Claims for Patent: 8,580,511

Summary for Patent: 8,580,511

Title:	Two-color fluorescent reporter for alternative pre-mRNA splicing
Abstract:	The present invention provides reporter constructs for in vivo or in vitro monitoring of alternative pre-mRNA splicing events. The reporter constructs described herein are also particularly useful for high-throughput screening of compounds that affect alternative pre-mRNA splicing. Kits comprising the reporter constructs of the present invention find utility in a wide range of applications including, for example, basic research, drug screening, and drug design.
Inventor(s):	Stoilov; Peter G. (Glendale, CA), Black; Douglas L. (Santa Monica, CA)
Assignee:	The Regents of the University of California (Oakland, CA)
Application Number:	12/305,764
Patent Claims:	1. An alternative pre-mRNA splicing reporter construct comprising a promoter operably linked to a nucleic acid sequence encoding first and second fluorescent proteins, wherein the nucleic acid sequence encoding the first fluorescent protein comprises a first start codon that resides in a Kozak consensus sequence and that is interrupted by a cassette comprising a nucleic acid with alternative splice sites, wherein the alternative splice sites produce at least two alternative splice products, wherein the nucleic acid sequence encoding the second fluorescent protein comprises a second start codon that resides in a Kozak consensus sequence, wherein the nucleic acid sequence encoding the second fluorescent protein is 3' to the nucleic acid sequence encoding the first fluorescent protein, wherein the first start codon is the only start codon in a Kozak consensus sequence that resides upstream of the second start codon. 2. The reporter construct of claim 1, wherein the nucleic acid with alternative splice sites is selected from the group consisting of an exon flanked by two introns, an intron with two 5' splice sites and one 3' splice site, and an intron with one 5' splice site and two 3' splice sites. 3. The reporter construct of claim 1, wherein the promoter comprises a human cytomegalovirus (CMV) promoter. 4. The reporter construct of claim 1, wherein the first and second fluorescent proteins are independently selected from the group consisting of a green fluorescent protein, red fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, and variants thereof. 5. The reporter construct of claim 4, wherein the green fluorescent protein comprises an enhanced green fluorescent protein (EGFP) or a variant thereof. 6. The reporter construct of claim 4, wherein the red fluorescent protein comprises a Discosoma striata red fluorescent protein (DsRed) or a variant thereof. 7. The reporter construct of claim 2, wherein the exon comprises exon 10 of the human tau gene. 8. A method for monitoring alternative pre-mRNA splicing in a test cell, the method comprising: (a) introducing the reporter construct of claim 1 into the test cell; (b) transcribing a pre-mRNA sequence from the reporter construct; and (c) detecting alternative splicing from the pre-mRNA sequence, wherein expression of the first fluorescent protein indicates that a first alternative splice product has been produced, and wherein expression of the second fluorescent protein indicates that a second alternative splice product has been produced. 9. The method of claim 8, further comprising introducing a control reporter construct into a control cell to detect the presence or absence of a difference in ribosome scanning processivity between the first and the second alternative splice products, wherein the control cell is a cell of the same or similar tissue type as the test cell; and wherein the control reporter constructs comprise a reporter construct without the alternative exon and one of the introns, or wherein the control reporter constructs comprise a reporter construct without any introns. 10. The method of claim 9, wherein two control reporter constructs are introduced into the control cell, a first control reporter detecting the presence or absence of a difference in ribosome scanning processivity in the first alternative splice product, and a second control reporter detecting the presence or absence of a difference in ribosome scanning processivity in the second alternative splice product. 11. The method of claim 8, further comprising calculating a ratio of the expression levels of the first and second fluorescent proteins in the test cell. 12. The method of claim 11, wherein the expression ratio in the test cell is compared to an expression ratio of the first and second fluorescent proteins in the control cell. 13. The method of claim 12, wherein a change in the expression ratio in the test cell relative to the control cell indicates a modulation in alternative pre-mRNA splicing. 14. The method of claim 13, wherein the modulation in alternative pre-mRNA splicing is associated with a disease characterized by alternative splicing. 15. The method of claim 14, wherein the disease is selected from the group consisting of tumorigenesis and cell transformation, neurodegenerative disorders, metabolic diseases and disorders, angiogenesis, muscular dystrophies, and inflammatory and autoimmune responses. 16. A method for identifying a compound that modulates alternative pre-mRNA splicing, the method comprising: (a) contacting a test cell expressing the reporter construct of claim 1 with a compound; and (b) determining the effect of the compound on the expression of the first and second fluorescent proteins, thereby identifying a compound that modulates alternative pre-mRNA splicing. 17. The method of claim 16, further comprising calculating a ratio of the expression levels of the first and second fluorescent proteins in the test cell. 18. A kit comprising the reporter construct of claim 1 and directions for introducing the reporter construct into a cell. 19. The kit of claim 18, further comprising directions for detecting the expression of the first and second fluorescent proteins.

Details for Patent 8,580,511

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2026-06-19
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2026-06-19
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2026-06-19