Genome editing without nucleases
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 of treating a medical condition associated with a gene deficiency, the method comprising steps of:
delivering to liver cells of a subject suffering from the medical condition a recombinant AAV (rAAV) vector, the rAAV vector comprising:
(i) a polynucleotide comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises a transgene whose expression corrects the gene deficiency; and the second nucleic acid sequence is positioned 5′ or 3′ to the first nucleic acid sequence and promotes production of two independent gene products upon integration of the polynucleotide into a target integration site in the genome of the liver cells;
(ii) a third nucleic acid sequence positioned 5′ to the polynucleotide of (i) in the rAAV vector and comprising a sequence that is substantially homologous to a genomic sequence 5′ of the target integration site; and
(iii) a fourth nucleic acid sequence positioned 3′ to the polynucleotide of (i) in the rAAV vector and comprising a sequence that is substantially homologous to a genomic sequence 3′ of the target integration site;
wherein the transgene encodes a therapeutic agent that has cell-intrinsic or cell-extrinsic activity that promotes a biological process to treat the medical condition;
wherein the delivering does not involve delivering a nuclease or nucleic acid encoding a nuclease;
wherein, upon delivering the rAAV vector, the transgene that corrects the gene deficiency is integrated and expressed in the liver cells of the subject to thereby treat the medical condition; and wherein the medical condition is selected from the group consisting of a branched-chain organic aciduria, maple syrup urine disease (MSUD), isovaleric acidaemia (IVA), propionic aciduria (PA), methylmalonic acidemia (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, hereditary tyrosinemia, Wilson disease, and Phenylketonuria (PKU) disease.
2. A method of treating a medical condition associated with a gene deficiency, the method comprising steps of:
delivering to liver cells of a subject suffering from the medical condition a recombinant AAV (rAAV) vector, the rAAV vector comprising:
(i) a polynucleotide comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises a transgene whose expression corrects the gene deficiency; and the second nucleic acid sequence is positioned 5′ or 3′ to the first nucleic acid sequence and promotes production of two independent gene products upon integration of the polynucleotide into a target integration site in the genome of the liver cells;
(ii) a third nucleic acid sequence positioned 5′ to the polynucleotide of (i) in the rAAV vector and comprising a sequence that is substantially homologous to a genomic sequence 5′ of the target integration site; and
(iii) a fourth nucleic acid sequence positioned 3′ to the polynucleotide of (i) in the rAAV vector and comprising a sequence that is substantially homologous to a genomic sequence 3′ of the target integration site;
wherein the transgene encodes a therapeutic agent that has cell-intrinsic or cell-extrinsic activity that promotes a biological process to treat the medical condition; wherein the transgene comprises a nucleotide sequence encoding α1-antitrypsin, α-L-iduronidase, methylmalonyl-CoA mutase, or UGT1A1;
wherein the delivering does not involve delivering a nuclease or nucleic acid encoding a nuclease; and
wherein, upon delivering the rAAV vector, the transgene that corrects the gene deficiency is integrated and expressed in the liver cells of the subject to thereby treat the medical condition.
3. A method of treating a medical condition associated with a gene deficiency, the method comprising steps of:
delivering to liver cells of a subject suffering from the medical condition a recombinant AAV (rAAV) vector, the rAAV vector comprising:
(i) a polynucleotide comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises a transgene whose expression corrects the gene deficiency; and the second nucleic acid sequence is positioned 5′ or 3′ to the first nucleic acid sequence and promotes production of two independent gene products upon integration of the polynucleotide into a target integration site in the genome of the liver cells;
(ii) a third nucleic acid sequence positioned 5′ to the polynucleotide of (i) in the rAAV vector and comprising a sequence that is substantially homologous to a genomic sequence 5′ of the target integration site; and
(iii) a fourth nucleic acid sequence positioned 3′ to the polynucleotide of (i) in the rAAV vector and comprising a sequence that is substantially homologous to a genomic sequence 3′ of the target integration site;
wherein the transgene encodes a therapeutic agent that has cell-intrinsic or cell-extrinsic activity that promotes a biological process to treat the medical condition;
wherein the therapeutic agent is secreted from the liver cell to convey therapeutic effect;
wherein the delivering does not involve delivering a nuclease or nucleic acid encoding a nuclease; and
wherein, upon delivering the rAAV vector, the transgene that corrects the gene deficiency is integrated and expressed in the liver cells of the subject to thereby treat the medical condition.
4. The method of claim 3 , wherein the second nucleic acid sequence 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 IX cleavage site; and an intronic splice donor/splice acceptor sequence.
5. The method of claim 3 , wherein expression and activity of an endogenous gene comprising the target integration site is not disrupted by the integration of the transgene.
6. The method of claim 5 , 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 sequence is substantially homologous to the DNA sequence downstream of the stop codon of the endogenous gene.
7. The method of claim 5 , 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 sequence is substantially homologous to the DNA sequence downstream of the start codon of the endogenous gene.
8. The method of claim 5 , wherein the endogenous gene is an albumin gene.
9. The method of claim 5 , wherein the transgene becomes operably linked to the promoter of the endogenous gene upon integration into the target integration site.
10. A method of treating a medical condition associated with a gene deficiency, the method comprising steps of:
delivering to liver cells of a subject suffering from the medical condition a recombinant AAV (rAAV) vector, the rAAV vector comprising:
(i) a polynucleotide comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises a transgene whose expression corrects the gene deficiency; and the second nucleic acid sequence is positioned 5′ or 3′ to the first nucleic acid sequence and promotes production of two independent gene products upon integration of the polynucleotide into a target integration site in the genome of the liver cells;
(ii) a third nucleic acid sequence positioned 5′ to the polynucleotide of (i) in the rAAV vector and comprising a sequence that is substantially homologous to a genomic sequence 5′ of the target integration site; and
(iii) a fourth nucleic acid sequence positioned 3′ to the polynucleotide of (i) in the rAAV vector and comprising a sequence that is substantially homologous to a genomic sequence 3′ of the target integration site;
wherein the transgene encodes a therapeutic agent that has cell-intrinsic or cell-extrinsic activity that promotes a biological process to treat the medical condition;
wherein the therapeutic agent has cell-intrinsic activity and substitutes for a mutant protein in the liver cell in which it is expressed;
wherein the delivering does not involve delivering a nuclease or nucleic acid encoding a nuclease; and
wherein, upon delivering the rAAV vector, the transgene that corrects the gene deficiency is integrated and expressed in the liver cells of the subject to thereby treat the medical condition.
11. The method of claim 10 , wherein the second nucleic acid sequence 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 IX cleavage site; and an intronic splice donor/splice acceptor sequence.
12. The method of claim 10 , wherein expression and activity of an endogenous gene comprising the target integration site is not disrupted by the integration of the transgene.
13. The method of claim 12 , 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 sequence is substantially homologous to the DNA sequence downstream of the stop codon of the endogenous gene.
14. The method of claim 12 , 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 sequence is substantially homologous to the DNA sequence downstream of the start codon of the endogenous gene.
15. The method of claim 12 , wherein the endogenous gene is an albumin gene.
16. The method of claim 12 , wherein the transgene becomes operably linked to the promoter of the endogenous gene upon integration into the target integration site.