Claims
- 1. A method of upregulating expression of a protein of interest in a cell, said cell containing a DNA encoding said protein, which DNA contains a mutation that causes downregulation of said protein by aberrant splicing in a pre-mRNA,
wherein said DNA encodes said pre-mRNA; wherein said pre-mRNA contains a native intron having a first set of splice elements, which native intron is removed by splicing when said mutation is absent to produce a first mRNA encoding said protein; and wherein said pre-mRNA further contains an aberrant intron different from said native intron having a second set of splice elements, which aberrant intron is removed by splicing when said mutation is present to produce an aberrant second mRNA different from said first mRNA; said method comprising:
administering to said cell a heterologous oligonucleotide, said heterologous oligonucleotide comprising a nuclear localization element joined to an antisense oligonucleotide, which antisense oligonucleotide hybridizes to said pre-mRNA in the nucleus of said cell to create a duplex thereof under conditions which permit splicing, and wherein said antisense oligonucleotide blocks a member of said aberrant second set of splice elements so that said native intron is removed by splicing and said protein of interest is produced.
- 2. A method according to claim 1, wherein said administering step is carried out in vivo.
- 3. A method according to claim 1, wherein said administering step is carried out in vitro.
- 4. A method according to claim 1, wherein said administering step is carried out by administering a vector that expresses said heterologous oligonucleotide in said cell.
- 5. A method according to claim 4, wherein said vector is a viral vector.
- 6. A method according to claim 5, wherein said heterologous oligonucleotide comprises RNA.
- 7. A method according to claim 1, wherein said administering step is carried out by administering an exogeneous oligonucleotide to said cell.
- 8. A method according to claim 1, wherein said nuclear localization element forms an snRNP complex in said cell.
- 9. A method according to claim 1, wherein said nuclear localization element comprises small nuclear RNA.
- 10. A method according to claim 9, wherein said nuclear localization element comprises U1 or U6 RNA.
- 11. A vector useful for upregulating expression of a protein of interest in a cell, said cell containing a DNA encoding said protein, which DNA contains a mutation that causes downregulation of said protein by aberrant splicing in a pre-mRNA,
wherein said DNA encodes said pre-mRNA; wherein said pre-mRNA contains a native intron having a first set of splice elements, which native intron is removed by splicing when said mutation is absent to produce a first mRNA encoding said protein; and wherein said pre-mRNA further contains an aberrant intron different from said native intron having a second set of splice elements, which aberrant intron is removed by splicing when said mutation is present to produce an aberrant second mRNA different from said first mRNA; said vector comprising:
a promoter operably associated with a nucleic acid sequence encoding a heterologous RNA, said heterologous RNA comprising a nuclear localization element joined to an antisense oligonucleotide, which antisense oligonucleotide hybridizes to said pre-mRNA in the nucleus of said cell to create a duplex thereof under conditions which permit splicing, and wherein said antisense oligonucleotide blocks a member of said aberrant second set of splice elements so that said native intron is removed by splicing and said protein of interest is produced.
- 12. A vector according to claim 11, wherein said vector is a viral vector.
- 13. A vector according to claim 11, wherein said nuclear localization element forms an snRNP complex in said cell.
- 14. A vector according to claim 11, wherein said nuclear localization element comprises small nuclear RNA.
- 15. A vector according to claim 11, wherein said nuclear localization element comprises U1 or U6 RNA.
- 16. An oligonucleotide useful for upregulating expression of a protein of interest in a cell, said cell containing a DNA encoding said protein, which DNA contains a mutation that causes downregulation of said protein by aberrant splicing in a pre-mRNA,
wherein said DNA encodes said pre-mRNA; wherein said pre-mRNA contains a native intron having a first set of splice elements, which native intron is removed by splicing when said mutation is absent to produce a first mRNA encoding said protein; and wherein said pre-mRNA further contains an aberrant intron different from said native intron having a second set of splice elements, which aberrant intron is removed by splicing when said mutation is present to produce an aberrant second mRNA different from said first mRNA; said oligonucleotide comprising a nuclear localization element joined to an antisense oligonucleotide, which antisense oligonucleotide hybridizes to said pre-mRNA in the nucleus of said cell to create a duplex thereof under conditions which permit splicing, and wherein said antisense oligonucleotide blocks a member of said aberrant second set of splice elements so that said native intron is removed by splicing and said protein of interest is produced.
- 17. An oligonucleotide according to claim 16, wherein said nuclear localization element forms an snRNP complex in said cell.
- 18. An oligonucleotide vector according to claim 16, wherein said nuclear localization element comprises small nuclear RNA.
- 19. An oligonucleotide according to claim 16, wherein said nuclear localization element comprises U1 or U6 RNA.
- 20. An oligonucleotide according to claim 16, wherein said oligonucleotide is about 50 to 500 nucleotides in length.
RELATED APPLICATIONS
[0001] This application claims priority from R. Kole et al., United States Provisional Application No. 60/082,510, filed Apr. 21, 1998, the disclosure of which is incorporated by reference herein in its entirety.
Government Interests
[0002] This invention was made with government support under grant number IR-1HL51940-1 from the National Institutes of Health. The Government has certain rights to this invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60082510 |
Apr 1998 |
US |