Promoters for expression of heterologous genes
The present disclosure relates generally to promoters derived from the AAV anti-sense strand, their use in the expression of one or more heterologous coding sequences, and isolated polynucleotides, vectors and recombinant viruses comprising the promoters. The present disclosure also relates to enhancers derived from the AAV anti-sense strand, their use in increasing the expression of one or more heterologous coding sequences, and isolated polynucleotides, vectors and recombinant viruses comprising the enhancers.
1. A vector, comprising, from 5′ to 3′, a 5′ ITR, a promoter, and a 3′ ITR, wherein:
the promoter is the only promoter between the 5′ ITR and the 3′ ITR; and
the promoter comprises a sequence of nucleotides that is the reverse, complement sequence of nucleotides selected from the group consisting of nucleotides 4398-4530 of the AAV2 genome of SEQ ID NO:1, nucleotides 4426-4580 of the AAV1 genome comprising the nucleotide sequence of SEQ ID NO:2, nucleotides 4412-4580 of the AAV3 genome comprising the nucleotide sequence of SEQ ID NO:3, nucleotides 4457-4623 of the AAV4 genome comprising the nucleotide sequence of SEQ ID NO:4, nucleotides 4411-4538 of the AAV6 genome comprising the nucleotide sequence of SEQ ID NO:6, nucleotides 4428-4576 of the AAV7 genome comprising the nucleotide sequence of SEQ ID NO:7, and nucleotides 4330-4393 of the AAV8 genome comprising the nucleotide sequence of SEQ ID NO:8.
2. The vector of claim 1 , wherein the 5′ ITR and the 3′ ITR are from AAV viruses of the same or different serotypes.
3. The vector of claim 1 , wherein the 5′ ITR and the 3′ ITR are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8 serotypes.
4. The vector of claim 1 , wherein the 5′ ITR or the 3′ ITR comprises a sequence having at least 90% sequence identity to the AAV2 ITR comprising the nucleotide sequence of SEQ ID NO:24, 25 or 32.
5. The vector of claim 1 , further comprising a spacer sequence immediately downstream of the promoter, wherein the total length of the promoter and the spacer is at least 60, 70, 80, 90, 100, 110, 120, or 130 nucleotides.
6. The vector of claim 1 , wherein the vector comprises one or more restriction enzyme sites positioned downstream of the promoter to facilitate insertion of a heterologous coding sequence that is operably linked to the promoter.
7. The vector of claim 1 , wherein the vector comprises a heterologous coding sequence operably linked to the promoter.
8. The vector of claim 7 , wherein the heterologous coding sequence encodes a peptide, a polypeptide, or a polynucleotide.
9. The vector of claim 8 , wherein the polynucleotide is an antisense oligonucleotide.
10. A method for expressing the heterologous coding sequence of claim 7 , comprising introducing directly into a host cell the vector of claim 7 , wherein the host cell mediates the expression of the heterologous coding sequence of claim 7 .
11. A vector, comprising, from 5′ to 3′, a 5′ ITR, a promoter, and a 3′ ITR, wherein:
the promoter is the only promoter between the 5′ ITR and the 3′ ITR, and
the promoter comprises the nucleotide sequence of any one of SEQ ID NOs:9-12, or a sequence having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs:9-12.
12. The vector of claim 11 , wherein the 5′ ITR and the 3′ ITR are from AAV viruses of the same or different serotypes.
13. The vector of claim 11 , wherein the 5′ ITR and the 3′ ITR are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8 serotypes.
14. The vector of claim 11 , wherein the 5′ ITR or the 3′ ITR comprises a sequence having at least 90% sequence identity to the AAV2 ITR comprising the nucleotide sequence of SEQ ID NO:24, 25 or 32.
15. The vector of claim 11 , further comprising a spacer sequence immediately downstream of the promoter, wherein the total length of the promoter and the spacer is at least 60, 70, 80, 90, 100, 110, 120, or 130 nucleotides.
16. The vector of claim 11 , wherein the vector comprises one or more restriction enzyme sites positioned downstream of the promoter to facilitate insertion of a heterologous coding sequence that is operably linked to the promoter.
17. The vector of claim 11 , wherein the vector comprises a heterologous coding sequence operably linked to the promoter.
18. The vector of claim 17 , wherein the heterologous coding sequence encodes a peptide, a polypeptide, or a polynucleotide.
19. The vector of claim 18 , wherein the polynucleotide is an antisense oligonucleotide.
20. A recombinant AAV having a genome that comprises, from 5′ to 3′, a 5′ ITR, a promoter, and a 3′ ITR, wherein:
the promoter is the only promoter between the 5′ ITR and the 3′ ITR; and
the promoter comprises:
(a) a sequence of nucleotides that is the reverse, complement sequence of nucleotides 4398-4530 of the AAV2 genome of SEQ ID NO:1, nucleotides 4426-4580 of the AAV1 genome comprising the nucleotide sequence of SEQ ID NO:2, nucleotides 4412-4580 of the AAV3 genome comprising the nucleotide sequence of SEQ ID NO:3, nucleotides 4457-4623 of the AAV4 genome comprising the nucleotide sequence of SEQ ID NO:4, nucleotides 4411-4538 of the AAV6 genome comprising the nucleotide sequence of SEQ ID NO:6, nucleotides 4428-4576 of the AAV7 genome comprising the nucleotide sequence of SEQ ID NO:7, and nucleotides 4330-4393 of the AAV8 genome comprising the nucleotide sequence of SEQ ID NO:8; or
(b) a sequence of any one of SEQ ID NOs:9-12, or a sequence having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs:9-12.
21. The recombinant AAV of claim 20 , wherein the promoter is operably linked to a heterologous coding sequence.
22. A method for expressing the heterologous coding sequence of claim 21 , comprising introducing directly into a host cell the recombinant AAV of claim 21 , wherein the host cell mediates the expression of the heterologous coding sequence of claim 21 .