Methods and compositions for improved homology directed repair

Abstract

The disclosure features methods of increasing repair of a DNA double stranded break (DSB) in a target gene by the homology-directed repair (HDR) pathway. The disclosure also features compositions for use in the methods.

1 . A method for increasing homology directed repair (HDR) of a double-strand break (DSB) in a target gene in a cell or a population of cells, the method comprising contacting a cell or a population of cells with a 53BP1 inhibitor, wherein the cell or the population of cells is a quiescent human cell or a population of quiescent human cells that is induced to divide, and wherein the DSB is mediated by a site-directed nuclease, thereby increasing HDR of the DSB in the target gene in the cell or the population of cells. 
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein the 53BP1 inhibitor is (i) a 53BP1 binding polypeptide that inhibits 53BP1 recruitment to the DSB in the cell or the population of cells; or (ii) a 53BP1 binding polypeptide comprising an amino acid sequence selected from a group consisting of: SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 83, or SEQ ID NO: 86. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the 53BP1 inhibitor comprises (i) a nucleic acid comprising a nucleotide sequence encoding a 53BP1 binding polypeptide that inhibits 53BP1 recruitment to the DSB site in the cell or the population of cells; or (ii) a nucleic acid comprising a nucleotide sequence selected from a group consisting of: SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 85, or SEQ ID NO: 88. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 5 , wherein the nucleic acid comprises (i) a vector comprising a nucleotide sequence encoding the 53BP1 binding polypeptide; or (ii) a vector comprising a nucleotide sequence selected from a group consisting of: SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 84, or SEQ ID NO: 87. 
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 7 , wherein the vector is an adeno associated viral vector (AAV). 
     
     
         10 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the cell or population of cells is isolated from a tissue sample obtained from a human donor, or a peripheral blood sample obtained from a human donor. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the cell or population of cells is:
 (i) a hematopoietic stem or progenitor cell (HSPC) or a population of HSPCs;   (ii) a long-term HSPC (LT-HSPC) or a population of LT-HSPCs;   (iii) a CD34 expressing cell or a population of CD34 expressing cells;   (iv) a white blood cell or a population of white blood cells;   (v) in the G0 phase of the cell cycle; or   (vi) a combination of (i)-(v).   
     
     
         15 - 18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein the cell or population of cells is induced to divide by treatment with one or more extrinsic signals, wherein the one or more extrinsic signals are selected from: an extrinsic signal that stimulates a cell signaling receptor, a mitogen, or an environmental factor. 
     
     
         20 - 23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein the DSB is mediated by a Cas9 DNA endonuclease and one or more guide RNAs (gRNA), or a  Streptococcus pyogenes  Cas9 (SpCas9) and one or more gRNAs. 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 1 , further comprising contacting the cell or population of cells with a donor polynucleotide comprising a nucleotide sequence that corrects or induces a mutation when incorporated at the site of the DSB, wherein the donor polynucleotide comprises a double-stranded DNA (dsDNA) or a single-stranded oligonucleotide (ssODN). 
     
     
         27 - 29 . (canceled) 
     
     
         30 . The method of  claim 26 , wherein the donor polynucleotide comprises a vector comprising the donor polynucleotide, wherein the vector comprises a recombinant single-stranded adeno-associated viral vector (rAAV). 
     
     
         31 . (canceled) 
     
     
         32 . The method of  claim 30 , wherein the rAAV is serotype 6 (AAV6). 
     
     
         33 . The method of  claim 1 , further comprising contacting the cell or population of cells with an inhibitor of DNA-PK. 
     
     
         34 . (canceled) 
     
     
         35 . The method of  claim 1 , wherein the cell or population of cells is induced to divide prior to contact with the 53BP1 inhibitor, is contacted with the 53BP1 inhibitor prior to being induced to divide, or is contacted with the 53BP1 inhibitor concurrent with being induced to divide. 
     
     
         36 - 37 . (canceled) 
     
     
         38 . The method of  claim 1 , wherein the cell or population of cells is contacted ex vivo and the method further comprises administering the cell or population of cells to a subject in need thereof, or wherein the cell or population of cells is contacted with the 53BP1 inhibitor in vivo. 
     
     
         39 - 82 . (canceled) 
     
     
         83 . The method of  claim 12 , wherein the tissue sample is obtained from a human donor, and wherein the human donor is:
 (i) administered an HSPC mobilizing agent prior to obtaining the tissue sample;   (ii) administered (i), wherein the HSPC mobilizing agent is Plurexifor;   (iii) administered a combination of HSPC mobilizing agents prior to obtaining the tissue sample; or   (iv) administered (iii), wherein the combination of HSPC mobilizing agents comprises Plerixafor and granulocyte colony stimulating factor (GCSF).   
     
     
         84 - 87 . (canceled) 
     
     
         88 . The method of  claim 19 , wherein the extrinsic signal is:
 (i) one or more cytokines;   (ii) one or more cytokines selected from interleukin-3 (IL-3), stem cell factor (SCF), Fms-like tyrosine kinase 3 (Flt3) ligand, and thrombopoietin;   (iii) a mitogen;   (iv) an environmental factor; or   (v) hypoxia.   
     
     
         89 - 110 . (canceled) 
     
     
         111 . The method of  claim 38 , wherein the population of cells is contacted ex vivo and the contacting results in an increase in HDR frequency in the cell population to 50% or more. 
     
     
         112 . (canceled) 
     
     
         113 . The method of  claim 111 , wherein contacting results in;
 (i) a 1-2 fold increase in HDR frequency in the cell population, or wherein contacting results in a 1.1-2 fold, 1.2-2 fold, 1.3-2 fold, 1.4-2 fold, 1.5-2 fold, 1.6-2 fold, 1.7-2 fold, 1.8-2 fold, or 1.9-2 fold increase in HDR frequency in the cell population;   (ii) a 1-2 fold decrease in indel frequency in the cell population, or wherein contacting results in a 1.1-2 fold, 1.2-2 fold, 1.3-2 fold, 1.4-2 fold, 1.5-2 fold, 1.6-2 fold, 1.7-2 fold, 1.8-2 fold, or 1.9-2 fold decrease in indel frequency in the cell population;   (iii) an increase in engraftment in vivo following administration;   (iv) an increase in chimerism in vivo following administration; or   (v) a combination of (i)-(iv).   
     
     
         114 - 117 . (canceled) 
     
     
         118 . A method for increasing HDR of a DSB in a target gene in a population of human CD34+ cells, the method comprising contacting the population of human CD34+ cells with
 (i) an inhibitor of 53BP1, wherein the inhibitor is nucleic acid comprising a nucleotide sequence encoding a 53BP1 binding polypeptide comprising the amino acid sequence of SEQ ID NO: 69, and   (ii) a Cas9 DNA endonuclease and one or more gRNAs to effect a DSB within the target gene in the population of cells.   
     
     
         119 . The method of  claim 118 , wherein the nucleic acid comprising a nucleotide sequence encoding the 53BP1 binding polypeptide is an RNA or a vector. 
     
     
         120 . (canceled) 
     
     
         121 . The method of  claim 119 , wherein the vector is an AAV or an AAV of the DJ serotype (AAV-DJ). 
     
     
         122 - 123 . (canceled) 
     
     
         124 . The method of  claim 118 , wherein the Cas9 DNA endonuclease is a polypeptide, and wherein the Cas9 DNA endonuclease is pre-complexed with one or more gRNAs. 
     
     
         125 . (canceled) 
     
     
         126 . The method of  claim 124 , wherein the Cas9 DNA endonuclease and one or more gRNAs are electroporated into the cells, and wherein the cell population is contacted with the nucleic acid encoding the 53BP1 polypeptide prior to electroporation, is contacted with the nucleic acid encoding the 53BP1 polypeptide during electroporation, or is contacted with the nucleic acid encoding the 53BP1 polypeptide subsequent to electroporation. 
     
     
         127 - 129 . (canceled) 
     
     
         130 . The method of  claim 118 , further comprising contacting the population of cells with a donor polynucleotide comprising a nucleotide sequence that corrects or induces a mutation when incorporated at the site of the DSB, wherein the donor polynucleotide comprises a dsDNA or a ssODN. 
     
     
         131 - 134 . (canceled) 
     
     
         135 . The method of  claim 130 , wherein the donor polynucleotide comprises a vector comprising the donor polynucleotide, wherein the vector comprises rAAV or AAV6. 
     
     
         136 - 137 . (canceled) 
     
     
         138 . The method of  claim 130 , wherein the donor polynucleotide is administered to the cells prior to electroporation or following electroporation. 
     
     
         139 . (canceled) 
     
     
         140 . The method of  claim 118 , further comprising contacting the population of cells with an inhibitor of DNA-PK. 
     
     
         141 . (canceled) 
     
     
         142 . The method of  claim 118 , wherein the cells are cultured with
 (i) one or more cytokines;   (ii) one or more cytokines selected from: thrombopoietin, Flt3 ligand, SCF, IL-3; or   (iii) a combination of cytokines comprising: thrombopoietin, Flt3 ligand, SCF, and IL-3.   
     
     
         143 - 147 . (canceled) 
     
     
         148 . The method of  claim 118 , wherein the cells are cultured under hypoxic conditions comprising atmospheric oxygen content less than 5%. 
     
     
         149 . The method of  claim 118 , wherein the population of cells is contacted ex vivo and the method further comprises administering the cells to a subject in need thereof, wherein contacting results in an increase in HDR frequency in the cell population to 50% or more. 
     
     
         150 . (canceled) 
     
     
         151 . The method of  claim 149 , wherein contacting results in:
 (i) a 1-2 fold increase in HDR frequency in the cell population, or wherein contacting results in a 1.1-2 fold, 1.2-2 fold, 1.3-2 fold, 1.4-2 fold, 1.5-2 fold, 1.6-2 fold, 1.7-2 fold, 1.8-2 fold, or 1.9-2 fold increase in HDR frequency in the cell population;   (ii) a 1-2 fold decrease in indel frequency in the cell population, or wherein contacting results in a 1.1-2 fold, 1.2-2 fold, 1.3-2 fold, 1.4-2 fold, 1.5-2 fold, 1.6-2 fold, 1.7-2 fold, 1.8-2 fold, or 1.9-2 fold decrease in indel frequency in the cell population;   (iii) an increase in engraftment in vivo following administration;   (iv) an increase in chimerism in vivo following administration; or   (v) a combination of (i)-(iv).   
     
     
         152 - 155 . (canceled) 
     
     
         156 . A method for increasing HDR of a DSB in a target gene in a population of human CD34+ cells, the method comprising contacting the population of human CD34+ cells with an effective amount of an inhibitor of the catalytic subunit of DNA-dependent protein kinase, and wherein the DSB is mediated by a site-directed nuclease, thereby increasing HDR of the DSB in the target gene in the population of human CD34+ cells. 
     
     
         157 - 228 . (canceled)