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. This disclosure also provides novel AAV capsid mutants. TRADE technology was used to identify novel AAV vectors that mediate neuronal transduction in the brain following intravenous administration. Application of TRADE in vivo resulted in the identification of new AAV capsids that can transduce neurons more efficiently and more specifically than AAV9 in the brain following administration of the new AAV capsids. The disclosed methods may be used to identify AAV capsids that target various cell populations.

1 - 19 . (canceled) 
     
     
         20 . A nucleic acid comprising:
 A Parvoviridae genome flanked by ITR sequences, wherein the Parvoviridae genome comprises a Parvoviridae intron, a Parvoviridae cap gene, and a first polyadenylation signal in a first orientation;   A first promoter in the first orientation that drives expression of the Parvoviridae cap gene in the presence of adenoviral helper functions; and   A second promoter in the first orientation that drives expression of the Parvoviridae cap gene in the absence of adenoviral helper functions.   
     
     
         21 . The nucleic acid of  claim 20 , wherein the second promoter is a cell type-specific promoter. 
     
     
         22 . The nucleic acid of  claim 20 , wherein the second promoter is a ubiquitous promoter. 
     
     
         23 . The nucleic acid of  claim 20 , wherein the Parvoviridae cap gene is a wild-type AAV cap gene. 
     
     
         24 . The nucleic acid of  claim 23 , wherein the AAV cap gene sequences is the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or other natural AAV isolate cap gene sequence. 
     
     
         25 . The nucleic acid of  claim 20 , wherein the Parvoviridae cap gene is an engineered AAV cap gene. 
     
     
         26 . The nucleic acid of  claim 20 , wherein the Parvoviridae cap gene is one of a library of diverse AAV cap genes. 
     
     
         27 . A nucleic acid library comprising a plurality of nucleic acids of  claim 20 , wherein the nucleic acids comprise a plurality of unique Parvoviridae cap gene sequences. 
     
     
         28 . The nucleic acid library of  claim 27 , wherein the nucleic acid library comprises greater than about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7  or 10 8  unique AAV cap gene sequences. 
     
     
         29 . The nucleic acid of  claim 20 , further comprising a gene of interest. 
     
     
         30 . A method for identifying an AAV vector with a cap gene sequence that has increased ability to transduce cells from a tissue of interest when compared to at least one other AAV vector with a different cap gene sequence, the method comprising:
 Preparing a first-round AAV TRADE vector library by introducing the nucleic acid library of  claim 27  into an AAV packaging cell line and recovering the first round AAV TRADE vector library from the packaging cell line;   Injecting one or more animals with the first-round AAV TRADE vector library;   Recovering cap gene sequences of AAV vectors that are enriched in cells of the tissue of interest in the animals;   Preparing a second-round AAV TRADE nucleic acid library comprising recovered cap gene sequences of the enriched AAV vectors and introducing this library into an AAV packaging cell line and recovering the second round AAV TRADE vector library from the packaging cell line;   Performing a second round of enrichment by injecting one or more animals with the second-round AAV TRADE vector library and recovering cap gene sequence that are enriched in cells of the tissue of interest in the animals; and   Identifying enriched AAV cap gene sequences after the first-round enrichment, after the second-round enrichment, and after any subsequent rounds of enrichment.   
     
     
         31 - 38 . (canceled) 
     
     
         39 . An AAV cap ORF sequence comprising one or more following mutations in the exon-intron junctions at splicing donor sites: 
 AAV1 VP1 cap ORF 1009-CTTAC(junction)CAGCA-1018* (SEQ ID NO:199)   AAV3 VP1 cap ORF 1006-CTTAC(junction)CAGCA-1015* (SEQ ID NO:199)   AAV1 VP1 cap ORF 1228-TTTAC(junction)CTTCA-1237 (SEQ ID NO:200)   AAV3 VP1 cap ORF 1237-TATAC(junction)CTTCG-1246 (SEQ ID NO:201)   AAV1 VP1 cap ORF 1331-ATTAC(junction)CTGAA-1340 (SEQ ID NO:202)   AAV1 VP1 cap ORF 1434-GCTAC(junction)CTGGA-1443 (SEQ ID NO:203)   AAV1 VP1 cap ORF 1502-TTTAC(junction)CTGGA-1510 (SEQ ID NO:204)   AAV1 VP1 cap ORF 1803-ATTAC(junction)CTGGC-1812 (SEQ ID NO:205)   AAV3 VP1 cap ORF 1803-CTTAC(junction)CTGGC-1812 (SEQ ID NO:206)   AAV1 VP1 cap ORF 1835-TGTAC(junction)CTGCA-1844 (SEQ ID NO:207)   AAV1 VP1 cap ORF 2189-GTTAC(junction)CTTAC-2198 (SEQ ID NO:208)   AAV9 VP1 cap ORF 2189-GATAC(junction)CTGAC-2198 (SEQ ID NO:209)   AAV1 VP1 cap ORF 2194-CTTAC(junction)CCGTC-2203 (SEQ ID NO:210)   AAV3 VP1 cap ORF 2194-CTCAC(junction)ACGAA-2203 (SEQ ID NO:211).   
     
     
         40 . (canceled) 
     
     
         41 . (canceled)