Methods and Compositions for Preparing Recombinant Adeno Associated Viruses and Uses Thereof
Disclosed herein are nucleic acid molecules and compositions of preparing recombinant adeno-associated viruses (rAAV) and uses thereof. Specifically, two-plasmid systems are provided for efficient rAAV production.
1 . An isolated nucleic acid molecule comprising
(a) a sequence encoding an AAV capsid, wherein isolated nucleic acid molecule does not comprise a sequence encoding AAV rep; and
(b) an exogenous gene flanked by inverted terminal repeat (ITR) sequences.
2 . The isolated nucleic acid molecule of claim 1 , wherein the AAV capsid comprises a capsid from AAV1, AAV2, AAV2 variant, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9 variant, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, AAV-DJ, LK03, NP59, or KP1.
3 . The isolated nucleic acid molecule of any one of claims 1-2 , wherein the exogeneous gene has a size from about 2 kilobases to about 7 kilobases.
4 . The isolated nucleic acid molecule of any one of claims 1-3 , further comprising a Ef1α promoter 5′ of the sequence coding the AAV capsid.
5 . The isolated nucleic acid molecule of any one of claims 1-4 , further comprising a p40 promoter 5′ of the sequence coding the AAV capsid.
6 . An isolated nucleic acid molecule comprising a sequence encoding an AAV rep protein, wherein the isolated nucleic acid molecule does not comprise a sequence encoding a capsid.
7 . The isolated nucleic acid molecule of claim 6 , wherein the AAV rep protein comprises REP2.
8 . The isolated nucleic acid molecule of any one of claim 6 or 7 , further comprising one or more helper elements.
9 . The isolated nucleic molecule of claim 8 , wherein the one or more helper elements comprises Ad5-VA, Ad5-E2A(DBP), or Ad5-E4.
10 . The isolated nucleic molecule of any one of claims 6-9 , further comprising a p5 promoter 5′ of the sequence encoding the AAV rep protein.
11 . The isolated nucleic molecule of any one of claims 6-9 , further comprising a sequence encoding a non-AUG translation start codon 5′ of the sequence encoding the AAV rep protein.
12 . The isolated nucleic molecule of claim 11 , wherein the non-AUG translation start codon is ACG.
13 . A composition comprising
(a) a first nucleic acid molecule comprising i) a sequence encoding an AAV rep protein or ii) a sequence comprising one or more helper elements; and
(b) a second nucleic acid molecule comprising i) sequence encoding an AAV capsid and ii) an exogenous gene flanked by inverted terminal repeat (ITR) sequences, wherein the first nucleic acid and the second nucleic acid are separate nucleic acid molecules.
14 . The composition of claim 13 , wherein the first nucleic acid molecule comprises the sequence encoding the AAV rep protein and the sequence comprising the one or more helper elements.
15 . The composition of claim 14 , wherein the one or more helper elements comprises Ad5-VA, Ad5-E2A(DBP), or Ad5-E4.
16 . The composition of any one of claims 13-15 , wherein the AAV rep protein comprises REP2.
17 . The composition of any one of claims 13-16 , wherein the AAV capsid comprises a capsid from AAV1, AAV2, AAV2 variant, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9 variant, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, AAV-DJ, LK03, NP59, or KP1.
18 . The composition of any one of claims 13-17 , wherein the exogeneous gene has a size from about 2 kilobases to about 7 kilobases.
19 . The composition of any one of claims 13-18 , wherein the first nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 2 or 3.
20 . The composition of any one of claims 13-19 , wherein the second nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 4, 5, 15, or 17.
21 . The composition of any one of claims 13-19 , wherein the second nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 14 or 18.
22 . The composition of any one of claims 13-19 , wherein the first nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 2, and wherein the second nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 15.
23 . The composition of any one of claims 13-22 , wherein the first nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 2, and wherein the second nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 17.
24 . A composition comprising
(a) a first isolated nucleic acid molecule comprising i) a sequence encoding an AAV rep protein or a sequence encoding an AAV capsid, and ii) an exogenous gene flanked by inverted terminal repeat (ITR) sequences; and
(b) a second isolated nucleic acid molecule comprising (A) one or more helper elements, and (B) a sequence encoding an AAV capsid complementing the AAV rep protein in (a) or a sequence encoding an AAV rep protein complementing the AAV capsid in (a);
wherein the first nucleic acid molecule and the second nucleic acid molecule are separate nucleic acid molecules.
25 . The composition of claim 24 , wherein the first nucleic acid molecule comprises the sequence encoding the AAV rep protein.
26 . The composition of claim 25 , wherein the AAV rep protein comprises REP2.
27 . The composition of claim 24 , wherein the first nucleic acid molecule comprises the sequence encoding the AAV capsid.
28 . The composition of claim 24 , wherein the first nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 14.
29 . The composition of claim 24 , wherein the first nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 18.
30 . The composition of claim 25 or 26 , wherein the second nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 2.
31 . The composition of claim 24 or 27 , wherein the AAV capsid comprises a capsid from AAV1, AAV2, AAV2 variant, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9 variant, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, AAV-DJ, LK03, NP59, or KP1.
32 . The composition of any one of claims 24-31 , wherein the exogeneous gene has a size from about 2 kilobases to about 7 kilobases.
33 . The composition of claim 24 , wherein the first nucleic acid molecule comprises the sequence encoding the AAV rep protein and the sequence encoding the AAV capsid.
34 . The composition of any one of claims 24-33 , wherein the one or more helper elements comprises Ad5-VA, Ad5-E2A(DBP), or Ad5-E4.
35 . The composition of claim 24 , wherein the first isolated nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 4.
36 . The composition of claim 24 , wherein the first isolated nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 4, 5, 15 or 17.
37 . The composition of claim 24 , wherein the second isolated nucleic acid molecule comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the sequence as set forth in SEQ ID NO: 7.
38 . A cell comprising the isolated nucleic acid molecule or the composition of any of the previous claims .
39 . The cell of claim 38 , wherein the cell comprises a HEK293 cell.
40 . The cell of claim 39 , wherein the HEK293 cell comprises a suspension HEK293 cell.
41 . A method comprising introducing the isolated nucleic acid molecule or the composition of any of the previous claims into a cell, thereby generating a cell comprising the isolated nucleic acid molecule or the composition.
42 . The method of claim 41 , wherein the introducing comprises transient transfection.
43 . The method of any one of claims 41-42 , wherein the cell comprises a HEK293 cell.
44 . The method of claim 43 , wherein the HEK293 cell comprises a suspension HEK293 cell.
45 . A method of producing AAVs, the method comprising using the cell of any one of claims 39-40 to generate the AAVs.
46 . The method of claim 45 , wherein the generated AAVs comprise a full/empty ratio of at least 5%, 10%, 20%, 30%, 40%, or 50% prior to any purification steps.
47 . The method of claim 45 or 46 , wherein a production yield of the generated AAVs is at least 1.1-fold, 1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, or 5.0-fold of a production yield of AAVs generated by a three-plasmid system.
48 . A composition of AAVs generated by the method of any one of claims 45-47 .
49 . A method of treating a condition in a subject in need thereof comprising administering the composition of AAVs of claim 48 to the subject, thereby treating the condition.
50 . Use of the composition of AAVs of claim 48 in manufacture of a medicament for treating a condition in a subject in need thereof.
51 . A kit comprising
the isolated nucleic acid molecule or the composition of any of the previous claims ; and instructions.