RNAi expression constructs
The present invention provides compositions and methods suitable for expressing 1-x RNAi agents against a gene or genes in cells, tissues or organs of interest in vitro and in vivo so as to treat diseases or disorders.
1. A genetic construct capable of modifying expression of one or more genes, the genetic construct comprising a 1-x RNAi expression cassette encoding x stem-loop structures, wherein x is two or more, and the stem-loop structures are separated from one another by one or more spacer regions, wherein the stem-loop structures are RNAi agents and at least one stem-loop structure is encoded by SEQ ID NO: 39.
2. The genetic construct of claim 1 , wherein x is 3, 4 or 5 stem-loop structures.
3. The genetic construct of claim 1 wherein at least one stem-loop structure is encoded by SEQ ID NO: 6.
4. The genetic construct of claim 1 wherein the one or more spacer regions are about 6 nucleotides.
5. The genetic construct of claim 1 wherein the stem of the stem-loop structures comprise about 19 base pairs.
6. The genetic construct of claim 1 wherein the loop of the stem-loop structures comprise about 5 to 9 nucleotides.
7. The genetic construct of claim 1 wherein the stem-loop structures target multiple regions of a HCV genome.
8. The genetic construct of claim 2 wherein the third, fourth and fifth stem-loop structures are encoded by one or more sequences as set forth in any of SEQ ID NO: 1-56.
9. The genetic construct of claim 1 wherein the RNAi expression cassette comprises a promoter and a terminator and the promoter and the terminator are taken from the same gene.
10. A 1-x RNAi expression cassette encoding x stem-loop structures, wherein x is two or more, and the stem-loop structures are separated from one another by one or more spacer regions, wherein the stem-loop structures are RNAi agents and at least one stem-loop structure is encoded by SEQ ID NO: 39.
11. The cassette of claim 10 wherein x is 3, 4 or 5 stem-loop structures.
12. The cassette of claim 11 wherein the third, fourth and fifth stem-loop structures are encoded by one or more sequences as set forth in any of SEQ ID NO: 1-56.
13. A method of modifying the expression of one or more genes, comprising:
introducing to a cell, tissue, or organ of interest a genetic construct comprising an RNAi expression cassette encoding x stem-loop structures, wherein x is two or more, and the stem-loop structures are separated from one another by one or more spacer regions, wherein the stem-loop structures are RNAi agents and at least one stem-loop structure is encoded by SEQ ID NO: 39.
14. The method of claim 13 , wherein x is 3, 4, or 5 stem-loop structures.
15. The method of claim 13 , wherein the one or more spacer regions are about 6 nucleotides.
16. The method of claim 13 , wherein the stem-loop structures target regions of gene sequences that are conserved.
17. The method of claim 13 , wherein the stem-loop structures target multiple regions on a HCV genome.
18. The method of claim 14 , wherein the third, fourth and fifth stem-loop structures are encoded by one or more sequences as set forth in any of SEQ ID NO: 1-56.
19. A method of modifying the expression of one or more genes, comprising:
introducing to a cell, tissue or organ of interest an RNAi agent that is an in vitro transcription product of a genetic construct encoding x stem-loop structures, wherein x is two or more, and the stem-loop structures are separated from one another by one or more spacer regions, wherein the stem-loop structures are RNAi agents and at least one stem-loop structure is encoded by SEQ ID NO: 39.
20. The method of claim 19 , wherein when x is 3 or more stem-loop structures, the third, fourth and fifth stem-loop structures are encoded by one or more sequences as set forth in any of SEQ ID NO: 1-56.
21. The method of claim 19 , wherein the RNAi agent comprises three stem-loop structures and the promoter is a T7 promoter.
22. The 1-x RNAi expression cassette of claim 10 , wherein at least one of the stem-loop structures is encoded by SEQ ID NO: 6.
23. The 1-x RNAi expression cassette of claim 22 , wherein when x is 3, 4 or 5 stem-loop structures, the third, fourth and fifth stem-loop structures are encoded by one or more sequences as set forth in any of SEQ ID NO: 1-56.
24. The genetic construct of claim 3 , wherein when x is 3, 4 or 5 stem-loop structures, the third, fourth and fifth stem-loop structures are encoded by one or more sequences as set forth in any of SEQ ID NO: 1-56.