Optimized promoter sequences, intron-free expression constructs and methods of use
The disclosure includes CpG reduced regulatory elements, polynucleotides comprising such CpG reduced regulatory elements, expression cassettes comprising such CpG reduced regulatory elements, and recombinant AAV vectors comprising such CpG reduced regulatory elements. The regulatory elements can be, for example, operably coupled to a transgene.
1. A polynucleotide comprising a regulatory element nucleic acid sequence at least 98% identical to the sequence of SEQ ID NO: 58 with or without 5′ and/or 3′ flanking 5-7 nucleotides or comprising the sequence of SEQ ID NO: 24.
2. A polynucleotide comprising a regulatory element nucleic acid sequence at least 98% identical to the sequence of SEQ ID NO: 58 or comprising the sequence of SEQ ID NO: 24.
3. A polynucleotide comprising a regulatory element nucleic acid sequence at least 98% identical to the sequence of SEQ ID NO: 58 and having reduced CpG(s) at the same positions as set forth in SEQ ID NO: 58; or comprising the sequence of SEQ ID NO: 24.
4. An expression cassette comprising the polynucleotide of claim 3 , and a transgene, wherein said regulatory element nucleic acid sequence is operably linked to said transgene.
5. The expression cassette of claim 4 , wherein said regulatory element nucleic acid sequence is positioned 5′ of said transgene.
6. The expression cassette of claim 5 , wherein said transgene encodes a therapeutic protein that is expressed in liver cells and secreted into the systemic circulation.
7. The expression cassette of claim 6 , wherein said therapeutic protein is a blood coagulation or clotting factor protein.
8. The expression cassette of claim 7 , wherein said blood coagulation or clotting factor protein is Factor IX (FIX), Factor VIII (FVIII), Factor VII (FVII) or Protein C.
9. The expression cassette of claim 6 , wherein said therapeutic protein is a lysosomal storage enzyme.
10. The expression cassette of claim 9 , wherein said lysosomal storage enzyme is acid alpha-glucosidase (GAA) or alpha-galactosidase (GLA).
11. An adeno-associated virus (AAV) vector comprising the expression cassette of claim 4 .
12. The AAV vector of claim 11 , wherein said AAV vector comprises:
a) an AAV capsid; and
b) AAV inverted terminal repeats (ITRs), wherein said AAV ITR(s) flank the 5′ or 3′ terminus of said expression cassette.
13. The AAV vector of claim 12 , wherein said AAV capsid comprises an amino acid comprising the sequence of SEQ ID NO:91 or SEQ ID NO:92.
14. The AAV vector of claim 11 , wherein said ITRs comprise one or more ITRs of any of: AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, or Rh74 AAV serotypes, or a combination thereof.
15. A pharmaceutical composition comprising a plurality of AAV vectors of claim 11 in a biologically compatible carrier or excipient.
16. The pharmaceutical composition of claim 15 , further comprising empty AAV capsids.
17. The AAV vector of claim 12 , wherein there is no more than 107 nucleotides of untranslated nucleic acid sequence between said regulatory element nucleic acid sequence and the 5′ end of said transgene.
18. A polynucleotide comprising a regulatory element nucleic acid sequence comprising the sequence of SEQ ID NO: 58.
19. An expression cassette comprising the polynucleotide of claim 18 and a transgene, wherein said regulatory element nucleic acid sequence is operably linked to said transgene and is positioned 5′ of said transgene.
20. The expression cassette of claim 19 , wherein said transgene encodes a therapeutic protein.
21. The expression cassette of claim 20 , wherein a non-coding nucleic acid is positioned between said regulatory element nucleic acid sequence and said transgene, and said non-coding nucleic acid sequence is not an intron.
22. The expression cassette of claim 20 , wherein said therapeutic protein is either Factor IX (FIX), Factor VIII (FVIII), Factor VII (FVII) or Protein C.
23. An adeno-associated virus (AAV) vector comprising the expression cassette of claim 19 .
24. The AAV vector of claim 23 , wherein said AAV vector comprises:
a) an AAV capsid; and
b) a 5′ AAV inverted terminal repeats (ITR) flanking the 5′ terminus of the expression cassette and a 3′ ITR flanking the 3′ terminus of the expression cassette.
25. The AAV vector of claim 24 , wherein said capsid comprises the amino acid sequence of SEQ ID NOs: 91 or 92.
26. The AAV vector of claim 24 , wherein there is no more than 107 nucleotides of untranslated nucleic acid sequence between said regulatory element nucleic acid sequence and the 5′ end of said transgene.
27. A polynucleotide comprising a regulatory element nucleic acid sequence comprising the sequence of SEQ ID NO: 24.
28. An expression cassette comprising the polynucleotide of claim 27 and a transgene, wherein said regulatory element nucleic acid sequence is operably linked to said transgene and is positioned 5′ of said transgene.
29. The expression cassette of claim 28 , wherein said transgene encodes a therapeutic protein.
30. The expression cassette of claim 29 , wherein a non-coding nucleic acid is positioned between said regulatory element nucleic acid sequence and said transgene, and said non-coding nucleic acid sequence is not an intron.
31. The expression cassette of claim 29 , wherein said therapeutic protein is either Factor IX (FIX), Factor VIII (FVIII), Factor VII (FVII) or Protein C.
32. An adeno-associated virus (AAV) vector comprising the expression cassette of claim 28 .
33. The AAV vector of claim 32 , wherein said AAV vector comprises:
a) an AAV capsid; and
b) a 5′ AAV inverted terminal repeats (ITR) flanking the 5′ terminus of the expression cassette and a 3′ ITR flanking the 3′ terminus of the expression cassette.
34. The AAV vector of claim 33 , wherein said capsid comprises the amino acid sequence of SEQ ID NOs: 91 or 92.
35. The AAV vector of claim 33 , wherein there is no more than 107 nucleotides of untranslated nucleic acid sequence between said regulatory element nucleic acid sequence and the 5′ end of said transgene.