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 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 of the RNAi agents is encoded by SEQ ID NO:42.
2. The 1-x RNAi expression cassette of claim 1 , wherein at least one of the RNAi agents is encoded by SEQ ID NO:6 or SEQ ID NO:39.
3. The 1-x RNAi expression cassette of claim 1 , wherein x is 3, 4 or 5 stem loop structures.
4. The 1-x RNAi expression cassette of claim 3 , wherein at least one of the RNAi agents is encoded by SEQ ID NO:6, and at least one of the RNAi agents is encoded by SEQ ID NO:39.
5. The 1-x RNAi expression cassette of claim 1 , wherein the spacer regions are about 6 nucleotides.
6. The 1-x RNAi expression cassette of claim 1 , wherein the stem of the stem-loop structures comprises about 19 base pairs.
7. The 1-x RNAi expression cassette of claim 1 , wherein the loop of the stem-loop structures comprises about 5 to 9 nucleotides.
8. The 1-x RNAi expression cassette of claim 1 , wherein the expression cassette further comprises a terminator sequence.
9. The 1-x RNAi expression cassette of claim 8 , wherein the promoter and the terminator are from the same gene.
10. A method of modifying the expression of one or more genes, comprising:
introducing to a cell, tissue, or organ of interest 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 of the RNAi agents is encoded by SEQ ID NO:42.
11. The method of claim 10 , wherein at least one of the RNAi agents is encoded by SEQ ID NO:6 or SEQ ID NO:39.
12. The method of claim 10 , wherein introducing the 1-x RNAi expression cassette to a cell, tissue, or organ of interest comprises:
ligating the 1-x RNAi expression cassette into a viral delivery vector to form a viral RNAi expression construct;
packaging the viral RNAi expression construct into viral particles; and
delivering the viral particles to the cell, tissue, or organ of interest.
13. The method of claim 10 , wherein at least one of the RNAi agents is encoded by SEQ ID NO:6, and at least one of the RNAi agents is encoded by SEQ ID NO: 39.
14. The method of claim 10 , wherein the stem of the stem-loop structures comprises about 19 base pairs.
15. The method of claim 10 , wherein the loop of the stem-loop structures comprises about 5 to 9 nucleotides.
16. The method of claim 10 , wherein the expression cassette further comprises a terminator sequence.
17. A method of modifying the expression of one or more genes, comprising:
introducing to a cell, tissue, or organ of interest an RNAi sequence that is an in vitro transcription product of a 1-x RNAi expression cassette 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 of the RNAi agents is encoded by SEQ ID NO: 42.
18. The method of claim 17 , wherein the 1-x RNAi expression cassette further comprises a terminator sequence.
19. The method of claim 17 , wherein the RNAi sequence comprises three stem-loop structures and the promoter is a T7 promoter.