Genome Editing without Nucleases

Abstract

Methods and compositions are provided for editing the genome of a cell without the use of an exogenously supplied nuclease. Aspects of the methods include contacting a cell with a targeting vector comprising nucleic acid sequence to be integrated into the target locus, where the cell is not also contacted with a nuclease. In addition, reagents, devices and kits thereof that find use in practicing the subject methods are provided.

1 . A method for the targeted integration of a transgene into the genome of a cell in the absence of an exogenously provided nuclease, the method comprising:
 contacting a cell with a recombinant viral vector, the recombinant viral vector comprising:   i. a polynucleotide comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes the transgene; and the second nucleic acid sequence is positioned 5′ or 3′ to the first nucleic acid sequence and promotes the production of two independent gene products upon integration into the target integration site in the genome of the cell;   ii. a third nucleic acid sequence positioned 5′ to the polynucleotide and comprising sequence that is substantially homologous to genomic sequence 5′ of a target integration site in the genome of the cell; and   iii. a fourth nucleic acid sequence positioned 3′ of the polynucleotide and comprising sequence that is substantially homologous to genomic sequence 3′ of a target integration site in the genome of the cell;   wherein the cell is not contacted with a nuclease or nucleic acid encoding a nuclease.   
     
     
         2 . The method according to  claim 1 , wherein the cell is a non-dividing cell. 
     
     
         3 . The method according to  claim 1 , wherein the contacting occurs in vivo. 
     
     
         4 . The method according to  claim 3 , wherein the method finds use in treating a medical condition associated with a gene deficiency. 
     
     
         5 . The method according to  claim 4 , wherein the medical condition is selected from the group consisting of hemophilia, hemophilia A, hemophilia B, a branched-chain organic aciduria, maple syrup urine disease (MSUD), isovaleric acidaemia (IVA), propionic aciduria (PA), methylmalonic aciduria (MMA), 3 methylcrotonyl glycinuria, 3-methylglutaconic Aciduria Type I, short/branched-chain Acyl-CoA dehydrogenase deficiency, 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency, isobutyryl-CoA dehydrogenase deficiency, 3-Hydroxyisobutyric aciduria, malonic aciduria, a long chained fatty acid oxidation disorder, a glycogen storage disease, Glycogen storage disease type I (GSD1), A carnitine cycle disorder, a urea cycle disorder, Crigler-Najjar syndrome, heraditary tyrosinemia, epidermolysis bullosa, Wilson disease, adenosine deaminase deficiency, sickle cell disease, X-Linked Severe Combined Immunodeficiency (SCID-X1), thalassemia, cystic fibrosis, alpha-1 anti-trypsin deficiency, diamond-blackfan anemia, Gaucher's disease, growth hormone deficiency, and Parkinson's Disease. 
     
     
         6 . The method according to  claim 3 , wherein the method finds use in promoting immunoprophylaxis. 
     
     
         7 . The method according to  claim 6 , wherein the transgene encodes an agent that promotes immunoprophylaxis. 
     
     
         8 . The method according to  claim 7 , wherein the agent that promotes immunoprophylaxis is an antibody or a chimeric polypeptide, and is specific for a pathogen selected from: human immunodeficiency virus (HIV), influenza virus, respiratory syncytial virus (RSV), hepatitis C virus (HCV), a plasmodium, plasmodium falciparum, plasmodium malariae, a fungus, and a bacterium. 
     
     
         9 . The method according to  claim 3 , wherein the method finds use in promoting wound healing. 
     
     
         10 . A recombinant viral vector for integrating a transgene into a target integration site in the genome of the cell, comprising:
 a polynucleotide cassette comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises the transgene; and the second nucleic acid sequence is positioned 5′ or 3′ to the first nucleic acid sequence and promotes the production of two independent gene products upon integration into the target integration site in the genome of the cell;   a third nucleic acid sequence positioned 5′ to the polynucleotide cassette and comprising sequence that is substantially homologous to genomic sequence 5′ of the target integration site in the genome of the cell; and   a fourth nucleic acid sequence positioned 3′ of the polynucleotide cassette and comprising sequence that is substantially homologous to genomic sequence 3′ of the target integration site in the genome of the cell.   
     
     
         11 . The recombinant viral vector according to  claim 10 , wherein the viral vector is an rAAV vector. 
     
     
         12 . The recombinant viral vector according to  claim 10 , wherein the nucleic acid sequence that promotes the production of two independent gene products at the target integration site is selected from: a sequence that encodes a 2A peptide; an IRES; an intein; a recognition sequence for a site specific protease; a sequence that encodes a cleavable linker that is cleaved as part of the coagulation cascade; a sequence that encodes a factor XI cleavage site; and an intronic splice donor/splice acceptor sequence. 
     
     
         13 . The recombinant viral vector according to  claim 12 , wherein the expression and activity of an endogenous gene comprising the target integration site is not disrupted by the integration of the transgene. 
     
     
         14 . The recombinant viral vector according to  claim 13 , wherein:
 the 3′ end of the endogenous gene comprises the target integration site,   the sequence of the third nucleic acid sequence is substantially homologous to the DNA sequence upstream of the stop codon of the endogenous gene; and   the sequence of the fourth nucleic acid sequences is substantially homologous to the DNA sequence downstream of the stop codon of the endogenous gene.   
     
     
         15 . The recombinant viral vector according to  claim 13 , wherein:
 the 5′ end of the endogenous gene comprises the target integration site,   the sequence of the third nucleic acid sequence is substantially homologous to the DNA sequence upstream of the start codon of the endogenous gene; and   the sequence of the fourth nucleic acid sequences is substantially homologous to the DNA sequence downstream of the start codon of the endogenous gene.   
     
     
         16 . The recombinant viral vector according to  claim 13 , wherein the endogenous gene is selected from the group consisting of the albumin gene, a collagen gene, and an actin gene. 
     
     
         17 . The recombinant viral vector according to  claim 10 , wherein the transgene complements a gene deficiency. 
     
     
         18 . The recombinant viral vector according to  claim 10 , wherein the transgene encodes an agent that promotes immunoprophylaxis. 
     
     
         19 . The recombinant viral vector according to  claim 18 , wherein the agent that promotes immunoprophylaxis is a polypeptide comprising an immunoglobulin domain and an effector domain. 
     
     
         20 . The recombinant viral vector according to  claim 18 , wherein the agent that promotes immunoprophylaxis is an antibody. 
     
     
         21 - 23 . (canceled)