Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-05-28
2003-07-15
Priebe, Scott D. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S455000, C536S023100, C536S023500
Reexamination Certificate
active
06593104
ABSTRACT:
1. BACKGROUND OF THE INVENTION
Macular degeneration is a clinical term that is used to describe a variety of diseases that are all characterized by a progressive loss of central vision associated with abnormalities of Bruch's membrane and the retinal pigment epithelium. These disorders include very common conditions that affect older patients (age related macular degeneration or AMD) as well as rarer, earlier-onset dystrophies that in some cases can be detected in the first decade of life
1-18
. The genes associated with some of these dystrophies have been mapped,
5-14
and in four cases, blue-cone monochromasy,
15
pattern dystrophy,
16,17
and Sorsby fundus dystrophy,
18
and Best Disease actually identified. However, none of the latter genes has been found to be responsible for a significant fraction of typical late-onset macular degeneration.
In developed countries, AMD is the most common cause of legal blindness in older patients.
19
The hallmark of this condition is the presence of drusen, which are ophthalmoscopically visible, yellow-white hyaline excrescences of Bruch's membrane. In some families, drusen are heritable in an autosomal dominant fashion.
In 1875, Hutchinson and Tay published a paper entitled “Symmetrical Central Choroido-Retinal Disease Occurring in Senile Persons”.
20
This paper includes one of the first descriptions of the constellation of clinical findings now known as age related macular degeneration (AMD). Specifically, three of the ten patients in the report were sisters affected with whitish spots (now referred to as drusen) in the macula. In 1899, Doyne
21
reported a similar disorder in which the abnormal spots were nearly confluent such that the macula had a “honeycomb” appearance. Histopathologic examination of one of Doyne's patients
22
revealed the abnormalities to be hyaline thickenings of Bruch's membrane. In 1925, Vogt
23
published the first description of the ophthalmoscopic appearance of a form of familial drusen that had been observed in patients living in the Leventine valley in the Ticino canton of southern Switzerland. Klainguti
24
fully characterized this condition in 1932 and demonstrated its autosomal dominant inheritance. This disorder eventually became known as malattia leventinese, “ML” (i.e., Leventine disease). In 1948, Waardenburg
25
stated that there was little reason to make a distinction between malattia leventinese and the condition described by Doyne, referred to as Doyne's Honeycomb Retinal Dystrophy (DHRD). This position was strengthened when Forni and Babel
26
found that the histopathologic features of malattia leventinese were indistinguishable from those of Doyne's honeycomb choroiditis. Piguet, Haimovici and Bird
27
recently reviewed the history of these conditions and also pointed out that the drusen in families with malattia leventinese are frequently distributed in a radical pattern. Choroidal neovascularization is uncommon in patients with radial drusen but does occur.
27
Although originally recognized in Switzerland, families affected with autosomal dominant radial drusen have been identified in Czechoslovakia,
28,29
and the United States.
30
In 1996, ML was mapped to chromosome 2p16-21
48
. Shortly thereafter, DHRD was mapped to the same locus
49
and the genetic interval was narrowed
48,49
. ML and DHRD are very similar phenotypically to AMD.
Currently, there is no therapy that is capable of significantly slowing the degenerative progression of macular degeneration, and treatment is limited to laser photocoagulation of the subretinal neovascular membranes that occur in 10-15% of affected patients.
2. SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of a novel human gene encoding a novel human protein, which has sequence homologies with fibulin (1 and 2), fibrillin, nidogen, notch, protein S and Factor IX. The newly identified proteins and nucleic acids described herein are referred to as “EFEMPs”. The human EFEMP1 gene (herein referred to as hEFEMP1) transcript is shown in FIG.
5
and includes 5′ and 3′ untranslated regions and a 1479 base pair open reading frame encoding a 493 amino acid polypeptide having SEQ ID NO. 1. Mouse EFEMP1 is expressed in eye, brain, heart, lung and kidney tissue.
In one aspect, the invention features isolated EFEMP1 nucleic acid molecules. In one embodiment, the EFEMP1 nucleic acid is from a vertebrate. In a preferred embodiment, the EFEMP1 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
FIG. 5
or a portion thereof. The disclosed molecules can be non-coding, (e.g. a probe, antisense, or ribozyme molecule) or can encode a functional EFEMP1 polypeptide (e.g. a polypeptide which specifically modulates biological activity, by acting as either an agonist or antagonist of at least one bioactivity of the human EFEMP1 polypeptide). In another embodiment, the nucleic acid of the present invention can hybridize to a vertebrate EFEMP1 gene or to the complement of a vertebrate EFEMP1 gene. In a further embodiment, the claimed nucleic acid can hybridize with a nucleic acid sequence shown in
FIG. 5
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 EFEMP1 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 NO: 1 or to the complement of the nucleic acid shown as FIG.
5
.
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
FIG. 5
or complements of the sequence set forth as
FIG. 5
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 EFEMP1 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 EFEMP 1 proteins by employing said expression vectors.
In another aspect, the invention features isolated EFEMP1 polypeptides, preferably substantially pure preparations, e.g. of plasma purified or recombinantly produced polypeptides. The EFEMP1 polypeptide can comprise a full length protein or can comprise smaller fragments corresponding to one or more particular motifs/domains, or fragments comprising at least about 6, 10, 25, 50, 75, 100, 125, 150, 200, 225, 250, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470 480 or 490 amino acids in length. In particularly preferred embodiments, the subject polypeptide has an EFEMP1 bioactivity.
In a preferred embodiment, the polypeptide is encoded by a nucleic acid which hybridizes with the nucleic acid sequence represented in FIG.
5
. In a further preferred embodiment, the EFEMP1 polypeptide is comprised of the amino acid sequence set forth in SEQ ID NO. 1. The subject EFEMP1 protein also includes within its scope modified proteins, e.g. proteins which are resistant to post-translational modification, for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycos
Sheffield Val C.
Stone Edwin M.
Kaushaz Sumesh
Needle & Rosenberg P.C.
Priebe Scott D.
University of Iowa Research Foundation
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