Claims
- 1. A method of producing a chimeric RNA molecule in a cell comprising: contacting a cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises:
a) one or more target binding domains that target binding of the nucleic acid molecule to a pre-mRNA expressed within the cell; b) a 3′ splice region comprising a branch point, a pyrimidine tract and a 3′ splice acceptor site; and c) a nucleotide sequence to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.
- 2. A method of producing a chimeric RNA molecule in a cell comprising: contacting a cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises:
a) one or more target binding domains that target binding of the nucleic acid molecule to a pre-mRNA expressed within the cell; b) a 3′ splice acceptor site; and c) a nucleotide sequence to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.
- 3. A method of producing a chimeric RNA molecule in a cell comprising contacting a cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises:
a) one or more target binding domains that target binding of the nucleic acid molecule to a pre-mRNA expressed within the cell; b) a 5′ splice site; and c) a nucleotide sequence to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.
- 4. The method of claim 1 wherein the nucleic acid molecule further comprises a 5′ donor site.
- 5. The method of claim 1, 2, 3 or 4 wherein said nucleic acid molecule further comprising a spacer region that separates the 3′ splice region from the target binding domain.
- 6. The method of claim 1, 2, 3, or 4 wherein said nucleic acid molecule further comprising a safety sequence comprising one or more complementary sequences that bind to one or both sides of the 3′ splice site.
- 7. The method of claim 1, 2, 3, or 4 wherein binding of said nucleic acid molecule binds to the target pre-mRNA is mediated by complementary, triple helix formation, or protein-nucleic acid interaction.
- 8. The method of claim 5 wherein binding of the nucleic acid molecule to the target pre-mRNA is mediated by complementary, triple helix formation, or protein-nucleic acid interaction.
- 9. The method of claim 6 wherein the binding of the nucleic acid molecule to the target pre-mRNA is mediated by complementary, triple helix formation, or protein-nucleic acid interaction.
- 10. The method of claim 1, 2, 3 or 4 wherein the nucleotide to be trans-spliced to the target pre-mRNA encodes a translatable polypeptide.
- 11. The method of claim 5 wherein the nucleotide to be trans-spliced to the target pre-mRNA encodes a translatable polypeptide.
- 12. The method of claim 6 wherein the nucleotide to be trans-spliced to the target pre-mRNA encodes a translatable polypeptide.
- 13. The method of claim 1, 2, 3 or 4 wherein the nucleotide sequence to be trans-spliced to the target pre-mRNA contains a nonsense mutation.
- 14. The method of claim 5 wherein the nucleotide sequence to be trans-spliced to the target pre-mRNA contains a nonsense mutation.
- 15. The method of claim 6 wherein the nucleotide sequence to be trans-spliced to the target pre-mRNA contains a nonsense mutation.
- 16. A method of producing a chimeric RNA molecule in a cell comprising: contacting a cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises:
a) one or more target binding domains that target binding of the nucleic acid molecule to a pre-mRNA expressed within the cell; b) a 5′ donor site; c) 3′ splice acceptor site; and c) a nucleotide sequence to be trans-spliced to the target pre-mRNA; wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.
- 17. The method of claim 16 wherein the nucleic acid molecule further comprises a spacer region that separates the 3′ splice region from the target binding domain.
- 18. The method of claim 16 further comprising a safety sequence comprising one or more complementary sequences that bind one of both sides of the 3′ splice site.
Government Interests
[0001] The present invention was made with government support under Grant Nos. SBIR R43DK56526-01 and SBIR5R44DK56526-03. The government has certain rights in the invention.