Glaucoma compositions

Details Glaucoma compositions Glaucoma compositions

1997-03-21

2001-08-07

Martin, Jill D. (Department: 1632)

Chemistry: molecular biology and microbiology

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

C435S320100, C435S455000, C435S456000, C435S069100, C536S023100, C536S023500, C536S024310

Reexamination Certificate

active

06271026

ABSTRACT:

2. BACKGROUND OF THE INVENTION
In the United States, glaucoma is the second leading cause of legal blindness overall and the leading cause of blindness in African-American individuals (Hiller, R and H. A. Kahn, (1975)
Am. J. Ophthalmol
80: 62 and Kahn, H. A. and H. B. Moorhead (1973)
US Public Health Service Publication
NIH73-427, 120). Primary open glaucoma (POAG) is the most common form of glaucoma affecting 1-2% of the population over age forty (J. M. Tielsch et al., (1990)
Arch Ophthalmol.
108: 286). Nearly 12,000 people in the United States are blinded annually by this disorder (H. B. Moorhead (1973)
US Public Health Service Publication
NIH73-427, 1202-4; J. M. Tielsch et al., (1990)
Arch Ophthalmol.
108: 286 and J. M. Tielsch, in
Transactions of the New Orleans Academy of Ophthalmology,
Ball, S. F. Franklin R. M., Eds (Kugler, Amsterdam, 1993), pp61-68).
One method of identifying genes involved in multifactorial disorders is to study Mendelian diseases with a similar phenotype. Juvenile open angle glaucoma (JOAG) is a term used to refer to a subset of POAG which has an earlier age of onset and a highly penetrant autosomal dominant mode of inheritance (A. T. Johnson et al., (1993)
Ophthalmology
100:524). On clinical examination, patients with juvenile onset open angle glaucoma are identical to patients with later onset disease in that both exhibit elevated intraocular pressure and optic nerve cupping in the presence of a biomicroscopically normal trabecular meshwork. Isolation of genes involved in JOAG are needed to develop therapeutics and diagnostics for glaucoma.
2. SUMMARY OF THE INVENTION
In one aspect, the invention features isolated GLC1A nucleic acid molecules. The disclosed molecules can be non-coding, (e.g. probe, antisense or ribozyme molecules) or can encode a functional polypeptide (e.g. a polypeptide which specifically modulates, e.g., by acting as either an agonist or antagonist, at least one bioactivity of the human GLC1A polypeptide).
In further embodiments, the nucleic acid molecule is a GLC1A nucleic acid that is at least 70%, preferably 80%, more preferably 85%, and even more preferably at least 95% homologous in sequence to the nucleic acids shown as SEQ ID Nos: 1 or 2 or to the complement of the nucleic acids shown as SEQ ID Nos: 1 or 2. In another embodiment, the nucleic acid molecule encodes a polypeptide that is at least 92% and more preferably at least 95% similar in sequence to the polypeptide shown in SEQ ID No: 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 6 consecutive nucleotides of the sequences set forth as SEQ ID Nos: 1 or 2 or complements of the sequences set forth as SEQ ID Nos: 1 or 2, or naturally occurring mutants thereof In preferred embodiments, the probe/primer further includes a label group attached thereto, which is capable of being detected.
For expression, the subject GLC1A nucleic acids can include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter (e.g., for constitutive expression or inducible expression) or transcriptional enhancer or suppressor sequence, which regulatory sequence is operably linked to the GLC1A gene sequence. Such regulatory sequences in conjunction with a GLC1A nucleic acid molecule can be useful vectors 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 GLC1A proteins by employing said expression vectors.
In another aspect, the invention features isolated GLC1A polypeptides, preferably substantially pure preparations e.g. of plasma purified or recombinantly produced GLC1A polypeptides. In one embodiment, the polypeptide is identical to or similar to a GLC1A protein represented in SEQ ID No: 3. Related members of the vertebrate and particularly the mammalian GLC1A family are also within the scope of the invention. Preferably, a GLC1A polypeptide has an amino acid sequence at least about 92% homologous and preferably at least about 95%, 96%, 97%, 98% or 99% homologous to the polypeptide represented in SEQ ID No: 3. In a preferred embodiment, the GLC1A polypeptide that is encoded by a nucleic acid which hybridizes with a nucleic acid sequence represented in one of SEQ ID Nos: 1 or 2. The subject GLC1A proteins also include modified protein, which are resistant to post-translational modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with intracellular proteins involved in signal transduction.
The GLC1A polypeptide can comprise a full length protein, such as represented in SEQ ID No: 3, or it can comprise a fragment corresponding to one or more particular motifs/domains, or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150, 175, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 480, 485, 490, 495, or 500 amino acids in length.
Another aspect of the invention features chimeric molecules (e.g. fusion proteins) comprised of a GLC1A protein. For instance, the GLC1A protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the GLC1A polypeptide (e.g. the second polypeptide portion is glutathione-S-transferase, an enzymatic activity such as alkaline phosphatase or an epitope tag).
Yet another aspect of the present invention concerns an immunogen comprising a GLC1A polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for a GLC1A polypeptide; e.g. a humoral response, an antibody response and/or cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g. a unique determinant, from the protein represented in SEQ ID No: 3.
A still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of the GLC1A protein. In preferred embodiments the antibody specifically binds to at least one epitope represented in SEQ ID No: 3.
The invention also features transgenic non-human animals which include (and preferably express) a heterologous form of a GLC1A gene described herein, or which misexpress an endogenous GLC1A gene (e.g., an animal in which expression of one or more of the subject GLC1A proteins is disrupted). Such a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed GLC1A alleles or for use in drug screening. Alternatively, such a transgenic animal can be useful for expressing recombinant GLC1A polypeptides.
In yet another aspect, the invention provides assays, e.g., for screening test compounds to identify inhibitors, or alternatively, potentiators, of an interaction between a GLC1A protein and, for example, a virus, an extracellular ligand of the GLC1A protein, or an intracellular protein which binds to the GLC1A protein. An exemplary method includes the steps of (i) combining a GLC1A polypeptide or bioactive fragments thereof, a GLC1A target molecule (such as a GLC1A ligand or a GLC1A substrate), and a test compound, e.g., under conditions wherein, but for the test compound, the GLC1A protein and target molecule are able to interact; and (ii) detecting the formation of a complex which includes the GLC1A protein and the target polypeptide either by directly quantitating the complex, by measuring inductive effects of the GLC1A protein, or, in the instance of a substrate, measuring the conversion to product. A statistically significant change, such as a decrease, in the interaction of the GLC1A and target molecule in the presence of a test compound (relative to what is detected in the absence of the test compound) is in

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