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 - 17 . (canceled)
18 . A transgene comprising from 5′ to 3′ orientation:
a first splice acceptor, a first coding sequence, a first terminator, a second terminator reverse complement, a second coding sequence reverse complement, and a second splice acceptor reverse complement, wherein the first coding sequence is operably linked to the first splice acceptor and first terminator, and the second coding sequence is operably linked to the second splice acceptor and second terminator,
wherein the first terminator is selected from an SV40 poly(A) or BGH poly(A),
wherein the first and second coding sequences differ in nucleic acid sequence but encode the same amino acids, wherein said amino acids encoded by the first and second coding sequences are amino acids encoded by the corresponding endogenous gene for alpha-1 antitrypsin deficiency, and
wherein the transgene is equal to or less than 4.7 kb.
19 . The transgene of claim 18 , wherein the second terminator is selected from an SV40 poly(A) or BGH poly(A).
20 . The transgene of claim 19 , wherein the amino acids encoded by the first and second coding sequences have at least 80% sequence identity to the amino acids encoded by the endogenous gene for alpha-1 antitrypsin deficiency, wherein the percent sequence identity is calculated by matching amino acids encoded by the first and second coding sequence with amino acids encoded by an endogenous gene for alpha-1 antitrypsin deficiency and dividing the number of matches by the length of the amino acids encoded by the first and second coding sequence, followed by multiplying the resulting value by 100.
21 . The transgene of claim 20 , wherein the amino acids encoded by the first and second coding sequences have about 98% sequence identity to the amino acids encoded by the endogenous gene for alpha-1 antitrypsin deficiency.
22 . The transgene of claim 20 , wherein the amino acids encoded by the first and second coding sequences have about 99% sequence identity to the amino acids encoded by an endogenous gene for alpha-1 antitrypsin deficiency.
23 . The transgene of claim 20 , wherein the transgene is harbored on a viral vector.
24 . The transgene of claim 23 , wherein the viral vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, and a lentivirus vector.
25 . The method of claim 24 , wherein the viral vector is an adeno-associated viral vector.
26 . The transgene of claim 25 , wherein the viral vector is incorporated into a viral particle.
27 . The transgene of claim 26 , wherein the transgene does not comprise homology arms.
28 . The transgene of claim 27 , wherein the first splice acceptor comprises a splice acceptor sequence from an intron of the endogenous gene for alpha-1 antitrypsin deficiency.
29 . An adeno-associated viral vector comprising:
(i) a transgene comprising from 5′ to 3′ orientation a first splice acceptor, a first coding sequence, a first terminator, a second terminator reverse complement, a second coding sequence reverse complement, and a second splice acceptor reverse complement, wherein the first coding sequence is operably linked to the first splice acceptor and first terminator, and the second coding sequence is operably linked to the second splice acceptor and second terminator,
wherein the first and second coding sequences differ in nucleic acid sequence but encode the same amino acids, wherein said amino acids encoded by the first and second coding sequences correspond to amino acids encoded by an endogenous gene, and
wherein the transgene is equal to or less than 4.7 kb; and
(ii) adeno-associated virus inverted terminal repeats flanking the transgene.
30 . The adeno-associated viral vector of claim 29 , wherein the amino acids encoded by the first and second coding sequences are amino acids encoded by the corresponding endogenous gene for alpha-1 antitrypsin deficiency.
31 . The adeno-associated viral vector of claim 30 , wherein the first and second coding sequences encode amino acids having at least 80% sequence identity to the amino acids encoded by the endogenous gene for alpha-1 antitrypsin deficiency, wherein the percent sequence identity is calculated by matching amino acids encoded by the first and second coding sequence with amino acids encoded by an endogenous gene for alpha-1 antitrypsin deficiency and dividing the number of matches by the length of the amino acids encoded by the first and second coding sequence, followed by multiplying the resulting value by 100.
32 . The adeno-associated viral vector of claim 31 , wherein the amino acids encoded by the first and second coding sequences have about 98% sequence identity to the amino acids encoded by the endogenous gene for alpha-1 antitrypsin deficiency.
33 . The adeno-associated viral vector of claim 31 , wherein the amino acids encoded by the first and second coding sequences have about 99% sequence identity to the amino acids encoded by an endogenous gene for alpha-1 antitrypsin deficiency.
34 . The adeno-associated viral vector of claim 30 , wherein the first terminator is selected from an SV40 poly(A) or BGH poly(A),
35 . The adeno-associated viral vector of claim 34 , wherein the second terminator is selected from an SV40 poly(A) or BGH poly(A).
36 . The adeno-associated viral vector of claim 35 , wherein the viral vector is incorporated into a viral particle.
37 . The adeno-associated viral vector of claim 35 , wherein the transgene does not comprise homology arms.
38 . The adeno-associated viral vector of claim 37 , wherein the first splice acceptor comprises splice acceptor sequence from an intron of the endogenous gene for alpha-1 antitrypsin deficiency.
39 . The adeno-associated viral vector of claim 37 , wherein the first terminator is SV40 poly(A) and the second terminator is BGH poly(A).