Improved peptide inhibitors of the P53-binding protein 53BP1

Abstract

Compositions and methods are provided for enhancing homologous recombination repair in gene editing applications using improved inhibitors of 53BP1. The present disclosure provides compositions and methods for enhancing HDR-mediated integration of polynucleotides in gene editing applications. The methods utilize polypeptide compositions that bind to p53 binding protein 1 (53BP1) and inhibit 53BP1 promotion of the NHEJ DNA repair pathway, favoring HDR-mediated DNA repair.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 476,120, filed December 19, 2022, and U.S. Provisional Application No. 63 / 490,447, filed March 15, 2023, each of which is incorporated by reference in its entirety for all purposes.

[0002] Provided herein are methods and compositions for increasing homology-directed repair, including homologous recombination, using inhibitors of 53BP1. [Background technology]

[0003] Gene editing modifies the genome of individual cells at one or more target loci. In some cases of therapeutic gene editing, such modifications are made at one or more target loci associated with a disease. Given that each human cell has two copies of the genome, gene editing (e.g., using the CRISPR / Cas system) can result in various genomic outcomes in a target cell population, such as cells with no changes to either allele of the target locus, cells with changes to only one allele, or cells with changes to both alleles. Modifications in such alleles with changes can include the insertion or deletion of one or more nucleotides (indels), or the insertion by homology-directed repair (HDR) of a donor polynucleotide intended to address (e.g., precisely correct) one or more mutations at the target locus. The edited cell population can be administered to a patient (e.g., a patient whose hematopoietic stem cells have been harvested and edited ex vivo for the purpose of treating or curing an underlying disease).

[0004] Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells, competing with repair via the homologous recombination repair pathway. DSB repair is regulated by p53-binding protein 1 (53BP1), which promotes NHEJ, and BRCA1, which promotes HR. 53BP1 prevents long-distance DNA end resection and inhibits BRCA1 recruitment to DSBs. 53BP1 also suppresses the formation of 3' single-stranded DNA tails, a rate-limiting step in HR initiation. Inhibition of 53BP1 function can increase HR activity, which could improve gene editing efficiency when HR is required. Therefore, improved 53BP1 inhibitors are needed to improve the efficiency of HDR-based gene editing approaches.

[0005] Sickle cell disease (SCD) is a genetic disease caused by a single point mutation at codon 6 in both copies of the beta globin (HBV) gene. This results in the E6V mutation, which causes the production of sickle (S) hemoglobin (HbS) instead of adult (A) hemoglobin (HbA). Gene-edited autologous hematopoietic stem cell-based therapies in clinical development for SCD are designed to reduce HbS production and restore HbA expression by directly correcting the causative point mutation through HDR-mediated incorporation of a corrective nucleic acid sequence. Increasing the rate of HDR in such gene editing methods is desirable to improve the efficiency of autologous cell product production for the treatment of sickle cell disease (SCD) and for any other disease treated by gene-edited autologous cell therapy (or even allogeneic cell therapy). Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides compositions and methods for enhancing HDR-mediated integration of polynucleotides in gene editing applications, utilizing polypeptide compositions that bind to p53 binding protein 1 (53BP1) and inhibit 53BP1-mediated stimulation of the NHEJ DNA repair pathway in favor of HDR-mediated DNA repair.

[0007] Thus, in one aspect, a polypeptide is provided, the polypeptide comprising an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 1 and having one or more modifications relative to SEQ ID NO: 1, wherein the one or more modifications include a modification at position 67 of SEQ ID NO: 1, a modification at position 68 of SEQ ID NO: 1, or a combination thereof. In a preferred embodiment, the polypeptide is capable of inhibiting the activity of 53BP1. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 having one or more modifications relative to SEQ ID NO: 1, wherein the one or more modifications include a modification at position 67 of SEQ ID NO: 1, a modification at position 68 of SEQ ID NO: 1, or a combination thereof. In some embodiments, the modification at position 67 of SEQ ID NO: 1 is Arg(L67R), His(L67H), Lys(L67K), Ser(L67S), Thr(L67T), Gln(L67Q), Asn(L67N), or His(L67H). In some embodiments, the modification at position 68 of SEQ ID NO:1 is Trp(H68W), Tyr(H68Y), or Phe(H68F).

[0008] In some embodiments, the polypeptide comprises one or more modifications selected from the group consisting of L67R, L67H, L67K, L67S, L67T, L67Q, L67N, L67H, H68W, H68Y, H68F, and combinations thereof. In some embodiments, the one or more modifications are: (a) Arg (L67R) at position 67 of SEQ ID NO: 1 and Trp (H68W) at position 68 of SEQ ID NO: 1; (b) His (L67H) at position 67 of SEQ ID NO: 1 and Tyr (H68Y) at position 68 of SEQ ID NO: 1; (c) His (L67H) at position 67 of SEQ ID NO: 1 and Phe (H68F) at position 68 of SEQ ID NO: 1; (d) His (L67H) at position 67 of SEQ ID NO: 1 and Trp (H68W) at position 68 of SEQ ID NO: 1; (e) Arg (L67R) at position 67 of SEQ ID NO: 1 and Tyr (H68Y) at position 68 of SEQ ID NO: 1; (f) Arg (L67R) at position 67 of SEQ ID NO: 1 and Phe (H68F) at position 68 of SEQ ID NO: 1; (g) Ser (L67R) at position 67 of SEQ ID NO: 1; (h) Thr (L67T) at position 67 of SEQ ID NO: 1 and Trp (H68W) at position 68 of SEQ ID NO: 1; (i) Ser (L67S) at position 67 of SEQ ID NO: 1 and Trp (H68W) at position 68 of SEQ ID NO: 1; (j) Gln (L67Q) at position 67 of SEQ ID NO: 1 and Tyr (H68Y) at position 68 of SEQ ID NO: 1; (k) Asn (L67N) at position 67 of SEQ ID NO: 1 and Tyr (H68Y) at position 68 of SEQ ID NO: 1; (l) Asn (L67N) at position 67 of SEQ ID NO: 1 and Trp (H68W) at position 68 of SEQ ID NO: 1; and (m) Lys (L67K) at position 67 of SEQ ID NO: 1 and Tyr (H68Y) at position 68 of SEQ ID NO: 1.

[0009] In some embodiments, the polypeptide comprises the sequence of any one of SEQ ID NOs: 2-72. In some embodiments, the polypeptide consists of the sequence of any one of SEQ ID NOs: 2-72. In particular embodiments, the polypeptide comprises or consists of the sequence of SEQ ID NO: 2. In other particular embodiments, the polypeptide comprises or consists of the sequence of SEQ ID NO: 3.

[0010] In some embodiments, the polypeptide further comprises one or more additional modifications relative to SEQ ID NO: 1. In some embodiments, the one or more additional modifications of the polypeptide relative to SEQ ID NO: 1 comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more modifications at any amino acid position relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more additional modifications of the polypeptide are selected from the group consisting of (a) a Gln (L2Q) at position 2 of SEQ ID NO: 1, (b) an Ile (A44I) at position 44 of SEQ ID NO: 1, (c) a Gln (S49Q) at position 49 of SEQ ID NO: 1, (d) a Gln (L62Q) at position 62 of SEQ ID NO: 1, (e) a Glu (D64E) at position 64 of SEQ ID NO: 1, (f) a Thr (K66T) at position 66 of SEQ ID NO: 1, (g) a Leu (P69L) at position 69 of SEQ ID NO: 1, (h) a Val (L70V) at position 70 of SEQ ID NO: 1, and combinations thereof. In some embodiments, the polypeptide further comprises one or more modifications at (a) position 12 of SEQ ID NO: 1, (b) position 14 of SEQ ID NO: 1, (c) position 65 of SEQ ID NO: 1, (d) position 66 of SEQ ID NO: 1, (e) position 67 of SEQ ID NO: 1, and / or (f) combinations thereof. In some embodiments, the modification at position 12 of SEQ ID NO:1 is Tyr (T12Y). In some embodiments, the modification at position 12 of SEQ ID NO:1 is Val (T12V). In some embodiments, the modification at position 14 of SEQ ID NO:1 is Glu (T14E). In some embodiments, the modification at position 14 of SEQ ID NO:1 is His (T14H). In some embodiments, the modification at position 65 of SEQ ID NO:1 is Lys (S65K). In some embodiments, the modification at position 66 of SEQ ID NO:1 is Gly (K66G). In some embodiments, the modification at position 67 of SEQ ID NO:1 is His (L67H). In some embodiments, the polypeptide further comprises one or two Gly at the C-terminus of the polypeptide.

[0011] In some embodiments, the polypeptide is 0.5 to 500 × 10 relative to the Tudor domain of 53BP1.-9 It has a binding affinity of M.

[0012] Also provided herein are compositions comprising the polypeptides in admixture with a carrier, excipient, or diluent. Also provided herein are polynucleotides comprising nucleic acid sequences encoding the polypeptides, as well as expression vectors comprising the polynucleotides.

[0013] In another aspect, provided herein are compositions comprising a polypeptide (or polynucleotide or its expression vector) described herein and one or more components of a gene editing system. In some embodiments, the one or more components of the gene editing system comprise (i) a nuclease capable of generating a double-stranded break within a locus of a cell, and (ii) a donor polynucleotide. In some embodiments, the donor polynucleotide comprises non-overlapping 5' and 3' homologous arms, wherein each homologous arm is homologous to a portion of the locus, and upon generation of a double-stranded break within the locus by the nuclease, the donor polynucleotide sequence is integrated into the locus by homology-directed repair (HDR). In some embodiments, the nuclease comprises a CRISPR nuclease and a single guide RNA (sgRNA) capable of hybridizing to a target sequence within the locus, wherein the sgRNA guides the CRISPR nuclease to the target sequence. In some embodiments, the CRISPR nuclease is a Cas protein. In some embodiments, the Cas protein is Cas9 or a high-fidelity variant thereof. In some embodiments, the sgRNA and CRISPR nuclease form a ribonucleoprotein (RNP) complex. In some embodiments, the sgRNA comprises one or more chemically modified nucleotides. In some embodiments, the modified nucleotides are selected from the group consisting of 2'-O-methyl nucleotides, 2'-O-methyl 3'-phosphorothioate nucleotides, and 2'-O-methyl 3'-thio PACE nucleotides. In some embodiments, the 5' end, 3' end, or a combination thereof of the modified sgRNA comprises modified nucleotides. In some embodiments, the donor polynucleotide is comprised in a viral vector, a plasmid, or a single-stranded oligodeoxynucleotide (ssODN). In some embodiments, the donor polynucleotide is comprised in an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.In some embodiments, the AAV vector is an AAV6 vector.

[0014] In another aspect, provided herein is a host cell comprising a polypeptide (or polynucleotide or expression vector thereof) described herein. In some embodiments, the host is a mammal. In some embodiments, the mammal is human. In some embodiments, the cell is a primary cell. In some embodiments, the primary cell is selected from the group consisting of primary blood cells and primary mesenchymal cells. In some embodiments, the primary cell is selected from the group consisting of primary stem cells, primary progenitor cells, and primary somatic cells. In some embodiments, the primary stem cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and organ stem cells. In some embodiments, the primary progenitor cell is selected from the group consisting of hematopoietic progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, multipotent progenitor cells, oligopotent progenitor cells, and lineage-restricted progenitor cells. In some embodiments, the primary somatic cell is selected from the group consisting of fibroblasts, hepatocytes, cardiac cells, liver cells, pancreatic cells, muscle cells, skin cells, blood cells, neural cells, and immune cells. In some embodiments, the immune cells are selected from the group consisting of T lymphocytes (T cells), B lymphocytes (B cells), small lymphocytes, natural killer cells (NK cells), natural killer T cells, macrophages, monocytes, monocyte precursors, eosinophils, neutrophils, basophils, megakaryocytes, myeloblasts, mast cells, and dendritic cells. In some embodiments, the primary cells are CD34+ hematopoietic stem cells or CD34+ hematopoietic progenitor cells.

[0015] In another aspect, provided herein is a kit comprising a polypeptide (or polynucleotide or expression vector thereof) described herein and one or more components of a gene editing system. In some embodiments, the one or more components of the gene editing system comprise (i) a nuclease capable of generating a double-stranded break within a locus in a cell, and (ii) a donor polynucleotide. In some embodiments, the kit further comprises an inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In some embodiments, the DNA-PKcs inhibitor is selected from the group consisting of AZD7648, M3814 / nedisertib, CC-115, and BAY-8400.

[0016] In another aspect, provided herein are methods for increasing homologous recombination in a cell, the method comprising administering a polypeptide described herein. In some embodiments, the method further comprises introducing one or more components of a gene editing system into the cell. In some embodiments, the one or more components of the gene editing system comprise (i) a nuclease capable of generating a double-stranded break within a locus in the cell, and (ii) a donor polynucleotide.

[0017] In another aspect, provided herein is a method for stably integrating an exogenous polynucleotide sequence into the genome of a cell, the method comprising introducing into the cell (a) a nuclease capable of generating a double-strand break in a locus of the cell, (b) a donor polynucleotide, and a polypeptide described herein; wherein upon generation of a double-strand break in the locus by the nuclease, the donor polynucleotide sequence is integrated into the locus by homology-directed repair (HDR), resulting in a gene-edited cell. In some embodiments, the method is performed ex vivo. In some embodiments, the locus of the cell contains one or more mutations associated with a disease or encodes an aberrant protein. In some embodiments, integration of the donor polynucleotide sequence into the host cell genome corrects a mutation in the cell associated with a disease. In some embodiments, integration of the donor polynucleotide sequence replaces a mutant allele in the cell with a wild-type allele. In some embodiments, the disease is selected from the group consisting of hemoglobinopathies, viral infections, X-linked severe combined immunodeficiency, Fanconi anemia, hemophilia, neoplasms, cancer, alpha-1 antitrypsin deficiency, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, cystic fibrosis, blood diseases and disorders, inflammation, immune system diseases or disorders, metabolic diseases, liver diseases and disorders, kidney diseases and disorders, muscle diseases and disorders, bone or cartilage diseases and disorders, nerve and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and lysosomal storage disorders. In some embodiments, the hemoglobinopathies are sickle cell disease, α-thalassemia, β-thalassemia, or δ-thalassemia. In some embodiments, the method further comprises administering the gene-edited cells to a patient in need thereof. In some embodiments, the administering comprises autologous transplantation of the gene-edited cells into the patient. In some embodiments, the administering comprises allogeneic transplantation of the gene-edited cells into the patient.

[0018] In another aspect, provided herein is a method for identifying a mutant polypeptide of a 53BP1 inhibitory polypeptide having the amino acid sequence of SEQ ID NO: 1, wherein the mutant polypeptide is capable of mediating integration of a donor polynucleotide by improved homology-directed repair (HDR), the method comprising:

[0019] (a) contacting a population of CD34+ hematopoietic stem and progenitor cells (HSPCs) with (i) a control polypeptide comprising the amino acid sequence of SEQ ID NO:1; (ii) one or more mutant polypeptides, each mutant polypeptide comprising the amino acid sequence of SEQ ID NO:1 with one or more modifications relative to SEQ ID NO:1, wherein the one or more modifications include a modification at position 67 of SEQ ID NO:1, a modification at position 68 of SEQ ID NO:1, or a combination thereof; (iii) a nuclease capable of generating a double-stranded break within a genetic locus of the HSPCs; and (iv) a donor polynucleotide comprising a reporter gene;

[0020] (b) separating (i) a first subpopulation of the contacted HSPCs that express at least a reference amount of the reporter gene, and (ii) a second subpopulation of the contacted HSPCs that do not express the reference amount of the reporter gene;

[0021] (c) determining the relative abundance of the control polypeptide and the variant polypeptide in both the first HSPC subpopulation and the second HSPC subpopulation, respectively.

[0022] In some embodiments, a mutant polypeptide is identified as being capable of mediating improved integration by homology-directed repair (HDR) relative to a control polypeptide if (i) the mutant polypeptide is more abundant in a first subpopulation than the control polypeptide, and / or (ii) the ratio of the mutant polypeptide:control polypeptide in the first subpopulation is higher than the ratio of the mutant polypeptide:control polypeptide in the second subpopulation. In some embodiments, each of the one or more mutant polypeptides further comprises a modification at position 12 of SEQ ID NO:1, a modification at position 14 of SEQ ID NO:1, or a combination thereof. In some embodiments, each of the one or more mutant polypeptides further comprises a modification at position 65 of SEQ ID NO:1, a modification at position 66 of SEQ ID NO:1, or a combination thereof. In some embodiments, contacting the first and second HSPC populations with the control polypeptide and the mutant polypeptide, respectively, comprises contacting the first and second HSPC populations with lentiviral vectors encoding the control polypeptide and the mutant polypeptide, respectively. In some embodiments, measuring the relative abundance of the control polypeptide and the mutant polypeptide comprises sequencing the lentiviral vectors encoding the control polypeptide and the mutant polypeptide. In some embodiments, each modification of the mutant polypeptide comprises a single amino acid substitution. In some embodiments, each lentiviral vector encoding a mutant polypeptide comprises a polynucleotide comprising an NNK codon that encodes the modification of the mutant polypeptide.

[0023] In some embodiments, the donor polynucleotide comprises non-overlapping 5' and 3' homologous arms, each homologous arm being homologous to a portion of the locus, and upon generation of a double-stranded break within the locus by the nuclease, the donor polynucleotide sequence is integrated into the locus by homology-directed repair (HDR). In some embodiments, the nuclease comprises a CRISPR nuclease and a single guide RNA (sgRNA) capable of hybridizing to a target sequence within the locus, wherein the sgRNA guides the CRISPR nuclease to the target sequence. In some embodiments, the sgRNA and the CRISPR nuclease form a ribonucleoprotein (RNP) complex. In some embodiments, the donor polynucleotide is comprised in a viral vector, a plasmid, or a single-stranded oligodeoxynucleotide (ssODN). In some embodiments, the donor polynucleotide is comprised in an adeno-associated viral (AAV) vector. In some embodiments, the method further comprises isolating a mutant polypeptide having improved HDR activity from the second HSPC population. [Brief explanation of the drawings]

[0024] [Figure 1] Schematic diagram outlining the possible impact of inhibiting the DNA repair enzyme 53BP1 on the various editing outcomes that can occur after nuclease-mediated site-specific double-strand breaks (DSBs). NHEJ: non-homologous end joining; MMEJ: microhomology-mediated end joining; HDR: homology-directed repair.

[0025] [Figure 2] Figure 1 shows a schematic outlining the development of a lentiviral-based pooled functional screening system in HSPCs, which was used to identify HDR-promoting proteins for a Cas9-mediated cleavage site of interest.

[0026] [Figure 3]Figure 1 shows sequences (A) and results (B) associated with the validation of a functional screening system to identify improved mutants of i53. Sequences included: i53 (positive control), a previously reported inactive mutant of i53 ("DM" = P67L L70V, negative control), and three mutants of i53 ("mut1" = L2Q, "mut2" = D64E, and "mut3" = L62Q). The three mutants have previously been reported to have reduced (but detectable) binding to the Tudor domain of 53BP1 compared to i53.

[0027] [Figure 4] A schematic diagram showing the binding interface between i53 and the 53BP1 Tudor domain at positions 67 and 68 of i53. Positions 67 and 68 of i53 were targeted for mutagenesis (A). A lentiviral-based pooled functional screen identified a single amino acid mutation in i53 that showed enhanced HDR-promoting ability compared to i53 at the HBB locus in HPSCs (B).

[0028] [Figure 5] Figure 1 shows results demonstrating validation of the top two hits, which were obtained from functional screening of the NNK library at residues 67 and 68 (L67R, L67H) by lentiviral expression.

[0029] [Figure 6] FIG. 1 shows the results of size exclusion chromatography, illustrating the binding of i53 mutants L67R and L67H to the 53BP1 Tudor domain.

[0030] [Figure 7] FIG. 1 shows the results of biolayer interferometry (BLI), depicting the binding kinetics of i53 mutants to the Tudor domain of immobilized 53BP1.

[0031] [Figure 8]FIG. 1 shows the structure obtained from crystallography, showing i53 mutants L67R and L67H complexed to the 53bp1 Tudor domain.

[0032] [Figure 9] Schematic diagram detailing the integration of purified i53 mutants, identified from functional screening as HDR-promoting factors into HSPC-based gene editing platforms.

[0033] [Figure 10] FIG. 1 shows the results of editing CD34+ HSPCs using the HBB-UBC-GFP AAV donor and purified L67R and L67H i53 mutants compared to i53 and i53DM mutants.

[0034] [Figure 11] FIG. 1 shows HDR rates (A) and DNA repair outcomes (B) after CD34+ HSPC cell editing using the HBB-SNP AAV donor (designed to correct the sequence encoding the sickle cell E6V mutation) and purified L67R and L67H compared to i53 and i53DM mutants.

[0035] [Figure 12] FIG. 1 shows dose-response curves of purified i53 mutants L67R and L67H compared to parental control i53 after CD34+ HSPC editing using an HBB-UBC-GFP AAV donor.

[0036] [Figure 13] FIG. 1 shows dose-response curves of purified i53 mutants L67R and L67H compared to parental control i53 after editing of CD34+ HSPCs using HBB-SNP AAV donors.

[0037] [Figure 14] FIG. 1 shows HDR rates after CD34+ HSPC cell editing using increasing MOIs of HBB-SNP AAV donor and purified i53 mutant L67R.

[0038] [Figure 15A] 1 shows the results of a lentiviral-based pooled functional screen of a combinatorial library targeting residues at the 53bp1 / i53 interface. [Figure 15B] 1 shows the results of a lentiviral-based pooled functional screen of a combinatorial library targeting residues at the 53bp1 / i53 interface.

[0039] [Figure 16]

[0033] Figure 1 shows results demonstrating validation of top hits by pooled lentiviral expression. Top hits were obtained from functional screening of combinatorial libraries at residues 67 and 68.

[0040] [Figure 17] FIG. 1 shows dose-response curves for purified i53 mutants L67R, L67H, and two representative purified double mutants of i53 (L67K H68F and L67H H68Y) after CD34+ HSPC cell editing using HBB-SNP AAV donors.

[0041] [Figure 18A] 1 shows additional HDR-promoting mutants of i53. Additional HDR-promoting mutants of i53 were identified by repeating the process on the L67R i53 mutant and screening combinatorial libraries targeting additional residues at the 53bp1 / i53 interface. [Figure 18B] 1 shows additional HDR-promoting mutants of i53. Additional HDR-promoting mutants of i53 were identified by repeating the process on the L67R i53 mutant and screening combinatorial libraries targeting additional residues at the 53bp1 / i53 interface.

[0042] [Figure 19A] 1 shows additional HDR-promoting mutants of i53. Additional HDR-promoting mutants of i53 were identified by repeating the process on the L67H i53 mutant and screening combinatorial libraries targeting additional residues at the 53bp1 / i53 interface. [Figure 19B] 1 shows additional HDR-promoting mutants of i53. Additional HDR-promoting mutants of i53 were identified by repeating the process on the L67H i53 mutant and screening combinatorial libraries targeting additional residues at the 53bp1 / i53 interface.

[0043] [Figure 20] Validation of top hits by pooled lentiviral expression. Top hits were obtained from combinatorial libraries at residues 12 and 14 using L67H as parent.

[0044] [Figure 21A] 1 shows additional HDR-promoting mutants of i53. Additional HDR-promoting mutants of i53 were identified by repeating the process on the L67H.H68Y i53 mutant and screening combinatorial libraries targeting additional residues at the 53bp1 / i53 interface. [Figure 21B] 1 shows additional HDR-promoting mutants of i53. Additional HDR-promoting mutants of i53 were identified by repeating the process on the L67H.H68Y i53 mutant and screening combinatorial libraries targeting additional residues at the 53bp1 / i53 interface.

[0045] [Figure 22A] 1 shows additional HDR-promoting mutants of i53. Additional HDR-promoting mutants of i53 were identified by repeating the process on the L67K.H68F i53 mutant and screening combinatorial libraries targeting additional residues at the 53bp1 / i53 interface. [Figure 22B] 1 shows additional HDR-promoting mutants of i53. Additional HDR-promoting mutants of i53 were identified by repeating the process on the L67K.H68F i53 mutant and screening combinatorial libraries targeting additional residues at the 53bp1 / i53 interface.

[0046] [Figure 23A] Validation of top hits by pooled lentiviral expression. Top hits were obtained from combinatorial libraries at residues 65 and 66 using T12Y.T14E.L67R as parent. [Figure 23B] Validation of top hits by pooled lentiviral expression. Top hits were obtained from combinatorial libraries at residues 65 and 66 using T12Y.T14E.L67R as parent.

[0047] [Figure 24A] Figure 1 shows GFP knock-in into CD34+ HSPC cells using three representative purified "hit" mutants of i53 (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and AAV donors targeting the HBB, HBA, CCR5, and Il2RG12 loci, respectively. [Figure 24B] Figure 1 shows GFP knock-in into CD34+ HSPC cells using three representative purified "hit" mutants of i53 (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and AAV donors targeting the HBB, HBA, CCR5, and Il2RG12 loci, respectively. [Figure 24C]Figure 1 shows GFP knock-in into CD34+ HSPC cells using three representative purified "hit" mutants of i53 (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and AAV donors targeting the HBB, HBA, CCR5, and Il2RG12 loci, respectively. [Figure 24D] Figure 1 shows GFP knock-in into CD34+ HSPC cells using three representative purified "hit" mutants of i53 (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and AAV donors targeting the HBB, HBA, CCR5, and Il2RG12 loci, respectively. [Figure 24E] Figure 1 shows GFP knock-in into CD34+ HSPC cells using three representative purified "hit" mutants of i53 (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and AAV donors targeting the HBB, HBA, CCR5, and Il2RG12 loci, respectively. [Figure 24F] Figure 1 shows GFP knock-in into CD34+ HSPC cells using three representative purified "hit" mutants of i53 (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and AAV donors targeting the HBB, HBA, CCR5, and Il2RG12 loci, respectively. [Figure 24G] Figure 1 shows GFP knock-in into CD34+ HSPC cells using three representative purified "hit" mutants of i53 (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and AAV donors targeting the HBB, HBA, CCR5, and Il2RG12 loci, respectively. [Figure 24H]Figure 1 shows GFP knock-in into CD34+ HSPC cells using three representative purified "hit" mutants of i53 (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and AAV donors targeting the HBB, HBA, CCR5, and Il2RG12 loci, respectively.

[0048] [Figure 25A] Figure 1 shows editing of CD34+ HSPC cells from three donors using three representative purified "hit" mutants of i53 identified in the screen above (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and an AAV donor template designed to correct the mutation encoding sickle cell disease E6V. [Figure 25B] Figure 1 shows editing of CD34+ HSPC cells from three donors using three representative purified "hit" mutants of i53 identified in the screen above (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and an AAV donor template designed to correct the mutation encoding sickle cell disease E6V. [Figure 25C] Figure 1 shows editing of CD34+ HSPC cells from three donors using three representative purified "hit" mutants of i53 identified in the screen above (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and an AAV donor template designed to correct the mutation encoding sickle cell disease E6V. [Figure 25D]Figure 1 shows editing of CD34+ HSPC cells from three donors using three representative purified "hit" mutants of i53 identified in the screen above (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and an AAV donor template designed to correct the mutation encoding sickle cell disease E6V. [Figure 25E] Figure 1 shows editing of CD34+ HSPC cells from three donors using three representative purified "hit" mutants of i53 identified in the screen above (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and an AAV donor template designed to correct the mutation encoding sickle cell disease E6V. [Figure 25F] Figure 1 shows editing of CD34+ HSPC cells from three donors using three representative purified "hit" mutants of i53 identified in the screen above (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) and an AAV donor template designed to correct the mutation encoding sickle cell disease E6V.

[0049] [Figure 26] FIG. 1 shows dose-response curves of purified "hit" mutants of i53 (L67R, L67H.H68Y, T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) relative to parental i53 using an AAV donor template designed to correct the sickle cell E6V-encoding mutation.

[0050] [Figure 27A] FIG. 10 shows the results of biolayer interferometry (BLI) measuring the binding kinetics of additional i53 mutants to immobilized 53BP1 Tudor domain. [Figure 27B]FIG. 10 shows the results of biolayer interferometry (BLI) measuring the binding kinetics of additional i53 mutants to immobilized 53BP1 Tudor domain. [Figure 27C] FIG. 10 shows the results of biolayer interferometry (BLI) measuring the binding kinetics of additional i53 mutants to immobilized 53BP1 Tudor domain. [Figure 27D] FIG. 10 shows the results of biolayer interferometry (BLI) measuring the binding kinetics of additional i53 mutants to immobilized 53BP1 Tudor domain. [Figure 27E] FIG. 10 shows the results of biolayer interferometry (BLI) measuring the binding kinetics of additional i53 mutants to immobilized 53BP1 Tudor domain. [Figure 27F] FIG. 10 shows the results of biolayer interferometry (BLI) measuring the binding kinetics of additional i53 mutants to immobilized 53BP1 Tudor domain. [Figure 27G] FIG. 10 shows the results of biolayer interferometry (BLI) measuring the binding kinetics of additional i53 mutants to immobilized 53BP1 Tudor domain.

[0051] [Figure 28] FIG. 1 shows the evaluation of stability and multiplex reactivity of a subset of purified i53 mutant proteins using dynamic light scattering (DLS). DETAILED DESCRIPTION OF THE INVENTION

[0052] The present disclosure provides compositions and methods for increasing homology-directed repair using inhibitors of 53BP1.

[0053] In certain gene editing applications, the inherent cellular repair mechanism, homology-directed repair (HDR), enables precise templated repair of nuclease-induced double-strand breaks (DSBs) in cells (e.g., hematopoietic stem and progenitor cells (HSPCs)) using donor sequences. Such donor sequences can be delivered by vectors such as adeno-associated viruses (e.g., AAV6) to correct, replace, or insert genes anywhere within the genome. However, other DSB repair pathways exist within cells, and religation of DSB ends can also occur through pathways such as non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ). NHEJ and MMEJ can compete with HDR and result in unwanted insertions and deletions (indels) in addition to the desired donor template outcome. The ratio of HDR to indel outcomes can be improved by increasing the level of donor template. However, high titers of donor vectors, such as AAV6, can induce the DNA damage response (DDR) pathway in cells, limiting the growth, yield, and engraftment of edited cells in ex vivo editing applications. Another route to improving HDR without increasing viral vector titer (or alternatively, allowing for a reduction in viral vector titer while maintaining a desired HDR level) is to inhibit key DNA repair enzymes involved in NHEJ and MMEJ repair. To improve HDR and reduce NHEJ in HDR-based gene editing applications, we have demonstrated that the polypeptides described herein specifically and efficiently inhibit the recruitment of 53BP1 (a key enzyme involved in NHEJ repair) to Cas9-mediated DSBs.

[0054] definition For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0055] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated or contradicted in the context of this disclosure.

[0056] As used herein, "or" means "and / or" unless otherwise indicated or contradicted by the context of the present disclosure.

[0057] As used herein, the words "comprising," "including," "containing," "having," and the like, unless otherwise specified, are intended to be open-ended and not exclusive, and therefore do not exclude additional, unrecited elements.

[0058] The term "about," as used herein when referring to a measurable value such as an amount, time, and the like, is intended to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the particular value, where such variation is appropriate to perform the disclosed methods.

[0059] Throughout this disclosure, various aspects of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Thus, a description of a range should be construed as including not only each individual numerical value within that range, but also all possible subranges specifically disclosed. For example, a description of a range such as 1 to 6 should be construed as including not only each individual numerical value within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, but also specifically disclosed subranges, e.g., 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This is true regardless of the breadth of the range.

[0060] "Homologous recombination" and "HR" refer to a type of genetic recombination in which DNA strands with similar or identical nucleotide sequences are exchanged. Cells can use HR to repair DNA double-strand breaks (DSBs) through the following general steps: HR is initiated when the DSB is resected by nucleases and helicases, generating a 3' single-stranded DNA (ssDNA) overhang onto which RAD51 recombinase assembles as a nucleoprotein filament. This structure can invade a homologous double-stranded DNA to serve as a template for repair DNA synthesis. The resulting intermediate can undergo differential metabolism to generate crossover or non-crossover products (San Filippo et al., Annu. Rev. Biochem. 2008. 77:229-57). Following a double-strand break, a fragment near the 5' end of the break is excised by nucleases and helicases, generating a 3' single-stranded DNA overhang onto which RAD51 recombinase assembles as a nucleoprotein filament. This structure then invades homologous double-stranded DNA, which is used as a template for DNA repair synthesis. The resulting intermediate can be metabolized to generate non-crossover products, restoring the damaged DNA molecule to the state that existed before the double-strand break. The term also encompasses recombination using single-stranded oligonucleotides (ssODNs), particularly those that require excision and can be activated by 53BP1 inhibitors.

[0061] As used herein, "HDR" or "homologous recombination repair" refers to the repair process of DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, e.g., a sister chromatid, or an exogenous nucleic acid, e.g., a template nucleic acid such as a donor polynucleotide described herein). Standard HDR typically operates when there is significant excision at the double-strand break, forming at least one single-stranded portion of DNA. In normal cells, HDR usually involves a series of steps, such as break recognition, break stabilization, excision, stabilization of single-stranded DNA, formation of a DNA crossover intermediate, resolution of the crossover intermediate, and ligation. This process requires RAD51 and BRCA2, and the homologous nucleic acid is usually double-stranded. This process can be induced by several site-specific nuclease systems that generate double-strand breaks. Such site-specific nuclease systems include, for example, meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas gene editing systems. In certain embodiments, HDR involves a double-strand break induced by a CRISPR-Cas nuclease, such as Cas9.

[0062] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0063] As used herein, the term "diagnosis" or "diagnosing" refers to the process of identifying a disease, such as cancer, by its signs, symptoms, and / or the results of various tests. The conclusion reached through such a process is a diagnosis. Common forms of testing include blood tests, medical imaging, urine tests, biopsies, etc.

[0064] The term "therapeutically effective amount" or simply "effective amount" refers to an amount of an agent or composition (e.g., a composition comprising an agent) that elicits the biological or medicinal response in a tissue, system, animal, or subject that is being sought by a researcher, veterinarian, physician, or other medical professional. The term "therapeutically effective amount" encompasses an amount of an agent or an amount of a composition comprising an agent that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the signs or symptoms of, the disorder or disease (e.g., a hematological or solid tumor) being treated. A "therapeutically effective amount" will vary depending on the composition, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0065] The term "treating" a disease, as used herein, means reducing or diminishing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. In one example, a therapy (e.g., administration of a therapeutic agent of the present disclosure) treats a disease or disorder by reducing one or more signs or symptoms associated with the disease or disorder, for example, compared to a response without the therapy. For example, administration of a therapeutic agent can result in an anti-tumor effect that reduces one or more signs or symptoms associated with cancer.

[0066] As used herein, the term "administration" means providing or giving one or more agents to a subject by any effective route, such agents treating one or more signs or symptoms associated with a disease / disorder or condition, including, but not limited to, cancer (e.g., lymphoma), viral infection, bacterial infection, etc. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes. Administration "in combination with" one or more additional therapeutic agents includes simultaneous administration (concurrent administration) and sequential administration in any order.

[0067] As used herein, the term "pharmaceutically acceptable" refers to a substance (including, but not limited to, salts, carriers, or diluents) that does not interfere with the biological activity or properties of a compound and is relatively non-toxic. That is, such a substance can be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which the compound is contained. Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of one or more agents, such as one or more modulators. Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations can contain an injectable fluid as a vehicle, including pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like. In addition to biologically neutral carriers, the administered pharmaceutical preparations may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate.

[0068] As used herein, the term "autologous" is intended to refer to any material that originates from an individual and is later reintroduced into the same individual.

[0069] As used herein, the term "allogeneic" is intended to refer to material derived from an individual that is subsequently reintroduced into a different individual of the same species.

[0070] As used herein, the term "donor polynucleotide" refers to a polynucleotide sequence comprising a donor sequence (e.g., including coding, non-coding, and / or regulatory sequences), which is flanked by 5' and 3' homologous arms, respectively, and which has sequence similarity to a target nucleic acid sequence. In some embodiments, the donor polynucleotide can be contained in a vector, e.g., a circular plasmid, a linearized plasmid, or a plasmid that is linearized by a cleavage process.

[0071] As used herein, the term "Cas molecule" refers to a Cas polypeptide, a Cas protein, or a nucleic acid encoding a Cas9 polypeptide. A "Cas polypeptide" or "Cas protein" is a polypeptide capable of interacting with a gRNA molecule and coordinating localization with the gRNA molecule to a site comprising a target domain and, in certain embodiments, a PAM sequence. Cas molecules include both naturally occurring Cas molecules and engineered, engineered, or modified Cas molecules or Cas polypeptides that differ from a reference sequence (e.g., the most similar naturally occurring Cas molecule), e.g., by at least one amino acid residue. (The term "engineered, engineered, or modified" as used in this context simply refers to differences from a reference sequence or a naturally occurring sequence and does not impose limitations to a particular process or source.) A Cas molecule can be a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide. A Cas molecule can be a nuclease (an enzyme that cleaves both strands of a double-stranded nucleic acid), a nickase (an enzyme that cleaves one strand of a double-stranded nucleic acid), or an enzymatically inactive (or ineffective) Cas molecule. Exemplary Cas molecules include high-fidelity Cas mutants with improved on-target specificity and reduced off-target activity. Examples of high-fidelity Cas9 mutants include, but are not limited to, those described in PCT Publications WO / 2018 / 068053 and WO / 2019 / 074542, each of which is incorporated by reference in its entirety.

[0072] As used herein, the terms "gRNA molecule" and "gRNA" refer to a guide RNA capable of guiding a Cas molecule to a target nucleic acid. In one embodiment, the term "gRNA molecule" refers to a guide ribonucleic acid. In another embodiment, the term "gRNA molecule" refers to a nucleic acid encoding a gRNA. In one embodiment, a gRNA molecule is not naturally occurring. In one embodiment, a gRNA molecule is a synthetic gRNA molecule. As used herein, the term "gRNA molecule" encompasses single guide RNA molecules, which include, on a single nucleic acid molecule, a first nucleotide sequence that is complementary to a target nucleic acid (e.g., within a locus) and a second nucleotide sequence that interacts with a CRISPR / Cas polypeptide, where the modified sgRNA guides the CRISPR / Cas polypeptide to the target nucleic acid.

[0073] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a particular sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a particular sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein when it is produced in a cell or other biological system by transcription and translation of its corresponding mRNA. Both the coding strand (the strand whose nucleotide sequence is identical to the mRNA sequence and is usually shown in a sequence listing) and the non-coding strand (used as a template for transcription of a gene or cDNA) can be said to encode a protein or other product of that gene or cDNA.

[0074] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that is normally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment, such as a host cell.

[0075] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are mutually degenerate and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may contain introns.

[0076] "Expression cassette" refers to a nucleic acid comprising expression control sequences operably linked to a nucleic acid encoding a transcript or polypeptide to be expressed. An expression cassette contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. An expression cassette can be a component of a vector, such as a cosmid, a plasmid (e.g., naked or contained in a liposome), or a virus (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus). An expression cassette can be present within a host cell.

[0077] "Amino acid" includes both natural and synthetic amino acids, and both D and L amino acids.

[0078] As used herein, " gene editing system " refers to a system for targeting and editing nucleic acid sequences (e.g., in gene loci). Non-limiting exemplary systems for gene editing include CRISPR (clustered regularly interspaced short palindromic repeats) system, zinc finger nuclease (ZFN) system, and transcription activator-like effector nuclease (TALEN) system. The components and methods of use of such systems are known in the art. An exemplary CRISPR system is disclosed in U.S. Patent No. 11,193,141, which is incorporated herein by reference in its entirety.

[0079] A "polypeptide fragment" is a portion of a polypeptide that includes a region substantially identical to a polypeptide of the present disclosure and has at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more of the binding activity to the Tudor domain of 53BP1 (the binding activity of the polypeptide from which the fragment is derived). By way of example and without limitation, a polypeptide fragment can have at least 1, 5, 10, 15, 20, 25, 30, 40, or more amino acids fewer than the full-length polypeptide. For example, a polypeptide of the present disclosure can be a polypeptide fragment of SEQ ID NO: 1 or a modified sequence thereof as described herein.

[0080] The percent "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to those in the reference sequence after aligning the two sequences and, if necessary, introducing gaps to achieve the maximum percent sequence identity. Alignment to determine percent amino acid sequence identity can be achieved by various methods. Such methods are within the skill of the art, for example, using BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences. Such alignment includes any algorithm necessary to achieve maximum alignment across the entire length of the sequences being compared. In certain embodiments, default parameters are used.

[0081] The term "amino acid" refers to the 20 common naturally occurring amino acids, including alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0082] "Modifications" to the polypeptides of the present disclosure can include substitutions, insertions, deletions, and chemical modifications. Techniques for preparing such polypeptides and polynucleotides encoding them are known in the art.

[0083] "Binding affinity" or "binding ability" of 53BP1 to the Tudor domain (residues 1484-1603) can be measured by any suitable assay in the art, such as by measuring the recruitment of 53BP1 to DSB sites as described in Canny, et al., "Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency," Nat Biotechnol. 36(1):95-102 (2018) (incorporated herein by reference in its entirety).

[0084] It should be understood that reference to a "position" within a polypeptide refers to the amino acid residue corresponding to that position in the reference polypeptide sequence. For example, position 68 refers to the position in the polypeptides of the present disclosure that corresponds to position 68 in SEQ ID NO: 1, regardless of whether that position in the polypeptides of the present disclosure is the 68th amino acid position.

[0085] Polypeptides In some embodiments, a polypeptide is provided, the polypeptide comprising an amino acid sequence having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 and having one or more modifications relative to SEQ ID NO: 1. In certain embodiments, the one or more modifications comprise a modification at position 67 of SEQ ID NO: 1, a modification at position 68 of SEQ ID NO: 1, or a combination thereof. In certain embodiments, the polypeptide comprises a modification at position 67. In certain embodiments, the polypeptide comprises a modification at position 68. In certain embodiments, the polypeptide comprises a modification at position 67 and a modification at position 68. In certain embodiments, the polypeptide comprises one or more additional modifications relative to the amino acid sequence of SEQ ID NO: 1.

[0086] In any of the above embodiments, the polypeptide can comprise an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity or higher to the amino acid sequence of SEQ ID NO:1, and further comprises one or more modifications relative to SEQ ID NO:1.

[0087] In some embodiments, a polypeptide is provided, the polypeptide comprising the sequence of SEQ ID NO: 1 with one or more modifications relative to SEQ ID NO: 1, wherein the one or more modifications comprise a modification at position 67 of SEQ ID NO: 1, a modification at position 68 of SEQ ID NO: 1, or a combination thereof. In certain embodiments, the polypeptide comprises a modification at position 67. In certain embodiments, the polypeptide comprises a modification at position 68. In certain embodiments, the polypeptide comprises a modification at position 67 and a modification at position 68. In certain embodiments, the polypeptide comprises one or more additional modifications relative to the amino acid sequence of SEQ ID NO: 1.

[0088] In any of the above embodiments, the modification at position 67 of SEQ ID NO: 1 can be (a) Arg(L67R), (b) His(L67H), (c) Lys(L67K) (provided that the polypeptide further comprises a modification at position 68 (L67K, H68*)), (d) Ser(L67S), (e) Thr(L67T), (f) Gln(L67Q), or (g) Asn(L67N). In certain embodiments, the modification at position 67 of SEQ ID NO: 1 can be (a) Arg(L67R), (b) His(L67H), (c) Ser(L67S), (d) Thr(L67T), (e) Gln(L67Q), or (f) Asn(L67N). In certain embodiments, the modification at position 67 of SEQ ID NO:1 can be (a) Arg (L67R), (b) His (L67H), or (c) Lys (L67K), provided that the polypeptide further comprises a modification at position 68 (L67K, H68*). In certain embodiments, the modification at position 67 of SEQ ID NO:1 can be (a) Arg (L67R) or (b) His (L67H). In certain embodiments, the modification at position 67 of SEQ ID NO:1 is not L67K.

[0089] In any of the above embodiments, the modification at position 68 of SEQ ID NO: 1 can be (a) Trp (H68W), (b) Tyr (H68Y), or Phe (H68F).

[0090] In any of the above embodiments, the one or more modifications are (a) Arg(L67R) at position 67 of SEQ ID NO:1 and Trp(H68W) at position 68 of SEQ ID NO:1; (b) His(L67H) at position 67 of SEQ ID NO:1 and Tyr(H68Y) at position 68 of SEQ ID NO:1; (c) His(L67H) at position 67 of SEQ ID NO:1 and Phe(H68F) at position 68 of SEQ ID NO:1; (d) His(L67H) at position 67 of SEQ ID NO:1 and Trp(H68W) at position 68 of SEQ ID NO:1; (e) Arg(L67R) at position 67 of SEQ ID NO:1 and Tyr(H68Y) at position 68 of SEQ ID NO:1; (f) Arg(L67R) at position 67 of SEQ ID NO:1 and Phe(H68F) at position 68 of SEQ ID NO:1; (g) (h) Thr (L67T) at position 67 of SEQ ID NO: 1 and Trp (H68W) at position 68 of SEQ ID NO: 1; (i) Ser (L67S) at position 67 of SEQ ID NO: 1 and Trp (H68W) at position 68 of SEQ ID NO: 1; (j) Gln (L67Q) at position 67 of SEQ ID NO: 1 and Tyr (H68Y) at position 68 of SEQ ID NO: 1; (k) Asn (L67N) at position 67 of SEQ ID NO: 1 and Tyr (H68Y) at position 68 of SEQ ID NO: 1; (l) Asn (L67N) at position 67 of SEQ ID NO: 1 and Trp (H68W) at position 68 of SEQ ID NO: 1; or (m) Lys (L67K) at position 67 of SEQ ID NO: 1 and Tyr (H68Y) at position 68 of SEQ ID NO: 1.

[0091] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 2. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 2.

[0092] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 3. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 3.

[0093] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 4. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 4.

[0094] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 5. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 5.

[0095] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 6. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 6.

[0096] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 7. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 7.

[0097] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 8. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 8.

[0098] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 9. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 9.

[0099] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 10. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 10.

[0100] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 11. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 11.

[0101] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 12. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 12.

[0102] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 13. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 13.

[0103] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 14. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 14.

[0104] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 15. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 15.

[0105] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 16. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 16.

[0106] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 17. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 17.

[0107] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 18. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 18.

[0108] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 19. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 19.

[0109] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 20. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 20.

[0110] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 21. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 21.

[0111] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 22. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 22.

[0112] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 23. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 23.

[0113] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 24. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 24.

[0114] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 25. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 25.

[0115] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 26. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 26.

[0116] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 27. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 27.

[0117] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 28. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 28.

[0118] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 29. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 29.

[0119] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 30. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 30.

[0120] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 31. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 31.

[0121] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 32. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 32.

[0122] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 33. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 33.

[0123] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 34. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 34.

[0124] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 35. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 35.

[0125] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 36. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 36.

[0126] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 37. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 37.

[0127] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 38. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 38.

[0128] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 39. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 39.

[0129] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 40. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 40.

[0130] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 41. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 41.

[0131] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 42. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 42.

[0132] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 43. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 43.

[0133] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 44. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 44.

[0134] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 45. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 45.

[0135] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 46. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 46.

[0136] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 47. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 47.

[0137] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 48. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 48.

[0138] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 49. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 49.

[0139] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 50. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 50.

[0140] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 51. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 51.

[0141] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 52. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 52.

[0142] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 53. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 53.

[0143] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 54. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 54.

[0144] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 55. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 55.

[0145] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 56. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 56.

[0146] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 57. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 57.

[0147] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 58. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 58.

[0148] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 59. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 59.

[0149] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 60. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 60.

[0150] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 61. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 61.

[0151] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 62. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 62.

[0152] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 63. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 63.

[0153] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 64. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 64.

[0154] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 65. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 65.

[0155] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 66. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 66.

[0156] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 67. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 67.

[0157] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 68. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 68.

[0158] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 69. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 69.

[0159] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 70. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 70.

[0160] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 71. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 71.

[0161] In some embodiments, a polypeptide is provided that comprises the sequence of SEQ ID NO: 72. In some embodiments, a polypeptide is provided that consists of the sequence of SEQ ID NO: 72.

[0162] In some embodiments, the polypeptide further comprises one or more additional modifications to the amino acid sequence of SEQ ID NO: 1. For example, and without limitation, the polypeptide further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more additional modifications to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the one or more additional modifications are selected from the group consisting of: (a) Gln (L2Q) at position 2 of SEQ ID NO:1; (b) Ile (A44I) at position 44 of SEQ ID NO:1; (c) Gln (S49Q) at position 49 of SEQ ID NO:1; (d) Gln (L62Q) at position 62 of SEQ ID NO:1; (e) Glu (D64E) at position 64 of SEQ ID NO:1; (f) Thr (K66T) at position 66 of SEQ ID NO:1; (g) Leu (P69L) at position 69 of SEQ ID NO:1; (h) Val (L70V) at position 70 of SEQ ID NO:1; and combinations thereof. In any of the above embodiments, the one or more additional modifications may include (a) position 12 of SEQ ID NO:1, (b) position 14 of SEQ ID NO:1, (c) position 65 of SEQ ID NO:1, (d) position 66 of SEQ ID NO:1, (e) position 67 of SEQ ID NO:1, and (f) combinations thereof, or may be selected from the group consisting of (a) position 12 of SEQ ID NO:1, (b) position 14 of SEQ ID NO:1, (c) position 65 of SEQ ID NO:1, (d) position 66 of SEQ ID NO:1, (e) position 67 of SEQ ID NO:1, and (f) combinations thereof. In certain embodiments, the one or more additional modifications may include Tyr at position 12 of SEQ ID NO:1 (T12Y) and / or Glu at position 14 of SEQ ID NO:1 (T14E). In certain embodiments, the one or more additional modifications may include Val at position 12 of SEQ ID NO:1 (T12V) and / or His at position 14 of SEQ ID NO:1 (T14H). In certain embodiments, the one or more additional modifications may include a Tyr at position 12 of SEQ ID NO: 1 (T12Y) and / or a Glu at position 14 of SEQ ID NO: 1 (T14E). In certain embodiments, the one or more additional modifications may include a Tyr at position 12 of SEQ ID NO: 1 (T12Y) and / or a Glu at position 14 of SEQ ID NO: 1 (T14E), a Lys at position 65 of SEQ ID NO: 1 (S65K), and / or a Gly at position 66 of SEQ ID NO: 1 (K66G).In certain embodiments, the modification at position 67 of SEQ ID NO: 1 is His(L67H). In certain embodiments, the modification at position 67 is Arg(L67R). For example, and without limitation, one or more additional modifications can be made at a position(s) selected from the group consisting of: 2, 4, 6, 8, 10, 11, 12, 14, 44, 46, 47, 48, 49, 62, 63, 64, 65, 66, 69, 70, 71, 72, 73, 74, and combinations thereof, corresponding to positions of SEQ ID NO: 1.

[0163] In any of the above embodiments, the polypeptide may further comprise one or two Gly residues at its C-terminus. Of course, the polypeptide may further comprise additional residues at its N-terminus or C-terminus. In any of the above embodiments, the polypeptide may comprise a Met residue at its N-terminus. Alternatively, in any of the above embodiments, the polypeptide does not comprise a Met residue at its N-terminus.

[0164] In any of the above embodiments, the polypeptide is 0.5 to 500 × 10 -9 For example, without limitation, the polypeptide may have a binding affinity of 0.5 to 15×10 M to the Tudor domain of 53BP1. -9 M, 0.5-25 x 10 -9 M, 0.5-50×10 -9 M, 0.5-100 x 10 -9 M, 0.5-200 x 10 -9 M, 1-200 x 10 -9 M, 1-300 x 10 -9 M, 1-400 x 10 -9 M, 1-500 x 10 -9 M, 100-300 x 10 -9 M, 100-250 x 10 -9 M, or 200-250 x 10 -9 M.

[0165] It will be appreciated that in any of the above embodiments, the polypeptide may be a fragment of a full-length polypeptide and may not include the entire sequence of SEQ ID NO: 1 or a modified version thereof. In any of the above embodiments, the polypeptide or polypeptide fragment is capable of binding to the Tudor domain of 53BP1 (residues 1484-1603).

[0166] fusion proteins In some embodiments, the polypeptides of the present disclosure may be conjugated to enzymes, toxins, or other binding proteins (e.g., biotin, digoxigenin, GFP, Flag, and fluorescent and / or luminescent substances) while retaining binding to the Tudor domain of 53BP1.

[0167] Additional amino acids or peptides or substitutions of individual amino acids or peptides (particularly at the amino and / or carboxy termini) may be introduced by chemical coupling using suitable reagents to obtain fusion proteins. Fusion polypeptides may also be prepared by genetic engineering, in which the gene for a polypeptide disclosed herein is linked to the gene for a fusion partner. Bivalent or bispecific polypeptides can be obtained by site-specifically and covalently linking the polypeptides disclosed herein to polypeptides of the same or different specificity (e.g., via an additionally introduced cysteine ​​or via a positively or negatively charged amino acid at the carboxy terminus of the fusion partner).

[0168] Polynucleotides In some embodiments, an isolated polynucleotide encoding a polypeptide of any of the above embodiments is provided. Of course, the isolated polynucleotide may be the polynucleotide itself or may be incorporated into a vector or cell.

[0169] vector In some embodiments, a vector is provided, the vector comprising a polynucleotide encoding the polypeptide of any of the above embodiments. In certain aspects, the vector is an expression vector.

[0170] In any of the above embodiments, the vector may be any suitable vector for delivering a polynucleotide to a host cell. For example, and without limitation, such vectors include prokaryotic vectors, viral vectors, or eukaryotic vectors, such as mammalian vectors. Exemplary viral vectors include adenoviruses, adeno-associated viruses, retroviruses, herpes viruses, lentiviruses, poxviruses, and cytomegaloviruses.

[0171] host cell In some embodiments, a host cell is provided comprising the polypeptide of any of the above embodiments. The host cells described herein can be used in any of the compositions, kits, and methods of the disclosure.

[0172] In some embodiments, a host cell is provided comprising the polypeptide of any of the above embodiments. In any of the above embodiments, the host cell may comprise the polypeptide of any of the above embodiments mixed with a carrier, additive, or diluent.

[0173] In some embodiments, a host cell is provided comprising the polynucleotide of any of the above embodiments.

[0174] In some embodiments, a host cell is provided comprising the vector of any of the above embodiments. In certain aspects, the vector is an expression vector.

[0175] In one embodiment, an isolated cell or cell line is provided that comprises any of the polypeptides and / or polynucleotides disclosed herein. The cell or cell line can comprise one or more transcribed and / or translated exogenous sequences, which are stably or transiently introduced into the cell. Non-limiting examples of cells include bacterial cells such as E. coli cells, insect cells, yeast cells, or mammalian cells. In some embodiments, the host cells are bone marrow cells, umbilical cord blood cells, hematopoietic stem and progenitor cells (HSPCs), peripheral blood CD34 cells, or the like. + Cells, peripheral blood CD34 + and CD90 + The subject matter may be a cell, a mammalian cell, or any combination thereof.

[0176] In preferred embodiments, the host cell is a mammalian cell. In more preferred embodiments, the mammalian cell is a human cell. In certain embodiments, the host cell is a stem cell. In certain embodiments, the host cell is a primary cell. In certain embodiments, the host cell is selected from the group consisting of primary blood cells and primary mesenchymal cells. In certain embodiments, the primary cell is selected from the group consisting of primary stem cells, primary progenitor cells, and primary somatic cells. In certain embodiments, the primary stem cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and organ stem cells. In certain embodiments, the progenitor cell is selected from the group consisting of hematopoietic progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, multipotent progenitor cells, oligopotent progenitor cells, and lineage-restricted progenitor cells. In certain embodiments, the somatic cell is selected from the group consisting of fibroblasts, hepatocytes, cardiac cells, liver cells, pancreatic cells, muscle cells, skin cells, blood cells, neural cells, and immune cells. In certain embodiments, the immune cells are selected from the group consisting of T lymphocytes (T cells), B lymphocytes (B cells), small lymphocytes, natural killer cells (NK cells), natural killer T cells, macrophages, monocytes, monocyte precursors, eosinophils, neutrophils, basophils, megakaryocytes, myeloblasts, mast cells, and dendritic cells. In certain embodiments, the primary cells are CD34 + are hematopoietic cells or progenitor cells.

[0177] In any of the above embodiments, the genetic locus of the cell may contain one or more mutations associated with a disease or encode an aberrant protein. In certain embodiments, the disease is selected from the group consisting of hemoglobinopathies, viral infections, X-linked severe combined immunodeficiency, Fanconi anemia, hemophilia, neoplasms, cancer, alpha-1 antitrypsin deficiency, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, cystic fibrosis, blood diseases and disorders, inflammation, immune system diseases or disorders, metabolic diseases, liver diseases and disorders, kidney diseases and disorders, muscle diseases and disorders, bone or cartilage diseases and disorders, nerve and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and lysosomal storage disorders. In certain embodiments, the hemoglobinopathies are sickle cell disease, α-thalassemia, β-thalassemia, or δ-thalassemia.

[0178] Gene editing system In any of the above embodiments, the host cell can further comprise one or more components of a gene editing system. In any of the above embodiments, the one or more components of the gene editing system can comprise a nuclease capable of generating a double-stranded break within a locus of the cell. In any of the above embodiments, the one or more components of the gene editing system can further comprise a donor polynucleotide.

[0179] In any of the above embodiments, the nuclease can comprise a CRISPR nuclease or a polynucleotide or expression vector encoding the CRISPR nuclease, and a single guide RNA (sgRNA) or a polynucleotide or expression vector encoding the sgRNA that can hybridize to the target sequence in the locus.In certain embodiments, the CRISPR nuclease can be a Cas protein.For example, but not limited to, the Cas protein can be Cas9 or its high-fidelity mutant. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, Csax, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or variants thereof. In certain embodiments, the sgRNA and CRISPR nuclease can form into a ribonucleoprotein (RNP) complex.

[0180] In any of the above embodiments, the sgRNA may comprise one or more chemically modified nucleotides. In such embodiments, the one or more chemically modified nucleotides may be selected from the group consisting of 2'-O-methyl nucleotides, 2'-O-methyl 3'-phosphorothioate nucleotides, and 2'-O-methyl 3'-thio PACE nucleotides. In any of the above embodiments, the 5'-end, 3'-end, or a combination thereof of the modified sgRNA comprises modified nucleotides. In some embodiments, the modified sgRNA comprises one, two, or three consecutive or non-consecutive modified nucleotides at or near its 5'-end and / or one, two, or three consecutive or non-consecutive modified nucleotides at or near its 3'-end. In some embodiments, the modified sgRNA comprises three consecutive modified nucleotides at its 5'-end and three consecutive modified nucleotides at its 3'-end. Modified sgRNAs can be chemically synthesized using methods known in the art.

[0181] In any of the above embodiments, the donor polynucleotide can be present in a viral vector.In a particular aspect, the viral vector can be an adeno-associated virus (AAV) vector containing the donor polynucleotide.Other viral vectors useful for gene therapy methods are known in the art.For example, the construct of the present disclosure can include alphavirus, herpesvirus, retrovirus, lentivirus, or vaccinia virus.The polynucleotide can be integrated into the genome of a cell by using a system such as the CRISPR system.

[0182] The donor polynucleotide sequence typically encodes a recombinant molecule to be expressed in the cells, e.g., for use in cell therapy. The processing steps of the method can additionally or alternatively include all or part of washing, diluting, selecting, isolating, separating, culturing, stimulating, packaging, and / or formulating the cells. Such methods typically allow for processing, e.g., selection or separation and / or transduction, of cells on a large scale (e.g., in a composition volume of about 50 mL or greater).

[0183] In some embodiments, the gene editing system utilizes site-specific nucleases to knock out target genomic sequences or knock in exogenous sequences, and to transfer exogenous sequences into cells by viral transduction using recombinant viral vectors.

[0184] In some embodiments, the gene editing system comprises a nuclease introduced into a cell, the nuclease capable of generating a double-strand break within or near a genomic target site. This can be useful for increasing the frequency of HDR and homologous recombination at or near the break site. Gene editing nucleases useful in the methods provided herein include, but are not limited to, TAL-effector DNA-binding domain-nuclease fusion proteins (TALENs), site-specific recombinases (e.g., serine recombinases or tyrosine recombinases), integrases (FLP, Cre, lambda integrase) or resolvases, transposases, zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or variants thereof.

[0185] In some embodiments, the Cas nuclease can be in the form of a protein. In some embodiments, the Cas nuclease can be in the form of a plasmid, such that cells harboring this expression construct can express the Cas nuclease. In other embodiments, the Cas nuclease is pre-complexed with a guide RNA and introduced into cells as a ribonucleoprotein (RNP). In some embodiments, the Cas nuclease and guide polynucleotide sequence are introduced into CD34+ cells by electroporation.

[0186] Introduction of the donor polynucleotide can be achieved by viral transduction using a delivery vector such as an adeno-associated virus (AAV). Any serotype or pseudotype of AAV can be used. Certain AAV vectors are derived from single-stranded (ss) DNA parvoviruses that are nonpathogenic to mammals. Briefly, the rep and cap viral genes, which can account for 96% of a typical wild-type AAV genome, can be removed in the generation of certain AAV vectors, leaving adjacent inverted terminal repeats (ITRs) that can be used to initiate viral DNA replication, packaging, and integration. Wild-type AAV integrates into the human host cell genome with preferential site specificity at chromosome 19q13.3. Alternatively, AAV can be maintained episomally. To date, at least 12 human AAV serotypes (AAV serotype 1 (AAV-1) to AAV-12) and over 100 non-human primate-derived serotypes have been discovered. Any of these serotypes, or any combination thereof, can be used within the scope of the present disclosure. The serotype of the viral vector can be selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the serotype is AAV6.

[0187] In some embodiments in which a donor polynucleotide is delivered into cells by a viral vector (e.g., an AAV vector), the use of the 53BP1 inhibitory polypeptides provided herein can advantageously increase the rate of integration of the donor sequence by HDR at a given multiplicity of infection (MOI) of the viral vector, compared to the rate of integration achieved without the use of the 53BP1 inhibitory polypeptide. Without being bound by theory, it is believed that the higher integration rate achieved by the 53BP1 inhibitory polypeptides provided herein allows for a lower viral MOI, and therefore, a reduced DNA damage response (DDR) to the introduction of the viral vector into host cells.

[0188] composition In some embodiments, a composition is provided, the composition comprising a polypeptide of any of the above embodiments in admixture with a carrier, additive, and / or diluent. In some embodiments, a composition is provided, the composition comprising a polynucleotide of any of the above embodiments in admixture with a carrier, additive, and / or diluent. In some embodiments, a composition is provided, the composition comprising a vector of any of the above embodiments in admixture with a carrier, additive, and / or diluent.

[0189] In some embodiments, the compositions of the present disclosure can further comprise an inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In certain embodiments, the DNA-PKcs inhibitor can be ADAD7648, M3814 / nedisertib, CC-115, or BAY-8400.

[0190] In any of the above embodiments, the composition can be a pharmaceutically acceptable composition. In any of the above embodiments, the carrier additive and / or diluent can be pharmaceutically acceptable. Guidance for preparing pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, (20th ed.) ed. AR Gennaro AR, 2000, Lippencott Williams & Wilkins.

[0191] Preparation method The polypeptides of the present disclosure can be prepared by standard methods in the art. For example, a polynucleotide encoding the polypeptide can be introduced into a host cell, for example, as part of an expression vector or expression cassette, and expressed in the host cell. In certain aspects, the host cell can be incubated in a medium under conditions sufficient to result in expression of the polypeptide, and the polypeptide can then be purified from the host cell or cell culture medium by methods known in the art.

[0192] Homologous recombination methods and compositions In some embodiments, a method for increasing homologous recombination in a cell is provided, the method comprising introducing into the cell the polypeptide, polynucleotide, or vector of any of the above embodiments. In any of the above embodiments, the method can further comprise introducing into the cell one or more components of a gene editing system described herein.

[0193] In some embodiments, the typical number of cells harvested from a donor prior to gene editing is about 1 x 10 4 ~1×10 5 , 1×10 5 ~1×10 6 , 1×10 6 ~1×10 7 In some embodiments, the cell number is at least about 1 x 10 cells / kg, or more, prior to gene editing. 5 ~1×10 7 In some embodiments, at least about 1 x 10 cells / kg are harvested. 4 , 2 × 10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2 × 10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 107 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , or approximately 1 × 10 8 The cells / kg are harvested, and then gene editing of the harvested cells proceeds. In some embodiments, the cells to be edited can be any type of host cell described herein.

[0194] Methods for introducing polypeptides, nucleic acids, and viral vectors (e.g., viral particles) into primary cells, target cells, or host cells are known in the art. Any known method can be used to introduce a polypeptide or nucleic acid (e.g., a nucleotide sequence encoding a DNA nuclease or a modified sgRNA) into primary cells, such as human primary cells. Non-limiting examples of suitable methods include electroporation (e.g., nucleofection), viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun bombardment, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery transfection, and the like.

[0195] Clearly, the cells so produced may be useful as gene-edited cells in therapeutic applications.

[0196] Uses of Polypeptides The polypeptides and / or polynucleotides of the present disclosure can be used in a wide range of applications. The polypeptides and / or polynucleotides can be used for the detection and quantitative measurement, as well as the separation and isolation of 53BP1. The present disclosure also provides uses of the polypeptides and / or polynucleotides disclosed herein for pharmaceutical use, particularly for the treatment of genetic disorders, and for the gene therapy and cell therapy methods described herein.

[0197] The polypeptides and polynucleotides disclosed herein can be used for genome engineering, epigenome engineering, genome targeting, and genome editing. The polypeptides and polynucleotides disclosed herein can be used to modify repair pathways, activate or stimulate HR or homology-based genome editing, inhibit 53BP1 recruitment to DSB sites or damaged chromatin in cells, or regulate DNA end resection. In one aspect, the polypeptides and polynucleotides disclosed herein can be used in combination with a gene editing system. In some such embodiments, the gene editing system includes a nuclease capable of generating a double-strand break within a locus in a cell and a donor polynucleotide. In embodiments, the polypeptides and / or polynucleotides are administered in combination with one or more elements or components of a CRISPR system. In embodiments, the polypeptides and / or polynucleotides are administered before, simultaneously with, or after one or more elements or components of a CRISPR system. In embodiments, the polypeptide and / or polynucleotide is administered before, simultaneously with, or after one or more elements or components of the CRISPR / Cas system. In embodiments, the polypeptide and / or polynucleotide is administered before, simultaneously with, or after one or more elements or components of the CRISPR / Cpf1 system.

[0198] In one aspect, there is provided a method for manipulating a DSB repair pathway in a cell during a genome engineering reaction, the method comprising introducing into the cell a polypeptide and / or polynucleotide disclosed herein.

[0199] In one aspect, there is provided a method of stimulating homology directed repair of a DSB in a cell, the method comprising introducing into the cell a polypeptide and / or polynucleotide disclosed herein.

[0200] In one aspect, a method for inhibiting 53BP1 recruitment to DSB sites in a cell is provided, the method comprising introducing into the cell a polypeptide and / or polynucleotide disclosed herein. Suppression of 53BP1 recruitment to DSB sites by the polypeptide can be monitored by methods known in the art, such as ionizing radiation focus formation.

[0201] In one aspect, a method is provided for inhibiting 53BP1 recruitment to damaged chromatin in a cell, the method comprising introducing into the cell a polypeptide and / or polynucleotide disclosed herein.

[0202] In one aspect, a method of inhibiting the function of 53BP1 in a cell is provided, the method comprising introducing into the cell a polypeptide and / or polynucleotide disclosed herein.

[0203] In one aspect, a method for increasing HR in a cell is provided, the method comprising introducing into the cell a polypeptide, polynucleotide, and / or expression vector disclosed herein. In certain embodiments, the method can further comprise introducing into the cell one or more components of a gene editing system described herein. Of course, the cell can be any host cell described herein.

[0204] In one aspect, there is provided a method of inducing DNA end resection in a cell, the method comprising introducing into the cell a polypeptide and / or polynucleotide disclosed herein.

[0205] In one aspect, there is provided a method of inducing BRCA1 recruitment to DSB sites in a cell, the method comprising introducing into the cell a polypeptide and / or polynucleotide disclosed herein.

[0206] In one aspect, a method for inhibiting the function of 53BP1 at a DSB site in a cell is provided, the method comprising introducing into the cell a polypeptide and / or polynucleotide disclosed herein.

[0207] In one aspect, there is provided a method of inducing gene conversion in a cell, the method comprising introducing into the cell a polypeptide and / or polynucleotide disclosed herein.

[0208] In one aspect, provided herein is a method of increasing the efficiency of gene editing by HDR in a cell contacted with a CRISPR system, the method comprising introducing into the cell a polypeptide and / or polynucleotide disclosed herein.

[0209] In one aspect, a method for improving gene targeting in a cell is provided, the method comprising introducing into a cell a polypeptide and / or polynucleotide disclosed herein in combination with an inhibitor of DNA-PK.

[0210] In one aspect, a method for modulating DNA end resection in a cell is provided, the method comprising introducing into a cell a polypeptide and / or polynucleotide disclosed herein in combination with an inhibitor of DNA-PK.

[0211] In one embodiment, the DNA-PK inhibitor is NU7441 (8-dibenzothiophen-4-yl-2-morpholin-4-yl-chromen-4-one, also known as KU-57788, Leahy JJ, et al. Bioorg Med Chem Lett, 2004, 14(24), 6083-608), KU-0060648 (Munck JM, et al. Mol Cancer Ther, 2012, 11(8), 1789-1798), NU7026 (Willmore E, et al. Blood, 2004, 103(12), 4659-466), or PIK-75 (WO / 2003 / 072557, September 4, 2003). In some embodiments, the DNA-PK inhibitor is ADAD7648, M3814 / nedisertib, CC-115, or BAY-8400.

[0212] In certain embodiments, the methods of the present disclosure are used to treat cells in the G1 phase of the cell cycle (G1) or the G0 phase of the cell cycle.

[0213] In one aspect, a method of stimulating HR in a non-dividing cell is provided, the method comprising introducing into the cell a polypeptide and / or polynucleotide disclosed herein.

[0214] In other aspects, the methods of the disclosure are performed on or used to treat cells that contain DSBs engineered for the purposes of genome modification or gene editing.

[0215] The present disclosure also contemplates the use of the methods, compositions, and kits disclosed herein in genome modification, provided that such use is not a method for treating the human or animal body by surgery or therapy, nor is such use a process for modifying the genetic identity of the human germline. Genome modification may include modifying a target polynucleotide sequence in a cell, modifying the expression of a polynucleotide sequence in a cell, generating model cells containing a mutant disease gene, or knocking out a gene. The use of the present disclosure may further include repairing or editing a cut target polynucleotide by inserting an exogenous template polynucleotide, where the repair or editing results in a mutation including an insertion, deletion, or substitution of one or more nucleotides in the target polynucleotide.

[0216] The methods, compositions, and kits disclosed herein are also contemplated for use in genome engineering, provided that such use is not a method for treating the human or animal body by surgery or therapy, nor is such use a process for altering the genetic identity of the human germline. Genome engineering can include modifying a target polynucleotide sequence in a cell, modifying the expression of a polynucleotide sequence in a cell, generating model cells containing a mutant disease gene, knocking in or out a gene, or correcting one or more mutations within a gene locus. The use of the present disclosure can further include repairing or editing a cut target polynucleotide by inserting an exogenous template polynucleotide, where the repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides in the target polynucleotide.

[0217] Methods for homologous recombination repair of engineered DSBs in cells for genome modification are provided, the methods comprising introducing into the cells the polypeptides and / or polynucleotides disclosed herein.

[0218] The present disclosure relates to the use of the polypeptides and / or polynucleotides disclosed herein for the purpose of genome modification in homologous recombination repair of engineered DSBs.

[0219] The present disclosure relates to the use of the polypeptides and / or polynucleotides disclosed herein in homology directed repair using single-stranded oligonucleotides (ssODNs).

[0220] The present disclosure also relates to the use of the polypeptides and / or polynucleotides disclosed herein to stimulate homology directed repair using single-stranded oligonucleotides (ssODNs).

[0221] In one embodiment, the method disclosed herein for activating or stimulating HR in a cell further comprises a gene editing system. In one embodiment, the gene editing system comprises contacting the cell with a nuclease. Examples of nucleases include, but are not limited to, zinc finger nucleases (ZFNs), engineered meganucleases, transcription activator-like effector nucleases (TALENs), megaendonucleases or homing endonucleases, clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nucleases, Cpf1 nucleases, Ttago nucleases, and fusions between nucleases, such as megaTALs and compact TALENs. In one embodiment, the gene editing step comprises a CRISPR / Cas9 system. In one embodiment, the gene editing step comprises a CRISPR / Cpf system.

[0222] A method for stimulating homology-based genome editing in a cell is provided, the method comprising introducing into the cell a polypeptide disclosed herein.

[0223] In some embodiments, the gene editing system can correct a genomic mutation. The genetic mutation can include at least one mutation in a polynucleotide sequence having a locus associated with a genetic disorder. In one aspect, the genomic mutation is selected from the group consisting of an insertion, a deletion, and a combination thereof. In some embodiments, the genetic disorder is a monogenic disorder. In some embodiments, the disorder is a polygenic disorder. In some embodiments, the disorder is associated with one or more SNPs. In certain embodiments, the genomic modification corrects a point mutation.

[0224] In one aspect of the disclosed method of correcting a genomic mutation, the gene editing system includes contacting a cell with a clustered regularly interspaced short palindromic repeats (CRISPR) associated (Cas) protein and one or two ribonucleic acids, where the ribonucleic acids guide the Cas protein to a selected motif in a target polynucleotide sequence associated with the genetic disorder and hybridize to the selected motif in the target polynucleotide sequence associated with the genetic disorder, and the target polynucleotide sequence is cleaved.

[0225] Targeted gene insertion and integration The present disclosure provides compositions and methods for introducing an exogenous polynucleotide sequence into a target site of an endogenous polynucleotide sequence at a genetic locus. In some embodiments, the polynucleotide sequence may contain at least one mutation. In some embodiments, the mutation may cause aberrant expression, which may manifest as a disease symptom. Described herein are methods for correcting or ameliorating aberrant expression caused by a mutation associated with a disease state.

[0226] The CRISPR-Cas system has rapidly emerged as an attractive tool for introducing double-strand breaks. Briefly, the CRISPR-Cas system utilizes a guide RNA or guide polynucleotide to guide the Cas nuclease to the target site, where it introduces a double-strand break in the sequence.

[0227] Can utilize HDR mechanism by simultaneously using donor template or donor polynucleotide sequence.HDR mechanism can excise donor polynucleotide sequence through the region of donor polynucleotide with high homology or sequence identity to make endogenous sequence.In this way, target gene insertion can be achieved by administering site-specific nuclease system in combination with donor polynucleotide.

[0228] In embodiments, the donor polynucleotide comprises an exogenous sequence (including coding and non-coding regulatory sequences). The exogenous sequence is flanked by regions of high homology with the targeted endogenous locus. In some embodiments, the target gene insert can replace at least a portion of the endogenous polynucleotide sequence. In certain embodiments, the exogenous sequence is integrated into the translation start site of the target locus. In certain embodiments, the exogenous sequence integrated into the host cell genome is expressed under the control of the native promoter sequence used by the target locus.

[0229] The endogenous polynucleotide may contain a polymorphism or mutation that results in the expression of an abnormal protein that results in disease symptoms. In some embodiments, the endogenous polynucleotide sequence contains a mutation. Such mutations include, but are not limited to, missense and nonsense mutations. In some embodiments, the endogenous polynucleotide sequence may contain an insertion, deletion, or truncation.

[0230] Donor polynucleotide In any of the above embodiments, donor polynucleotide can be contained in a virus vector, a plasmid, a single-stranded oligodeoxynucleotide (ssODN) or any other suitable vector.In certain embodiments, donor polynucleotide can be contained in an adeno-associated virus (AAV) vector.In certain embodiments, the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12.In a preferred embodiment, the AAV vector is AAV6.

[0231] In any of the above embodiments, the donor polynucleotide comprises non-overlapping 5' and 3' homologous arms, where each homologous arm is homologous to a portion of the locus, and upon generation of a double-stranded break within the locus by a nuclease, the donor polynucleotide sequence is integrated into the locus by homology-directed repair (HDR).

[0232] In certain embodiments, integration of the donor polynucleotide sequence into the host cell genome can correct a mutation in the cell that is associated with disease, hi certain embodiments, integration of the donor polynucleotide sequence replaces a mutant allele in the host cell with a wild-type allele.

[0233] Homologous arms In preferred embodiments, the 5' and 3' homologous arms of the donor polynucleotide each have at least 95% sequence identity with distinct regions of the target locus, such that HDR of the exogenous polynucleotide occurs exclusively via the 5' and 3' homologous arms. The entire exogenous polynucleotide sequence between the homologous arms is then integrated into the target locus. In some embodiments, the homologous arms contain sequences that direct integration of the donor polynucleotide immediately downstream of the target gene's native promoter, thereby ensuring that the integrated donor sequence is transcribed from and regulated by the target gene's native promoter sequence. In other embodiments, the homologous arms contain sequences that direct integration of the donor polynucleotide into the locus, thereby replacing the target gene in whole or in part, e.g., only the region of the target gene that harbors a mutation. In some such embodiments, the target gene promoter remains intact to control transgene expression.

[0234] The homology arms can be of various lengths. In some embodiments, the 5' and 3' homology arms can be the same length. In some embodiments, the 5' and 3' homology arms can be different lengths.

[0235] In some embodiments, the 5' homologous arm comprises about 50 base pairs to about 1,000 base pairs. In some embodiments, the 5' homologous arm comprises at least about 50 base pairs. In some embodiments, the 5' homologous arm comprises at most about 1,000 base pairs. In some embodiments, the 5' homologous arm comprises about 50 base pairs to about 100 base pairs, about 50 base pairs to about 150 base pairs, about 50 base pairs to about 200 base pairs, about 50 base pairs to about 250 base pairs, about 50 base pairs to about 300 base pairs, about 50 base pairs to about 350 base pairs, about 50 base pairs to about 400 base pairs, about 50 base pairs to about 450 base pairs, about 50 base pairs to about 500 base pairs, about 50 base pairs to about 750 base pairs, about 50 base pairs to about 1,000 base pairs, about 100 base pairs to about 150 base pairs, about 100 base pairs to about 200 base pairs, about 100 base pairs to about 250 base pairs, about 100 base pairs to about 300 base pairs, about 100 base pairs to about 350 base pairs, about 100 base pairs to about 400 base pairs, about 100 base pairs to about 450 base pairs, about 100 base pairs to about 500 base pairs, about 100 base pairs to about 750 base pairs, about 100 base pairs to about 1,000 base pairs, about 150 base pairs to about 200 base pairs, about 150 base pairs to about 250 base pairs, about 150 base pairs to about 300 base pairs, about 150 base pairs to about 350 base pairs, about 15 0 base pairs to about 400 base pairs, about 150 base pairs to about 450 base pairs, about 150 base pairs to about 500 base pairs, about 150 base pairs to about 750 base pairs, about 150 base pairs to about 1,000 base pairs, about 200 base pairs to about 250 base pairs, about 200 base pairs to about 300 base pairs, about 200 base pairs to about 350 base pairs, about 200 base pairs to about 400 base pairs, about 200 base pairs to about 450 base pairs, about 200 base pairs to about 500 base pairs, about 200 base pairs to about 750 base pairs, about 200 base pairs to about 1,000 base pairs, about 250 base pairs to about 300 base pairs, about 250 base pairs to about 350 base pairs, about 250 base pairs to about 400 base pairs, about 250 base pairs to about 450 base pairs, about 250 base pairs to about 500 base pairs, about 250 base pairs to about 750 base pairs, about 250 base pairs to about 1,000 base pairs, about 300 base pairs to about 350 base pairs, about 300 base pairs to about 400 base pairs, about 300 base pairs to about 450 base pairs, about 300 base pairs to about 500 base pairs, about 300 base pairs to about 750 base pairs, about 300 base pairs to about 1,000 base pairs, about 350 base pairs to about 400 base pairs, about 350 base pairs to about 450 base pairs, about 350 base pairs to about 500 base pairs, about 350 base pairs to about 750 base pairs, about 350 base pairs to about 1,000 base pairs, about 400 base pairs to about 450 base pairs, about 400 base pairs to about 500 base pairs, about 400 base pairs to about 750 base pairs, about 400 base pairs to about 1,000 base pairs, about 450 base pairs to about 500 base pairs, about 450 base pairs to about 750 base pairs, about 450 base pairs to about 1,000 base pairs, about 500 base pairs to about 750 base pairs, about 500 base pairs to about 1,000 base pairs, or about 750 base pairs to about 1,000 base pairs.

[0236] In some embodiments, the 3' homologous arm comprises about 50 base pairs to about 1,000 base pairs. In some embodiments, the 3' homologous arm comprises at least about 50 base pairs. In some embodiments, the 3' homologous arm comprises at most about 1,000 base pairs. In some embodiments, the 3' homologous arm comprises about 50 base pairs to about 100 base pairs, about 50 base pairs to about 150 base pairs, about 50 base pairs to about 200 base pairs, about 50 base pairs to about 250 base pairs, about 50 base pairs to about 300 base pairs, about 50 base pairs to about 350 base pairs, about 50 base pairs to about 400 base pairs, about 50 base pairs to about 450 base pairs, about 50 base pairs to about 500 base pairs, about 50 base pairs to about 750 base pairs, about 50 base pairs to about 1,000 base pairs, about 100 base pairs to about 150 base pairs, about 100 base pairs to about 200 base pairs, about 100 base pairs to about 250 base pairs, about 100 base pairs to about 300 base pairs, about 100 base pairs to about 350 base pairs, about 100 base pairs to about 400 base pairs, about 100 base pairs to about 450 base pairs, about 100 base pairs to about 500 base pairs, about 100 base pairs to about 750 base pairs, about 100 base pairs to about 1,000 base pairs, about 150 base pairs to about 200 base pairs, about 150 base pairs to about 250 base pairs, about 150 base pairs to about 300 base pairs, about 150 base pairs to about 350 base pairs, about 15 0 base pairs to about 400 base pairs, about 150 base pairs to about 450 base pairs, about 150 base pairs to about 500 base pairs, about 150 base pairs to about 750 base pairs, about 150 base pairs to about 1,000 base pairs, about 200 base pairs to about 250 base pairs, about 200 base pairs to about 300 base pairs, about 200 base pairs to about 350 base pairs, about 200 base pairs to about 400 base pairs, about 200 base pairs to about 450 base pairs, about 200 base pairs to about 500 base pairs, about 200 base pairs to about 750 base pairs, about 200 base pairs to about 1,000 base pairs, about 250 base pairs to about 300 base pairs, about 250 base pairs to about 350 base pairs, about 250 base pairs to about 400 base pairs, about 250 base pairs to about 450 base pairs, about 250 base pairs to about 500 base pairs, about 250 base pairs to about 750 base pairs, about 250 base pairs to about 1,000 base pairs, about 300 base pairs to about 350 base pairs, about 300 base pairs to about 400 base pairs, about 300 base pairs to about 450 base pairs, about 300 base pairs to about 500 base pairs, about 300 base pairs to about 750 base pairs, about 300 base pairs to about 1,000 base pairs, about 350 base pairs to about 400 base pairs, about 350 base pairs to about 450 base pairs, about 350 base pairs to about 500 base pairs, about 350 base pairs to about 750 base pairs, about 350 base pairs to about 1,000 base pairs, about 400 base pairs to about 450 base pairs, about 400 base pairs to about 500 base pairs, about 400 base pairs to about 750 base pairs, about 400 base pairs to about 1,000 base pairs, about 450 base pairs to about 500 base pairs, about 450 base pairs to about 750 base pairs, about 450 base pairs to about 1,000 base pairs, about 500 base pairs to about 750 base pairs, about 500 base pairs to about 1,000 base pairs, or about 750 base pairs to about 1,000 base pairs.

[0237] CRISPR systems In some embodiments, a CRISPR enzyme combined with (and optionally complexed with) a guide sequence is delivered to a cell. In some embodiments, methods for introducing protein components (e.g., Cas9 / gRNA RNP) into cells according to the present disclosure can be by physical delivery methods (e.g., electroporation, particle gun, calcium phosphate bombardment, cell compaction, or cell squeezing), liposomes, or nanoparticles. In some embodiments, a target polynucleotide is modified in a eukaryotic cell. In some embodiments, the method includes binding a CRISPR complex to the target polynucleotide to effect cleavage of the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes to a target sequence within the target polynucleotide, the guide sequence being linked to a tracr mate sequence, which in turn hybridizes to the tracr sequence.

[0238] Binding of the polynucleotide sequence recruits a Cas protein and promotes double-stranded cleavage of the polynucleotide sequence by the Cas nuclease. In some embodiments, the guide polynucleotide sequence binds to a region of a gene corresponding to a coding sequence. In some embodiments, the coding sequence is an exon. In some embodiments, the guide polynucleotide can bind to a region of a gene corresponding to a non-coding region. In some embodiments, the non-coding region is an intron or untranslated region (UTR).

[0239] In some embodiments, the guide polynucleotide sequence comprises a chemical modification. In some embodiments, the guide polynucleotide sequence comprises a 2'-O-methyl-3'-phosphorothioate modification. Examples of chemical modifications for guide polynucleotide sequences that enhance the stability and cleavage efficiency of the CRISPR-Cas system include, but are not limited to, those described in PCT Publications WO / 2016164356 and WO2016 / 089433 (each of which is incorporated herein by reference in its entirety).

[0240] Delivery Vector The donor polynucleotide sequences described herein can be incorporated into a variety of gene therapy constructs, for example, to deliver nucleic acids encoding proteins to subjects in need thereof. A vector construct refers to a polynucleotide molecule containing all or part of a viral genome and an exogenous polynucleotide sequence. In some cases, gene transfer can be mediated by a DNA viral vector such as an adenovirus (Ad) or an adeno-associated virus (AAV). Other vectors useful for gene therapy methods are known in the art. For example, the constructs of the present disclosure can include alphaviruses, herpesviruses, retroviruses, lentiviruses, or vaccinia viruses.

[0241] Adenoviruses are a relatively well-characterized group of viruses, including more than 50 serotypes. Adenoviruses are easy to handle by applying molecular biology techniques and do not require integration into the host cell genome. Recombinant Ad-derived vectors have been constructed, including vectors that reduce the possibility of recombination and generation of wild-type viruses. Wild-type AAV has high infectivity and can be integrated into the host genome with high specificity.

[0242] Any serotype or pseudotype of AAV can be used. Certain AAV vectors are derived from single-stranded (ss) DNA parvoviruses that are nonpathogenic to mammals. Briefly, the rep and cap viral genes, which can account for 96% of a typical wild-type AAV genome, can be removed in the generation of certain AAV vectors, leaving adjacent inverted terminal repeats (ITRs) that can be used to initiate viral DNA replication, packaging, and integration. Wild-type AAV integrates into the human host cell genome with preferential site specificity at chromosome 19q13.3. Alternatively, AAV can be maintained episomally.

[0243] To date, at least 12 human serotypes of AAV (AAV serotype 1 (AAV-1) through AAV-12) and over 100 serotypes from non-human primates have been discovered. Any of these serotypes, or any combination thereof, can be used within the scope of the present disclosure.

[0244] The serotype of the viral vector used in certain embodiments of the present invention can be selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. Other serotypes are known in the art or described herein and are applicable to the present disclosure. In certain instances, the present invention includes an AAV9 viral vector comprising a glucocerebrosidase nucleic acid of the present invention.

[0245] Treatment methods In some embodiments, the polypeptides or compositions of the present disclosure can be used to treat diseases by introducing the polypeptide into cells targeted for HDR gene editing. In certain aspects, the method is not a process for altering the genetic identity of the human germline. By way of example and without limitation, the disease can be selected from the group consisting of cancer, cardiovascular disease (e.g., heart failure, hypertension, and atherosclerosis), respiratory disease, kidney disease, gastrointestinal disease (e.g., inflammatory bowel disease, e.g., Crohn's disease and ulcerative colitis), liver, gallbladder, and bile duct diseases (e.g., hepatitis and cirrhosis), blood diseases, metabolic diseases, endocrine diseases, and reproductive diseases (e.g., diabetes), bone and bone mineral metabolism diseases, immune system diseases (e.g., autoimmune diseases, e.g., rheumatoid arthritis, lupus erythematosus, and other autoimmune diseases), musculoskeletal and connective tissue diseases (e.g., arthritis, achondroplasia), infectious diseases, and neurological diseases (e.g., Alzheimer's disease, Huntington's disease, and Parkinson's disease).

[0246] Embodiments of the present disclosure provide for the treatment of various cancers, including but not limited to carcinoma, melanoma, lymphoma, sarcoma, blastoma, leukemia, myeloma, osteosarcoma, neuro-oncology, and cancers of organs such as breast, ovarian, and prostate.

[0247] In embodiments, treatments are provided for cancers associated with BRCA-1 deficiency, BRCA-2 deficiency, BRCA-1 / BRCA-2 double deficiency, and Fanconi anemia. In some embodiments, the cancer is breast cancer, particularly invasive ductal carcinoma and invasive lobular carcinoma. In some embodiments, the cancer is ovarian cancer, particularly epithelial ovarian tumors, germ cell ovarian tumors, and sex cord-stromal tumors.

[0248] The method disclosed herein for activating homologous recombination can be used to genetically modify the polynucleotide associated with genetic disorders.In some embodiments, the genetic disorder is a single-gene disorder.In some embodiments, the genetic disorder is a polygenic disorder.In some embodiments, the genetic disorder is associated with one or more SNPs.In certain embodiments, the genome modification corrects point mutations.

[0249] Examples of genetic disorders and polynucleotide sequences associated with genetic disorders can be found on the World Wide Web (see, e.g., the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Mass.) or the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.)), in published patents and applications (see, e.g., U.S. Published Application No. 2015 / 0247150), and in publications (see, e.g., Turitz Cox DB et al, Nature Medicine 21, 121-131, 2015, and O'Connor TP and RG Crystal, Nature Reviews / Genetics Volume 7, April 2006, pages 261-276 (including supplementary information and publications cited therein)).

[0250] In one embodiment, the genetic disorder is a genetic disorder of muscle. In one embodiment, the genetic disorder is myotonic dystrophy type 1. In one embodiment, the genetic disorder is myotonic dystrophy type 2. In one embodiment, the genetic disorder is Duchenne muscular dystrophy (DMD). In one embodiment, the genetic disorder is Becker muscular dystrophy.

[0251] In one aspect, the genetic disorder is a genetic disorder of the liver, such as alpha-1 antitrypsin deficiency, Wilson's disease, hereditary hemochromatosis, tyrosinemia type I, glycogen storage disease type IV, argininosuccinate lyase deficiency, citrin deficiency, cholesterol ester storage disease, and hereditary fructose intolerance.

[0252] In one aspect, the genetic disorder is alpha-1 antitrypsin deficiency, which is an autosomal recessive (codominant) disease caused by mutations in the SERPINA1 gene, which encodes the serine protease inhibitor AAT.

[0253] In one embodiment, the genetic disease is Wilson's disease, which depends on mutations in the gene encoding ATP7B Cu translocase, a protein expressed primarily by hepatocytes and which regulates copper levels in the liver.

[0254] In one aspect, the genetic disorder is a genetic disorder of the lung.

[0255] In one aspect, the genetic disorder is cystic fibrosis, which is an autosomal recessive genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein (a member of the ATP-binding cassette superfamily of transmembrane proteins).

[0256] In other aspects, the genetic disorder can be hemophilia, alpha 1-antitrypsin deficiency, Canavan disease, adenosine deaminase deficiency, X-linked severe combined immunodeficiency, familial amyloidotic polyneuropathy, thalassemia, Tay-Sachs disease, late-onset infantile ceroid lipofuscinosis, mucopolysaccharidoses, Niemann-Pick disease, achondroplasia, Huntington's disease, spinocerebellar ataxia, Friedreich's ataxia, amyotrophic lateral sclerosis, monogenic hypercholesterolemia, and other monogenic disorders.

[0257] In certain embodiments, the genetic disorder is sickle cell anemia, and the methods disclosed herein involve correcting the mutant HBB hemoglobin gene by gene conversion with its paralog HBD.

[0258] In some embodiments, the gene-edited cells are hematopoietic stem or progenitor cells. Genetically modified hematopoietic stem or progenitor cells produced using the compositions and methods provided herein can be used as part of a treatment plan for any disease or condition for which HSC transplantation (HSCT) is useful. HSCT can be used to treat many diseases, including congenital and acquired diseases. In some embodiments, acquired diseases treatable with HSCT include (1) malignancies (e.g., hematological malignancies (e.g., leukemias (e.g., acute lymphoblastic leukemia (ALL)), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), lymphomas (e.g., Hodgkin's disease, non-Hodgkin's lymphoma), myelomas (e.g., multiple myeloma (Kahler's disease)), solid tumor cancers (e.g., neuroblastoma, desmoplastic small round cell tumor, Ewing's sarcoma, choriocarcinoma), (2) hematological disorders (e.g., phagocytic disorders (e.g., chronic granulomatous disease), bone marrow deficiency, etc.), and (3) hematological disorders (e.g., phagocytic disorders (e.g., chronic granulomatous disease), bone marrow deficiency, etc.). (1) amyloidosis (e.g., amyloid light chain (AL) amyloidosis), (2) anemia (e.g., erythroderma, erythrocytosis congenita), (3) amyloidosis (e.g., amyloid light chain (AL) amyloidosis), (4) anemia (e.g., erythroderma, erythrocytosis congenita), (5) anemia (e.g., erythroderma, erythrocytosis vera), (6) anemia (e.g., erythroderma, erythrocytosis vera), (7) anemia (e.g., erythroderma, erythrocytosis vera), (8) anemia (e.g., erythroderma, erythrocytosis vera), (9) anemia (e.g., erythroderma, erythrocytosis vera), (10) anemia (e.g., erythroderma, erythroderma vera), (11) anemia (e.g., erythroderma, erythroderma vera), (12) anemia (e.g., erythroderma, erythroderma vera), (13) anemia (e.g., erythroderma, erythroderma vera), (14) anemia (e.g., erythroderma vera), (15) anemia (e.g., erythroderma vera), (16) anemia (e.g., erythroderma vera), (17) anemia (e.g., erythroderma vera), (18) anemia (e.g., erythroderma vera), (19) anemia (e.g., erythroderma vera), (19) anemia (e

[0259] In some embodiments, congenital diseases treatable with HSCT include (1) lysosomal storage diseases (e.g., lipidoses (disorders of lipid storage, e.g., neuronal ceroid lipofuscinosis (e.g., infantile neuronal ceroid lipofuscinosis (INCL, Santavuori disease) and Jansky-Bierschowski disease (late-onset infantile neuronal ceroid lipofuscinosis))), sphingolipidoses (e.g., Niemann-Pick disease and Gaucher disease), leukodystrophies (e.g., adrenoleukodystrophy, metachromatic leukodystrophy, Krabbe disease (globoid cell leukodystrophy)), mucopolysaccharidoses (e.g., Hurler syndrome (MPS IH, α-L-iduronidase deficiency), Scheie syndrome (MPS IS), Hurler-Scheie syndrome (MPS I HS), Hunter syndrome (MPS II, iduronidase sulfate deficiency), Sanfilippo syndrome (MPS (1) immunodeficiencies (e.g., T-cell deficiencies (e.g., ataxia-telangiectasia and DiGeorge syndrome), combined T-cell and B-cell deficiencies (e.g., severe combined immunodeficiency (SCID) of all types), well-defined syndromes (e.g., Wiskott-Aldrich syndrome), phagocytic disorders (e.g., Kostmann syndrome, Shwachman-Diamond syndrome), immune dysregulation disorders (e.g., , Griscelli syndrome, type II), innate immune deficiencies (e.g., NF-kappa-B essential modulator (NEMO) deficiency (inhibitor of kappa light polypeptide gene enhancer in B-cell gamma kinase deficiency)), (3) blood disorders (e.g., hemoglobinopathies (e.g., sickle cell disease, thalassemia (e.g., β-thalassemia)), anemias (e.g., aplastic anemias, e.g., Diamond-Blackfan anemia and Fanconi anemia), cytopenias (e.g., amegakaryocytic thrombocytopenia), and hemophagocytic syndromes (e.g., hemophagocytic lymphohistiocytosis (HLH))).

[0260] In some embodiments, the disease or condition is selected from the group consisting of hemoglobinopathies, viral infections, X-linked severe combined immunodeficiency, Fanconi anemia, hemophilia, neoplasms, cancer, amyotrophic lateral sclerosis, alpha antitrypsin deficiency, Alzheimer's disease, Parkinson's disease, cystic fibrosis, blood diseases and disorders, inflammation, immune system diseases or disorders, metabolic diseases, liver diseases and disorders, kidney diseases and disorders, muscle diseases and disorders, bone or cartilage diseases and disorders, nerve and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and lysosomal storage disorders. In some embodiments, the hemoglobinopathies are selected from the group consisting of sickle cell disease, α-thalassemia, β-thalassemia, and δ-thalassemia.

[0261] In one aspect, the target polynucleotide sequence is related to a genetic lung disorder.In one embodiment, the target polynucleotide sequence is related to cystic fibrosis, and in particular, the polynucleotide sequence is related to the cystic fibrosis transmembrane conductor receptor (CFTR) gene locus.CFTR mutations (for example, the deletion of phenylalanine at position 508 of exon 11) cause cystic fibrosis.

[0262] In one embodiment, the target polynucleotide sequence is associated with a genetic muscle disorder. In one embodiment, the target polynucleotide sequence is associated with muscular dystrophy. In one embodiment, the target polynucleotide sequence is associated with Duchenne muscular dystrophy (DMD) (mutation of the dystrophin gene). In one embodiment, the target polynucleotide sequence is associated with Becker muscular dystrophy (mutation of the dystrophin gene). In one embodiment, the target polynucleotide sequence is associated with myotonic dystrophy type 1 (mutation of the DMPK gene) or myotonic dystrophy type 2 (mutation of the CNBP gene).

[0263] In one embodiment, the target polynucleotide sequence is associated with sickle cell anemia (mutant HBB hemoglobin).

[0264] In one embodiment, the target polynucleotide sequence is associated with a genetic disorder of the liver. In one embodiment, the target polynucleotide sequence is associated with alpha-1 antitrypsin deficiency (mutation in the SERPINA1 gene). In one embodiment, the target polynucleotide sequence is associated with Wilson's disease (mutation in the gene encoding the ATP7B Cu translocase).

[0265] In one aspect, the methods of the present disclosure further include providing a functional protein having improved properties relative to its naturally occurring counterpart (particularly a functional protein that is missing or defective in a subject), e.g., to treat a genetic disorder. In embodiments, the methods include incorporating a sequence encoding the functional protein into cells of a subject in need thereof by sequentially administering a gene editing system and one or more transgene(s) encoding a non-naturally occurring protein having enhanced properties relative to its naturally occurring counterpart. In other embodiments, the methods include administering to the subject genetically modified cells that express a functional form of one or more proteins that are aberrantly expressed in the subject. Thus, isolated cells may be introduced into the subject (ex vivo cell therapy) or cells that are part of the subject may be modified (in vivo). In certain embodiments, the transgene(s) are delivered using viral vectors, non-viral vectors, and / or combinations thereof.

[0266] The components of the disclosed methods can be delivered by delivery systems known in the art. Such delivery systems include, but are not limited to, viral-based or non-viral-based systems (see, e.g., Sambrook et al., supra; Findeis, Mark A., editor, Nonviral vectors for gene therapy: methods and protocols (Totowa, NJ: Humana Press, c2001); Rolland, Alain and Sullivan, Sean M., editors, Pharmaceutical gene delivery systems (New York: Marcel Decker, c2003); Rolland, Alain, editor, Advanced gene delivery: from concepts to pharmaceutical products (Amsterdam: Harwood Academic, c1999); K. Kataoka, Taira, K., Niidome, T. editors, Non-viral gene therapy: gene design and delivery (Tokyo; New York: Springer, c2005); and S. Lasic, Danilo D., Liposomes in gene delivery (Boca Raton, Fla.: CRC Press, c2006). Press, 1997).

[0267] Typical virus-based systems may include, for example, retrovirus, lentivirus, adenovirus, adeno-associated virus, SV40, polyomavirus, papillomavirus, picornavirus, poxvirus, helper-dependent adenovirus, and herpes simplex virus gene transfer vectors. In one embodiment, the virus-based system is an adenovirus vector or an adeno-associated virus vector. Suitable plasmid expression vectors can also be used. Examples of plasmid expression vectors include, but are not limited to, expression vectors commercially available from Novagen (e.g., pET vectors, Rosetta™ (D3), Origami™ (DE3)), expression vectors commercially available from New England Labs, Inc. (e.g., pMAL™ vector), expression vectors commercially available from Invitrogen Inc. (e.g., pAd / CMV / VS-DEST™, pAd-DEST™ vector, pLenti4 / V5-DEST™), and expression vectors commercially available from Clontech (e.g., pAdeno X™ and pAd5F35).

[0268] Examples of non-viral-based systems include lipofection, nucleofection, electroporation, microinjection, sonoporation, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, mRNA, nanomaterial-based delivery, artificial virions, and drug-enhanced DNA uptake.

[0269] In one aspect, the component is a polypeptide disclosed herein, which is delivered into the cell by electroporation, sonoporation, microinjection, liposome delivery, or nanomaterial-based delivery.

[0270] In one aspect, the component is a polynucleotide disclosed herein, which is delivered into a cell using a vector (e.g., an adenoviral vector, a retroviral vector, an adeno-associated viral vector, a lentiviral vector, a herpes viral vector, an SV40 vector, a polyoma viral vector, a papilloma viral vector, a picorna viral vector, a pox viral vector, or a helper-dependent adenoviral vector).

[0271] In certain aspects, a method for integrating an exogenous polynucleotide sequence into the genome of a cell is provided. The method can include introducing into the cell (a) a nuclease capable of generating a double-strand break within a locus in the cell, (b) a donor polynucleotide, and (c) a 53BP1 inhibitory polypeptide or polynucleotide described herein or an expression vector thereof, wherein upon generation of the double-strand break within the locus by the nuclease, the donor polynucleotide sequence is integrated into the locus by homology-directed repair (HDR), resulting in a gene-edited cell. In some embodiments, the method is performed ex vivo. The nuclease, donor polynucleotide sequence, and 53BP1 inhibitory polypeptide can be a 53BP1 inhibitory polypeptide of any of the above embodiments. In such embodiments, the cell can be any host cell of the present disclosure. In some embodiments, the host cell is isolated from a donor, e.g., a healthy donor or a donor suffering from a genetic disease. In certain aspects, the resulting gene-edited cells can be administered to a patient, thereby allowing the patient to use the gene-edited cells for further cell therapy. In some embodiments, administering comprises autologous transplantation of a pharmaceutical composition comprising cells genetically modified according to the methods described herein. In other embodiments, administering comprises allogeneic transplantation of a pharmaceutical composition comprising cells genetically modified according to the methods described herein. As a non-limiting example, gene-edited cells can be administered to a patient suffering from sickle cell disease and edited to correct a mutation associated with sickle cell disease or to incorporate a wild-type allele. In certain aspects, integration of the donor polynucleotide sequence into the host cell genome corrects a mutation in the cell associated with the disease. In certain aspects, the genetic locus of the cell can contain one or more mutations associated with the disease or encode an aberrant protein. In certain embodiments, integration of the donor polynucleotide sequence replaces a mutant allele in the cell with a wild-type allele. The disease can be any disease disclosed herein.Non-limiting examples of diseases include hemoglobinopathies, viral infections, X-linked severe combined immunodeficiency, Fanconi anemia, hemophilia, neoplasms, cancer, alpha-1 antitrypsin deficiency, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, cystic fibrosis, blood diseases and disorders, inflammation, immune system diseases or disorders, metabolic diseases, liver diseases and disorders, kidney diseases and disorders, muscle diseases and disorders, bone or cartilage diseases and disorders, nerve and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and lysosomal storage disorders. Preferably, the hemoglobinopathies are sickle cell disease, α-thalassemia, β-thalassemia, or δ-thalassemia.

[0272] kit The present disclosure further provides kits for carrying out the assays or methods disclosed herein.

[0273] In some embodiments, a kit is provided, the kit comprising the polypeptide, polynucleotide, or vector of any of the above embodiments.

[0274] In some embodiments, a kit is provided, the kit comprising the polypeptide, polynucleotide, or vector of any of the above embodiments and one or more components of the gene editing system described herein. In any of the above embodiments, the kit further comprises an inhibitor of DNA-PKcs.

[0275] In any of the above embodiments, the kit may further comprise an activator of DNA end resection and an activator of homologous recombination as described herein. [Example]

[0276] The following examples are put forth so as to provide those of ordinary skill in the art with a full disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0277] Example 1: Development of a functional screen to identify improved peptide inhibitors of 53BP1

[0278] Materials and Methods

[0279] Molecular cloning:

[0280] Constructs for lentiviral-based expression and screening of i53 mutants were cloned from a third-generation lentiviral plasmid (Lenti SFFV) purchased from Twist Biosciences (SEQ ID NO: 73). An empty vector was constructed to contain BamHI and NsiI restriction enzyme sites upstream of the T2A-mCherry-WPRE cassette (to enable fluorescence-based monitoring of cells expressing i53 mutants). i53 mutant sequences were ordered as gene fragments from Twist Biosciences or IDT, or amplified from pre-constructed plasmids using primers designed to introduce the desired amino acid mutation(s). Pooled NNK and combinatorial libraries were constructed using NNK primers (IDT) or oligopools (Twist Biosciences), respectively. Combinatorial libraries were designed using one codon per amino acid (A = GCA, C = TGC, D = GAC, E = GAG, F = TTC, G = GGT, H = CAC, I = ATA, K = AAA, L = CTC, M = ATG, N = AAT, P = CCT, Q = CAG, R = CGG, S = AGC, T = ACC, V = GTC, W = TGG, Y = TAT). For certain library compositions, cysteine ​​(C) and methionine (M) were excluded due to their inherent reactivity and susceptibility to oxidation (N = 324–400 for any given two-position combinatorial library). Mutants and libraries were cloned into digested empty vector at the BamHI / NsiI cleavage sites using standard Gibson assembly protocols.

[0281] cell culture

[0282] Lenti-X HEK293T cells (Takara Bio) were cultured in DMEM (1X) + GlutaMAX-I (Gibco) supplemented with 10% FBS (Sigma). K562 cells (ATCC) were cultured in RPMI (Gibco) supplemented with 10% FBS and 1X penicillin-streptomycin (Gibco). All cells were grown in a humidified incubator at 37°C with 5% CO2 and subcultured every 3–5 days.

[0283] Lentivirus production:

[0284] Lenti-X HEK293T cells (Takara Bio) were transfected at 4.5 × 10 cells per 10-cm dish 18–24 h before transfection. 6 Cells were seeded at a density of 1000 x 1000 cells. The prepared cells were co-transfected with MISSION® Genomics Lentivirus Packaging Mix (Mirus Bio) and lentiviral plasmids containing the i53 mutants / library of interest using TransIT®-Lenti Transfection Reagent (Mirus Bio). Viral supernatants were collected 48 hours post-transfection, passed through a 0.45 μm filter (Cytiva), flash-frozen, and stored at -80°C until use. Viral titers were measured by FACS in K562 cells and typically ranged from approximately 0.5 to 1.5 x 10 7 It was TU / mL.

[0285] AAV production:

[0286] The HBB-targeting AAV6 vectors HBB-SNP and HBB-UBC-GFP were prepared as previously described. 1,2 HBB-SNP AAV6 was produced by Viralgen, and HBB-UBC-GFP AAV6 was produced by Vigene. Titers used in CD34+ HSPC editing experiments were measured using droplet digital PCR (ddPCR).

[0287] Culture of CD34+ HSPCs:

[0288] Human CD34 HSPCs were cultured as previously described. 1,2 CD34 HSPCs were purchased from AllCells and isolated from G-CSF-mobilized peripheral blood from healthy donors. CD34 HSPCs were cultured at 2.5 x 10 in StemSpan™-AOF (Stemcell) supplemented with stem cell factor (SCF) (100 ng / mL), thrombopoietin (TPO) (100 ng / mL), FLT3-ligand (100 ng / mL), IL-6 (100 ng / mL) (all Peprotech), and UM171 (35 nM) (Selleckchem). 5 ~5×10 5 Cells were cultured at 1000 cells / mL. Cells were cultured at 37°C, 5% CO2, and 5% O2.

[0289] Lentiviral transduction of CD34+ HSPCs:

[0290] One day after thawing, CD34+ HSPC cells were transduced with lentivirus at an MOI of 0.25 to 1. Cells were concentrated using centrifugation (180g x 7 min), counted, and then 4 x 10 cells were transduced in medium containing lentivirus, cyclosporin A (5 μM, Sigma Aldrich), and Synperonic F108 (0.5 mg / mL, Sigma). 6 After 4 hours of incubation, the cells were spun down, washed once with medium, and added at a concentration of 3.5 x 10 cells / mL in lentivirus-free medium. 5 Cells were seeded at a density of 1000 cells / mL.

[0291] Genome editing of CD34+ HSPCs:

[0292] The chemically modified single guide RNA (sgRNA) used to edit CD34+ HSPCs at the HBB locus was purchased from Synthego. The sgRNA sequence was modified by adding 2'-O-methyl-3'-phosphorothioate to the three terminal nucleotides at the 5' and 3' ends. The target sequence of the HBB sgRNA is as follows: HBB: 5'CTTGCCCCACAGGGCAGTAA-3' (SEQ ID NO: 74). Cas9 protein (SpyFi Cas9) was purchased from Aldevron. Prior to electroporation, RNP complexes were formed at a Cas9:sgRNA molar ratio of 1:2.5 at 25°C for 10-15 minutes. Cells were harvested, counted, and pelleted at 180g for 7 minutes. The cell pellet was resuspended in Maxcyte buffer containing the RNP complex and electroporated using a Maxcyte ExPERT ATx Nucleofector. After electroporation, cells were cultured at 3.5 x 10 in cytokine-supplemented medium. 5 Seed the desired AAV donor at 5.0 x 10 cells / mL. 2 ~2.5×10 4 Twenty-four hours after nucleofection, cells were spun down, washed once with medium, and resuspended in AAV-free medium at 3.5 × 10 vector genomes / cell. 5 Cells were seeded at a density of 1000 cells / mL.

[0293] Pooled library screening:

[0294] The lentiviral-based i53 mutant library was transduced at an MOI of approximately 0.2–0.5 (targeting approximately 30% transduction and coverage of >500 cells per library member in the mCherry+ / GFP+ cell population for each replicate tested). Three days after transduction, the HBB-UBB-GFP donor AAV6 (2.5 × 10) was transduced. 4Cells were edited in triplicate or quadruplicate in HBB as described above using a 100-μL MOI (1000 μg / mL vector genome / cell). Three days after editing, cells were pelleted and resuspended in medium containing DAPI (Miltenyi Biotec). Live mCherry+ / GFP+ and mCherry+ / GFP- single cells were collected using a FACSAria cell sorter (Becton Dickinson). Population purity was confirmed by a post-sort purity check. After sorting, genomic DNA was harvested from each sorted cell population using the Quick-DNA 96 Plus kit (Zymo Research). The DNA concentration of each sample was measured using the Qubit 1X dsDNA BR Assay Kit (ThermoFisher).

[0295] Next generation sequencing (NGS) of pooled libraries:

[0296] An amplicon sequencing workflow was designed to sequence and quantify i53 variants in the starting and post-selection pools. Primers and PCR conditions were optimized to specifically amplify the entire mutant coding sequence from plasmids, lentiviral libraries, and genomic DNA containing lentiviral vector inserts. After the initial amplification, the i53 amplicons were subjected to further PCR amplification to add sequencing adapters and sample indexes to enable sample multiplexing. The resulting sequencing library was then sequenced on an Illumina MiSeq instrument using paired-end reads to cover the entire i53 coding sequence.

[0297] NGS analysis:

[0298] Sequencing results were analyzed to identify and quantify all mutations within the coding sequences of the i53 pooled library. Tests using individual mutants combined to obtain the frequency of specific mutant alleles demonstrated that the sequencing and analysis workflow could accurately detect and quantify various i53 variants. This approach was used to confirm sequence diversity in the plasmid and lentiviral libraries prior to screening. For data analysis screening, a first quality control was performed on all runs to verify good values ​​for the number of mapped reads (>1e5 reads per sample), the percentage of reads inherited from the parent (from cloning), and the overall sequence distribution (free from bias). The i53 allele frequencies in the GFP+ and GFP- populations were then normalized by their parental abundance, and the enrichment fold change was calculated by dividing the normalized frequency of GFP+ cells by the normalized frequency of GFP- cells. All datasets included an internal standard (NNK-generated parental sequence). A final quality control of the datasets was performed using the internal standard, and sequencing runs in which the abundance of the internal standard differed from the parental carryover control by more than 10% were excluded.

[0299] Data processing and visualization were performed using R (v4.1.2) and the ggplot2 package. Mutants were ranked by fold change relative to the parent, and any mutant with an average greater than 1.0 was scored as "better than the parent." Additionally, a paired Student's t-test was performed for each mutant relative to the control to determine statistical significance (cutoff 0.05).

[0300] result

[0301] Figure 2 outlines the development of a lentivirus-based pooled screening system in HSPCs used to identify HDR-enhancing proteins at a desired Cas9-mediated cleavage site. As shown in Figure 2A, protein variants were cloned into a lentiviral backbone, and the resulting plasmid library was then used to transfect Hek293 cells to generate lentiviruses packaging the desired protein library. Freshly thawed CD34+ HSPCs were then transduced with lentivirus encoding the library at a level resulting in approximately one protein variant copy per transduced cell. Bulk transduced cell populations were then edited using Cas9, guide RNA, and an AAV6 template (encoding a GFP insert at the desired cleavage site). Several days after editing, cells were sorted by flow cytometry into GFP+ and GFP- populations. Genomic DNA was extracted from each sorted cell population, sequenced by NGS, and analyzed to determine the distribution of variants relative to the control. Mutants enriched in the GFP+ population relative to the control were then individually validated and evaluated. To establish this screening system, we constructed a lentivirus-based construct containing a restriction enzyme cleavage site upstream of the T2A-mCherry-WPRE cassette to facilitate cloning of the protein of interest, as shown in Figure 2B. Placing a T2A-mCherry tag at the 3' end of the protein variant allowed for independent expression of both the protein variant and mCherry after transcription and translation, enabling fluorescence-based monitoring of cells expressing the protein variant of interest.

[0302] The screening system was designed to detect known inhibitors of 53BP1 (i53 4This was validated using a previously characterized mutant of 53BP1. i53 is an engineered ubiquitin mutant identified by a binding-based screen and found to selectively bind to the Tudor domain of 53BP1. When transiently expressed in cells, i53 was found to block 53BP1 recruitment to DSBs and improve HDR editing outcomes. When the use of i53 as a protein-based additive was evaluated for enhancing HDR incorporation of donor sequences, a mild increase (10–20%, using purified i53 at 1.5 mg / mL in nucleofection solution) was observed, but relatively inconsistent results were obtained (data not shown).

[0303] To validate the lentivirus-based screening system in HSPCs, various mutants of i53 were cloned into the lentiviral vector: i53 (positive control), a previously reported inactive mutant of i53 ("DM" = P67L L70V, negative control), and three mutants of i53 ("mut1" = L2Q, "mut2" = D64E, and "mut3" = L62Q, which are i53 mutants). 4 We previously reported that i53 mutants exhibited reduced (but detectable) binding to the Tudor domain of 53BP1 compared to i53 mutants. After verifying that these mutants had different effects on HDR when expressed by lentiviral transduction in HSPCs (data not shown), we pooled together the plasmids encoding these five i53 mutants to generate a "mock" library. CD34+ HSPCs were transduced with lentivirus containing the "mock" library and duplicated editing was performed at the HBB locus using the HBB-UBC-GFP AAV (MOI = 2500). As shown in Figure 3B, next-generation sequencing (NGS) analysis of gDNA purified from the sorted mCherry+GFP+ and mCherry+GFP- populations revealed the expected enrichment of differential mutations in the mCherry+GFP+ population compared to the positive and negative controls (i53 and DM).

[0304] Example 2: Functional screening yields improved 53BP1 inhibitors that promote HDR

[0305] Materials and Methods

[0306] Validation of hits by lentiviral expression:

[0307] The sequences of individual i53 mutants of interest were cloned into the lentiviral-based expression plasmids described above. Hits were either validated as a pooled "validation library" (mutant and control plasmids manually mixed to generate pools of 5-25 mutants) or validated individually. Lentiviruses generated from these plasmids were used to transduce CD34+ HSPC cells at an MOI of 0.5-1 on day 1 after thawing. On day 4, transduced cells were transduced at 1.25-2.5 x 10 4 Cells were edited with HBB-UBC-GFP AAV6 at a concentration of 100 vector genomes / cell. Cells transduced with the pooled validation library were edited in triplicate or quadruplicate. Cells transduced with individual mutants were edited in duplicate.

[0308] For each mutant tested, the integration rate of the HBB-UBC-GFP donor was measured using a Beckman Coulter CytoFLEX. DAPI (Miltenyi Biotec) was used to distinguish live from dead cells. mCherry expression was used to distinguish transduced from untransduced cells, and GFP integration rates were compared between the two populations to quantify the impact of lentiviral-based mutant expression on HDR rates. Flow cytometry data were analyzed using FlowJo 10 software.

[0309] For the pooled validation library, cells were sorted and analyzed as described above. Next-generation sequencing (NGS) analysis of purified gDNA from the sorted mCherry+GFP+ and mCherry+GFP- populations determined differences in mutation enrichment and validated the effect of individual variants on HDR rates relative to controls.

[0310] result

[0311] Using a validated lentiviral-based pooled functional screening system (described in Example 1), we identified single amino acid variants of i53 that showed enhanced HDR-promoting ability for i53 at the HBB locus of HPSCs. To separately alter the amino acid identity of residues 67 and 68 of i53, we generated a focused saturation mutagenesis library using NNK primers. As shown in Figure 4A, these residues (shown in red) are located at the Tudor domain-binding interface of i53 / 53BP1 (PDB=5JS6). 4 Based on their location in the i53 genome, these i53 surface residues were targeted for saturation mutagenesis. We hypothesized that the chemical properties of these i53 surface residue side chains directly influence the interaction of i53 with the 53bp1 Tudor domain, potentially resulting in functional effects on HDR levels in cells. This focused NNK library was transduced into CD34+ HSPC cells using lentivirus packaging, and triplicate editing was performed at the HBB locus using the HBB-UBC-GFP AAV (MOI = 1250). Next-generation sequencing (NGS) analysis of gDNA purified from the sorted mCherry+GFP+ and mCherry+GFP- populations revealed differential enrichment of mutations in the mCherry+GFP+ population compared to i53. Consistent enrichment was observed at both residue 67 (Figure 4B) and residue 68 (Figure 4C) across unique codon overlaps and across amino acid categories in the NNK library. The positive charge at residue 67 appeared to be particularly beneficial for HDR levels at HBB compared to the parent L67. Therefore, the top two hits (candidates) from the screening, L67R and L67H, were subjected to further validation and characterization.

[0312] The L67R and L67H i53 mutants and controls (parental control i53 and negative control i53 inactivation mutant "DM" = P67L L70V) were individually cloned into the lentiviral vector described above. Each construct was packaged and transduced into CD34+ HSPC cells, and duplicate editing was performed at the HBB locus using HBB-UBC-GFP AAV (MOI = 1250). Edited cells were analyzed by flow cytometry, and GFP integration rates were compared between live mCherry+ cells (transduced, expressing the mutant of interest) and live mCherry- cells (untransduced, control). As shown in Figure 5, cell populations expressing the L67R and L67H i53 mutants exhibited higher GFP integration rates than cell populations expressing i53 and DM relative to the corresponding control cells. These results demonstrate differential enrichment of L67R and L67H relative to i53 in pooled NNK screening.

[0313] Example 3: Validation of improved i53 mutants containing alterations at positions 67 and 68

[0314] Materials and Methods

[0315] Production and purification of i53 mutant proteins:

[0316] The sequences of the different i53 mutants were cloned into bacterial expression plasmids to generate N-terminal His-tagged fusion proteins with a protease cleavage site between the 6x-His tag and the i53 mutant sequence. For protein expression, the resulting plasmids were transformed into E. coli BL21(DE3)-RIL. Cells were grown in LB medium supplemented with 0.4% glucose at 30°C to an OD600 of 0.8 and induced with 0.4 mM IPTG at 16°C for 18 h. Cells were harvested by centrifugation and resuspended in 50 mM potassium phosphate pH 8.0, 500 mM NaCl, 20 mM imidazole, and 3 mM β-mercaptoethanol. Cells were lysed using a microfluidic device. 0.2 mM phenylmethylsulfonyl fluoride (PMSF) was immediately added to the crude lysate and centrifuged at 20,000 g for 20 min. The soluble fraction was then incubated with 2 ml of Ni-NTA (GE Healthcare) per 1,000 OD for 1 hour at 4°C. After incubation with the Ni-NTA resin, the lysate was removed by pelleting the resin at 2,500 g for 3 minutes and washed three times with nine bed volumes of 50 mM potassium phosphate pH 8.0, 500 mM NaCl, 20 mM imidazole, and 3 mM β-mercaptoethanol. After batch washing, the Ni-NTA resin was loaded onto a gravity-flow column, and the His-tagged i53 mutant protein was eluted with six bed volumes of 50 mM potassium phosphate pH 8.0, 300 mM NaCl, 500 mM imidazole, and 3 mM β-mercaptoethanol. The eluted protein was dialyzed overnight against 10 mM Tris / HCl pH 8.0, 200 mM NaCl, and 1 mM DTT, and the 6xHis tag was cleaved with protease. The protein was purified by anion exchange chromatography on a HiTrapQ column (GE Healthcare) with a linear NaCl gradient and further purified twice by size exclusion chromatography using a Superdex S200 26 / 60 column (GE Healthcare) eluted with 10 mM Tris / HCl pH 8.0, 200 mM NaCl, and 1 mM DTT. The protein was concentrated to approximately 20 mg / mL and flash-frozen for storage.

[0317] Size exclusion chromatography: Identification of the 53BP1 Tudor domain:i53 mutant complex

[0318] Recombinantly purified 53BP1 Tudor domain (53BP1 residues 1484-1603) was mixed with recombinantly purified i53 mutants at a concentration of 0.5 mg / mL. The proteins were incubated at room temperature for 30 minutes and then injected into an HPLC (Agilent, 1260 Infinity II). 5 μL of the protein complex was injected onto a MAbPac 4 x 300 mm SEC column with a 5 μm particle size and a 300 Å pore size. HPLC was performed at 0.2 mL per minute using PBS as the mobile phase, and absorbance at 280 nm was continuously measured for approximately 1 total column volume. The retention time of the 53BP1 Tudor domain alone was 14.6 minutes. The retention time of the i53 mutants was approximately 15.5 minutes. A stable complex of the 53BP1 Tudor domain:i53 mutants was found to have a retention time of 14.3 minutes.

[0319] Biolayer Interferometry (BLI):

[0320] Data were collected using an Octet R8 system (Sartorius). Purified 53BP1 Tudor domain was labeled with NHS-biotin at exposed primary amine groups using the ChromaLINK NHS-Biotin Protein Labeling Kit (Vector Laboratories). One equivalent of ChromaLink biotin was incubated with the 53BP1 Tudor domain for 2 hours, and the buffer was then exchanged with fresh PBS. The labeling efficiency was calculated to be approximately one biotin per 53BP1 Tudor domain molecule. An Octet SA biosensor chip (Sartorius) was incubated with the biotin-labeled 53BP1 Tudor domain (ligand) for 60–80 seconds. The labeled chip was then immersed in 1x binding buffer (Sartorius) for 60 seconds to remove excess ligand and establish a baseline. The labeled chip was then introduced to the i53 mutant (analyte) for 500–600 seconds, and the response was continuously monitored to detect association. A range of analyte concentrations was tested, from the highest to lowest nM (i.e., 200, 100, 50, 25, 12.5, 6.25, 3.125). The chip was then introduced into 1x binding buffer for 5 min, and the response was continuously monitored to detect dissociation. The dissociation constant (K D ) was calculated using a 1:1 binding model, and the on-rate (k a ) and off-rate (k d ) was calculated as the change in response (nm) over time (seconds).

[0321] Editing CD34+ HSPCs with i53 mutant purified protein:

[0322] Purified i53 mutant proteins were added to CD34+ HSPC cells at concentrations of 0.0125–1.6 mg / mL (volume of added protein ≤ 1 / 10 of the Maxcyte cuvette volume) as part of the nucleofection mixture. CD34+ hPSCs were edited with the protein mutants 3 days after thawing and harvested 2 days after nucleofection for NGS analysis or 3–5 days after nucleofection for GFP expression analysis. Editing reactions using a given concentration of protein were repeated in duplicate or triplicate (either using the same CD34+ HSPC donor or across multiple donors).

[0323] For editing in combination with a DNAPK small molecule inhibitor ("DNAPKi," AZD7648, Sellekchem #58832), cells nucleofected with i53 mutant proteins were added to medium containing both AAV and a 0.5uM concentration of DNAPKi. 24 hours after nucleofection, cells were spun down, washed with medium, and resuspended in 3.5x10 cells in medium without AAV or DNAPKi. 5 Cells were seeded at a density of 1000 cells / mL.

[0324] Crystallography:

[0325] The purified human i53:53BP1 complex was subjected to crystallization in 10 mM Tris 8.0, 200 mM NaCl, and 1 mM DTT under previously published conditions. 4The i53:53BP1 complex was crystallized in 0.1 M MES (2-(N-morpholino)ethanesulfonic acid) pH 6.0, 0.2 M trimethylamine N-oxide, and 25% (w / v) PEG MME (polyethylene glycol monomethyl ether) 2000 at room temperature at a protein concentration of 30 mg / mL. Crystals were grown by sitting-drop vapor diffusion at 23 °C within 7 days. Crystals were cryoprotected by adding glycerol to the reservoir solution at a final concentration of 20% (v / v) and then flash-frozen in liquid nitrogen. The i53:53BP1 complex crystallized in the P212121 space group, with one i53:53BP1 complex molecule per asymmetric unit cell with dimensions of 40.3 Å × 46.8 Å × 89.7 Å.

[0326] Structure determination:

[0327] The structure of the human i53:53BP1 Tudor domain (WT, L67H, L67R) was solved using molecular replacement and the previously published structure of the WT i53:53BP1 Tudor domain (PDB code: 5J26). A final model of the human i53:53BP1 Tudor domain (WT, L67H, L67R) was constructed using the original data and refined to an extended resolution of approximately 1.1 Å for each dataset. 5 was used to build a full model of the I53:53BP1 complex and complete further refinements.

[0328] Measurement of targeted integration of HBB-UBC-GFP (flow cytometry-based analysis):

[0329] Targeted integration rates of the HBB UBC GFP donor were measured using a Beckman Coulter CytoFLEX. DAPI (Miltenyi Biotec) was used to distinguish live from dead cells. Flow cytometry data were analyzed using FlowJo 10 software.

[0330] Measurement of targeted integration of HBB-SNP (NGS-based analysis):

[0331] The frequency of homology-directed repair (HDR) and other editing outcomes at HBB were measured using next-generation sequencing (NGS). An NGS assay was developed to quantify the total number of alleles, determining their frequency at the HBB locus. Such alleles were either (1) unedited (WT%), (2) modified by HDR and incorporating a different sequence present in the AAV repair template (HR%), or (3) mutated during the genome correction process, resulting in a gene that produces mutant β-globin (indel%).

[0332] For this assay, genomic DNA was harvested from cells using a Quick-DNA 96 Plus kit (Zymo Research). DNA concentration was measured using a Qubit 1X dsDNA BR Assay Kit (ThermoFisher). The purified genomic DNA was then used to amplify the HBB locus by polymerase chain reaction (PCR). The PCR product was diluted using nuclease-free water and used as template DNA for targeted NGS library preparation. A second PCR using primers with a partial Illumina adapter was performed to amplify a 142-base pair sequence. This 142-base pair sequence contains the region of the HBB locus to be corrected during the genome correction process. The PCR product was diluted again and used as a template in a third PCR reaction using Nextera XT index primers. This third PCR reaction was used to assign a unique identifier to each sample and add the full-length adapter sequence required for Illumina sequencing. PCR product size was assessed using an Agilent BioAnalyzer. The PCR products were then pooled and purified using a Qiagen PCR purification kit, quantified using PicoGreen, and the PCR products were prepared for sequencing.

[0333] Based on the PicoGreen concentration, the pooled PCR product library was diluted to a final concentration of 4 nM. Sequencing was performed on a MiSeq system using the Illumina MiSeq Sequencing Reagent Kit (V2, 300 cycles). A 10% Phix control library was added to the sample library to improve sequence diversity and allow for error rate measurement. The library was denatured and loaded onto the sequencing reagent cartridge at 8–12 pM. Sequencing involved paired-end 150 base pair reads and dual-indexed reads. Sequencing data was demultiplexed based on the provided sample index, generating FASTQ files for each sample.

[0334] CRISPResso2 Pipeline 3 FASTQ files were processed using RT-PCR. Data were recorded as %WT, %HR correction, and %indels. Sequences containing indels were further analyzed to obtain an overview of the size of insertions and deletions resulting from the gene editing process. A subset of indels was classified as "HBD" based on sequence homology to the HBD gene (approximately 93% sequence homology to HBB) and considered to have arisen from homologous recombination with the endogenous HBD. The remaining indels were classified as either NHEJ or MMEJ. This classification was performed by leveraging data from previous experiments using shRNA knockdown of POLQ (a key protein in MMEJ) (not shown) to infer the impact on individual indel profiles. Indels affected by POLQ knockdown (centered around the prominent -9 deletion) were classified as MMEJ. The remaining unaffected indels were classified as NHEJ.

[0335] A positive control with known editing results, a negative control with no editing, and a no-template control were processed in parallel with each set of samples.

[0336] result

[0337] Size-exclusion chromatography was performed to verify the binding of i53 mutants to the 53BP1 Tudor domain. Both the i53 mutants and the 53BP1 Tudor domain were run on a size-exclusion column (SEC) and then mixed to monitor and evaluate complex formation, validating L67H and L67R as 53BP1 binders. As shown in Figure 6, the retention time of the i53 mutant alone is approximately 15.5 min. The retention time of the 53BP1 Tudor domain is approximately 14.6 min. When the two proteins are mixed with a two-fold excess of i53, they form a complex, shifting the retention time to approximately 14.3 min. Figure 6A shows the SEC traces of i53 alone, the 53BP1 Tudor domain alone, and the mixture. Figure 6B shows the SEC traces of the i53 mutant L67R, the 53BP1 Tudor domain, and the mixture. Figure 6C shows the SEC traces of the i53 mutant L67H, the 53BP1 Tudor domain, and the mixture. i53, the L67R mutant, and the L67H mutant all appear to bind and shift the retention time of the 53BP1:i53 mutant complex in a similar manner, suggesting that the L67R and L67H mutants retain 53BP1 Tudor domain binding and may therefore increase levels of HDR through a mechanism of action similar to that of the parent i53.

[0338] Biolayer interferometry (BLI) was performed to measure the binding kinetics of i53 mutants with immobilized 53BP1 Tudor domain (Figure 7). Using BLI, kinetic measurements (on-rate and off-rate) were collected for the binding of i53 mutants to biotin-labeled 53BP1 Tudor domain. The on-rate and off-rate measured at various solution concentrations were used to determine the dissociation constant (K) of each listed mutant. D ) was calculated. D A 1:1 binding model was used for the calculation of K D >50 nM, ++ indicates a K between 5 and 50 nM DFigure 7A shows the binding of i53 to immobilized 53BP1 Tudor domain. Figure 7B shows the binding of i53 inactivated mutant ("DM", P69L L70V) to immobilized 53BP1 Tudor domain. Figure 7C shows the binding of i53 mutant L67R to immobilized 53BP1 Tudor domain. Figure 7D shows the binding of i53 mutant L67H to immobilized 53BP1 Tudor domain. Figure 7E shows that both L67R and L67H have clearly increased binding rates compared to parent i53, suggesting that these proteins likely have an enhancing effect on HDR levels compared to i53.

[0339] To further evaluate the binding interactions between i53 mutants L67R and L67H and 53bp1, we performed X-ray crystallography on the structures of i53 mutants complexed with the 53bp1 Tudor domain. Figure 8A shows the structural alignment of the 53BP1 Tudor domain (yellow) bound to the i53 mutants wild-type (WT) (blue), L67R (green), and L67H (pink) with RMSD <0.5 Å. Zoom-in views of the complexes for i53 (Figure 8B), L67R (Figure 8C), and L67H (Figure 8D) are shown in the solvent-exposed loop preceding β5. The amino acid changes L67R and L67H in i53 were found to form new hydrogen bonds (shown by the black dotted lines). These hydrogen bonds were a 2.8 Å H-bond between L67R and D1550 of the 53BP1 Tudor domain, a 2.9 Å H-bond between L67H and D64 of i53, and a water-mediated H-bond between L67H and S1554 of the 53BP1 Tudor domain. These new interactions likely contribute to the increased affinity of these two mutations for the 53BP1 Tudor domain.

[0340] The L67R and L67H mutants were then evaluated as purified proteins for enhanced HDR integration. Figure 9 shows a detailed schematic diagram of the integration of purified i53 mutants as HDR-promoting factors into the HSPC gene editing workflow. Editing was performed 3 days after thawing CD34+ HSPC cells. Purified i53 mutants were incorporated at various concentrations into a nucleofection solution containing Cas9 and guide RNA. After nucleofection, cells were incubated for 24 hours in medium containing various concentrations of AAV and harvested for analysis 24–96 hours after AAV washout.

[0341] Using HBB-UBC-GFP AAV as a donor template, we performed editing of CD34+ HSPC cells in the absence and presence of purified i53 mutants L67R and L67H, as well as parental control i53 and a negative control i53 inactivated mutant ("DM" = P67LL70V). Purified proteins were incorporated into the nucleofection solution at a concentration of 0.4 mg / mL. After nucleofection, cells were incubated with AAV at an MOI of 1250. Cells were analyzed by flow cytometry. As shown in Figure 10, the use of L67R and L67H significantly affected the GFP incorporation rate in live cells compared to parental i53. L67R and L67H increased the HDR% by an average of 69% relative to DM, whereas i53 only increased the HDR% by 19%. Furthermore, these results support the results of the pooled NNK screen (described in Examples 1 and 2), suggesting that this functional screening platform can identify protein variants that can significantly increase HDR levels when used as protein-based additives for HDR gene editing.

[0342] Editing of CD34+ HSPC cells was further evaluated using purified i53 mutants L67R and L67H and HBB-SNP AAV, a donor template designed to correct the sequence encoding the E6V mutation in the HBB gene responsible for sickle cell disease. 1,2Protein variants (including the parental control i53 and the negative control i53 inactivation variant ("DM" = P67L L70V)) were incorporated into the nucleofection solution at a concentration of 0.4 mg / mL. After nucleofection, cells were incubated with AAV at MOIs of 312.5 and 2500. To quantify the editing results, gDNA was extracted from the edited cells and analyzed by NGS. As shown in Figure 11A, L67R and L67H were found to significantly affect the proportion of HDR-corrected alleles compared to the parental i53 at both MOIs of 312.5 and 2500. L67R and L67H were found to increase the level of HDR-corrected alleles by an average of 56% and 44%, respectively, relative to DM when edited at MOIs of 312.5 and 2500. In contrast, i53 editing at MOIs of 312.5 and 2500 only increased the % HDR-corrected allele levels by 14% and 15%, respectively. Notably, the incorporation of L67R and L67H and an MOI of 312.5 resulted in HDR-corrected allele levels similar to those achieved with only 2500 MOI (8x AAV). As shown in Figure 11B, edited alleles from each sample were further characterized by the type of editing (unedited WT (wild-type), MMEJ, NHEJ, HBD, or HDR). The results are shown below. i53 mutants were found to decrease NHEJ% and increase HDR% (MMEJ% and HBD% were unchanged by i53 mutant treatment). L67R and L67H were found to have a more pronounced effect on NHEJ% compared to i53. These results are consistent with the established mechanism of action of i53 mutants and the relative 53BP1 binding rates described above.

[0343] Next, we used HBB-UBC-GFP AAV to generate dose-response curves for purified i53 mutants L67R and L67H compared with the parental control i53. Protein mutants were incorporated into the nucleofection solution at concentrations of 0.1, 0.2, 0.4, and 0.8 mg / mL. After nucleofection, cells were incubated with AAV at an MOI of 2500. Cells were analyzed by flow cytometry. As shown in Figure 12, cells treated with L67R and L67H maintained high levels of GFP incorporation across all editing conditions in live cells compared with the control (cells edited without protein-based additives; the average GFP incorporation rate for two replicates is shown as a gray dotted line) and parental i53. L67R and L67H were found to increase the level of GFP incorporation by an average of 70% across all concentrations tested. The level of GFP incorporation in cells treated with the highest concentration of i53 (0.8 mg / mL) was increased by 45% compared with control-edited cells. However, the effect of i53 on the level of GFP incorporation decreased significantly with decreasing protein concentration (29% at 0.4 mg / mL, 18% at 0.2 mg / mL, and 5% at 0.1 mg / mL). These results suggest that L67R and L67H are significantly more potent than parental i53 and are in good agreement with the relative 53BP1 binding rates shown above.

[0344] Similarly, purified i53 mutants L67R and L67H were identified as E6V sickle cell mutations. 1,2Using a modified donor, a dose-response curve was determined comparing parental control i53 cells with HBB-SNP AAV. Protein variants were incorporated into the nucleofection solution at concentrations of 0.1, 0.2, and 0.4 mg / mL. After nucleofection, cells were incubated with AAV at MOIs of 312.5 and 2500. To quantify editing results, gDNA was extracted from edited cells and analyzed by NGS. As shown in Figure 13, cells treated with L67R and L67H retained high levels of HDR-corrected alleles across all editing conditions compared to control (cells edited without protein-based additives; average HDR% for four replicates is shown as a gray dotted line) and parental i53 cells. L67R and L67H were found to increase HDR% by an average of 62% and 27%, using MOIs of 312.5 and 2500, respectively, across all concentrations tested. As previously observed, all conditions incorporating L67R and L67H and an MOI of 312.5 yielded levels of % HDR-corrected alleles similar to those obtained using only an MOI of 2500. The levels of HDR-corrected alleles in cells treated with the highest concentration of i53 (0.4 mg / mL) were increased by 45% and 18% over control-edited cells at MOIs of 312.5 and 2500, respectively. However, the effect of i53 on % HDR levels was found to decrease significantly with decreasing protein concentration (312.5 MOI: 24% at 0.2 mg / mL, 18% at 0.1 mg / mL; 2500 MOI: 5% at 0.2 mg / mL, 3% at 0.1 mg / mL). These results support previous findings that L67R and L67H are significantly more potent than the parental i53 and continue to closely match the relative 53BP1 binding rates described above.

[0345] Editing of CD34+ HSPC cells was further evaluated using increasing MOIs of HBB-SNP AAV donor and purified i53 mutant L67R. L67R was incorporated into the nucleofection solution at 0.8 mg / mL. After nucleofection, cells were incubated with AAV at a range of MOIs (78.125-2500) with or without the addition of the additional NHEJ small molecule inhibitor DNAPKi (AZD7648, 0.5 μM). To quantify induction of the DNA damage response (DDR) pathway, a subset of edited cells was harvested 24 hours after nucleofection and subjected to immunofluorescence analysis of the DDR markers p21 and yH2AX. 5 The percentage of HDR was quantified. 48 hours after nucleofection, DNA was extracted from the remaining edited cells and analyzed by NGS to quantify the editing results. As shown in Figure 14A, cells treated with L67R retained higher levels of HDR-corrected alleles across all editing conditions compared to the control (cells edited without protein-based additives). The effect of L67R application on HDR% was significantly higher at low MOI (156.25: 225%, 78.125: 220%) than at high MOI (2500: 47%, 625: 57%, 312.5: 58%). As shown in Figure 14B, adding DNAPKi to the culture medium containing cells edited using L67R further increased the levels of HDR-corrected alleles compared to controls (2500: 69%, 625: 83%, 312.5: 99%, 156.25: 313%, 78.125: 315%). These results suggest that the use of L67R and other high-affinity i53 mutants in combination with DNAPKi can dramatically increase baseline editing rates by inhibiting the NHEJ effect.

[0346] Quantification of these DDR markers in edited HSPCs revealed an AAV concentration-dependent increase in yH2AX (Figure 14C) and p21 (Figure 14C) expression. Addition of L67R was not observed to significantly increase yH2Ax and p21 responses using any given MOI. These results suggest that L67R and other high-affinity i53 mutants can be utilized to maintain high levels of editing while allowing for favorable reduction of AAV6.

[0347] Example 4: Functional screening of combinatorial libraries yields further improved HDR-promoting factors

[0348] We further utilized our validated lentivirus-based pooled functional screening system to identify additional HDR-promoting mutants by screening a combinatorial library targeting residues at the 53bp1 / i53 interface. A combinatorial library was designed to explore all amino acid combinations at residues L67 and H68, which we previously screened. This combinatorial library was packaged into CD34+ HSPC cells, and editing at the HBB locus was performed in quadruplicate using the HBB-UBC-GFP AAV (MOI = 2500). As shown in Figure 15A, next-generation sequencing (NGS) analysis of gDNA purified from the sorted mCherry+GFP+ and mCherry+GFP- populations revealed differential enrichment of mutations in the mCherry+GFP+ population compared to the i53 population. (Cysteine, C, and methionine, M, were excluded to avoid potential issues due to their inherent reactivity and susceptibility to oxidation.) Hits significantly enriched compared to i53 (shown in purple) included L67H (shown in green) and L67R (shown in blue). When the results were analyzed by amino acid category, clear trends emerged among the hits, as shown in Figure 15B. In addition to the previously identified hits (L67R and L67H), combining a positively charged and / or polar uncharged amino acid at residue 67 with an aromatic amino acid at residue 68 appears to be particularly beneficial for HDR levels in HBB compared to the original L67 and H68 variants. Therefore, a large subset of these variants (i.e., L67H, H68Y, L67S, H68W, etc.) was further validated and characterized.

[0349] The top mutants identified from the combinatorial screen were individually cloned into the lentiviral vectors described above. The resulting plasmids were pooled with controls (parental control i53 and negative control i53 inactivation mutant "DM" = P67L L70V) to generate a small validation library. This library was packaged and transduced into CD34+ HSPC cells, and editing at the HBB locus was performed in quadruplicate using HBB-UBC-GFP AAV (MOI = 2500). As shown in Figure 16, NGS analysis of gDNA purified from the sorted mCherry+GFP+ and mCherry+GFP- populations confirmed positive enrichment for the majority of double mutants tested compared to i53.

[0350] Editing of CD34+ HSPC cells was evaluated using the HBB-SNP AAV donor (described above) and two representative purified double mutants of i53 (L67K H68F and L67H H68Y). Protein mutants (including L67R, L67H, parental control i53, and negative control i53 inactivation mutant ("DM" = P67L L70V)) were incorporated into the nucleofection solution at concentrations of 0.1 and 0.4 mg / mL. After nucleofection, cells were incubated with AAV at MOIs of 312.5 and 2500. gDNA was extracted from edited cells and analyzed by NGS. As shown in Figure 17, cells treated with both i53 single and double mutants retained high levels of HDR-corrected alleles across all editing conditions compared to the control (cells edited without protein-based additives; average HDR% for four replicates is shown as a gray dotted line) and parental i53. The four i53 mutants tested were found to increase HDR% by an average of 70% and 35% across all concentrations, using MOIs of 312.5 and 2500, respectively. Incorporation of i53 also increased HDR% compared to the control, but at a significantly lower level than the i53 mutants (312.5 MOI: 47% at 0.4 mg / mL, 22% at 0.1 mg / mL; 2500 MOI: 22% at 0.4 mg / mL, 8% at 0.1 mg / mL). These results further support the results of combinatorial library and validation screening, again suggesting that functional screening platforms (described in Examples 1 and 2) can identify protein mutants that can significantly increase HDR levels when used as protein-based additives for HDR-mediated gene editing.

[0351] Example 5: Further improved HDR-promoting factors containing alterations at positions 67 and / or 68

[0352] Materials and Methods

[0353] AAV production (HBA-UBC-GFP, CCR5-UBC-GFP, and IL2RG-UBC-GFP):

[0354] All AAV6 vectors were cloned into the pAAV-MCS plasmid (Agilent Technologies), which contains inverted terminal repeats (ITRs) derived from AAV2. The left and right homologous arms (LHA / RHA) were derived from human genomic DNA and corresponded to specific lengths at each knock-in site. The lengths of the left and right homologous arms for the HBA, CCR5, and IL2RG donors were as follows: HBA LHA: 976 bp, HBA RHA: 879 bp, CCR5 LHA: 502 bp, CCR5 RHA: 500 bp, IL2RG LHA: 400 bp, and IL2RG RHA: 414 bp. Each vector contained the UBC promoter, CopGFP, and BGH poly(A). UBC-GFP-BGH was synthesized as a gene fragment (Twist Bioscience) and cloned into pAAV along with the corresponding LHA and RHA using the standard Gibson assembly protocol. The constructed LHA-UBC-GFP-BGH-RHA sequences for the HBA-UBC-GFP, CCR5-UBC-GFP, and IL2RG-UBC-GFP AAV donors are shown herein as SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively.

[0355] HBA-UBC-GFP AAV6 was produced by Packgene. CCR5-UBC-GFP AAV6 and IL2RG-UBC-GFP AAVG were produced by Vigene. Titers used in CD34+ HSPC editing experiments were measured using droplet digital PCR (ddPCR).

[0356] Genome editing of CD34+ HSPCs (HBA, CCR5, Il2RG):

[0357] Chemically modified single guide RNAs (sgRNAs) were used to edit CD34+ HSPCs at the HBA, CCR5, and IL2RG loci. The sgRNA sequences were modified by adding 2'-O-methyl-3'-phosphorothioate to the three terminal nucleotides at the 5' and 3' ends. The target sequences for the HBA, CCR5, and IL2RG sgRNAs were as follows: HBA: 5'-GGCAAGAAGCATGGCCACCG-3' (SEQ ID NO: 78), CCR5: 5'-GCAGCATAGTGAGCCCAGAA-3' (SEQ ID NO: 79), IL2RG: 5'-TGGTAATGATGGCTTCAACA-3' (SEQ ID NO: 80). Cas9 protein (SpyFi Cas9) was purchased from Aldevron. Before electroporation, RNP complexes were formed at a Cas9:sgRNA molar ratio of 1:2.5 at 25°C for 10–15 minutes. Cells were harvested, counted, and pelleted at 180 g for 7 minutes. The cell pellet was resuspended in Maxcyte buffer containing RNP complex and electroporated using a Maxcyte ExPERT ATx Nucleofector. After electroporation, cells were diluted to 3.5 x 10 in cytokine-supplemented medium. 5 Seed the desired AAV donor at 5.0 x 10 cells / mL. 2 ~2.5×10 4 Twenty-four hours after nucleofection, cells were spun down, washed once with medium, and resuspended in AAV-free medium at 3.5 × 10 vector genomes / cell. 5 Cells were seeded at a density of 1000 cells / mL.

[0358] Purified i53 mutant proteins were added to CD34+ HSPC cells at a concentration of 0.4 mg / mL (volume of added protein ≤ 1 / 10 of the Maxcyte cuvette volume) as part of the nucleofection mixture. CD34+ hPSCs were edited with the protein mutants 3 days after thawing and harvested for GFP expression analysis 3–5 days after nucleofection. Editing reactions using a given concentration of protein were repeated in duplicate or triplicate (either using the same CD34+ HSPC donor or across multiple donors).

[0359] Measurement of targeted integration of HBB-UBC-GFP, CCR5-UBC-GFP, and IL2RG-UBC-GFP (flow cytometry-based analysis):

[0360] Targeted integration rates of HBA-UBC-GFP, CCR5-UBC-GFP, and IL2RG-UBC-GFP donors were measured using a Beckman Coulter CytoFLEX. DAPI (Miltenyi Biotec) was used to distinguish live from dead cells. Flow cytometry data were analyzed using FlowJo 10 software.

[0361] Measurement of OT-1 editing (NGS-based analysis):

[0362] The off-target (OT) editing site, OT-1, can be assessed using an assay very similar to the one described above that measures the targeted integration of the HBB-SNP. This off-target editing site can be identified using three different methods: in silico prediction, 13 Circle -Seq 14 , and Guide-Seq 15 ) and confirmed as a significant off-target editing site by amplicon sequencing. The workflow of the OT-1 amplicon sequencing assay is very similar to that of the on-target sequencing assay, but involves one less PCR step. A small 166-base pair sequence encompassing the OT-1 editing site is amplified directly from genomic DNA in the first PCR and then labeled with a barcode and adapter sequence in the second PCR. The resulting PCR products are sized, quantified, and prepared for sequencing in the same way as the HBB on-target sequencing assay. Sequencing data are also processed using the Crispresso2 pipeline. 3 In contrast to the on-target sequencing assay, there are no homologous direct repair results in OT-1, and only WT% and indel% are recorded.

[0363] Multiplexed Reactivity Assay:

[0364] ELISA plates were each coated with 50 μL of one of the following antigens diluted in 50 mM carbonate, pH 9.0: cardiolipin (200 μg / mL, C0563, Sigma), KLH (20 μg / mL, H8283, Sigma), LPS (5 μg / mL, tlrl-eblps, InvivoGen), dsDNA (4 μg / mL, D4522, Sigma), insulin (20 μg / mL, I9278, Sigma), and RNA (4 μg / mL). 50 μL of purified 53BP1 Tudor domain was also coated onto the ELISA plate, diluted (5 μg / mL) in PBS. The coated plates were incubated overnight at 4°C and blocked the next day with PBS + 0.5% BSA for 1 hour at room temperature (RT), followed by three washes with PBST (PBS + 0.1% Tween® 20). Fifty microliters of 100 nM anti-his hrp (1:2000 dilution in blocking buffer, 43360, Qiagen) was added to each well and incubated for 1 hour at room temperature. The plate was then washed with PBST, and 100 μL of chemiluminescent imaging reagent (SuperSignal ELISA Pico Chemiluminescent Substrate, 37069, Thermo) was added to measure the signal. The signal / background was calculated as follows:

number

[0365] Differential scanning fluorimetry and dynamic light scattering:

[0366] For nano-DSF (differential scanning fluorimetry) and nano-DLS (dynamic light scattering) measurements, i53 mutants were diluted to 0.5 mg / mL in 1x PBS pH 7.4 (catalog no. 10010023, ThermoFisher Scientific) and analyzed with a Prometheus Panta (NanoTemper) using a high-sensitivity capillary (catalog no. PR-C006, NanoTemper). Melting temperatures were calculated from a two-state model between 25 °C and 95 °C using PR.PantaAnalysis (NanoTemper) and averaged from three replicates.

[0367] result

[0368] We repeated the process with the L67R i53 mutant and identified additional HDR-promoting mutants of i53 by screening combinatorial libraries targeting additional residues at the 53bp1 / i53 interface. Using L67R as the parent sequence, we designed a combinatorial library to explore all amino acid combinations at positions 12 and 14. Lentiviruses packaged with this combinatorial library were used to transduce CD34+ HSPC cells, and editing at the HBB locus was performed in triplicate using the HBB-UBC-GFP AAV (MOI = 2500). As shown in Figure 18A, NGS analysis of gDNA purified from the sorted mCherry+GFP+ and mCherry+GFP- populations revealed enrichment of distinct mutations (T12 and T14, shown in blue) in the mCherry+GFP+ population compared to the parent L67R. As shown in Figure 18B, clear trends emerged among the hits when the results were analyzed by amino acid category. The combination of aromatic and / or hydrophobic amino acids at residue 12 with a negative charge at residue 14 appeared to be particularly beneficial for HDR levels in HBB compared to the parent T12 T14 L67R. Therefore, a subset of these mutants (i.e., T12Y.T14E.L67R and T12I.T14D.L67R) were further validated and characterized.

[0369] We repeated the process with the L67H i53 mutant and identified additional HDR-promoting mutants of i53 by screening combinatorial libraries targeting additional residues at the 53bp1 / i53 interface. Using L67R as the parent sequence, we designed a combinatorial library to explore all amino acid combinations at positions 12 and 14. Lentiviruses packaged with this combinatorial library were used to transduce CD34+ HSPC cells, and editing at the HBB locus was performed in triplicate using the HBB-UBC-GFP AAV (MOI = 2500). As shown in Figure 19A, NGS analysis of gDNA purified from the sorted mCherry+GFP+ and mCherry+GFP- populations revealed enrichment of distinct mutations (T12 and T14, shown in blue) in the mCherry+GFP+ population compared to the parent L67H. As shown in Figure 19B, analysis of the results by amino acid category revealed similar trends in hits as in the analog library with the parent L67R (Figure 18, i.e., combinations of aromatic and / or hydrophobic amino acids at residue 12 with a negative charge at residue 14), but additional beneficial amino acid combinations at residues 12 and 14 emerged as hits relative to the parent L67H (i.e., combinations of a hydrophobic amino acid at residue 12 with a histidine at residue 14, or replacement of residue 14 with a negatively charged amino acid while retaining the original threonine at residue 12). Therefore, a subset of these mutants (i.e., T12V.T14H.L67H and T14D.L67H, etc.) were further validated and characterized.

[0370] The top hits from a combinatorial library at residues 12 and 14 using L67H as the parent were validated by pooled lentiviral expression. The top mutants identified from the combinatorial screen were individually cloned into the lentiviral vectors described above. The resulting plasmids were pooled with controls (parental control L67H and negative control i53 inactivation mutant "DM" = P67L L70V) to generate a small validation library. Lentiviruses packaged with this library were used to transduce CD34+ HSPC cells, and editing at the HBB locus was performed in triplicate using HBB-UBC-GFP AAV (MOI = 2500). As shown in Figure 20, NGS analysis of gDNA purified from the sorted mCherry+GFP+ and mCherry+GFP- populations confirmed positive enrichment relative to L67H for the majority of the mutants tested.

[0371] We repeated the process with the L67H.H68Y i53 mutant to identify additional HDR-promoting i53 variants by screening combinatorial libraries targeting additional residues at the 53bp1 / i53 interface. Using L67H.H68Y as the parent sequence, we designed a combinatorial library to explore all amino acid combinations at positions 12 and 14. Lentiviruses containing this combinatorial library were used to transduce CD34+ HSPC cells, and editing at the HBB locus was performed in triplicate using the HBB-UBC-GFP AAV (MOI = 2500). As shown in Figure 21A, next-generation sequencing (NGS) analysis of gDNA purified from the sorted mCherry+GFP+ and mCherry+GFP- populations revealed enrichment of distinct mutations (T12 and T14, shown in blue) in the mCherry+GFP+ population compared to the parent L67H.H68Y. As shown in Figure 21B, when the results were analyzed by amino acid category, similar trends in hits were observed as in the analogous libraries with parent L67R and L67H (Figures 18 and 20, i.e., combinations of aromatic and / or hydrophobic amino acids at residue 12 with a negative charge at residue 14), while several additional unique and beneficial amino acid combinations emerged as hits for parent L67H.H68Y (i.e., T12I.T14N.L67H.H68Y).

[0372] We repeated the process with the L67K.H68F i53 mutant to identify additional HDR-promoting mutants of i53 by screening combinatorial libraries targeting additional residues at the 53bp1 / i53 interface. Using L67K.H68F as the parent sequence, we designed a combinatorial library to explore all amino acid combinations at positions 12 and 14. Lentiviruses packaged with this combinatorial library were used to transduce CD34+ HSPC cells, and editing at the HBB locus was performed in triplicate using the HBB-UBC-GFP AAV (MOI = 2500). As shown in Figure 22A, NGS analysis of gDNA purified from the sorted mCherry+GFP+ and mCherry+GFP- populations revealed enrichment of distinct mutations (T12 and T14, shown in blue) in the mCherry+GFP+ population compared to the parent L67K.H68F. As shown in Figure 22B, when the results were analyzed by amino acid category, similar trends in hits were observed as in the analogous libraries with the parents L67R, L67H, and L67H.H68Y (Figures 18, 20, and 22, i.e., combinations of aromatic and / or hydrophobic amino acids at residue 12 with a negative charge at residue 14), while additional beneficial amino acid combinations at residues 12 and 14 emerged as hits for the parent L67K.H68F (i.e., combinations of tyrosine at position 12 with a polar, uncharged residue (including the original threonine) at position 14).

[0373] The top hits from a combinatorial library at residues 65 and 66 using T12Y.T14E.L67R as the parent were validated by pooled lentiviral expression. The top mutants identified from the combinatorial screen were individually cloned into the lentiviral vectors described above. The resulting plasmids were pooled with controls (parental controls T12Y.T14E.L67R, L67R, and i53, and a negative control i53 inactivation mutant "DM" = P67L L70V) to generate a small validation library. Lentiviruses containing this library were used to transduce CD34+ HSPC cells, and editing at the HBB locus was performed in triplicate using HBB-UBC-GFP AAV (MOI = 2500). NGS analysis of gDNA purified from the sorted mCherry+GFP+ and mCherry+GFP- populations confirmed positive enrichment for T12Y.T14E.L67R for many of the mutants tested, as shown in Figure 23. A subset of these mutants, including the top hits identified in the above screen, were further validated and characterized.

[0374] Multiple clinically important HBB loci 1,2 , HBA 10 , CCR5 11 , and Il2RG 12We performed GFP knock-in in CD34+ HSPC cells using AAV targeting i53 and three representative purified "hit" mutants of i53 identified in the above screen (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R). Protein mutants (including L67R and the parental control i53) were incorporated into the nucleofection solution at a concentration of 0.4 mg / mL. After nucleofection, cells were incubated with AAV at MOIs of 625 and 2500. As shown in Figure 24, the four i53 mutants tested (L67R, T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) were found to increase % GFP integration at the (A) HBB, (B) HBA, (C) CCR5, and (D) IL2RG loci compared to no protein controls in both CD34+ HSPC donors and at both MOIs tested. Integration of parental i53 also increased % GFP knock-in compared to no protein controls, although at a consistently lower level than the i53 mutants identified in the functional screen. The fold increase in HDR% with the addition of i53 mutants varied across the loci tested, averaging approximately 57% for (E) HBB, 96% for (F) HBA, 49% for (G) CCR5, and 82% for (H) IL2RG across two donors and two MOIs (slightly higher fold changes were observed at low MOIs than at high MOIs for all AAVs tested). In comparison, i53 increased HDR% by approximately 33% for HBB, 36% for HBA, 27% for CCR5, and 39% for IL2RG. These results suggest that additional potent mutants identified by repeating the above screen with the L67 / H68 mutant may significantly increase HDR compared to i53 at multiple clinically important loci.

[0375] Next, we performed editing of CD34+ HSPC cells from three donors using HBB-SNP AAV (a sickle cell mutation-correcting AAV template) and three representative i53 "hit" variants identified in the above screen (T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R). Protein variants (including L67R and the parental control i53) were incorporated into the nucleofection solution at a concentration of 0.8 mg / mL. After nucleofection, cells were incubated with AAV at MOIs of 312.5 and 2500. gDNA was extracted from the edited cells and analyzed by next-generation sequencing. As shown in Figure 25A, using these editing conditions, all tested variants significantly affected the proportion of HDR-corrected alleles compared to the control at both MOIs of 312.5 and 2500. As shown in Figure 25B, edited alleles from each sample were further characterized by the type of editing (unedited wild-type (WT), MMEJ, NHEJ, HBD, or HDR). The resulting analysis results are shown. Figure 25C shows the breakdown of NHEJ and HDR editing contributions normalized by total edited alleles. The increase in HDR% observed for the i53 mutant was accompanied by a corresponding decrease in NHEJ%, with the i53 mutant showing a more pronounced effect on HDR% and NHEJ% compared to i53. As shown in Figure 25D, incorporating i53 mutants and / or wild-type i53 into the editing protocol did not significantly increase the frequency of indels at the off-target editing site OT-1, as assessed by NGS. Furthermore, a subset of edited cells was collected to assess the relative amount of DDR by quantifying (Figure 25E) yH2Ax phosphorylation and (Figure 25F) p21 expression. Higher amounts of yH2Ax phosphorylation and p21 expression were observed in cells edited at an MOI of 2500 compared to cells edited at an MOI of 312.5, whereas addition of i53 mutants and / or wild-type i53 at both MOIs did not significantly increase yH2AX and p21 responses compared to no-protein controls.These results suggest that the high-affinity i53 mutants identified in the above screen can be utilized to favorably reduce AAV6 and DDR while maintaining high levels of editing.

[0376] Dose-response curves were generated for the purified "hit" mutants of i53 identified in the above screen (L67R, L67H.H68Y, T12Y.T14E.L67R, T12V.T14H.L67H, and T12Y.T14E.S65K.K66G.L67R) against parental i53 using HBB-SNP AAV (a sickle cell mutation-corrected AAV template). 1,2 The protein variants were incorporated into the nucleofection solution at concentrations of 0.2, 0.4, and 0.8 mg / mL. After nucleofection, cells were incubated with AAV at MOIs of 312.5 and 2500. To quantify the editing results, gDNA was extracted from the edited cells and analyzed by NGS. As shown in Figure 26, cells treated with the i53 variants retained high levels of HDR-corrected alleles (normalized to total edited alleles) across all editing conditions compared to the control (cells edited without protein-based additives; the average HDR% for two replicates is shown as a gray dotted line) and the parental i53. While a promotion was observed at 0.8 mg / mL, its effect on HDR% levels was found to significantly decrease as the protein concentration decreased. These results confirm previous findings that the high-affinity i53 variants identified in the above screen are significantly more potent than the parental i53.

[0377] Biolayer interferometry (BLI) was performed to measure the binding kinetics of additional i53 mutants with immobilized 53BP1 Tudor domain. BLI was used to collect kinetic measurements (on-rate and off-rate) for the binding of i53 mutants to biotin-labeled 53BP1 Tudor domain. The on-rate and off-rate measured at various solution concentrations were used to determine the dissociation constant (K) of each listed mutant. D ) was calculated. D A 1:1 binding model was used for the calculation of K D>50 nM, ++ indicates a K between 5 and 50 nM D Figure 27A shows ubiquitin binding to immobilized 53BP1 Tudor domain. Figure 27B shows i53 binding to immobilized 53BP1 Tudor domain. Figure 27C shows mutant i53L67R binding to immobilized 53BP1 Tudor domain. Figure 27D shows mutant i53L67H binding to immobilized 53BP1 Tudor domain. Figure 27E shows mutant i53T12Y.T14E.L67R binding to immobilized 53BP1 Tudor domain. Figure 27F shows mutant i53T12V.T14H.L67H binding to immobilized 53BP1 Tudor domain. All purified "hit" mutants of i53 showed distinct binding kinetics compared to parent i53, demonstrating a clear increase in binding rate. Therefore, compared to i53, these proteins likely have an enhancing effect on HDR levels.

[0378] The stability and polyreactivity of a subset of purified i53 mutant proteins were further evaluated. As shown in Figure 28A, the i53 mutant proteins were purified to high quality and exhibited monodisperse properties when analyzed using dynamic light scattering (DLS). As shown in Figure 28B, an ELISA-based assay revealed that the purified high-affinity i53 mutants exhibited low polyreactivity and highly specific binding to the 53BP1 Tudor domain.

[0379] References

[0380] 1.Dever,D.,Bak,R.,Reinisch,A.et al.CRISPR / Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539,384-389(2016)

[0381] 2.Wilkinson,A.C.,Dever,D.P.,Baik,R.et al.Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice. Nat Commun 12,686 (2021)

[0382] 3.Clement,K.,Rees,H.,Canver,M.C.et al.CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol 37,224-226(2019)

[0383] 4.Canny,M.,Moatti,N.,Wan,L.et al.Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat Biotechnol 36,95-102(2018)

[0384] 5.Emsley,P.,Cowtan,K.Coot:model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60,2126-2132(2004)

[0385] 6.Ranjha,L.,Howard,S.M.& Cejka,P.Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes. Chromosoma 127,187-214(2018)

[0386] 7.Schiroli,G.,Conti,A.,Ferrari,S.,et al.Precise gene editing preserves hematopoietic stem cell function following transient p53-Mediated DNA damage response. Cell Stem Cell 24,551-565(2019)

[0387] 8.Nakamura,K.,Sakai,W.,Kawamoto,T.,et al.Genetic dissection of vertebrate 53BP1: a major role in non- homologous end joining of DNA double strand breaks. DNA Repair 5,741-749(2006)

[0388] 9.Bunting,S.F.Callen,E.,Wong,N.,et al.53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141,243-254(2010)

[0389] 10. Cromer,M.K.,Camarena,J.,Martin,R.M.et al.Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells. Nat Med 27,677-687(2021)

[0390] 11. Gomez-Ospina,N.,Scharenberg,S.G.,Mostrel,N.et al.Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I.Nat Commun 10,4045(2019)

[0391] 12. Pavel-Dinu,M.,Wiebking,V.,Dejene,B.T.et al.Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nat Commun 10,1634(2019)

[0392] 13. Cradick,T.J.,Qiu,P.,Lee,C.M.,et al.COSMID:A Web-based Tool for Identifying and Validating CRISPR / Cas Off-target Sites. Mol Ther Nucleic Acids 3,e214(2014)

[0393] 14. Tsai,S.,Nguyen,N.,Malagon-Lopez,J.et al.CIRCLE-seq:a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods 14,607-614(2017)

[0394] 15. Tsai,S.,Zheng,Z.,Nguyen,N.et al.GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33,187-197(2015)

[0395] All publications and patent applications cited herein are hereby incorporated by reference as if each such publication or patent application were specifically and individually indicated to be so incorporated by reference. While the claimed subject matter has been described in various embodiments, it will be apparent to those skilled in the art that various modifications, substitutions, omissions, and alterations can be made therein without departing from the spirit thereof. Accordingly, the scope of the subject matter is intended to be limited only by the following claims, including equivalents thereof. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10]

Claims

1. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1, having one or more modifications relative to SEQ ID NO: 1, wherein the one or more modifications include a modification at position 67 of SEQ ID NO: 1, a modification at position 68 of SEQ ID NO: 1, or a combination thereof.

2. A polypeptide comprising an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the polypeptide has one or more modifications relative to SEQ ID NO: 1, wherein the one or more modifications include a modification at position 67 of SEQ ID NO: 1, a modification at position 68 of SEQ ID NO: 1, or a combination thereof.

3. The modification at position 67 of SEQ ID NO: 1 is (a) Arg(L67R), (b) His(L67H), (c) Lys(L67K), wherein the polypeptide further comprises an alteration at position 68 of SEQ ID NO: 1; (d) Ser (L67S), (e) Thr (L67T), (f) Gln (L67Q), (g) Asn(L67N), or The polypeptide according to any one of claims 1 to 2, wherein (h) is His(L67H).

4. The modification at position 68 of SEQ ID NO: 1 is (a) Trp (H68W), (b) Tyr(H68Y), or (c) Phe(H68F).

5. The one or more modifications are: (a) Arg at position 67 of SEQ ID NO: 1; (b) His at position 67 of SEQ ID NO: 1; (c) Lys at position 67 of SEQ ID NO: 1 (L67K), wherein the polypeptide further comprises an alteration at position 68 of SEQ ID NO: 1; (d) Ser at position 67 of SEQ ID NO: 1 (L67S); (e) Thr at position 67 of SEQ ID NO: 1 (L67T); (f) Gln at position 67 of SEQ ID NO: 1 (L67Q); (g) Asn at position 67 of SEQ ID NO: 1 (L67N); (h) His at position 67 of SEQ ID NO: 1 (L67H); (i) Trp at position 68 of SEQ ID NO: 1 (H68W); (j) Tyr at position 68 of SEQ ID NO: 1 (H68Y); (k) Phe (H68F) at position 68 of SEQ ID NO: 1, and (l) The polypeptide according to any one of claims 1 to 2, selected from the group consisting of a combination thereof.

6. The one or more modifications are: (a) Arg at position 67 of SEQ ID NO: 1 (L67R) and Trp at position 68 of SEQ ID NO: 1 (H68W); (b) His at position 67 of SEQ ID NO: 1 (L67H) and Tyr at position 68 of SEQ ID NO: 1 (H68Y); (c) His at position 67 of SEQ ID NO: 1 (L67H) and Phe at position 68 of SEQ ID NO: 1 (H68F); (d) His at position 67 of SEQ ID NO: 1 (L67H) and Trp at position 68 of SEQ ID NO: 1 (H68W); (e) Arg at position 67 of SEQ ID NO: 1 (L67R) and Tyr at position 68 of SEQ ID NO: 1 (H68Y); (f) Arg at position 67 of SEQ ID NO: 1 (L67R) and Phe at position 68 of SEQ ID NO: 1 (H68F); (g) Ser (L67S) at position 67 of SEQ ID NO: 1 and Phe (H68F) at position 68 of SEQ ID NO: 1; (h) Thr at position 67 of SEQ ID NO: 1 (L67T) and Trp at position 68 of SEQ ID NO: 1 (H68W); (i) Ser at position 67 of SEQ ID NO: 1 (L67S) and Trp at position 68 of SEQ ID NO: 1 (H68W); (j) Gln (L67Q) at position 67 of SEQ ID NO: 1 and Tyr (H68Y) at position 68 of SEQ ID NO: 1; (k) Asn at position 67 of SEQ ID NO: 1 (L67N) and Tyr at position 68 of SEQ ID NO: 1 (H68Y); (l) an Asn at position 67 of SEQ ID NO: 1 (L67N) and a Trp at position 68 of SEQ ID NO: 1 (H68W); or (m) Lys at position 67 of SEQ ID NO: 1 (L67K) and Tyr at position 68 of SEQ ID NO: 1 (H68Y).

7. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

2.

8. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

3.

9. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

4.

10. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

5.

11. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

6.

12. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

7.

13. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

8.

14. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

9.

15. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

10.

16. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

11.

17. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

12.

18. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

13.

19. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

14.

20. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

15.

21. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

16.

22. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

17.

23. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

18.

24. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

19.

25. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

20.

26. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

21.

27. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

22.

28. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

23.

29. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

24.

30. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

25.

31. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

26.

32. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

27.

33. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

28.

34. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

29.

35. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

30.

36. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

31.

37. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

32.

38. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

33.

39. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

34.

40. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

35.

41. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

36.

42. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

37.

43. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

38.

44. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

39.

45. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

40.

46. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

41.

47. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

42.

48. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

43.

49. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

44.

50. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

45.

51. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

46.

52. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

47.

53. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

48.

54. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

49.

55. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

50.

56. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

51.

57. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

52.

58. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

53.

59. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

54.

60. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

55.

61. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

56.

62. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

57.

63. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

58.

64. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

59.

65. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

60.

66. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

61.

67. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

62.

68. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

63.

69. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

64.

70. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

65.

71. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

66.

72. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

67.

73. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

68.

74. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

69.

75. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

70.

76. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

71.

77. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:

72.

78. The polypeptide of any one of claims 1 to 6, further comprising one or more additional modifications relative to SEQ ID NO:

1.

79. 31. The polypeptide of claim 30, wherein the one or more additional modifications to SEQ ID NO: 1 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more modifications at any amino acid position relative to the amino acid sequence of SEQ ID NO:

1.

80. The one or more additional modifications are (a) Gln(L2Q) at position 2 of SEQ ID NO: 1; (b) Ile at position 44 of SEQ ID NO: 1 (A44I); (c) Gln at position 49 of SEQ ID NO: 1 (S49Q); (d) Gln at position 62 of SEQ ID NO: 1 (L62Q); (e) Glu at position 64 of SEQ ID NO: 1 (D64E); (f) Thr at position 66 of SEQ ID NO: 1 (K66T); (g) Leu at position 69 of SEQ ID NO: 1 (P69L); (h) Val at position 70 of SEQ ID NO: 1 (L70V), and 31. The polypeptide of claim 30, selected from the group consisting of (i) a combination thereof.

81. (a) position 12 of SEQ ID NO: 1; (b) position 14 of SEQ ID NO: 1; (c) position 65 of SEQ ID NO: 1; (d) position 66 of SEQ ID NO: 1; (e) position 67 of SEQ ID NO: 1, and (f) The polypeptide of any one of claims 1 to 6 or 30 to 32, further comprising one or more additional modifications in combination therewith.

82. 82. The polypeptide of claim 81, wherein the modification at position 12 of SEQ ID NO: 1 is Tyr (T12Y).

83. 82. The polypeptide of claim 81, wherein the modification at position 12 of SEQ ID NO: 1 is Val (T12V).

84. The polypeptide of any one of claims 81 to 82, wherein the modification at position 14 of SEQ ID NO: 1 is Glu (T14E).

85. The polypeptide of any one of claims 81 to 82, wherein the modification at position 14 of SEQ ID NO: 1 is His (T14H).

86. The polypeptide of any one of claims 81 to 85, wherein the modification at position 65 of SEQ ID NO: 1 is Lys (S65K).

87. 87. The polypeptide of any one of claims 81 to 86, wherein the modification at position 66 of SEQ ID NO: 1 is Gly (K66G).

88. The polypeptide of any one of claims 81 to 87, wherein the modification at position 67 of SEQ ID NO: 1 is His (L67H).

89. 89. The polypeptide of any one of claims 1 to 88, further comprising one or two Gly at the C-terminus of the polypeptide.

90. The polypeptide has a specific binding affinity of 0.5 to 500×10 to the Tudor domain of 53BP1. －９ 90. The polypeptide of any one of claims 1 to 89, having a binding affinity of M.

91. A composition comprising a polypeptide according to any one of claims 1 to 90 in admixture with a carrier, excipient or diluent.

92. A polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of claims 1 to 90.

93. 93. An expression vector comprising the polynucleotide of claim 92.

94. 94. A composition comprising the polypeptide of any one of claims 1 to 90, the composition of claim 91, the polynucleotide of claim 92, or the expression vector of claim 93, and one or more components of a gene editing system.

95. The one or more components of the gene editing system include: (i) a nuclease capable of generating a double-strand break within a cellular locus; and (ii) the composition of Claim 94, comprising a donor polynucleotide.

96. 96. The composition of claim 95, wherein the donor polynucleotide comprises non-overlapping 5' and 3' homologous arms, wherein each homologous arm is homologous to a portion of the locus, and wherein upon generation of the double-stranded break within the locus by the nuclease, the sequence of the donor polynucleotide is integrated into the locus by homology-directed repair (HDR).

97. 97. The composition of any one of claims 95-96, wherein the nuclease comprises a CRISPR nuclease and a single guide RNA (sgRNA) capable of hybridizing to a target sequence within the locus, wherein the sgRNA guides the CRISPR nuclease to the target sequence.

98. 98. The composition of claim 97, wherein the CRISPR nuclease is a Cas protein.

99. 99. The composition of claim 98, wherein the Cas protein is Cas9 or a high-fidelity variant thereof.

100. 100. The composition of any one of claims 97 to 99, wherein the sgRNA and the CRISPR nuclease are formed into a ribonucleoprotein (RNP) complex.

101. 101. The composition of any one of claims 97 to 100, wherein the sgRNA comprises one or more chemically modified nucleotides.

102. 102. The composition of claim 101, wherein the modified nucleotides are selected from the group consisting of 2'-O-methyl nucleotides, 2'-O-methyl 3'-phosphorothioate nucleotides, and 2'-O-methyl 3'-thio PACE nucleotides.

103. 103. The composition of any one of claims 101-102, wherein the 5' end, 3' end, or a combination thereof of the modified sgRNA comprises modified nucleotides.

104. 104. The composition of any one of claims 95 to 103, wherein the donor polynucleotide is comprised in a viral vector, a plasmid, or a single-stranded oligodeoxynucleotide (ssODN).

105. 105. The composition of any one of claims 95 to 104, wherein the donor polynucleotide is comprised in an adeno-associated virus (AAV) vector.

106. 106. The composition of claim 105, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

107. The composition of claim 105, wherein the AAV vector is an AAV6 vector.

108. 108. The composition of any one of claims 94 to 107, further comprising an inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs).

109. 109. The composition of claim 108, wherein the DNA-PKcs inhibitor is selected from the group consisting of AZD7648, M3814 / nedisertib, CC-115, and BAY-8400.

110. The composition of any one of claims 95 to 109, wherein the locus is the HBB gene.

111. A host cell comprising a polypeptide according to any one of claims 1 to 90 or a composition according to claim 91.

112. 94. A host cell comprising the polynucleotide of claim 92 or the expression vector of claim 93.

113. A host cell comprising the composition of any one of claims 94 to 110.

114. The host cell of any one of claims 111 to 113, wherein the host is a mammal.

115. The host cell of claim 114, wherein the mammal is a human.

116. 116. The host cell of any one of claims 111 to 115, wherein the cell is a primary cell.

117. 117. The host cell of claim 116, wherein the primary cells are selected from the group consisting of primary blood cells and primary mesenchymal cells.

118. 117. The host cell of claim 116, wherein the primary cell is selected from the group consisting of a primary stem cell, a primary progenitor cell, and a primary somatic cell.

119. The host cell of claim 118, wherein the primary stem cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and organ stem cells.

120. 119. The host cell of claim 118, wherein the primary progenitor cells are selected from the group consisting of hematopoietic progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, multipotent progenitor cells, oligopotent progenitor cells, and lineage-restricted progenitor cells.

121. 119. The host cell of claim 118, wherein the primary somatic cell is selected from the group consisting of a fibroblast, a hepatocyte, a cardiac cell, a liver cell, a pancreatic cell, a muscle cell, a skin cell, a blood cell, a neuronal cell, and an immune cell.

122. 122. The host cell of claim 121, wherein the immune cell is selected from the group consisting of T lymphocytes (T cells), B lymphocytes (B cells), small lymphocytes, natural killer cells (NK cells), natural killer T cells, macrophages, monocytes, monocyte precursors, eosinophils, neutrophils, basophils, megakaryocytes, myeloblasts, mast cells, and dendritic cells.

123. The host cell of claim 116, wherein the primary cell is a CD34+ hematopoietic stem cell or a CD34+ hematopoietic progenitor cell.

124. 124. The host cell of any one of claims 111 to 123, wherein the genetic locus of the cell contains one or more mutations associated with a disease or encodes an aberrant protein.

125. 125. The host cell of any one of claims 113 to 124, wherein integration of the donor polynucleotide sequence into the host cell allows correction of a disease-associated mutation in the cell.

126. 126. The host cell of any one of claims 124-125, wherein the disease is selected from the group consisting of hemoglobinopathies, viral infections, X-linked severe combined immunodeficiency, Fanconi anemia, hemophilia, neoplasms, cancer, alpha-1 antitrypsin deficiency, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, cystic fibrosis, blood diseases and disorders, inflammation, diseases or disorders of the immune system, metabolic diseases, liver diseases and disorders, kidney diseases and disorders, muscle diseases and disorders, bone or cartilage diseases and disorders, nerve and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and lysosomal storage disorders.

127. 127. The host cell of claim 126, wherein the hemoglobinopathy is sickle cell disease, alpha-thalassemia, beta-thalassemia, or delta-thalassemia.

128. 125. The host cell of any one of claims 113 to 124, wherein integration of the donor polynucleotide sequence is capable of replacing a mutant allele in the host cell with a wild-type allele.

129. A polypeptide according to any one of claims 1 to 90, a polynucleotide according to claim 92, or an expression vector according to claim 93; and one or more components of a gene editing system.

130. The one or more components of the gene editing system include: (i) a nuclease capable of generating a double-strand break within a cellular locus; and 130. The kit of claim 129, comprising (ii) a donor polynucleotide.

131. 131. The kit of Claim 130, wherein the donor polynucleotide comprises non-overlapping 5' and 3' homologous arms, wherein each homologous arm is homologous to a portion of the locus, and wherein upon generation of the double-stranded break in the locus by the nuclease, a sequence of the donor polynucleotide is integrated into the locus by homology-directed repair (HDR).

132. 132. The kit of any one of claims 130-131, wherein the nuclease comprises a CRISPR nuclease and a single guide RNA (sgRNA) capable of hybridizing to a target sequence within the locus, wherein the sgRNA guides the CRISPR nuclease to the target sequence.

133. 133. The kit of Claim 132, wherein the CRISPR nuclease is a Cas protein.

134. 134. The kit of claim 133, wherein the Cas protein is Cas9 or a high-fidelity variant thereof.

135. The kit of any one of claims 132 to 134, wherein the sgRNA and the CRISPR nuclease are formed into a ribonucleoprotein (RNP) complex.

136. 136. The kit of any one of claims 132 to 135, wherein the sgRNA comprises one or more chemically modified nucleotides.

137. 137. The kit of claim 136, wherein the modified nucleotides are selected from the group consisting of 2'-O-methyl nucleotides, 2'-O-methyl 3'-phosphorothioate nucleotides, and 2'-O-methyl 3'-thio PACE nucleotides.

138. 138. The kit of any one of claims 136-137, wherein the 5' end, 3' end, or a combination thereof of the modified sgRNA comprises modified nucleotides.

139. 139. The kit of any one of claims 130 to 138, wherein the donor polynucleotide is contained in a viral vector, a plasmid, or a single-stranded oligodeoxynucleotide (ssODN).

140. 140. The kit of any one of claims 130 to 139, wherein the donor polynucleotide is contained in an adeno-associated virus (AAV) vector.

141. 141. The kit of claim 140, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

142. 141. The kit of claim 140, wherein the AAV vector is an AAV6 vector.

143. 143. The kit of any one of claims 129 to 142, further comprising an inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs).

144. 144. The kit of claim 143, wherein the DNA-PKcs inhibitor is selected from the group consisting of AZD7648, M3814 / nedisertib, CC-115, and BAY-8400.

145. 145. The kit of any one of claims 130 to 144, wherein the locus is the HBB gene.

146. A method for increasing homologous recombination in a cell, the method comprising administering to said cell a polypeptide according to any one of claims 1 to 90.

147. 147. The method of Claim 146, further comprising introducing into the cell one or more components of a gene editing system.

148. The one or more components of the gene editing system include: (i) a nuclease capable of generating a double-strand break within a cellular locus; and (ii) the method of claim 147, comprising a donor polynucleotide.

149. 1. A method for stably integrating an exogenous polynucleotide sequence into the genome of a cell, comprising administering to said cell: (a) a nuclease capable of generating a double-stranded break within a gene locus in said cell; (b) a donor polynucleotide, and (c) introducing a polypeptide according to any one of claims 1 to 90, When the nuclease generates a double-stranded break within the locus, the sequence of the donor polynucleotide is integrated into the locus by homology-directed repair (HDR), resulting in a gene-edited cell.

150. 150. The method of Claim 149, wherein the donor polynucleotide comprises non-overlapping 5' and 3' homologous arms, wherein each homologous arm is homologous to a portion of the locus, and wherein upon generation of the double-stranded break within the locus by the nuclease, the sequence of the donor polynucleotide is integrated into the locus by homology-directed repair (HDR).

151. 151. The method of any one of claims 149-150, wherein the nuclease comprises a CRISPR nuclease and a single guide RNA (sgRNA) capable of hybridizing to a target sequence within the locus, wherein the sgRNA guides the CRISPR nuclease to the target sequence.

152. 152. The method of Claim 151, wherein the CRISPR nuclease is a Cas protein.

153. 153. The method of Claim 152, wherein the Cas protein is Cas9 or a high-fidelity variant thereof.

154. 154. The method of any one of claims 151 to 153, wherein the sgRNA and the CRISPR nuclease are formed into a ribonucleoprotein (RNP) complex.

155. 155. The method of any one of claims 151 to 154, wherein the sgRNA comprises one or more chemically modified nucleotides.

156. 156. The method of claim 155, wherein said modified nucleotides are selected from the group consisting of 2'-O-methyl nucleotides, 2'-O-methyl 3'-phosphorothioate nucleotides, and 2'-O-methyl 3'-thio PACE nucleotides.

157. 157. The method of Claim 155 or 156, wherein the 5' end, 3' end, or a combination thereof, of the modified sgRNA comprises modified nucleotides.

158. 158. The method of any one of claims 149 to 157, wherein the donor polynucleotide is contained in a viral vector, a plasmid, or a single-stranded oligodeoxynucleotide (ssODN).

159. 159. The method of any one of claims 149 to 158, wherein the donor polynucleotide vector is comprised in an adeno-associated virus (AAV) vector.

160. 160. The method of claim 159, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

161. 160. The method of claim 159, wherein the AAV vector is an AAV6 vector.

162. 162. The method of any one of claims 149 to 161, further comprising introducing into said cells an inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs).

163. 163. The method of claim 162, wherein the DNA-PKcs inhibitor is selected from the group consisting of AZD7648, M3814 / nedisertib, CC-115, and BAY-8400.

164. 164. The method of any one of claims 149 to 163, wherein the locus is the HBB gene.

165. 165. The method of any one of claims 149 to 164, wherein the host is a mammal.

166. 166. The method of claim 165, wherein the mammal is a human.

167. 167. The method of any one of claims 149 to 166, wherein the cells are primary cells.

168. 168. The method of claim 167, wherein the primary cells are selected from the group consisting of primary blood cells and primary mesenchymal cells.

169. 168. The method of claim 167, wherein the primary cells are selected from the group consisting of primary stem cells, primary progenitor cells, and primary somatic cells.

170. 170. The method of claim 169, wherein the stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and organ stem cells.

171. 170. The method of claim 169, wherein the progenitor cells are selected from the group consisting of hematopoietic progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, multipotent progenitor cells, oligopotent progenitor cells, and lineage-restricted progenitor cells.

172. 170. The method of claim 169, wherein the somatic cells are selected from the group consisting of fibroblasts, hepatocytes, cardiac cells, liver cells, pancreatic cells, muscle cells, skin cells, blood cells, nerve cells, and immune cells.

173. 170. The method of claim 169, wherein the immune cells are selected from the group consisting of T lymphocytes (T cells), B lymphocytes (B cells), small lymphocytes, natural killer cells (NK cells), natural killer T cells, macrophages, monocytes, monocyte precursors, eosinophils, neutrophils, basophils, megakaryocytes, myeloblasts, mast cells, and dendritic cells.

174. 168. The method of claim 167, wherein the primary cells are CD34+ hematopoietic stem cells or CD34+ hematopoietic progenitor cells.

175. 175. The method of any one of claims 149 to 174, wherein the genetic locus of the cell contains one or more mutations associated with disease or encodes an aberrant protein.

176. 175. The method of any one of claims 149 to 174, wherein the sequence of the donor polynucleotide is integrated into the genome of the host cell, thereby correcting a disease-associated mutation in the cell.

177. 175. The method of any one of claims 149 to 174, wherein the integration of the donor polynucleotide sequence replaces a mutant allele in the cell with a wild-type allele.

178. 177. The method of claim 175 or 176, wherein the disease is selected from the group consisting of hemoglobinopathies, viral infections, X-linked severe combined immunodeficiency, Fanconi anemia, hemophilia, neoplasms, cancer, alpha-1 antitrypsin deficiency, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, cystic fibrosis, blood diseases and disorders, inflammation, diseases or disorders of the immune system, metabolic diseases, liver diseases and disorders, kidney diseases and disorders, muscle diseases and disorders, bone or cartilage diseases and disorders, nerve and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and lysosomal storage disorders.

179. 179. The method of claim 178, wherein the hemoglobinopathy is sickle cell disease, alpha-thalassemia, beta-thalassemia, or delta-thalassemia.

180. 180. The method of any one of claims 149-179, further comprising administering the gene-edited cells to a patient in need thereof.

181. 1. A method for identifying a mutant polypeptide of a 53BP1 inhibitory polypeptide having the amino acid sequence of SEQ ID NO:1, wherein the mutant polypeptide is capable of mediating integration of a donor polynucleotide by improved homology-directed repair (HDR), the method comprising: a. A CD34+ hematopoietic stem and progenitor cell (HSPC) population is isolated from i. a control polypeptide comprising the amino acid sequence of SEQ ID NO: 1; ii. one or more mutant polypeptides, each mutant polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with one or more modifications relative to SEQ ID NO: 1, wherein the one or more modifications comprise a modification at position 67 of SEQ ID NO: 1, a modification at position 68 of SEQ ID NO: 1, or a combination thereof; iii. a nuclease capable of generating a double-stranded break within the gene locus of said HSPC, and iv. contacting with a donor polynucleotide comprising a reporter gene; b. i. a first subpopulation of contacted HSPCs that express at least a reference amount of the reporter gene; and ii. isolating a second subpopulation of contacted HSPCs that do not express the reference amount of the reporter gene; c. measuring the relative abundance of a control polypeptide and a variant polypeptide in both the first HSPC subpopulation and the second HSPC subpopulation, respectively.

182. 182. The method of Claim 181, wherein the mutant polypeptide is identified as being capable of mediating improved integration by homology-directed repair (HDR) relative to the control polypeptide if (i) the mutant polypeptide is more abundant than the control polypeptide in the first subpopulation, and / or (ii) the ratio of mutant polypeptide:control polypeptide in the first subpopulation is higher than the ratio of mutant polypeptide:control polypeptide in the second subpopulation.

183. 183. The method of claim 181 or 182, wherein each of the one or more mutant polypeptides further comprises a modification at position 12 of SEQ ID NO: 1, a modification at position 14 of SEQ ID NO: 1, or a combination thereof.

184. 184. The method of any one of claims 181 to 183, wherein each of the one or more mutant polypeptides further comprises a modification at position 65 of SEQ ID NO:1, a modification at position 66 of SEQ ID NO:1, or a combination thereof.

185. 185. The method of any one of claims 183 to 184, wherein contacting the first and second HSPC populations with the control polypeptide and the mutant polypeptide, respectively, comprises contacting the first and second HSPC populations with lentiviral vectors encoding the control polypeptide and the mutant polypeptide, respectively.

186. 186. The method of any one of claims 181 to 185, wherein measuring the relative abundance of a control polypeptide and a mutant polypeptide comprises sequencing the lentiviral vector encoding the control polypeptide and the mutant polypeptide.

187. 187. The method of any one of claims 181 to 186, wherein each modification of the mutant polypeptide comprises a single amino acid substitution.

188. The method of claim 185, wherein each lentiviral vector encoding a mutant polypeptide comprises a polynucleotide comprising an NNK codon that encodes an alteration in the mutant polypeptide.

189. 189. The method of any one of claims 181-188, wherein the donor polynucleotide comprises non-overlapping 5' and 3' homologous arms, wherein each homologous arm is homologous to a portion of the locus, and wherein upon generation of the double-stranded break in the locus by the nuclease, the sequence of the donor polynucleotide is integrated into the locus by homology-directed repair (HDR).

190. 190. The method of any one of claims 181 to 189, wherein the nuclease comprises a CRISPR nuclease and a single guide RNA (sgRNA) capable of hybridizing to a target sequence within the locus, wherein the sgRNA guides the CRISPR nuclease to the target sequence.

191. 191. The method of Claim 190, wherein the sgRNA and the CRISPR nuclease are formed into a ribonucleoprotein (RNP) complex.

192. 192. The method of any one of claims 181 to 191, wherein the donor polynucleotide is comprised in a viral vector, a plasmid, or a single-stranded oligodeoxynucleotide (ssODN).

193. 193. The method of claim 192, wherein the donor polynucleotide is contained in an adeno-associated virus (AAV) vector.

194. 194. The method of any one of claims 181-193, further comprising isolating a mutant polypeptide with improved HDR activity from said second population of HSPCs.

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