Compositions and methods for enhancing donor oligonucleotide-based gene editing

Abstract

Compositions for enhanced gene editing and methods of use thereof are. The composition contains a cell-penetrating antibody and a donor oligonucleotide containing a sequence that can correct a mutation in a cell's genome. Preferably, the composition does not contain a nuclease, PNA, or nanoparticle. The compositions are used to modify the genome of a cell by contacting the cell with an effective amount of the composition. Genomic modification occurs at a higher frequency both ex vivo and in vivo, when cells are contacted with the cell-penetrating antibody and donor oligonucleotide as compared to the absence of the cell-penetrating antibody.

1 - 48 . (canceled) 
     
     
         49 . A composition comprising a non-covalent complex of
 (i) a donor oligonucleotide comprising a sequence that can correct at least one mutation in a cell's genome; and,   (ii) a 3E10 antibody or a cell-penetrating fragment thereof.   
     
     
         50 . The composition of  claim 49 , wherein the 3E10 antibody or cell-penetrating fragment thereof is selected from the group consisting of a cell-penetrating monovalent, divalent, or multivalent single chain variable fragment (scFv) of 3E10, a cell-penetrating diabody of 3E10, and a cell-penetrating humanized form or variant of 3E10. 
     
     
         51 . The composition of  claim 50 , wherein the 3E10 antibody or cell-penetrating fragment thereof incorporates an aspartic acid (Asp) to asparagine (Asn) substitution in a position corresponding to the Asp31 position in the heavy chain of 3E10. 
     
     
         52 . The composition of  claim 49 , wherein the 3E10 antibody or a cell-penetrating fragment thereof comprises:
 (a) a heavy chain variable region (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 15;   (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 17;   (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 18;   (d) a light chain variable region (VL) CDR1 comprising the amino acid sequence of SEQ ID NO: 24;   (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and,   (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 26;   or   (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 16;   (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 17;   (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 18;   (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 24;   (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and,   (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 26.   
     
     
         53 . The composition of  claim 49 , wherein the 3E10 antibody or a cell-penetrating fragment thereof comprises a VH sequence set forth in SEQ ID NOS: 1, 2, 3, 4, 5, or 6. 
     
     
         54 . The composition of  claim 49 , wherein the 3E10 antibody or a cell-penetrating fragment thereof comprises a VL sequence set forth in SEQ ID NOS: 7, 8, 9, 10, or 11. 
     
     
         55 . The composition of  claim 49 , wherein the 3E10 antibody or a cell-penetrating fragment thereof comprises a VH sequence set forth in SEQ ID NOS: 1, 2, 3, 4, 5, or 6, and a VL sequence set forth in SEQ ID NOS: 7, 8, 9, 10, or 11. 
     
     
         56 . The composition of  claim 49 , wherein the donor oligonucleotide comprises single stranded or double stranded DNA. 
     
     
         57 . The composition of  claim 49 , wherein the donor oligonucleotide comprises one or more inserted, substituted, or deleted nucleotides relative to a target gene in the cell's genome. 
     
     
         58 . The composition of  claim 49 , wherein the cell's genome has a mutation underlying a disease or disorder selected from the group consisting of hemophilia, muscular dystrophy, globinopathies, cystic fibrosis, xeroderma pigmentosum, lysosomal storage diseases, immune deficiency syndromes, tyrosinemia, Fanconi anemia, spherocytosis, alpha-1-anti-trypsin deficiency, Wilson's disease, Leber's hereditary optic neuropathy, and chronic granulomatous disorder. 
     
     
         59 . The composition of  claim 58 , wherein the immune deficiency syndromes is X-linked severe combined immunodeficiency or ADA deficiency. 
     
     
         60 . The composition of  claim 58 , wherein the mutation is in a gene encoding coagulation factor VIII, a gene encoding coagulation factor IX, a gene encoding dystrophin, a gene encoding beta-globin, a CFTR gene, an XPC gene, an XPD gene, a gene encoding DNA polymerase eta, a FANCA gene, a FANCB gene, a FANCC gene, a FANCD1 gene, a FANCD2 gene, a FANCE gene, a FANCF gene, a FANCG gene, a FANCI gene, a FANCJ gene, a FANCL gene, a SPTA1 gene or other spectrin genes, an ANK1 gene, a SERPINA1 gene, an ATP7B gene, an IL2RG gene, an ADA gene, an FAH gene, a CYBA gene, a CYBB gene, an NCF1 gene, an NCF2 gene, or an NCF4 gene. 
     
     
         61 . The composition of  claim 49 , wherein the composition does not comprise a nuclease, triplex-forming peptide nucleic acid oligomer, or nanoparticles. 
     
     
         62 . The composition of  claim 49 , wherein the 3E10 antibody or a cell-penetrating fragment thereof can be transported into the nucleus of the cell without the aid of a carrier or conjugate. 
     
     
         63 . A pharmaceutical composition comprising the composition of  claim 49  and a pharmaceutically acceptable excipient. 
     
     
         64 . The pharmaceutical composition of  claim 63 , wherein the composition is packaged in a polymeric nanoparticle. 
     
     
         65 . The pharmaceutical composition of  claim 64 , wherein the polymeric nanoparticle comprises a polyhydroxy acid polymer. 
     
     
         66 . The pharmaceutical composition of  claim 65 , wherein the polyhydroxy acid polymer is as poly(lactic-co-glycolic acid) (PLGA). 
     
     
         67 . The pharmaceutical composition of  claim 64 , wherein the polymeric nanoparticle comprises a targeting moiety, a cell penetrating peptide, or a combination thereof, which is associated with, linked, conjugated, or otherwise attached directly or indirectly to the polymeric nanoparticle. 
     
     
         68 . A method of modifying the genome of a cell comprising contacting the cell with an effective amount of a composition comprising a non-covalent complex of
 (i) a donor oligonucleotide comprising a sequence that can correct at least one mutation in a cell's genome; and,   (ii) a 3E10 antibody or a cell-penetrating fragment thereof,   wherein the 3E10 antibody or a cell-penetrating fragment thereof comprises   (a) a heavy chain variable region (V H ) complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 15,   (b) a V H  CDR2 comprising the amino acid sequence of SEQ ID NO: 17,   (c) a V H  CDR3 comprising the amino acid sequence of SEQ ID NO: 18,   (d) a light chain variable region (V L ) CDR1 comprising the amino acid sequence of SEQ ID NO: 24,   (e) a V L  CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and   (f) a V L  CDR3 comprising the amino acid sequence of SEQ ID NO: 26,   or   (a) a V H  CDR1 comprising the amino acid sequence of SEQ ID NO: 16;   (b) a V H  CDR2 comprising the amino acid sequence of SEQ ID NO: 17;   (c) a V H  CDR3 comprising the amino acid sequence of SEQ ID NO: 18;   (d) a V L  CDR1 comprising the amino acid sequence of SEQ ID NO: 24;   (e) a V L  CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and,   (f) a V L  CDR3 comprising the amino acid sequence of SEQ ID NO: 26.   
     
     
         69 . The method of  claim 68 , wherein the method is conducted in vivo in a subject. 
     
     
         70 . The method of  claim 68 , wherein the method is conducted ex vivo. 
     
     
         71 . The method of  claim 68 , wherein the cell is a hematopoietic stem cell. 
     
     
         72 . A modified cell obtained according to the method of  claim 68 . 
     
     
         73 . The method of  claim 68 , wherein the donor oligonucleotide comprises single stranded or double stranded DNA. 
     
     
         74 . The method of  claim 68 , wherein the donor oligonucleotide comprises one or more inserted, substituted, or deleted nucleotides relative to a target gene in the cell's genome. 
     
     
         75 . A method to treat a disease or disorder comprising administering a therapeutically effective amount of a modified cell obtained according to the method of  claim 68  to a subject in need thereof. 
     
     
         76 . The method of  claim 75 , wherein the disease or disorder is selected from the group consisting of hemophilia, muscular dystrophy, globinopathies, cystic fibrosis, xeroderma pigmentosum, lysosomal storage diseases, immune deficiency syndromes, tyrosinemia, Fanconi anemia, spherocytosis, alpha-1-anti-trypsin deficiency, Wilson's disease, Leber's hereditary optic neuropathy, and chronic granulomatous disorder. 
     
     
         77 . The method of claim  81 , wherein the immune deficiency syndromes is X-linked severe combined immunodeficiency or ADA deficiency. 
     
     
         78 . The method of  claim 68 , wherein the at least one mutation is in a gene encoding coagulation factor VIII, a gene encoding coagulation factor IX, a gene encoding dystrophin, a gene encoding beta-globin, a CFTR gene, an XPC gene, an XPD gene, a gene encoding DNA polymerase eta, a FANCA gene, a FANCB gene, a FANCC gene, a FANCD1 gene, a FANCD2 gene, a FANCE gene, a FANCF gene, a FANCG gene, a FANCI gene, a FANCJ gene, a FANCL gene, a SPTA1 gene or other spectrin genes, an ANK1 gene, a SERPINA1 gene, an ATP7B gene, an IL2RG gene, an ADA gene, an FAH gene, a CYBA gene, a CYBB gene, an NCF1 gene, an NCF2 gene, or an NCF4 gene.