Transposition-based therapies

Abstract

Gene therapy compositions and methods are provided that make use of novel transposases and/or chimeric transposases for targeted transposition.

1 - 151 . (canceled) 
     
     
         152 . A composition comprising a nucleic acid encoding a transposase enzyme, wherein the transposase enzyme comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 and has a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 2, or a position corresponding thereto. 
     
     
         153 . The composition of  claim 152 , wherein the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P). 
     
     
         154 . The composition of  claim 153 , wherein the non-polar aliphatic amino acid is alanine (A). 
     
     
         155 . The composition of  claim 152 , wherein the transposase enzyme further comprises a substitution S 8 X 1 , wherein X 1  is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P), or a substitution corresponding thereto. 
     
     
         156 . The composition of  claim 155 , wherein X 1  is proline (P). 
     
     
         157 . The composition of  claim 152 , wherein the transposase enzyme further comprises a substitution C13RX 2 , wherein X 2  is selected from lysine (K), arginine (R), and histidine (H), or a substitution corresponding thereto. 
     
     
         158 . The composition of  claim 157 , wherein X 2  is arginine (R). 
     
     
         159 . The composition of  claim 152 , wherein the transposase enzyme further comprises a substitution N125X 3 , wherein X 3  is selected from is selected from lysine (K), arginine (R), and histidine (H), or a substitution corresponding thereto. 
     
     
         160 . The composition of  claim 159 , wherein X 3  is lysine (K). 
     
     
         161 . The composition of  claim 152 , wherein the transposase enzyme further comprises two or more of substitutions S8P, C13R, and N125K, or a substitution corresponding thereto. 
     
     
         162 . The composition of  claim 152 , wherein the transposase enzyme does not have additional amino acid residues at the C terminus relative to SEQ ID NO: 2. 
     
     
         163 . The composition of  claim 152 , wherein the transposase enzyme comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 2. 
     
     
         164 . The composition of  claim 152 , wherein the transposase enzyme comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 2. 
     
     
         165 . The composition of  claim 152 , further comprising a transcription activator-like effector (TALE) DNA binding domain (DBD), or a nuclease-deficient Cas9 (dCas9)/gRNA connected to the transposase enzyme. 
     
     
         166 . The composition of  claim 165 , wherein the transposase enzyme is capable of inserting a transposon at a TA dinucleotide site or a TTAA (SEQ ID NO: 1) tetranucleotide site in a genomic safe harbor site (GSHS) of a nucleic acid molecule. 
     
     
         167 . The composition of  claim 166 , wherein the TALE DBD or dCas9/gRNA is suitable for directing the transposase enzyme to the GSHS sequence. 
     
     
         168 . The composition of  claim 152 , wherein the nucleic acid is co-formulated with a nucleic acid encoding a transposon. 
     
     
         169 . The composition of  claim 168 , wherein the composition is in the form of a lipid nanoparticle (LNP). 
     
     
         170 . A composition comprising the transposase enzyme polypeptide encoded by the nucleic acid of  claim 152 . 
     
     
         171 . A method for inserting a gene into the genome of a cell, comprising contacting a cell with the composition of  claim 168 .