Claims for Patent: 8,795,962

Summary for Patent: 8,795,962

Title:	Expression vectors based on modified ribosomal protein promoters and uses thereof in post-transcriptional assessment
Abstract:	The present invention relates to expression vector comprising (a) a promoter region comprising a non-inducible constitutively active ribosomal protein gene promoter, (b) an operably linked reporter or gene sequence, and (c) a 3\' untranslated region (3\' UTR), which are suitable means for an selective assessment of post-transcriptional regulation, post-transcriptional control elements and factors as well as for identifying compounds that effect post-transcription. The present invention furthermore relates to arrays, expression vector libraries and cell lines containing the expression vector(s). The present invention furthermore relates to a method and kit for identifying compounds that affect post-transcriptional regulation of reporter(s) or gene(s), that utilize the expression vector(s).
Inventor(s):	Abu Khabar; Khalid S. (Riyadh, SA)
Assignee:	King Faisal Specialist Hospital and Research Center (Riyadh, SA)
Application Number:	13/000,556
Patent Claims:	1. An in vitro method for assessing a post-transcriptional effect in a cell wherein said method comprises: (i) providing an expression vector comprising: (a) a promoter region comprising a non-inducible constitutively active ribosomal protein gene promoter and at least one sp1 site-containing sequence, wherein the ribosomal protein gene promoter comprises the promoter of ribosomal protein S23 (RPS23) gene or the promoter of ribosomal protein S30 (RPS30) gene, or a fragment of said RPS23 promoter or said RPS30 promoter wherein said fragment has at least 50 nucleotides; (b) a reporter gene or heterologous gene; and (c) a 3' untranslated region (3' UTR), wherein said reporter gene or heterologous gene is operably linked to said promoter region and said 3' UTR; (ii) introducing the expression vector into a cell; and (iii) assaying reporter gene or heterologous gene expression in the transfected cell of step (ii) wherein expression of the reporter gene or heterologous gene is responsive to a post-transcriptional effect, and wherein the expression of said reporter gene or heterologous gene is independent of transcriptional induction by TNF-.alpha. or okadaic acid. 2. The method according to claim 1, wherein the post-transcriptional effect is post-transcriptional regulation of genes. 3. An in vitro method for identifying compounds that affect post-transcriptional regulation of reporter(s) or gene(s), comprising the following steps: 1) transfecting a cell with at least one expression vector or linear expression cassette comprising: (a) a promoter region comprising a non-inducible constitutively active ribosomal protein gene promoter and at least one sp 1 site-containing sequence, wherein the ribosomal protein gene promoter comprises the promoter of ribosomal protein S23 (RPS23) gene or the promoter of ribosomal protein S30 (RPS30) gene, or a fragment of said RPS23 promoter or said RPS30 promoter wherein said fragment has at least 50 nucleotides; (b) a reporter gene or heterologous gene; and (c) a 3' untranslated region (3' UTR), wherein said reporter gene or heterologous gene is operably linked to said promoter region and said 3' UTR; in order to create a stable cell line harbouring said expression vector(s); 2) providing at least one compound to be tested; 3) incubating the cells created in step 1) with one or more compounds to be tested; and 4) determining the effect of the compound(s) on the post-transcriptional regulation by determining the mRNA level and/or the expression level of the reporter gene or heterologous gene, wherein the expression of said reporter gene or heterologous gene is independent of transcriptional induction by TNF-.alpha. or okadaic acid. 4. The method according to claim 1, wherein the RPS23 promoter, the RPS30 promoter, or said fragment of the RPS23 promoter or the RPS30 promoter is modified for high expression by mutating a TATA like sequence. 5. The method according to claim 1, wherein the RPS23 promoter or the RPS30 promoter has been truncated. 6. The method according to claim 1, wherein the promoter region further comprises intron sequence(s) of genes encoding ribosomal proteins, exon sequence(s) of genes encoding ribosomal proteins, tetracycline operator (tetO) sequences, and/or modified sequences wherein the modification eliminates a restriction site. 7. The method according to claim 1, wherein the expression vector comprises the nucleic acid sequence of any of SEQ ID NOs. 3 to 8 or a sequence complementary thereto. 8. The method according to claim 1, wherein the 3' UTR comprises an mRNA destabilization or stabilization element from a 3' UTR of a cellular mRNA, wherein the mRNA destabilization or stabilization element is selected from AU-rich elements, GU-rich elements, and U-rich sequences. 9. The method according to claim 3, wherein the RPS23 promoter, the RPS30 promoter, or said fragment of the RPS23 promoter or the RPS30 promoter is modified for high expression by mutating a TATA like sequence. 10. The method according to claim 3, wherein the RPS23 promoter or the RPS30 promoter has been truncated. 11. The method according to claim 3, wherein the promoter region further comprises intron sequence(s) of genes encoding ribosomal proteins, exon sequence(s) of genes encoding ribosomal proteins, tetracycline operator (tetO) sequences, and/or modified sequences wherein the modification eliminates a restriction site. 12. The method according to claim 3, wherein the expression vector comprises the nucleic acid sequence of any of SEQ ID NOs. 3 to 8 or a sequence complementary thereto. 13. The method according to claim 3, wherein the 3' UTR comprises an mRNA destabilization or stabilization element from a 3' UTR of a cellular mRNA, wherein the mRNA destabilization or stabilization element is selected from AU-rich elements, GU-rich elements, and U-rich sequences. 14. An in vitro method for assessing a post-transcriptional effect in a cell wherein said method comprises: (i) providing an expression vector comprising: (a) a promoter region, wherein the promoter region consists of a non-inducible constitutively active ribosomal protein gene promoter that is from the promoter of ribosomal protein S23 (RPS23) gene or the promoter of ribosomal protein S30 (RPS30) gene, and at least one sequence selected from sp1 site-containing sequences, intron sequences of genes encoding ribosomal proteins, exon sequences of genes encoding ribosomal proteins, and tetracycline operator (tetO) sequences; (b) a reporter gene or heterologous gene; and (c) a 3' untranslated region (3' UTR), wherein said reporter gene or heterologous gene is operably linked to said promoter region and said 3' UTR; (ii) introducing the expression vector into a cell; and (iii) assaying reporter gene or heterologous gene expression in the transfected cell of step (ii) wherein expression of the reporter gene or heterologous gene is responsive to a post-transcriptional effect, and wherein the expression of said reporter gene or heterologous gene is independent of transcriptional induction by TNF-.alpha. or okadaic acid. 15. An in vitro method for identifying compounds that affect post-transcriptional regulation of reporter(s) or gene(s), comprising the following steps: 1) transfecting a cell with at least one expression vector or linear expression cassette comprising: (a) a promoter region, wherein the promoter region consists of a non-inducible constitutively active ribosomal protein gene promoter that is from the promoter of ribosomal protein S23 (RPS23) gene or the promoter of ribosomal protein S30 (RPS30) gene, and, optionally, at least one sequence selected from sp1 site-containing sequences, intron sequences of genes encoding ribosomal proteins, exon sequences of genes encoding ribosomal proteins, and tetracycline operator (tetO) sequences; (b) a reporter gene or heterologous gene; and (c) a 3' untranslated region (3' UTR), wherein said reporter gene or heterologous gene is operably linked to said promoter region and said 3' UTR; in order to create a stable cell line harbouring said expression vector(s); 2) providing at least one compound to be tested; 3) incubating the cells created in step 1) with one or more compounds to be tested; and 4) determining the effect of the compound on the post-transcriptional regulation by determining the mRNA level and/or the expression level of the reporter gene or heterologous gene, wherein the expression of said reporter gene or heterologous gene is independent of transcriptional induction by TNF-.alpha. or okadaic acid. 16. An in vitro method for assessing a post-transcriptional effect in a cell wherein said method comprises: (i) providing an expression vector comprising: (a) a promoter region comprising a non-inducible constitutively active ribosomal protein gene promoter that comprises the promoter of the human RPS23 gene that has the nucleic acid sequence of SEQ ID NO:2 or the promoter of the human RPS30 gene that has the nucleic acid sequence of SEQ ID NO:1, or a fragment of said human RPS23 promoter or said human RPS30 promoter wherein said fragment has at least 50 nucleotides; (b) a reporter gene or heterologous gene; and (c) a 3' untranslated region (3' UTR), wherein said reporter gene or heterologous gene is operably linked to said promoter region and said 3' UTR; (ii) introducing the expression vector into a cell; and (iii) assaying reporter gene or heterologous gene expression in the transfected cell of step (ii) wherein expression of the reporter gene or heterologous gene is responsive to a post-transcriptional effect, and wherein the expression of said reporter gene or heterologous gene is independent of transcriptional induction by TNF-.alpha. or okadaic acid. 17. An in vitro method for identifying compounds that affect post-transcriptional regulation of reporter(s) or gene(s), comprising the following steps: 1) transfecting a cell with at least one expression vector comprising: (a) a promoter region comprising a non-inducible constitutively active ribosomal protein gene promoter that comprises the promoter of the human RPS23 gene that has the nucleic acid sequence of SEQ ID NO:2 or the promoter of the human RPS30 gene that has the nucleic acid sequence of SEQ ID NO:1, or a fragment of said human RPS23 promoter or said human RPS30 promoter wherein said fragment has at least 50 nucleotides; (b) a reporter gene or heterologous gene; and (c) a 3' untranslated region (3' UTR), wherein said reporter gene or heterologous gene is operably linked to said promoter region and said 3' UTR; in order to create a stable cell line harbouring said expression vector(s); 2) providing at least one compound to be tested; 3) incubating the cells created in step 1) with one or more compounds to be tested; and 4) determining the effect of the compound(s) on the post-transcriptional regulation by determining the mRNA level and/or the expression level of the reporter gene or heterologous gene, wherein the expression of said reporter gene or heterologous gene is independent of transcriptional induction by TNF-.alpha. or okadaic acid. 18. The method according to claim 1, wherein the non-inducible constitutively active ribosomal protein gene promoter comprises the promoter of the human RPS23 gene that has the nucleic acid sequence of SEQ ID NO:2 or the promoter of the human RPS30 gene that has the nucleic acid sequence of SEQ ID NO:1, or a fragment of said human RPS23 promoter or said human RPS30 promoter wherein said fragment has at least 50 nucleotides. 19. The method according to claim 3, wherein the non-inducible constitutively active ribosomal protein gene promoter comprises the promoter of the human RPS23 gene that has the nucleic acid sequence of SEQ ID NO:2 or the promoter of the human RPS30 gene that has the nucleic acid sequence of SEQ ID NO:1, or a fragment of said human RPS23 promoter or said human RPS30 promoter wherein said fragment has at least 50 nucleotides.

Details for Patent 8,795,962

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		2028-06-27
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2028-06-27
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2028-06-27