METHODS FOR TARGETED INSERTION OF DNA IN GENES
Methods and compositions for modifying the coding sequence of endogenous genes using rare-cutting endonucleases and transposases. The methods and compositions described herein can be used to modify the coding sequence of endogenous genes.
1 . A method of integrating a transgene into an endogenous gene, the method comprising:
a. administering a transgene, wherein the transgene comprises
i. a first and second splice acceptor sequence,
ii. a first and second partial coding sequence, and
iii. one bidirectional terminator or a first and second terminator;
b. administering at least one rare-cutting endonuclease targeted to a site within the endogenous gene,
wherein the transgene is integrated within the endogenous gene.
2 . The method of claim 1 , wherein the first splice acceptor is operably linked to the first partial coding sequence and the second splice acceptor is operably linked to the second partial coding sequence.
3 . The method of claim 2 , wherein the first partial coding sequence is operably linked to the first terminator, and the second partial coding sequence is operably linked to the second terminator.
4 . The method of claim 2 , wherein the first and second partial coding sequences are operably linked to the bidirectional terminator.
5 . The method of claim 3 , wherein the first and second splice acceptors, first and second coding sequences, and first and second terminators are oriented in a tail-to-tail orientation.
6 . The method of claim 5 , wherein the transgene further comprises a first and second target site for one or more rare-cutting endonucleases, wherein the target sites flank the first and second splice acceptors.
7 . The method of claim 5 , wherein the transgene further comprises a left and right homology arm which flank the first and second splice acceptors.
8 . The method of claim 7 , wherein the transgene is harbored within an adeno-associated viral vector.
9 . The method of claim 7 , wherein the transgene further comprises a first and second target site for the one or more rare-cutting endonucleases, wherein the target sites flank the first and second splice acceptors.
10 . The method of claim 9 , wherein the first and second target sites flank the first and second homology arms.
11 . The method of claim 1 , wherein the transgene is integrated within an intron of the endogenous gene or at an intron-exon junction.
12 . The method of claim 1 , wherein the transgene is integrated within an intron, or at the intron-exon junction of the ATXN3 gene or CACNA1A gene.
13 . The method of claim 12 , wherein the transgene comprises a first and second partial coding sequence encoding the peptide produced by exon 10 of a non-pathogenic ATXN3 gene and is targeted to intron 9, or the intron 9 exon 10 junction, of a pathogenic ATXN3 gene.
14 . The method of claim 12 , wherein the transgene comprises a first and second partial coding sequence encoding the peptide produced by exon 47 of a non-pathogenic CACNA1A gene and is targeted to intron 46, or the intron 46 exon 47 junction, of a pathogenic CACNA1A gene.
15 . The method of claim 1 , wherein the nuclease is a CRISPR/Cas12a nuclease or a CRISPR/Cas9 nuclease.
16 . The method of claim 1 , wherein the first and second partial coding sequences encode the same amino acids.
17 . The method of claim 1 , wherein the first and second coding sequences differ in nucleic acid sequence but encode the same amino acids.
18 . The method of claim 1 , wherein the transgene is harbored on a vector, wherein the vector format is selected from double-stranded linear DNA, double-stranded circular DNA, or a viral vector.
19 . The method of claim 18 , wherein the viral vector is selected from an adenovirus vector, an adeno-associated virus vector, or a lentivirus vector.
20 . The method of claim 19 , wherein the transgene is equal to or less than 4.7 kb.
21 . The method of claim 1 , wherein said endogenous gene is the wild type gene of said partial coding sequences.
22 . The method of claim 21 , wherein said endogenous gene is aberrant and the partial coding sequences encode a partial protein from a functional version of said endogenous gene.
23 . A DNA polynucleotide comprising:
a. a first and second splice acceptor sequence,
b. a first and second partial coding sequence,
c. one bidirectional terminator or a first and second terminator,
d. optionally, a first and second homology arm, and
e. optionally, a first and second rare-cutting endonuclease target site.
24 . A method of integrating a transgene into an endogenous gene, the method comprising:
a. administering a transgene, wherein the transgene comprises
i. a left and right transposon end,
ii. a first and second splice acceptor sequence,
iii. a first and second partial coding sequence, and
iv. one bidirectional terminator or a first and second terminator;
b. administering a transposase
wherein the transgene is integrated within the endogenous gene.
25 . A method of integrating a transgene into an endogenous gene, the method comprising:
a. administering a transgene, wherein the transgene comprises
i. a first and second splice acceptor sequence,
ii. a first and second coding sequence, and
iii. one bidirectional terminator or a first and second terminator;
b. administering at least one rare-cutting endonuclease targeted to a site within the endogenous gene,
wherein the transgene is integrated within the endogenous gene.
26 . A method of integrating a transgene into an endogenous gene, the method comprising:
a. administering a transgene, wherein the transgene comprises
i. a first and second splice acceptor sequence,
ii. a first and second coding sequence,
iii. one bidirectional terminator or a first and second terminator, and
iv. a first and second homology arm
wherein the transgene is integrated within the endogenous gene.