Glaucoma therapeutics and diagnostics based on a novel human...

Details Glaucoma therapeutics and diagnostics based on a novel human... Glaucoma therapeutics and diagnostics based on a novel human...

1998-05-22

2001-03-27

Schwartzman, Robert A. (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S320100, C536S023500

Reexamination Certificate

active

06207450

ABSTRACT:

1. BACKGROUND OF THE INVENTION
Glaucoma is the second leading cause of vision loss worldwide (Quigley, H. A.
Br. J Ophthalmol.
80: 389-393 (1996)). By the year 2000, an estimated 66.8 million people will have primary glaucoma and 6.7 million will be bilaterally blind due to this disorder (Quigley, H. A.
Br. J. Ophthalmol.
80: 389-393 (1996)). In the United States, glaucoma is the second leading cause of permanent blindness and the leading cause among African Americans(Leske, M. C.
Am. J. Epidemiol
118:166-191(1983)). Glaucoma is an optic neuropathy characterized by optic nerve head excavation (cupping) which can lead to loss of peripheral vision and sometimes loss of central vision. While glaucoma is treatable and vision loss can be prevented, once vision loss occurs it is irreversible.
Elevated intraocular pressure (IOP) is a major risk factor for the development of glaucoma, but it is not found in all patients with the disease (Sommer, A. et al.,
Arch. Ophthalmol.
109: 1090-1095 (1991) and is not included in the definition. The glaucoma associated with elevated IOP is divided into three major categories: open angle, closed angle and developmental. Each of these categories is further divided into primary and secondary forms, and by age of onset.
Recently, genes have been identified which cause juvenile-onset primary open angle glaucoma (MYOC and the GLCIA locus on chromosome 1) (Stone, E. M. et al.,
Science
275: 668-670 (1997)), PCG (CYPIBI at the GLC3A locus on chromosome 2) (Stoilov, I. et al.,
Hum. Mol. Genet.
6: 641-647 (1997)), Rieger Syndrome (PITX2 at the RIEG1 locus on chromosome 4) (Semina, E. V. et al.,
Nat. Genet.
14: 392-399 (1996) and IH (PITX2 at the IRID2 locus on chromosome 4) (Alward, W. L. M. et al.,
Am. J. Ophthalmol.
125: 98-100 (1998)). MYOC also appears to be involved in approximately 3% of adult-onset open angle glaucoma (Stone, E. M. et al.,
Science
275: 668-670 (1997)).
Several loci involved in glaucoma or associated phenotypes have been genetically mapped including that of a second PCG locus (GLC3B) to 1p36 (Akarsu, A. N. et al.
Hum. Mol Genet.
5: 1199-1203 (1996)), a second Rieger syndrome locus (RIEG2) to 13q14 (Phillips, J. C. et. al.,
Am. J. Hum. Genet.
59: 613-619 (1996)) and two loci for adult-onset open angle glaucoma (GLCIB and GLCIC) to chromosomes 2 (Stoilova, D et al.,
Genomics,
36: 142-150 (1996) and 3 (Wirtz, M. K.
Am. J. Hum. Genet.
60:296-304 (1997)). In addition, a group of dominant disorders involving changes in the anterior segment of the eye have been mapped to 6p25 (Mears, A. J. et al.,
Am. J Hum. Genet.
59:1321-1327 (1996); Gould, D. B. et al.,
Am. J. Hum. Genet.
61:765-768 (1997); Jordan, T. et al.,
Am. J. Hum. Genet.
61:882-888 (1997); and Graff C. et al.,
Hum. Genet.
101:130-134 (1997)). These disorders all have glaucoma as part of their phenotype and have beer postulated to be allelic (Jordan, T. et al.,
Am. J. Hum. Genet.
61:882-888 (1997)).
2. SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of a novel human gene, which encodes a novel human protein. The newly identified protein and nucleic acid described herein are referred to as “FKHL7”. FKHL7 is a monomeric DNA binding protein that shares a core binding site (RTAAYA; SEQ ID NO:22) with four other FKHL7-like proteins. The forkhead domain shows strong homology to the human gene, FKHL 14 and the mouse genes, Fkh1 and Fkh14, by BLASTN analysis.
A 9.8 kb subclone of BAC471g19 was partially sequenced and determined to contain the entire coding region of FKHL7 as well as 5′ and 3′ untranslated sequences (SEQ ID NO. 1). The human FKHL7 coding sequence is 1.7 kb in size and contains no introns. The 1659 bp open reading frame (SEQ ID NO. 3) encodes a 553 amino acid polypeptide (SEQ ID NO. 2). The COOH-terminal domain contains several stretches of homopolymeric runs of alanine and glycine. The FKHL7 coding region contains 5 recognition sites for the restriction enzyme NotI. A BLASTN screen of the public dbEST database with the FKHL7 genomic sequence yields only partial human and mouse cDNA coverage of this gene. Based on the analysis of cDNA clones identified in the public databases, there is evidence for the utilization of at least two different polyadenylation signals within the 3′ untranslated region.
Human FKHL7 is most abundantly expressed during embryogenesis and of the adult tissue tested, the most abundant expression occurred in the eye. Significant expression was also observed in adult heart, kidney and lung, while relatively little to no expression was observed in adult skeletal muscle, spleen or liver.
In one aspect, the invention features isolated FKHL7 nucleic acid molecules;. In one embodiment, the FKHL7 nucleic acid is from a vertebrate. In a preferred embodiment, the FKHL7 nucleic acid is from a mammal, e.g. a human. In an even more preferred embodiment, the nucleic acid has the nucleic acid sequence set forth in SEQ ID NO. 1 or 3 or a portion thereof. The disclosed molecules can be non-coding, (e.g. a probe, antisense, or ribozyme molecule) or can encode a functional FKHL7 polypeptide (e.g. a polypeptide which functions as either an agonist or antagonist of at least one bioactivity of the human FKHL7 polypeptide). In one embodiment, the nucleic acid of the presents invention can hybridize to a vertebrate FKHL7 gene or to the complement of a vertebrate FKHL7 gene. In a further embodiment, the claimed nucleic acid can hybridize with a nucleic: acid sequence shown in
FIG. 1
(SEQ ID NOS. 1 and 3) or a complement thereof. In a preferred embodiment, the hybridization is conducted under mildly stringent or stringent conditions.
In further embodiments, the nucleic acid molecule is an FKHL7 nucleic acid that is at least about 70%, preferably about 80%, more preferably about 85%, and even more preferably at least about 90% or 95% homologous to the nucleic acid shown as SEQ ID NOS: 1 or 3 or to the complement of the nucleic acid shown as SEQ ID NOS: 1 or 3.
The invention also provides probes and primers comprising substantially purified oligonucleotides, which correspond to a region of nucleotide sequence which hybridizes to at least about 6, at least about 10, at least about 15, at least about 20, or preferably at least about 25 consecutive nucleotides of the sequence set forth as SEQ ID NO. 1 or SEQ ID NO. 3 or complements of the sequence set forth as SEQ ID NOS. 1 or 3 or naturally occurring mutants or allelic variants thereof In preferred embodiments, the probe/primer further includes a label group attached thereto, which is capable of being detected.
For expression, the subject nucleic acids can be operably linked to a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter (e.g., for constitutive expression or inducible expression) or transcriptional enhancer sequence. Such regulatory sequences in conjunction with an FKHL7 nucleic acid molecule can provide a useful vector for gene expression. This invention also describes host cells transfected with said expression vector whether prokaryotic or eukaryotic and in vitro (e.g. cell culture) and in vivo (e.g. transgenic) methods for producing FKHL7 proteins by employing said expression vectors.
In another aspect, the invention features isolated FKHL7 polypeptides, preferably substantially pure preparations, e.g. of plasma purified or recombinantly produced polypeptides. The FKHL7 polypeptide can comprise a full length protein or can comprises smaller fragments corresponding to one or more particular motifs/domains, or fragments comprising at least about 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 510, 520, 530 or 540 amino acids in length. In particularly preferred embodiments, the subject polypeptide is capable of binding to an upstream region of a gene and/or otherwise regulating expression of a gene.
In a preferred embodiment, the polypeptide is encoded by a nucleic acid:, which hybridizes with the nucleic acid sequence represented in

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