Claims for Patent: 6,066,726

Summary for Patent: 6,066,726

Title:	Neuron-specific transcriptional promoter
Abstract:	The 5\'-flanking region and core regulatory domains that underlie neuronal specific expression of the human .gamma.-aminobutyric acid type A (GABA.sub.A) receptor .beta.1 subunit gene are identified herein. Sequence analysis, mapping of transcriptional initiation sites, and transfection of reporter gene constructs into primary cultures demonstrate that neuronal and region specific activity resides in a TATA-less minimal promoter of 186 bp, comprising an initiator, the major transcriptional start site, a presumptive TFIID binding site, and an enhancer. Enhancer sequence contained within a 26 bp region at the 5\'-end of the minimal promoter is essential for activity but not for tissue specificity. Moreover, .beta.1 promoter activity is subject to autologous inhibition, indicating that GABA-induced receptor mRNA downregulation results from an inhibition of gene transcription. Regulation of neurotransmitter receptor gene expression plays an important role in nervous system development and function, and impaired gene regulation may underlie the etiology of certain neurological diseases.
Inventor(s):	Farb; David H. (Cambridge, MA), Russek; Shelley J. (Cambridge, MA)
Assignee:	Trustees of Boston University (Boston, MA)
Application Number:	08/889,502
Patent Claims:	1. An isolated DNA molecule comprising: a) a neuron-specific transcriptional promoter, which is more transcriptionally active in hippocampal cells than in neocortical cells, the promoter comprising a transcriptional initiation sequence element 5'-GCGCAGGTCCATTCGGGAAT-3' SEQ ID NO: 18, and sequence elements 5'-ACTAGGAATATTGTTTG-3' SEQ ID NO: 17 and 5'-GGACATGGAGCAC-3' SEQ ID NO: 19 positioned at least 35 nucleotide base pairs upstream from the transcription initiation sequence element; and b) a collection of one or more cis-acting elements, wherein said elements are CAAT boxes that confer orientation specificity on the transcriptional activity of the promoter, and are functionally located upstream from the promoter. 2. The isolated DNA molecule of claim 1, further comprising at least 4 sequential nucleotides from nucleotides 130 to 309 in SEQ ID NO: 37. 3. The isolated DNA molecule of claim 1, wherein said collection of cis-acting elements comprises a first sequence element upstream from a second sequence element, and both said first and said second sequence elements are 5'-CAAT-3'. 4. The isolated DNA molecule of claim 1, further comprising nucleotides 263 to 287 listed in SEQ ID NO: 37. 5. An isolated DNA molecule consisting essentially of: a) a neuron-specific transcriptional promoter, which is more transcriptionally active in hippocampal cells than in neocortical cells, the promoter consisting essentially of a transcription initiation site sequence element 5'-GCGCAGGTCCATTCGGGAAT-3' SEQ ID NO: 18, and sequence elements 5'-ACTAGGAATATTGTTTG-3' SEQ ID NO: 17 and 5'-GGACATGGAGCAC-3' SEQ ID NO: 19 positioned at least 35 nucleotide base pairs upstream from the transcription initiation site; b) a cis-acting element that is a CAAT box and is an orientation specific transcriptional stimulators element for the promoter, the cis-acting element being functionally located upstream from the promoter and comprising at least 4 sequential nucleotides corresponding to 4 sequential nucleotides from nucleotides 130 to 309 in SEQ ID NO: 37; c) an open reading frame comprising at least one exon of a protein coding sequence which is transcribed under control of the promoter; and d) a negative regulatory sequence element downstream from the promoter, the negative regulatory sequence element comprising all or a portion of Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor. 6. The isolated DNA molecule of claim 5, wherein the negative regulatory sequence element is part of an intron downstream from at least one exon in the open reading frame. 7. The isolated DNA molecule of claim 5, wherein the negative regulatory sequence element has transcriptional promoter activity for transcription in opposite orientation from RNA transcribed under control of the promoter. 8. The isolated DNA molecule of claim 7, wherein the transcriptional promoter activity of the negative regulatory sequence element is greater in neocortical cells than in hippocampal cells. 9. An isolated DNA molecule comprising: a) the orientation dependent neuron-specific transcriptional promoter and enhancer sequence corresponding to nucleotide 1-541 of SEQ ID NO: 37; and b) an heterologous open reading frame functionally linked to the promoter. 10. An isolated DNA molecule comprising: a) the orientation dependent neuron-specific transcriptional promoter and enhancer sequence corresponding to nucleotide 1-541 of SEQ ID NO: 37; b) an heterologous open reading frame functionally linked to the promoter; and c) Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor functionally linked in reverse orientation downstream of at least one exon in the heterologous open reading frame so as to function as a negative regulatory sequence element of the heterologous open reading frame in non-hippocampal cells. 11. An isolated DNA molecule comprising: a) the orientation dependent neuron-specific transcriptional promoter and enhancer sequence corresponding to nucleotide 130-541 of SEQ ID NO: 37; and b) an heterologous open reading frame functionally linked to the promoter. 12. An isolated DNA molecule comprising: a) the orientation dependent neuron-specific transcriptional promoter and enhancer sequence corresponding to nucleotide 130-541 of SEQ ID NO: 37; b) an heterologous open reading frame functionally linked to the promoter; and c) Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor functionally linked in reverse orientation downstream of at least one exon in the heterologous open reading frame so as to function as a negative regulatory sequence element of the heterologous open reading frame in non-hippocampal cells. 13. The isolated DNA molecule of claim 12 wherein Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor is part of an intron downstream from at least one exon in the heterologous open reading frame. 14. An isolated DNA molecule comprising: a) the orientation dependent neuron-specific transcriptional promoter and enhancer sequence corresponding to nucleotide 145-541 of SEQ ID NO: 37; and b) an heterologous open reading frame functionally linked to the promoter. 15. An isolated DNA molecule comprising: a) the orientation dependent neuron-specific transcriptional promoter and enhancer sequence corresponding to nucleotide 145-541 of SEQ ID NO: 37; b) an heterologous open reading frame functionally linked to the promoter; and c) Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor functionally linked in reverse orientation downstream of at least one exon in the heterologous open reading frame so as to function as a negative regulatory sequence element of the heterologous open reading frame in non-hippocampal cells. 16. The isolated DNA molecule of claim 15 wherein Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor is part of an intron downstream from at least one exon in the heterologous open reading frame which it negatively regulates. 17. An isolated DNA molecule comprising: a) the orientation dependent neuron-specific transcriptional promoter and enhancer sequence corresponding to nucleotide 217-541 of SEQ ID NO: 37; and b) an heterologous open reading frame functionally linked to the promoter. 18. An isolated DNA molecule comprising: a) the orientation dependent neuron-specific transcriptional promoter and enhancer sequence corresponding to nucleotide 217-541 of SEQ ID NO: 37; b) an heterologous open reading frame functionally linked to the promoter; and c) Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor functionally linked in reverse orientation downstream of at least one exon in the heterologous open reading frame so as to function as a negative regulatory sequence element of the heterologous open reading frame in non-hippocampal cells. 19. The isolated DNA molecule of claim 18 wherein Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor is part of an intron downstream from at least one exon in the heterologous open reading frame which it negatively regulates. 20. An isolated DNA molecule comprising: a) the orientation dependent neuron-specific transcriptional promoter and enhancer sequence corresponding to nucleotide 241-541 of SEQ ID NO: 37; and b) an heterologous open reading frame functionally linked to the promoter. 21. An isolated DNA molecule comprising: a) the orientation dependent neuron-specific transcriptional promoter and enhancer sequence corresponding to nucleotide 241-541 of SEQ ID NO: 37; b) an heterologous open reading frame functionally linked to the promoter; and c) Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor functionally linked in reverse orientation downstream of at least one exon in the heterologous open reading frame so as to function as a negative regulatory sequence element of the heterologous open reading frame in non-hippocampal cells. 22. The isolated DNA molecule of claim 21 wherein Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor is part of an intron downstream from at least one exon in the heterologous open reading frame which it negatively regulates. 23. An isolated DNA molecule comprising: a) the orientation dependent neuron-specific transcriptional promoter and enhancer sequence corresponding to nucleotide 130-509 of SEQ ID NO: 37; and b) an heterologous open reading frame functionally linked to the promoter. 24. An isolated DNA molecular comprising: a) the orientation dependent neuron-specific transcriptional promoter and enhancer sequence corresponding to nucleotide 130-509 of SEQ ID NO: 37; b) an heterologous open reading frame functionally linked to the promoter; and c) Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor functionally linked in reverse orientation downstream of at least one exon in the heterologous open reading frame so as to function as a negative regulatory sequence element of the heterologous open reading frame in non-hippocampal cells. 25. The isolated DNA molecule of claim 24 wherein Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor is part of an intron downstream from at least one exon in the heterologous open reading frame which it negatively regulates. 26. An isolated DNA molecular comprising: a) The neuron-specific transcriptional promoter corresponding to nucleotide 152-541 of SEQ ID NO 37; and b) an heterologous open reading frame functionally linked to the promoter. 27. An isolated DNA molecular comprising: a) The neuron-specific transcriptional promoter corresponding to nucleotide 152-541 of SEQ ID NO 37; b) an heterologous open reading frame functionally linked to the promoter; and c) Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor functionally linked in reverse orientation downstream of at least one exon in the heterologous open reading frame so as to function as a negative regulatory sequence element of the heterologous open reading frame in non-hippocampal cells. 28. The isolated DNA molecule of claim 27 wherein Intron-1 of the .beta.-1 subunit gene of GABA.sub.A receptor is part of an intron downstream from at least one exon in the heterologous open reading frame which it negatively regulates.

Details for Patent 6,066,726

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		2015-11-09
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2015-11-09
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2015-11-09