High activity regulatory elements
Provided herein are compositions and methods for driving high expression of a transgene. Compositions and methods for driving high expression of a transgene comprising one or more human-derived regulatory elements, which, when operably linked to a transgene, can result in high expression of the transgene in one or more cell types or tissues.
1. An expression cassette comprising a regulatory element operably linked to a therapeutic transgene, wherein the regulatory element comprises:
(i) a sequence selected from the group consisting of SEQ ID NOS: 13-17 and 22-41; or
(ii) a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and a combination of SEQ ID NOS: 1 and 2;
wherein the regulatory element of (ii) is operably linked to a promoter.
2. The expression cassette of claim 1 , wherein the regulatory element is non-naturally occurring.
3. The expression cassette of claim 1 , wherein the regulatory element comprises SEQ ID NO: 3 or SEQ ID NO: 4.
4. The expression cassette of claim 1 , wherein the regulatory element of (ii) is located between the promoter and the therapeutic transgene.
5. The expression cassette of claim 1 , wherein the regulatory element comprises a sequence selected from the group consisting of SEQ ID NOS: 13-17 and 22-41.
6. The expression cassette of claim 5 , wherein the regulatory element is the only promoter in the expression cassette.
7. The expression cassette of claim 1 , wherein the regulatory element is no more than 120 base pairs (bp).
8. The expression cassette of claim 1 , wherein the regulatory element is no more than 60 bp.
9. The expression cassette of claim 1 , wherein the regulatory element is no more than 100 bp.
10. The expression cassette of claim 1 , wherein the expression cassette is part of a recombinant adeno-associated virus (rAAV).
11. The expression cassette of claim 10 , wherein the rAAV is an rAAV1, rAAV2, rAAV3, rAAV3b, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rAAV-DJ, or rscAAV.
12. The expression cassette of claim 1 , wherein the therapeutic transgene is a DNA binding protein, a gene editing protein, a transcriptional activator, or a transcriptional repressor.
13. The expression cassette of claim 1 , wherein the therapeutic transgene is ATP7A, ATP7B, ATP8B1, ABCB4, ABCB11, CKDL5, CNTNAP2, ZEB2, Factor 1, Factor 2, Factor 3, Factor 4, Factor 5, Factor 6, Factor 7, Factor 8, Factor 9, Factor 10, Factor 11, or Factor 12.
14. The expression cassette of claim 1 , wherein the therapeutic transgene is ATP7B.
15. An expression vector comprising the expression cassette of claim 1 .
16. A viral particle comprising the expression cassette of claim 1 .
17. The viral particle of claim 16 , wherein the viral particle is an adeno-associated virus (AAV).
18. The viral particle of claim 17 , wherein the AAV is selected from the group consisting of: AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, and scAAV.
19. The expression cassette of claim 1 , wherein the regulatory element is the sequence of SEQ ID NO: 2 operably linked to the promoter.
20. The expression cassette of claim 1 , wherein the regulatory element is the sequence of SEQ ID NO: 14.
21. An adeno-associated virus (AAV) expression cassette comprising a human-derived regulatory element of no more than 120 bp operably linked to a transgene of at least 3 kb, wherein the regulatory element results in increased transgene expression by at least 2 fold as compared to expression of the transgene when operably linked to a CMV promoter, and wherein the human-derived regulatory element comprises:
(i) a sequence selected from the group consisting of SEQ ID NOS: 13-17 and 22-41; or
(ii) a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and a combination of SEQ ID NOS: 1 and 2;
wherein the human-derived regulatory element of (ii) is operably linked to a promoter.
22. The AAV expression cassette of claim 21 , wherein the increased transgene expression is at least 50 fold as compared to expression of the transgene when operably linked to a CMV promoter.
23. The AAV expression cassette of claim 21 , wherein the increased transgene expression is at least 100 fold as compared to expression of the transgene when operably linked to a CMV promoter.
24. The AAV expression cassette of claim 21 , wherein the human-derived regulatory element exhibits a size-normalized expression activity that is at least 1.5 fold as compared to size-normalized expression activity of a CMV promoter operably linked to the transgene.
25. The AAV expression cassette of claim 21 , wherein the increased transgene expression occurs in at least 2 different cell types.
26. The AAV expression cassette of claim 25 , wherein the at least 2 different cell types are selected from the group consisting of: kidney cells, neurons, and liver cells.
27. The AAV expression cassette of claim 21 , wherein the human-derived regulatory element comprises any one or more of: SEQ ID NOS: 13-17 and 22-41, and wherein no other promoter sequences are present in the expression cassette.
28. The AAV expression cassette of claim 21 , wherein the transgene is ATP7A, ATP7B, ATP8B1, ABCB4, ABCB11, CDKL5, CNTNAP2, ZEB2, Factor 1, Factor 2, Factor 3, Factor 4, Factor 5, Factor 6, Factor 7, Factor 8, Factor 9, Factor 10, Factor 11, or Factor 12.
29. The AAV expression cassette of claim 21 , wherein the transgene is ATP7B.
30. The AAV expression cassette of claim 21 , wherein the AAV is selected from the group consisting of: AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, and scAAV.
31. The AAV expression cassette of claim 30 , wherein the AAV is AAV8 or AAV5.
32. An adeno-associated virus (AAV) expression cassette comprising a human-derived regulatory element of no more than 120 bp operably linked to a transgene of at least 3 kb; wherein the regulatory element results in increased transgene expression by at least 2 fold as compared to expression of the transgene when operably linked to a CMV promoter, wherein the human-derived regulatory element comprises any one or more of SEQ ID NOS: 13-17 and 22-41, and wherein no other promoter sequences are present in the expression cassette.