Claims for Patent: 5,604,114

Summary for Patent: 5,604,114

Title:	Cis-acting repression sequences, cis-acting antirepression sequences, vectors, methods of preparation and use
Abstract:	Cis-acting repression sequences which are able to provide a cis-acting inhibitory effect on the expression of a gene when placed downstream of the gene in its untranslated message are dislcosed. Cis-acting anti-repression sequences which can relieve the cis-acting repression in the presence of the art gene product are also disclosed. These sequences correspond to a sufficient number of nucleotides from the HIV-I, HIV-2, STLV-3 or HTLV-IV genomes to provide the repression or anti-repression effects. The use of the sequences in vectors and systems to control the expression of a desired gene product is also described.
Inventor(s):	Haseltine; William A. (Cambridge, MA), Rosen; Craig A. (Glen Ridge, NJ), Sodroski; Joseph G. (Cambridge, MA), Terwilliger; Ernest (Boston, MA), Goh; Wei C. (Stanford, CA)
Assignee:	Dana-Farber Cancer Institute (Boston, MA)
Application Number:	08/041,887
Patent Claims:	1. A nucleotide region which contains both cis-acting repression sequences (CRS) and a cis-acting anti-repression sequence (RRE), said region consisting of the intron located between a major splice donor site in the env gene and a major splice acceptor site in the env gene, said env gene selected from the group consisting of HIV-I, HIV-2, HTLV-IV, STLV-3, and a sufficient fragment of said nucleotide region to suppress expression of a heterologous gene when said region is inserted downstream of said heterologous gene in said gene's untranslated message. 2. The nucleotide region of claim 1, wherein the cis-acting anti-repression sequence (RRE) present is sufficient to counteract the cis-acting repression sequences (CRS), and wherein said counteraction occurs in the presence of an effective amount of the rev (art) gene product. 3. A cis-acting repression sequence (CRS) selected from the group consisting of HIV-I nucleotides 6376-6725, 7283-7325, 5893-6538, 8204-8597, and a sufficient fragment thereof to suppress the expression of a heterologous gene, when said CRS is inserted downstream of said heterologous gene in said gene's untranslated message. 4. The cis-acting repression sequence (CRS) of claim 3, which consists of nucleotides 6376-6725 of HIV-I. 5. The cis-acting repression sequence (CRS) of claim 3, which consists of nucleotides 7283-7325 of HIV-I. 6. The cis-acting repression sequence (CRS) of claim 3, which consists of nucleotides 5893-6538 of HIV-I. 7. The cis-acting repression sequence (CRS) of claim 3, which consists of nucleotides 8204-8597 of HIV-I. 8. A cis-acting anti-repression sequence (RRE) which consists of HIV-I nucleotides 7283-7760. 9. The cis-acting anti-repression sequence (RRE) of claim 8, or a sufficient fragment thereof which counteracts the cis-acting repression sequence (CRS) inserted downstream of said heterologous gene in said gene's untranslated message, wherein said CRS is selected from the group consisting of HIV-I nucleotides 6376-6725, 7283-7325, 5893-6538, 8204-8597, and a sufficient fragment thereof to suppress the expression of said heterologous gene, and wherein said counteraction occurs in the presence of an effective amount of the rev (art) gene product. 10. A cis-acting anti-repression sequence (RRE) which comprises a sufficient fragment of a region consisting of the intron located between a major splice donor site and a major splice acceptor site in the env gene, said env gene selected from the group consisting of HIV-I, HIV-2, HTLV-IV and STLV-3, to counteract the cis-acting repression sequence (CRS) inserted downstream of a heterologous gene in said gene's untranslated message, wherein said CRS is selected from the group consisting of HIV-I nucleotides 6376-6725, 7283-7325, 5893-6538, 8204-8597, and a sufficient fragment thereof to suppress the expression of said heterologous gene, and wherein said counteraction occurs in the presence of an effective amount of rev (art) gene product. 11. A vector containing: (a) a gene which is heterologous to an HIV-I, HIV-2, HTLV-IV or STLV-3 genome, wherein downstream, in said heterologous gene's untranslated message, is inserted (b) a nucleotide region consisting of cis-acting repression sequence (CRS) and a cis-acting anti-repression sequence (RRE), said nucleotide region found within the intron located between a major splice donor site in the env gene and a major splice acceptor site in the env gene, said env gene selected from the group consisting of HIV-I, HIV-2, HTLV-IV, STLV-3, and a sufficient fragment thereof to suppress expression of said heterologous gene, and wherein the RRE present in the nucleotide region is sufficient to counteract this cis-acting repression effect on repression of the heterologous gene caused by the CRS, said counteraction occurring in the presence of an effective amount of the rev (art) gene product, and (c) a promotor operatively-lined 5' to the heterologous gene. 12. The vector of claim 11, wherein the CRS and the RRE are from HIV-I. 13. The vector of claim 11, wherein the CRS and RRE are from HIV-2. 14. The vector of claim 11, wherein the CRS and the RRE are from HTLV-IV. 15. The vector of claim 11, wherein the CRS and the RRE are from STLV-3. 16. The vector of claim 12 in which the promoter is an HIV-I LTR, and the vector further contains: (d) an HIV-I LTR 3' to the CRS and RRE. 17. The vector of claim 13 in which the promoter is an HIV-2 LTR, and the vector further contains: (d) an HIV-2 LTR 3' to the CRS and RRE. 18. The vector of claim 14 in which the promoter is an HTLV-IV LTR, and the vector further contains: (d) an HTLV-IV LTR 3' to the CRS and RRE. 19. The vector of claim 15 in which the promoter is an STLV-3 LTR, and the vector further contains: (d) an STLV-3 LTR 3' to the CRS and RRE. 20. A vector containing: (a) a gene which is heterologous to an HIV-I genome, wherein downstream, in said heterologous gene's untranslated message, is inserted (b) a nucleotide region consisting of nucleotides 6376-7760 of the HIV-I genome, said region containing both cis-acting repression sequences (CRS) and a cis-acting anti-repression sequence (RRE), and (c) a promoter operatively-linked 5' to the heterologous gene. 21. A method for expressing a heterologous gene product which comprises: (a) transfecting a pre-selected cell line with the vector of claim 11, and (b) contacting the RRE with an effective amount of the rev (art) gene product to activate the RRE and counteract the cis-acting repression effect on expression of the heterologous gene caused by the CRS, thereby expressing the heterologous gene product. 22. The method of claim 21, wherein the pre-selected cell line is a tat cell line containing a tat gene obtained from a viral species selected from the group consisting of HIV-I, HIV-2, HTLV-IV and STLV-3, and wherein the promoter operatively-linked 5' to the heterologous gene is an LTR from the same viral species as that from which the tat gene was obtained. 23. The method of claim 22, wherein the RRE is contacted with the rev (art) gene product in said cell line. 24. The method of claim 23, wherein said second vector expresses the rev (art) gene product under the control of a regulatable promoter which can be activated to express an effective amount of the rev (art) gene product to activate the RRE. 25. A method for expressing a heterologous gene product which comprises: (a) transfecting a pre-selected cell line with the vector of claim 20, and (b) contacting the RRE with an effective amount of the rev (art) gene product to activate the RRE and counteract the cis-acting repression effect on expression of the heterologous gene caused by the CRS thereby expressing the heterologous gene product. 26. The method of claim 25, wherein the promoter is the HIV-I LTR. 27. The method of claim 26, wherein the pre-selected cell line is a tat.sub.III cell line.

Details for Patent 5,604,114

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		2014-02-18
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2014-02-18
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2014-02-18