U.S. patent application number 12/985741 was filed with the patent office on 2011-05-05 for disease-associated proteins.
Invention is credited to Carlos Alvarez, David Hicks, Thirry Leveillard, Saddek Mohand-Said, Olivier Poch, Jose Alain Sahel.
Application Number | 20110105411 12/985741 |
Document ID | / |
Family ID | 34945897 |
Filed Date | 2011-05-05 |
United States Patent
Application |
20110105411 |
Kind Code |
A1 |
Leveillard; Thirry ; et
al. |
May 5, 2011 |
Disease-Associated Proteins
Abstract
Disclosed are methods and compositions for early diagnosis,
monitoring and treatment of an ocular disorder. In particular, the
invention relates to a novel protein, that is differentially
transcribed and expressed in subjects suffering from retinal
dystrophies and the like, such as retinal dystrophy and age-related
macular degeneration compared with healthy subjects, antibodies
which recognize this protein, and methods for diagnosing such
conditions.
Inventors: |
Leveillard; Thirry;
(Maisons-Alfort, FR) ; Sahel; Jose Alain; (Paris,
FR) ; Mohand-Said; Saddek; (Paris, FR) ; Poch;
Olivier; (Strasbourg, FR) ; Hicks; David;
(Strasbourg, FR) ; Alvarez; Carlos; (Riehen,
CH) |
Family ID: |
34945897 |
Appl. No.: |
12/985741 |
Filed: |
January 6, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11596327 |
Sep 9, 2008 |
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PCT/EP2005/005184 |
May 12, 2005 |
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12985741 |
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Current U.S.
Class: |
514/20.8 ;
435/320.1; 435/325; 435/69.1; 435/7.21; 436/94; 530/350; 530/387.9;
536/23.1 |
Current CPC
Class: |
C07K 14/47 20130101;
C07K 14/435 20130101; Y10T 436/143333 20150115; A61P 25/00
20180101; A61P 27/02 20180101 |
Class at
Publication: |
514/20.8 ;
530/350; 536/23.1; 435/325; 435/320.1; 436/94; 435/7.21; 530/387.9;
435/69.1 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 14/00 20060101 C07K014/00; C07H 21/00 20060101
C07H021/00; C12N 5/10 20060101 C12N005/10; C12N 15/63 20060101
C12N015/63; G01N 33/50 20060101 G01N033/50; G01N 33/53 20060101
G01N033/53; C07K 16/00 20060101 C07K016/00; C12P 21/00 20060101
C12P021/00; A61P 27/02 20060101 A61P027/02 |
Foreign Application Data
Date |
Code |
Application Number |
May 13, 2004 |
FR |
0405224 |
Claims
1. An isolated polypeptide consisting of an amino acid sequence
selected from the group consisting of the polypeptides set forth in
SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and
SEQ18.
2. An isolated nucleic acid molecule comprising a nucleotide
sequence that encodes a polypeptide according to claim 1.
3. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of SEQ1, SEQ3, SEQ 5,
SEQ 7, SEQ 9, SEQ 11, SEQ 13, SEQ15 and SEQ17.
4. A host cell comprising an isolated nucleic acid molecule
according to claim 2.
5. A vector molecule comprising at least a fragment of an isolated
DNA according to claims 2.
6. A vector molecule according to claim 5 comprising
transcriptional control sequences.
7. An isolated nucleic acid molecule comprising a nucleotide
sequence that hybridizes under high stringency conditions to the
isolated DNA of claims 2.
8. A host cell comprising a vector molecule according to claim
5.
9. A vertebrate host cell which can be propagated in vitro and
which is capable upon growth in culture of producing a polypeptide
according to claim 1, wherein said cell comprises at least one
transcriptional control sequence, wherein said one or more
transcriptional control sequences control transcription of DNA
encoding a polypeptide according to claim 1.
10. A vertebrate cell according to claim 9 wherein said one or more
transcriptional control DNA sequences are non-human transcriptional
control sequences.
11. A method for the diagnosis of retinal dystrophy in a human
which comprises: detecting the decreased transcription of messenger
RNA transcribed from the DNA encoding for a RDCVF1 or RDCVF2
polypeptide, in the eye of a human, wherein said decreased
transcription is diagnostic of said human's suffering from retinal
dystrophy.
12. A method for the diagnosis of retinal dystrophy in a human
which comprises: measuring the amount of an RDCVF1 or RDCVF2
polypeptide or fragments thereof, in rod cells of a human, wherein
the presence of an decreased amount of said polypeptide or
fragments thereof, relative to the amount of said polypeptide or
fragments thereof in normal eye tissue, is diagnostic of said
human's suffering from retinal dystrophy.
13. The method of claim 12, wherein said detecting step comprises
contacting said tissue with an antibody which specifically binds to
an RDCVF1 or RDCVF2 polypeptide, or a fragment thereof and
detecting specific binding of said antibody with a polypeptide in
said tissue, wherein detection of specific binding to a polypeptide
indicates the presence of an RDCVF1 or RDCVF2 polypeptide, or a
fragment thereof.
14. A purified antibody or a fragment thereof which specifically
binds to a polypeptide that comprises the amino acid sequence set
forth in SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and
SEQ18.
15. An antibody fragment according to claim 14 which is an Fab or
F(ab').sub.2 fragment.
16. An antibody according to claim 14 which is a polyclonal
antibody.
17. An antibody according to claim 14 which is a monoclonal
antibody.
18. A kit for the diagnosis of retinal dystrophy in a human
comprising a means for collecting a sample and an antibody
according to claim 14.
19. A method for producing RDCVF1 or RDCVF2 polypeptides
comprising: culturing a host cell having incorporated therein an
expression vector containing recombinant RDCVF1 or RDCVF2 -encoding
polynucleotide under conditions sufficient for expression of RDCVF1
or RDCVF2 polypeptides in the host cell, thereby causing the
production of an expressed polypeptide; and recovering the
polypeptide produced by said cell.
20. A method for preventing or protecting neuronal cells from
degeneration comprising transfecting said cells with a vector
comprising a nucleic acid sequence encoding an RDCVF1 or RDCVF2
polypeptide operably linked to a transcription control sequence,
and expressing said polypeptide in said cells.
21. The method of claim 20 wherein said cells are retinal
cells.
22. The method of claim 20 wherein the vector is derived from an
adenovirus or an adeno-associated virus.
23. A retinoprotective agent comprising a polypeptide selected from
the group of SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16
and SEQ18 and, optionally, a pharmaceutically acceptable
carrier.
24. A retinoprotective agent comprising a therapeutically effective
amount of a nucleic acid selected from the group of SEQ1, SEQ3, SEQ
5, SEQ 7, SEQ 9, SEQ 11, SEQ13, SEQ15 and SEQ17 and, optionally, a
pharmaceutically acceptable carrier.
25. A pharmaceutical composition comprising a therapeutically
effective amount of a polypeptide selected from the group of SEQ2,
SEQ 4, SEQ 6,SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and SEQ18 and a
pharmaceutically acceptable carrier.
26. A pharmaceutical composition comprising a therapeutically
effective amount of a nucleic acid selected from the group of SEQ1,
SEQ3, SEQ 5,SEQ 7, SEQ 9, SEQ 11, SEQ13, SEQ15 and SEQ17 and a
pharmaceutically acceptable carrier.
27. A method for the treatment of retinal dystrophy comprising
administering a therapeutically effective amount of a polypeptide
selected from the group of SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12,
SEQ14, SEQ16 and SEQ18, and optionally a pharmaceutically
acceptable carrier, to a subject in need of said treatment.
28. A method according to claim 27 said retinal dystrophy is
retinitis pigmentosa, age-related macular degeneration,
Bardet-Biedel syndrome, Bassen-kornzweig syndrome, best disease,
choroidema, gyrate atrophy, congenital amourosis, refsun syndrome,
stargardt disease or Usher syndrome.
29. Use of a polypeptide selected from the group of SEQ2, SEQ 4,
SEQ 6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and SEQ18 and, optionally,
a pharmaceutically acceptable carrier, in the manufacture of a
medicament for the treatment of retinal dystrophy.
30. Use of claim 29 wherein said retinal dystrophy is retinitis
pigmentosa, age-related macular degeneration, Bardet-Biedel
syndrome, Bassen-Kornzweig syndrome, Best disease, choroidema,
gyrate atrophy, congenital amourosis, Refsun syndrome, Stargardt
disease or Usher syndrome.
Description
FIELD OF THE INVENTION
[0001] This invention relates to methods and compositions for
detection and treatment of retinal degenerative diseases. In
particular, the invention relates to a protein that protects
against cone degeneration, nucleic acid molecules that encode such
a protein, antibodies that recognize the protein, and methods for
diagnosing retinal degenerative diseases.
BACKGROUND OF THE INVENTION
[0002] Photoreceptors are a specialized subset of retinal neurons,
that are responsible for vision. Photoreceptors consist of rods and
cones which are the photosensitive cells of the retina. Each rod
and cone elaborates a specialized cilium, referred to as an outer
segment, that houses the phototransduction machinery. The rods
contain a specific light-absorbing visual pigment, rhodopsin. There
are three classes of cones in humans, characterized by the
expression of distinct visual pigments: the blue cone, green cone
and red cone pigments. Each type of visual pigment protein is tuned
to absorb light maximally at different wavelengths. The rod
rhodopsin mediates scotopic vision (in dim light), whereas the cone
pigments are responsible for photopic vision (in bright light). The
red, blue and green pigments also form the basis of color vision in
humans. The visual pigments in rods and cones respond to light and
generate an action potential in the output cells, the rod bipolar
neurons, which is then relayed by the retinal ganglion neurons to
produce a visual stimulus in the visual cortex.
[0003] In humans, a number of diseases of the retina involve the
progressive degeneration and eventual death of photoreceptors,
leading inexorably to blindness. Degeneration of photoreceptors,
such as by inherited retinal dystrophies (e.g., retinitis
pigmentosa), age-related macular degeneration and other
maculopathies, or retinal detachment, are all characterized by the
progressive atrophy and loss of function of photoreceptor outer
segments. In addition, death of photoreceptors or loss of
photoreceptor function results in partial deafferentation of second
order retinal neurons (rod bipolar cells and horizontal cells) in
patients with retinal dystrophies, thereby decreasing the overall
efficiency of the propagation of the electrical signal generated by
photoreceptors. Secondary glial and pigment epithelium changes
secondary to photoreceptors degeneration result in vascular changes
leading to ischemia and gliosis. Trophic factors that are capable
of rescuing photoreceptors from cell death and/or restoring the
function of dysfunctional (atrophic or dystrophic) photoreceptors
may represent useful therapies for the treatment of such
conditions.
[0004] The progression of these conditions points to a sequential
loss of the two classes of photoreceptors: initially rods are lost
as a direct result of a genetic or environmental or unknown lesion,
resulting in night blindness and a reduction in visual field
followed inevitabley by loss of cones leading to total blindness.
Thus, cones die indirectly since they do not express the primary
lesion.
[0005] Not all of the genes associated with retinal dystrophy have
yet been identified. Identification of such genes would make
possible to both diagnose the disease and identify effective
therapies.
SUMMARY OF THE INVENTION
[0006] The invention relates generally to a novel gene family,
Rod-derived Cone Viability Factor (Rdcvf). In a first aspect, the
invention provides an isolated polypeptide with an amino acid
sequence as set forth in SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12,
SEQ14, SEQ16 and SEQ18.
[0007] In a second aspect, the invention provides an isolated
nucleic acid molecule comprising a nucleotide sequence as set forth
in SEQ1, SEQ3, SEQ 5, SEQ 7, SEQ 9, SEQ 11, SEQ 13, SEQ15 and
SEQ17.
[0008] In a preferred embodiment, the invention provides an
isolated nucleic acid molecule that encodes for a corresponding
polypeptide selected from the group of SEQ2, SEQ 4, SEQ 6, SEQ 8,
SEQ10, SEQ12, SEQ14, SEQ16 and SEQ18.
[0009] A third aspect of the present invention encompasses a method
for the diagnosis of retinal dystrophy in a human which includes
detecting the decreased transcription of messenger RNA transcribed
from Rdcvf1 or Rdcvf2-encoding DNA in the eye from a mammalian
organism, preferably a human, where such decreased transcription is
diagnostic of the organisms' affliction with retinal dystrophy or
pathological aging (ARMD). Another embodiment of the assay aspect
of the invention provides a method for the diagnosis of retinal
dystrophy in a mammalian organism, preferably a human, which
requires measuring the amount of a Rdcvf1 or Rdcvf2 polypeptide or
fragments thereof in the eye of a human suspected of suffering form
a retinal dystrophy, where the presence of a decreased amount of
the polypeptide or fragments thereof, relative to the amount of the
polypeptide or fragments thereof in the eye of an individual not
suffering from a retinal dystrophy, is diagnostic of the human's
suffering from retinal dystrophy.
[0010] In accordance with another aspect of the invention there are
provided anti-sense polynucleotides that regulate transcription of
the Rdcvf1 or Rdcvf2 gene; in another embodiment, double stranded
RNA is provided that can regulate the transcription of the Rdcvf1
or Rdcvf2 gene.
[0011] Another aspect of the invention provides a process for
producing the aforementioned polypeptides.
[0012] In a preferred embodiment of this aspect of the invention
there are provided methods for producing the aforementioned Rdcvf1
polypeptides comprising culturing host cells having incorporated
therein an expression vector containing recombinant Rdcvf1 or
Rdcvf2-encoding polynucleotide under conditions sufficient for
expression of Rdcvf1 or Rdcvf2 polypeptides in the host and then
recovering the expressed polypeptide.
[0013] In accordance with another aspect of the invention there are
provided products, compositions, processes and methods that utilize
the aforementioned polypeptides and polynucleotides for, inter
alia, research, biological, clinical and therapeutic purposes.
[0014] In certain additional preferred aspects of the invention
there are provided an antibody or a fragment thereof which
specifically binds to a polypeptide that comprises the amino acid
sequence set forth in SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12,
SEQ14, SEQ16 and SEQ18.
[0015] In certain particularly preferred aspects in this regard,
the antibodies are highly selective for mammalian, preferably mouse
and in particular human Rdcvf1 or Rdcvf2 polypeptides or portions
of such Rdcvf1 or Rdcvf2 polypeptides. In a related aspect, an
antibody or fragment thereof is provided that binds to a fragment
or portion of the amino acid sequence set forth in SEQ2, SEQ 4, SEQ
6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and SEQ18.
[0016] In another aspect, methods of treating a disease in a
subject, where the disease is mediated by or associated with a
change in Rdcvf1 or Rdcvf2 gene expression e.g. a decrease in the
presence of RDCVF1 or RDCVF2 polypeptide in the eye, by the
administration of a therapeutically effective amount of a RDCVF1 or
RDCVF2 protein as set out in SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10,
SEQ12, SEQ14, SEQ16 and SEQ18 to the subject is provided.
[0017] Also provided are methods for the diagnosis of a disease or
condition associated with an decrease in Rdcvf1 or Rdcvf2 gene
expression or decrease in the presence of RDCVF1 or RDCVF2
polypeptide in a subject, which comprises utilizing an antibody
that binds to a polypeptide with the amino acid sequence set out in
SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12, SEQ 14, SEQ16 and SEQ18 in
an immunoassay.
[0018] In yet another aspect, the invention provides cells which
can be propagated in vitro, preferably vertebrate cells, which are
capable upon growth in culture of producing a polypeptide that
comprises the amino acid sequence set forth in SEQ2, SEQ 4, SEQ 6,
SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and SEQ18, where the cells
contain transcriptional control DNA sequences, other than mouse or
human Rdcvf1 or Rdcvf2 transcriptional control sequences, where the
transcriptional control sequences control transcription of DNA
encoding a polypeptide with the amino acid sequence according to
SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and
SEQ18.
[0019] In a related aspect, the present invention provides a method
for producing Rdcvf1 or Rdcvf2 polypeptides which comprises
culturing a host cell having incorporated therein an expression
vector containing recombinant Rdcvf1 or Rdcvf2-encoding
polynucleotide under conditions sufficient for expression of Rdcvf1
or Rdcvf2 polypeptides in the host cell, thereby causing the
production of an expressed polypeptide, and recovering the
expressed polypeptide.
[0020] In yet another aspect of the present invention there are
provided assay methods and kits comprising the components necessary
to detect abnormal, e.g. below-normal expression of Rdcvf1 or
Rdcvf2 polynucleotides or polypeptides or fragments thereof in body
tissue samples derived from a patient, such kits comprising e.g.,
antibodies that bind to Rdcvf1 or Rdcvf2 or oligonucleotide probes
that hybridize with polynucleotides of the invention. In a
preferred embodiment, such kits also comprise instructions
detailing the procedures by which the kit components are to be
used.
[0021] In another aspect, the invention is directed to an Rdcvf1 or
Rdcvf2 polypeptide for use in the treatment of a human or animal
body. A related aspect is directed to the use of an Rdcvf1 or
Rdcvf2 polypeptide or fragment thereof, nucleotide encoding Rdcvf1
or Rdcvf2 or a fragment thereof, or antibody that binds to Rdcvf1
or Rdcvf2 or a fragment thereof in the manufacture of a medicament
to treat a retinal dystrophy.
[0022] In another aspect, the invention provides a retinoprotective
agent comprising a polypeptide selected from the group of SEQ2, SEQ
4, SEQ 6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and SEQ18 and,
optionally, a pharmaceutically acceptable carrier. In a related
aspect the invention provides a pharmaceutical compositions
comprising a Rdcvf1 or Rdcvf2 polypeptide or fragment thereof,
nucleotide encoding Rdcvf1 or Rdcvf2 or a fragment thereof, for the
treatment of a retinal dystrophy. In another related aspect, the
invention provides a pharmaceutical composition comprising a
therapeutically effective amount of a polypeptide selected from the
group of SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and
SEQ18 and a pharmaceutically acceptable carrier.
[0023] In a related aspect, the invention provides a method for the
treatment of retinal dystrophy comprising administering a
therapeutically effective amount of a polypeptide selected from the
group of SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and
SEQ18 and pharmaceutically acceptable carrier, to a subject in
need.
[0024] Methods for the identification of molecules that can bind to
Rdcvf1 or Rdcvf2 and/or modulate the activity of Rdcvf1 or Rdcvf2
or molecules that can bind to nucleic acid sequences that modulate
the transcription or translation of Rdcvf1 or Rdcvf2 are disclosed
in the prior art, e.g. WO 02/081513.
[0025] In yet another aspect, the invention is directed to methods
for the introduction of nucleic acids of the invention into one or
more tissues of a subject in need of treatment with the result that
one or more proteins encoded by the nucleic acids are expressed and
or secreted by cells within the tissue.
[0026] In another aspect the invention provides a method of
providing photoreceptor cells for implantation wherein the
photoreceptor cells are cultured together with RdCVF1 or
RdCVF2.
[0027] Other objects, features, advantages and aspects of the
present invention will become apparent to those of skill from the
following description. It should be understood, however, that the
following description and the specific examples, while indicating
preferred embodiments of the invention, are given by way of
illustration only. Various changes and modifications within the
spirit and scope of the disclosed invention will become readily
apparent to those skilled in the art from reading the following
description and from reading the other parts of the present
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1: mouse RdCVF1 nucleotide sequence from the expression
cloning and mouse RdCVF1 amino acid sequence.
[0029] FIG. 2: mouse RdCVF1L nucleotide sequence. and amino acid
sequence.
[0030] FIG. 3: human RdCVF1 and human RdCVF1 amino acids
sequence.
[0031] FIG. 4: human RdCVF1L nucleotide sequence. human RdCVF1L
amino acids sequence.
[0032] FIG. 5: mouse RdCVF2 nucleotide sequence and: mouse RdCVF2
amino acids.
[0033] FIG. 6: mouse RdCVF2L nucleotide sequence and mouse RdCVF2L
amino acid.
[0034] FIG. 7: human RdCVF2 nucleotide sequence. and human RdCVF2
amino acids sequence.
[0035] FIG. 8: rat RdCVF1 and rat RdCVF1 amino acids sequence
[0036] FIG. 9: rat RdCVF2 and rat RdCVF2 amino acids sequence
DETAILED DESCRIPTION OF THE INVENTION
[0037] All patent applications, patents and literature references
cited herein are hereby incorporated by reference in their
entirety.
[0038] In practicing the present invention, many conventional
techniques in molecular biology, microbiology, and recombinant DNA
are used. These techniques are well known and are explained in, for
example, Current Protocols in Molecular Biology, Volumes I, II, and
III, 1997 (F. M. Ausubel ed.); Sambrook et al., 1989, Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A
Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.);
Oligonucleotide Synthesis, 1984 (M. L. Gait ed.); Nucleic Acid
Hybridization, 1985, (Hames and Higgins); Transcription and
Translation, 1984 (Hames and Higgins eds.); Animal Cell Culture,
1986 (R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL
Press); Perbal, 1984, A Practical Guide to Molecular Cloning; the
series, Methods in Enzymology (Academic Press, Inc.); Gene Transfer
Vectors for Mammalian Cells, 1987 (J. H. Miller and M. P. Calos
eds., Cold Spring Harbor Laboratory); and Methods in Enzymology
Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds.,
respectively).
[0039] As used herein, "differentially expressed gene" refers to
(a) a gene containing at least one of the DNA sequences disclosed
herein (e.g., as shown in SEQ1, SEQ3, SEQ 5, SEQ 7, SEQ 9, SEQ 11,
SEQ 13, SEQ15 and SEQ17); (b) any DNA sequence that encodes the
amino acid sequence disclosed herein (e.g., as shown in SEQ2, SEQ
4, SEQ 6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and SEQ 18); or (c) any
DNA sequence that is substantially similar to the coding sequences
disclosed herein.
[0040] In its broadest sense, the term "substantially similar",
when used herein with respect to a nucleotide sequence, means a
nucleotide sequence corresponding to a reference nucleotide
sequence, wherein the corresponding sequence encodes a polypeptide
having substantially the same structure and function as the
polypeptide encoded by the reference nucleotide sequence, e.g.
where only changes in amino acids not affecting the polypeptide
function occur. Desirably the substantially similar nucleotide
sequence encodes the polypeptide encoded by the reference
nucleotide sequence. The percentage of identity between the
substantially similar nucleotide sequence and the reference
nucleotide sequence desirably is at least 90%, more preferably at
least 95%, still more preferably at least 99%, even more preferably
99.9%. Sequence comparisons are carried out using a Smith-Waterman
sequence alignment algorithm (see e.g. Waterman, M. S. Introduction
to Computational Biology: Maps, sequences and genomes. Chapman
& Hall. London: 1995. ISBN 0-412-99391-0, or at
http://www-hto.usc.edu/software/seqaln/index.html). The localS
program, version 1.16, is used with following parameters: match: 1,
mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty:
2. A nucleotide sequence "substantially similar" to reference
nucleotide sequence hybridizes to the reference nucleotide sequence
in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at
50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree.
C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC,
0.1% SDS at 50.degree. C., more desirably still in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C.,
preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1
mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at
50.degree. C., more preferably in 7% sodium dodecyl sulfate (SDS),
0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in
0.1.times.SSC, 0.1% SDS at 65.degree. C., yet still encodes a
functionally equivalent gene product.
[0041] The differentially expressed genes disclosed herein are
expressed in eye tissue and in particular is produced in rod cells
however, in a human afflicted with a retinal dystrophy such as
retinitis pigmentosa, age-related macular degeneration,
Bardet-Biedel syndrome, Bassen-Kornzweig syndrome, Best disease,
choroidema, gyrate atrophy, congenital amourosis, Refsun syndrome,
Stargardt disease and Usher syndrome it is produced in decreases
amounts relative to, i.e., to a lesser extent than in the
corresponding tissues of humans who do not suffer from retinal
dystrophy. Messenger RNA transcribed from the differentially
expressed genes, and protein translated from such mRNA, is present
in rod tissues and/or associated with such tissues in an amount at
least about half, preferably at least about five times, more
preferably at least amount ten times, most preferably at least
about 100 times less than the levels of mRNA and protein found in
corresponding tissues found in humans who do not suffer from a
retinal dystrophy. Such decreases transcription of Rdcvf1 or Rdcvf2
mRNA is referred to herein as "decreased transcription."
[0042] A "host cell," as used herein, refers to a prokaryotic or
eukaryotic cell that contains heterologous DNA that has been
introduced into the cell by any means, e.g., electroporation,
calcium phosphate precipitation, microinjection, transformation,
viral infection, and the like.
[0043] "Heterologous" as used herein means "of different natural
origin" or represent a non-natural state. For example, if a host
cell is transformed with a DNA or gene derived from another
organism, particularly from another species, that gene is
heterologous with respect to that host cell and also with respect
to descendants of the host cell which carry that gene. Similarly,
heterologous refers to a nucleotide sequence derived from and
inserted into the same natural, original cell type, but which is
present in a non-natural state, e.g. a different copy number, or
under the control of different regulatory elements.
[0044] A vector molecule is a nucleic acid molecule into which
heterologous nucleic acid may be inserted which can then be
introduced into an appropriate host cell. Vectors preferably have
one or more origin of replication, and one or more site into which
the recombinant DNA can be inserted. Vectors often have convenient
means by which cells with vectors can be selected from those
without, e.g., they encode drug resistance genes. Common vectors
include plasmids, viral genomes, and (primarily in yeast and
bacteria) "artificial chromosomes."
[0045] "Plasmids" generally are designated herein by a lower case p
preceded and/or followed by capital letters and/or numbers, in
accordance with standard naming conventions that are familiar to
those of skill in the art. Starting plasmids disclosed herein are
either commercially available, publicly available on an
unrestricted basis, or can be constructed from available plasmids
by routine application of well known, published procedures. Many
plasmids and other cloning and expression vectors that can be used
in accordance with the present invention are well known and readily
available to those of skill in the art. Moreover, those of skill
readily may construct any number of other plasmids suitable for use
in the invention. The properties, construction and use of such
plasmids, as well as other vectors, in the present invention will
be readily apparent to those of skill from the present
disclosure.
[0046] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated, even if subsequently reintroduced into the natural
system. Such polynucleotides could be part of a vector and/or such
polynucleotides or polypeptides could be part of a composition, and
still be isolated in that such vector or composition is not part of
its natural environment.
[0047] As used herein, the term "transcriptional control sequence"
refers to DNA sequences, such as initiator sequences, enhancer
sequences, and promoter sequences, which induce, repress, or
otherwise control the transcription of protein encoding nucleic
acid sequences to which they are operably linked.
[0048] As used herein, "Rdcvf1 transcriptional control sequences"
or "Rdcvf2 transcriptional control sequences" are any of those
transcriptional control sequences normally found associated with a
mammalian Rdcvf1 or Rdcvf2 gene, preferably with the Rdcvf2 gene as
found in the mouse or human genome.
[0049] As used herein, "non-human transcriptional control sequence"
is any transcriptional control sequence not found in the human
genome.
[0050] The term "polypeptide" is used interchangeably herein with
the terms "polypeptides" and "protein(s)".
[0051] As used herein, a "chemical derivative" of a polypeptide of
the invention is a polypeptide of the invention that contains
additional chemical moieties not normally a part of the molecule.
Such moieties may improve the molecule's solubility, absorption,
biological half life, etc. The moieties may alternatively decrease
the toxicity of the molecule, eliminate or attenuate any
undesirable side effect of the molecule, etc. Moieties capable of
mediating such effects are disclosed, for example, in Remington's
Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa.
(1980).
[0052] As used herein, a "neuroprotective agent" is a compound that
prevents or protects neuronal cells from degeneration. A
"retinoprotective agent" is a compound that prevents or protects
retinal cells from degeneration.
[0053] The invention includes nucleic acid molecules, preferably
DNA molecules, such as (1) an isolated comprising a nucleotide
sequence as set forth in SEQ1, SEQ3, SEQ 5, SEQ 7, SEQ 9, SEQ 11,
SEQ 13, SEQ15 and SEQ17, (2) isolated nucleic acid molecules that
comprise nucleic acid sequences that hybridize under high
stringency conditions to the isolated DNA as set forth in SEQ1,
SEQ3, SEQ 5, SEQ 7, SEQ 9, SEQ 11, SEQ 13, SEQ15 and SEQ17, and (3)
nucleic acid sequences that hybridize to (1) or (2), above. Such
hybridization conditions may be highly stringent or less highly
stringent, as described above. In instances wherein the nucleic
acid molecules are deoxyoligonucleotides ("oligos"), highly
stringent conditions may refer, e.g., to washing in
6.times.SSC/0.05% sodium pyrophosphate at 37.degree. C. (for
14-base oligos), 48.degree. C. (for 17-base oligos), 55.degree. C.
(for 20-base oligos), and 60.degree. C. (for 23-base oligos).
Suitable ranges of such stringency conditions for nucleic acids of
varying compositions are described in Krause and Aaronson (1991)
Methods in Enzymology, 200:546-556 in addition to Maniatis et al.,
cited above.
[0054] These nucleic acid molecules may act as target gene
antisense molecules, useful, for example, in target gene regulation
and/or as antisense primers in amplification reactions of target
gene nucleic acid sequences. Further, such sequences may be used as
part of ribozyme and/or triple helix sequences, also useful for
target gene regulation. Still further, such molecules may be used
as components of diagnostic methods whereby the presence of a
RdCVF1 or RdCVF2 disease-causing allele, may be detected.
[0055] The invention also encompasses (a) vectors that contain any
of the foregoing coding sequences (i.e., sense) and/or their
complements (i.e., antisense); (b) expression vectors that contain
any of the foregoing coding sequences operatively associated with a
regulatory element that directs the expression of the coding
sequences; and (c) genetically engineered host cells that contain
any of the foregoing coding sequences operatively associated with a
regulatory element that directs the expression of the coding
sequences in the host cell. As used herein, regulatory elements
include but are not limited to inducible and non-inducible
promoters, enhancers, operators and other elements known to those
skilled in the art that drive and regulate expression.
[0056] The invention includes fragments of any of the nucleic acid
sequences disclosed herein. Fragments of the full length Rdcvf1 or
Rdcvf2 gene may be used as a hybridization probe for a cDNA library
to isolate the full length gene and to isolate other genes which
have a high sequence similarity to the Rdcvf1 or Rdcvf2 gene and
similar biological activity. Probes of this type preferably have at
least about 30 bases and may contain, for example, from about 30 to
about 50 bases, about 50 to about 100 bases, about 100 to about 200
bases, or more than 200 bases (e.g. 300). The probe may also be
used to identify a cDNA clone corresponding to a full length
transcript and a genomic clone or clones that contain the complete
Rdcvf1 or Rdcvf2 gene including regulatory and promoter regions,
exons, and introns. An example of a screen comprises isolating the
coding region of the Rdcvf1 or Rdcvf2 gene by using the known DNA
sequence to synthesize an oligonucleotide probe or random priming
of the isolated sequence disclosed in table 1. Labeled
oligonucleotides having a sequence complementary to that of the
gene of the present invention are used to screen a library of human
cDNA, genomic DNA to determine which individual clones of the
library the probe hybridizes to.
[0057] In addition to the gene sequences described above, orthologs
of such sequences, as may, for example be present in other species,
may be identified and may be readily isolated, without undue
experimentation, by molecular biological techniques well known in
the art. Further, there may exist genes at other genetic loci
within the genome that encode proteins which have extensive
homology (homologs) to one or more domains of such gene products.
These genes may also be identified via similar techniques.
[0058] For example, the isolated expressed gene sequence may be
labeled and used to screen a cDNA library constructed from mRNA
obtained from the organism of interest. Hybridization conditions
will be of a lower stringency when the cDNA library was derived
from an organism different from the type of organism from which the
labeled sequence was derived. Alternatively, the labeled fragment
may be used to screen a genomic library derived from the organism
of interest, again, using appropriately low stringent conditions.
Such low stringency conditions will be well known to those of skill
in the art, and will vary predictably depending on the phylogeny of
specific organisms from which the library and the labeled sequences
are derived. For guidance regarding such conditions see, for
example, Sambrook et al. cited above.
[0059] Further, a previously unknown expressed gene-type sequence
may be isolated by performing PCR using two degenerated
oligonucleotide primer pools designed on the basis of amino acid
sequences within the gene of interest. The template for the
reaction may be cDNA obtained by reverse transcription of mRNA
prepared from human or non-human cell lines or tissue known or
suspected to express a homologue or splicing variants.
[0060] The PCR product may be subcloned and sequenced to ensure
that the amplified sequences represent the sequences of a expressed
gene-like nucleic acid sequence. The PCR fragment may then be used
to isolate a full length cDNA clone by a variety of methods. For
example, the amplified fragment may be labeled and used to screen a
bacteriophage cDNA library. Alternatively, the labeled fragment may
be used to screen a genomic library.
[0061] PCR technology may also be utilized to isolate full length
cDNA sequences. For example, RNA may be isolated, following
standard procedures, from an appropriate cellular or tissue source.
A reverse transcription reaction may be performed on the RNA using
an oligonucleotide primer specific for the most 5' end of the
amplified fragment for the priming of first strand synthesis. The
resulting RNA/DNA hybrid may then be "tailed" with guanines using a
standard terminal transferase reaction, the hybrid may be digested
with RNAase H, and second strand synthesis may then be primed with
a poly-C primer. Thus, cDNA sequences upstream of the amplified
fragment may easily be isolated. For a review of cloning strategies
which may be used, see e.g., Sambrook et al., 1989, supra.
[0062] In cases where the differentially expressed gene identified
is the normal, or wild type, gene, this gene may be used to isolate
mutant alleles of the gene. Such an isolation is preferable in
processes and disorders which are known or suspected to have a
genetic basis. Mutant alleles may be isolated from individuals
either known or suspected to have a genotype which contributes to
disease symptoms. Mutant alleles and mutant allele products may
then be utilized in the diagnostic assay systems described
below.
[0063] A cDNA of the mutant gene may be isolated, for example, by
using RT-PCR, a technique which is well known to those of skill in
the art. In this case, the first cDNA strand may be synthesized by
hybridizing an oligo-dT oligonucleotide (or random hexamers) to
mRNA isolated from tissue known or suspected to be expressed in an
individual putatively carrying the mutant allele, and by extending
the new strand with reverse transcriptase. The second strand of the
cDNA is then synthesized using an oligonucleotide that hybridizes
specifically to the 5' end of the normal gene (or by any other
means). Using these two primers, the product is then amplified via
PCR, cloned into a suitable vector, and subjected to DNA sequence
analysis through methods well known to those of skill in the art.
By comparing the DNA sequence of the mutant gene to that of the
normal gene, the mutation(s) responsible for the loss or alteration
of function of the mutant gene product can be ascertained.
[0064] Alternatively, a genomic or cDNA library can be constructed
and screened using DNA or RNA, from a tissue known to or suspected
of expressing the gene of interest in an individual suspected of to
carry the mutant allele. The normal gene or any suitable fragment
thereof may then be labeled and used as a probe to identify the
corresponding mutant allele in the library. The clone containing
this gene may then be purified through methods routinely practiced
in the art, and subjected to sequence analysis as described
above.
[0065] Additionally, an expression library can be constructed
utilizing DNA isolated from or cDNA synthesized from a tissue known
to or suspected of expressing the gene of interest in an individual
suspected of to carry the mutant allele. In this manner, gene
products made by the putatively mutant tissue may be expressed and
screened using standard antibody screening techniques in
conjunction with antibodies raised against the normal gene product,
as described, below. (For screening techniques, see, for example,
Harlow, E. and Lane, eds., 1988, "Antibodies: A Laboratory Manual",
Cold Spring Harbor Press, Cold Spring Harbor.) In cases where the
mutation results in an expressed gene product with altered function
(e.g., as a result of a missense mutation), a polyclonal set of
antibodies are likely to cross-react with the mutant gene product.
Library clones detected via their reaction with such labeled
antibodies can be purified and subjected to sequence analysis as
described above.
[0066] Differentially expressed gene products include those
proteins encoded by nucleotide sequence set forth in SEQ1, SEQ3,
SEQ 5, SEQ 7, SEQ 9, SEQ 11, SEQ 13, SEQ15 and SEQ17 in particular,
a polypeptide that is or includes the amino acid sequence set out
in SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and
SEQ18.
[0067] In addition, expressed gene products may include proteins
that represent functionally equivalent gene products. Such an
equivalent gene product may contain deletions, additions or
substitutions of amino acid residues within the amino acid sequence
encoded by the differentially expressed gene sequences described,
above, but which result in a silent change, thus producing a
functionally equivalent differentially expressed gene product
(polymorphisms). Amino acid substitutions may be made on the basis
of similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues
involved and on comparison with amino-acids sequence from other
species.
[0068] For example, nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and methionine; polar neutral amino acids include
glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine; positively charged (basic) amino acids include arginine,
lysine, and histidine; and negatively charged (acidic) amino acids
include aspartic acid and glutamic acid. "Functionally equivalent,"
as utilized herein, may refer to a protein or polypeptide capable
of exhibiting a substantially similar in vivo or in vitro activity
as the endogenous differentially expressed gene products encoded by
the differentially expressed gene sequences described above.
"Functionally equivalent" may also refer to proteins or
polypeptides capable of interacting with other cellular or
extracellular molecules in a manner similar to the way in which the
corresponding portion of the endogenous differentially expressed
gene product would. For example, a "functionally equivalent"
peptide would be able, in an immunoassay, to diminish the binding
of an antibody to the corresponding peptide (i.e., the peptidic
amino acid sequence of which was modified to achieve the
"functionally equivalent" peptide) of the endogenous protein, or to
the endogenous protein itself, where the antibody was raised
against the corresponding peptide of the endogenous protein. An
equimolar concentration of the functionally equivalent peptide will
diminish the aforesaid binding of the corresponding peptide by at
least about 5%, preferably between about 5% and 10%, more
preferably between about 10% and 25%, even more preferably between
about 25% and 50%, and most preferably between about 40% and
50%.
[0069] The differentially expressed gene products may be produced
by recombinant DNA technology using techniques well known in the
art. Thus, methods for preparing the differentially expressed gene
polypeptides and peptides of the invention by expressing nucleic
acid encoding differentially expressed gene sequences are described
herein. Methods which are well known to those skilled in the art
can be used to construct expression vectors containing expressed
gene protein coding sequences and appropriate
transcriptional/translational control signals. These methods
include, for example, in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. See, for example, the techniques described in
Sambrook et al., 1989, supra, and Ausubel et al., 1989, supra.
Alternatively, RNA or cDNA capable of encoding expressed gene
protein sequences may be chemically synthesized using, for example,
synthesizers. See, for example, the techniques described in
"Oligonucleotide Synthesis", 1984, Gait, M. J. ed., IRL Press,
Oxford, which is incorporated by reference herein in its
entirety.
[0070] A variety of host-expression vector systems may be utilized
to express the differentially expressed gene coding sequences of
the invention. Such host-expression systems represent vehicles by
which the coding sequences of interest may be produced and
subsequently purified, but also represent cells which may, when
transformed or transfected with the appropriate nucleotide coding
sequences, express the differentially expressed gene protein of the
invention in situ. These include but are not limited to
microorganisms such as bacteria (e.g., E. coli, B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DNA or
cosmid DNA expression vectors containing differentially expressed
gene protein coding sequences; yeast (e.g. Saccharomyces, Pichia)
transformed with recombinant yeast expression vectors containing
the differentially expressed gene protein coding sequences; insect
cell systems infected with recombinant virus expression vectors
(e.g., baculovirus) containing the differentially expressed gene
protein coding sequences; plant cell systems infected with
recombinant virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed with
recombinant plasmid transformation vectors (e.g., Ti plasmid)
containing differentially expressed gene protein coding sequences;
or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3) harboring
recombinant expression constructs containing promoters derived from
the genome of mammalian cells (e.g., metallothioneine promoter) or
from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter; Cytomegalovirus Early gene
promoter).
[0071] Expression of RDCVF1 or RDCVF2 by a cell from an Rdcvf1 or
Rdcvf2 gene that is native to a the cell can also be performed.
Methods for such expression are detailed in, e.g., U.S. Pat. Nos.
5,641,670; 5,733,761; 5,968,502; and 5,994,127, all of which are
expressly incorporated by reference herein in their entirety. Cells
that have been induced to express RDCVF1 or RDCVF2 by the methods
of any of U.S. Pat. Nos. 5,641,670; 5,733,761; 5,968,502; and
5,994,127 can be implanted into a desired tissue in a living animal
in order to increase the local concentration of RDCVF1 or RDCVF2 in
the tissue.
[0072] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
differentially expressed gene protein being expressed. For example,
when a large quantity of such a protein is to be produced, for the
generation of antibodies or to screen peptide libraries, for
example, vectors which direct the expression of high levels of
fusion protein products that are readily purified may be desirable.
Such vectors include, but are not limited, to the E. coli
expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in
which the differentially expressed gene protein coding sequence may
be ligated individually into the vector in frame with the lac Z
coding region so that a fusion protein is produced; pIN vectors
(Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van
Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the
like. PGEX vectors may also be used to express foreign polypeptides
as fusion proteins with glutathione S-transferase (GST). In
general, such fusion proteins are soluble and can easily be
purified from lysed cells by adsorption to glutathione-asephagarose
beads followed by elution in the presence of free glutathione. The
PGEX vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene protein can be
released from the GST moiety by using these endopeptidases.
[0073] Promoter regions can be selected from any desired gene using
vectors that contain a reporter transcription unit lacking a
promoter region, such as a chloramphenicol acetyl transferase
("cat") or luciferase transcription unit, downstream of restriction
site or sites for introducing a candidate promoter fragment; i.e.,
a fragment that may contain a promoter. As is well known,
introduction into the vector of a promoter-containing fragment at
the restriction site upstream of the cat or luciferase gene
engenders production of CAT or luciferase activity, which can be
detected by standard CAT assays or luminometry. Vectors suitable to
this end are well known and readily available. Three such vectors
are pKK232-8, -pCM7 and pGL3 (Promega, E1751, Genebank Ass n.sup.o
u47295). Thus, promoters for expression of polynucleotides of the
present invention include not only well known and readily available
promoters, but also promoters that readily may be obtained by the
foregoing technique, using a reporter gene assay.
[0074] Among known bacterial promoters suitable for expression of
polynucleotides and polypeptides in accordance with the present
invention are the E. coli lacI and lacZ promoters, the T3 and T7
promoters, the T5 tac promoter, the lambda PR, PL promoters and the
trp promoter. Among known eukaryotic promoters suitable in this
regard are the CMV immediate early promoter, the HSV thymidine
kinase promoter, the early and late SV40 promoters, the promoters
of retroviral LTRs, such as those of the Rous sarcoma virus
("RSV"), and metallothionein promoters, such as the mouse
metallothionein-I promoter.
[0075] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is one of several insect systems that
can be used as a vector to express foreign genes. The virus grows
in Spodoptera frugiperda cells. The differentially expressed gene
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter). Successful insertion of differentially expressed gene
coding sequence will result in inactivation of the polyhedrin gene
and production of non-occluded recombinant virus (i.e., virus
lacking the proteinaceous coat coded for by the polyhedrin gene).
These recombinant viruses are then used to infect Spodoptera
frugiperda cells in which the inserted gene is expressed. (E.g.,
see Smith et al., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No.
4,215,051).
[0076] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the differentially expressed gene coding
sequence of interest may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric gene may then be
inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable and capable of expressing differentially expressed
gene protein in infected hosts. (e.g., See Logan & Shenk, 1984,
Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation
signals may also be required for efficient translation of inserted
differentially expressed gene coding sequences. These signals
include the ATG initiation codon and adjacent sequences. In cases
where an entire differentially expressed gene, including its own
initiation codon and adjacent sequences, is inserted into the
appropriate expression vector, no additional translational control
signals may be needed. However, in cases where only a portion of
the differentially expressed gene coding sequence is inserted,
exogenous translational control signals, including, perhaps, the
ATG initiation codon, must be provided. Furthermore, the initiation
codon must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals (Kozack sequence) and
initiation codons can be of a variety of origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of appropriate transcription enhancer elements,
transcription terminators, etc. (see Bittner et al, 1987, Methods
in Enzymol. 153:516-544).
[0077] Selection of appropriate vectors and promoters for
expression in a host cell is a well known procedure and the
requisite techniques for expression vector construction,
introduction of the vector into the host and expression in the host
per se are routine skills in the art.
[0078] Generally, recombinant expression vectors will include
origins of replication, a promoter derived from a highly-expressed
gene to direct transcription of a downstream structural sequence,
and a selectable marker to permit isolation of vector containing
cells after exposure to the vector.
[0079] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins. Appropriate cell lines or host systems can be chosen
to ensure the correct modification and processing of the foreign
protein expressed. To this end, eukaryotic host cells which possess
the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the gene product
may be used. Such mammalian host cells include but are not limited
to CHO, VERO, BHK, HeLa, COS, MOCK, 293, 3T3, WI38, etc.
[0080] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the differentially expressed protein may be
engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the differentially expressed protein. Such
engineered cell lines may be particularly useful in screening and
evaluation of compounds that affect the endogenous activity of the
differentially expressed protein. These stable cell lines might by
used as a way of cellular therapy directly or after
encapsulation.
[0081] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for dhfr, which confers resistance to
methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567;
O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt,
which confers resistance to mycophenolic acid (Mulligan & Berg,
1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers
resistance to the aminoglycoside G-418 (Colberre-Garapin, et al.,
1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to
hygromycin (Santerre, et al., 1984, Gene 30:147) genes.
[0082] An alternative fusion protein system allows for the ready
purification of non-denatured fusion proteins expressed in human
cell lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:
8972-8976). In this system, the gene of interest is subcloned into
a vaccinia recombination plasmid such that the gene's open reading
frame is translationally fused to an amino-terminal tag consisting
of six histidine residues. Extracts from cells infected with
recombinant vaccinia virus are loaded onto Ni.sup.2+ nitriloacetic
acid-agarose columns and histidine-tagged proteins are selectively
eluted with imidazole-containing buffers.
[0083] When used as a component in assay systems such as those
described below, the differentially expressed protein may be
labeled, either directly or indirectly, to facilitate detection of
a complex formed between the differentially expressed protein and a
test substance. Any of a variety of suitable labeling systems may
be used including but not limited to radioisotopes such as
.sup.125I; enzyme labeling systems that generate a detectable
calorimetric signal or light when exposed to substrate; and
fluorescent labels.
[0084] Where recombinant DNA technology is used to produce the
differentially expressed protein for such assay systems, it may be
advantageous to engineer fusion proteins that can facilitate
labeling, immobilization and/or detection.
[0085] Indirect labeling involves the use of a protein, such as a
labeled antibody, which specifically binds to either a
differentially expressed gene product. Such antibodies include but
are not limited to polyclonal, monoclonal, chimeric, single chain,
Fab fragments and fragments produced by an Fab expression
library.
[0086] In another embodiment, nucleic acids comprising a sequence
encoding RDCVF1 or RDCVF2 protein or functional derivative thereof,
are administered to promote cone function, by way of gene therapy.
Gene therapy refers to therapy performed by the administration of a
nucleic acid to a subject. In this embodiment of the invention, the
nucleic acid produces its encoded protein that mediates a
therapeutic effect by promoting cone function.
[0087] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0088] In a preferred aspect, the therapeutic comprises a Rdcvf1 or
Rdcvf2 nucleic acid that is part of an expression vector that
expresses a RDCVF1 or RDCVF2 protein or fragment or chimeric
protein thereof in a suitable host. In particular, such a nucleic
acid has a promoter operably linked to the Rdcvf1 or Rdcvf2 coding
region, said promoter being inducible or constitutive, and,
optionally, tissue-specific. In another particular embodiment, a
nucleic acid molecule is used in which the Rdcvf1 or Rdcvf2 coding
sequences and any other desired sequences are flanked by regions
that promote homologous recombination at a desired site in the
genome, thus providing for intrachromosomal expression of the
Rdcvf1 or Rdcvf2 nucleic acid (Koller and Smithies, 1989, Proc.
Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature
342:435-438).
[0089] Delivery of the nucleic acid into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vector, or indirect, in which
case, cells are first transformed with the nucleic acid in vitro,
then transplanted into the patient. These two approaches are known,
respectively, as in vivo or ex vivo gene therapy.
[0090] In a specific embodiment, the nucleic acid is directly
administered in vivo, where it is expressed to produce the encoded
product. This can be accomplished by any of numerous methods known
in the art, e.g., by constructing it as part of an appropriate
nucleic acid expression vector and administering it so that it
becomes intracellular, e.g., by infection using a defective or
attenuated retroviral or other viral vector (adenovirus,
adeno-associated virus and lentivirus) (see, e.g., U.S. Pat. No.
4,980,286 and others mentioned infra), or by direct injection of
naked DNA, or by use of microparticle bombardment (e.g., a gene
gun; Biolistic, Dupont), or coating with lipids or cell-surface
receptors or transfecting agents, encapsulation in liposomes,
microparticles, or microcapsules, or by administering it in linkage
to a peptide which is known to enter the nucleus, by administering
it in linkage to a ligand subject to receptor-mediated endocytosis
(see e.g., U.S. Pat. Nos. 5,166,320; 5,728,399; 5,874,297; and
6,030,954, all of which are incorporated by reference herein in
their entirety) (which can be used to target cell types
specifically expressing the receptors), etc. In another embodiment,
a nucleic acid-ligand complex can be formed in which the ligand
comprises a fusogenic viral peptide to disrupt endosomes, allowing
the nucleic acid to avoid lysosomal degradation. In yet another
embodiment, the nucleic acid can be targeted in vivo for cell
specific uptake and expression, by targeting a specific receptor
(see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316;
WO93/14188; and WO 93/20221). Alternatively, the nucleic acid can
be introduced intracellularly and incorporated within host cell DNA
for expression, by homologous recombination (see, e.g., U.S. Pat.
Nos. 5,413,923; 5,416,260; and 5,574,205; and Zijlstra et al.,
1989, Nature 342:435-438).
[0091] In a specific embodiment, a viral vector that contains the
Rdcvf1 or Rdcvf2 nucleic acid is used. For example, a retroviral
vector can be used (see, e.g., U.S. Pat. Nos. 5,219,740; 5,604,090;
and 5,834,182). These retroviral vectors have been modified to
delete retroviral sequences that are not necessary for packaging of
the viral genome and integration into host cell DNA. The Rdcvf1 or
Rdcvf2 nucleic acid to be used in gene therapy is cloned into the
vector, which facilitates delivery of the gene into a patient.
[0092] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Methods for conducting adenovirus-based gene therapy are
described in, e.g., U.S. Pat. Nos. 5,824,544; 5,868,040; 5,871,722;
5,880,102; 5,882,877; 5,885,808; 5,932,210; 5,981,225; 5,994,106;
5,994,132; 5,994,134; 6,001,557; and 6,033,8843, all of which are
incorporated by reference herein in their entirety.
[0093] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy. Adeno-associated viruses are especially attractive
vehicles for delivering genes to the retina. Methods for producing
and utilizing AAV are described, e.g., in U.S. Pat. Nos. 5,173,414;
5,252,479; 5,552,311; 5,658,785; 5,763,416; 5,773,289; 5,843,742;
5,869,040; 5,942,496; and 5,948,675, all of which are incorporated
by reference herein in their entirety.
[0094] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient directly or after encapsulation.
[0095] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells and may be
used in accordance with the present invention, provided that the
necessary developmental and physiological functions of the
recipient cells are not disrupted. The technique should provide for
the stable transfer of the nucleic acid to the cell, so that the
nucleic acid is expressible by the cell and preferably heritable
and expressible by its cell progeny.
[0096] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. In a preferred
embodiment, epithelial cells are injected, e.g., subcutaneously. In
another embodiment, recombinant skin cells may be applied as a skin
graft onto the patient. Recombinant blood cells (e.g.,
hematopoietic stem or progenitor cells) are preferably administered
intravenously. The amount of cells envisioned for use depends on
the desired effect, patient state, etc., and can be determined by
one skilled in the art.
[0097] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0098] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0099] In an embodiment in which recombinant cells are used in gene
therapy, a Rdcvf1 or Rdcvf2 nucleic acid is introduced into the
cells such that it is expressible by the cells or their progeny,
and the recombinant cells are then administered in vivo for
therapeutic effect. In a specific embodiment, stem or progenitor
cells are used. Any stem-and/or progenitor cells which can be
isolated and maintained in vitro can potentially be used in
accordance with this embodiment of the present invention. Such stem
cells include but are not limited to hematopoietic stem cells
(HSC), stem cells of epithelial tissues such as the skin and the
lining of the gut, embryonic heart muscle cells, liver stem cells
(see, e.g., WO 94/08598), and neural stem cells (Stemple and
Anderson, 1992, Cell 71:973-985).
[0100] Epithelial stem cells (ESCs) or keratinocytes can be
obtained from tissues such as the skin and the lining of the gut by
known procedures (Rheinwald, 1980, Meth. Cell Bio. 21A:229). In
stratified epithelial tissue such as the skin, renewal occurs by
mitosis of stem cells within the germinal layer, the layer closest
to the basal lamina. Stem cells within the lining of the gut
provide for a rapid renewal rate of this tissue. ESCs or
keratinocytes obtained from the skin or lining of the gut of a
patient or donor can be grown in tissue culture (Pittelkow and
Scott, 1986, Mayo Clinic Proc. 61:771). If the ESCs are provided by
a donor, a method for suppression of host versus graft reactivity
(e.g., irradiation, drug or antibody administration to promote
moderate immunosuppression) can also be used. Retinal stem cells
(Tropepe et al., 2000, Science, 287: 2032).
[0101] With respect to hematopoietic stem cells (HSC), any
technique which provides for the isolation, propagation, and
maintenance in vitro of HSC can be used in this embodiment of the
invention. Techniques by which this may be accomplished include (a)
the isolation and establishment of HSC cultures from bone marrow
cells isolated from the future host, or a donor, or (b) the use of
previously established long-term HSC cultures, which may be
allogeneic or xenogeneic. Non-autologous HSC are used preferably in
conjunction with a method of suppressing transplantation immune
reactions of the future host/patient. In a particular embodiment of
the present invention, human bone marrow cells can be obtained from
the posterior iliac crest by needle aspiration (see, e.g., Kodo et
al., 1984, J. Clin. Invest. 73:1377-1384). In a preferred
embodiment of the present invention, the HSCs can be made highly
enriched or in substantially pure form. This enrichment can be
accomplished before, during, or after long-term culturing, and can
be done by any techniques known in the art. Long-term cultures of
bone marrow cells can be established and maintained by using, for
example, modified Dexter cell culture techniques (Dexter et al.,
1977, J. Cell Physiol. 91:335) or Witlock-Witte culture techniques
(Witlock and Witte, 1982, Proc. Natl. Acad. Sci. USA
79:3608-3612).
[0102] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[0103] Described herein are methods for the production of
antibodies capable of specifically recognizing one or more
differentially expressed gene epitopes. Such antibodies may
include, but are not limited to polyclonal antibodies, monoclonal
antibodies (mAbs), humanized or chimeric antibodies, single chain
antibodies, Fab fragments, F(ab').sub.2 fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies, and epitope-binding fragments of any of the above. Such
antibodies may be used, for example, in the detection of a
fingerprint, target, gene in a biological sample, or,
alternatively, as a method for the inhibition of abnormal target
gene activity. Thus, such antibodies may be utilized as part of
disease treatment methods, and/or may be used as part of diagnostic
techniques whereby patients may be tested for abnormal levels of
Rdcvf1 or Rdcvf2, or for the presence of abnormal forms of Rdcvf1
or Rdcvf2 by sampling the aqueous humor and/or vitreous by methods
familiar to those of skill in the art (e.g. Forster, R K, Abbott, R
L, Gelender, H. (1980) Management of infectious endophthalmitis
Ophthalmology 87, 313-319)].
[0104] For the production of antibodies to a differentially
expressed gene, various host animals may be immunized by injection
with a differentially expressed protein, or a portion thereof or by
DNA immunization. Such host animals may include but are not limited
to rabbits, mice, and fats, to name but a few. Various adjuvants
may be used to increase the immunological response, depending on
the host species, including but not limited to Freund's (complete
and incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum.
[0105] Polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of animals immunized with
an antigen, such as target gene product, or an antigenic functional
derivative thereof. For the production of polyclonal antibodies,
host animals such as those described above, may be immunized by
injection with differentially expressed gene product supplemented
with adjuvants as also described above.
[0106] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, may be obtained by any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to the hybridoma technique of Kohler and Milstein, (1975,
Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell
hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72;
Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and
the EBV-hybridoma technique (Cole et at, 1985, Monoclonal
Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such
antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. The hybridoma producing the
mAb of this invention may be cultivated in vitro or in vivo.
Production of high titers of mAbs in vivo makes this the presently
preferred method of production.
[0107] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608;
Takeda et al., 1985, Nature, 314:452-454) by splicing the genes
from a mouse antibody molecule of appropriate antigen specificity
together with genes from a human antibody molecule of appropriate
biological activity can be used. A chimeric antibody is a molecule
in which different portions are derived from different animal
species, such as those having a variable or hypervariable region
derived from a murine mAb and a human immunoglobulin constant
region.
[0108] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci.
USA 85:5879-5883; and Ward et at, 1989, Nature 334:544-546) can be
adapted to produce differentially expressed gene-single chain
antibodies. Single chain antibodies are formed by linking the heavy
and light chain fragments of the Fv region via an amino acid
bridge, resulting in a single chain polypeptide.
[0109] Most preferably, techniques useful for the production of
"humanized antibodies" can be adapted to produce antibodies to the
polypeptides, fragments, derivatives, and functional equivalents
disclosed herein. Such techniques are disclosed in U.S. Pat. Nos.
5,932,448; 5,693,762; 5,693,761; 5,585,089; 5,530,101; 5,910,771;
5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,545,580; 5,661,016;
and 5,770,429, the disclosures of all of which are incorporated by
reference herein in their entirety.
[0110] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, such fragments include
but are not limited to: the F(ab').sub.2 fragments which can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments. Alternatively, Fab expression
libraries may be constructed (Huse et al., 1989, Science,
246:1275-1281) to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity.
[0111] Particularly preferred, for ease of detection, is the
sandwich assay, of which a number of variations exist, all of which
are intended to be encompassed by the present invention.
[0112] For example, in a typical forward assay, unlabeled antibody
is immobilized on a solid substrate and the sample to be tested
brought into contact with the bound molecule. After a suitable
period of incubation, for a period of time sufficient to allow
formation of an antibody-antigen binary complex. At this point, a
second antibody, labeled with a reporter molecule capable of
inducing a detectable signal, is then added and incubated, allowing
time sufficient for the formation of a ternary complex of
antibody-antigen-labeled antibody. Any unreacted material is washed
away, and the presence of the antigen is determined by observation
of a signal, or may be quantitated by comparing with a control
sample containing known amounts of antigen. Variations on the
forward assay include the simultaneous assay, in which both sample
and antibody are added simultaneously to the bound antibody, or a
reverse assay in which the labeled antibody and sample to be tested
are first combined, incubated and added to the unlabeled surface
bound antibody. These techniques are well known to those skilled in
the art, and the possibility of minor variations will be readily
apparent. As used herein, "sandwich assay" is intended to encompass
all variations on the basic two-site technique. For the
immunoassays of the present invention, the only limiting factor is
that the unlabeled and the labeled antibodies be an RdCVF1- or
RdCVF2-specific antibody.
[0113] The most commonly used reporter molecules in this type of
assay are either enzymes, fluorophore- or radionuclide-containing
molecules. In the case of an enzyme immunoassay an enzyme is
conjugated to the second antibody, usually by means of
glutaraldehyde or periodate. As will be readily recognized,
however, a wide variety of different ligation techniques exist,
which are well-known to the skilled artisan. Commonly used enzymes
include horseradish peroxidase, glucose oxidase, beta-galactosidase
and alkaline phosphatase, among others. The substrates to be used
with the specific enzymes are generally chosen for the production,
upon hydrolysis by the corresponding enzyme, of a detectable color
change. For example, p-nitrophenyl phosphate is suitable for use
with alkaline phosphatase conjugates; for peroxidase conjugates,
1,2-phenylenediamine or toluidine are commonly used. It is also
possible to employ fluorogenic substrates, which yield a
fluorescent product rather than the chromogenic substrates noted
above. A solution containing the appropriate substrate is then
added to the tertiary complex of antibody-RdCVF1- or RdCVF2-labeled
antibody. The substrate reacts with the enzyme linked to the second
antibody, giving a qualitative visual signal, which may be further
quantitated, usually spectrophotometrically, to give an evaluation
of the amount of Rdcvf1 or Rdcvf2 which is present in the serum
sample.
[0114] Alternately, fluorescent compounds, such as fluorescein and
rhodamine, may be chemically coupled to antibodies without altering
their binding capacity. When activated by illumination with light
of a particular wavelength, the fluorochrome-labeled antibody
absorbs the light energy, inducing a state of excitability in the
molecule, followed by emission of the light at a characteristic
longer wavelength. The emission appears as a characteristic color
visually detectable with a light microscope. Immunofluorescence and
EIA techniques are both very well established in the art and are
particularly preferred for the present method. However, other
reporter molecules, such as radioisotopes, chemiluminescent or
bioluminescent molecules may also be employed. It will be readily
apparent to the skilled artisan how to vary the procedure to suit
the required use.
[0115] This invention also relates to the use of polynucleotides of
the present invention as diagnostic reagents. Detection of a
mutated form of the gene encoding a polypeptide selected from the
group consisting of the polypeptides set forth in SEQ2, SEQ 4, SEQ
6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and SEQ18 which is associated
with a dysfunction will provide a diagnostic tool that can add to,
or define, a diagnosis of a disease, or susceptibility to a
disease, which results from under-expression, over-expression or
altered spatial or temporal expression of the gene. Individuals
carrying mutations in the gene may be detected at the DNA level by
a variety of techniques.
[0116] Nucleic acids for diagnosis may be obtained from a subject's
cells, such as from blood, urine, saliva, tissue biopsy or autopsy
material. The genomic DNA may be used directly for detection or may
be amplified enzymatically by using PCR or other amplification
techniques prior to analysis. RNA or cDNA may also be used in
similar fashion. Deletions and insertions can be detected by a
change in size of the amplified product in comparison to the normal
genotype. Point mutations can be identified by hybridizing
amplified DNA to labeled nucleotide sequences. Perfectly matched
sequences can be distinguished from mismatched duplexes by RNase
digestion or by differences in melting temperatures. DNA sequence
differences may also be detected by alterations in electrophoretic
mobility of DNA fragments in gels, with or without denaturing
agents (SSCP), or by direct DNA sequencing (e.g., Myers et al.,
Science (1985) 230:1242). Sequence changes at specific locations
may also be revealed by nuclease protection assays, such as RNase
and S1 protection or the chemical cleavage method (see Cotton et
al., Proc Natl Acad Sci USA (1985) 85: 4397-4401). In another
embodiment, an array of oligonucleotides probes comprising the
Rdcvf1 or Rdcvf2 nucleotide sequence or fragments thereof can be
constructed to conduct efficient screening of e.g., genetic
mutations. Array technology methods are well known and have general
applicability and can be used to address a variety of questions in
molecular genetics including gene expression, genetic linkage, and
genetic variability (see for example: M. Chee et al., Science, Vol
274, pp 610-613 (1996)).
[0117] The diagnostic assays offer a process for diagnosing or
determining a susceptibility to disease through detection of
mutation in the Rdcvf1 or Rdcvf2 gene by the methods described. In
addition, such diseases may be diagnosed by methods comprising
determining from a sample derived from a subject an abnormal Rdcvf1
or Rdcvf2 expression: Expression can be measured at the RNA level
using any of the methods well known in the art for the quantitation
of polynucleotides, such as, for example, nucleic acid
amplification, for instance PCR, RT-PCR, RNase protection, Northern
blotting and other hybridization methods. Assay techniques that can
be used to determine levels of a protein, such as a polypeptide of
the present invention, in a sample derived from a host are
well-known to those of skill in the art. Such assay methods include
radioimmunoassays, competitive-binding assays, Western Blot
analysis and ELISA assays.
[0118] Thus in another aspect, the present invention relates to a
diagnostic kit which comprises:
[0119] (a) a polynucleotide of the present invention, preferably
the nucleotide sequence encoding a polypeptide selected from the
group consisting of the polypeptides set forth in SEQ2, SEQ 4, SEQ
6, SEQ 8, SEQ10, SEQ12, SEQ14, SEQ16 and SEQ18 or a fragment
thereof;
[0120] (b) a nucleotide sequence complementary to that of (a);
[0121] (c) a polypeptide of the present invention, preferably the
polypeptide of SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12, SEQ14,
SEQ16 and SEQ18 or a fragment thereof; or
[0122] (d) an antibody to a polypeptide of the present invention,
preferably to a polypeptide selected from the group consisting of
the polypeptides set forth in SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10,
SEQ12, SEQ14, SEQ16 and SEQ18.
[0123] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component. Such a kit will be of
use in diagnosing a disease or susceptibility to a disease,
particularly retinitis pigmentosa, age-related macular
degeneration, Bardet-Biedel syndrome, Bassen-kornzweig syndrome,
best disease, choroidema, gyrate atrophy, congenital amourosis,
refsun syndrome, stargardt disease and Usher syndrome. The
nucleotide sequences of the present invention are also valuable for
chromosome localization. The chromosomal location can be obtained
by PCR on DNA prepared from a panel of hybrid cell lines
(mouse-hamster). The chromosomal location of the human gene can be
predicted from the location of the mouse gene by syntheny (McCarthy
et al.(1997), Genome research, 7, 1153). The sequence is
specifically targeted to, and can hybridize with, a particular
location on an individual chromosome, including a mouse or a human
chromosome. The mapping of relevant sequences to chromosomes
according to the present invention is an important first step in
correlating those sequences with gene associated disease. Once a
sequence has been mapped to a precise chromosomal location, the
physical position of the sequence on the chromosome can be
correlated with genetic map data. Such data are found in, for
example, V. McKusick, Mendelian Inheritance in Man (available
on-line through Johns Hopkins University Welch Medical Library,
RetNet). The relationship between genes and diseases that have been
mapped to the same chromosomal region are then identified through
linkage analysis (coinheritance of physically adjacent genes).
[0124] The differences in the cDNA or genomic sequence between
affected and unaffected individuals can also be determined. If a
mutation is observed in some or all of the affected individuals but
not in any normal individuals, then the mutation is likely to be
the causative agent of the disease.
[0125] An additional embodiment of the invention relates to the
administration of a pharmaceutical composition, in conjunction with
a pharmaceutically acceptable carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may
consist of Rdcvf1 or Rdcvf2, mimetics or agonists. The compositions
may be administered alone or in combination with at least one other
agent, such as stabilizing compound, which may be administered in
any sterile, biocompatible pharmaceutical carrier, including, but
not limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs or hormones.
[0126] The pharmaceutical compositions encompassed by the invention
may be administered by any number of routes: e.g. Transscleral
delivery of bioreactive protein to the choroid and retina Ambati et
al. (2000) Investigative Ophthalmology and Visual Science, 41,
1186. The formulation of topical ophthalmic preparations, including
ophthalmic solutions, suspensions and ointments is well known to
those skilled in the art (see Remington's Pharmaceutical Sciences,
18th Edition, Chapter 86, pages 1581-1592 Mack Publishing Company,
1990). Other modes of administration are available, including
intracameral injections (which may be made directly into the
anterior chamber or directly into the vitreous chamber),
subconjunctival injections and retrobulbar injections, and methods
and means for producing ophthalmic preparations suitable for such
modes of administration are also well known.
[0127] As used in this application, "extraocular" refers to the
ocular surface and the (external) space between the eyeball and the
eyelid. Examples of extraocular regions include the eyelid fornix
or cul-de-sac, the conjunctival surface and the corneal surface.
This location is external to all ocular tissue and an invasive
procedure is not required to access this region. Examples of
extraocular systems include inserts and "topically" applied drops,
gels or ointments which may be used to deliver therapeutic material
to these regions. Extraocular devices are generally easily
removable, even by the patient.
[0128] The following patents disclose extraocular systems which are
used to administer drugs to the extraocular regions. Higuchi et al.
discloses in U.S. Pat. Nos. 3,981,303, 3,986,510 and 3,995,635, a
biodegradable ocular insert which contains a drug. The insert can
be made in different shapes for retention in the cul-de-sac of the
eyeball, the extraocular space between the eyeball and the eyelid.
Several common biocompatible polymers are disclosed as suitable for
use in fabricating this device. These polymers include zinc
alginate, poly (lactic acid), poly (vinyl alcohol), poly
(anhydrides) and poly (glycolic acid). The patents also describe
membrane coated devices with reduced permeation to the drug and
hollow chambers holding the drug formulation.
[0129] Theeuwes, U.S. Pat. No. 4,217,898, discloses microporous
reservoirs which are used for controlled drug delivery. These
devices are placed extraocularly in the ocular cul-de-sac. Among
the polymer systems of interest include poly
(vinylchloride)-co-poly (vinyl acetate) copolymers. Kaufman
discloses in U.S. Pat. Nos. 4,865,846 and 4,882,150 an ophthalmic
drug delivery system which contains at least one bio-erodible
material or ointment carrier for the conjunctival sac. The patent
discloses polymer systems, such as, poly (lactide), poly
(glycolide), poly (vinyl alcohol) and cross linked collagen, as
suitable delivery systems.
[0130] In the presently described use of RDCVF1 or RDCVF2 protein
product for the treatment of retinal disease or injury it is also
advantageous that a topically applied ophthalmic formulation
include an agent to promote the penetration or transport of the
therapeutic agent into the eye. Such agents are known in the art.
For example, Ke et al., U.S. Pat. No. 5,221,696 disclose the use of
materials to enhance the penetration of ophthalmic preparations
through the cornea.
[0131] Intraocular systems are those systems which are suitable for
use in any tissue compartment within, between or around the tissue
layers of the eye itself. These locations include subconjunctival
(under the ocular mucous membrane adjacent to the eyeball), orbital
(behind the eyeball), and intracameral (within the chambers of the
eyeball itself). In contrast to extraocular systems, an invasive
procedure consisting of injection or implantation is required to
access these regions.
[0132] The following patents disclose intraocular devices. Wong,
U.S. Pat. No. 4,853,224, discloses microencapsulated drugs for
introduction into the chamber of the eye. Polymers which are used
in this system include polyesters and polyethers. Lee, U.S. Pat.
No. 4,863,457, discloses a biodegradable device which is surgically
implanted intraocularly for the sustained release of therapeutic
agents. The device is designed for surgical implantation under the
conjunctiva (mucous membrane of the eyeball). Krezancaki, U.S. Pat.
No. 4,188,373, discloses a pharmaceutical vehicle which gels at
human body temperature. This vehicle is an aqueous suspension of
the drug and gums or cellulose derived synthetic derivatives.
Haslam et al. discloses in U.S. Pat. Nos. 4,474,751 and 4,474,752 a
polymer-drug system which is liquid at room temperature and gels at
body temperature. Suitable polymers used in this system include
polyoxyethylene and polyoxy propylene. Davis et al. disclose in
U.S. Pat. No. 5,384,333 a biodegradable injectable drug delivery
polymer which provides long term drug release. The drug composition
is made up of a pharmaceutically active agent in a biodegradable
polymer matrix, where the polymer matrix is a solid at temperatures
in the range 20 degree to 37 degree C. and is flowable at
temperatures in the range 38 degree to 52 degree C. The drug
delivery polymer is not limited to the delivery of soluble or
liquid drug formulations. For example, the polymer can be used as a
matrix for stabilizing and retaining at the site of injection
drug-containing microspheres, liposomes or other particulate-bound
drugs.
[0133] A particularly suitable vehicle for intraocular injection is
sterile distilled water in which the RDCVF1 or RDCVF2 protein
product is formulated as a sterile, isotonic solution, properly
preserved. Yet another ophthalmic preparation may involve the
formulation of the RDCVF1 or RDCVF2 protein product with an agent,
such as injectable microspheres or liposomes, that provides for the
slow or sustained release of the protein which may then be
delivered as a depot injection. Other suitable means for the
intraocular introduction of RDCVF1 or RDCVF2 protein product
includes, implantable drug delivery devices or which contain the
RDCVF1 or RDCVF2 protein product.
[0134] The ophthalmic preparations of the present invention,
particularly topical preparations, may include other components,
for example ophthalmically acceptable preservatives, tonicity
agents, cosolvents, wetting agents, complexing agents, buffering
agents, antimicrobials, antioxidants and surfactants, as are well
known in the art. For example, suitable tonicity enhancing agents
include alkali metal halides (preferably sodium or potassium
chloride), mannitol, sorbitol and the like. Sufficient tonicity
enhancing agent is advantageously added so that the formulation to
be instilled into the eye is hypotonic or substantially isotonic.
Suitable preservatives include, but are not limited to,
benzalkonium chloride, thimerosal, phenethyl alcohol,
methylparaben, propylparaben, chlorhexidine, sorbic acid and the
like. Hydrogen peroxide may also be used as preservative. Suitable
cosolvents include, but are not limited to, glycerin, propylene
glycol and polyethylene glycol. Suitable complexing agents include
caffeine, polyvinylpyrrolidone, beta-cyclodextrin or
hydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting
agents include, but are not limited to, sorbitan esters,
polysorbates such as polysorbate 80, tromethamine, lecithin,
cholesterol, tyloxapol and the like. The buffers can be
conventional buffers such as borate, citrate, phosphate,
bicarbonate, or Tris-HCl.
[0135] The formulation components are present in concentrations
that are acceptable to the extraocular or intraocular site of
administration. For example, buffers are used to maintain the
composition at physiological pH or at slightly lower pH, typically
within a pH range of from about 5 to about 8. Additional
formulation components may include materials which provide for the
prolonged ocular residence of the extraocularly administered
therapeutic agent so as to maximize the topical contact and promote
absorbtion. Suitable materials include polymers or gel forming
materials which provide for increased viscosity of the ophthalmic
preparation. Chitosan is a particularly suitable material as an
ocular release-rate controlling agent in sustained release liquid
ophthalmic drug formulations (see U.S. Pat. No. 5,422,116, Yen, et.
al.) The suitability of the formulations of the instant invention
for controlled release (e.g., sustained and prolonged delivery) of
an ophthalmic treating agent in the eye can be determined by
various procedures known in the art, e.g., as described in Journal
of Controlled Release, 6:367-373, 1987, as well as variations
thereof.
[0136] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.).
[0137] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0138] Pharmaceutical preparations for oral use can be obtained
through combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers, such as sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums including arabic and tragacanth; and proteins such as gelatin
and collagen. If desired, disintegrating or solubilizing agents may
be added, such as the cross-linked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate.
[0139] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0140] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with a
filler or binders, such as lactose or starches, lubricants, such as
talc or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0141] Pharmaceutical formulations suitable for parenteral
administration may be formulated m aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents which
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions.
[0142] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0143] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0144] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
etc. Salts tend to be more soluble in aqueous or other protonic
solvents than are the corresponding free base forms. In other
cases, the preferred preparation may be a lyophilized powder which
may contain any or all of the following: 1-50 mM histidine, 0.1%-2%
sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is
combined with buffer prior to use.
[0145] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of Rdcvf1 or Rdcvf2,
such labeling would include amount, frequency, and method of
administration.
[0146] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0147] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually mice, rabbits, dogs,
or pigs. The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0148] A therapeutically effective dose refers to that amount of
active ingredient, for example Rdcvf1 or Rdcvf2 or fragments
thereof, antibodies to Rdcvf1, agonists, which ameliorates the
symptoms or condition. Therapeutic efficacy and toxicity may be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., ED50 (the dose therapeutically
effective in 50% of the population) and LD50 (the dose lethal to
50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index, and it can be
expressed as the ratio, LD50/ED50. Pharmaceutical compositions
which exhibit large therapeutic indices are preferred. The data
obtained from cell culture assays and animal studies is used in
formulating a range of dosage for human use. The dosage contained
in such compositions is preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage varies within this range depending upon the dosage form
employed, sensitivity of the patient, and the route of
administration.
[0149] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active moiety or to maintain the desired effect. Factors
which may be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0150] Normal dosage amounts may vary from 0.1 to 100,000
micrograms, up to a total dose of about 1 g, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc. Pharmaceutical formulations
suitable for oral administration of proteins are described, e.g.,
in U.S. Pat. Nos. 5,008,114; 5,505,962; 5,641,515; 5,681,811;
5,700,486; 5,766,633; 5,792,451; 5,853,748; 5,972,387; 5,976,569;
and 6,051,561.
[0151] The following Examples illustrate the present invention,
without in any way limiting the scope thereof.
EXAMPLES
1. Expression Cloning:
[0152] 1) Total ARN Purification from Five Weeks Normal Mouse
Retinas:
[0153] The cDNA library was constructed from retinas of Five weeks
old C57BL/6@N mice generally according to the method of Glissin et
al (1974), Biochemistry, 13, 2633-2637. Briefly, after killing,
animals were enucleated and the eyes were first placed in Phosphate
Buffer Saline (PBS) supplemented with 0.1% DiEthyl PyroCarbonate
(DEPC). Neural retina was quickly dissected (the retinal pigmented
epithelium was omitted in this tissue preparation). Quickly after
each dissection, tissues were homogenised in fresh 6 M Guanidinium
Chloride. Ten retinas were pooled in 2.4 ml GC in 4 ml sterile
tubes, and the tissue was disrupted completely by strong
homogenisation for 1 minute at room temperature.
[0154] Messenger RNA (mRNA) Purification from Five Weeks Normal
Mouse Retina:
[0155] mRNA was isolated on oligo-dT coated porous beads (Oligotex,
Qiagen) under stringent conditions according to the method of
Kuribayashi et al, (1988). Nucleic Acids Res. Symposium series, 19,
61-64. Briefly, 100-150 .mu.g total mouse retina RNA were mixed
with 15 .mu.l of oligo-dT beads in Binding Buffer [10 mM Tris pH
7.5; 0.3 M NaCl; 0.1 M EDTA; 0.5% w/v Sodium Duodecyl Sulfate
(SDS)] and incubated 6 minutes at 65.degree. C. in 0.5 l becher of
water, then progressively cooled to room temperature for about 3-4
hours, then centrifuged at room temperature to recover the agarose
beads. They were then washed twice by incubating 10 minutes in 0.4
ml of (0.1 M Tris pH 7.5; 0.1 M NaCl; 1 mM EDTA; 0.5% w/v SDS).
Bounded RNA (mRNA) were eluted in two steps with 50 .mu.l of
70.degree. C.-warmed RNAse-free water, precipitated with 10 .mu.l
Sodium Acetate pH 5.2 and 0.25 ml ethanol and incubated 12 hours at
-70.degree. C. mRNA were collected by centrifugation (1 hour at
15.000 rpm, followed by two washes with 70% ethanol) and
resuspended in 20 .mu.l of RNAse-free water. mRNA concentration was
measured at 260 nm and the absence of rRNA contaminations was
checked by gel electrophoresis under denaturating condition as
above.
[0156] cDNA Synthesis:
[0157] was carried out according to the method of Okayama and Berg,
(1982), Mol Cell Biol., 2, 161-170. First strand synthesis was
primed with 2.5 .mu.g of NotI adaptor oligonucleotide
(5'TGTTACCAATCTGAAGTGGGAGCGGCCGACAA(T).sub.18 3') and incubated for
2 hours with 50 units of modified Moloney Murine Leukaemia virus
(M-MLV) reverse transcriptase (Superscript II, Life Technology)
under the conditions recommended by the supplier. For second strand
synthesis, reaction was incubated 4 hours at 14.degree. C. with 4
units of RNAseH and 100 units of DNA polymerase I in SS Buffer [40
mM Tris pH 7.2; 85 mM Potassium Chloride; 4.4 mM Magnesium
chloride; 3 mM DTT; 5 .mu.g/ml Bovin Serum Albumin (BSA)] in a
final volume of 0.25 ml. EcoRI adapters (5'-OH AATTCGGCACGAGG
3'-OH/3'-OH GCCGTGCTCC5'-PO.sub.4) were ligated at both ends of
double strand cDNA in 14 hours at 16.degree. C. using 40 units of
T4 DNA ligase (Promega, Madison, USA) in a total volume of 20 .mu.l
under the conditions recommended by the supplier. The products of
this reaction are dscDNA that have an EcoRI half site in 5' and
NotI half site in 3' that can be oriented in the cloning
vector.
[0158] Ligation of dscDNA in pcDNA3:
[0159] was performed according to Maniatis T. (1992), Molecular
cloning: a laboratory manual, 2nd ed., using 10 .mu.g of pcDNA3
plasmid (Invitrogen) prepared by cutting with EcoRI and NotI
(Promega, Madison, USA) under the conditions specified by the
supplier.
[0160] Propagation of Recombinant Clones:
[0161] was performed generally according to the method of Birnboim
et al (1979), Nucleic Acids Res., 7, 1513-1523. Briefly, In order
to make pools of 100 primary clones, we slightly modified the XL1
Gold (Strategene) transformation protocol (provided by Stratagene)
as followed. After incubation in growth medium, the transformation
reaction was brought to 20% (vol./vol.) glycerol and 8% (vol./vol.)
horse serum albumin (HAS, Life Technologies). HAS and glycerol
prevent death followed freezing thawing. A titration was done by
platting on agar plates (100 .mu.g/ml ampicillin) increasing volume
of each transformation reaction to calculate the volume giving 100
colonies, while the bulk transformation reaction was stored at
-80.degree. C. Recombinant plasmids from the library were purified
by 96 at once. In order to prepare 96 pools of 100 clones, the
calculated volume corresponding to 100 clones was platted on agar
and grown 20 hours at 37.degree. C. The DNA was purified directly
from colonies striped from agar plates. A stock of each culture at
23% glycerol was stored at -80.degree. C. The DNA was purified
using Qiawell ultra (Qiagen,) using a protocol recommended by the
supplier. Typically, 10 .mu.g of purified plasmid were obtained,
the concentration of each preparation was measured using optical
density at 260 nm. To sub-divide selected pools of 100 into pools
of 10, 50 .mu.l of a 1/250.000 dilution of the glycerol stock from
the original pool was plate on an agar plate at 100 .mu.g/ml
ampicillin. After growth 16 hours at 37.degree. C., individual 160
colonies were replicated on 16 agar plates (10 per plate) and grown
16 hours at 37.degree. C. The 10 colonies from each plate were
harvested and grown in liquid medium [Luria Broth (LB), 100
.mu.g/ml ampicillin] for 3 hours at 37.degree. C. A stock of these
cultures at 30% glycerol was stored at -80.degree. C. Plasmid DNA
was prepared as before. To divide sub-pools of 10 into individual
clones, 50 .mu.l of a 1/250.000 dilution of the glycerol stock of
the sub-pools of 10 was plated onto agar plate with 100 .mu.g/ml
ampicillin. After growth 16 hours at 37.degree. C., 16 individual
colonies were picked and grown 16 hours in 2 ml LB 100 .mu.g/ml
ampicillin. A stock of these cultures at 30% glycerol was stored at
-80.degree. C. Plasmid DNA was prepared as before.
[0162] Transient Transfection in Cos-1 Cells:
[0163] was performed using the method of Chen and Okayama, (1987).
High-efficiency transformation of mammalian cells by plasmid DNA
(Mol Cell Biol., 7, 2745-2752).
[0164] Chick Embryo Retinal Cultures:
[0165] The protocol was adapted from Adler and Hatlee [(1989)
Science, 243, 391]. Chick embryonic retina (6 days in ovo) is
dissociated and plated in monolayer culture. Under these culture
conditions with the absence of differentiation signals, cones
represent 60-80% of cells. We have produced polyclonal antibodies
into rabbit against visinin (a chicken cone marker, Genbank
accession number M84729) and verified that the proportion of cones
in our culture is of 60-80%. The simple environment of our model
(chemically defined medium, absence of cell to cell contacts) in
addition to the ease and speed of the method make it a very
appropriate system to study trophic factors involved in cone
survival. Briefly, Retinas from embryos issued from a control
isolate of progenitors, are dissected after six days of development
in ovo, cells dissociated and plated at low density (10.sup.5
cells/cm.sup.2). During ten days cell viability (60-80% cones) was
followed using LIVE/DEAD assay (Molecular probes, Eugene, USA) an
assay that quantifies live and dead cells. The number of cells
alive decreases down to 8% of initial cells number after seven days
in culture in chemically defined medium. When performed in the
presence of conditioned media from COS1 cells transfected with
pools of clones from the library, live cells are counted after
seven days in vitro.
Chicken progenitors (strain 657 red label) were maintained in a
separated compartment for the purpose of this experiment in a
hatching facility 25 km from the laboratory. Fertilized eggs
obtained naturally were collected weekly, and maintained at
17.degree. C. (their biological zero) in the laboratory after
hatching. Daily, 5 eggs are incubated for 24 hours at 20.degree. C.
then 136 hours at 37.degree. C. with intermittent reversion of the
inclination of the eggs in a humidified chamber. The day of the
culture, eggs surface are washed with Mucocit-A then broken, the
chicken embryos are transferred in PBS. The stage of development of
the each embryo is verified be the 29.sup.th by visual comparison
to Hamburger and Hamilton (1951), in (Essential Development
Biology, Stem and Holland Ed.). Two of the embryos were chosen and
enucleated, the eyes transferred in CO.sub.2-independent medium
(Life technologies). Retinas were dissected and transferred in
Ringer buffer and washed twice. Retinas are cut in small pieces and
treated 20 minutes at 37.degree. C. with a solution of trypsin
(0.25% w/v). The reaction is stopped by addition of culture media
(M199, Life Technologies) supplemented with 10% inactivated FCS.
The cell suspension is treated for few minutes in 25 .mu.l of DNAse
I (1 mg/ml, Sigma). The cell suspension is then washed twice in
Chemical Defined Culture Media [CDCM, equal volumes of DMEM and
M199 media (Life Technologies) and AB with Supplements (5 .mu.g/ml
Insulin; 5 .mu.g/ml Transferring; 64 nM Progesterone; 0.1 mM
Putrescin; 5 ng/ml Selenium; 3 mM Taurine; 2.7 .mu.M Cytidine
5'-diphosphoethanolamine; 5.2 .mu.M Cytidine 5'-diphosphocholine,
0.2 .mu.g/ml Hydrocortisone; 30 nM 3,3'-5-triiodo-L-thyronine; 1 mM
Sodium Pyruvate), 0.3 .mu.M Prostaglandin D.sub.2; 0.1 mg/ml
linoleic acid] in order to remove the FCS. The concentration of
cells stained with trypan blue is measured with Mallassez'cell and
brought to two concentrations (5.6 and 1.12 10.sup.5 cells/ml)
corresponding to the two plating densities (2 and 4 10.sup.5
cells/cm.sup.2). Conditioned media from Cos-1 transfected cells are
thaw on ice and 50 .mu.l transferred to two 96 wells tissue culture
treated black plate (Corning Costar) that have been coated a
solution of 100 .mu.g/ml Poly-L-lysine (Sigma) according the
plan:
First Round of Screening:
TABLE-US-00001 [0166] 1 1 1 1 2 C 2 2 2 3 3 P C 3 3 4 4 4 C 4 5 5 5
5 6 C 6 6 6 7 7 C 7 7 8 8 8 8 C 9 9 9 9 10 C 10 10 10 11 11 11 C 11
12 12 12 12 C 13 13 13 13 14 14 C 14 14 15 15 15 C 15 16 16 16 16
17 C 17 17 17 18 18 C 18 18 19 19 19 19 P 20 20 20 C 20
Where numbers refer to n.sup.o of pools of 100 clones, C to
conditioned media from Cos-1 cells transfected with the empty
vector (pcDNA3) and P a positive control (conditioned media
transfected with pcDNA-mouseGDNF.
Second and Third Round of Screening:
TABLE-US-00002 [0167] x.(y).01 x.(y).01 x.(y).01 x.(y).01 x.(y).02
C x.(y).02 x.(y).02 x.(y).02 x.(y).03 x.(y).03 P C x.(y).03
x.(y).03 x.(y).04 x.(y).04 x.(y).04 C x.(y).04 x.(y).05 x.(y).05
x.(y).05 x.(y).05 x.(y).06 C x.(y).06 x.(y).06 x.(y).06 x.(y).07
x.(y).07 C x.(y).07 x.(y).07 x.(y).08 x.(y).08 x.(y).08 x.(y).08 C
x.(y).09 x.(y).09 x.(y).09 x.(y).09 x.(y).10 C x.(y).10 x.(y).10
x.(y).10 x.(y).11 x.(y).11 x.(y).11 C x.(y).11 x.(y).12 x.(y).12
x.(y).12 x.(y).12 C x.(y).13 x.(y).13 x.(y).13 x.(y).13 x.(y).14
x.(y).14 C x.(y).14 x.(y).14 x.(y).15 x.(y).15 x.(y).15 C x.(y).15
x.(y).16 x.(y).16 x.(y).16 x.(y).16 x.(y) C x.(y) x.(y) x.(y) C57
C57 C C57 C57 C3H C3H C3H C3H P 0 0 0 C 0
Where x (second round) and y (third round) represent the n.sup.o of
the pools selected in the first and second round respectively. 01
to 16 represent the sub pools used. x(y) represents the parental
pool from which the 16 pools derived. C is as is first round. P was
modified as pCMVScript-CNTF in the second round and progressively
to pcDNA-939.09.08 in the third round. 0 represents chick cone
cells in CDCM media only. C57 and C3H, conditioned media from
explants of retinas prepared as described in (preparing conditioned
media from explants of mouse retinas) from C57BL/6@N and C3H/He@N
mouse retinas aged of 5 weeks respectively. 50 .mu.l of cell
suspensions corresponding to two densities (2 and 4 10.sup.5
cells/cm.sup.2) were added to the well of the two 96 wells plates
filled conditioned media with a 8 channel motorized pipette
(Biohit) in order to minimized experimental errors. Cells were
incubated for 7 days at 37.degree. C. in 5% CO.sub.2.
[0168] Preparing Conditioned Media from Explants of Mouse
Retinas:
[0169] The second and third round screening included positive
controls adapted from Mohand-Said et al. (1998). Five weeks aged
mouse 5C57BL/6@N (wild type) and C3H/He@N (rd1) were sacrificed and
enucleated. Two retinas were dissected and incubated in 24 hours at
37.degree. C. in 5% CO.sub.2 in 1.5 ml of CDCM in 12 wells plates.
Conditioned media were recovered and concentrated by a factor 40 by
ultra-filtration on Vivaspin (Sartorius, cutting point 10 kDa).
Conditioned media were frizzed in liquid nitrogen and stored in
aliquots at -20.degree. C. before used. The day of use, conditioned
media were thawed on ice, diluted 10 times in CDCM and sterilized
by filtration on 0.22 .mu.m filter (Acrodisk 13, Gelman
Sciences).
[0170] Functional Assay, Live/Dead Assay:
[0171] The functional assay is based on the number of chicken
retinal cells alive after 7 days incubation in vitro. We used
Live/Dead assay kit (Molecular probes, Eugene, USA) that is based
on the use of two fluorogenic dyes (Calcein AM and Ethidium dimer)
that stain live and dead cells respectively. A cell that is alive
processes a metabolic activity (here an esterase activity) that
coverts the substrate (Calcein AM) in its fluorescent product
emitting at 520 nm. The membrane permeability of a dead cell is
altered and permits the DNA staining of the nucleus by Ethidium
dimmer emitting at 635 nm. A cell is alive: emitting at 520 nm
after excitation at 485 nm, or dead: emitting at 635 nm after
excitation at 520 nm. Using epifluorescence microscopy, the two
types of fluorescent cells can be visualized separately. After 7
days in vitro, cells were incubated for 30 minutes at room
temperature in the dark with 2.7 .mu.M Calcein-AM and 0.3 mM
Ethidium dimer.
[0172] Image Acquisition:
[0173] Briefly, image acquisition consisted of autofocussing each
well, automatic cell counting in two fluorescence's followed by
processing of the raw data using specialist software e.g. Metamorph
(Universal Imaging Corporation, West Chester, USA) to obtain
digitalised pictures of each well of the plate. We used a inverted
microscope (Nikon TE 200) equipped with a mercury epifluorescent
lamp with two excitation filters 485 and 520 nm, two emission
filters 520 and 635 nm, an object (.times.10), a computer driven
motorized platine (Multicontrol 2000, Martzauzer and a CCD camera
(Cohu).
To record the plate, it is positioned on the motorised platine, and
the focus done manually on the first well and this plane is
recorded (z origin). The threshold of the image dead and live is
set from the first well. The centre of the first well is adjusted
using white light by aligning manually the bottom of the first well
to the bottom of the image on the computer monitor, then by
aligning the extreme right of the first well to the right of the
image on the computer screen and to record the two positions. The
centre of the first well is calculated and gives the position of
the centre of each well of the plate. We have noticed in the
development process that there is a slight higher density of cells
at the edge of the well and excluded the edge from the
acquisitions. It is important that the image from each well be
centred perfectly in order to avoid any misleading results. When
set-up, the first scan of the plate executes a recording of dead
cells. The dead cells density is the less variable under these
conditions. The application executes an auto focusing by taking
images at different focal plans and choosing the brightest one, the
right focus. This z position is stored and platine executes
programmed movements in the x and y axes taking a total of 4 images
that when reconstituted in one image represents 2/3 of the surface
of the well. A pile of images from the focus plans is stored for
control. The platine executes an auto focusing and four
acquisitions for each well of the plate starting by wells A1 to
A12, then B12 to B1, C1 to C12 etc . . . At the end, the platine
moves outside the plate in order to overexpose the last well (H1).
The scanning of dead cells takes 30 minutes. The second scan (live
cells) is executed after switching the filter. This second scan is
using the recorded z positions of each well from the dead scan.
Four images are taken from each well as for dead cells. At the end
of the second scan (22 minutes) dead and live reconstituted images
are stored in a file that is named automatically with the date of
the day. Cells numbers (dead and line) are counted automatically
with pre-set morphometric parameters (average) and displayed on the
computer monitor in order to check if the experiment is correct. It
is important to check on a daily basis that the number of cells
alive is not too high. We have observed that if plated at too
important density, the chicken retinal cells survived longer most
likely by producing their own survival factor. We screened cells in
the absence of this effect. Before scanning the second plate (same
experiment with twice density of cells plated), we add an a at the
end of the name of the log file from the first plate. Images of
each experiment were stored on CD-ram. We have generated a library
of more than 250 CD-rom.
[0174] Cells Counting and Selection of the Pools:
[0175] The cells numbers (live and dead) were counted using images
of each experiment stored on CD-rom. The log file from an
experiment was first loaded on a computer (counting off-line) and
opened using Metamorph software. In a first step, the images
corresponding to the 14 wells C (conditioned media from Cos-1 cells
transfected with the empty vector) are opened. After adjusting the
threshold of the image, the command Integrated Morphometry Analysis
is used to measure the distribution of the areas between 10 and 250
of the object (number of live cells for each total area) for these
control wells. The distribution follows a Gaussian curve with the
maximal number of objects corresponding to an isolated cell. This
standard value (SV) is then used to calculate the value of the area
above which an object will be counted as two cells (standard object
cut, SOC) through our empirical function: SOC=29/20.74 SV. The SOC
value of each individual plate is used to count the number of live
cells of the plate. These numbers are then transferred into a excel
table.
For the first round of screening, the value were plotted for each
pool as fold difference (increase or decrease live cells numbers)
versus the average of the 14 control wells+the standard deviation.
In order to redress the variations coming from the position
difference within the plate, we calculated the average of the fold
difference individually between the 80 wells corresponding to
positions where the pools are tested and the 14 wells control for
200 independent plates. In average, the differences observed are
due only to position difference and cells numbers were corrected
with this coefficient. In order to discriminate in more stringent
way the pools to be selected, the live cells counts were plotted as
fold difference versus as control all values not exceeding 1.3 but
exceeding 0.4. In this manner, all pools were fold difference
versus the 14 wells corresponding to empty vector are in the
interval of 0.4 to 1.3 are considered as having no effect and used
as control. After correction, the fold difference versus control of
the two plates was multiplied and the result sorted by decreasing
fold difference. The pools on the top of that list were further
checked by visual inspection of the graph corresponding to the 20
pools of the experiment (both plates) and of the images of live and
dead cells to avoid to screen misleading pools. For the second and
third round, the plates screening for sub-pools include additional
control. We prepared conditioned media from retina explants of 5
weeks of age. We selected experiments where positive effects were
recorded for retina explants from C57BL/6@N and dismissed the
others. The results were plotted as fold difference versus the 14
control wells, no recalculation of the control was made. The fold
difference versus control of the two plates was multiplied and the
results were sorted by decreasing fold difference for the 16
sub-pools. Isolated cDNA were sequenced using T7 primer (5'
GTAATACGACTCACTATAGGGC 3') on a capillary sequencer (CEQ2000,
Beckman Coulter). DNA sequence was compared to databases using
Basic Local Alignment Search Tool (BLAST).
[0176] Identification of Rdcvf2 and Human Homologues:
[0177] Using the Rdcvf1 sequence (the nucleotide sequence encoding
the polypeptides set forth SEQ2, SEQ 4, SEQ 6, SEQ 8, SEQ10, SEQ12,
SEQ14, SEQ16 and SEQ18 and BLAST, homologous murine and human
polypeptides were identified. EST clones with homology to mouse
RdCVF2 (GenBank Accession No: bc016199) are identified:
[0178] Real-time RT-PCR Analysis of Rdcvf1 Expression:
[0179] The retinal expression of Rdcvf1 by mouse C57BL/6@N and
C3H/He@N aged as well as congenic C3H (+/+ and rd/rd) of 5 weeks is
studied using real-time RT-PCR on a lightcycler (Roche) with
sybergreen PCR kit (Roche). cDNAs are produced by priming with a
random hexamer oligonucleotide (pdN6, Amersham), M-MLV reverse
transcriptase (superscript II, Life Technologies) and total RNA
from mouse retina prepared as in 1). cDNAs are normalized using a
ubiquitous messenger Glucose-6-Phosphate DeHydrogenase (G6PDH). 0.2
.mu.l of first strand cDNAs synthesis (an equivalent of 10 ng of
total RNA) is amplified with 2 .mu.M of the oligonucleotides 5'
TCTATGTGTCCCAGGACCCTACAG 3' and 5' TTTATGCACAAGTAGTACCAGGACAG 3' in
triple in a total volume of 25 .mu.l using the following program:
30 seconds at 95.degree. C., and 35 cycles of a sequence (1 second
at 95.degree. C., 18 seconds at 55.degree. C., 10 seconds at
72.degree. C.).
[0180] RT-PCR Analysis of Rdcvf2
[0181] RT-PCR for Rdcvf2 expression is carried out using primers 5'
GCCAGCGTTTTCTGCCTTTTAC 3' and 5' AAGCCCTGCCTGCTCTAACATC 3'. The
analysis shows that RdCVF2 is expressed in a rod-dependent manner
and that Rdcvf2 expression is not restricted to the retina, but
that also other neuronal cells express Rdcvf2 whereas the
expression of Rdcvf1 seems to be restricted to retinal cells.
[0182] Live/Death Assays of Rdcvf1 or Rdcvf2
[0183] COS-1 cells are transfected with a suitable expression
vector carrying the Rdcvf1 or Rdcvf2 under control of a inducible
promoter. Control cells are transfected with the empty vector. The
cells are incubated for a suitable period of time upon induction of
Rdcvf1 or Rdcvf2 expression. Subsequently, the number of surviving
cone cells incubated with conditioned media from COS-1 cells
transfected with Rdcvf1 or Rdcvf2 and the number of surviving
control cells are counted according to the above described method.
The cells expressing Rdcvf1 or Rdcvf2 show a significantly higher
amount of surviving cells.
Sequence CWU 1
1
261468DNAMus musculus 1atcggatccc tctctgggtc cccagctcct tgcatactgc
taccatggca tctctcttct 60ctggacgcat cttgatcagg aacaacagcg accaggatga
agtggagaca gaggcagagc 120tgagccgtag gttagagaat cgtctggtgt
tgctgttctt cggcgccggc gcctgtcccc 180agtgccaggc ctttgcccca
gtcctcaaag acttcttcgt gcggctcact gacgagttct 240acgtgctgcg
ggcagcacag ctggccctgg tctatgtgtc ccaggaccct acagaggagc
300aacaggacct cttcctcagg gacatgcctg aaaaatggct cttcctgccg
ttccatgatg 360aactgaggag gtgaggcccc agggaagacc agggagggct
tcctggagaa ggcatttccc 420tggaggttta ctgtcctggt actacttgtg
cataaagagg tattcctc 4682109PRTMus musculus 2Met Ala Ser Leu Phe Ser
Gly Arg Ile Leu Ile Arg Asn Asn Ser Asp1 5 10 15Gln Asp Glu Val Glu
Thr Glu Ala Glu Leu Ser Arg Arg Leu Glu Asn 20 25 30Arg Leu Val Leu
Leu Phe Phe Gly Ala Gly Ala Cys Pro Gln Cys Gln 35 40 45Ala Phe Ala
Pro Val Leu Lys Asp Phe Phe Val Arg Leu Thr Asp Glu 50 55 60Phe Tyr
Val Leu Arg Ala Ala Gln Leu Ala Leu Val Tyr Val Ser Gln65 70 75
80Asp Pro Thr Glu Glu Gln Gln Asp Leu Phe Leu Arg Asp Met Pro Glu
85 90 95Lys Trp Leu Phe Leu Pro Phe His Asp Glu Leu Arg Arg 100
1053675DNAMus musculus 3tgctaccatg gcatctctct tctctggacg catcttgatc
aggaacaaca gcgaccagga 60tgaagtggag acagaggcag agctgagccg taggttagag
aatcgtctgg tgttgctgtt 120cttcggcgcc ggcgcctgtc cccagtgcca
ggcctttgcc ccagtcctca aagacttctt 180cgtgcggctc actgacgagt
tctacgtgct gcgggcagca cagctggccc tggtctatgt 240gtcccaggac
cctacagagg agcaacagga cctcttcctc agggacatgc ctgaaaaatg
300gctcttcctg ccgttccatg atgaactgag gagggacctc gggcgccagt
tctctgtccg 360tcaactgcca gcggttgtgg tacttaagcc tggtggggac
gtgctgacaa gcgacgccac 420ggaggagatc cagcgtctgg gacccgcctg
ctttgccaac tggcaggagg ccgcagagct 480cctggaccgc agcttcctgc
aaccggagga tttggatgag cctgcgcggc gcagcatcac 540cgagcctctg
cgccgtcgca agtaccgagt agaccgggat gtcggccggg agcggggccg
600aaacggccgc gactctggtg acccccaggg ggacgcgggt acaagggcgg
agctctggtg 660agcggccgct cgagt 6754217PRTMus musculus 4Met Ala Ser
Leu Phe Ser Gly Arg Ile Leu Ile Arg Asn Asn Ser Asp1 5 10 15Gln Asp
Glu Val Glu Thr Glu Ala Glu Leu Ser Arg Arg Leu Glu Asn 20 25 30Arg
Leu Val Leu Leu Phe Phe Gly Ala Gly Ala Cys Pro Gln Cys Gln 35 40
45Ala Phe Ala Pro Val Leu Lys Asp Phe Phe Val Arg Leu Thr Asp Glu
50 55 60Phe Tyr Val Leu Arg Ala Ala Gln Leu Ala Leu Val Tyr Val Ser
Gln65 70 75 80Asp Pro Thr Glu Glu Gln Gln Asp Leu Phe Leu Arg Asp
Met Pro Glu 85 90 95Lys Trp Leu Phe Leu Pro Phe His Asp Glu Leu Arg
Arg Asp Leu Gly 100 105 110Arg Gln Phe Ser Val Arg Gln Leu Pro Ala
Val Val Val Leu Lys Pro 115 120 125Gly Gly Asp Val Leu Thr Ser Asp
Ala Thr Glu Glu Ile Gln Arg Leu 130 135 140Gly Pro Ala Cys Phe Ala
Asn Trp Gln Glu Ala Ala Glu Leu Leu Asp145 150 155 160Arg Ser Phe
Leu Gln Pro Glu Asp Leu Asp Glu Pro Ala Arg Arg Ser 165 170 175Ile
Thr Glu Pro Leu Arg Arg Arg Lys Tyr Arg Val Asp Arg Asp Val 180 185
190Gly Arg Glu Arg Gly Arg Asn Gly Arg Asp Ser Gly Asp Pro Gln Gly
195 200 205Asp Ala Gly Thr Arg Ala Glu Leu Trp 210 2155353DNAHomo
sapiens 5cccagcaccc aacccaggtt accatggcct ccctgttctc tggccgcatc
ctgatccgca 60acaatagcga ccaggacgag ctggatacgg aggctgaggt cagtcgcagg
ctggagaacc 120ggctggtgct gctgttcttt ggtgctgggg cttgtccaca
gtgccaggcc ttcgtgccca 180tcctcaagga cttcttcgtg cggctcacag
atgagttcta tgtactgcgg gcggctcagc 240tggccctggt gtacgtgtcc
caggactcca cggaggagca gcaggacctg ttcctcaagg 300acatgccaaa
gaaatggctt ttcctgccct ttgaggatga tctgaggagg tga 3536109PRTHomo
sapiens 6Met Ala Ser Leu Phe Ser Gly Arg Ile Leu Ile Arg Asn Asn
Ser Asp1 5 10 15Gln Asp Glu Leu Asp Thr Glu Ala Glu Val Ser Arg Arg
Leu Glu Asn 20 25 30Arg Leu Val Leu Leu Phe Phe Gly Ala Gly Ala Cys
Pro Gln Cys Gln 35 40 45Ala Phe Val Pro Ile Leu Lys Asp Phe Phe Val
Arg Leu Thr Asp Glu 50 55 60Phe Tyr Val Leu Arg Ala Ala Gln Leu Ala
Leu Val Tyr Val Ser Gln65 70 75 80Asp Ser Thr Glu Glu Gln Gln Asp
Leu Phe Leu Lys Asp Met Pro Lys 85 90 95Lys Trp Leu Phe Leu Pro Phe
Glu Asp Asp Leu Arg Arg 100 1057686DNAHomo sapiens 7ccggggacca
cacgccgcgc tgtccccagc acccaaccca ggttaccatg gcctccctgt 60tctctggccg
catcctgatc cgcaacaata gcgaccagga cgagctggat acggaggctg
120aggtcagtcg caggctggag aaccggctgg tgctgctgtt ctttggtgct
ggggcttgtc 180cacagtgcca ggccttcgtg cccatcctca aggacttctt
cgtgcggctc acagatgagt 240tctatgtact gcgggcggct cagctggccc
tggtgtacgt gtcccaggac tccacggagg 300agcagcagga cctgttcctc
aaggacatgc caaagaaatg gcttttcctg ccctttgagg 360atgatctgag
gagggacctc gggcgccagt tctcagtgga gcgcctgccg gcggtcgtgg
420tgctcaagcc ggacggggac gtgctcactc gcgacggcgc cgacgagatc
cagcgcctgg 480gcaccgcctg cttcgccaac tggcaggagg cggccgaggt
gctggaccgc aacttccagc 540tgccagagga cctggaggac caggagccac
ggagcctcac cgagtgcctg cgccgccaca 600agtaccgcgt ggaaaaggcg
gcgcgaggcg ggcgcgaccc cgggggaggg ggtggggagg 660agggcggggc
cggggggctg ttctga 6868212PRTHomo sapiens 8Met Ala Ser Leu Phe Ser
Gly Arg Ile Leu Ile Arg Asn Asn Ser Asp1 5 10 15Gln Asp Glu Leu Asp
Thr Glu Ala Glu Val Ser Arg Arg Leu Glu Asn 20 25 30Arg Leu Val Leu
Leu Phe Phe Gly Ala Gly Ala Cys Pro Gln Cys Gln 35 40 45Ala Phe Val
Pro Ile Leu Lys Asp Phe Phe Val Arg Leu Thr Asp Glu 50 55 60Phe Tyr
Val Leu Arg Ala Ala Gln Leu Ala Leu Val Tyr Val Ser Gln65 70 75
80Asp Ser Thr Glu Glu Gln Gln Asp Leu Phe Leu Lys Asp Met Pro Lys
85 90 95Lys Trp Leu Phe Leu Pro Phe Glu Asp Asp Leu Arg Arg Asp Leu
Gly 100 105 110Arg Gln Phe Ser Val Glu Arg Leu Pro Ala Val Val Val
Leu Lys Pro 115 120 125Asp Gly Asp Val Leu Thr Arg Asp Gly Ala Asp
Glu Ile Gln Arg Leu 130 135 140Gly Thr Ala Cys Phe Ala Asn Trp Gln
Glu Ala Ala Glu Val Leu Asp145 150 155 160Arg Asn Phe Gln Leu Pro
Glu Asp Leu Glu Asp Gln Glu Pro Arg Ser 165 170 175Leu Thr Glu Cys
Leu Arg Arg His Lys Tyr Arg Val Glu Lys Ala Ala 180 185 190Arg Gly
Gly Arg Asp Pro Gly Gly Gly Gly Gly Glu Glu Gly Gly Ala 195 200
205Gly Gly Leu Phe 2109600DNAMus musculus 9ataaaataga gggtgggaga
ggttgatggc gtggctctgc tttttggtgc ggggcaccca 60gctgtcatcg ctgctgtcgc
agcttctgga gtggccactg tgctctctcc tcccttcggc 120tcaaggtgag
ctgttccagc agaaggcggg gctgagaggc gcctagtgct gcgggaggct
180cagtgtcatc ttccagctaa caggtggccg tgcagcccag ggctcgtctc
tccactgtgt 240cctcttcacg ccgagctcgt ggcgatggtg gacgtgctgg
gcgggcggcg cctggtgacc 300cgggagggca cggtggtgga ggccgaggtg
gcgctgcaga acaaggtggt agctttgtac 360tttgcggcgg gccggtgctc
gcccagccgc gacttcacgc cgctgctctg cgacttctac 420acggagctgg
tgagcgaggc gcggcggccc gctcccttcg aggtggtttt cgtgtcggca
480gacggcagtg cggaggagat gttggacttc atgcgcgagc tgcacggctc
ctggctggca 540ttgcccttcc acgaccccta ccggcagtga gtggggaccc
aggggtcatg gggctggcgc 60010101PRTMus musculus 10Met Val Asp Val Leu
Gly Gly Arg Arg Leu Val Thr Arg Glu Gly Thr1 5 10 15Val Val Glu Ala
Glu Val Ala Leu Gln Asn Lys Val Val Ala Leu Tyr 20 25 30Phe Ala Ala
Gly Arg Cys Ser Pro Ser Arg Asp Phe Thr Pro Leu Leu 35 40 45Cys Asp
Phe Tyr Thr Glu Leu Val Ser Glu Ala Arg Arg Pro Ala Pro 50 55 60Phe
Glu Val Val Phe Val Ser Ala Asp Gly Ser Ala Glu Glu Met Leu65 70 75
80Asp Phe Met Arg Glu Leu His Gly Ser Trp Leu Ala Leu Pro Phe His
85 90 95Asp Pro Tyr Arg Gln 100111181DNAMus musculus 11aaggactctg
gtgggtagag agggtttgca aggcaggata aaatagaggg tgggagaggt 60tgatggcgtg
gctctgcttt ttggtgcggg gcacccagct gtcatcgctg ctgtcgcagc
120ttctggagtg gccactgtgc tctctcctcc cttcggctca aggtgagctg
ttccagcaga 180aggcggggct gagaggcgcc tagtgctgcg ggaggctcag
tgtcatcttc cagctaacag 240gtggccgtgc agcccagggc tcgtctctcc
actgtgtcct cttcacgccg agctcgtggc 300gatggtggac gtgctgggcg
ggcggcgcct ggtgacccgg gagggcacgg tggtggaggc 360cgaggtggcg
ctgcagaaca aggtggtagc tttgtacttt gcggcgggcc ggtgctcgcc
420cagccgcgac ttcacgccgc tgctctgcga cttctacacg gagctggtga
gcgaggcgcg 480gcggcccgct cccttcgagg tggttttcgt gtcggcagac
ggcagtgcgg aggagatgtt 540ggacttcatg cgcgagctgc acggctcctg
gctggcattg cccttccacg acccctaccg 600gcatgaactg aagaagaggt
acgaaatcac cgccatcccc aagctggtgg tcatcaagca 660gaacggagct
gtcatcacca acaaagggcg gaagcagatc cgagagcgcg ggctagcttg
720ctttcagaac tgggtggaag cagccgatgt tttccaaaac ttctcggggt
gaccagggca 780gttgctggaa gttcagggca actatcttca aaaagggctt
agctggttcc cttctctgct 840gaggaatgtc attgtagagt caccatgctg
tgacagagag cataaactgc tcaggaaaga 900actacgtctg ccccctgtgg
gtcctagagc tccgttgaat gtttatttct tacacctttc 960tccaccggtg
cctaggatcc aggacacatc agccacgagt taacagaact ctatgcaaga
1020tgctctttcc tacaggaaat ttctttgata aattgaccta tggaggtgat
acattttctg 1080atgacatttt tgtgatgctt tggtaaacgt atttattact
caggtttgta gactgtgtaa 1140tttaataaac caacactcac actttgaaaa
aaaaaaaaaa a 118112156PRTMus musculus 12Met Val Asp Val Leu Gly Gly
Arg Arg Leu Val Thr Arg Glu Gly Thr1 5 10 15Val Val Glu Ala Glu Val
Ala Leu Gln Asn Lys Val Val Ala Leu Tyr 20 25 30Phe Ala Ala Gly Arg
Cys Ser Pro Ser Arg Asp Phe Thr Pro Leu Leu 35 40 45Cys Asp Phe Tyr
Thr Glu Leu Val Ser Glu Ala Arg Arg Pro Ala Pro 50 55 60Phe Glu Val
Val Phe Val Ser Ala Asp Gly Ser Ala Glu Glu Met Leu65 70 75 80Asp
Phe Met Arg Glu Leu His Gly Ser Trp Leu Ala Leu Pro Phe His 85 90
95Asp Pro Tyr Arg His Glu Leu Lys Lys Arg Tyr Glu Ile Thr Ala Ile
100 105 110Pro Lys Leu Val Val Ile Lys Gln Asn Gly Ala Val Ile Thr
Asn Lys 115 120 125Gly Arg Lys Gln Ile Arg Glu Arg Gly Leu Ala Cys
Phe Gln Asn Trp 130 135 140Val Glu Ala Ala Asp Val Phe Gln Asn Phe
Ser Gly145 150 155131472DNAHomo sapiens 13gtgtgggcgg ggcgcagttg
ggggagggtg cagagacctg agggcttgag gttgcctggc 60tggccccgct cccagaggcg
ggtgccgcgc tgtcgcccag gtatctgggg tctctggtgt 120ctgagtgtct
cattgtcggc gcgaacacaa ttgctccagc cacaggcgag gcctggccaa
180ggtgtgggcg catctagggc aggtcttgag aggtccagcg cccggtggtg
cggacagagg 240cggggcaccg cggcgctcgc cgccgcctcc ccgcaggtga
tcatcctcct gcaggtgtcc 300tcgggtctca ggtggctgcg tgtctgcgcc
atggttgaca ttctgggcga gcggcacctg 360gtgacctgta agggcgcgac
ggtggaggcc gaggcggcgc tgcagaacaa ggtggtggca 420ctgtacttcg
cggcggcccg gtgcgcgccg agccgcgact tcacgccgct gctctgcgac
480ttctatacgg cgctggtggc cgaggcgcgg cggcccgcgc ccttcgaagt
ggtcttcgtg 540tcagccgacg gcagctccca ggagatgctg gacttcatgc
gcgagctgca tggcgcctgg 600ctggcgctgc ccttccacga cccctaccgg
caacggagtc tcgctctgtt gcccaggctg 660gagtgcagtg gcgtgatctt
agctcactgc aacctttgcc tcctgggttc aagtgattct 720ctagccttag
cctcctgagc atctgggact acagccattg ctgtgaatta cgtgagggaa
780agatattgaa gaggagttgg acactccgag agtgcagctg ttctcccccc
gcaccatccg 840tgtcctgcat tctgcgagtc tgtgctcatt aacaatgtgc
tgtgaccatg tgactcagca 900atcctgctgc tgggtatata cccgaaagaa
aggaaaagga agccagtata ttgaagaggt 960atctgcaccc ccatgtttat
tgcagcactg ttcacaacag ccaagatttg gaagcaacct 1020aagtgtccat
caacagatga atggataaag aaaacgtggt acatatacac aatggagtac
1080tcttcagcca ttaaaaaaat gagattctgt catttgcaat aatatagatg
gaaaaggagg 1140cccttatgtg aagtgaaata agccaggcac agaaagacaa
acatcacatg ttctcactta 1200tttgtgggat ctaatgatca aaacaattga
actcttggac atagagagta gaaggttggt 1260taccagaagc tggaaaggaa
agtggggttg ggaggaaggt gggaatggtt aataggtaca 1320aaaaaataca
aagaataaat aagacctaat atttgatagc acaacagtgt gactactgtc
1380aataatcatt taattgtaca tttaaaaata actataattg cattgtttgt
aacacaaaag 1440ataaatgctt gaggagaaaa aaaaaaaaaa aa 147214135PRTHomo
sapiens 14Met Val Asp Ile Leu Gly Glu Arg His Leu Val Thr Cys Lys
Gly Ala1 5 10 15Thr Val Glu Ala Glu Ala Ala Leu Gln Asn Lys Val Val
Ala Leu Tyr 20 25 30Phe Ala Ala Ala Arg Cys Ala Pro Ser Arg Asp Phe
Thr Pro Leu Leu 35 40 45Cys Asp Phe Tyr Thr Ala Leu Val Ala Glu Ala
Arg Arg Pro Ala Pro 50 55 60Phe Glu Val Val Phe Val Ser Ala Asp Gly
Ser Ser Gln Glu Met Leu65 70 75 80Asp Phe Met Arg Glu Leu His Gly
Ala Trp Leu Ala Leu Pro Phe His 85 90 95Asp Pro Tyr Arg Gln Arg Ser
Leu Ala Leu Leu Pro Arg Leu Glu Cys 100 105 110Ser Gly Val Ile Leu
Ala His Cys Asn Leu Cys Leu Leu Gly Ser Ser 115 120 125Asp Ser Leu
Ala Leu Ala Ser 130 13515654DNARattus sp. 15atggtgtctc tcttctctgg
ccgcatcttg atcaggaaca acagcgacca ggatgaagtg 60gagacagagg cagagctgag
ccgccggtta gagaatcgtc ttgtgctact gttcttcggt 120gctggggcct
gtccccagtg ccaggccttc gccccagtcc tcaaagactt cttcgtgcgg
180ctcactgatg agttctacgt gctacgggca gcacagctgg ccctggtcta
tgtgtcccag 240gaccctacag aggagcaaca ggacctgttc ctccgggaca
tgcctgaaaa gtggctcttc 300ctgccgttcc atgatgacct gaggagagac
ctcgggcgcc agttctccgt gcgtcaactg 360ccagcggtcg tggtactcaa
gccaggcggg gacgtgctga cgagcgacgc cacggacgaa 420atccagcgtc
tgggacccgc ctgcttcgcc aactggcagg aggcggcaga gctgttggac
480cgcagcttcc tgcagccgga ggacctggat gagcccgcgc ggcgcagcat
cacggaacct 540ctgcgtcgtc gcaagtaccg cgtagaccgg gatgctggcc
gggggcgggg ccgaaacgag 600tgcgactcta gaaatcccca ggggggcgcg
ggtgcaagga cggagctctg gtga 65416217PRTRattus sp. 16Met Val Ser Leu
Phe Ser Gly Arg Ile Leu Ile Arg Asn Asn Ser Asp1 5 10 15Gln Asp Glu
Val Glu Thr Glu Ala Glu Leu Ser Arg Arg Leu Glu Asn 20 25 30Arg Leu
Val Leu Leu Phe Phe Gly Ala Gly Ala Cys Pro Gln Cys Gln 35 40 45Ala
Phe Ala Pro Val Leu Lys Asp Phe Phe Val Arg Leu Thr Asp Glu 50 55
60Phe Tyr Val Leu Arg Ala Ala Gln Leu Ala Leu Val Tyr Val Ser Gln65
70 75 80Asp Pro Thr Glu Glu Gln Gln Asp Leu Phe Leu Arg Asp Met Pro
Glu 85 90 95Lys Trp Leu Phe Leu Pro Phe His Asp Asp Leu Arg Arg Asp
Leu Gly 100 105 110Arg Gln Phe Ser Val Arg Gln Leu Pro Ala Val Val
Val Leu Lys Pro 115 120 125Gly Gly Asp Val Leu Thr Ser Asp Ala Thr
Asp Glu Ile Gln Arg Leu 130 135 140Gly Pro Ala Cys Phe Ala Asn Trp
Gln Glu Ala Ala Glu Leu Leu Asp145 150 155 160Arg Ser Phe Leu Gln
Pro Glu Asp Leu Asp Glu Pro Ala Arg Arg Ser 165 170 175Ile Thr Glu
Pro Leu Arg Arg Arg Lys Tyr Arg Val Asp Arg Asp Ala 180 185 190Gly
Arg Gly Arg Gly Arg Asn Glu Cys Asp Ser Arg Asn Pro Gln Gly 195 200
205Gly Ala Gly Ala Arg Thr Glu Leu Trp 210 21517471DNARattus sp.
17atggtggacg tgctgggcgg gcggcgcctg atgacccgcg agggcacgct ggtggaggcc
60gaggcggcgc tgcagaacaa ggtggtagct ctgtactttg ccgcaggccg gtgcgcgccc
120agccgcgact tcacgccgct gctctgcgac ttctacacgg agctggtgag
cgaggcgcgg 180cggcccgcac ccttcgaggt ggttttcgtg tcggcggacc
gcagcgccga agagatgctg 240gacttcatgc gcgagctgca cggctcctgg
ctggcattgc ccttccacga cccctaccgg 300catgagctga agaagaggta
tgacatcacc gtcatcccca aggtcgtggt catcaagcag 360aatggagccg
tcatcaccaa caaagggcgg aaacagatcc gggagcgcgg actagcttgc
420tttcagaact gggtggaagc agctgatgtt ttccaaaact tctcagggtg a
47118156PRTRattus 18Met Val Asp Val Leu Gly Gly Arg Arg Leu Met Thr
Arg Glu Gly Thr1 5 10 15Leu Val Glu Ala Glu Ala Ala Leu Gln Asn Lys
Val Val Ala Leu Tyr 20 25 30Phe Ala Ala Gly Arg Cys Ala Pro Ser Arg
Asp Phe Thr Pro Leu Leu 35 40 45Cys Asp Phe Tyr Thr Glu Leu Val Ser
Glu Ala Arg Arg Pro Ala Pro 50 55 60Phe Glu Val Val Phe Val Ser
Ala
Asp Arg Ser Ala Glu Glu Met Leu65 70 75 80Asp Phe Met Arg Glu Leu
His Gly Ser Trp Leu Ala Leu Pro Phe His 85 90 95Asp Pro Tyr Arg His
Glu Leu Lys Lys Arg Tyr Asp Ile Thr Val Ile 100 105 110Pro Lys Val
Val Val Ile Lys Gln Asn Gly Ala Val Ile Thr Asn Lys 115 120 125Gly
Arg Lys Gln Ile Arg Glu Arg Gly Leu Ala Cys Phe Gln Asn Trp 130 135
140Val Glu Ala Ala Asp Val Phe Gln Asn Phe Ser Gly145 150
1551950DNAartificialPrimer 19tgttaccaat ctgaagtggg agcggccgac
aatttttttt tttttttttt 502014DNAartificialPrimer 20aattcggcac gagg
142110DNAartificialPrimer 21cctcgtgccg 102222DNAartificialPrimer
22gtaatacgac tcactatagg gc 222324DNAartificialPrimer 23tctatgtgtc
ccaggaccct acag 242426DNAartificialPrimer 24tttatgcaca agtagtacca
ggacag 262522DNAartificialPrimer 25gccagcgttt tctgcctttt ac
222622DNAartificialPrimer 26aagccctgcc tgctctaaca tc 22
* * * * *
References