Methods for targeted insertion of dna in genes

Abstract

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.

What is claimed is: 
     
         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.