CRISPR/CAS-MEDIATED GENE EDITING OF HUMAN STEM CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Pat. Appl. No. 63/119,547, filed on November 30, 2020, which application is incorporated herein by reference in its entirety .

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under Grant No. R01 HL135607-01 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] A new class of medicines through DNA editing has been revolutionized by the development and advancement of CRISPR systems (1). Following a targeted break on both strands of DNA (a double-stranded break (DSB)), the lesion on DNA is repaired in one of two primary ways: ligation of the two ends together by nonhomologous end-joining (NHEJ) and microhomology-mediated end joining (MMEJ), or the homology-directed repair (HDR) pathway. NHEJ and MMEJ are used in all cells to repair spontaneous breaks and can result in insertions or deletions (indels) of one to several bases at the site of the break. Alternatively, in HDR, the molecular homologous recombination machinery is used and results in precise changes to the DNA. HDR requires a homologous donor DNA to template the precise changes that are made in the genomic DNA (1, 2). In sum, genome editing harnesses endogenous DNA repair processes to generate precise genomic modifications.

[0004] Currently many preclinical studies involving HDR-mediated gene editing highlight that the rates of HDR can be remarkably high, with frequencies as high as 30-70% in primary human cells such as hematopoietic stem and progenitor cells (HSPCs), T-cells, induced pluripotent stem cells (IPSCs), basal cells, and mesenchymal stromal cells (MSCs). HSCs have the ability to repopulate an entire hematopoietic system and thus strategies aimed at developing cell-based therapies involving genome editing for various hematological diseases such as sickle cell disease, p-thalassemia, and X-linked severe combined immunodeficiency are progressing towards clinical trials. We have shown that recombinant adeno-associated viral vectors of serotype 6 (rAAV6) can efficiently deliver single-stranded DNA cargos to serve as a gene-targeting donor template (3-6). However, current xenograft studies support the idea that HSCs are more resistant to HDR-mediated editing, perhaps one mechanistic explanation for the observation that HDR-edited cells engraft less efficiently following transplantation in immunodeficient mice. Reductions in HDR frequency during long-term engraftment have been observed previously and therefore remains a major impediment to bringing HDR-mediated therapies to clinic (3, 7-9).

[0005] One of the key questions in the metabolism of DSBs is how a cell chooses to repair the break. A key step is how the end is processed. The NHEJ pathway is activated if proteins such as 53BP1 bind the end and the Ku70/Ku80 dimer is recruited to bind the end. In contrast, end resection to generate 3’ single strand tails is a key early step in activating the recombination pathway of repair and is facilitated by the protein CtIP. One of the mechanisms by which 53BP1 biases repair towards NHEJ is by inhibiting binding of BRCAl, a protein required for homologous recombination (10). It has been previously shown that inhibiting 53BP1 through an engineered ubiquitin variant called i53 delivered by either plasmid transfection or AAV delivery could increase the frequency of Cas9 mediated HDR in human cancer cell lines. i53 was shown to inhibit accumulation of 53BP1 at DSBs and was thus thought to block NHEJ and promote breaks being repaired by alternative mechanisms such as HDR (11). Such an approach would face important limitations in primary cells, however, as the transfection of naked DNA plasmids into primary human cells results in the induction of a toxic Type I interferon response, and the kinetics of expression via AAV transduction might not be effective.

[0006] There exists therefore a need for new and efficient methods for promoting HDR- mediated genomic editing in primary cells, and particularly in hematopoietic stem cells (HSCs) or hematopoietic stem and progenitor cells (HSPCs). The present disclosure addresses these needs and provides other advantages as well. BRIEF SUMMARY

[0007] In one aspect, the present disclosure provides a method of genetically modifying a primary human cell, the method comprising: (i) introducing into the cell an RNA-guided nuclease and a single guide RNA (sgRNA) targeting a genetic locus of interest; (ii) introducing a homologous donor template into the cell, wherein the homologous donor template comprises a nucleotide sequence that is homologous to the locus of interest; and (iii) introducing a purified i53 peptide into the cell; wherein the sgRNA directs the RNA-guided nuclease to the locus of interest, the RNA-guided nuclease cleaves the locus at the target sequence of the sgRNA, and the homologous donor template is integrated at the site of the cleaved locus by homology directed repair (HDR).

[0008] In some embodiments of the method, the primary human cell is a cell selected from the group consisting of a CD34 ⁺ hematopoietic stem and progenitor cell (HSPC), a T cell, a mesenchymal stem cell (MSC), an airway basal stem cell, and an induced pluripotent stem cell (IPSC). In some embodiments, the locus of interest is a gene selected from the group consisting of Hemoglobin Subunit Beta (HBB), C-C Motif Chemokine Receptor 5 (CCR5), Interleukin 2 Receptor Subunit Gamma (IL2RG), Hemoglobin Subunit Alpha 1 (HBA1), or Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). In some embodiments, the sgRNA comprises 2'-O-methyl-3'-phosphorothioate (MS) modifications at one or more nucleotides. In some embodiments, the 2'-O-methyl-3'-phosphorothioate (MS) modifications are present at the three terminal nucleotides of the 5' and 3' ends. In some embodiments, the RNA-guided nuclease is Cas9.

[0009] In some embodiments, the sgRNA and RNA-guided nuclease are introduced into the cell as a ribonucleoprotein (RNP). In some embodiments, the RNP is introduced into the cell by electroporation. In some embodiments, the i53 peptide is introduced into the cell by electroporation. In some embodiments, the i53 peptide and the RNP are introduced together into the cell. In some embodiments, the level of the i53 peptide in the cell four hours after electroporation is less than 0.1% of the level in the cell immediately after electroporation. In some embodiments, the amino acid sequence of the i53 peptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the i53 peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, the i53 peptide is recombinant. [0010] In some embodiments, the homologous repair template is introduced into the cell using an adeno-associated virus serotype 6 (AAV6) vector. In some embodiments, the AAV vector is transduced into the cell at a multiplicity of infection (MOI) of less than about 2500, 1250, or 625. In some embodiments, the MOI is about 625. In some embodiments, the concentration of the i53 peptide used for electroporation is about 1-2 mg/ml. In some embodiments, the concentration of the i53 peptide is about 1.5 mg/ml.

[0011] In some embodiments, the frequency of HDR at the locus of interest in the cell is higher than the frequency in an equivalent cell in the presence of the sgRNA, RNA-guided nuclease, and homologous donor template, but in the absence of the i53 peptide. In some embodiments, the frequency of HDR at the locus of interest in the cell is at least about 10%, 20%, 30%, 40%, or more higher than the frequency in an equivalent cell in the presence of the sgRNA, RNA-guided nuclease, and homologous donor template, but in the absence of the i53 peptide. In some embodiments, the frequency of indels at the locus of interest in the cell is lower than the frequency in an equivalent cell in the presence of the sgRNA, RNA-guided nuclease, and homologous donor template, but in the absence of the i53 peptide. In some embodiments, the frequency of indels at the locus of interest in the cell is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more lower than the frequency in an equivalent cell in the presence of the sgRNA, RNA-guided nuclease, and homologous donor template, but in the absence of the i53 peptide.

[0012] In some embodiments, the method further comprises introducing a second sgRNA into the cell targeting a second genetic locus, and introducing a second homologous donor template comprising a nucleotide sequence that is homologous to the second genetic locus, wherein the second sgRNA directs the RNA-guided nuclease to the second genetic locus, the RNA-guided nuclease cleaves the second genetic locus at the target sequence of the second sgRNA, and the second homologous donor template is integrated at the site of the cleaved second genetic locus by HDR. In some embodiments, the frequency of HDR is higher at both the locus of interest and at the second genetic locus in the presence of the i53 peptide than in the absence of the i53 peptide. In some embodiments, the frequency of indels is lower at both the locus of interest and at the second genetic locus in the presence of the i53 peptide than in the absence of the i53 peptide.

[0013] In another aspect, the present disclosure provides a method of treating a genetic disorder in a human subject in need thereof, the method comprising: isolating a primary cell from the subject; genetically modifying the primary cell using the method of any of the herein-described methods, wherein the integration of the homologous donor template at the locus of interest in the cell corrects a mutation at the locus or leads to the expression of a therapeutic protein in the cell that is absent or deficient in the subject; and reintroducing the genetically modified cell into the subject.

[0014] In some embodiments of the method, the genetic disorder is a disorder selected from the group consisting of p-thalassemia, sickle cell disease (SCD), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1, Gaucher disease, Cystic Fibrosis, Krabbe disease, and X-linked chronic granulomatous disease (X-CGD).

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A-1B. Cas9-RNP and AAV6-mediated targeting in different CD34 ⁺ donors. FIG. 1A: CD34 ⁺ HSPCs were sorted into sub-populations which were subsequently electroporated with Cas9-RNP and AAV6 and with or without i53 peptide. HDR-mediaied outcomes were assessed by ddPCR. Data from n=4 independent biological replicates with median+range graphed, unless indicated otherwise. FIG. IB: Four donors of CD34 ⁺ HSPCs and sub-populations were electroporated with Cas9-RNP and AAV6 and with or without i53 peptide. HDR-mediated outcomes were assessed by ddPCR. Data from n=4 independent biological replicates.

[0016] FIGS. 2A-2F. Cas9-RNP and AAV6-mediated targeting of human primary stem cells using i53 recombinant peptide. FIG. 2A: Schematic of DNA repair pathways (NHEJ and HDR) illustrating transient inhibition of NHEJ by i53 peptide. FIG. 2B: CD34 ⁺ HSPCs were electroporated with Cas9-RNP and AAV6 and with or without i53 peptide. HDR- mediated outcomes were assessed by ddPCR. Data from n≥4 independent biological replicates with mean±SD graphed, unless indicated otherwise. FIG. 2C: Indel rates were determined PCR amplicon analyses through ICE or TIDE. Data from n≥3 independent biological replicates with mean+SD graphed, unless indicated otherwise. FIG. 2D: HDR rates in airway stem cells were determined by ICE or TIDER analyses. HDR in MSCs were determined by the read out of GFP expressing cells via flow cytometry. Data from n≥3 biological replicates with meaniSD graphed. FIG. 2E: HSPCs were electroporated with Cas9-RNP targeting HBB gene with or without i53 peptide. Cells were collected at time points 0, 2, 4, 6, 8, 24, 48, and 72 hours post-electroporation and indel rates were analyzed by ICE. Data from n=2 biological replicates with meantSD graphed. FIG. 2F: Indels (NHEJ and MMEJ) were assessed by Sanger sequencing and ICE analysis 72 hours post- electroporation. Data from n=2 biological replicates. MeantSD shown.

[0017] FIGS. 3A-3B. FIG. 3A. Indels (NHEJ and MMEJ) were assessed by Sanger sequencing and ICE analysis 72 hours post-electroporation. Data from n=2 biological replicates. MeantSD shown. FIG. 3B shows the distribution of indels identified with or without i53.

[0018] FIGS. 4A-4G. Optimizing the use of i53 peptide for targeting HBB locus in CD34 ⁺ HSPCs. FIG. 4A: Experimental layout for targeting CD34 ⁺ HSPCs at HBB locus using i53 peptide. FIG. 4B: Heatmap illustrating HDR rates in response to various doses of AAV6 and i53 peptide. CD34 ⁺ HSPCs were electroporated with Cas9-RNP, 625 MOI to 5000 MOIs of AAV6 and 0 μg/ml to 1500 μg/ml of i53 peptide. HDR-mediated outcomes were assessed by ddPCR Data from n≥2 independent biological replicates with mean values graphed. FIG. 4C: Indel rates were determined by ddPCR or ICE. Data from n≥3 independent biological replicates with meantSD graphed. FIG. 4D: Edited HSPCs were plated on methylcellulose and scored as CFU-E, BFU-E, CFU-GM, or CFU-GEMM based on morphology 14 days after plating. Data from n≥4 independent biological replicates with meantSD graphed. *:p<0.05 by unpaired t-test. FIG. 4E: X-Y linear correlation between HDR frequency and % colonies formed on methocult. FIG. 4F: Allele spectra and corresponding percentages of alleles generated by ICE following editing with Cas9-RNP. FIG. 4G: Representative FACS plots of biallelic targeting using HBB-mCheny and HBB-GFP encoding AAV donors.

[0019] FIGS. 5A-5C. Determining the dosage of i53 peptide for targeting HBB locus in CD34 ⁺ HSPCs. FIG. 5A: CD34 ⁺ HSPCs were electroporated with Cas9-RNP, 625 MOI of AAV6 and 0 μg/ml to 5000 μg/ml of i53 peptide. HDR-mediated outcomes were assessed by ddPCR Data from n≥3 independent biological replicates with mean values graphed. Indel rates were determined by ddPCR or ICE. Data from n≥3 independent biological replicates with meantSD graphed. FIG. SB: CD34 ⁺ HSPCs were electroporated with Cas9-RNP, AAV6 with or without i53 peptide. Viability of CD34 HSPCs was determined 72 hours-post editing. FIG. 5C: Edited HSPCs were plated on methylcellulose and scored as CFU-E, BFU- E, CFU-GM, or CFU-GEMM based on morphology 14 days after plating. Data from n≥4 independent biological replicates with mean±SD graphed. *:p<0.05 by impaired t-test. [0020] FIGS. 6A-6G. Determining the toxicity of the use of i53 peptide during genome editing in CD34 ⁺ HSPCs. FIG. 6A: Immunoblot showing the expression of His-i53 peptide in CD34 ⁺ cells at Ohr, Ihr, 2hrs, 3hrs, 4hrs, 24hrs and 48hrs post-electroporation. GAPDH served as a loading control. FIG. 6B: HSPCs were electroporated with Cas9-RNP and AAV with or without i53 peptide. Cells were collected at time points 0, 2, 4, 6, 8, 24, 48, and 72 hours post-electroporation and HDR and indel rates were analyzed by ddPCR and ICE. Data from n=2 biological replicates with two technical replicates. HDR represented in blue and indels are represented in red. Mean±SD graphed. FIG. 6C: Indels consisting of NHEJ and MMEJ are represented. Data from n=2 biological replicates with two technical replicates. Mean+SD graphed. FIG. 6D and FIG. 6E: Expression of p21 assessed by ddPCR. FIG. 6F: HSPCs were edited with indicated MOI of AAV6 and with or without i53 peptide. Expression of p21 assessed by ddPCR. FIG. 6G: Measuring translocation after HBB and AAVS1 di-genic targeting.

[0021] FIGS. 7A-7C. Determining the kinetics of i53 peptide during genome editing in CD34 ⁺ HSPCs. FIG. 7A: Immunoblot showing the expression of His-i53 peptide in CD34 ⁺ cells at Ohr, 4hrs, 24hrs and 48hrs post-electroporation. GAPDH served as a loading control. FIG. 7B: Quantification of the immunoblot in FIG 7A. FIG. 7C: Allele spectra and corresponding percentages of alleles generated by ICE following editing with Cas9-RNP.

[0022] FIGS. 8A-8G. HBB-gene targeted CD34 ⁺ HSPCs display improved long-term and multi-lineage reconstitution in NSG mice. FIG. 8A: Experimental layout. FIG. 8B: HBB gene editing outcomes in CD34 ⁺ HSPCs in vitro. Data from two biological donors (donors A and B). FIG. 8C: Human engraftment (14 weeks post-transplantation) in NSG mice from all experimental groups. Data for donor A and donor B represented in a separate panel. Median values reported. FIG. 8D: Percentage of human cells representing B cells (yellow circle), myeloid cells (red square) and other cells (brown triangle). Bars represent median. FIG. 8E: Percentage of HDR alleles in the human cells in the bone marrow of NSG mice. Median values reported. FIG. 8F: Percentage of HDR alleles in the human B cells in the bone marrow of NSG mice. FIG. 8G: Percentage of HDR alleles in the human myeloid cells in the bone marrow of NSG mice.

[0023] FIG. 9. Analysis of human CD34 ⁺ HSPC engraftment in NSG mice. DETAILED DESCRIPTION

1. Introduction

[0024] The present disclosure provides methods for improving the efficiency of homology directed repair (HDR)-mediated modification of genomic sequences in primary cells. The methods involve the introduction into cells of single guide RNAs (sgRNAs), RNA-guided nucleases (e.g., Cas9), homologous repair templates, and recombinant i53 polypeptide. The methods can be used, e.g., to integrate cDNAs encoding functional proteins into cells to correct or compensate for mutations in cells from a subject with a genetic disorder, or to modify endogenous genomic sequences for any purpose using HDR.

[0025] In particular embodiments, the sgRNA and nuclease are delivered to cells as ribonucleoprotein (RNP) complexes, and both the RNPs and i53 peptide are delivered together by electroporation, followed by the transduction of the homologous repair template using an AAV6 viral vector. The introduction of the i53 peptide transiently increases the rate of HDR and reduces non-homologous end-joining (NHEJ) in the primary cells, and also permits the use of lower amounts of donor template (e.g., reduced MOIs when using viral vectors such as AAV6) than is possible in the absence of i53 peptide, while still achieving high levels of HDR in the cells and high levels of engrafhnent in vivo. This system can be used to modify any human cell, and in particular embodiments CD34 ⁺ HSPCs are used.

2. Definitions

[0026] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0027] 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,” 6 “6a, n,” 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 so forth.

[0028] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Any reference to “about X” specifically indicates at least the values X, 0.8X, 0.8 IX, 0.82X, 0.83X, 0.84X, 0.85X, 0.86X, 0.87X, 0.88X, 0.89X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, LUX, 1.12X, 1.13X, 1.14X, 1.15X, 1.16X, 1.17X, 1.18X, 1.19X, and 1.2X. Thus, “about X” is intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”

[0029] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0030] The term “gene” means the segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

[0031] A "promoter" is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The promoter can be a heterologous promoter.

[0032] “i53” or “i53 peptide” is a peptide variant of ubiquitin that can specifically inhibit 53BP1 (see, e.g., UniProt Ref. Q12888; NCBI ID Gene ID 7158). 53BP1 binds to double stranded breaks in the DNA and promotes non-homologous end-joining (NHEJ). In particular embodiments, the i53 peptide used in the present methods comprises or consists of the sequence of SEQ ID NO: 1 or SEQ ID NO:2, or a derivative, variant, and/or fragment thereof that maintains 53BPl-inhibiting activity. i53 can be, e.g., about 74 amino acids in length (see, e.g., SEQ ID NO:1), or longer, e.g., if the peptide contains a tag such as a His or FLAG tag (see, e.g., SEQ ID NO:2). In some embodiments, the i53 peptide comprises a His tag and is 85 amino acids in length (approx. 9.6 kDa). For example, i53 peptides that comprise the amino acid sequence of SEQ ID NO: 1 and that also contain a tag such as a His tag (as shown, e.g., in SEQ ID NO:2) or a FLAG tag can be used. In some embodiments, the i53 peptide comprises an amino acid sequence that comprises 1, 2, 3 or 4 amino acid substitutions relative to SEQ ID NO: 1 or SEQ ID NO:2.

[0033] An "expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter. The promoter can be a heterologous promoter. In the context of promoters operably linked to a polynucleotide, a “heterologous promoter” refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism).

[0034] As used herein, a first polynucleotide or polypeptide is "heterologous" to an organism or a second polynucleotide or polypeptide sequence if the first polynucleotide or polypeptide originates from a foreign species compared to the organism or second polynucleotide or polypeptide, or, if from the same species, is modified from its original form. For example, when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence).

[0035] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including foil-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. [0036] The terms “expression” and “expressed” refer to the production of a transcriptional and/or translational product, e.g., of an introduced cDNA or encoded protein. In some embodiments, the term refers to the production of a transcriptional and/or translational product encoded by a gene or a portion thereof. The level of expression of a DNA molecule in a cell may be assessed on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell.

[0037] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a poly-peptide also describes every- possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0038] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. In some cases, conservatively modified variants of a protein can have an increased stability, assembly, or activity as described herein. [0039] The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins, W. H. Freeman and Co., N. Y. (1984)).

[0040] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0041] In the present application, amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1, in an unmodified wild- type polypeptide sequence.

[0042] As used in herein, the terms “identical” or percent “identity,” in the context of describing two or more polynucleotide or amino acid sequences, refer to two or more sequences or specified subsequences that are the same. Two sequences that are “substantially identical” have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection where a specific region is not designated. With regard to polynucleotide sequences, tills definition also refers to the complement of a test sequence. With regard to amino acid sequences, in some cases, the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.

[0043] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST 2.0 algorithm and the default parameters discussed below are used.

[0044] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0045] An algorithm for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, which is described in Altschul et al., (1990) J. Mol. Biol. 215: 403- 410. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0046] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. [0047] The “CRISPR-Cas” system refers to a class of bacterial systems for defense against foreign nucleic acids. CRISPR-Cas systems are found in a wide range of bacterial and archaeal organisms. CRISPR-Cas systems fall into two classes with six types, I, II, III, IV, V, and VI as well as many sub-types, with Class 1 including types I and III CRISPR systems, and Class 2 including types II, IV, V and VI; Class 1 subtypes include subtypes I-A to I-F, for example. See, e.g., Fonfara et al., Nature 532, 7600 (2016); Zetsche et al., Cell 163, 759- 771 (2015); Adli et al. (2018). Endogenous CRISPR-Cas systems include a CRISPR locus containing repeat clusters separated by non-repeating spacer sequences that correspond to sequences from viruses and other mobile genetic elements, and Cas proteins that carry out multiple functions including spacer acquisition, RNA processing from the CRISPR locus, target identification, and cleavage. In class 1 systems these activities are effected by multiple Cas proteins, with Cas3 providing the endonuclease activity, whereas in class 2 systems they are all carried out by a single Cas, Cas9.

[0048] A “homologous repair template” or “homologous donor template” refers to a polynucleotide sequence that can be used to repair a double stranded break (DSB) in the DNA, e.g., a CRISPR/Cas9-mediated break at a locus targeted by a herein-described sgRNA as induced using the herein-described methods and compositions. The homologous repair template comprises homology to the genomic sequence surrounding the DSB, i.e., comprising target locus homology arms as described herein. In some embodiments, two distinct homologous regions are present on the template, with each region comprising at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or more nucleotides or more of homology with the corresponding genomic sequence. In particular embodiments, the templates comprise two homology arms comprising about 500 nucleotides of homology extending from either site of the sgRNA target site. The repair template can be present in any form, e.g., on a plasmid that is introduced into the cell, as a free-floating doubled stranded DNA template (e.g., a template that is liberated from a plasmid in the cell), or as single-stranded DNA. In particular embodiments, the template is present within a viral vector, e.g., an adeno- associated viral vector such as AAV6. In some embodiments, the templates of the disclosure a codon-optimized, e.g., full-length, codon-optimized cDNAs, as well as, typically, a polyadenylation signal such as from bovine growth hormone or rabbit beta-globin. In some embodiments, the cDNA comprises a promoter, operably linked to the cDNA. In some embodiments, the template comprises a sequence other than a cDNA, e.g., a sequence designed to correct a specific mutation in a genomic locus, or to introduce a specific deletion or insertion into a locus. The process of repairing a double-stranded break using a homologous donor template is referred to as Homology Directed Repair (HDR).

[0049] As used herein, “homologous recombination” or “HR” refers to insertion of a nucleotide sequence during repair of double-strand breaks in DNA via homology-directed repair (HDR) mechanisms. This process uses a “donor template” or “homologous repair template” with homology to nucleotide sequence in the region of the break as a template for repairing a double-strand break. The presence of a double-stranded break facilitates integration of the donor sequence. The donor sequence may be physically integrated or used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence. This process is used by a number of different gene editing platforms that create the double-strand break, such as meganucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas9 gene editing systems. In particular embodiments, HR involves double-stranded breaks induced by CRISPR-Cas9.

3. Methods of enhancing HDR

[0050] The present disclosure provides methods for improving the efficiency of genomic editing through homology-directed repair (HDR), e.g., for editing genomic sequences or integrating cDNAs into endogenous loci in cells, through the administration of purified i53 peptide to the cells. The present methods and compositions allow genomic editing to be performed with higher rates of HDR and with lower rates of non-homologous end-joining (NHEJ) and, as a result, of insertions and deletions (indels). Further, the methods allow for high levels of HDR and cell engraftment to be achieved with lower levels of administered donor templates, e.g., using lower multiplicities of infection (MOI) when donor templates are introduced using viral vectors such as adeno-associated viral vectors (AAV) such as AAV6. The effects observed using i53 peptides in cells is transient, allowing HDR to be achieved without introducing longer-term genomic instability as might be observed, e.g. using nucleic acids encoding i53.

[0051] In particular embodiments, the cells are primary human cells, including stem cells such as CD34+ hematopoietic stem and progenitor cells (HSPCs) or hematopoietic stem cells (HSCs). In some embodiments, cells from a subject are modified using the methods described herein and then reintroduced into the subject. For example, the cells can be taken from a subject with a genetic condition and the methods used to integrate a functional cDNA into the genome of the cells, wherein the expression of the cDNA in the modified cells in vivo restores protein activity that is missing or deficient in the subject or is otherwise beneficial to the subject.

[0052] The present disclosure is based in part on the identification that purified i53 peptide, e.g., purified recombinant i53 peptide, can effectively and safely increase HDR, decrease NHEJ, and decrease indels, when introduced together with a guide RNA and RNA-guided nuclease such as Cas9, and with a homologous donor template. In particular embodiments, the guide RNA and RNA-guided nuclease are introduced as a ribonucleoprotein (RNP), for example by electroporation. In particular embodiments, the i53 peptide is introduced together with the RNP.

4. Preparation of i53

[0053] In some embodiments, the i53 peptide used in the present methods comprises (or consists of) the sequence of SEQ ID NO: 1 or SEQ ID NO:2, or comprises (or consists of) the sequence of SEQ ID NO: 1 or SEQ ID NO:2 with 1, 2, 3, 4 or more amino acid substitutions (e.g., conservative amino acid substitutions). The i53 sequence can also be found, e.g., at ww ^, w.addgene.org/92170/sequences/, which is herein incorporated by reference in its entirety. In some embodiments, the i53 peptide used in the present methods comprises (or consists of) an i53 amino acid sequence as disclosed in www.addgene.org/92170/sequences/, or comprises (or consists of) an i53 amino acid sequence as disclosed in www.addgene.org/92170/sequences/ with 1, 2, 3, 4, or more amino acid substitutions (e.g., a conservative amino acid substitution). In some embodiments, the i53 peptide used in the present methods is a derivative, variant, or fragment of SEQ ID NO: 1 or SEQ ID NO:2 or an i53 amino acid sequence as disclosed in www.addgene.org/92170/sequences/ that comprises 53BPl-inhibiting activity.

[0054] In some embodiments, the i53 peptide is about 74 amino acids long. In some embodiments, the i53 peptide is shorter than 74 amino acids long, e.g., 50, 55, 60, 65, 70, 71, 72, or 73 amino acids long. In some embodiments, the i53 peptide is 75 amino acids long or longer, e.g., 76, 77, 78, 79, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the i53 peptide comprises additional elements such as a label such as a His tag or a FLAG tag. In some embodiments, the i53 peptide comprises a His tag and is 85 amino acids long. [0055] The present i53 peptides can comprise non-natural or non-proteinogenic amino acids, such as chemical mimetics of corresponding naturally occurring amino acids, non- standard amino acids such as D-amino acids, β-alanine, GABA, ornithine, citrulline, hydroxyproline, norleucine, 3-nitrotyrosine, nitroarginine, naphtylalanine, Abu, DAB, methionine sulfoxide, methionine sulfone, and more generally, P-amino acids (i.e., β3 and β2), homo-amino acids, beta-homo-amino acids, proline and pyruvic acid derivatives, 3- substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, N-methyl amino acids, alpha-methyl amino acids, ACHC, peptoids, and others.

Recombinant production and purification of i53

[0056] In particular embodiments, the i53 peptide is produced recombinantly and purified for use in the present methods. The synthesis of i53 for use in the present methods can be accomplished using standard molecular biology methods. For example, the nucleotide sequences encoding i53 can be synthesized using standard methods and cloned into a suitable expression vector, e.g., the His-tag expression vector pET30(a)+. Recombinant i53 can then be expressed in suitable cells, e.g., E. coli, and purified, and the protein concentrations and purities determined by, e.g., BCA assay and SDS-PAGE, respectively.

[0057] Basic texts disclosing general methods and techniques in the field of recombinant genetics include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel etal., eds., Current Protocols in Molecular Biology (1994).

[0058] For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

[0059] Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Lett. 22 1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12: 6159-6168 (1984). Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255: 137-149 (1983).

[0060] The sequence of a polynucleotide encoding an i53 peptide can be verified after cloning or subcloning using, e.g., the drain termination method for sequencing double- stranded templates of Wallace et al, Gene 16: 21-26 (1981). Nucleotide sequences encoding i53 peptides can be determined based on their encoded amino acid sequences (e.g., as shown in SEQ ID NO: 1 or SEQ ID NO:2, at www.addgene.org/92170/sequences/, and as described, e.g., in Canny et al. (2018) and in US Patent App. Pub. No. US 2019/0010196, the entire disclosures of each of which are herein incorporated by reference). Nucleotide sequences encoding i53 peptide can also be found, e.g., at www.addgene.org/92170/sequences/. Nucleic acid sequences encoding i53 can be isolated using standard cloning techniques such as polymerase chain reaction (PCR). Most commonly used techniques for this purpose are described in standard texts, e.g., Sambrook and Russell, supra.

[0061] Upon acquiring a nucleic acid sequence encoding an i53 peptide, the coding sequence can be modified as appropriate (e.g., adding a coding sequence for a heterologous tag, such as an affinity tag, for example, 6 x His tag or GST tag) and then be subcloned into a vector, for instance, an expression vector, so that recombinant i53 can be produced from the resulting construct, for example, after transfection and culturing host cells under conditions permitting recombinant protein expression directed by a promoter operably linked to the coding sequence.

[0062] In some embodiments, the polynucleotide sequence encoding an i53 peptide can be further altered to coincide with the preferred codon usage of a particular host. For example, the preferred codon usage of one strain of bacterial cells can be used to derive a polynucleotide that encodes an i53 peptide and includes the codons favored by this strain. The frequency of preferred codon usage exhibited by a host cell can be calculated by averaging frequency of preferred codon usage in a large number of genes expressed by the host cell (e.g., calculation service is available from web site of the Kazusa DNA Research Institute, Japan). This analysis is preferably limited to genes that are highly expressed by the host cell.

Expression of recombinant peptide

[0063] To obtain high level expression of a nucleic acid encoding an i53 peptide, a polynucleotide encoding the peptide can be subcloned into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator and a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook and Russell, supra, and Ausubel et al., supra. Bacterial expression systems for expressing a recombinant polypeptide are available in, e.g., E. coli, Bacillus sp., Salmonella, and Caulobacter. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. In one embodiment, the eukaryotic expression vector is an adenoviral vector, an adeno-associated vector, or a retroviral vector.

[0064] The promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is optionally positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function. In one embodiment, the promoter is an IPTG-inducible promoter.

[0065] In addition to the promoter, the expression vector typically includes a transcription unit or expression cassette that contains all the additional elements required for the expression of the peptide in host cells. A typical expression cassette thus contains a promoter operably linked to the coding sequence and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. The nucleic acid sequence encoding the peptide is typically linked to a cleavable signal peptide sequence to promote secretion of the recombinant peptide by the transformed cell. Such signal peptides include, among others, the signal peptides from tissue plasminogen activator, insulin, and neuron growth factor, and juvenile hormone esterase of Heliothis virescens. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.

[0066] In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.

[0067] The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, pET30(a)+, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., His, FLAG, or c-myc.

[0068] Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr vims. Other exemplary eukaryotic vectors include pMSG, pAV009/A ⁺ , pMTO10/A ⁺ , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0069] Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as a baculovirus vector in insect cells, with a polynucleotide sequence encoding the peptide under the direction of the polyhedrin promoter or other strong baculovirus promoters.

[0070] The elements that are typically included in expression vectors also include a replicon that functions in E. colt, a gene encoding a protein that provides antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable. The prokaryotic sequences are optionally chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary. Similar to antibiotic resistance selection markers, metabolic selection markers based on known metabolic pathways may also be used as a means for selecting transformed host cells.

[0071] When periplasmic expression of a recombinant peptide (e.g., i53 peptide) is desired, the expression vector further comprises a sequence encoding a secretion signal, such as the E. coli OppA (Periplasmic Oligopeptide Binding Protein) secretion signal or a modified version thereof, which is directly connected to 5' of the coding sequence of the protein to be expressed. This signal sequence directs the recombinant protein produced in cytoplasm through the cell membrane into the periplasmic space. The expression vector may further comprise a coding sequence for signal peptidase 1, which is capable of enzymatically cleaving the signal sequence when the recombinant protein is entering the periplasmic space. More detailed description for periplasmic production of a recombinant protein can be found in, e.g., Gray et al., Gene 39: 247-254 (1985), U.S. Patent Nos. 6,160,089 and 6,436,674.

Transfection

[0072] Standard transfection methods are used to produce bacterial, mammalian, yeast, insect, or plant cell lines that express large quantities of a recombinant polypeptide (e.g., i53 peptide), which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264: 17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bact. 132: 349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101: 347-362 (Wu etal., eds, 1983).

[0073] Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, e.g., Sambrook and Russell, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the recombinant polypeptide.

Detection of expression in host cells

[0074] After the expression vector is introduced into appropriate host cells, the transfected cells are cultured under conditions favoring expression of the peptide. The cells are then screened for the expression of the recombinant peptide, which is subsequently recovered from the culture using standard techniques (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al., supra; and Sambrook and Russell, supra).

[0075] Several general methods for screening gene expression are well known among those skilled in the art. First, gene expression can be detected at the nucleic acid level. A variety of methods of specific DNA and RNA measurement using nucleic acid hybridization techniques are commonly used (e.g., Sambrook and Russell, supra). Some methods involve an electrophoretic separation (e.g., Southern blot for detecting DNA and northern blot for detecting RNA), but detection of DNA or RNA can be carried out without electrophoresis as well (such as by dot blot). The presence of nucleic acid encoding an i53 peptide in transfected cells can also be detected by PCR or RT-PCR using sequence-specific primers.

[0076] Second, gene expression can be detected at the polypeptide level. Various immunological assays are routinely used by those skilled in the art to measure the level of a gene product, particularly using polyclonal or monoclonal antibodies that react specifically with an i53 peptide (e.g., Harlow and Lane, Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988; Kohler and Milstein, Nature, 256: 495-497 (1975)). Such techniques require antibody preparation by selecting antibodies with high specificity against the peptide. The methods of raising polyclonal and monoclonal antibodies are well established and their descriptions can be found in the literature, see, e.g., Harlow and Lane, supra, Kohler and Milstein, Eur. J. Immunol., 6: 511-519 (1976).

Purification of Recombinantly Produced Peptides

[0077] Once the expression of a recombinant i53 peptide in transfected host cells is confirmed, the host cells are then cultured in an appropriate scale for the purpose of purifying the recombinant polypeptide.

[0078] When polypeptides such as the i53 peptide are produced recombinantly by transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the polypeptides may form insoluble aggregates. There are several protocols that are suitable for purification of protein inclusion bodies. For example, purification of aggregate proteins (hereinafter referred to as inclusion bodies) typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells, e.g., by incubation in a buffer of about 100-150 μg/ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent. The cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY). Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel et al. and Sambrook and Russell, both supra, and will be apparent to those of skill in the art.

[0079] The cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible. The remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers will be apparent to those of skill in the art.

[0080] Following the washing step, the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties). The proteins that farmed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%, vohime/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents that are capable of solubilizing aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, may be inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re- formation of the immunologically and/or biologically active protein of interest. After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques. For further description of purifying recombinant polypeptides from bacterial inclusion body, see, e.g., Patra et al., Protein Expression and Purification 18: 182- 190 (2000).

[0081] Alternatively, it is possible to purify recombinant polypeptides, e.g., i53 peptide, from bacterial periplasm. Where the recombinant protein is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see e.g., Ausubel et al, supra). To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO» and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art. Protein Separation Techniques for Purification

[0082] When a recombinant polypeptide is expressed in host cells in a soluble form, its purification can follow a standard protein purification procedure as described herein. Such standard purification procedures are also suitable for purifying a polypeptide obtained from chemical synthesis.

Solubility Fractionation

[0083] Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfete concentrations. A typical protocol is to add saturated ammonium sulfete to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic proteins. The precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfete is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diafiltration. Other methods that rely on solubility' of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.

Size Differential Filtration

[0084] Based on a calculated molecular weight, a protein of greater and lesser size can be isolated using ultrafiltration through membranes of different pore sizes (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of a protein of interest, e.g., an i53 peptide. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below. Column Chromatography

[0085] Proteins of interest (such as an i53 peptide) can also be separated from other proteins on the basis of their size, net surface charge, hydrophobicity, or affinity for ligands. In addition, antibodies raised against i53 can be conjugated to column matrices and the corresponding peptide immunopurified. All of these methods are well known in the art. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).

Chemical synthesis

[0086] The i53 peptide can also be synthesized chemically using peptide synthesis or other protocols well known in the art. i53 peptides may be synthesized by solid-phase peptide synthesis methods using procedures similar to those described by Merrifield et al, J. Am. Chem. Soc., 85:2149-2156 (1963); Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology Gross and Meienhofer (eds.), Academic Press, N.Y., vol. 2, pp. 3-284 (1980); and Stewart et al., Solid Phase Peptide Synthesis 2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). During synthesis, N-a-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C- terminal and to a solid support, i.e., polystyrene beads. The peptides are synthesized by linking an amino group of an N-a-deprotected amino acid to an a-carboxy group of an N-a- protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation. The most commonly used N-a-protecting groups include Boc, which is acid labile, and Fmoc, which is base labile.

5. Introduction of i53 into cells

[0087] The purified i53 can be introduced into cells in any of a number of ways, e.g., by electroporation, microinjection, lipofection, electroporation, nanoparticle bombardment, the use of cell-penetrating peptide (CPP) tags, and the like. In particular embodiments, the i53 peptide is introduced into cells by electroporation. In particular embodiments, the i53 is introduced by electroporation together with RNPs comprising an sgRNA and RNA-guided nuclease.

[0088] The i53 can be introduced into cells at any suitable concentration, i.e., a concentration sufficient to increase HDR in the cell and decrease NHEJ, indels, etc. The precise concentration used will depend upon the cell type, the targeted locus, the nature of genetic modification desired, and other factors known to one of skill in the art. The effect of i53 peptide is concentration dependent, and HDR in HSPCs, for example, increases in a dose dependent manner. In some embodiments, the i53 peptide is present at a concentration of from 10 μg/ml to 10 mg/ml, from 100 μg/ml to 5 mg/ml, from 250 μg/ml to 2.5 mg/ml, or at about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5 or more mg/ml. In particular embodiments, the i53 peptide is introduced at about 1.5 mg/ml.

[0089] In particular embodiments, the i53 peptide introduced into cells is transient. For example, in some embodiments, there is a reduction of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the i53 peptide in the cells as detected, e.g., by immunoblot, within 4 hours after introduction of the peptide into the cells, relative to the amount present immediately after introduction. Without being bound by the following theory, it is believed that the transient nature of purified i53 peptide in cells is advantageous in that it transiently promotes HDR upon introduction of the peptide into the cells, but does not persist long enough to promote longer-term instability in the cells.

6. Other components sgRNAs

[0090] The i53 peptides as described herein are introduced into cells in conjunction with single guide RNAs (sgRNAs). sgRNAs interact with a site-directed nuclease such as Cas9 and specifically bind to or hybridize to a target nucleic acid within the genome of a cell, such that the sgRNA and the site-directed nuclease co-localize to the target nucleic acid in the genome of the cell. The sgRNAs as used herein comprise a targeting sequence comprising homology (or complementarity) to a target DNA sequence, and a constant region that mediates binding to Cas9 or another RNA-guided nuclease. The sgRNA can target any sequence within the target gene adjacent to a PAM sequence. The sgRNAs used in the present methods and compositions can target any locus that is to be modified or edited. In some embodiments, the target gene or locus is a safe harbor locus such as CCR5 or a locus associated with a genetic disorder, such as sickle cell disease, β-thalassemia, X-linked severe combined immunodeficiency (e.g., SCID-X1), X-linked chronic granulomatous disease (X- CGD), cystic fibrosis, lysosomal storage disorders such as mucopolysaccharidosis type 1, Gaucher’s disease, or Krabbe disease, and others, and the methods are used to correct a mutated copy of the gene in a patient. A non-limiting list of genes that can be targeted or introduced using the present methods includes HBB, CYBB, CCR5, IL2RG, HBA1, HBA2, CFTR, and others.

[0091] The targeting sequence of the sgRNAs may be, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or 15-25, 18-22, or 19-21 nucleotides in length, and shares homology with a targeted genomic sequence, in particular at a position adjacent to a CRISPR PAM sequence. The sgRNA targeting sequence is designed to be homologous to the target DNA, i.e., to share the same sequence with the non-bound strand of the DNA template or to be complementary to the strand of the template DNA that is bound by the sgRNA. The homology or complementarity of the targeting sequence can be perfect (i.e., sharing 100% homology or 100% complementarity to the target DNA sequence) or the targeting sequence can be substantially homologous (i.e., having less than 100% homology or complementarity, e.g., with 1-4 mismatches with the target DNA sequence).

[0092] Each sgRNA also includes a constant region that interacts with or binds to the site- directed nuclease, e.g., Cas9. In the nucleic acid constructs provided herein, the constant region of an sgRNA can be from about 70 to 250 nucleotides in length, or about 75-100 nucleotides in length, 75-85 nucleotides in length, or about 80-90 nucleotides in length, or 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides in length. The overall length of the sgRNA can be, e.g., from about 80-300 nucleotides in length, or about 80-150 nucleotides in length, or about 80-120 nucleotides in length, or about 90-110 nucleotides in length, or, e.g, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 nucleotides in length.

[0093] It will be appreciated that it is also possible to use two-piece gRNAs (crtracrRNAs) in the present methods, i.e., with separate crRNA and tracrRNA molecules in which the target sequence is defined by the crispr RNA (crRNA), and the tracrRNA provides a binding scaffold for the Cas nuclease.

[0094] In some embodiments, e.g., when the methods are used to introduce a functional full-length cDNA to the genome, the target sequence is located near the translational start site of the gene, such that the full-length cDNA can be expressed under the control of the endogenous promoter. In other embodiments, the target sequence can be elsewhere in a gene or locus, e.g., to modify the sequence at the site of a mutation, to introduce a regulatory element, to introduce a deletion to remove protein function, to introduce an expression cassette comprising a coding sequence operably linked to a promoter, etc. It will be understood that the present methods can be used to enhance the rate of HDR for any purpose, and using sgRNAs targeting any part of a gene or genome.

[0095] In some embodiments, the sgRNAs comprise one or more modified nucleotides. For example, the polynucleotide sequences of the sgRNAs may also comprise RNA analogs, derivatives, or combinations thereof. For example, the probes can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone (e.g., phosphorothioates). In some embodiments, the sgRNAs comprise 3’ phosphorothiate intemucleotide linkages, 2’-O- methyl-3 ’-phosphoacetate modifications, 2 ’-fluoro-pyrimidines, S-constrained ethyl sugar modifications, or others, at one or more nucleotides. In particular embodiments, the sgRNAs comprise 2'-O-methyl-3'-phosphorothioate (MS) modifications at one or more nucleotides (see, e.g., Hendel et al. (2015) Nat. Biotech. 33(9):985-989, the entire disclosure of which is herein incorporated by reference). In particular embodiments, the 2'-O-methyl-3'- phosphorothioate (MS) modifications are at the three terminal nucleotides of the 5' and 3' ends of the sgRNA.

[0096] The sgRNAs can be obtained in any of a number of ways. For sgRNAs, primers can be synthesized in the laboratory using an oligo synthesizer, e.g., as sold by Applied Biosystems, Biolytic Lab Performance, Sierra Biosystems, or others. Alternatively, primers and probes with any desired sequence and/or modification can be readily ordered from any of a large number of suppliers, e.g., ThermoFisher, Biolytic, IDT, Sigma-Aldritch, GeneScript, etc.

RNA-guided nucleases

[0097] The sgRNAs are used together with an RNA-guided nuclease, e.g. a CRISPR-Cas nuclease. Any CRISPR-Cas nuclease can be used in the method, i.e., a CRISPR-Cas nuclease capable of interacting with a guide RNA and cleaving the DNA at the target site as defined by the guide RNA. In some embodiments, the nuclease is Cas9 or Cpfl. In particular embodiments, the nuclease is Cas9. The Cas9 or other nuclease used in the present methods can be from any source, so long that it is capable of binding to an sgRNA of the present disclosure and being guided to and cleaving the specific sequence targeted by the targeting sequence of the sgRNA. In particular embodiments, the Cas9 is from Streptococcus pyogenes. In some embodiments, a high fidelity Cas9 nuclease is used. [0098] Also disclosed herein are CRISPR/Cas or CRISPR/Cpfl systems that target and cleave DNA at a locus of interest. An exemplary CRISPR/Cas system comprises (a) a Cas (e.g., Cas9) or Cpfl polypeptide or a nucleic acid encoding said polypeptide, (b) an sgRNA that hybridizes specifically to the locus of interest, or a nucleic acid encoding said guide RNA, (c) a donor template as described herein, and (d) an i53 peptide. In particular embodiments, the CRISPR/Cas system comprises an RNP comprising an sgRNA targeting the locus of interest and a Cas protein such as Cas9.

[0099] In addition to the CRISPR/Cas9 platform (which is a type II CRISPR/Cas system), alternative systems exist including type I CRISPR/Cas systems, type III CRISPR/Cas systems, and type V CRISPR/Cas systems. Various CRISPR/Cas9 systems have been disclosed, including Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Campylobacter jejuni Cas9 (CjCas9) and Neisseria cinerea Cas9 (NcCas9) to name a few. Alternatives to the Cas system include the Francisella novicida Cpfl (FnCpfl), Acidaminococcus sp. Cpfl (AsCpfl), and Lachnospiraceae bacterium ND2006 Cpfl (LbCpfl) systems. Any of the above CRISPR systems may be used to induce a single or double stranded break at the locus of interest to carry out the methods disclosed herein.

Introducing the sgRNA and RNA-guided nuclease into cells

[0100] The sgRNA and nuclease can be introduced into a cell using any suitable method, e.g., by introducing one or more polynucleotides encoding the sgRNA and the nuclease into the cell, e.g., using a vector such as a viral vector or delivered as naked DNA or RNA, such that the sgRNA and nuclease are expressed in the cell. In some embodiments, one or more polynucleotides encoding the sgRNA, the nuclease or a combination thereof are included in an expression cassette. In some embodiments, the sgRNA, the nuclease, or both sgRNA and nuclease are expressed in the cell from an expression cassette. In some embodiments, the sgRNA, the nuclease, or both sgRNA and nuclease are expressed in the cell under the control of a heterologous promoter. In some embodiments, one or more polynucleotides encoding the sgRNA and the nuclease are operatively linked to a heterologous promoter. In particular embodiments, the sgRNA and nuclease are assembled into ribonucleoproteins (RNPs) prior to delivery to the cells. The RNPs can be introduced into the cell using any suitable method, e.g., microinjection, electroporation, or other chemical transfection (e.g., lipid vesicles, osmocytosis, soluporation or other permeabilization techniques, etc.) or physical transfection methods (e.g., mechanical transfection, membrane disruption or permeabilization, etc.). In particular embodiments, the RNPs are introduced into the cell by electroporation.

[0101] Techniques for insertion of transgenes, including large transgenes, capable of expressing functional proteins, including enzymes, cytokines, antibodies, and cell surface receptors are known in the art (See, e.g. Bak and Porteus, Cell Rep. 2017 Jul 18; 20(3): 750- 756 (integration of EGFR); Kanojia et al., Stem Cells. 2015 Oct;33(10):2985-94 (expression of anti-Her2 antibody); Eyquem et al., Nature. 2017 Mar 2;543(7643): 113-117 (site-specific integration of a CAR); O’Connell et al., 2010 PLoS ONE 5(8): e 12009 (expression of human IL-7); Tuszynski et al., Nat Med. 2005 May;ll(5):551-5 (expression of NGF in fibroblasts); Sessa et al., Lancet. 2016 Jul 30;388(10043):476-87 (expression of arylsulfatase A in ex vivo gene therapy to treat MLD); Rocca et al., Science Translational Medicine 25 Oct 2017: Vol. 9, Issue 413, eaaj2347 (expression of fiataxin); Bak and Porteus, Cell Reports, Vol. 20, Issue 3, 18 July 2017, Pages 750-756 (integrating large transgene cassettes into a single locus), Dever et al., Nature 17 November 2016: 539, 384-389 (adding tNGFR into hematopoietic stem cells (HSC) and HSPCs to select and enrich for modified cells); each of which is herein incorporated by reference in its entirety.

Homologous Repair Templates

[0102] The homologous repair template used in the present methods can be any template used for genomic editing purposes, e.g., to integrate a cDNA or other sequence into a corresponding endogenous locus or a safe harbor locus, to introduce a deletion, insertion, or sequence modification into a targeted genomic locus, or for any other method wherein a genomic locus is cleaved using an sgRNA and RNA-guided nuclease such as Cas9, and the cleaved sequence is modified via HDR using a homologous donor template .

[0103] In some embodiments, the methods are used to introduce a cDNA into a targeted genomic locus. For example, in some embodiments, the methods can be used to integrate a cDNA such as a functional, codon-optimized cDNA into the genome of cells of a subject with a genetic disorder caused by a deficit or absence in the protein encoded by the cDNA, or a genetic or other disorder that can be treated or ameliorated in any way by the expression of the cDNA.

[0104] In some embodiments, the cDNA is integrated, e.g., at the translational start site of the endogenous locus, such that the cDNA is expressed under the control of the endogenous promoter and other regulatory elements. In other embodiments, the template comprises a promoter, operably linked to the cDNA, e.g., when the cDNA is integrated in a safe harbor locus such as the C-C chemokine receptor type 5 (CCR5) locus. In such embodiments, any promoter that can induce expression of the therapeutic protein in the modified cells can be used, including endogenous and heterologous promoters, inducible promoters, constitutive promoters, cell-specific promoters, and others. In some embodiments, the promoter is the phosphoglycerate kinase (PGK) promoter, the spleen focus-forming virus (SFFV) promoter, or the CD68 promoter.

[0105] In some instances, in addition to the promoter, the transgene is optionally linked to one or more regulatory elements such as enhancers or post-transcriptional regulatory- sequences. For example, one can include regulatory sequences (microRNA (miRNA) target sites) in the RNA to avoid expression in certain tissues (post-transcriptional targeting). In some instances, the expression control sequence functions to express the therapeutic transgene following the same expression pattern as in normal individuals (physiological expression) (See Toscano et al., Gene Therapy (2011) 18, 117-127 (2011), incorporated herein by reference in its entirety for its references to promoters and regulatory sequences).

[0106] In some embodiments, the cDNA in the homologous repair template is codon- optimized, e.g., comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or more homology to the wild-type cDNA sequence, or to a fiagment thereof.

[0107] In particular embodiments, the template further comprises a polyA sequence or signal, e.g., a bovine growth hormone polyA sequence or a rabbit beta-globin polyA sequence, at the 3’ end of the cDNA. In particular embodiments, a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) is included within the 3’UTR of the template, e.g., between the 3’ end of the cDNA coding sequence and the 5’ end of the polyA sequence, so as to increase the expression of the cDNA. Any suitable WPRE sequence can be used; See, e.g., Zufferey et al. (1999) J. Virol. 73(4):2886-2892; Donello, et al. (1998). J Virol 72: 5085-5092; Loeb, et al. (1999). Hum Gene Ther 10: 2295-2305; the entire disclosures of which are herein incorporated by reference).

[0108] In particular embodiments, the cDNA (or cDNA and polyA signal) is flanked in the template by homology regions corresponding to the targeted locus. For example, an exemplary template can comprise, in linear order: a first genomic homology region, an optional promoter, a cDNA, a polyA sequence, and a second genomic homology region, where the first and second homology regions are homologous to the genomic sequences extending in either direction from the sgRNA target site. The homology regions can be of any size, e.g., 100-1000 bp, 300-800 bp, 400-600 bp, or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more base pairs.

Introduction of donor templates into cells

[0109] Any suitable method can be used to introduce the polynucleotide, or donor construct, into the primary cells. In particular embodiments, the polynucleotide is introduced using a recombinant adeno-associated viral vector, e.g., rAAV6. In some instances, the donor template is single stranded, double stranded, a plasmid or a DNA fragment. In some instances, plasmids comprise elements necessary for replication, including a promoter and optionally a 3’ UTR.

[0110] Further disclosed herein are vectors comprising (a) one or more nucleotide sequences homologous to the locus of interest, and (b) a cDNA as described herein. The vector can be a viral vector, such as a retroviral, lentiviral (both integration competent and integration defective lentiviral vectors), adenoviral, adeno-associated viral or herpes simplex viral vector. Viral vectors may further comprise genes necessary for replication of the viral vector.

[0111] In some embodiments, the targeting construct comprises: (1) a viral vector backbone, e.g. an AAV backbone, to generate virus; (2) arms of homology to the target site of at least 200 bp but ideally at least 400 bp on each side to assure high levels of reproducible targeting to the site (see, Porteus, Annual Review of Pharmacology and Toxicology, Vol. 56:163-190 (2016); which is hereby incorporated by reference in its entirety); (3) a cDNA encoding a functional protein and capable of expressing the functional protein, optionally a promoter, a polyA sequence, and optionally a WPRE element; and optionally (4) an additional marker gene to allow for enrichment and/or monitoring of the modified host cells. Any AAV known in the art can be used. In some embodiments the primary AAV serotype is AAV6. In some embodiments, the vector, e.g., rAAV6 vector, comprising the donor template is from about 1 -2 kb, 2-3 kb, 3-4 kb, 4-5 kb, 5-6 kb, 6-7 kb, 7-8 kb, or larger.

[0112] In some embodiments, viral vectors, e.g., AAV6 vector, is transduced at a multiplicity of infection (MOI) of, e.g., about 1x10 ³ , 5x10 ³ , 1x10 ⁴ , 5x10 ⁴ , 1x10 ⁵ , between 2x10 ⁴ and 1x10 ⁵ viruses per cell, or less than 1x10 ⁵ . In particular embodiments, the viral vector is introduced at an MOI of less than about 2500, e.g., about 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 850, 800, 750, 700, 675, 650, 625, 600, 550, 500, 450, 400, or less. In particular embodiments, the viral vector is introduced at an MOI of about 625 in the presence of the i53 peptide. In some embodiments, the viral vector is administered in the presence of the i53 peptide at an MOI that is 1-fold, 2-fold, 3-fold, 4-fold, or more lower than a standard or recommended MOI in the absence of the i53 peptide.

[0113] Suitable marker genes are known in the art and include Myc, HA, FLAG, GFP, truncated NGFR, truncated EGFR, truncated CD20, truncated CD 19, as well as antibiotic resistance genes. In some embodiments, the homologous repair template and/or vector (e.g., AAV6) comprises an expression cassette comprising a coding sequence for truncated nerve growth factor receptor (tNGFR), operably linked to a promoter such as the Ubiquitin C promoter.

[0114] The inserted construct can also include other safety switches, such as a standard suicide gene into the locus (e.g. iCasp9) in circumstances where rapid removal of cells might be required due to acute toxicity. The present disclosure provides a robust safety switch so that any engineered cell transplanted into a body can be eliminated, e.g.. by removal of an auxotrophic factor. This is especially important if the engineered cell has transformed into a cancerous cell.

[0115] The present methods allow for the efficient integration of the donor template at the endogenous locus of interest. In some embodiments, the present methods allow for the insertion of the donor template in 20%, 25%, 30%, 35%, 40%, or more cells, e.g., cells fiom an individual with a condition to be treated using the present methods and/or compositions. The methods also allow for high levels of expression of protein in cells, e.g., cells fiom an individual with an integrated cDNA as described herein, e.g., levels of expression that are at least about 70%, 75%, 80%, 85%, 90%, 95%, or more relative to the expression in healthy control cells.

Cells

[0116] Animal cells, mammalian cells, preferably human cells, modified ex vivo, in vitro, or in vivo are contemplated. Also included are cells of other primates; mammals, including commercially relevant mammals, such as cattle, pigs, horses, sheep, cats, dogs, mice, rats; birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys. In particular embodiments, the cells are human cells, e.g., human cells from a subject with a genetic disorder or condition. [0117] In particular embodiments, the cells used in the present methods are primary cells, i.e., cells taken directly from a living tissue (e.g., biopsy, blood sample, etc.). In some embodiments, the cell is an embryonic stem cell, a stem cell, a progenitor cell, a pluripotent stem cell, an induced pluripotent stem cell (iPSC), a somatic stem cell, a differentiated cell, a mesenchymal stem cell or a mesenchymal stromal cell, an airway basal stem cell, a neural stem cell, a hematopoietic stem cell or a hematopoietic progenitor cell, an adipose stem cell, a keratinocyte, a skeletal stem cell, a muscle stem cell, a fibroblast, an NK cell, a B-cell, a T cell, or a peripheral blood mononuclear cell (PBMC). In particular embodiments, the cells are CD34 ⁺ hematopoietic stem and progenitor cells (HSPCs), e.g., cord blood-derived (CB), adult peripheral blood-derived (PB), or bone marrow derived HSPCs. HSPCs can be isolated from a subject, e.g., by collecting mobilized peripheral blood and then enriching the HSPCs using the CD34 marker.

[0118] To avoid immune rejection of the modified cells when administered to a subject, the cells to be modified are preferably derived from the subject’s own cells. Thus, preferably the mammalian cells are autologous cells from the subject to be treated with the modified cells. In some embodiments, however, the cells are allogeneic, i.e., isolated from an HLA-matched or HLA-compatible, or otherwise suitable, donor.

[0119] In some embodiments, cells are harvested from the subject and modified according to the methods disclosed herein, which can include selecting certain cell types, optionally expanding the cells and optionally culturing the cells, and which can additionally include selecting cells that contain a transgene integrated into the targeted locus. In particular embodiments, such modified cells are then reintroduced into the subject.

[0120] Further disclosed herein are methods of using said nuclease systems to produce the modified host cells described herein, comprising introducing into a mammalian cell: (a) an RNP comprising a Cas nuclease such as Cas9 and an sgRNA specific to a locus of interest, (b) an i53 peptide, and (c) a homologous donor template or vector as described herein. Each component can be introduced into the cell directly or can be expressed in the cell by introducing a nucleic acid encoding the components of said one or more nuclease systems.

[0121] In some embodiments, the present methods target integration of a functional cDNA at the corresponding endogenous locus or at a safe harbor locus in a host cell ex vivo. In some embodiments, the methods target the modification of a genomic sequence, e.g., the alteration of a genomic sequence, or the introduction of a deletion or insertion, at an endogenous locus. Such methods can further comprise (a) optionally expanding said cells, and/or (b) optionally culturing the cells.

[0122] In any of these methods, the nuclease can produce one or more single stranded breaks within the locus of interest, or a double stranded break within the locus of interest. In these methods, the locus is modified by homologous recombination with said donor template or vector to result in insertion of the transgene into the locus. The methods can further comprise (c) selecting cells that contain the transgene integrated into the locus of interest.

7. Detecting i53 peptide activity

[0123] The activity of i53 peptide and/or the efficacy of the present methods can be assessed in any of a number of ways. For example, the activity of i53 peptide can be assessed by measuring the rate of HDR in cells such as CD34 ⁺ HSPCs, e.g., the rate of integration of a cDNA at genomic loci such as HBB, CCR5, 1L2RG, HBA1, or CFTR when an i53 peptide is introduced together with an sgRNA, RNA-guided nuclease, and homologous donor template. In some embodiments, the rate of HDR in such cells is increased by at least about 10%, 20%, 30%, 40%, 50%, or more relative to the rate in equivalent cells but in the absence of i53. In some embodiments, the activity of i53 peptide can be assessed by measuring the rate of NHEJ or indels in cells such as CD34 ⁺ HSPCs. In some embodiments, the rate of indels is decreased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more relative to the rate in equivalent cells in the absence of i53 peptide. In some embodiments, the activity of i53 peptide is assessed by determining the MOI for a viral vector comprising a homologous donor template that is required to achieve a given level of HDR For example, in some embodiments, the presence of i53 can allow a decrease in the MOI used of, e.g., 1-fold, 2- fold, 3-fold, 4-fold, or more, while still maintaining similar rates of HDR as compared to in an equivalent cell in the absence of i53. In some embodiments, the activity of i53 peptide can be assessed by determining, e.g., the ability of modified cells to achieve a given rate of engrafhnent in animal models. For example, the presence of i53 can allow the use of an MOI that is, e.g., 1-fold, 2-fold, 3-fold, 4-fold, or more lower than the MOI needed in the absence of i53 peptide, to achieve a given rate of engrafhnent. The activity of i53 can also be assessed in cells by examining, e.g., the activity of 53BP1, such as the binding of 53BP1 to the ends of double-stranded DNA breaks. 8. Methods of treatment

[0124] In some embodiments, following the modification of the genome in cells from a subject using the herein-described methods, and, e.g., confirming expression of a protein encoded by an introduced cDNA, a plurality of modified cells can be reintroduced into the subject, such that they can repopulate and differentiate, and due to the expression of the integrated cDNA (or other genetic modification), can improve one or more abnormalities or symptoms in the subject with the genetic disorder. In some embodiments, the cells are expanded, selected, and/or induced to undergo differentiation, prior to reintroduction into the subject.

[0125] Disclosed herein, in some embodiments, are methods, including therapeutic methods and methods of administration. Although the descriptions of methods provided herein are principally directed to administration to humans, it will be understood by the skilled artisan that they are generally suitable for administration to any animals.

[0126] The modified host cells of the present disclosure included in the pharmaceutical compositions described above may be administered by any delivery route, systemic delivery or local delivery, which results in a therapeutically effective outcome. These include, but are not limited to, enteral, gastroenteral, epidural, oral, transdermal, intracerebral, intracerebroventricular, epicutaneous, intradermal, subcutaneous, nasal, intravenous, intra- arterial, intramuscular, intracardiac, intraosseous, intrathecal, intraparenchymal, intraperitoneal, intravesical, intravitreal, intracavemous), interstitial, intra-abdominal, intralymphatic, intramedullary, intrapulmonary, intraspinal, intrasynovial, intrathecal, intratubular, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, soft tissue, and topical. In particular embodiments, the cells are administered intravenously.

[0127] In some embodiments, a subject will undergo a conditioning regime before cell transplantation. For example, before hematopoietic stem cell transplantation, a subject may undergo myeloablative therapy, non-myeloablative therapy or reduced intensity conditioning to prevent rejection of the stem cell transplant even if the stem cell originated from the same subject. The conditioning regime may involve administration of cytotoxic agents. The conditioning regime may also include immunosuppression, antibodies, and irradiation. Other possible conditioning regimens include antibody-mediated conditioning (see, e.g., Czechowicz et al., 318(5854) Science 1296-9 (2007); Palchaudari et al., 34(7) Nature Biotechnology 738-745 (2016); Chhabra et al, 10:8(351) Science Translational Medicine 351ral05 (2016)) and CAR T-mediated conditioning (see, e.g., Arai et al., 26(5) Molecular Therapy 1181-1197 (2018); each of which is hereby incorporated by reference in its entirety). For example, conditioning needs to be used to create space in the brain for microglia derived from engineered hematopoietic stem cells (HSCs) to migrate in to deliver the protein of interest (as in recent gene therapy trials for ALD and MLD). The conditioning regimen is also designed to create niche “space” to allow the transplanted cells to have a place in the body to engraft and proliferate. In HSC transplantation, for example, the conditioning regimen creates niche space in the bone marrow for the transplanted HSCs to engraft. Without a conditioning regimen, the transplanted HSCs cannot engraft.

[0128] The present disclosure additionally provides methods of administering modified host cells in accordance with the disclosure to a subject in need thereof. Pharmaceutical compositions including the modified host cell may be administered to a subject using any amount and any route of administration effective for preventing, treating, or managing the condition in question. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The subject may be a human, a mammal, or an animal. The specific therapeutically or prophylactically effective dose level for any particular individual will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific payload employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration; the duration of the treatment; drugs used in combination or coincidental with the specific modified host cell employed; and like factors well known in the medical arts.

[0129] In certain embodiments, modified host cell pharmaceutical compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from, e.g., about 1 x 10 ⁴ to 1 x 10 ⁵ , 1 x 10 ⁵ to 1 x 10 ⁶ , 1 x 10 ⁶ to 1 x 10 ⁷ , or more modified cells to the subject, or any amount sufficient to obtain the desired therapeutic or prophylactic, effect. The desired dosage of the modified host cells of the present disclosure may be administered one time or multiple times. In some embodiments, delivery of the modified host cell to a subject provides a therapeutic effect for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years.

[0130] The modified host cells may be used in combination with one or more other therapeutic, prophylactic, research or diagnostic agents, or medical procedures, either sequentially or concurrently. In general, each agent w-ill be administered at a dose and/or on a time schedule determined forthat agent.

9. Examples

[0131] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the disclosure in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

Example 1. Transient Inhibition of 53BP1 Safely Increases the Frequency of Genome Editing by Homologous Recombination in Primary Human Cells

[0132] In this Example, we discovered that delivery- of i53 recombinant peptide is an effective method of increasing the frequency of HDR genome editing at a variety of loci in human HSPCs and also increases HDR in a variety- of therapeutically relevant human primary- cell types including T-cells, MSCs, airway stem cells (basal cells) and IPSCs. The inhibition is transient because the peptide is rapidly degraded and thus this approach could become an important method to increase the frequency- of HDR for therapeutic purposes. Moreover, i53 peptide also reduces the formation of INDELs and enables the multiplicity of infection (MOI) of AAV6 needed to achieve high frequencies of gene targeting to be significantly reduced (4- fold).

Results

25 Reproducibility of Gene Targeting in Different CD 34' Donors Across Different CD34 ⁺ Sub- Populations

[0133] We used the previously described gene targeting system combining RNP electroporation with AAV6 transduction to modify exon 1 of the HBB gene by HDR in CD34 ⁺ hematopoietic stem and progenitor cells (HSPCs) and tested the efficiency of the system across different CD34 ⁺ sub-populations and from different CD34 ⁺ donors (3, 12, 13). We found that the highest levels of gene targeting occurred in bulk CD34 ⁺ HSPCs (median=75.4%) (FIG. 1A). The frequency of gene targeting was highly reproducible across four different donors with a range of 67.7-77.8% (FIG. 1A). To determine the frequency of gene targeting in different CD34 ⁺ sub-populations, we sorted CD34 ⁺ cells based on previously defined cell surface markers (CD34, CD38, CD90, and CD45RA) for progenitor cells (CD38 ⁺ ; CD34 ⁺ and CD38 ⁺ ), multi-potent progenitor cells (MPP; CD34*, CD38-, CD90-, CD45RA-), hematopoietic stem cells (HSCs; CD34 ⁺ , CD38-, CD90 ⁺ , CD45RA-) and long-term multi-potent progenitor cells (LMPP, CD34 ⁺ , CD38-, CD90-, CD45RA ⁺ ). We found that across all of the CD34 ⁺ sub-populations the frequency of gene targeting remained high (>50% allele correction) but was slightly decreased in the more primitive cells (HSCs (median= 60.1%; range= 46.2-64.3% ) and LMPP’s (median= 59.1%; range= 43.5-72.9% ) (FIG. 1A). We also discovered that while there was variability between different donors in each sub-population, across all donors the frequency of gene targeting remained high (FIG. IB). This data demonstrates the robustness of the system in generating high frequencies of gene targeting at the HBB locus across a range of different donors and across a variety of CD34 ⁺ sub-populations, including those that have been characterized as the most primitive. i53 recombinant protein is efficient at improving HDR in human primary cells.

[0134] It has been previously reported in cancer cell lines that HDR could be increased by inhibiting 53BP1 using plasmid transfection of inhibitory peptide (11). The rationale was that this would block NHEJ based repair and bias cells to repair using the HDR pathway (FIG. 2A). Plasmid transfection, however, is not tolerated in primary human cells because of the activation of the Type I interferon response from naked cytosolic DNA sensing. In addition, the prolonged inhibition of 53BP1 might cause genomic instability that would counter- balance any potential increase in HDR frequency. It is known, for example, that mice with knockouts of 53BP1 are cancer prone, including lymphomas derived from the hematopoietic system. We tested, therefore, whether delivering the i53 molecule as a purified peptide, which could bind an inhibit 53BP1, could increase HDR in primary human HSPCs. We reasoned that hypothesized that i53 would be effective when the Cas9 RNP induced DSBs were formed but would not be needed at later time points. We tested gene targeting at four different loci (ABB, CCR5, IL2RG and HBA1) (3, 13, 14) in CD34 ⁺ HSPCs from either cord blood or plerixafor mobilized healthy donor cells. The cells were electroporated with Cas9 RNP using previously described chemically modified sgRNAs (15) either with or without i53 recombinant protein and transduced with adeno-associated virus type 6 (AAV6) homologous donor templates (5). For four different clinically relevant loci targeted (HBB, CCR5, Il2RG, and HBA1), we observed an increase (25-33% increase over without peptide) in HDR frequencies (FIG. 2B). Simultaneously as an additional measurement of DSB repair, we investigated the capacity of i53 peptide to suppress NHEJ in HSPCs. We found that the indels were efficiently inhibited in the targeted cells with the use of i53 peptide (69.0% decrease for HBB, 20.6% decrease for CCR5 and 15.3% decrease for lL2RG) (FIG. 2C). Because HBA1 is essentially identical to HBA2 for measuring indels, we were unable to measure the specific indel formation frequency at that locus.

[0135] The HBB gene with the R2 guide is a good candidate to extend the evaluation for the efficacy and safety of using i53 recombinant peptide because the HBB gene is of immediate clinical relevance. It is also a gene target in which increasing HDR and decreasing indels could both have clinical benefit. After induction of DSBs by Cas9 RNP, we observed that CD34 ⁺ cells take 24 hours to repair 50% of DSBs by NHEJ and 48 hours for 80% completion and up to 72 hours for full completion (FIG. 2E). Therefore, in order to have i53 recombinant peptide present in the cells at the time of inducing the DSBs, we transiently delivered the i53 peptide at the time of Cas9 RNP delivery into HSPCs (FIG. 2F). Interestingly for these cells undergoing DNA repair without a homologous repair template, no difference was found in the kinetics and total frequencies of 1NDEL formations between the cells treated with or without i53 peptide. The cells treated with i53 peptide also took 24 hours to repair 50% of DSBs and 72 hours for full completion, and the total indel frequencies were at 72.0% for RNP only and 69.5% for RNP + i53 peptide treated cells. However, through the comparison of the Sanger sequencing traces analyzed by ICE, we noted that the indel spectrum for cells with or without i53 peptide were noticeably different. In the absence of i53 peptide, 25.5% of the indels were insertions or deletions of sizes equal to less than 7bp in lengths, which we call NHEJ, and 46.5% of the indels were greater than 7bp, which we called as MMEJ. With the i53 peptide treatment, NHEJ was decreased to 7.5% with a resultant compensatory increase in MMEJ to 62% (FIG. 2F).

Optimizing the use of i53 peptide in CD34 ⁺ HSPCs

[0136] The effect of i53 peptide is concentration dependent as HDR in HSPCs increased in a dose dependent manner: ~10% increase at 250 μg/ml; 46% increase at 1500 μg/ml by approximately 46% and 38% increase at 5000 μg/ml (FIG. 5A). i53 peptide concentrations up to 5000 μg/ml did not result in notable decrease in viability of HSPCs and dosage of i53 peptide beyond 1500 μg/ml did not result in any additional improvement in HDR and we therefore used 1500 μg/ml of peptide in subsequent experiments unless noted otherwise.

[0137] We have described in our previous studies that gene-specific AAV6 donor MOIs in the range of 2500 to 10,000 can be used in order to achieve clinically relevant and high levels of HDR in HSPCs. It has been described that using AAV6 can impact cell fitness, affecting both cell viability and stem cell function. To determine the extent to which AAV6 impairs colony forming potential of progenitors cells, we conducted a colony-forming unit (CFU) assay using HSPCs transduced with a range of MOIs of AAV6 fiom 625 to 5000. HSPCs transduced with AAV6 were plated to semisolid methylcellulose media, which supports the growth of multiple progenitor cells (myeloid: CFU-GM; erythroid: BFU-E and CFU-E; and mixed myeloid and erythroid: CFU-GEMM). After 14 days of plating, we observed that only approximately 42.7% of HSPCs transduced with 5000 MOI of AAV6 are capable of forming colonies but with lower MOIs of 2500, 1250, and 625, approximately 65.6%, 85.0% and 90.5% of HSPCs, respectively, were able to form colonies (FIG. 5C). Therefore, we used i53 peptide to determine if it could facilitate using lower MOI’s without compromising the high frequencies of HDR in HSPCs. We determined the levels of HDR using RNP and a variety- of AAV6 MOIs ranging fiom 625 to 5000 in cells treated with or without i53 peptide and found that we could significantly enhance HDR while reducing the amount of AAV6 donors by 8 fold (67.1% HDR by 625 MOI vs 64.9% HDR by 5000 MOI) in CD34+ HSPCs (FIG. 4B). As we drastically improved HDR in a AAV6 dose-dependent manner from 67.1% to 86.2% using MOIs from 625 to 5000 and we also correspondingly inhibited indel formations (FIG. 4C). Furthermore, the CFU assays conducted using edited HSPCs with RNP and 625 MOI AAV6 or 2500 MOI AAV6 suggest that we can achieve higher HDR (~70%) with 625 MOI and i53 peptide without decreasing the colony forming potential of the edited cells (FIGS. 4D, 4E).

Increasing Bi-Allelic Gene Targeting using i53

[0138] To determine if i53 could increase the frequency of bi-allelic gene targeting, we targeted the HBB gene with R2 guide and simultaneously provided GFP and mCherry expressing AAV 6 donors (16). Three days after electroporation and transduction, 13.3% of cells were double positive for GFP and mCherry without i53. The frequency increased to 18.8% (an increase of 41%) using i53. These results demonstrate that i53 can increase the frequency of cells that have undergone bi-allelic homologous recombination editing (FIG. 4G). i53 Peptide is Only Transiently Present in CD34~ HSPCs Following Electroporation

[0139] When regulation of DNA repair pathways fails (53BP1), translocations and other genome rearrangements can result, diminishing cell viability and increasing the chance of tumorigenic changes. To determine if the duration of inhibition of 53BP1 plays a role in this, we investigated the kinetics of the inhibitor in HSPCs. Following the electroporation of HSPCs with RNP + AAV6 and i53 peptide, we collected cells at one-hour intervals from 0 to 4 hours and at 24 hours and 48 hours. The i53 protein is tagged at its N-terminus with a His- tag and we measured the amount of i53 by Western blotting by probing with an anti-His tag antibody. We found that the peptide is rapidly degraded and by 4 hours is at 0.09% of the hour 0 levels, thus decreasing concerns about the potential long-term genotoxicity coming from 53BP1 inhibition by i53 peptide (FIGS. 6A, 7A-7B). After confirming that i53 peptide is transiently present, we assessed its impact on the kinetics of both HDR and INDELS. CD34 ⁺ HSPCs were edited with Cas9 RNP and AAV6 donor with or without i53 peptide. Cells were collected at different time points (0, 2, 4, 6, 8, 24, 48, and 72 hours) over 72 hours. ddPCR analyses of the samples show that indel formation is suppressed by i53 peptide for at least 24 hours allowing DNA repair pathway to be biased towards HDR in the presence of AAV donor. The i53 peptide treated cells fully completed HDR by 48 hours post-editing whereas with no treatment the cells were at 80% completion with a lower average HDR rate (FIG. 6B).

[0140] One of the positive features of using i53 peptide is to reduce the amount of AAV loaded onto the cells without compromising HDR frequency. Upon editing of HSPCs with RNP and AAV transduction, we found that using higher amounts of AAV (MOI of 5000) induces higher p21 expression while the lower amounts of AAV (MOI of 625) results in lower p21 expression, in a AAV-dose dependent manner (FIGS. 6D-6E). Strong p53 transcriptional response, resulting in p21 upregulation triggered by AAV transduction during genome editing has previously been reported (17) and our findings showing dose responses of p21 upregulation to various AAV doses further corroborate the finding that the use of AAV during gene editing can trigger a p53 transcriptional response. Through the use of i53 peptide while lowering the AAV MOI down to 625, we can achieve similar rates of HDR (56% for 625 MOI vs 59% for 5000), while reducing p21 upregulation (FIG. 6F). The reduction of the p21 response would be predicted to result in a higher quality HSPC cell product.

[0141] To assess whether transient inhibition of 53BP1 changes the frequency of translocations, we used a combination of two sgRNAs to target HSPCs at HBB and AAVS1 and quantified the enrichment of oncogenic translocations using droplet digital PCR (ddPCR) (FIG. 6G), as previously described (6). The mean translocation frequency of 0.45% and 0.48% for cells treated with or without i53, suggesting that transient i53 exposure does not result in an increase in translocation.

Transient inhibition of 53BP1 Results in Improved Engraftment of Gene Edited Cells

[0142] The CFU assay gives a measure of HSPC function. To evaluate potential HSPC and HSC function we performed transplantation experiments in NSG mice (FIG. 8A). We evaluated the efficiency of engrafting HSPCs targeted with low and high MOIs (625 MOI for low vs 2500 MOI for high) of AAV6 with i53 peptide, treatment. We gene edited and then transplanted frozen mobilized peripheral blood (mPB) CD34 ⁺ HSPCs derived from two healthy donors (donor A and donor B). For cells obtained from donor A, we observed in vitro HDR frequencies of 13.7% (625 MOI), 31.8% (625 MOI + i53), 29.0% (2500 MOI), and 67.8% (2500 MOI + i53), and for donor B, HDR fiequencies of 18.4% (625 MOI), 44.5% (625 MOI + i53), 38.4% (2500 MOI), and 51.6% (2500 MOI + i53), as measured by digital droplet PCR (ddPCR) (FIG. 8B). After confirming that the percentages of HDR-edited cells were consistently higher among HSPCs edited with 625 MOI AAV and i53 peptide in comparison to 2500 MOI AAV, the gene-edited cells were transplanted by retro-orbital injection into sublethally irradiated adult immunodeficient non-obese diabetic (NOD)-severe combined immunodeficiency (SCJD)-Il2Rg ^-/- (NSG) recipient mice (1 x 10 ⁵ cells per mouse, n=2 mPB donors). Upon analysis of the NSG mice 14 weeks post-engraftment, we observed human multi-lineage engraftment measured by the presence of HLA-ABC positive cells in the bone marrow of all transplanted mice for all six groups (Mock, Mock + i53, 625, 625+ i53, 2500, 2500 + i53) (FIG. 8C). The CD34 ⁺ HSPC donor variability resulted in profound differences in human chimaerism levels between the two donors: donor A (4.6% to 25.8%) and donor B (6.0% to 55.1%) and these differences can be explained by transplantation of few total cells (1 x 10 ⁵ cells per mouse) and fewer phenotypically identified long-term HSCs. While we observed a particularly lower human chimaerism for donor A than donor B, we found a notable benefit in both donors from reducing the AAV load down to 625 MOI as displayed by similar or high levels of human chimaerism compared to mock treated groups. Using a lower AAV load of 625 MOI results in reduction of in vitro HDR rates in comparison to high AAV MOI of 2500, albeit no significant differences were observed in HDR rates in vivo upon analysis of human cells in the bone marrow of NSG mice. Interestingly, among the four groups tested, we found the highest median rates of HDR in vivo in the 625 MOI + i53 peptide groups of 19.1% for donor A and 49.1% for donor B (FIG. 8E). Collectively, these findings highlight the major advantages of using combination of i53 peptide and low AAV load in improving HDR rates both in vitro and in vivo while preserving HSC engrafhnent potential during genome editing.

Discussion

[0143] Towards the goal of harnessing genome editing through Cas9/sgRNA and AAV transduction to develop a potentially curative therapy for various monogenic blood disorders, it is now well established that CD34 ⁺ HSPCs with engrafting and repopulation potentials can be genome-modified. However, many challenges remain as LT-HSCs are particularly more resistant to HDR-mediated editing and thereby require more rigorous conditioning during gene editing including using higher AAV load and improved culture conditions. We have shown that increasing the amount of AAV loaded (i.e., using high MOI of AAV) onto HSCs leads to improvement of the overall HDR rate in cells in vitro. This strategy comes at the expense of edited HSPCs having reduced reconstitution potential in NSG mice, and we and others have alluded to this effect from mainly AAV transduction, which triggers strong p53 transcriptional response including p21 upregulation. To mitigate this effect, we proposed the use of short-duration i53 peptide which can be expressed rapidly in the cells to improve HDR while simultaneously using significantly less amount of AAV. i53 in the form of protein can easily be delivered transiently into the cells, where it binds to 53BP1 and suppresses 53BP1 accumulation to DSBs. The suppression of 53BP1 by i53 peptide allows the cells to bias the cell repair machinery towards HDR over NHEJ during DNA repair given that AAV donors are available to the cells.

[0144] 53BP1, a DNA damage response sensor, has been shown to accumulate at site of DSBs within 24 hours and the percentage of 53BP1 -positive cells correlate well with the fraction of NHEJ measured at targeted locus (17). Therefore, we believe that the timing and the rapid expression of i53 in the cells during genome editing to be highly critical and have shown that the delivery of i53 recombinant peptide is an effective method of increasing the frequency of HDR genome editing. We have also observe that i53 peptide works more efficiently in certain primary cell types such as airway stem cells. However, the biology and mechanisms to explain this observation is currently unknown. One of our hypothesis is that the positive correlation between 53BP1 foci-bearing cells and of modified targeted alleles by NHEJ suggests that i53 peptide may work more efficiently for cells or loci that bears higher levels of 53BP1 upon induction of DSBs.

[0145] The majority of i53 peptide quickly gets degraded by 4 hours post-electroporation, suggesting that 53BP1 suppression is only transient and therefore safe to use. However, further investigation to assess the long-term safety of using i53 peptide to transiently inhibit 53BP1 in HSCs is needed. While the His-tag on N-terminus of i53 protein does not seem to affect its binding properties, our goal is to remove the His-tag in order to incorporate into our clinical efforts.

[0146] In conclusion, we suggest a modified approach to using less stringent conditions of AAV transduction during genome editing while using i53 recombinant peptide to improve HDR in HSCs. Our data suggest that our approach results in enrichment of targeted cells in the transplanted population of cells in NSG mice, and can thereby ameliorate the problem of inefficient HSC targeting.

References

1. Porteus, M. H. A New Class of Medicines through DNA Editing. N. Engl. J. Med. 380, 947-959 (2019).

2. Yeh, C. D., Richardson, C. D. & Com, J. E. Advances in genome editing through control of DNA repair pathways. Nat. Cell Biol. 21, 1468-1478 (2019).

3. Dever, D. P. et al. CRISPR/Cas9 p-globin gene targeting in human haematopoietic stem cells. Nature 539, 384-389 (2016).

4. Pavel-Dinu, M. et al. Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nat. Commun. 10, 1634 (2019).

5. Bak, R. O., Dever, D. P. & Porteus, M. H. CRISPR/Cas9 genome editing in human hematopoietic stem cells. Nat. Protoc. 13, 358-376 (2018).

6. Bak, R. O. et al. Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6. Elife 6, (2017). 7. DeWitt, M. A. et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci. Transl. Med. 8, 360ral34 (2016).

8. Genovese, P. et al. Targeted genome editing in human repopulating haematopoietic stem cells. Nature 510, 235-240 (2014).

9. Wang, J. et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat. Biotechnol. 33, 1256-1263 (2015).

10. Escribano-Diaz, C. et al. A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCAl-CtIP controls DNA repair pathway choice. Mol. Cell 49, 872-883 (2013).

11. Canny, M. D. et al. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat. Biotechnol. 36, 95-102 (2018).

12. Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216-1224 (2018).

13. Charlesworth, C. T. et al. Priming human repopulating hematopoietic stem and progenitor cells for cas9/sgmagene targeting. Mol. Ther. Nucleic Acids 12, 89-104 (2018).

14. Damian, M. & Porteus, M. H. A crisper look at genome editing: RNA-guided genome modification. Mol. Ther. 21, 720-722 (2013).

15. Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985-989 (2015).

16. Bak, R. O. & Porteus, M. H. CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors. Cell Rep. 20, 750-756 (2017).

17. Schiroli, G. et al. Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response. Cell Stem Cell 24, 551-565 ,e8 (2019).

[0147] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

EXEMPLARY EMBODIMENTS

[0148] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:

1. A method of genetically modifying a primary human cell, the method comprising:

(i) introducing into the cell an RNA-guided nuclease and a single guide RNA (sgRNA) targeting a genetic locus of interest;

(ii) introducing a homologous donor template into the cell, wherein the homologous donor template comprises a nucleotide sequence that is homologous to the locus of interest; and

(iii) introducing a purified i53 peptide into the cell; wherein the sgRNA directs the RNA-guided nuclease to the locus of interest, the RNA-guided nuclease cleaves the locus at the target sequence of the sgRNA, and the homologous donor template is integrated at the site of the cleaved locus by homology directed repair (HDR).

2. The method of embodiment 1, wherein the primary human cell is a cell selected from the group consisting of a CD34+ hematopoietic stem and progenitor cell (HSPC), a T cell, a mesenchymal stem cell (MSC), an airway basal stem cell, and an induced pluripotent stem cell (IPSC).

3. The method of embodiment 1 or 2, wherein the locus of interest is a gene selected from the group consisting of Hemoglobin Subunit Beta (HBB), C-C Motif Chemokine Receptor 5 (CCR5), Interleukin 2 Receptor Subunit Gamma (IL2RG), Hemoglobin Subunit Alpha 1 (HBA1), and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

4. The method of any one of embodiments 1 to 3, wherein the sgRNA comprises 2'-O-methyl-3'-phosphorothioate (MS) modifications at one or more nucleotides.

5. The method of embodiment 4, wherein the 2'-O-methyl-3'- phosphorothioate (MS) modifications are present at the three terminal nucleotides of the 5' and 3' ends. 6. The method of any one of embodiments 1 to 5, wherein the RNA- guided nuclease is Cas9.

7. The method of any one of embodiments 1 to 6, wherein the sgRNA and RNA-guided nuclease are introduced into the cell as a ribonucleoprotein (RNP).

8. The method of embodiment 7, wherein the RNP is introduced into the cell by electroporation.

9. The method of any one of embodiments 1 to 8, wherein the i53 peptide is introduced into the cell by electroporation.

10. The method of embodiment 9, wherein the i53 peptide and the RNP are introduced together into the cell.

11. The method of embodiment 9 or 10, wherein the level of the i53 peptide in the cell four hours after electroporation is less than 0.1% of the level in the cell immediately after electroporation.

12. The method of any one of embodiments 1 to 11, wherein the amino acid sequence of the i53 peptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1 or SEQ ID NO:2.

13. The method of embodiment 12, wherein the i53 peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2.

14. The method of any one of embodiments 1 to 13, wherein the i53 peptide is recombinant.

15. The method of any one of embodiments 1 to 14, wherein the homologous repair template is introduced into the cell using an adeno-associated virus serotype 6 (AAV6) vector.

16. The method of embodiment 15, wherein the AAV vector is transduced into the cell at a multiplicity of infection (MOI) of less than about 2500, 1250, or 625.

17. The method of embodiment 16, wherein the MOI is about 625. 18. The method of any one of embodiments 1 to 17, wherein the concentration of the i53 peptide used for electroporation is about 1-2 mg/ml.

19. The method of embodiment 18, wherein the concentration of the i53 peptide is about 1.5 mg/ml.

20. The method of any one of embodiments 1 to 19, wherein the frequency of HDR at the locus of interest in the cell is higher than the frequency in an equivalent cell in the presence of the sgRNA, RNA-guided nuclease, and homologous donor template, but in the absence of the i53 peptide.

21. The method of embodiment 20, wherein the frequency of HDR at the locus of interest in the cell is at least about 10%, 20%, 30%, 40%, or more higher than the frequency in an equivalent cell in the presence of the sgRNA, RNA-guided nuclease, and homologous donor template, but in the absence of the i53 peptide.

22. The method of any one of embodiments 1 to 21, wherein the frequency of indels at the locus of interest in the cell is lower than the frequency in an equivalent cell in the presence of the sgRNA, RNA-guided nuclease, and homologous donor template, but in the absence of the i53 peptide.

23. The method of embodiment 22, wherein the frequency of indels at the locus of interest in the cell is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more lower than the frequency in an equivalent cell in the presence of the sgRNA, RNA-guided nuclease, and homologous donor template, but in the absence of the i53 peptide.

24. The method of any one of embodiments 1 to 23, further comprising introducing a second sgRNA into the cell targeting a second genetic locus, and introducing a second homologous donor template comprising a nucleotide sequence that is homologous to the second genetic locus, wherein the second sgRNA directs the RNA-guided nuclease to the second genetic locus, the RNA-guided nuclease cleaves the second genetic locus at the target sequence of the second sgRNA, and the second homologous donor template is integrated at the site of the cleaved second genetic locus by HDR. 25. The method of embodiment 24, wherein the frequency of HDR is higher at both the locus of interest and at the second genetic locus in the presence of the i53 peptide than in the absence of the i53 peptide.

26. The method of embodiment 24 or 25, wherein the frequency of indels is lower at both the locus of interest and at the second genetic locus in the presence of the i53 peptide than in the absence of the i53 peptide.

27. A method of treating a genetic disorder in a human subject in need thereof, the method comprising: isolating a primary cell from the subject; genetically modifying the primary cell using the method of any one of claims 1 to 26, wherein the integration of the homologous donor template at the locus of interest in the cell corrects a mutation at the locus or leads to the expression of a therapeutic protein in the cell that is absent or deficient in the subject; and reintroducing the genetically modified cell into the subject.

28. The method of embodiment 27, wherein the genetic disorder is a disorder selected from the group consisting of β-thalassemia, sickle cell disease (SCD), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1, Cystic Fibrosis, Gaucher disease, Krabbe disease, and X-linked chronic granulomatous disease (X-CGD).