Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-08-13
2002-05-28
Nguyen, Dave T. (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S325000, C536S023500
Reexamination Certificate
active
06395548
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to vascular therapy.
Angiogenesis results from endothelial cell proliferation induced by angiogenic factors. Angiogenic factors bind to receptors on endothelial cells which line blood vessels. This event triggers signals which cause the cells to proliferate; the proliferating endothelial cells secrete proteases which digest the basement membrane surrounding a vessel. The junctions between the endothelial cells are altered, allowing projections from the cells to pass through the space created. These outgrowths then become new blood vessels, e.g., capillaries.
Vascular endothelial cell growth factor (VEGF) and VEGF receptors (VEGF-Rs) play a role in vasculogenesis and angiogenesis. Although VEGF is secreted by a variety of cell types, including vascular smooth muscle cells, osteoblasts, fibroblasts, and macrophages, its proliferative and chemotactic activities are restricted to endothelial cells. VEGF signaling is mediated by two VEGF-Rs, the endothelial cell-specific tyrosine kinase receptors, flt-1 and KDR/flk-1. Despite its importance in VEGF signaling, the molecular mechanisms of VEGF and VEGF-R expression have not been elucidated.
SUMMARY OF THE INVENTION
The invention is based on the discovery that endothelial PAS domain protein-1 (EPAS1) binds to cis-acting regulatory sequences associated with genes encoding such angiogenic factors as VEGF and VEGF-Rs such as KDR/flk-1 and flt-1, thereby transactivating the promoters of such genes. Accordingly, the invention features a method of increasing the level of EPAS1 in a cell, e.g., an endothelial cell. An increase in the level of EPAS1 leads to increased promoter transactivation and increased transcription of genes encoding angiogenic factors which participate in the blood vessel formation.
The invention also includes a novel basic helix-loop-helix/Per-AhR-Arnt-Sim (bHLH/PAS) protein which binds to EPAS1 and forms a heterodimer which transactivates transcription of genes encoding angiogenic factors. Increasing the level of ARNT4 in a cell, e.g., an endothelial cell also leads to increased promoter transactivation and increased expression of angiogenic factors which participate in the blood vessel formation.
Angiogenic factors are proteins or polypeptides and ligands thereof that participate in the process of new blood vessel formation. For example, angiogenic factors include VEGF, VEGF-Rs, and other signalling proteins such as intracellular tyrosine kinases which participate in the angiogenic process. Preferably, the angiogenic factors are expressed in endothelial cells, e.g., VEGF, VEGF-Rs such as KDR/flk-1 or flt-1, and tyrosine kinases such as Tie2.
A method of inhibiting angiogenesis in a mammal is carried out by administering to the mammal a compound which inhibits binding of EPAS1 to cis-acting transcription regulatory DNA associated with a gene encoding an angiogenic factor. Angiogenesis is also inhibited by administering a compound which inhibits binding of EPAS1 to ARNT4, i.e., a compound which inhibits the formation of a functional heterodimer that can transactivate a promoter of gene encoding an angiogenic factor. The angiogenic factor is preferably VEGF, a VEGF-R such as KDR/flk-1 or flt-1. For example, the compound inhibits transcription of the angiogenic factor by binding to a cis-acting regulatory sequence such as the sequence 5′ GCCCTACGTGCTGTCTCA 3′ (SEQ ID NO:1) in VEGF promoter DNA. For example, the compound is an EPAS1 polypeptide that binds to a cis-acting regulatory sequence of a gene but fails to transactivate the promoter of the gene, e.g, a polypeptide lacking a transactivation domain (amino acids 486-690 of EPAS1).
Table 1: Transactivation Domain of Human EPAS1 EDYYTSLDNDLKIEVIEKLFAMDTEAKDQCSTQTDFNELDLETLAPYIPMDGEDFQLSPI CPEERLLAENPQSTPQHCFSAMTNIFQPLAPVAPHSPFLLDKFQQQLESKKTEPEHRPMS SIFFDAGSKASLPPCCGQASTPLSSMGGRSNTQWPPDPPLHFGPTKWAVGDQRTEFLGAA PLGPPVSPPHVSTFKTRSAKGFGAR (SEQ ID NO:2)
When such an EPAS1 mutant is bound to a cis-acting regulatory DNA, it prevents wild type EPAS1 binding and thereby inhibits transcription of a gene encoding an angiogenic factor (and, in turn, angiogenesis). For example, the EPAS1 polypeptide contains the N-terminal binding domain (amino acids 14-67 of EPAS1; RRKEKSRDAARCRRSKETEVFYELAHELPLPHSVSSHLDKASIMRLEISFLRTH; SEQ ID NO:3) more preferably the EPAS polypeptide contains amino acids 1-485 of human EPAS1. The amino acid sequence of such an EPAS1 dominant negative mutant polypeptide and DNA encoding such a mutant polypeptide is provided below.
TABLE 2
EPAS1 dominant negative mutant
1
MTADKEKKRS SSERRKEKSR DAARCRRSKE TEVFYELAHE LPLPHSVSSH
(SEQ ID NO:4)
51
LDKASIMRLE ISFLRTHKLL SSVCSENESE AEADQQMDNL YLKALEGFIA
101
VVTQDGDMIF LSENISKFMG LTQVELTGHS IFDFTHPCDH EEIRENLSLK
151
NGSGFGKKSK DMSTERDFFM RMKCTVTNRG RTVNLKSATW KVLHCTGQVK
201
VYNNCPPHNS LCGYKEPLLS CLIIMCEPIQ HPSHMDIPLD SKTFLSRHSM
251
DMKFTYCDDR ITELIGYHPE ELLGRSAYEF YHALDSENMT KSHQNLCTKG
301
QVVSGQYRML AKHGGYVWLE TQGTVIYNPR NLQPQCIMCV NYVLSEIEKN
351
DVVFSMDQTE SLFKPHLMAM NSIFDSSGKG AVSEKSNFLF TKLKEEPEEL
401
AQLAPTPGDA IISLDFGNQN FEESSAYGKA ILPPSQPWAT ELRSHSTQSE
451
AGSLPAFTVP QAAAPGSTTP SATSSSSSCS TPNSP
TABLE 3
DNA encoding EPAS1 Dominant Negative Mutant
cctgactgcgcggggcgctcgggacctgcgcgcacctcggaccttcaccacccgcccggg
(SEQ ID NO:5)
ccgcggggagcggacgagggccacagccccccacccgccagggagcccaggtgctcggcg
tctgaacgtctcaaagggccacagcgacaatgacagctgacaaggagaagaaaaggagta
gctcggagaggaggaaggagaagtcccgggatgctgcgcggtgccggcggagcaaggaga
cggaggtgttctatgagctggcccatgagctgcctctgccccacagtgtgagctcccatc
tggacaaggcctccatcatgcgactggaaatcagcttcctgcgaacacacaagctcctct
cctcagtttgctctgaaaacgagtccgaagccgaagctgaccagcagatggacaacttgt
acctgaaagccttggagggtttcattgccgtggtgacccaagatggcgacatgatctttc
tgtcagaaaacatcagcaagttcatgggacttacacaggtggagctaacaggacatagta
tctttgacttcactcatccctgcgaccatgaggagattcgtgagaacctgagtctcaaaa
atggctctggttttgggaaaaaaagcaaagacatgtccacagagcgggacttcttcatga
ggatgaagtgcacggtcaccaacagaggccgtactgtcaacctcaagtcagccacctgga
aggtcttgcactgcacgggccaggtgaaagtctacaacaactgccctcctcacaatagtc
tgtgtggctacaaggagcccctgctgtcctgcctcatcatcatgtgtgaaccaatccagc
acccatcccacatggacatccccctggatagcaagaccttcctgagccgccacagcatgg
acatgaagttcacctactgtgatgacagaatcacagaactgattggttaccaccctgagg
agctgcttggccgctcagcctatgaattctaccatgcgctagactccgagaacatgacca
agagtcaccagaacttgtgcaccaagggtcaggtagtaagtggccagtaccggatgctcg
caaagcatgggggctacgtgtggctggagacccaggggacggtcatctacaaccctcgca
acctgcagccccagtgcatcatgtgtgtcaactacgtcctgagtgagattgagaagaatg
acgtggtgttctccatggaccagactgaatccctgttcaagccccacctgatggccatga
acagcatctttgatagcagtggcaagggggctgtgtctgagaagagtaacttcctattca
ccaagctaaaggaggagcccgaggagctggcccagctggctcccaccccaggagacgcca
tcatctctctggatttcgggaatcagaacttcgaggagtcctcagcctatggcaaggcca
tcctgcccccgagccagccatgggccacggagttgaggagccacagcacccagagcgagg
ctgggagcctgcctgccttcaccgtgccccaggcagctgccccgggcagcaccaccccca
gtgccaccagcagcagcagcagctgctccacgcccaatagcccttga
Rather than administering EPAS1 polypeptides or ARNT4 polypeptides, the method may be carried out by administering DNA encoding such polypeptides. For example, the compound is a nucleic acid encoding an EPAS1 polypeptide lacking amino acids 486-690 of EPAS1. For example, the nucleic acid encodes a dominant negative mutant of EPAS1 which contains amino acids 1-485 of wild type EPAS1, i.e., SEQ ID NO:5.
For antisense therapy, the compound is a antisense nucleic acid molecule containing at least 10 nucleotides the sequence of which is complementary to an mRNA encoding all or part of a wild type EPAS1 polypeptide. Preferably, the compound, e.g., an antisense oligonucleotide or antisense RNA produced from an antisense template, inhibits EPAS1 expression. For example, the compound may inhibit EPAS1 expression by inhibiting translation of EPAS1 mRNA. For example, antisense therapy is carried out by administering a single stranded nucleic acid complementary at least a portion of EPAS1 MRNA to interfere with the translation of MRNA into protein, thus reducing the amount of functional EPAS1 produced in the cell. A reduction in the amount o
Hsieh Chung-Ming
Lee Mu-En
Maemura Koji
Beattie Ingrid A.
Levin Mintz
Nguyen Dave T.
President and Fellows of Harvard College
Shukla Ram R.
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