This frequency suggests there is an in vivo enrichment of gene-corrected erythroid progenitors, which might have a better fitness compared to the non-corrected sickle cells or cells with bi-allelic INDELs (bi-allelic INDEL cells would lead to a beta-thalassemia major phenotype because the INDELs are frameshifting). The non-mobilized CD34+ HSPCs retained long-term engraftment potential following transplantation into NSG mice. These results demonstrate the therapeutic potential of the gene correction platform to treat sickle cell disease (and directly revert the codon 6 mutation). [0195] For tetramer analysis, approximately 1×10⁶ differentiated erythrocytes were lysed using water equivalent to three volumes of pelleted cells. The mixture was incubated at room temperature for 15min, followed by 30s sonication. To separate lysate from erythrocyte ghosts, cells underwent centrifugation at 13,000 RPM for 5min. HPLC analysis of hemoglobin in their native form were analyzed on a weak cation-exchange PolyCAT A column (100 × 4.6-mm, 3μm, 1,000Å) (PolyLC Inc., Columbia, MD, USA) using a Shimadzu UFLC system at room temperature. Mobile phase A (MPA) consists of 20mM Bis-tris + 2mM KCN, pH 6.96. Mobile phase B (MPB) consists of 20mM Bis-tris + 2mM KCN + 200mM NaCl, pH 6.55. Clear hemolysate was diluted four times in MPA, and then 20μL was injected onto the column. A flow rate of 1.5mL/min and the following gradients were used in time (min)/%B organic solvent: (0/10%; 8/40%; 17/90%; 20/10%; 30/stop). [0196] For assessment of human engraftment, 16 post-transplantation of edited CD34+ HSPCs, mice were euthanized, and cells were harvested from 2xfibula, 2x tibia, 2x femurs, homers, sternum, and spine using a pestle and mortar. Mononuclear cells were enriched using a Ficoll gradient centrifugation (Ficoll-Paque Plus, GE Healthcare, Chicago, IL) for 25min at 2,000g at room temperature. The samples were then stained for 30min at 4°C with the following antibodies: monoclonal anti-human CD33 V450 (WM53; BD Biosciences, San Jose, CA, USA); HLA-ABC FITC (W6/32; BioLegend, San Diego, CA, USA); CD19 PerCp-Cy5.5 (HIB19; BD Biosciences); anti-mouse PE-Cy5 mTer119 (TER-119; eBiosciences, San Diego, CA, USA); anti-mouse CD45.1 PE-Cy7 (A20; eBiosciences, San Diego, CA, USA); hGPA PE (HIR2; eBiosciences, San Diego, CA, USA); hCD34 APC (581; BioLegend, San Diego, CA, USA); and CD10 APC-Cy7 (HI10a; BioLegend, San Diego, CA, USA). Multi-lineage engraftment was defined by the bi-lineage reconstitution of myeloid cells (CD33+) and B-cells (CD19+) of engrafted human cells (CD45+; HLA-A/B/C+ cells). For secondary NSG mice transplantations, only a portion of the primary mouse mononuclear population was stained and analyzed for lineage-specific populations (B cells, myeloid cells, T/NK cells, and HSPCs) and targeted allele analysis. The rest of the cells (2.5×105 cells-1.3×106 cells) were transplanted into sub-lethally irradiated six- to eight-week-old NSG mice. Cells were the assessed in same aforementioned manner 14 weeks post-transplantation into secondary mice. Example 4: Manufacturing at Larger Scales of Gene Corrected CD34+ cells [0197] A gcHBB-SCD clinical-scale manufacturing protocol was developed that met specifications for cell product potency (gcHBB alleles), identity and purity (CD34+), and cell viability. To match the planned clinical manufacturing process, current good manufacturing practice (cGMP) compliant cell culture reagents were used with key contract development and manufacturing organizations (CDMOs) for large-scale provisions of key manufacturing reagents. A protocol for rAAV6 production using a 2-plasmids system technology was utilized. The scale- up production process was increased up to 3L scale in a suspension culture of HEK-293T. Several mid-scale rAAV6 lots were produced at Stanford Laboratory of Cell and Gene Medicine (LCGM). The following parameters were assessed for clinical-scale manufactured gcHBB-SCD: 1) feasibility of CD34+ harvest and manufacturing from plerixafor mobilized healthy donors, 2) efficacy of gcHBB-SCD cell product manufactured at large-scale, and 3) long-term toxicological and tumorigenic potential in NSG mice. [0198] Apheresis products of plerixafor mobilized-peripheral blood (on average 50-billion of total nucleated cells) were purchased and freshly enriched for CD34+ HSPCs. Once plated, cells were cultured for two days at a concentration of 2.5 x 10⁵ cells/mL in cytokine-supplemented media. At day 2, cells were collected and underwent the gene correction protocol adapted for large-scale manipulation. The final gcHBB-SCD product was then harvested at day 4 and underwent quality control testing and cryopreservation. The feasibility of manufacturing was tested at clinically relevant cell numbers (2.3 x 10⁷ – 1.3 x 10⁸ CD34+ HSPCs) with a mean of 77% viability, 94% CD34+ cells, and 45% gcHBB alleles (FIG.3A). The colony forming-unit ability (CFU-assay) of edited cells was 2-fold lower compared to untreated controls without lineage skewing (FIG.3B). Genotype analysis of colonies showed a higher percent of gene- corrected HSPCs than gcHBB alleles (FIG.3C), with an average 56% of cells having at least one corrected gcHBB allele (FIG.3D) of which 32% and 24% were mono- and bi-allelic events, respectively (FIG.3D). Other Tested Parameters [0199] Optimizing the genome editing protocol was necessary to achieve higher in vivo retention of gene correction alleles with minimal off-target activity. The following parameters were assessed: 1) the addition of the UM171 small molecule to the culture media, which has been shown to promote HSC cycling and self-renewal, to increase rAAV6 transduction efficiency and HSCs recovery, and to reduce reactive oxidative species (ROS) generation during cell cycle; 2) implementation of the low-density culture condition to promote HSC cycling; and 3) use of a high-fidelity Cas9 (R691A) variant that works well in the RNP format. [0200] While the addition of UM171 to the culture medium did not impact the in vitro gene targeting frequencies (FIG.4A) nor the in vivo engraftment potential of human CD34+ cells (FIG.4B), it increased in vivo retention of gcHBB alleles with respect to the in vitro population (median ratio of 0.75 (in vivo divided by in vitro)) in the NSG bone marrow of transplanted mice (FIG.4C). When calculating the absolute number of human cells with corrected alleles on a mouse-by-mouse basis, a ~3-fold increase was measured in the total number of engrafted gcHBB-SCD cells in the UM171 treated group. Because of the large and known heterogeneity of human engraftment in the NSG model, the result was not statistically significant (FIG.4D). All gcHBB-SCD cell populations were also able to engraft in secondary transplant experiments (FIG.4E). [0201] As previously demonstrated, low-density cell culture condition primes multi-lineage repopulating HSPCs for HR (FIG.5A-5B). These findings corresponded to a decrease in pro- inflammatory cytokines in the cell culture media (FIG.5C). Cytokines such as TGFA, IL-8, IL- 1RA, IP-10, HGF and MCP-1 were at significantly lower concentration in the media at day 2, indicating a potential decreased effect of inhibitory feedback signals on cell growth. [0202] Finally, to improve the editing safety profile by using a high-fidelity (HiFi) Cas9 protein. This modified Cas9 variant has been shown to retain the high on-target activity of WT Cas9 while reducing off-target editing. When used in the gene targeting protocol, HiFi Cas9 was equivalent to WT protein in on-target activity at the HBB gene, generating an average of 71% HR when coupled with rAAV6 transduction (FIG.5D). Importantly, HiFi Cas9 dramatically decreased INDEL frequency at off target 1 (OT1) below the limit of detection in both the in vitro culture and the in vivo engrafted HSPCs (FIG.5E). Example 5: Distribution of HBB Gene Editing [0203] Using the disclosed method above to generate CD34+ cells from a subject, the efficiency of HBB gene correction was assessed. A summary schematic of the method is shown in FIG.6. [0204] Following electroporation and transduction of the Cas9 nuclease, guide polynucleotide, and the AAV6 harboring the donor polynucleotide sequence, digital droplet PCR (ddPCR) was used to assess the success of the donor polynucleotide integration. Using primers that are specific for the HBB locus, it can be determined whether the editing resulted in (1) %HR, where the donor has integrated via HDR; (2) %WT, where no editing occurred, or (3) % INDEL, where the donor template was not integrated and resulted in an insertion or deletion at the target site. FIG. 7A shows the distribution of editing on 6 different process development runs. As shown in FIG. 7A, the % of HR mediated editing was consistent across the different runs, however, PD7 showed the greatest % HR compared to the other runs, with greater than 50% HR. [0205] In FIG.7B, the same data is shown but grouped by the 3 different classifications of editing. FIG.7B shows that over the 6 PD runs, the average is approximately 46% editing efficiency derived from editing of healthy donor cells. [0206] For ddPCR, HBB-edited HSPCs (gcHBB-SCD) were harvested within 2-days post- electroporation, together with mock control (electroporation only or untreated HSPCs), and then analyzed for modification frequencies of the HBB alleles. To collect gDNA, QuickExtract DNA extraction solution (Epicentre, Madison, WI, USA) was used, an in–out PCR was done to amplify solely the chromosomal HBB locus, and to exclude the episomal, unintegrated AAV6 from analysis. The following primers were designed to create an HBB-specific 1.4-kb gel-band: forward (out): 5ƍ-AGGAAGCAGAACTCTGCACTTCA-3ƍ (SEQ ID NO: 11) and reverse (in): 5ƍ-AGTCAGTGCCTATCAGAAACCCAAGAG-3ƍ (SEQ ID NO: 12). The PCR product was purified and diluted to 10 fg/^L in nuclease-free water. ddPCR analysis was carried out as described above with the PCR product generated as the template (20 fg total amplicon). This strategy, as previously described(28), utilizes a three-probe set (HEX/FAM/HEX) on the sample amplicon, where a probe binds the HR sequence (HR), another binds the reference sequence downstream of the Cas9 break site (REF), and the third binds to the Cas9 break site (WT). To gather allelic event percentages, the Poisson-corrected copies/^L for HR and WT counts per REF counts were divided; the remaining counts out of HR and WT events were counted as NHEJ. Gate threshold were set on mock control. The following primers and probes were used in the ddPCR reaction: [0207] HR probe (HEX) 5’-TGACTCCTGAGGAAAAATCCGCAGTCA-3’ (SEQ ID NO: 6), [0208] Ref probe (HEX) 5’-ACGTGGATGAAGTTGGTGGTGAGG-3’ (SEQ ID NO: 7), [0209] WT probe (FAM) 5’-CCCCACAGGGCAGTAACGGCAGACTTC-3’ (SEQ ID NO: 8), [0210] FWD ddPCR primer- 5’-TCACTAGCAACCTCAAACAGAC-3’ (SEQ ID NO: 9), [0211] REV ddPCR primer-5’-CCTGTCTTGTAACCTTGATACC-3’(SEQ ID NO: 10). Example 6: Assessing Purity and Viability of the Drug Product [0212] To assess whether the genetically modified cells of Example 5 were of good quality, the purity and the viability were assessed. [0213] For measuring viability, cells were stained using Acridine Orange (AO) and propidium iodide (PI), both are nuclear staining agents for quantifying live/dead cell ratios. AO is permeable to both live and dead cells and stains all nucleated cells to generate green fluorescence. PI enters dead cells with compromised membranes and stains all dead nucleated cells to generate red fluorescence. Cells stained with both AO and PI fluoresce red due to Förster resonance energy transfer (FRET), so all live nucleated cells fluoresce green and all dead nucleated cells fluoresce red. [0214] FIG.8A shows cells that have been stained with AO/PI and demonstrates that the edited cells have high viability after the editing. [0215] To determine the purity, cells were incubated with a CD34 antibody and assessed by flow cytometry to determine the presence of any non-CD34+ cells that might be present in the edited cell composition. FIG.8B demonstrates that the drug product contains few non-CD34+ cells. Example 7: In vivo Engraftment in Mice [0216] The previous examples have demonstrated the drug product containing the HBB-edited CD34+ cells are both viable and pure. To assess whether these cells can be used to correct sickle cell disease, an NSG humanized mouse model was used to determine whether the edited cells can demonstrate engraftment. [0217] FIG.9A shows a schematic of the engraftment protocol into 16+ weeks old NSG mice. Cells from 3 different lots were combined and thawed for preparation into NSG mice. In FIG.9B and FIG.9C, NSG mice were dosed with a cells derived from PD1, PD3, and PD5. The cells residing in the bone marrow were assessed to determine the engraftment efficiency of the drug product. [0218] FIG.9B shows the percent of cells quantified from bone marrow that were determined to be (a) human engrafted cells, (b) B-cells, or (c) myeloid cells. Each dot represents each animal. The average for each cell is summarized: human engraftment (~40%), B cells (~ 55%), and myeloid cells (~30%). [0219] To determine whether the HBB correction (gcHBB) was integrated, ddPCR was performed on the isolated cells from the bone marrow of dosed NSG mice. FIG.9C shows a comparison of the in vivo engraftment versus the in vitro gcHBB allele for the B-cells, myeloid cells, and a bulk population of cells (not purified). [0220] Table 2 summarizes the parameters evaluated for the different quality control runs of the different process development runs as well as showing the different development runs met criteria needed to proceed into manufacturing. Table 2: Quality Control Parameters for Different Process Development Runs

L

Example 8: Manufacturing of gcHBB Drug Product [0221] Based on the results on the process development runs, a scaled up protocol including use in a semi-closed manufacturing system was used to assess whether drug products manufactured in this manner would be consistent with previous runs as disclosed in the previous examples. [0222] FIG.10 and Table 3 show the results of two different runs (RUN-01 and RUN-02) and the percent of alleles of HBB were measured by HBB and shown as (1) % WT, (2) %HR, and (3) % NHEJ, where integration occurred nonspecifically through non-homologous end joining. Table 3: Summary results of different runs

Example 9: Cryopreservation of CD34+ HSPCs Post Isolation [0223] Gene edited gcHBB-SCD drug products manufactured during the tumor/tox study included the subsequent culture of freshly isolated CD34+ HSPCs at Day 0 immediately after CliniMACS selection. While this method yielded gcHBB-SCD drug products that met all release criteria, it limited the ability to process multiple apheresis collections in the unlikely event of collecting less than the threshold number of cells needed to proceed with gene correction. Some of the challenges associated with this direct culture step also included the introduction of residual RBCs from the CD34+ cell isolation procedure that may have been a factor that contributes to extensive cell loss prior to gene correction. The introduction of a cryopreservation step has the added advantage of banking cells prior to commencement of the gcHBB-SCD cell manufacturing until the minimum starting dose is achieved. This step also essentially reduces the number of residual RBCs by their loss during the freeze-thaw process itself in addition to the two centrifugation steps for cryopreservation and the cell thaw. Post isolation of CD34+ HSPCs using the CliniMACS instrument and CliniMACS CD34 Reagent kits as described in Example 1, the cells collected are counted using the Cellometer Auto-2000 using AO/PI staining, pelleted by centrifugation and then resuspended in CryoStor CS5 at a cell concentration of 5-20 x 10⁶ cells/mL. Samples are then collected for phenotype analysis and the cells are cryopreserved in cryopreservation bags and stored in the LN₂ vapor phase until an optimal dose of CD34+ cells is achieved. Based on the data collected from tumor/tox studies, process development runs, as well as the clinical-scale engineering runs, the cell viability and cell recovery at harvest were comparable when gcHBB-SCD cells were produced with both methods. A summary of the data is presented in Table 4, which represents the arithmetic mean of two to six replicates as specified. The cell recovery is measured by calculating the number of cells in the gcHBB-SCD drug substance at harvest as a percentage of the number of cells cultured on Day 0. Cell recovery improved when cryopreservation was introduced following isolation. Table 4: Comparison of In-process and Final Cell Viability and Recovery of gcHBB-SCD Cells Manufactured with Freshly Isolated and Cryopreserved CD34+HSPCs

[0224] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Table 5: Sequence Table

Claims

CLAIMS WHAT IS CLAIMED IS: 1. A large scale ex vivo method of generating a population of genetically modified stem cells from a population of stem cells, wherein the population of stem cells comprises at least one mutation in the HBB gene, the method comprising: 1) obtaining the population of stem cells from a subject; 2) culturing the population of stem cells; 3) introducing into the population of stem cells: i) a CRISPR-associated Cas9 nuclease; and ii) a guide polynucleotide sequence that hybridizes to a target sequence in the HBB gene; and 4) contacting the population of stem cells with an AAV vector comprising a donor polynucleotide sequence comprising SEQ ID NO: 5 flanked by a 5’ homology and 3’ homology arm having at least 75% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4, respectively; thereby generating the population of genetically modified stem cells.

2. The large scale ex vivo method of claim 1, wherein the target sequence comprises SEQ ID NO: 1.

3. The large scale ex vivo method of claim 1 or 2, wherein the CRISPR-associated Cas9 nuclease is a high-fidelity CRISPR-associated Cas9 nuclease.

4. The large scale ex vivo method of any one of claims 1-3, wherein the CRISPR-associated Cas9 nuclease comprises a mutation at position R691.

5. The large scale ex vivo method of claim 4, wherein the mutation at position R691 is an alanine.

6. The large scale ex vivo method of any one of claims 1-5, wherein the population of stem cells is cultured in cytokine-rich cell culture media.

7. The large scale ex vivo method of claim 6, wherein the cytokine-rich media comprises human SCF, human TPO, human Flt3 ligand, human IL-6, or any combination thereof.

8. The large scale ex vivo method of any one of claims 1-7, where the culturing is performed in the presence of UM171.

9. The large scale ex vivo method of any one of claims 1-8, wherein the culturing is performed at a low-cell density.

10. The large scale ex vivo method of claim 9, wherein the low-cell density comprises 2.5-5.0 x 10⁵ cells/ml.

11. The large scale ex vivo method of any one of claims 1-10, wherein the population of stem cells obtained in step 1) is frozen.

12. The large scale ex vivo method of any one of claims 1-11, wherein the population of stem cells obtained in step 1) is an apheresis product.

13. The large scale ex vivo method of claim 12, wherein the population of stem cells cultured in step 2) is enriched from the apheresis product.

14. The large scale ex vivo method of any one of claims 1-13, wherein step 2) can further comprise cryopreservation of the population of stem cells.

15. The large scale ex vivo method of any one of claims 1-14, wherein the cryopreservation is performed in cryopreservation media comprising heparin and pulmozyme.

16. The large scale ex vivo method of claim 15, wherein the cryopreservation media further comprises human serum albumin.

17. The large scale ex vivo method of any one of claims 1-16, wherein the method results in at least 20% gene edited stem cells.

18. The large scale ex vivo method of any one of claims 1-17, where in the CRISPR-associated Cas9 nuclease introduces a double stranded break.

19. The large scale ex vivo method of any one of claims 1-18, wherein the genetically modified stem cell comprises at least 10%, 20%, 30%, 40%, 50% greater expression of wild-type beta globin compared to a cell that was not genetically modified. 20. The large scale ex vivo method of any one of claims 1-19, wherein the genetically modified stem cell comprises at least 10%,

20%, 30%, 40%, 50% less expression of a mutant form of beta globin compared to a cell that was not genetically modified.

21. The large scale ex vivo method of any one of claims 1-20, wherein the AAV vector is AAV6.

22. The large scale ex vivo method of any one of claims 1-21, wherein the culturing is performed at low oxygen conditions.

23. The large scale ex vivo method of claim 22, wherein the low oxygen conditions comprise 5% O₂.

24. The large scale ex vivo method of claim 23, wherein the low oxygen conditions further comprise 5% CO₂.

25. The large scale ex vivo method of any one of claims 1-24, wherein step 4) occurs within 20 minutes of step 3).

26. The large scale ex vivo method of claim 25, wherein step 4) occurs within 15 minutes of step 3).

27. The large scale ex vivo method of claim 26, wherein step 4) occurs within 6 minutes of step 3).

28. The large scale ex vivo method of any one of claims 1-27 , wherein the culturing of step 2) results in reduced production of pro-inflammatory cytokines.

29. The large scale ex vivo method of claim 28, wherein the culturing of step 2) results in at least 50% reduced production of pro-inflammatory cytokines.

30. The large scale ex vivo method of any one of claims 1-29, wherein the population of stem cells comprises a population of human stem and progenitor cells (HSPCs).

31. The large scale ex vivo method of any one of claims 1-30, wherein the population of stem cells is mobilized by plerixafor.

32. The large scale ex vivo method of any one of claims 1-31, wherein the method generates a yield of at least 10%- 25% genetically modified stem cells.

33. The large scale ex vivo method of any one of claims 1-32, wherein the method generates at least 50-150 x 10⁶ genetically modified stem cells.

34. The large scale ex vivo method of any one of claims 1-33, wherein the method generates the genetically modified stem cells in at least 5L, 10L, 50L, 100L, 300L, or 500L.

35. The large scale ex vivo method of any one of claim 1-34, wherein the donor polynucleotide sequence comprises SEQ ID NO: 2.

36. A large scale ex vivo method of generating a population of genetically modified stem cells from a population of stem cells, wherein the population of stem cells comprises at least one mutation in the HBB gene, the method comprising: 1) obtaining the population of stem cells from a subject; 2) culturing the population of stem cells; 3) introducing into the population of stem cells: i) a Cas9 nuclease; and ii) a guide polynucleotide sequence that hybridizes to a target sequence in the HBB gene, wherein the target sequence comprises SEQ ID NO: 1; and 4) contacting the population of stem cells with an AAV vector comprising a donor polynucleotide sequence comprising SEQ ID NO: 5 flanked by a 5’ homology and 3’ homology arm having at least 75% sequence identity to SEQ ID NO: 3 and SEQ ID NO: 4, respectively; thereby generating the population of genetically modified stem cells.

37. The large scale ex vivo method of claim 36, wherein the CRISPR-associated Cas9 nuclease is a high-fidelity CRISPR-associated Cas9 nuclease.

38. The large scale ex vivo method of claim 36 or 37, wherein the CRISPR-associated Cas9 nuclease comprises a mutation at position R691.

39. The large scale ex vivo method of claim 38, wherein the mutation at position R691 is an alanine.

40. The large scale ex vivo method of any one of claims 36-39, wherein the population of stem cells is cultured in cytokine-rich cell culture media.

41. The large scale ex vivo method of claim 40, wherein the cytokine-rich media comprises human SCF, human TPO, human Flt3 ligand, human IL-6, or a combination thereof.

42. The large scale ex vivo method of any one of claims 36-41, where the culturing is performed in the presence of UM171.

43. The large scale ex vivo method of any one of claims 36-42, wherein the culturing is performed at a low-cell density.

44. The large scale ex vivo method of claim 43, wherein the low-cell density comprises 2.5-5.0 x 10⁵ cells/ml.

45. The large scale ex vivo method of claim 36, wherein the population of stem cells obtained in step 1) is frozen.

46. The large scale ex vivo method of claim 36, wherein the population of stem cells obtained in step 1) is an apheresis product.

47. The large scale ex vivo method of claim 46, wherein the population of stem cells cultured in step 2) is enriched from the apheresis product.

48. The large scale ex vivo method of any one of claims 36-47, wherein step 2) can further comprise cryopreservation of the population of stem cells.

49. The large scale ex vivo method of any one of claims 36-48, wherein the cryopreservation is performed in cryopreservation media comprising heparin and pulmozyme.

50. The large scale ex vivo method of claim 49, wherein the cryopreservation media further comprises human serum albumin.

51. The large scale ex vivo method of any one of claims 36-50, wherein the method results in at least 20% of gene edited cells.

52. The large scale ex vivo method of any one of claims 36-51, where in the CRISPR-associated Cas9 nuclease introduces a double stranded break.

53. The large scale ex vivo method of any one of claims 36-52, wherein the genetically modified stem cell comprises at least 10%, 20%, 30%, 40%, 50% greater expression of wild-type beta globin compared to a cell that was not genetically modified.

54. The large scale ex vivo method of any one of claims 36-53, wherein the genetically modified stem cell comprises at least 10%, 20%, 30%, 40%, 50% less expression of a mutant form of beta globin compared to a cell that was not genetically modified.

55. The large scale ex vivo method of any one of claims 36-54, wherein the AAV vector is AAV6.

56. The large scale ex vivo method of any one of claims 36-55, wherein the culturing is performed at low oxygen conditions.

57. The large scale ex vivo method of claim 56, wherein the low oxygen conditions comprise 5% O₂.

58. The large scale ex vivo method of claim 57, wherein the low oxygen conditions further comprise 5% CO₂.

59. The large scale ex vivo method of any one of claims 36-58, wherein step 4) occurs within 20 minutes of step 3).

60. The large scale ex vivo method of claim 59, wherein step 4) occurs within 15 minutes of step 3).

61. The large scale ex vivo method of claim 60, wherein step 4) occurs within 6 minutes of step 3).

62. The large scale ex vivo method of any one of claims 36-61, wherein the culturing of step 2) results in reduced production of pro-inflammatory cytokines.

63. The large scale ex vivo method of claim 62, wherein the culturing of step 2) results in at least 50% reduced production of pro-inflammatory cytokines.

64. The large scale ex vivo method of any one of claims 36-63, wherein the population of stem cells comprises a population of human stem and progenitor cells (HSPCs).

65. The large scale ex vivo method of any one of claims 36-64, wherein the population of stem cells is mobilized by plerixafor.

66. The large scale ex vivo method of any one of claims 36-65, wherein the method generates a yield of at least 10%-25% genetically modified stem cells.

67. The large scale ex vivo method of any one of claims 36-66, wherein the method generates at least 50-150 x 10⁶ genetically modified stem cells.

68. The large scale ex vivo method of any one of claims 36-67, wherein the method generates the genetically modified stem cells in at least 5L, 10L, 50L, 100L, 300L, or 500L.

69. The large scale ex vivo method of any one of claim 36-67, wherein the donor polynucleotide sequence comprises SEQ ID NO: 2.

70. A method of treating a hematological disease in a subject in need thereof, the method comprising: 1) generating a population of genetically modified stem cells according to any one of claims 1-69; and 2) administering the population of genetically modified stem cells to the subject in need thereof.

71. The method of claim 70, wherein the population of genetically modified cells administered in step 2) comprises 50-150 x 10⁶ genetically modified stem cells.

72. The method of claim 70 or 71, wherein the subject has sickle cell disease (SCD).

73. The method of any one of claims 70-72, wherein the administering is performed intravenously.

74. A large scale ex vivo method for generating genetically modified stem cells at a clinical scale for use in treating sickle cell disease in a subject in need thereof, the method comprising: 1) obtaining a population of cells from the subject; 2) isolating a population of hematopoietic stem cells from the population of cells; 3) culturing the population of stem cells; 4) introducing into the population of stem cells: a) a CRISPR-associated Cas9 nuclease; and b) a guide polynucleotide sequence that hybridizes to a target sequence comprising SEQ ID NO: 1; and 5) contacting the population of stem cells with an AAV vector comprising a donor polynucleotide sequence comprising SEQ ID NO 2; thereby generating the genetically modified stem cells.

75. An ex vivo method of generating a population of genetically modified CD34+ hematopoietic stem and progenitor cells (HSPCs) from a population of stem cells, the method comprising: (a) isolating the population of stem cells from a subject, wherein the isolating comprises one or more steps selected from mobilizing the stems cells in the subject and collecting the mobilized stem cells from the subject; (b) selecting for a CD34+ HSPC enriched stem cell population from the isolated stem cells; (c) cryopreserving the CD34+ HSPC-enriched stem cell population; (d) optionally, repeating steps (a) to (c) until a threshold number of CD34+ HSPCs required for gene editing has been isolated from the subject; (e) thawing the one or more cryopreserved CD34+ HSPC-enriched stem cell populations to obtain a thawed population of CD34+ HSPCs, wherein the thawed population of CD34+ HSPCs comprises the threshold number of CD34+ HSPCs of step (d); and (f) gene editing one or more target genes of the CD34+ HSPCs; thereby generating the population of genetically modified CD34+ HSPCs.

76. The ex vivo method of claim 75, wherein the gene editing comprises: (a) introducing into the thawed population of CD34+ HSPCs: i) a CRISPR-associated Cas9 nuclease; and ii) a guide polynucleotide sequence that hybridizes to a target sequence in the genome of the CD34+ HSPCs; and (b) contacting the thawed population of CD34+ HSPCs with an AAV vector comprising a donor polynucleotide sequence; whereupon induction of a double-strand break by the Cas9 nuclease and guide polynucleotide, the donor polynucleotide sequence is integrated into the genome of the cell by homology directed repair.

77. The ex vivo method of claim 75, wherein the population of stem cells comprises at least one mutation in a target gene.

78. The ex vivo method of claim 75 or 76, further comprising the step of culturing the thawed population of CD34+ HSPCs.

79. The ex vivo method of any one of claims 75 to 78, wherein the target sequence comprises SEQ ID NO: 1.

80. The ex vivo method of any one of claims 75 to 79, wherein the CRISPR-associated Cas9 nuclease is a high-fidelity CRISPR-associated Cas9 nuclease.

81. The ex vivo method of claim 80, wherein the CRISPR-associated Cas9 nuclease comprises a mutation at position R691.

82. The ex vivo method of claim 81, wherein the mutation at position R691 is an alanine.

83. The ex vivo method of any one of claim 78, wherein the population of stem cells is cultured in cytokine-rich cell culture media.

84. The ex vivo method of claim 83, wherein the cytokine-rich media comprises human SCF, human TPO, human Flt3 ligand, human IL-6, or any combination thereof.

85. The ex vivo method of claim 78, where the culturing is performed in the presence of UM171.

86. The ex vivo method of any one of claims 78 to 85, wherein the culturing is performed at a low-cell density.

87. The ex vivo method of claim 86, wherein the low-cell density comprises 2.5-5.0 x 10⁵ cells/ml.

88. The ex vivo method of any one of claims 75 to 87, wherein collecting the mobilized stem cells from the subject comprises apheresis.

89. The ex vivo method of any one of claims 75 to 88, wherein the cryopreservation is performed in cryopreservation media comprising heparin and pulmozyme.

90. The ex vivo method of claim 89, wherein the cryopreservation media further comprises human serum albumin.

91. The ex vivo method of any one of claims 75 to 90, wherein the method results in at least 20% gene edited CD34+ HSPCs within the thawed population of CD34+ HSPCs.

92. The ex vivo method of any one of claims 76 to 91, wherein the AAV vector is AAV6.

93. The ex vivo method of any one of claims 78 to 92, wherein the culturing is performed at low oxygen conditions.

94. The ex vivo method of claim 93, wherein the low oxygen conditions comprise 5% O2.

95. The ex vivo method of claim 94, wherein the low oxygen conditions further comprise 5% CO2.

96. The ex vivo method of any one of claims 78 to 95, wherein the culturing results in reduced production of pro-inflammatory cytokines.

97. The ex vivo method of claim 96, wherein the culturing results in at least 50% reduced production of pro-inflammatory cytokines.

98. The ex vivo method of any one of claims 75 to 97, wherein the population of stem cells is mobilized by plerixafor.

99. The ex vivo method of any one of claims 75 to 98, wherein the method generates a yield of at least 10%-25% genetically modified stem cells.

100. The ex vivo method of any one of claims 75 to 99, wherein the method generates at least 50-150 x 10⁶ genetically modified stem cells.

101. The ex vivo method of any one of claims 75 to 100, wherein the method generates the genetically modified stem cells in at least 5L, 10L, 50L, 100L, 300L, or 500L.

102. The ex vivo method of any one of claims 75 to 101, wherein the percentage of cells recovered following gene editing relative to the threshold number of cells is at least 65%.

103. The ex vivo method of claim 76, wherein the donor polynucleotide sequence comprises SEQ ID NO: 2.^