Methods for using transcription-dependent directed evolution of aav capsids

Abstract

Disclosed are methods for performing transcription-dependent directed evolution (TRADE) and novel AAV capsids selected using such methods.

1 . A nucleic acid molecule comprising a Parvoviridae cap gene sequence in an antisense orientation, wherein the Parvoviridae cap gene sequence in the antisense orientation comprises a messenger ribonucleic acid (mRNA) splicing suppression mutation. 
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein the nucleic acid molecule further comprises a regulatory sequence. 
     
     
         3 . The nucleic acid molecule of  claim 2 , wherein the regulatory sequence drives expression of the Parvoviridae cap gene sequence in the antisense orientation. 
     
     
         4 . The nucleic acid molecule of  claim 3 , wherein the regulatory sequence is a promoter sequence. 
     
     
         5 . The nucleic acid molecule of  claim 4 , wherein the promoter is a cell type-specific promoter, a tissue-specific promoter, or a ubiquitous promoter. 
     
     
         6 . The nucleic acid molecule of  claim 1 , wherein the Parvoviridae cap gene sequence is derived from an adeno-associated virus (AAV) genome comprising an AAV cap gene sequence. 
     
     
         7 . The nucleic acid molecule of  claim 6 , wherein the AAV cap gene sequence is a wild-type AAV cap gene sequence. 
     
     
         8 . The nucleic acid molecule of  claim 6 , wherein the AAV cap gene sequence is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or other natural AAV isolate cap gene sequence. 
     
     
         9 . The nucleic acid molecule of  claim 6 , wherein the AAV cap gene sequence is an engineered AAV cap gene sequence. 
     
     
         10 . The nucleic acid molecule of  claim 6 , wherein the AAV cap gene sequence is one of a library of diverse AAV cap gene sequences. 
     
     
         11 . The nucleic acid molecule of  claim 1 , wherein the messenger ribonucleic acid (mRNA) splicing suppression mutation comprises a mutation within an exon-intron junction, wherein the exon-intron junction comprises a sequence as set forth in any one of SEQ ID NOs: 199-220 or 463. 
     
     
         12 . The nucleic acid molecule of  claim 1 , wherein the Parvoviridae cap gene sequence in the antisense orientation comprises two or more messenger ribonucleic acid (mRNA) splicing suppression mutations. 
     
     
         13 . A nucleic acid molecule comprising:
 (a) a first nucleic acid sequence comprising a first regulatory sequence and a Parvoviridae cap gene sequence in a sense orientation;   (b) a second nucleic acid sequence comprising a second regulatory sequence and the Parvoviridae cap gene sequence in an antisense orientation, wherein the Parvoviridae cap gene sequence in the antisense orientation comprises a messenger ribonucleic acid (mRNA) splicing suppression mutation.   
     
     
         14 . The nucleic acid molecule of  claim 13 , wherein the second regulatory sequence drives expression of an antisense transcript. 
     
     
         15 . The nucleic acid molecule of  claim 13 , wherein the second regulatory sequence is a cell type-specific promoter, a tissue-specific promoter, or a ubiquitous promoter. 
     
     
         16 . The nucleic acid molecule of claim of  claim 13 , wherein the first regulatory sequence drives expression of the Parvoviridae cap gene sequence in the sense orientation. 
     
     
         17 . The nucleic acid molecule of  claim 13 , wherein the Parvoviridae cap gene sequence is derived from an adeno-associated virus (AAV) genome comprising an AAV cap gene sequence. 
     
     
         18 . The nucleic acid molecule of  claim 17 , wherein the AAV cap gene sequence is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or other natural AAV isolate cap gene sequence. 
     
     
         19 . The nucleic acid molecule of  claim 17 , wherein the AAV cap gene sequence is an engineered AAV cap gene sequence. 
     
     
         20 . The nucleic acid molecule of  claim 17 , wherein the AAV cap gene sequence is one of a library of diverse AAV cap gene sequences. 
     
     
         21 . (canceled) 
     
     
         22 . (canceled)