METHODS FOR USING TRANSCRIPTION-DEPENDENT DIRECTED EVOLUTION OF AAV CAPSIDS
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)