Claims for Patent: 6,458,538

Summary for Patent: 6,458,538

Title:	Methods of assaying for compounds that inhibit premature translation termination and nonsense-mediated RNA decay
Abstract:	The present application provides methods of assaying for compounds that inhibit premature translation termination and nonsense mediated RNA decay in cells.
Inventor(s):	Beckmann; Holger (El Cerrito, CA), Learned; Marc (El Granada, CA), Peltz; Stuart (Piscataway, NJ), Czaplinski; Kevin (Summerset, NJ)
Assignee:	PTC Therapeutics, Inc. (Piscataway, NJ) Tularik Incorporated (South San Francisco, CA)
Application Number:	09/461,508
Patent Claims:	1. A method of screening for compounds that inhibit premature translation termination thereby increasing the amount of full-length polypeptide, the method comprising the steps of: (i) expressing in a culture cell a nucleic acid encoding a chimeric polypeptide, wherein the coding sequence for the polypeptide comprises a premature stop codon and the nucleic acid comprises a promoter operably linked to a heterologous nucleic acid encoding the chimeric polypeptide, which heterologous nucleic acid comprises an intron and at least two exons comprising coding sequence; (ii) contacting the cell with a candidate compound; and (iii) detecting an increase in the amount of the full-length polypeptide encoded by nucleic acid. 2. The method of claim 1, wherein the premature stop codon is located in a last exon. 3. The method of claim 1, wherein the heterologous nucleic acid encodes a chimeric polypeptide comprising luciferase-encoding and .beta.-globin-encoding nucleic acids. 4. The method of claim 1, wherein the heterologous nucleic acid encodes an enzyme. 5. The method of claim 1, wherein the heterologous nucleic acid encodes luciferase, green fluorescent protein, red fluorescent protein, phosphatase, peroxidase, kinase, chloramphenicol transferase, or .beta.-galactosidase. 6. The method of claim 1, wherein the heterologous nucleic acid encodes an immunoglobulin. 7. The method of claim 1, wherein the heterologous nucleic acid comprises a nucleic acid sequence encoding a .alpha.-globin, .beta.-globin, factor VIII, factor IX, vWF, p53, dystrophin, CFTR, Rb, MSH1, MSH2, APC, Wt1, hexosaminidase A, neurofibromin 1, or neurofibromin 2. 8. The method of claim 1, wherein the polypeptide is detected by ELISA, light emission, colorimetric measurements, enzymatic activity, drug resistance, FACS, or radioactivity. 9. The method of claim 1, wherein the assay is performed in a well of a microtiter dish. 10. The method of claim 1, wherein the microtiter dish has 96 or 384 wells. 11. The method of claim 9, further comprising repeating steps (i)-(iii) in parallel in a microtiter plate format, wherein between at least about 100 and at least about 6,000 different compounds are tested. 12. The method of claim 1, wherein the assay is performed in a high throughput integrated system comprising an automatic pipetting station, a robotic armature, and a robotic controller. 13. The method of claim 1, wherein the cell is adhered to a solid substrate. 14. The method of claim 13, wherein the solid support is selected from the group consisting of a bead, a membrane, and a microtiter plate. 15. The method of claim 11, wherein the cell is a human cell or a mouse cell. 16. The method of claim 1, wherein the cell is stably transfected with the nucleic acid. 17. A kit for screening for compounds that inhibit translation termination and nonsense-mediated mRNA decay, the kit comprising a nucleic acid encoding a chimeric polypeptide, wherein the polypeptide coding sequence comprises a premature stop codon, and the nucleic acid comprises a promoter operably linked to a heterologous nucleic acid encoding the chimeric polypeptide, which heterologous nucleic acid comprises an intron and at least two exons comprising coding sequence; instructions for practicing a method of screening for compounds that inhibit translation termination at premature stop codons and nonsense mediated RNA decay; and a control compound that inhibits translation termination and nonsense-mediated RNA decay. 18. The kit of claim 17, wherein the control compound is G418or gentamycin sulfate. 19. The kit of claim 17, wherein the premature stop codon is located in a last exon. 20. The kit of claim 17, wherein the nucleic acid encodes luciferase, green fluorescent protein, red fluorescent protein, phosphatase, peroxidase, kinase, chloramphenicol transferase, or .beta.-galactosidase. 21. The method of claim 1, wherein contacting the cell with the candidate compound increases the amount of RNA comprising the premature stop codon that is present in the cell.

Details for Patent 6,458,538

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		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26