U.S. patent application number 13/152077 was filed with the patent office on 2011-11-24 for mg53 compositions and methods of use.
This patent application is currently assigned to UNIVERSITY OF MEDICINE AND DENTISTRY OF NEW JERSEY. Invention is credited to Chuanxi Cai, Jianjie Ma, Noah Weisleder.
Application Number | 20110287015 13/152077 |
Document ID | / |
Family ID | 39344823 |
Filed Date | 2011-11-24 |
United States Patent
Application |
20110287015 |
Kind Code |
A1 |
Ma; Jianjie ; et
al. |
November 24, 2011 |
MG53 COMPOSITIONS AND METHODS OF USE
Abstract
Disclosed herein are nucleic acid sequences that encode novel
polypeptides. Also disclosed are polypeptides encoded by these
nucleic acid sequences, and antibodies, which
immunospecifically-bind to the polypeptide, as well as derivatives,
variants, mutants, or fragments of the aforementioned polypeptide,
polynucleotide, or antibody. The invention further discloses
therapeutic, diagnostic and research methods for diagnosis,
treatment, and prevention of disorders involving any one of these
novel human nucleic acids and proteins.
Inventors: |
Ma; Jianjie; (Belle Mead,
NJ) ; Weisleder; Noah; (Elizabeth, NJ) ; Cai;
Chuanxi; (Highland Park, NJ) |
Assignee: |
UNIVERSITY OF MEDICINE AND
DENTISTRY OF NEW JERSEY
Somerset
NJ
|
Family ID: |
39344823 |
Appl. No.: |
13/152077 |
Filed: |
June 2, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12307303 |
Jan 2, 2009 |
7981866 |
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PCT/US2007/015815 |
Jul 11, 2007 |
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13152077 |
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60830013 |
Jul 11, 2006 |
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60876871 |
Dec 22, 2006 |
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Current U.S.
Class: |
424/139.1 ;
435/252.3; 435/252.33; 435/254.2; 435/254.21; 435/331; 435/375;
435/419; 435/7.1; 435/7.92; 530/387.3; 530/387.9; 536/23.53 |
Current CPC
Class: |
A01K 67/0276 20130101;
A61P 21/00 20180101; A61P 13/08 20180101; C12N 15/8509 20130101;
A61P 9/04 20180101; A61P 25/18 20180101; A61P 37/06 20180101; A01K
2227/105 20130101; A61P 33/00 20180101; A61P 37/02 20180101; A61P
17/06 20180101; A61P 17/14 20180101; A61P 1/14 20180101; A61P 37/08
20180101; C40B 30/06 20130101; A61P 3/04 20180101; A61P 13/02
20180101; A61P 1/02 20180101; A61P 29/00 20180101; A61P 17/02
20180101; C07K 2319/10 20130101; A61P 15/00 20180101; A61P 3/14
20180101; A61P 9/12 20180101; A01K 2267/0375 20130101; A61P 25/04
20180101; C07K 16/18 20130101; A01K 2217/075 20130101; A61P 9/00
20180101; C07K 14/4702 20130101; A61P 25/22 20180101; A61P 39/06
20180101; A61P 41/00 20180101; C40B 40/02 20130101; A61P 25/16
20180101; A61P 31/04 20180101; A61P 35/00 20180101; A61P 9/02
20180101; A61P 11/02 20180101; A61P 25/00 20180101; A61P 25/14
20180101; A61P 17/00 20180101; A61P 43/00 20180101; A61P 1/04
20180101; A61P 11/06 20180101; A61P 25/24 20180101; A61P 25/28
20180101; A61P 31/18 20180101; A61P 37/04 20180101; A61P 13/12
20180101; A61P 3/10 20180101; A61P 17/10 20180101; A61P 9/10
20180101; A61P 15/08 20180101; A61P 19/10 20180101; A61P 25/08
20180101; A61P 7/02 20180101; A61P 21/04 20180101; A61P 31/12
20180101; A61P 17/16 20180101; A61P 7/04 20180101 |
Class at
Publication: |
424/139.1 ;
530/387.9; 530/387.3; 435/375; 435/7.1; 536/23.53; 435/7.92;
435/252.3; 435/419; 435/331; 435/252.33; 435/254.21; 435/254.2 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 5/071 20100101 C12N005/071; G01N 33/566 20060101
G01N033/566; A61P 43/00 20060101 A61P043/00; C12N 1/21 20060101
C12N001/21; C12N 5/10 20060101 C12N005/10; C12N 1/19 20060101
C12N001/19; C07K 16/18 20060101 C07K016/18; C07H 21/00 20060101
C07H021/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] The U.S. Government has certain rights in this invention
pursuant to the following grants: RO1-CA095739; R.beta.1-AG015556;
RO1-HL069000 awarded to Dr. Jianjie Ma by the United States
National Institutes of Health (NIH).
Claims
1. An isolated anti-MG53 antibody comprising at least two
complementarity determining regions (CDRs), wherein the antibody
specifically binds to at least one amino acid residue selected from
residues 1-477 of the amino acid sequence of at least one of SEQ ID
NO: 1, 3, 5 or 7.
2. The antibody of claim 1, wherein the antibody specifically binds
to at least one amino acid residue selected from residues 1-144 of
the amino acid sequence of at least one of SEQ ID NO: 1, 3, 5 or
7.
3. The antibody of claim 1, wherein the antibody specifically binds
to at least one amino acid residue selected from residues 2-62 of
the amino acid sequence of at least one of SEQ ID NO: 1, 3, 5 or
7.
4. The antibody of claim 1, wherein the antibody specifically binds
to at least one amino acid residue selected from residues 2-268 of
the amino acid sequence of at least one of SEQ ID NO: 1, 3, 5 or
7.
5. The antibody of claim 1, wherein the antibody specifically binds
to at least one amino acid residue selected from residues 246-477
of the amino acid sequence of at least one of SEQ ID NO: 1, 3, 5 or
7.
6. The antibody of claim 1, wherein the anti-MG53 antibody is a
member selected from the group consisting of a polyclonal antibody,
a monoclonal antibody, and a bispecific antibody.
7. The antibody of claim 6, wherein the antibody is monoclonal.
8. The antibody of claim 7, wherein the monoclonal antibody is at
least one of chimeric, humanized, or a fully human antibody.
9. The antibody of claim 8, wherein the antibody is at least one of
a single chain, a single domain, or a antibody fragment.
10. The antibody of claim 9, wherein the fragment is selected from
the group consisting of an Fv fragment, an Fab fragment, an Fab'
fragment, and an F(ab').sub.2 fragment.
11. The antibody of claim 6, wherein the bispecific antibody
comprises at least one variable region capable of specifically
binding MG53.
12. The antibody of claim 9, wherein the single chain, single
domain or antibody fragment comprises at least one heavy (H) chain
variable region (VH) CDR.
13. The antibody of claim 9, wherein the single chain, single
domain or antibody fragment comprises at least one light (L) chain
variable region (VL) CDR.
14. The antibody of claim 13, wherein the light chain is a kappa
chain or a lambda chain.
15. The antibody of claim 1, wherein the antibody is a type
selected from the group consisting of IgG, IgM, IgA, IgE and
IgD.
16. A composition comprising an effective amount of the antibody of
claim 1 and a pharmaceutically acceptable carrier or excipient.
17. A method of treating or diagnosing an MG53-related condition
comprising contacting or administering the composition of claim 16
to a cell, tissue, organ or individual, wherein the composition is
effective for treating or diagnosing the MG53-related
condition.
18. The method of claim 17 wherein an amount of antibody
administered to an the individual is from about 0.1 mg/kg to about
2000 mg/kg.
19. The method of claim 17, wherein the antibody is administered by
at least one route selected from intravenously, intraperitoneally,
intramuscularly, and subcutaneously.
20. An isolated and/or recombinant anti-MG53 antibody wherein the
antibody specifically binds to an epitope within a polypeptide
having at least 90% sequence identity to the amino acid sequence of
SEQ ID NO: 1.
21. The antibody of claim 20, wherein the antibody specifically
binds to an epitope within residues 1-144 of the amino acid
sequence of SEQ ID NO: 1.
22. An isolated nucleic acid encoding the antibody of claim 1 or a
portion thereof.
23. A host cell comprising the nucleic acid of claim 22.
24. The antibody of claim 1, wherein the antibody comprises at
least one heavy chain or light chain CDR.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 12/307,303 filed Jan. 2, 2009, which claims the benefit
under 35 U.S.C. .sctn.119 to PCT/US2007/015815 filed Jul. 11, 2007,
which claims the benefit under 35 U.S.C. .sctn.119(e) of U.S.
Provisional Applications Nos. 60/830,013 filed Jul. 11, 2006; and
60/876,871 filed Dec. 22, 2006, all of which are hereby
incorporated by reference in their entirety for all purposes.
INCORPORATION BY REFERENCE
[0003] In compliance with 37 C.F.R. .sctn.1.52(e)(5), the sequence
information contained on compact disc, file name:
Ma.sub.--2007utility SEQ List_ST25.txt; size 61 KB; created on:
Jun. 29, 2007; using PatentIn-3.4, and Checker 4.4.0 is hereby
incorporated by reference in its entirety. The Sequence Listing
information recorded in computer readable form (CRF) is identical
to the written Sequence Listing provided herewith. The data in the
paper copy of the Sequence Listing, and Computer Readable Form of
the Sequence Listing submitted herewith contain no new matter, and
are fully supported by the priority applications, U.S. Provisional
Patent Applications Nos. 60/830,013; and 60/876,871.
FIELD OF THE INVENTION
[0004] This invention relates to polypeptides, nucleic acids
encoding the same, antibodies that immunospecifically-bind to the
polypeptides and associated methods of use.
BACKGROUND
[0005] In response to external damage and internal degeneration,
the cells of the body must repair the membrane surrounding the each
individual cell in order to maintain their function and the health
of the organism. Defects in the ability of the cell to repair
external membranes have been linked to many diseases and
pathological conditions, for example, neurodegenerative diseases
(e.g., Parkinson's Disease), heart attacks, heart failure, muscular
dystrophy, bed sores, diabetic ulcers, oxidative damage, and tissue
damage such as sinusitis that occurs as side effect from the
administration of chemotherapeutic agents. Also, the muscle
weakness and atrophy associated with various diseases, as well as
the normal aging process, has been linked to altered membrane
repair. In order for these cells to repair their membranes in
response to acute damage they make use of small packets of membrane
that are inside of the cell, referred to as vesicles. These
vesicles are normally found within the cell, but upon damage to the
cell membrane, these vesicles move to the damage site and form a
patch to maintain the cell integrity. Without this essential
function, the cell can die and the cumulative effect of this
cellular injury can eventually result in dysfunction of the tissue
or organ.
[0006] Many companies are interested in approaches to improve the
regenerative capacity of various tissues. For example, the wound
repair market, alone, is expected to exceed $11 billion by 2009.
Therefore, there exists an ongoing need for the development of
pharmaceutical modulators of the cell membrane repair process for
the treatment of conditions related to acute and chronic cellular
and tissue damage.
SUMMARY
[0007] The present invention relates to the surprising and
unexpected discovery of proteins involved in the repair of cell
membrane damage. The invention generally relates to nucleic acids,
and includes polypeptides encoded from nucleic acids of the
invention. More specifically, the invention relates to
compositions, for example, nucleic acids, which are useful for
inhibiting transcription or translation of target nucleic acids;
nucleic acids encoding cytoplasmic, nuclear, membrane bound, and
secreted polypeptides; as well as vectors, host cells, antibodies,
recombinant proteins, pseudopeptides, fusion proteins, chemical
compounds, and methods for producing the same.
[0008] In certain aspects, the present invention also relates to
compositions useful as therapeutics for treating and prevention of
diseases and disorders. Therapeutic compositions of the invention
comprise nucleic acids, including an interfering nucleic acids, and
nucleic acids encoding polypeptides corresponding to the protein of
SEQ ID NO. 1 (herein, "MG53"), MG53 polypeptides, homologs and
portions thereof, MG53 psuedopeptides, MG53 peptide analogs and
MG53 peptidomimetics; as well as compounds that can modulate the
activity of MG53 or intermolecular interactions involving MG53, and
for example, caveolin-3 (SEQ ID NO. 8). As described herein, MG53
mediates the repair of damage to cellular membranes, and therefore,
the targeting and modulating MG53 gene expression, polypeptide
synthesis, activity or protein-protein interactions represent a
novel therapeutic intervention for tissue repair.
[0009] In certain additional aspects the invention relates to
compositions and methods related to the treatment of tissue damage.
In certain exemplary embodiments, the invention encompasses, for
example, the administration of an effective amount of a therapeutic
composition of the invention for the promotion of wound healing;
for ameliorating surgical trauma, for treatment and/or prevention
of age-related deficiencies in tissue repair that occur as a
natural side-effect of the aging process; for treatment and/or
prevention of injury to any type of muscle tissue, such as those
occurring in subjects suffering from cardiovascular diseases and/or
sports-related injuries; as well as the repair and regeneration of
body tissues through cosmetic or personal care use.
[0010] In addition, the invention relates to nucleic acids,
including interfering nucleic acids, and polypeptides encoding MG53
interacting proteins, for example, caveolin-3 (SEQ ID NO. 8)
polypeptides and homologs thereof; psuedopeptides and
peptidomimetics; as well as compounds that can modulate the
activity of caveolin-3 or its intermolecular interactions with
MG53. Therefore, in additional aspects, the present invention
encompasses methods for the targeting of caveolin-3 gene
expression, activity, and/or intermolecular interactions for the
treatment and/or prevention of a disease or disorder in a subject,
for example, for the promotion of tissue repair as described
above.
[0011] The preceeding general areas of utility are given by way of
example only and are not intended to be limiting on the scope of
the present disclosure and appended claims. Additional objects and
advantages of the present invention will be appreciated by one of
ordinary skill in the art in light of the instant claims,
description, and examples. These additional objects and advantages
are expressly included within the scope of the present
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1: MG53 is a muscle specific member of the TRIM protein
family. An alignment of the protein sequence of MG53 from various
organisms (See SEQ ID NOs.: 1, 3, 5, 9-16) reveals this protein to
be a member of the TRIM family. Functional domains are boxed in
grey while arrows indicate the domain continues onto another line
of the sequence.
[0013] FIG. 2: Illustrates an examplary domain comparison of some
homologous proteins that contain one or more of the conserved
tripartite motifs which are present in MG53. MG53 is unique in it's
ability to translocate to an injury site at the cell membrane
following multiple forms of insult and mediate repair of the
damaged membrane--a function which is not exhibited by the other
TRIM family proteins listed.
[0014] FIG. 3: MG53 contains unique TRIM and SPRY motifs and is
predominantly expressed in muscle cells. A. Diagram of motif
structure of MG53. From the results of cDNA cloning and homology
searches, several motif sequences are detected in MG53 as shown.
The sequences of rabbit and mouse MG53 cDNAs have been deposited in
the databases under accession numbers AB231473 and AB231474,
respectively. B. Western blot analysis shows the specific
expression of MG53 in skeletal and cardiac muscles. Lysate (20
.mu.g total protein per lane) from mouse tissues (lung, kidney,
skeletal muscle, liver, heart, brain) were analyzed using
anti-mouse MG53 polyclonal antibody. C. Immunofluorescence staining
of longitudinal transverse sections from mouse skeletal muscle
cells. Scale bar is 125 .mu.m.
[0015] FIG. 4. Induction of filapodia-like structure with
overexpression of MG53 in both muscle and non-muscle cells A.
Western blot analysis shows the overexpression level of MG53 in
C2C12 myoblasts (left panel) and CHO (middle panel) cells, and also
GFP-MG53 and MG53-GFP (right panel) in C2C12 myoblasts (20 .mu.g
total protein per lane). B. Typical confocal images of CHO (upper
panel) and C2C12 myoblasts (lower panel) transfected with GFP (left
panel), or GFP+MG53 (right panel), revealing filapodia-like
structures after overexpression of MG53. Scale bar is 5 .mu.m. C.
Confocal images of GFP-MG53 (left panel) and MG53-GFP (right panel)
expressed in CHO cells (upper panel) and C2C12 (lower panel)
myoblasts, revealing membrane targeting and intracellular vesicular
distribution of MG53, as well as the appearance of filapodia-like
structure. Scale bar is 5 .mu.m. D. Magnified confocal images
illustrating the intracellular vesicles, budding vesicles (left
panel) on the plasma membrane and extracellular vesicles (right
panel). Scale bar is 1 .mu.m
[0016] FIG. 5. MG53 contributes to skeletal muscle myogenesis by
regulating myoblast differentiation. A. Western blot analysis shows
the shRNA mediated down regulation of MG53 in CHO cells. Lysates
were prepared from CHO cells transfected with a MG53 expression
vector and either shRNA or scrambled shRNA plasmids targeting MG53.
Immunoblotting was performed with polyclonal anti-mouse antibody
for MG53 (upper panel) or monoclonal antibody for .cndot.-actin
(lower panel). B. Representative fluorescent microscope images of
C2C12 cells at different days of differentiation (Day 0, upper
panel; Day 5, middle panel; Day 10, lower panel.) to illustrate the
absence of myotube formation in cells transfected with shRNA
against MG53 (right panel) compared to the scrambled shRNA as
control (left panel). Scale bar is 50 .mu.m. C. Statistical
analysis of the down-regulation of MG53 inhibiting myotube
formation at 5 days or 10 days (*p<0.01 and **p<0.001 by t
test) compared to the control. The ratio of green myotubes to all
green cells was defined as the percentage of myotubes. Data are
represented as mean with SEM.
[0017] FIG. 6. Functional interaction between MG53 and caveolin-3
regulates dynamic membrane budding process in skeletal muscle. A.
Western blot analysis of the expression level of MG53 (upper
panel), caveolin-3 (middle panel) and caveolin-1 (lower panel)
during C2C12 cell differentiation at the indicated time following
induction of differentiation (day 0, 2, 5, 8, 10). B. Whole cell
lysate from mouse gastrocnemius skeletal muscle was subjected to
co-IP with anti-MG53 (rabbit polyclonal antibody), anti-caveolin-3
(mouse monoclonal antibody), normal rabbit IgG as a negative
control and cell lysate as a positive control. C. Confocal images
to illustrate the disappearance of filapodia-like structures during
the process of C2C12 myotube formation (right panel) compared to
myoblasts (left panel). Notice that intracellular vesicles positive
for GFP-MG53 are still present in transfected C2C12 myotubes. D.
Overexpression of caveolin-3 in C2C12 myoblast cells prevents
MG53-induced filapodia-like structures from forming. CHO cells
(upper panel) or C2C12 myoblast cells (lower panel) were
co-transfected with pcDNA-Cav-3 and GFP-MG53 (10:1) (right panel),
or co-transfected with pcDNA vector and GFP-MG53 (10:1) as control
(left panel). Confocal images were taken at 48 hours after
transfection. Scale bar is 10 .mu.m. E and F. Statistical analysis
for C and D. The ratio of cells displaying filapodia-like
structures to all green cells was defined as the filapodia-like
structure percentage. Data are represented as mean with SEM.
(*p<0.01 by t test).
[0018] FIG. 7. shRNA-mediated suppression of caveolin-3 expression
affects the myotube formation. A. The down-regulation level of
caveolin-3 was analyzed by Western blot after transfection with
shRNA plasmid for caveolin-3 in C2C12 myotubes (6 days after
differentiation). Cells transfected with the scrambled shRNA
plasmid acted as a control. B. Down-regulation of caveolin-3 (right
panel) by shRNA inhibits myotube formation compared to the control
shRNA (left panel). Red fluorescence indicates the transfected
cells. Fluorescence microscopy images were taken at 6 days after
differentiation induction. Scale bar is 20 .mu.m C. Statistical
analysis shows that down-regulation of caveolin-3 significantly
inhibits myotube formation at 6 days (*p<0.001 by t test)
compared to the control. The ratio of red fluorescent myotubes to
all red fluorescent cells served as the percentage of myotubes.
Data are represented as mean with SEM. D. Confocal images of C2C12
myoblasts with co-expression of both GFP-MG53 and shRNA for
caveolin-3 (right panel) reveal no affect on the filapodia-like
structures induced by GFP-MG53 or on the distribution of GFP-MG53
compared to the control shRNA (left panel). Scale bar is 5
.mu.m.
[0019] FIG. 8. Treatment of cells with methyl-.-cyclodextrin leads
to increased exocytosis and solubilization of GFP-MG53 in C2C12
myoblasts. A. Representative confocal images that illustrate the
spontaneous vesicles fusion and budding off from the membrane at
the indicated time points (0 minute, left panel; 15 minutes, right
panel). Scale bar is 5 .mu.m. B. Confocal images to illustrate the
GFP-MG53 induced vesicles budding off from the membrane quickly
after treatment with 10 mM M-.CD at the indicated time points (0
second, left panel; 16 seconds, middle panel; 32 seconds, right
panel). C. Confocal images to show the solubilization of GFP-MG53
after prolonged treatment with 10 mM M-.CD at room temperature for
1 hour (right panel) compared to the same cell before treatment
(left panel). Scale bar is 5 .mu.m.
[0020] FIG. 9. MG53 knockout mice are susceptible to cardiac
damage. Paraffin-embedded sections of myocardium from unexercised
wild type mice show normal morphology (left) and no Evans blue
staining (right). In contrast, and mg53-/- mice display a Evans
blue infiltration into myocytes, indicating that there are
significant defects in membrane integrity in the mg53-/- heart.
[0021] FIG. 10. Progressive pathology is seen in mg53-/- skeletal
muscle due to increased damage of cell membranes. A. Haematoxylin
and Eosin (H/E) staining illustrates increased number of central
nuclei (arrows) in aging mg53-/- muscle (10m) versus young (3m)
wild type (wt) or mg53-/- mice. B. The diameter of muscle fibers in
aged (8-10 month) mg53-/- mice (blue, n=541) decreased compared to
aged (8-10 month) wild type controls (black, n=562) while there is
no difference in young (3-5 months) wt (n=765) versus mg53-/-
(n=673) muscle. Percentage of muscle fibers that display central
nuclei in mg53-/- skeletal muscle increases with age when compared
to wt. Data is mean.+-.s.e.m., *p<0.05 by ANOVA. C. Trace
recordings of contractile performance of intact soleus muscle
obtained from mice subjected to 30 min down-hill exercise running
was assessed using an in vitro voltage stimulation protocol,
following described procedures. Black trace represents wt muscle,
blue trace corresponds to mg53-/- muscle. D. Prior to fatigue
stimulation (Pre, open bars), the maximal tetanic force, normalized
in g/mg total protein, was significantly lower in aging mg53-/-
muscle (blue) versus wt (black) (n=4). At 6 min after fatigue
stimulation (After, closed bars), the wt muscle recovered
significantly more than mg53-/- muscle. *p<0.05 by ANOVA. E.
Extensive Evans blue staining reveals serve damage in mg53-/-
skeletal muscle subjected to down-hill running when compared to
minimal staining in wt muscles. F. Chart of the quantity of Evans
blue dye extracted by formamide from aging mg53-/- (blue) and wt
(black) skeletal muscle following exercise. The data represents
mean value of Evans blue (ng) per g of muscle.+-.s.e.m. n=8-12,
*p<0.005 by Student's t-test.
[0022] FIG. 11. Ablation of MG53 leads to defective muscle membrane
repair function. (a) Immunostaining of MG53 in isolated wt FDB
fibers to illustrate their co-localization at the injury site.
These are representative images from >20 different muscle fibers
which display damage during isolation. (b) Exclusion of
membrane-impermeable FM-143 fluorescent dye in a FDB muscle fibers
isolated from the wt mice following laser-induced damage of the
sarcolemmal membrane. (c) Entry of FM-143 fluorescent dye into a
FDB muscle fiber isolated from the mg53-/- mice following
laser-induced damage. Times after laser injury were indicated. (d)
Time-dependent accumulation of FM-143 inside the FDB muscle fiber
induced by a laser damage of the sarcolemmal membrane. Data are
means.+-.s.e.m. for n=30 fibers obtained from wt mice and n=18
fibers from mg53-/- mice.
[0023] FIG. 12. MG53 containing vesicles form a patch in the plasma
membrane following physical insult. A. Damage of a C2C12 myoblast
membrane using a micropipette leads to rapid accumulation of
GFP-MG53 at the injury site (arrow). Images were representative of
n=40 separate cells. B. Recovery of a mature C2C12 myotube in
response to a severe damage, e.g. separation of the cell membrane,
is associated with recruitment of GFP-MG53 toward the healing site
(n=28).
[0024] FIG. 13. Role of TRIM and SPRY domains in targeting of MG53
to the cell surface membrane of muscle cells. A. Scheme of the MG53
deletion fusion protein constructs with GFP fused to the N-terminus
or C-terminus. With reference to SEQ ID NO. 1, "TRIM" represents
a.a. 1-287 and "SPRY" represents a.a. 288-477 and includes both the
PRY and SPRY motifs. B. Representative confocal images showing
intracellular localization of each deletion construct in C2C12
cells. Scale bar is 5 .mu.m. C. MG53 interacts with caveolin-3
through the TRIM motif. Cell lysate from CHO cells co-transfected
with GFP-MG53 or GFP-TRIM and pcDNA-Cav-3 was subjected to IP with
anti-caveolin-3 (mouse monoclonal antibody). (Lane 1, mixed cell
lysate as positive control; Lane 2, normal mouse IgG as negative
control; lane 3, lysate from cells overexpressing GFP-MG53; Lane 4,
lysate from cells overexpressing GFP-TRIM).
[0025] FIG. 14. Role of TRIM and SPRY domains in targeting of MG53
to the cell surface membrane in non-muscle CHO cells.
Representative confocal images showing that GFP-MG53 exhibits
intracellular vesicle, membrane targeting and budding, however
MG53-GFP is mainly soluble in nature (upper panel); SPRY-GFP and
GFP-SPRY are cytosolic (middle panel); TRIM-GFP and GFP-TRIM are
mainly intracellular vesicle, and do not target to plasma membrane
(lower panel). "TRIM" represents a.a. 1-287 and "SPRY" represents
a.a. 288-477 and includes both the PRY and SPRY motifs. Scale bar
is 5 .mu.m.
[0026] FIG. 15. Purification of recombinant TAT-MG53 and mutant
constructs. (a) Representation of the TAT-MG53 recombinant protein
construct and associated deletion constructs. (b) Coomassie blue
staining of a denaturing gel showing the purification steps for
TAT-MG53. Gel lanes were loaded with a molecular weight marker (M),
E. coli supernatant (Sup), immunoaffinity column flow through (FT),
wash flow through (W1,2) and elution fractions (E1-5). (c)
Coomassie stained denaturing gel of recombinant mutant TAT-MG53
proteins isolated from E. coli.
[0027] FIG. 16: Stable HEK293 (Human Embryonic Kidney) cell lines
were generated that express RFP-MG53. (a) Cell lines that stably
express an RFP (red fluorescent protein) control protein that shows
a cytosolic expression pattern. (b) Injury of HEK293 cells
expressing RFP only with a microelectrode results in no
translocation of RFP to the injury site (arrow). Some bleaching of
RFP fluorescence occurs from excessive entry of extracellular
buffer (*). (c) HEK293 cells that are stably expressing RFP-MG53
show localization to intracellular vesicles. (d) Injury of HEK293
cells expressing RFP-MG53 results in massive translocation of MG53
to the injury site (arrow) in less than 90 seconds. Limited buffer
entry into the cell by rapid repair of the plasma membrane prevents
bleaching of the RFP-MG53 fluorescence.
[0028] FIG. 17: Recombinant human TAT-MG53 (See HIV-1 TAT protein,
SEQ ID NO. 17) can penetrate cells of different origins. HL-1
cardiomyocytes and 3T3 fibroblasts were incubated with 4 or 8 pg/mL
recombinant human TAT-MG53 for 15 minutes at 37.degree. C. Cells
were washed three times in a buffered salt solution and then lysed
for western blot analysis. Western blot shows that control cells
(control) do not contain endogenous MG53, however those incubated
with TAT-MG53 contain ample intracellular TAT-MG53. Note that
TAT-MG53 is slightly larger than MG53 visualized from skeletal
muscle extract (muscle) due to the addition of the TAT cell
penetrating peptide to the protein.
[0029] FIG. 18: Recombinant expression of MG53. (a) Coomassie blue
stained gel of recombinant human MG53 protein (arrow) fractions
isolated from Sf9 cells with a Ni-NTA column. Input=cell extract,
FT=flow through, M=marker, E=elution number. (b) Coomassie blue
stained gel of recombinant human TAT-MG53 (arrow) isolated from Sf9
cells. (c) Coomassie blue stained gel of recombinant mouse TAT-MG53
(arrow) isolated from E. coli.
[0030] FIG. 19: MG53 interacts with cellular membranes through an
association with phosphatidylserine to mediate vesicular
trafficking. (A) PIP.sub.2-Strip lipid dot blot analysis reveals
recombinant MG53 (1 .mu.g/ml) specifically binds phosphatidylserine
(PS) and not other membrane lipids, including sphingosine-1-P,
phosphatidic acid, phosphotidylcholine, phosphatidylethanolamine
and various phosphainositol metabolites. (B) Annexin-V-GFP (a
molecule with well defined ability to bind PS) transfected into
C2C12 myoblasts (left) displays minimal translocation following
cell wounding with a microelectrode (arrow), while co-expression of
Annexin-V-GFP with RFP-MG53 (right) results in accelerated
accumulation of Annexin-V-GFP. Data represent mean.+-.s.e.m.
(n=10). *p<0.01 by Student's t-test.
[0031] FIG. 20: Illustration demonstrating the inventors' current
hypothesis on the mechanism of membrane repair mediated by MG53.
While not being limited to any particular theory, experimental
evidence indicates that MG53 is likely localized to the inner
surface of the plasma membrane due to its association with
phosphatidylserine-containing vesicles. Under normal conditions
MG53 is likely monomeric and sequestered proximal to the membrane
surface due to associations with caveolin-3. Following damage to
the cellular membrane MG53, which is normally in its reduced form,
is exposed to a localized oxidative environment which triggers the
formation of disulfide cross-bridges and intermolecular MG53
oligomerization. The oligomerization of MG53 brings
phosphatidylserine-containing vesicles together at the damage site.
The lipid vesicles are then able to patch the damaged
membrane--likely mediated by simple hydrophobic forces.
DETAILED DESCRIPTION
[0032] The present invention provides novel nucleotides and
polypeptides encoded thereby. Included in the invention are the
novel nucleic acid sequences and their encoded polypeptides. The
sequences are collectively referred to herein as "MG53 nucleic
acids" or "MG53 polynucleotides" and the corresponding encoded
polypeptides are referred to as "MG53 polypeptides" or "MG53
proteins." Unless indicated otherwise, "MG53" is meant to refer to
any of the novel sequences disclosed herein.
[0033] Dynamic membrane repair is essential not only for long-term
maintenance of cellular integrity but also for recovery from acute
cell injury. Membrane repair defects have been linked to numerous
disease states including muscular dystrophy, heart failure and
neurodegeneration. Repair of the cell membrane requires
intracellular vesicular trafficking that is associated with
accumulation of vesicles at the plasma membrane.
[0034] The present invention relates to the discovery that
vesicular fusion during acute membrane repair is driven by
mitsugumin53 (MG53) (SEQ ID NO. 1), a novel muscle-specific
tri-partite motif (TRIM) family protein. MG53 expression is
necessary to allow intracellular vesicles trafficking to and fusion
with the plasma membrane. Acute injury of the cellular membrane
leads to recruitment of MG53-containing vesicles to patch the
membrane at the injury site. Cells that are null for MG53 display
defects in membrane repair in response to multiple stresses,
including laser-induced injury, muscle damage induced by exercise,
and compromised recovery of muscle contractile function after
fatigue. Thus, MG53 is a critical component of the vesicular
trafficking events that underlie the acute repair and remodeling of
cellular membranes.
[0035] The invention is based in part upon the discovery of nucleic
acid sequences encoding novel polypeptides. The novel nucleic acids
and polypeptides are referred to herein as MG53 nucleic acids and
polypeptides.
[0036] In one aspect, the invention provides an isolated MG53
nucleic acid molecule encoding a MG53 polypeptide that includes a
nucleic acid sequence that has at least 30%, 40%, 50%, 60%, 70%,
80%, 90% or 100% identity to the nucleic acids disclosed in SEQ ID
NOS: 2, 4, and 6. In certain embodiments, the isolated MG53 nucleic
acid molecule will hybridize under stringent conditions to a
nucleic acid sequence complementary to a nucleic acid molecule that
includes a protein-coding sequence of a MG53 nucleic acid sequence.
The invention also includes an isolated nucleic acid that encodes a
MG53 polypeptide, or a fragment, homolog, analog, fusion protein,
pseudopeptide, peptidomimetic or derivative thereof. For example,
the nucleic acid can encode a polypeptide at least 30%, 40%, 50%,
60%, 70%, 80%, 90% or 100% identity to a polypeptide comprising the
amino acid sequences of SEQ ID NOS: 1, 3, 5, and 7. The nucleic
acid can be, for example, a genomic DNA fragment or a cDNA molecule
that includes the nucleic acid sequence of any of SEQ ID NOS: 2, 4,
and 6.
[0037] Also included in the invention is an oligonucleotide, e.g.,
an oligonucleotide which includes at least 6 contiguous nucleotides
of a MG53 nucleic acid (e.g., SEQ ID NOS: 2, 4, and 6) or a
complement of said oligonucleotide.
[0038] Also included in the invention are substantially purified
MG53 polypeptides (SEQ ID NOS: 1, 3, 5, and 7). In certain
embodiments, the MG53 polypeptides include an amino acid sequence
that is substantially identical to the amino acid sequence of a
human MG53 polypeptide.
[0039] The invention also features antibodies that
immunoselectively-bind to MG53 polypeptides, or fragments,
homologs, analogs, pseudopeptides, peptidomimetics or derivatives
thereof.
[0040] In another aspect, the invention includes pharmaceutical
compositions that include therapeutically- or
prophylactically-effective amounts of a therapeutic and a
pharmaceutically-acceptable carrier. The therapeutic can be, e.g.,
a MG53 nucleic acid, for example, a peptide nucleic acid, a cDNA,
or RNA, such as for example, a small inhibitory RNA; a MG53
polypeptide; or an antibody specific for a MG53 polypeptide. In a
further aspect, the invention includes, in one or more containers,
a therapeutically- or prophylactically-effective amount of this
pharmaceutical composition.
[0041] In a further aspect, the invention includes a method of
producing a polypeptide by culturing a cell that includes an
endogenous or exogenously expressed MG53 nucleic acid, under
conditions allowing for expression of the MG53 polypeptide encoded
by the DNA. If desired, the MG53 polypeptide can then be
recovered.
[0042] In still another aspect the invention includes a method of
producing a polypeptide by culturing a cell that contains an
endogenous MG53 nucleic acid disposed upstream or downstream of an
exogenous promoter. In certain embodiments, the exogenous promoter
is incorporated into a host cell's genome through homologous
recombination, strand break or mismatch repair mechanisms.
[0043] In another aspect, the invention includes a method of
detecting the presence of a MG53 polypeptide in a sample. In the
method, a sample is contacted with a compound that selectively
binds to the polypeptide under conditions allowing for formation of
a complex between the polypeptide and the compound. The complex is
detected, if present, thereby identifying the MG53 polypeptide
within the sample.
[0044] The invention also includes methods to identify specific
cell or tissue types based on their expression of a MG53 nucleic
acid, polypeptide or MG53 fusion polypeptide. For example, in
certain embodiments the invention includes fusion proteins
comprising a "tag" or indicator portion and an MG53 portion. In
certain aspects the tag or indicator portion can be a peptide
adapted for purification purposes, for example, FLAG tag,
6.times.His tag, or the like. In other aspects, the tag peptide
comprises a peptide adapted for providing a signal such as an
antibody epitope or a fluorescent peptide. Still other aspects
include the fusion of the MG53 with a peptide that is adapted for
mediating subcellular localization or translocation across a
cellular membrane, for example, a TAT fusion protein from the HIV
virus.
[0045] Also included in the invention is a method of detecting the
presence of a MG53 nucleic acid molecule in a sample by contacting
the sample with a MG53 nucleic acid probe or primer, and detecting
whether the nucleic acid probe or primer bound to a MG53 nucleic
acid molecule in the sample.
[0046] In a further aspect, the invention provides a method for
modulating the activity of a MG53 polypeptide by contacting a cell
sample that includes the MG53 polypeptide with a compound that
binds to the MG53 polypeptide in an amount sufficient to modulate
the activity of said polypeptide. The compound can be, e.g., a
small molecule, such as a nucleic acid, peptide, polypeptide,
peptidomimetic, carbohydrate, lipid or other organic (carbon
containing) or inorganic molecule, as further described herein.
[0047] Also within the scope of the invention is the use of a
therapeutic of the invention in the manufacture of a medicament for
treating or preventing disorders or syndromes including, e.g.,
cardiovascular disease, cardiomyopathy, atherosclerosis,
hypertension, congenital heart defects, aortic stenosis, atrial
septal defect (ASD), atrioventricular (A-V) canal defect, ductus
arteriosus, pulmonary stenosis, subaortic stenosis, ventricular
septal defect (VSD), valve diseases, hypercoagulation, hemophilia,
ulcers, wounds, lesions, cuts, abrasions, oxidative damage,
age-related tissue degeneration, surgically related lesions, burns,
muscle weakness, muscle atrophy, connective tissue disorders,
idiopathic thrombocytopenic purpura, heart failure, secondary
pathologies caused by heart failure and hypertension, hypotension,
angina pectoris, myocardial infarction, tuberous sclerosis,
scleroderma, transplantation, autoimmune disease, lupus
erythematosus, viral/bacterial/parasitic infections, multiple
sclerosis, autoimmune disease, allergies, immunodeficiencies, graft
versus host disease, asthma, emphysema, ARDS, inflammation and
modulation of the immune response, viral pathogenesis,
aging-related disorders, Th1 inflammatory diseases such as
rheumatoid arthritis, multiple sclerosis, inflammatory bowel
diseases, AIDS, wound repair, heart attacks, heart failure,
muscular dystrophy, bed sores, diabetic ulcers, oxidative damage,
and tissue damage such as sinusitis or mucositis, wrinkles, eczema
or dermatitis, dry skin, obesity, diabetes, endocrine disorders,
anorexia, bulimia, renal artery stenosis, interstitial nephritis,
glomerulonephritis, polycystic kidney disease, systemic, renal
tubular acidosis, IgA nephropathy, nephrological diseases,
hypercalceimia, Lesch-Nyhan syndrome, Von Hippel-Lindau (VHL)
syndrome, trauma, regeneration (in vitro and in vivo),
Hirschsprung's disease, Crohn's Disease, appendicitis,
endometriosis, laryngitis, psoriasis, actinic keratosis, acne, hair
growth/loss, allopecia, pigmentation disorders, myasthenia gravis,
alpha-mannosidosis, beta-mannosidosis, other storage disorders,
peroxisomal disorders such as zellweger syndrome, infantile refsum
disease, rhizomelic chondrodysplasia (chondrodysplasia punctata,
rhizomelic), and hyperpipecolic acidemia, osteoporosis, muscle
disorders, urinary retention, Albright Hereditary Ostoeodystrophy,
ulcers, Alzheimer's disease, stroke, Parkinson's disease,
Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan
syndrome, multiple sclerosis, ataxia-telangiectasia, behavioral
disorders, addiction, anxiety, pain, neuroprotection, Stroke,
Aphakia, neurodegenerative disorders, neurologic disorders,
developmental defects, conditions associated with the role of GRK2
in brain and in the regulation of chemokine receptors,
encephalomyelitis, anxiety, schizophrenia, manic depression,
delirium, dementia, severe mental retardation and dyskinesias,
Gilles de la Tourette syndrome, leukodystrophies, cancers, breast
cancer, CNS cancer, colon cancer, gastric cancer, lung cancer,
melanoma, ovarian cancer, pancreatic cancer, kidney cancer, colon
cancer, prostate cancer, neuroblastoma, and cervical cancer,
Neoplasm; adenocarcinoma, lymphoma; uterus cancer, benign prostatic
hypertrophy, fertility, control of growth and
development/differentiation related functions such as but not
limited maturation, lactation and puberty, reproductive
malfunction, and/or other pathologies and disorders of the
like.
[0048] The therapeutic composition of the invention comprises, in
certain embodiments, for example, an MG53 nucleic acid; a nucleic
acid that binds an MG53 encoding nucleic acid; an MG53 polypeptide,
peptide analog, pseudopeptide or peptidomimetic based thereon; a
small molecule modulator of MG53 or a MG53 protein-protein
interaction; or a MG53-specific antibody or biologically-active
derivatives or fragments thereof. As described herein, MG53
mediates the repair of damage to cellular membranes. Therefore,
targeting the expression and/or activity of these nucleic acids,
polypeptides, and homologs thereof will allow for a novel treatment
of various acute and chronic diseases and conditions related to
tissue repair.
[0049] In certain other aspects, the invention includes methods for
the treatment of or amelioration of tissue damage and/or disorders
related to tissue damage comprising administering an effective
amount of the composition of the invention to a subject in need
thereof. In certain embodiments, the invention comprises methods
for treating tissue damage or wounds, for example, cuts, ebrasions,
lesions, ulcers, burns, bed sores, gum diseases, mucositis, and the
like, comprising administering an effective amount of the
therapeutic composition of the invention to a subject in need
thereof.
[0050] In still other embodiments, the invention comprises
therapeutic compositions useful as a surgical adjuvant. In any of
the embodiments described herein, the surgical adjuvant composition
of the invention can be used or applied as a stand alone
therapeutic directly to the surgical site or it can be integrally
associated with a surgical or medical implement, for example, the
therapeutic of the invention may be conjugated to a polymer-based
stent, tube or other implantable device, such that the therapeutic
diffuses to the site of action in a controlled manner to accelerate
healing and/or to minimize trauma from an invasive surgical
procedure. In another embodiment, the therapeutic composition of
the invention is applied as, for example, a film or coating to the
medical implement such that the therapeutic diffuses into the blood
stream or surrounding tissues and/or wears away, and is thereby
delivered directly to the site of tissue damage; minimizing or
ameliorating the amount of cellular damage that occurs due to the
use of the surgical implement.
[0051] In still other embodiments, the invention comprises methods
for the treatment and/or prevention of deficiencies in tissue
repair that occur as a natural side-effect of the aging process
(e.g., skin rejuvenation, receding gums, bone degeneration,
arthritis, Alzheimer's, Parkinson's, and the like). In certain
aspects of this embodiment, the invention comprises administering
an effective amount of a therapeutic composition of the invention
to a subject suffering from age-related deficiencies in tissue
repair capacity, tissue integrity, and/or tissue elasticity. In
certain embodiments, the age-related deficiency is at least one of
wrinkles, crows feet, facial lines, pot marks, scars, fibroids, sun
spots, and the like, or combinations thereof.
[0052] Furthermore, due to the muscle-specific nature of the
expression of the endogenous MG53 gene, the invention encompasses
methods for the treatment and/or prevention of any type of muscle
or vascular cell/tissue injury, for example, tissue injury that
occurs as a result of cardiovascular disease, for example,
myocardial infarction; or rigorous physical activity, for example,
sports-related injuries, comprising administering an effective
amount of the therapeutic of the invention to a subject in need
thereof.
[0053] In still other embodiments, the invention comprises a
cosmetic composition useful for the repair, regeneration, or
restoration of body tissues comprising the therapeutic of the
invention and a cosmetically suitable carrier or excipient. In one
aspect of this embodiment, the invention encompasses a method of
enhancing the appearance of skin comprising administering an
effective amount of the therapeutic composition of the invention in
a cosmetic to a subject.
[0054] In any aspect of the invention, the therapeutic composition
of the invention can be in any pharmaceutically acceptable form and
administered by any pharmaceutically acceptable route, for example,
the therapeutic composition can be administered as an oral dosage,
either single daily dose or unitary dosage form, for the treatment
of a muscle damage due to a myocardial infarction, sclerotic
lesion, or muscle tear due to sports-related activity to promote
the regeneration and repair of the damaged muscle tissue. Such
pharmaceutically acceptable carriers and excipients and methods of
administration will be readily apparent to those of skill in the
art.
[0055] In addition, the invention relates to nucleic acids,
including interfering nucleic acids, and polypeptides encoding MG53
interacting proteins, for example, caveolin-3 (SEQ ID NO. 8)
polypeptides and homologs thereof; psuedopeptides and
peptidomimetics; as well as compounds that can modulate the
activity of caveolin-3 or its intermolecular interactions with
MG53. Therefore, in additional aspects, the present invention
encompasses methods for the targeting of caveolin-3 gene
expression, activity, and/or intermolecular interactions for the
treatment and/or prevention of a disease or disorder in a subject,
for example, for the promotion of tissue repair as described
above.
[0056] For example, the compositions of the present invention will
have efficacy for treatment of patients suffering from the diseases
and disorders disclosed above and/or other pathologies and
disorders of the like. The polypeptides can be used as immunogens
to produce antibodies specific for the invention, and as vaccines.
They can also be used to screen for potential agonist and
antagonist compounds. In addition, a cDNA encoding MG53 may be
useful in gene therapy, and MG53 may be useful when administered to
a subject in need thereof. By way of non-limiting example, the
compositions of the present invention will have efficacy for
treatment of patients suffering from the diseases and disorders
disclosed above and/or other pathologies and disorders of the
like.
[0057] The invention further includes a method for screening for a
modulator of disorders or syndromes including, e.g., the diseases
and disorders disclosed above and/or other pathologies and
disorders of the like. The method includes contacting a test
compound with a MG53 polypeptide and determining if the test
compound binds to said MG53 polypeptide. Binding of the test
compound to the MG53 polypeptide indicates the test compound is a
modulator of activity, or of latency or predisposition to the
aforementioned disorders or syndromes.
[0058] Also within the scope of the invention is a method for
screening for a modulator of activity, or of latency or
predisposition to disorders or syndromes including, e.g., the
diseases and disorders disclosed above and/or other pathologies and
disorders of the like by administering a test compound to a test
animal at increased risk for the aforementioned disorders or
syndromes. The test animal expresses a recombinant polypeptide
encoded by a MG53 nucleic acid. Expression or activity of MG53
polypeptide is then measured in the test animal, as is expression
or activity of the protein in a control animal which
recombinantly-expresses MG53 polypeptide and is not at increased
risk for the disorder or syndrome. Next, the expression of MG53
polypeptide in both the test animal and the control animal is
compared. A change in the activity of MG53 polypeptide in the test
animal relative to the control animal indicates the test compound
is a modulator of latency of the disorder or syndrome.
[0059] In yet another aspect, the invention includes a method for
determining the presence of or predisposition to a disease
associated with altered levels of a MG53 polypeptide, a MG53
nucleic acid, or both, in a subject (e.g., a human subject). The
method includes measuring the amount of the MG53 polypeptide in a
test sample from the subject and comparing the amount of the
polypeptide in the test sample to the amount of the MG53
polypeptide present in a control sample. An alteration in the level
of the MG53 polypeptide in the test sample as compared to the
control sample indicates the presence of or predisposition to a
disease in the subject. Preferably, the predisposition includes,
e.g., the diseases and disorders disclosed above and/or other
pathologies and disorders of the like. Also, the expression levels
of the new polypeptides of the invention can be used in a method to
screen for various disorders as well as to determine the stage of
particular disorders.
[0060] In a further aspect, the invention includes a method of
treating or preventing a pathological condition associated with a
disorder in a mammal by administering to the subject a MG53
polypeptide, a MG53 nucleic acid, or a MG53-specific antibody to a
subject (e.g., a human subject), in an amount sufficient to
alleviate or prevent the pathological condition. In preferred
embodiments, the disorder, includes, e.g., the diseases and
disorders disclosed above and/or other pathologies and disorders of
the like.
[0061] In yet another aspect, the invention can be used in a method
to identity the cellular receptors and downstream effectors of the
invention by any one of a number of techniques commonly employed in
the art. These include but are not limited to the two-hybrid
system, affinity purification, co-precipitation with antibodies or
other specific-interacting molecules.
[0062] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0063] As used herein, the term "MG53 antagonist" or "antagonist of
MG53" is used generally to refer to an agent capable of direct or
indirect inhibition of MG53 expression, translation, and/or
activity. Also, as used herein "MG53 receptor" relates generally to
any protein or fragment thereof capable of undergoing binding to a
MG53 protein.
[0064] As used herein, the term "caveolin antagonist" or
"antagonist of caveolin" is used generally to refer to an agent
capable of direct or indirect inhibition of caveolin expression,
translation, and/or activity. Also, as used herein "caveolin
receptor" relates generally to any protein or fragment thereof
capable of undergoing binding to a caveolin protein.
[0065] In certain aspects, the modulation of MG53 activity is
accomplished by, for example, the use of or modulation of MG53
binding partners, i.e., factors that bind to MG53 and neutralize
its biological activities, such as neutralizing anti-MG53, MG53
receptors (for example, or caveolin-3), MG53 receptor fragments,
and MG53 receptor analogs; the use of MG53-receptor antagonists,
such as anti-caveolin-3 antibodies, pseudopeptides, peptide analogs
or peptidomimetics that bind and disrupt the MG53-receptor
interaction; small molecules that inhibit MG53 activity or
intermolecular interactions, or alter the normal configuration of
MG53, or inhibit productive MG53/MG53-receptor binding; or the use
of nucleotide sequences derived from MG53 gene and/or MG53 receptor
gene, including coding, non-coding, and/or regulatory sequences to
prevent or reduce MG53 expression by, for example, antisense,
ribozyme, and/or triple helix approaches.
[0066] In another aspect, the present invention features a nucleic
acid molecule, such as a decoy RNA, dsRNA, siRNA, shRNA, micro RNA,
aptamers, antisense nucleic acid molecules, which down regulates
expression of a sequence encoding MG53 or a MG53 receptor, for
example, caveolin-3. In an embodiment, a nucleic acid molecule of
the invention is adapted to treat and/or prevent tissue damage and
promote tissue repair. In another embodiment, a nucleic acid
molecule of the invention has an endonuclease activity or is a
component of a nuclease complex, and cleaves RNA having a MG53 or a
MG53 receptor nucleic acid sequence.
[0067] In one embodiment, a nucleic acid molecule of the invention
comprises between 12 and 100 bases complementary to RNA having a
MG53 or a MG53 receptor nucleic acid sequence. In another
embodiment, a nucleic acid molecule of the invention comprises
between 14 and 24 bases complementary to RNA having a MG53 or a
MG53 receptor nucleic acid sequence. In any embodiment described
herein, the nucleic acid molecule can be synthesized chemically
according to methods well known in the art.
[0068] In another aspect the present invention provides a kit
comprising a suitable container, the active agent capable of
inhibiting MG53 activity, expression or binding in a
pharmaceutically acceptable form disposed therein, and instructions
for its use.
[0069] In another aspect, the invention relates to a method for
diagnosing or monitoring disorder or disease or progression
comprising detecting for the presence of a nucleotide polymorphism
in the MG53 or a MG53 receptor structural gene associated with the
disease, through the detection of the expression level of a MG53 or
a MG53 receptor gene or protein or both. Polymorphisms have been
identified that correlate with disease severity. (See, Zhong et
al., Simultaneous detection of microsatellite repeats and SNPs in
the macrophage migration inhibitory factor (MG53) gene by thin-film
biosensor chips and application to rural field studies. Nucleic
Acids Res. 2005 Aug. 2; 33(13):e121; Donn et al., A functional
promoter haplotype of macrophage migration inhibitory factor is
linked and associated with juvenile idiopathic arthritis. Arthritis
Rheum. 2004 May; 50(5):1604-10; all of which are incorporated
herein by reference in their entirety for all purposes.). As used
herein, "MG53 or MG53 receptor gene" or "MG53 or MG53 receptor
structural gene" may include the 5' UTR, 3' UTR, promoter
sequences, enhancer sequences, intronic and exonic DNA of the MG53
or MG53 receptor gene as well as the MG53 or MG53 receptor gene
mRNA or cDNA sequence.
[0070] As one of ordinary skill will comprehend, the MG53 or MG53
receptor gene polymorphisms associated with tissue repair
disorders, and hence useful as diagnostic markers according to the
methods of the invention may appear in any of the previously named
nucleic acid regions. Techniques for the identification and
monitoring of polymorphisms are known in the art and are discussed
in detail in U.S. Pat. No. 6,905,827 to Wohlgemuth, which is
incorporated herein by reference in its entirety for all
purposes.
[0071] Certain aspects of the invention encompass methods of
detecting gene expression or polymorphisms with one or more DNA
molecules wherein the one or more DNA molecules has a nucleotide
sequence which detects expression of a gene corresponding to the
oligonucleotides depicted in the Sequence Listing. In one format,
the oligonucleotide detects expression of a gene that is
differentially expressed. The gene expression system may be a
candidate library, a diagnostic agent, a diagnostic oligonucleotide
set or a diagnostic probe set. The DNA molecules may be genomic
DNA, RNA, protein nucleic acid (PNA), cDNA or synthetic
oligonucleotides. Following the procedures taught herein, one can
identify sequences of interest for analyzing gene expression or
polymorphisms. Such sequences may be predictive of a disease
state.
[0072] Diagnostic Oligonucleotides of the Invention
[0073] As used herein, the term "oligonucleotide molecule" is
intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA
molecules (e.g., mRNA), analogs of the DNA or RNA generated using
nucleotide analogs, and derivatives, fragments and homologs
thereof. The nucleic acid molecule may be single-stranded or
double-stranded, but preferably is comprised double-stranded
DNA.
[0074] In certain aspects, the invention relates to diagnostic
oligonucleotides and diagnostic oligonucleotide set(s), for which a
correlation exists between the health status of an individual, and
the individual's expression of RNA or protein products
corresponding to the nucleotide sequence. In some instances, only
one oligonucleotide is necessary for such detection. Members of a
diagnostic oligonucleotide set may be identified by any means
capable of detecting expression or a polymorphism of RNA or protein
products, including but not limited to differential expression
screening, PCR, RT-PCR, SAGE analysis, high-throughput sequencing,
microarrays, liquid or other arrays, protein-based methods (e.g.,
western blotting, proteomics, mass-spectrometry, and other methods
described herein), and data mining methods, as further described
herein.
[0075] In the context of the invention, nucleic acids and/or
proteins are manipulated according to well known molecular biology
techniques. Detailed protocols for numerous such procedures are
described in, e.g., in Ausubel et al. Current Protocols in
Molecular Biology (supplemented through 2000) John Wiley &
Sons, New York ("Ausubel"); Sambrook et al. Molecular Cloning--A
Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989 ("Sambrook"), and Berger
and Kimmel Guide to Molecular Cloning Techniques, Methods in
Enzymology volume 152 Academic Press, Inc., San Diego, Calif.
("Berger").
[0076] Genotyping
[0077] In addition to, or in conjunction with the correlation of
expression profiles and clinical data, it is often desirable to
correlate expression patterns with the subject's genotype at one or
more genetic loci or to correlate both expression profiles and
genetic loci data with clinical data. The selected loci can be, for
example, chromosomal loci corresponding to one or more member of
the candidate library, polymorphic alleles for marker loci, or
alternative disease related loci (not contributing to the candidate
library) known to be, or putatively associated with, a disease (or
disease criterion). Indeed, it will be appreciated, that where a
(polymorphic) allele at a locus is linked to a disease (or to a
predisposition to a disease), the presence of the allele can itself
be a disease criterion.
[0078] Numerous well known methods exist for evaluating the
genotype of an individual, including southern analysis, restriction
fragment length polymorphism (RFLP) analysis, polymerase chain
reaction (PCR), amplification length polymorphism (AFLP) analysis,
single stranded conformation polymorphism (SSCP) analysis, single
nucleotide polymorphism (SNP) analysis (e.g., via PCR, Taqman or
molecular beacons), among many other useful methods. Many such
procedures are readily adaptable to high throughput and/or
automated (or semi-automated) sample preparation and analysis
methods. Most, can be performed on nucleic acid samples recovered
via simple procedures from the same sample as yielded the material
for expression profiling. Exemplary techniques are described in,
e.g., Sambrook, and Ausubel, supra.
[0079] The invention also features nucleic acid molecules, for
example enzymatic nucleic acid molecules, antisense nucleic acid
molecules, decoys, double stranded RNA, triplex oligonucleotides,
and/or aptamers, and methods to modulate gene expression of, for
example, genes encoding a MG53 protein, a MG53 protein or MG53
receptor binding protein or a MG53 receptor protein. In particular,
the instant invention features nucleic-acid based molecules and
methods to modulate the expression of a MG53 protein or MG53
receptor protein.
[0080] The invention features one or more enzymatic nucleic
acid-based molecules and methods that independently or in
combination modulate the expression of gene(s) encoding a MG53
protein, a MG53 protein or MG53 receptor binding protein, and/or a
MG53 receptor protein, for example, caveolin-3.
[0081] The description below of the various aspects and embodiments
is provided with reference to the exemplary MG53 and MG53 receptor
genes. However, the various aspects and embodiments are also
directed to genes which encode homologs, orthologs, and paralogs of
other MG53 proteins, MG53 binding proteins, and MMG53 receptor
genes and include all isoforms, splice variants, and polymorphisms.
Those additional genes can be analyzed for target sites using the
methods described for MG53 proteins, MG53 binding proteins, and
MG53 receptor genes. Thus, the inhibition and the effects of such
inhibition of the other genes can be performed as described
herein.
[0082] By "down-regulate" it is meant that the expression of the
gene, or level of RNAs or equivalent RNAs encoding one or more
proteins, or activity of one or more proteins, such as MG53 and
MG53 receptor genes, is reduced below that observed in the absence
of the nucleic acid molecules of the invention. In one embodiment,
inhibition or down-regulation with enzymatic nucleic acid molecule
preferably is below that level observed in the presence of an
enzymatically inactive or attenuated molecule that is able to bind
to the same site on the target RNA, but is unable to cleave that
RNA. In another embodiment, inhibition or down-regulation with
antisense oligonucleotides is preferably below that level observed
in the presence of, for example, an oligonucleotide with scrambled
sequence or with mismatches. In another embodiment, inhibition or
down-regulation of MG53 proteins, MG53 binding proteins, and MG53
receptor genes with the nucleic acid molecule of the instant
invention is greater in the presence of the nucleic acid molecule
than in its absence.
[0083] By "up-regulate" is meant that the expression of the gene,
or level of RNAs or equivalent RNAs encoding one or more protein
subunits, or activity of one or more protein subunits, such as MG53
proteins, MG53 binding proteins, and MG53 receptor genes, is
greater than that observed in the absence of the nucleic acid
molecules of the invention. For example, the expression of a gene,
such as MG53 proteins, MG53 binding proteins, and MG53 receptor
genes, can be increased in order to treat, prevent, ameliorate, or
modulate a pathological condition caused or exacerbated by an
absence or low level of gene expression. In one embodiment the
invention relates to a method for treating or preventing bladder
over activity by up-regulating the expression, release, and/or
activity of a MG53 proteins, MG53 binding proteins, and MG53
receptor genes.
[0084] By "modulate" is meant that the expression of the gene, or
level of RNAs or equivalent RNAs encoding one or more proteins, or
activity of one or more proteins is up-regulated or down-regulated,
such that the expression, level, or activity is greater than or
less than that observed in the absence of the nucleic acid
molecules of the invention.
[0085] By "gene" it is meant a nucleic acid that encodes RNA, for
example, nucleic acid sequences including but not limited to a
segment encoding a polypeptide.
[0086] "Complementarity" refers to the ability of a nucleic acid to
form hydrogen bond(s) with another RNA sequence by either
traditional Watson-Crick or other non-traditional types.
[0087] By "RNA" is meant a molecule comprising at least one
ribonucleotide residue. By "ribonucleotide" or "2'-OH" is meant a
nucleotide with a hydroxyl group at the 2' position of a
D-ribo-furanose moiety.
[0088] By "nucleotide" is meant a heterocyclic nitrogenous base in
N-glycosidic linkage with a phosphorylated sugar. Nucleotides are
recognized in the art to include natural bases (standard), and
modified bases well known in the art. Such bases are generally
located at the 1' position of a nucleotide sugar moiety.
Nucleotides generally comprise a base, sugar and a phosphate group.
The nucleotides can be unmodified or modified at the sugar,
phosphate and/or base moiety, (also referred to interchangeably as
nucleotide analogs, modified nucleotides, non-natural nucleotides,
non-standard nucleotides and other; see for example, Usman and
McSwiggen, supra; Eckstein et al., International PCT Publication
No. WO 92/07065; Usman et al., International PCT Publication No. WO
93/15187; Uhlman & Peyman, supra all are hereby incorporated by
reference herein). There are several examples of modified nucleic
acid bases known in the art as summarized by Limbach et al., 1994,
Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of
chemically modified and other natural nucleic acid bases that can
be introduced into nucleic acids include, for example, inosine,
purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,
2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,
naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),
5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,
5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,
wybutosine, wybutoxosine, 4-acetyltidine,
5-(carboxyhydroxymethyl)uridine,
5'-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,
1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,
3-methylcytidine, 2-methyladenosine, 2-methylguanosine,
N6-methyladenosine, 7-methylguanosine,
-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,
5-methylcarbonylmethyluridine, 5-methyloxyuridine,
5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,
beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,
threonine derivatives and others (Burgin et al., 1996,
Biochemistry, 35, 14090; Uhlman & Peyman, supra).
[0089] By "modified bases" in this aspect is meant nucleotide bases
other than adenine, guanine, cytosine and uracil at 1' position or
their equivalents; such bases can be used at any position, for
example, within the catalytic core of an enzymatic nucleic acid
molecule and/or in the substrate-binding regions of the nucleic
acid molecule.
[0090] By "enzymatic nucleic acid molecule" it is meant a nucleic
acid molecule which has complementarity in a substrate binding
region to a specified gene target, and also has or mediates an
enzymatic activity which is active to specifically cleave target
RNA. That is, the enzymatic nucleic acid molecule is able to
intermolecularly cleave RNA, alone or as a component of an
enzymatic complex, and thereby inactivate a target RNA molecule.
These complementary regions allow sufficient hybridization of the
enzymatic nucleic acid molecule to the target RNA and thus permit
cleavage. One hundred percent complementarity is preferred, but
complementarity as low as 50-75% can also be useful in this
invention (see for example Werner and Uhlenbeck, 1995, Nucleic
Acids Research, 23, 2092 2096; Hammann et al., 1999, Antisense and
Nucleic Acid Drug Dev., 9, 25 31). The nucleic acids can be
modified at the base, sugar, and/or phosphate groups. The term
"enzymatic nucleic acid" is used interchangeably with phrases such
as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme
or aptamer-binding ribozyme, regulatable ribozyme, catalytic
oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, siRNA, micro
RNA, short hairpin RNA, endoribonuclease, RNA-induced silencing
complexes, endonuclease, minizyme, leadzyme, oligozyme or DNA
enzyme. All of these terminologies describe nucleic acid molecules
with enzymatic activity.
[0091] The specific enzymatic nucleic acid molecules described in
the instant application are not limiting in the invention and those
skilled in the art will recognize that all that is important in an
enzymatic nucleic acid molecule of this invention is that it has a
specific substrate binding site which is complementary to one or
more of the target nucleic acid regions, and that it have
nucleotide sequences within or surrounding that substrate binding
site which impart a nucleic acid cleaving and/or ligation activity
to the molecule (Cech et al., U.S. Pat. No. 4,987,071; Cech et al.,
1988, 260 JAMA 3030).
[0092] Several varieties of enzymatic RNAs are known presently.
Each can catalyze the hydrolysis of RNA phosphodiester bonds in
trans (and thus can cleave other RNA molecules) under physiological
conditions. In general, enzymatic nucleic acids act by first
binding to a target RNA. Such binding occurs through the target
binding portion of a enzymatic nucleic acid which is held in close
proximity to an enzymatic portion of the molecule that acts to
cleave the target RNA. Thus, the enzymatic nucleic acid first
recognizes and then binds a target RNA through complementary
base-pairing, and once bound to the correct site, acts
enzymatically to cut the target RNA. Strategic cleavage of such a
target RNA will destroy its ability to direct synthesis of an
encoded protein. After an enzymatic nucleic acid has bound and
cleaved its RNA target, it is released from that RNA to search for
another target and can repeatedly bind and cleave new targets.
Thus, a single ribozyme molecule is able to cleave many molecules
of target RNA. In addition, the ribozyme is a highly specific
inhibitor of gene expression, with the specificity of inhibition
depending not only on the base-pairing mechanism of binding to the
target RNA, but also on the mechanism of target RNA cleavage.
Single mismatches, or base-substitutions, near the site of cleavage
can completely eliminate catalytic activity of a ribozyme.
[0093] By "nucleic acid molecule" as used herein is meant a
molecule having nucleotides. The nucleic acid can be single,
double, or multiple stranded and can comprise modified or
unmodified nucleotides or non-nucleotides or various mixtures and
combinations thereof.
[0094] By "equivalent" or "related" RNA to MG53 proteins, MG53
binding proteins, and MG53 receptor genes is meant to include those
naturally occurring --RNA molecules having homology (partial or
complete) to MG53 proteins, MG53 binding proteins, and MG53
receptor genes encoding for proteins with similar function as MG53
proteins, MG53 binding proteins, and MG53 receptor proteins in
various organisms, including human, rodent, primate, rabbit, pig,
protozoans, fungi, plants, and other microorganisms and parasites.
The equivalent RNA sequence also includes in addition to the coding
region, regions such as 5'-untranslated region, 3'-untranslated
region, introns, intron-exon junction and the like. By "homology"
is meant the nucleotide sequence of two or more nucleic acid
molecules is partially or completely identical. In certain
embodiments the homolgous nucleic acid has 30%, 40%, 50%, 60%, 70%,
80%, 90%, or 95% homology to MG53, MG53 binding protein, and/or
MG53 receptor gene.
[0095] By "antisense nucleic acid", it is meant a non-enzymatic
nucleic acid molecule that binds to target RNA by means of RNA-RNA
or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993
Nature 365, 566) interactions and alters the activity of the target
RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and
Woolf et al., U.S. Pat. No. 5,849,902). Typically, antisense
molecules are complementary to a target sequence along a single
contiguous sequence of the antisense molecule. However, in certain
embodiments, an antisense molecule can bind to substrate such that
the substrate molecule forms a loop or hairpin, and/or an antisense
molecule can bind such that the antisense molecule forms a loop or
hairpin. Thus, the antisense molecule can be complementary to two
(or even more) non-contiguous substrate sequences or two (or even
more) non-contiguous sequence portions of an antisense molecule can
be complementary to a target sequence or both. For a review of
current antisense strategies, see Schmajuk et al., 1999, J. Biol.
Chem., 274, 21783-21789, Delihas et al., 1997, Nature, 15, 751-753,
Stein et al., 1997, Antisense N. A. Drug Dev., 7, 151, Crooke,
2000, Methods Enzymol., 313, 3-45; Crooke, 1998, Biotech. Genet.
Eng. Rev., 15, 121-157, Crooke, 1997, Ad. Pharmacol, 40, 1-49,
which are incorporated herein by reference in their entirety. In
addition, antisense DNA can be used to target RNA by means of
DNA-RNA interactions, thereby activating RNase H, which digests the
target RNA in the duplex. The antisense oligonucleotides can
comprise one or more RNAse H activating region, which is capable of
activating RNAse H cleavage of a target RNA. Antisense DNA can be
synthesized chemically or expressed via the use of a single
stranded DNA expression vector or equivalent thereof.
[0096] Long double-stranded RNAs (dsRNAs; typically >200 nt) can
be used to silence the expression of target genes in a variety of
organisms and cell types (e.g., worms, fruit flies, and plants).
Upon introduction, the long dsRNAs enter a cellular pathway that is
commonly referred to as the RNA interference (RNAi) pathway. First,
the dsRNAs get processed into 20-25 nucleotide (nt) small
interfering RNAs (siRNAs) by an RNase III-like enzyme called Dicer
(initiation step). Then, the siRNAs assemble into
endoribonuclease-containing complexes known as RNA-induced
silencing complexes (RISCs), unwinding in the process. The siRNA
strands subsequently guide the RISCs to complementary RNA
molecules, where they cleave and destroy the cognate RNA (effecter
step). Cleavage of cognate RNA takes place near the middle of the
region bound by the siRNA strand. In mammalian cells, introduction
of long dsRNA (>30 nt) initiates a potent antiviral response,
exemplified by nonspecific inhibition of protein synthesis and RNA
degradation. The mammalian antiviral response can be bypassed,
however, by the introduction or expression of siRNAs.
[0097] Injection and transfection of dsRNA into cells and organisms
has been the main method of delivery of siRNA. And while the
silencing effect lasts for several days and does appear to be
transferred to daughter cells, it does eventually diminish.
Recently, however, a number of groups have developed expression
vectors to continually express siRNAs in transiently and stably
transfected mammalian cells. (See, e.g., Brummelkamp T R, Bernards
R, and Agami R. (2002). A system for stable expression of short
interfering RNAs in mammalian cells. Science 296:550-553; Lee N S,
Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, and Rossi
J. (2002). Expression of small interfering RNAs targeted against
HIV-1 rev transcripts in human cells. Nature Biotechnol.
20:500-505; Miyagishi M, and Taira K. (2002). U6-promoter-driven
siRNAs with four uridine 3' overhangs efficiently suppress targeted
gene expression in mammalian cells. Nature Biotechnol. 20:497-500;
Paddison P J, Caudy A A, Bernstein E, Hannon G J, and Conklin D S.
(2002). Short hairpin RNAs (shRNAs) induce sequence-specific
silencing in mammalian cells. Genes & Dev. 16:948-958; Paul C
P, Good P D, Winer I, and Engelke D R. (2002). Effective expression
of small interfering RNA in human cells. Nature Biotechnol.
20:505-508; Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester W
C, and Shi Y. (2002). A DNA vector-based RNAi technology to
suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci.
USA 99(6):5515-5520; Yu J-Y, DeRuiter S L, and Turner D L. (2002).
RNA interference by expression of short-interfering RNAs and
hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA
99(9):6047-6052, which are herein incorporated by reference in
their entirety).
[0098] Some vectors have been engineered to express small hairpin
RNAs (shRNAs), which get processed in vivo into siRNAs-like
molecules capable of carrying out gene-specific silencing. The
vectors contain the shRNA sequence between a polymerase III (pol
III) promoter (e.g., U6 or H1 promoters) and a 4-5 thymidine
transcription termination site. The transcript is terminated at
position 2 of the termination site (pol III transcripts naturally
lack poly(A) tails) and then folds into a stem-loop structure with
3' UU-overhangs. The ends of the shRNAs are processed in vivo,
converting the shRNAs into .about.21 nt siRNA-like molecules, which
in turn initiate RNAi. This latter finding correlates with recent
experiments in C. elegans, Drosophila, plants and Trypanosomes,
where RNAi has been induced by an RNA molecule that folds into a
stem-loop structure. The use of siRNA vectors and expression
systems is known and are commercially available from Ambion,
Inc..RTM. (Austin, Tex.), Lentigen, Inc. (Baltimore, Md.), Panomics
(Fremont, Calif.), and Sigma-Aldrich (ST. Louis, Mo.).
[0099] In another aspect of the invention, enzymatic nucleic acid
molecules or antisense molecules that interact with target RNA
molecules, and down-regulate MG53, MG53 binding proteins, and/or a
MG53 receptor gene activity are expressed from transcription units
inserted into DNA or RNA vectors. The recombinant vectors are
preferably DNA plasmids or viral vectors. Enzymatic nucleic acid
molecule or antisense expressing viral vectors can be constructed
based on, but not limited to, lenti virus, cytomegalovirus,
adeno-associated virus, retrovirus, adenovirus, or alphavirus.
Preferably, the recombinant vectors capable of expressing the
enzymatic nucleic acid molecules or antisense are delivered, and
persist in target cells. Alternatively, viral vectors can be used
that provide for expression of enzymatic nucleic acid molecules or
antisense. Such vectors can be repeatedly administered as
necessary. Once expressed, the enzymatic nucleic acid molecules or
antisense bind to the target RNA and down-regulate its function or
expression. Delivery of enzymatic nucleic acid molecule or
antisense expressing vectors can be systemic, such as by
intravenous or intramuscular administration, by administration to
target cells explanted from the patient or subject followed by
reintroduction into the patient or subject, or by any other means
that would allow for introduction into the desired target cell.
Antisense DNA can be expressed via the use of a single stranded DNA
intracellular expression vector.
[0100] By "vectors" is meant any nucleic acid-based technique used
to deliver a desired nucleic acid, for example, bacterial plasmid,
viral nucleic acid, HAC, BAC, and the like.
[0101] The nucleic acid molecules of the instant invention,
individually, or in combination or in conjunction with other drugs,
can be used to treat diseases or conditions discussed above. For
example, the subject can be treated, or other appropriate cells can
be treated, as is evident to those skilled in the art, individually
or in combination with one or more drugs under conditions suitable
for the treatment.
[0102] The use of specially designed vector constructs for inducing
RNA interference has numerous advantages over oligonucleotide-based
techniques. The most significant advantages are stability, and
induced transcription via inducible promoters. Promoter regions in
the vector ensure that shRNA transcripts are constantly expressed,
maintaining cellular levels at all times. Thus, the duration of the
effect is not as transient as with injected RNA, which usually
lasts no longer than a few days. And by using expression constructs
instead of oligo injection, it is possible to perform
multi-generational studies of gene knockdown because the vector can
become a permanent fixture in the cell line.
[0103] By "triplex forming oligonucleotides" or "triplex
oligonucleotide" is meant an oligonucleotide that can bind to a
double-stranded DNA in a sequence-specific manner to form a
triple-strand helix. Formation of such triple helix structure has
been shown to inhibit transcription of the targeted gene
(Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504;
Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al., 2000,
Biochim. Biophys. Acta, 1489, 181-206).
[0104] By "double stranded RNA" or "dsRNA" is meant a double
stranded RNA that matches a predetermined gene sequence that is
capable of activating cellular enzymes that degrade the
corresponding messenger RNA transcripts of the gene. These dsRNAs
are referred to as short intervening RNA (siRNA) and can be used to
inhibit gene expression. see for example Bass, 2001, Nature, 411,
428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer
et al., International PCT Publication No. WO 00/44895;
Zernicka-Goetz et al., International PCT Publication No. WO
01/36646; Fire, International PCT Publication No. WO 99/32619;
Plaetinck et al., International PCT Publication No. WO 00/01846;
Mello and Fire, International PCT Publication No. WO 01/29058;
Deschamps-Depaillette, International PCT Publication No. WO
99/07409; and Li et al., International PCT Publication No. WO
00/44914.
[0105] In one embodiment of the present invention, a nucleic acid
molecule of the instant invention can be between about 10 and 100
nucleotides in length. For example, enzymatic nucleic acid
molecules of the invention are preferably between about 15 and 50
nucleotides in length, more preferably between about 25 and 40
nucleotides in length (for example see Jarvis et al., 1996, J.
Biol. Chem., 271, 29107 29112). Exemplary antisense molecules of
the invention are preferably between about 15 and 75 nucleotides in
length, more preferably between about 20 and 35 nucleotides in
length (see for example Woolf et al., 1992, PNAS., 89, 7305 7309;
Milner et al., 1997, Nature Biotechnology, 15, 537 541). Exemplary
triplex forming oligonucleotide molecules of the invention are
preferably between about 10 and 40 nucleotides in length, more
preferably between about 12 and 25 nucleotides in length (see for
example Maher et al, 1990, Biochemistry, 29, 8820 8826; Strobel and
Dervan, 1990, Science, 249, 73 75). Those skilled in the art will
recognize that all that is required is that the nucleic acid
molecule be of sufficient length and suitable conformation for the
nucleic acid molecule to interact with its target and/or catalyze a
reaction contemplated herein. The length of the nucleic acid
molecules of the instant invention are not limiting within the
general limits stated. Preferably, a nucleic acid molecule that
modulates, for example, down-regulates MG53, MG53 binding protein,
and/or a MG53 receptor gene expression comprises between 12 and 100
bases complementary to a RNA molecule of a MG53 gene, a MG53
binding protein gene, and/or a MG53 receptor gene.
[0106] The invention provides a method for producing a class of
nucleic acid-based gene modulating agents which exhibit a high
degree of specificity for the RNA of a desired target. For example,
the enzymatic nucleic acid molecule is preferably targeted to a
highly conserved sequence region of target RNAs encoding a MG53,
MG53 binding protein, and/or a MG53 receptor gene such that
specific treatment of a disease or condition can be provided with
either one or several nucleic acid molecules of the invention. Such
nucleic acid molecules can be delivered exogenously to specific
tissue or cellular targets as required. Alternatively, the nucleic
acid molecules (e.g., ribozymes and antisense) can be expressed
from DNA and/or RNA vectors that are delivered to specific
cells.
[0107] As used in herein "cell" is used in its usual biological
sense, and does not refer to an entire multicellular organism. The
cell can, for example, be in vivo, in vitro or ex vivo, e.g., in
cell culture, or present in a multicellular organism, including,
e.g., birds, plants and mammals such as humans, cows, sheep, apes,
monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g.,
bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
[0108] The nucleic acid-based inhibitors of the invention are added
directly, or can be complexed with cationic lipids, packaged within
liposomes, or otherwise delivered to target cells or tissues. The
nucleic acid or nucleic acid complexes can be locally administered
to relevant tissues in vitro, ex vivo, or in vivo through injection
or infusion pump, with or without their incorporation in
biopolymers.
[0109] In another embodiment, the invention features an enzymatic
nucleic acid molecule having one or more non-nucleotide moieties,
and having enzymatic activity to cleave an RNA or DNA molecule.
[0110] In a further embodiment, the described nucleic acid
molecules, such as antisense or ribozymes, can be used in
combination with other known treatments to treat conditions or
diseases discussed above. For example, the described molecules can
be used in combination with one or more known therapeutic
agents.
[0111] Antisense molecules can be modified or unmodified RNA, DNA,
or mixed polymer oligonucleotides and primarily function by
specifically binding to matching sequences resulting in inhibition
of peptide synthesis (Wu-Pong, November 1994, BioPharm, 20-33). The
antisense oligonucleotide binds to target RNA by Watson Crick
base-pairing and blocks gene expression by preventing ribosomal
translation of the bound sequences either by steric blocking or by
activating RNase H enzyme. Antisense molecules can also alter
protein synthesis by interfering with RNA processing or transport
from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996,
Crit. Rev. in Oncogenesis 7, 151-190).
[0112] In addition, binding of single stranded DNA to RNA can
result in nuclease degradation of the heteroduplex (Wu-Pong, supra;
Crooke, supra). To date, the only backbone modified DNA chemistry
which acts as substrates for RNase H are phosphorothioates,
phosphorodithioates, and borontrifluoridates. Recently it has been
reported that 2'-arabino and 2'-fluoro-arabino-containing oligos
can also activate RNase H activity.
[0113] A number of antisense molecules have been described that
utilize novel configurations of chemically modified nucleotides,
secondary structure, and/or RNase H substrate domains (Woolf et
al., International PCT Publication No. WO 98/13526; Thompson et
al., International PCT Publication No. WO 99/54459; Hartmann et
al., U.S. Ser. No. 60/101,174 which was filed on Sep. 21, 1998) all
of these are incorporated by reference herein in their
entirety.
[0114] Several varieties of enzymatic RNAs are presently known. In
addition, several in vitro selection (evolution) strategies (Orgel,
1979, Proc. R. Soc. London, B 205, 435) have been used to evolve
new nucleic acid catalysts capable of catalyzing cleavage and
ligation of phosphodiester linkages (Joyce, 1989, Gene, 82, 83 87;
Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific
American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel
et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93;
Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op.
Biotech., 7, 442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94,
4262; Tang et al., 1997, RNA 3, 914; Nakacane & Eckstein, 1994,
supra; Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995,
supra; Vaish et al., 1997, Biochemistry 36, 6495; all of these are
incorporated by reference herein). Each can catalyze a series of
reactions including the hydrolysis of phosphodiester bonds in trans
(and thus can cleave other RNA molecules) under physiological
conditions.
[0115] The enzymatic nature of an enzymatic nucleic acid molecule
can allow the concentration of enzymatic nucleic acid molecule
necessary to affect a therapeutic treatment to be lower. This
reflects the ability of the enzymatic nucleic acid molecule to act
enzymatically. Thus, a single enzymatic nucleic acid molecule is
able to cleave many molecules of target RNA. In addition, the
enzymatic nucleic acid molecule is a highly specific inhibitor,
with the specificity of inhibition depending not only on the
base-pairing mechanism of binding to the target RNA, but also on
the mechanism of target RNA cleavage. Single mismatches, or
base-substitutions, near the site of cleavage can be chosen to
greatly attenuate the catalytic activity of a enzymatic nucleic
acid molecule.
[0116] Nucleic acid molecules having an endonuclease enzymatic
activity are able to repeatedly cleave other separate RNA molecules
in a nucleotide base sequence-specific manner. Such enzymatic
nucleic acid molecules can be targeted to virtually any RNA
transcript, and achieve efficient cleavage in vitro (Zaug et al.,
324, Nature 429 1986; Uhlenbeck, 1987 Nature 328, 596; Kim et al.,
84 Proc. Natl. Acad. Sci. USA 8788, 1987; Dreyfus, 1988, Einstein
Quart. J. Bio. Med., 6, 92; Haseloff and Gerlach, 334 Nature 585,
1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic
Acids Research 1371, 1989; Santoro et al., 1997 supra).
[0117] Because of their sequence specificity, trans-cleaving
enzymatic nucleic acid molecules can be used as therapeutic agents
for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem.
30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38,
2023-2037). Enzymatic nucleic acid molecules can be designed to
cleave specific RNA targets within the background of cellular RNA.
Such a cleavage event renders the RNA non-functional and abrogates
protein expression from that RNA. In this manner, synthesis of a
protein associated with a disease state can be selectively
inhibited (Warashina et al., 1999, Chemistry and Biology, 6,
237-250).
[0118] Enzymatic nucleic acid molecules of the invention that are
allosterically regulated ("allozymes") can be used to modulate
MG53, MG53 binding proteins, and/or MG53 receptor gene expression.
These allosteric enzymatic nucleic acids or allozymes (see for
example George et al, U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih
et al., U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No.
5,871,914, Nathan and Ellington, International PCT publication No.
WO 00/24931, Breaker et al., International PCT Publication Nos. WO
00/26226 and 98/27104, and Sullenger et al., International PCT
publication No. WO 99/29842) are designed to respond to a signaling
agent, which in turn modulates the activity of the enzymatic
nucleic acid molecule and modulates expression of MG53, MG53
binding proteins, and/or MG53 receptor gene. In response to
interaction with a predetermined signaling agent, the allosteric
enzymatic nucleic acid molecule's activity is activated or
inhibited such that the expression of a particular target is
selectively down-regulated. The target can comprise MG53, MG53
binding proteins, and/or MG53 receptor gene.
[0119] Oligonucleotides (eg; antisense, GeneBlocs) are synthesized
using protocols known in the art as described in Caruthers et al.,
1992, Methods in Enzymology 211, 3 19, Thompson et al.,
International PCT Publication No. WO 99/54459, Wincott et al.,
1995, Nucleic Acids Res. 23, 2677 2684, Wincott et al., 1997,
Methods Mol. Bio., 74, 59, Brennan et al, 1998, Biotechnol Bioeng.,
61, 33 45, and Brennan, U.S. Pat. No. 6,001,311. All of these
references are incorporated herein by reference. In a non-limiting
example, small scale syntheses are conducted on a 394 Applied
Biosystems, Inc. synthesizer. Alternatively, the nucleic acid
molecules of the present invention can be synthesized separately
and joined together post-synthetically, for example by ligation
(Moore et al., 1992, Science 256, 9923; Draper et al.,
International PCT publication No. WO 93/23569; Shabarova et al.,
1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997,
Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997,
Bioconjugate Chem. 8, 204).
[0120] The nucleic acid molecules of the present invention can be
modified extensively to enhance stability by modification with
nuclease resistant groups, for example, 2'-amino, 2'-C-allyl,
2'-fluoro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren,
1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31,
163).
[0121] While chemical modification of oligonucleotide
internucleotide linkages with phosphorothioate, phosphorothioate,
and/or 5'-methylphosphonate linkages improves stability, too many
of these modifications can cause some toxicity. Therefore when
designing nucleic acid molecules the amount of these
internucleotide linkages should be minimized. The reduction in the
concentration of these linkages should lower toxicity resulting in
increased efficacy and higher specificity of these molecules.
[0122] Nucleic acid molecules having chemical modifications that
maintain or enhance activity are provided. Such nucleic acid is
also generally more resistant to nucleases than unmodified nucleic
acid. Nucleic acid molecules are preferably resistant to nucleases
in order to function as effective intracellular therapeutic agents.
Improvements in the chemical synthesis of RNA and DNA (Wincott et
al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992,
Methods in Enzymology 211, 3-19 (incorporated by reference herein)
have expanded the ability to modify nucleic acid molecules by
introducing nucleotide modifications to enhance their nuclease
stability as described above. The use of the nucleic acid-based
molecules of the invention can lead to better treatment of the
disease progression by affording the possibility of combination
therapies (e.g., multiple antisense or enzymatic nucleic acid
molecules targeted to different genes, nucleic acid molecules
coupled with known small molecule inhibitors, or intermittent
treatment with combinations of molecules and/or other chemical or
biological molecules). The treatment of subjects with nucleic acid
molecules can also include combinations of different types of
nucleic acid molecules.
[0123] In one embodiment, nucleic acid catalysts having chemical
modifications that maintain or enhance enzymatic activity are
provided. Such nucleic acids are also generally more resistant to
nucleases than unmodified nucleic acid.
[0124] In one embodiment, the invention features modified enzymatic
nucleic acid molecules with phosphate backbone modifications
comprising one or more phosphorothioate, phosphorodithioate,
methylphosphonate, morpholino, amidate carbamate, carboxymethyl,
acetamidate, polyamide, sulfonate, sulfonamide, sulfamate,
formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a
review of oligonucleotide backbone modifications see Hunziker and
Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in
Modern Synthetic Methods, VCH, 331 417, and Mesmaeker et al., 1994,
Novel Backbone Replacements for Oligonucleotides, in Carbohydrate
Modifications in Antisense Research, ACS, 24 39. These references
are hereby incorporated by reference herein. Various modifications
to nucleic acid (e.g., antisense and ribozyme) structure can be
made to enhance the utility of these molecules. For example, such
modifications can enhance shelf-life, half-life in vitro,
bioavailability, stability, and ease of introduction of such
oligonucleotides to the target site, including e.g., enhancing
penetration of cellular membranes and conferring the ability to
recognize and bind to targeted cells.
[0125] Administration of Nucleic Acid Molecules. Methods for the
delivery of nucleic acid molecules are described in Akhtar et al.,
1992, Trends Cell Bio., 2, 139; and Delivery Strategies for
Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995 which are
both incorporated herein by reference. Sullivan et al., PCT WO
94/02595, further describes the general methods for delivery of
enzymatic RNA molecules. These protocols can be utilized for the
delivery of virtually any nucleic acid molecule. Nucleic acid
molecules can be administered to cells by a variety of methods
known to those familiar to the art, including, but not restricted
to, encapsulation in liposomes, by iontophoresis, or by a
incorporation into other vehicles, such as hydrogels,
cyclodextrins, biodegradable nanocapsules, and bioadhesive
microspheres. Alternatively, the nucleic acid/vehicle combination
is locally delivered by direct injection or by use of an infusion
pump. Other routes of delivery include, but are not limited to oral
(tablet or pill form) and/or intrathecal delivery (Gold, 1997,
Neuroscience, 76, 1153-1158). Other approaches include the use of
various transport and carrier systems, for example, through the use
of conjugates and biodegradable polymers. For a comprehensive
review on drug delivery strategies including CNS delivery, see Ho
et al., 1999, Curr. Opin. Mol. Ther., 1, 336-343 and Jain, Drug
Delivery Systems: Technologies and Commercial Opportunities,
Decision Resources, 1998 and Groothuis et al., 1997, J.
NeuroVirol., 3, 387-400.
[0126] The molecules of the instant invention can be used as
pharmaceutical agents. Pharmaceutical agents prevent, inhibit the
occurrence, or treat (alleviate a symptom to some extent,
preferably all of the symptoms) a disease state in a subject.
[0127] The negatively charged polynucleotides of the invention can
be administered (e.g., RNA, DNA or protein) and introduced into a
subject by any standard means, with or without stabilizers,
buffers, and the like, to form a pharmaceutical composition. When
it is desired to use a liposome delivery mechanism, standard
protocols for formation of liposomes can be followed. The
compositions of the present invention can also be formulated and
used as tablets, capsules or elixirs for oral administration;
suppositories for rectal administration; sterile solutions;
suspensions for injectable administration; and the other
compositions known in the art.
[0128] The present invention also includes pharmaceutically
acceptable formulations of the compounds described. These
formulations include salts of the above compounds, e.g., acid
addition salts, for example, salts of hydrochloric, hydrobromic,
acetic acid, and benzene sulfonic acid.
[0129] A pharmacological composition or formulation refers to a
composition or formulation in a form suitable for administration,
e.g., systemic administration, into a cell or subject, preferably a
human. By "systemic administration" is meant in vivo systemic
absorption or accumulation of drugs in the blood stream followed by
distribution throughout the entire body. Suitable forms, in part,
depend upon the use or the route of entry, for example oral,
transdermal, or by injection. Such forms should not prevent the
composition or formulation from reaching a target cell (i.e., a
cell to which the negatively charged polymer is desired to be
delivered to). For example, pharmacological compositions injected
into the blood stream should be soluble. Other factors are known in
the art, and include considerations such as toxicity and forms
which prevent the composition or formulation from exerting its
effect.
[0130] Administration routes which lead to systemic absorption
include, without limitations: intravenous, subcutaneous,
intraperitoneal, inhalation, oral, intrapulmonary and
intramuscular. The rate of entry of a drug into the circulation has
been shown to be a function of molecular weight or size. The use of
a liposome or other drug carrier comprising the compounds of the
instant invention can potentially localize the drug, for example,
in certain tissue types, such as the tissues of the reticular
endothelial system (RES). A liposome formulation which can
facilitate the association of drug with the surface of cells, such
as, lymphocytes and macrophages is also useful.
[0131] By pharmaceutically acceptable formulation is meant, a
composition or formulation that allows for the effective
distribution of the nucleic acid molecules of the instant invention
in the physical location most suitable for their desired activity.
Non-limiting examples of agents suitable for formulation with the
nucleic acid molecules of the instant invention include: PEG
conjugated nucleic acids, phospholipid conjugated nucleic acids,
nucleic acids containing lipophilic moieties, phosphorothioates,
P-glycoprotein inhibitors (such as Pluronic P85) which can enhance
entry of drugs into various tissues, for example the CNS
(Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13,
16-26); biodegradable polymers, such as poly
(DL-lactide-coglycolide) microspheres for sustained release
delivery after implantation (Emerich, D F et al, 1999, Cell
Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.; and loaded
nanoparticles, such as those made of polybutylcyanoacrylate, which
can deliver drugs across the blood brain barrier and can alter
neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol
Psychiatry, 23, 941-949, 1999). Other non-limiting examples of
delivery strategies, including CNS delivery of nucleic acid
molecules include material described in Boado et al., 1998, J.
Pharm. Sci., 87, 1308-1315; Tyler et al, 1999, FEBS Lett., 421,
280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado,
1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al.,
1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999,
PNAS USA., 96, 7053-7058. All these references are hereby
incorporated herein by reference.
[0132] The invention also features the use of the composition
comprising surface-modified liposomes containing poly (ethylene
glycol) lipids (PEG-modified, or long-circulating liposomes or
stealth liposomes). Nucleic acid molecules of the invention can
also comprise covalently attached PEG molecules of various
molecular weights. These formulations offer a method for increasing
the accumulation of drugs in target tissues. This class of drug
carriers resists opsonization and elimination by the mononuclear
phagocytic system (MPS or RES), thereby enabling longer blood
circulation times and enhanced tissue exposure for the encapsulated
drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al.,
Chem. Pharm. Bull. 1995, 43, 1005-1011). Long-circulating liposomes
are also likely to protect drugs from nuclease degradation to a
greater extent compared to cationic liposomes, based on their
ability to avoid accumulation in metabolically aggressive MPS
tissues such as the liver and spleen. All of these references are
incorporated by reference herein.
[0133] The present invention also includes compositions prepared
for storage or administration which include a pharmaceutically
effective amount of the desired compounds in a pharmaceutically
acceptable carrier or diluent. Acceptable carriers or diluents for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing Co. (A. R. Gennaro edit. 1985) hereby incorporated
by reference herein. For example, preservatives, stabilizers, dyes
and flavoring agents can be provided. These include sodium
benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In
addition, antioxidants and suspending agents can be used.
[0134] A pharmaceutically effective dose or pharmaceutically
effective amount is that dose required to prevent, inhibit the
occurrence, or treat (alleviate a symptom to some extent,
preferably all of the symptoms) of a disease state. The
pharmaceutically effective dose depends on the type of disease, the
composition used, the route of administration, the type of mammal
being treated, the physical characteristics of the specific mammal
under consideration, concurrent medication, and other factors which
those skilled in the medical arts will recognize. Generally, an
amount between 0.1 mg/kg and 1000 mg/kg body weight/day of active
ingredients is administered dependent upon potency of the
negatively charged polymer.
[0135] The formulations can be administered orally, topically,
parenterally, by inhalation or spray or rectally in dosage unit
formulations containing conventional non-toxic pharmaceutically
acceptable carriers, adjuvants and vehicles. The term parenteral as
used herein includes percutaneous, subcutaneous, intravascular
(e.g., intravenous), intramuscular, or intrathecal injection or
infusion techniques and the like. In addition, there is provided a
pharmaceutical formulation comprising a nucleic acid molecule of
the invention and a pharmaceutically acceptable carrier. One or
more nucleic acid molecules of the invention can be present in
association with one or more non-toxic pharmaceutically acceptable
carriers and/or diluents and/or adjuvants, and if desired other
active ingredients. The pharmaceutical compositions of the
invention can be in a form suitable for oral use, for example, as
tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules, emulsion, hard or soft capsules,
or syrups or elixirs.
[0136] Compositions intended for oral use can be prepared according
to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions can contain one
or more such sweetening agents, flavoring agents, coloring agents
or preservative agents in order to provide pharmaceutically elegant
and palatable preparations. Tablets contain the active ingredient
in admixture with non-toxic pharmaceutically acceptable excipients
that are suitable for the manufacture of tablets. These excipients
can be for example, inert diluents, such as calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example magnesium stearate,
stearic acid or talc. The tablets can be uncoated or they can be
coated by known techniques. In some cases such coatings can be
prepared by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monosterate or glyceryl distearate can be
employed.
[0137] Formulations for oral use can also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0138] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents can be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions can also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one
or more sweetening agents, such as sucrose or saccharin.
[0139] Oily suspensions can be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions can contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
and flavoring agents can be added to provide palatable oral
preparations. These compositions can be preserved by the addition
of an anti-oxidant such as ascorbic acid.
[0140] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents or suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, can also be present.
[0141] Pharmaceutical compositions of the invention can also be in
the form of oil-in-water emulsions. The oily phase can be a
vegetable oil or a mineral oil or mixtures of these. Suitable
emulsifying agents can be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions can also contain sweetening and flavoring
agents.
[0142] Syrups and elixirs can be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol, glucose or
sucrose. Such formulations can also contain a demulcent, a
preservative and flavoring and coloring agents. The pharmaceutical
compositions can be in the form of a sterile injectable aqueous or
oleaginous suspension. This suspension can be formulated according
to the known art using those suitable dispersing or wetting agents
and suspending agents that have been mentioned above. The sterile
injectable preparation can also be a sterile injectable solution or
suspension in a non-toxic parentally acceptable diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that can be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil can be
employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of
injectables.
[0143] Nucleic acid molecules of the invention can also be
administered in the form of suppositories, e.g., for rectal
administration of the drug or via a catheter directly to the
bladder itself These compositions can be prepared by mixing the
drug with a suitable non-irritating excipient that is solid at
ordinary temperatures but liquid at the rectal temperature and will
therefore melt in the rectum to release the drug. Such materials
include cocoa butter and polyethylene glycols.
[0144] Nucleic acid molecules of the invention can be administered
parenterally in a sterile medium. The drug, depending on the
vehicle and concentration used, can either be suspended or
dissolved in the vehicle. Advantageously, adjuvants such as local
anesthetics, preservatives and buffering agents can be dissolved in
the vehicle.
[0145] The amount of active ingredient that can be combined with
the carrier materials to produce a single dosage form varies
depending upon the host treated and the particular mode of
administration. Dosage unit forms generally contain between from
about 1 mg to about 1000 mg of an active ingredient.
[0146] It is understood that the specific dose level for any
particular patient or subject depends upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, and rate of excretion, drug combination
and the severity of the particular disease undergoing therapy.
[0147] For administration to non-human animals, the composition can
also be added to the animal feed or drinking water. It can be
convenient to formulate the animal feed and drinking water
compositions so that the animal takes in a therapeutically
appropriate quantity of the composition along with its diet. It can
also be convenient to present the composition as a premix for
addition to the feed or drinking water.
[0148] The composition can also be administered to a subject in
combination with other therapeutic compounds to increase the
overall therapeutic effect. The use of multiple compounds to treat
an indication can increase the beneficial effects while reducing
the presence of side effects.
[0149] Alternatively, certain of the nucleic acid molecules of the
instant invention can be expressed within cells from eukaryotic
promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345;
McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399;
Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591 5;
Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3 15; propulic
et al., 1992, J. Virol., 66, 1432 41; Weerasinghe et al., 1991, J.
Virol., 65, 5531 4; Ojwang et al., 1992, Proc. Natl. Acad. Sci.
USA, 89, 10802 6; Chen et al., 1992, Nucleic Acids Res., 20, 4581
9; Sarver et al., 1990 Science, 247, 1222 1225; Thompson et al,
1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene
Therapy, 4, 45; all of these references are hereby incorporated in
their totalities by reference herein). Those skilled in the art
realize that any nucleic acid can be expressed in eukaryotic cells
from the appropriate DNA/RNA vector.
[0150] In one aspect the invention features an expression vector
comprising a nucleic acid sequence encoding at least one of the
nucleic acid molecules of the instant invention. The nucleic acid
sequence encoding the nucleic acid molecule of the instant
invention is operably linked in a manner which allows expression of
that nucleic acid molecule.
[0151] Transcription of the nucleic acid molecule sequences are
driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA
polymerase II (pol II), or RNA polymerase III (pol III).
Transcripts from pol II or pol III promoters are expressed at high
levels in all cells; the levels of a given pol II promoter in a
given cell type depends on the nature of the gene regulatory
sequences (enhancers, silencers, etc.) present nearby. Prokaryotic
RNA polymerase promoters are also used, providing that the
prokaryotic RNA polymerase enzyme is expressed in the appropriate
cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87,
6743 7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867 72; Lieber
et al., 1993, Methods Enzymol., 217, 47 66; Zhou et al., 1990, Mol.
Cell. Biol., 10, 4529 37). All of these references are incorporated
by reference herein. Several investigators have demonstrated that
nucleic acid molecules, such as ribozymes expressed from such
promoters can function in mammalian cells (e.g. Kashani-Sabet et
al., 1992, Antisense Res. Dev., 2, 3 15; Ojwang et al., 1992, Proc.
Natl. Acad. Sci. USA, 89, 10802 6; Chen et al, 1992, Nucleic Acids
Res., 20, 4581 9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90,
6340 4; L'Huillier et al., 1992, EMBO J., 11, 4411 8; Lisziewicz et
al., 1993, Proc. Natl. Acad. Sci. U.S.A, 90, 8000 4; Thompson et
al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,
1993, Science, 262, 1566).
[0152] In another aspect the invention features an expression
vector comprising nucleic acid sequence encoding at least one of
the nucleic acid molecules of the invention, in a manner which
allows expression of that nucleic acid molecule. The expression
vector comprises in one embodiment; a) a transcription initiation
region; b) a transcription termination region; c) a nucleic acid
sequence encoding at least one said nucleic acid molecule; and
wherein said sequence is operably linked to said initiation region
and said termination region, in a manner which allows expression
and/or delivery of said nucleic acid molecule.
[0153] A further object of the present invention is to provide a
kit comprising a suitable container, the therapeutic of the
invention in a pharmaceutically acceptable form disposed therein,
and instructions for its use.
[0154] In another embodiment, an isolated nucleic acid molecule of
the invention comprises a nucleic acid molecule that is a
complement of the nucleotide sequence of MG53, a MG53 binding
protein, and/or a MG53 receptor. As used herein, the term
"complementary" refers to Watson-Crick or Hoogsteen base pairing
between nucleotides units of a nucleic acid molecule, and the term
"binding" means the physical or chemical interaction between two
polypeptides or compounds or associated polypeptides or compounds
or combinations thereof. Binding includes ionic, non-ionic, van der
Waals, hydrophobic interactions, and the like. A physical
interaction can be either direct or indirect.
[0155] As used herein, "fragments" are defined as sequences of at
least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino
acids, a length sufficient to allow for specific hybridization in
the case of nucleic acids or for specific recognition of an epitope
in the case of amino acids, and are at most some portion less than
a full length sequence.
[0156] The term "host cell" includes a cell that might be used to
carry a heterologous nucleic acid, or expresses a peptide or
protein encoded by a heterologous nucleic acid. A host cell can
contain genes that are not found within the native
(non-recombinant) form of the cell, genes found in the native form
of the cell where the genes are modified and re-introduced into the
cell by artificial means, or a nucleic acid endogenous to the cell
that has been artificially modified without removing the nucleic
acid from the cell. A host cell may be eukaryotic or prokaryotic.
General growth conditions necessary for the culture of bacteria can
be found in texts such as BERGEY'S MANUAL OF SYSTEMATIC
BACTERIOLOGY, Vol. 1, N. R. Krieg, ed., Williams and Wilkins,
Baltimore/London (1984). A "host cell" can also be one in which the
endogenous genes or promoters or both have been modified to produce
one or more of the polypeptide components of the complex of the
invention.
[0157] "Derivatives" are compositions formed from the native
compounds either directly, by modification, or by partial
substitution.
[0158] "Analogs" are nucleic acid sequences or amino acid sequences
that have a structure similar to, but not identical to, the native
compound.
[0159] Derivatives or analogs of the nucleic acids or proteins of
the invention include, but are not limited to, molecules comprising
regions that are substantially homologous to the nucleic acids or
proteins of the invention, in various embodiments, by at least
about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% identity (with a
preferred identity of 80-95%) over a nucleic acid or amino acid
sequence of identical size or when compared to an aligned sequence
in which the alignment is done by a computer homology program known
in the art, or whose encoding nucleic acid is capable of
hybridizing to the complement of a sequence encoding the proteins
of the invention under stringent, moderately stringent, or low
stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993.
Nucleic acid derivatives and modifications include those obtained
by gene replacement, site-specific mutation, deletion, insertion,
recombination, repair, shuffling, endonuclease digestion, PCR,
subcloning, and related techniques.
[0160] "Homologs" can be naturally occurring, or created by
artificial synthesis of one or more nucleic acids having related
sequences, or by modification of one or more nucleic acid to
produce related nucleic acids. Nucleic acids are homologous when
they are derived, naturally or artificially, from a common ancestor
sequence (e.g., orthologs or paralogs). If the homology between two
nucleic acids is not expressly described, homology can be inferred
by a nucleic acid comparison between two or more sequences. If the
sequences demonstrate some degree of sequence similarity, for
example, greater than about 30% at the primary amino acid structure
level, it is concluded that they share a common ancestor. For
purposes of the present invention, genes are homologous if the
nucleic acid sequences are sufficiently similar to allow
recombination and/or hybridization under low stringency
conditions.
[0161] As used herein "hybridization," refers to the binding,
duplexing, or hybridizing of a molecule only to a particular
nucleotide sequence under low, medium, or highly stringent
conditions, including when that sequence is present in a complex
mixture (e.g., total cellular) DNA or RNA.
[0162] Furthermore, one of ordinary skill will recognize that
"conservative mutations" also include the substitution, deletion or
addition of nucleic acids that alter, add or delete a single amino
acid or a small number of amino acids in a coding sequence where
the nucleic acid alterations result in the substitution of a
chemically similar amino acid. Amino acids that may serve as
conservative substitutions for each other include the following:
Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic
acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q);
hydrophilic: Glycine (G), Alanine (A), Valine (V), Leucine (L),
Isoleucine (I); Hydrophobic: Phenylalanine (F), Tyrosine (Y),
Tryptophan (W); Sulfur-containing Methionine (M), Cysteine (C). In
addition, sequences that differ by conservative variations are
generally homologous.
[0163] Descriptions of the molecular biological techniques useful
to the practice of the invention including mutagenesis, PCR,
cloning, and the like include Berger and Kimmel, GUIDE TO MOLECULAR
CLONING TECHNIQUES, METHODS IN ENZYMOLOGY, volume 152, Academic
Press, Inc., San Diego, Calif. (Berger); Sambrook et al., MOLECULAR
CLONING--A LABORATORY MANUAL (2nd Ed.), Vol. 1-3, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1989, and CURRENT
PROTOCOLS 1N MOLECULAR BIOLOGY, F. M. Ausubel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc.; Berger, Sambrook, and
Ausubel, as well as Mullis et al., U.S. Pat. No. 4,683,202 (1987);
PCR PROTOCOLS A GUIDE TO METHODS AND APPLICATIONS (Innis et al.
eds), Academic Press, Inc., San Diego, Calif. (1990) (Innis);
Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; Lueng, et
al.
[0164] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. For suitable expression systems for both prokaryotic and
eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,
MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0165] A polynucleotide can be a DNA molecule, a cDNA molecule,
genomic DNA molecule, or an RNA molecule. A polynucleotide as DNA
or RNA can include a sequence wherein T (thymidine) can also be U
(uracil). If a nucleotide at a certain position of a polynucleotide
is capable of forming a Watson-Crick pairing with a nucleotide at
the same position in an anti-parallel DNA or RNA strand, then the
polynucleotide and the DNA or RNA molecule are complementary to
each other at that position. The polynucleotide and the DNA or RNA
molecule are substantially complementary to each other when a
sufficient number of corresponding positions in each molecule are
occupied by nucleotides that can hybridize with each other in order
to effect the desired process.
[0166] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. By "transformation" is meant a permanent or
transient genetic change induced in a cell following incorporation
of new DNA (i.e., DNA exogenous to the cell).
[0167] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes
Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton,
1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell
receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and
immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and
Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc.
Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters
(Edlund, et al., 1985. Science 230: 912-916), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, e.g., the
murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379)
and the alpha-fetoprotein promoter (Campes and Tilghman, 1989.
Genes Dev. 3: 537-546).
[0168] In any of the embodiments, the nucleic acids encoding the
MG53, MG53 binding protein, and/or MG53 receptor can be present as:
one or more naked DNAs; one or more nucleic acids disposed in an
appropriate expression vector and maintained episomally; one or
more nucleic acids incorporated into the host cell's genome; a
modified version of an endogenous gene encoding the components of
the complex; one or more nucleic acids in combination with one or
more regulatory nucleic acid sequences; or combinations thereof.
The nucleic acid may optionally comprise a linker peptide or fusion
protein component, for example, His-Tag, FLAG-Tag, fluorescent
protein, GST, TAT, an antibody portion, a signal peptide, and the
like, at the 5' end, the 3' end, or at any location within the
ORF.
[0169] In a preferred embodiment, the nucleic acid of the invention
comprises a polynucleotide encoding the soluble (i.e., the
extracellular) portion of a MG53 receptor. Any of the embodiments
described herein, can be achieved using standard molecular
biological and genetic approaches well known to those of ordinary
skill in the art.
[0170] Where the host is prokaryotic, such as E. coli, competent
cells which are capable of DNA uptake can be prepared from cells
harvested after exponential growth phase and subsequently treated
by the CaCl.sub.2 method by procedures well known in the art.
Alternatively, MgCl.sub.2, RbCl, liposome, or liposome-protein
conjugate can be used. Transformation can also be performed after
forming a protoplast of the host cell or by electroporation. These
examples are not limiting on the present invention; numerous
techniques exist for transfecting host cells that are well known by
those of skill in the art and which are contemplated as being
within the scope of the present invention.
[0171] When the host is a eukaryote, such methods of transfection
with DNA include calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors, as
well as others known in the art, may be used. The eukaryotic cell
may be a yeast cell (e.g., Saccharomyces cerevisiae) or may be a
mammalian cell, including a human cell. For long-term, high-yield
production of recombinant proteins, stable expression is
preferred.
[0172] Polypeptides
[0173] By "MG53," "MG53 binding protein," and "MG53 receptor"
proteins is meant, a peptide or protein comprising a full length
MG53, MG53 binding protein or a MG53 receptor protein, domain,
fusion protein, chimera, or fragment thereof.
[0174] In other embodiments, the invention pertains to isolated
nucleic acid molecules that encode MG53, MG53 binding proteins,
and/or MG53 receptor polypeptides, antibody polypeptides, or
biologically active portions thereof. The polypeptides of the
complex can be formed, for example, using a peptide synthesizer
according to standard methods; or by expressing each polypeptide
separately in a cell or cell extract, then isolating and purifying
the polypeptide.
[0175] Antibodies
[0176] The term "antibody" as used herein refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
(Ig) molecules, i.e., molecules that contain an antigen-binding
site that specifically binds (immunoreacts with) an antigen,
comprising at least one, and preferably two, heavy (H) chain
variable regions (abbreviated herein as VH), and at least one and
preferably two light (L) chain variable regions (abbreviated herein
as VL). Such antibodies include, but are not limited to,
polyclonal, monoclonal, chimeric, single chain, Fab, Fab' and
F(ab')2 fragments, and an Fab expression library. The VH and VL
regions can be further subdivided into regions of hypervariability,
termed "complementarity determining regions" ("CDR"), interspersed
with regions that are more conserved, termed "framework regions"
(FR). The extent of the framework region and CDR's has been
precisely defined (see, Kabat, E. A., et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242, and
Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are
incorporated herein by reference). Each VH and VL is composed of
three CDR's and four FRs, arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, FR4. In general, antibody molecules obtained from humans
relates to any of the classes IgG, IgM, IgA, IgE and IgD, which
differ from one another by the nature of the heavy chain present in
the molecule. Certain classes have subclasses as well, such as
IgG.sub.1, IgG.sub.2, and others. Furthermore, in humans, the light
chain may be a kappa chain or a lambda chain. Reference herein to
antibodies includes a reference to all such classes, subclasses and
types of human antibody species.
[0177] Antibodies can be prepared from the intact polypeptide or
fragments containing peptides of interest as the immunizing agent.
A preferred antigenic polypeptide fragment is 15-100 contiguous
amino acids of MG53, MG53 binding protein, or MG53 receptor
protein. In one embodiment, the peptide is located in a
non-transmembrane domain of the polypeptide, e.g., in an
extracellular or intracellular domain. An exemplary antibody or
antibody fragment binds to an epitope that is accessible from the
extracellular milieu and that alters the functionality of the
protein. In certain embodiments, the present invention comprises
antibodies that recognize and are specific for one or more epitopes
of a MG53 protein, MG53 binding protein, and/or MG53 receptor
protein, variants, portions and/or combinations thereof. In
alternative embodiments antibodies of the invention may target and
interfere with the MG53/MG53 receptor interaction to inhibit
signaling.
[0178] The preparation of monoclonal antibodies is well known in
the art; see for example, Harlow et al., Antibodies: A Laboratory
Manual, page 726 (Cold Spring Harbor Pub. 1988). Monoclonal
antibodies can be obtained by injecting mice or rabbits with a
composition comprising an antigen, verifying the presence of
antibody production by removing a serum sample, removing the spleen
to obtain B lymphocytes, fusing the lymphocytes with myeloma cells
to produce hybridomas, cloning the hybridomas, selecting positive
clones that produce antibodies to the antigen, and isolating the
antibodies from the hybridoma cultures. Monoclonal antibodies can
be isolated and purified from hybridoma cultures by techniques well
known in the art.
[0179] In other embodiments, the antibody can be recombinantly
produced, e.g., produced by phage display or by combinatorial
methods. Phage display and combinatorial methods can be used to
isolate recombinant antibodies that bind to MG53, MG53 binding
proteins, and/or MG53 receptor proteins or fragments thereof (as
described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Fuchs et
al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum
Antibod Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; Clackson et al. (1991) Nature 352:624-628; Gram et
al. (1992) PNAS 89:3576-3580.
[0180] Human monoclonal antibodies can also be generated using
transgenic mice carrying the human immunoglobulin genes rather than
the mouse system. Splenocytes from these transgenic mice immunized
with the antigen of interest are used to produce hybridomas that
secrete human mAbs with specific affinities for epitopes from a
human protein (see, e.g., Wood et al. International Application WO
91/00906; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L.
et al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994
Proc. Natl. Acad. Sci. USA 81:6851-6855).
[0181] A therapeutically useful antibody to the components of the
complex of the invention or the complex itself may be derived from
a "humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse complementarity determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain, then substituting
human residues into the framework regions of the murine
counterparts. The use of antibody components derived from humanized
monoclonal antibodies obviates potential problems associated with
immunogenicity of murine constant regions. Techniques for producing
humanized monoclonal antibodies can be found in Jones et al.,
Nature 321: 522, 1986 and Singer et al., J. Immunol. 150: 2844,
1993. The antibodies can also be derived from human antibody
fragments isolated from a combinatorial immunoglobulin library;
see, for example, Barbas et al., Methods: A Companion to Methods in
Enzymology 2, 119, 1991. In addition, chimeric antibodies can be
obtained by splicing the genes from a mouse antibody molecule with
appropriate antigen specificity together with genes from a human
antibody molecule of appropriate biological specificity; see, for
example, Takeda et al., Nature 314: 544-546, 1985. A chimeric
antibody is one in which different portions are derived from
different animal species.
[0182] Anti-idiotype technology can be used to produce monoclonal
antibodies that mimic an epitope. An anti-idiotypic monoclonal
antibody made to a first monoclonal antibody will have a binding
domain in the hypervariable region that is the "image" of the
epitope bound by the first monoclonal antibody. Alternatively,
techniques used to produce single chain antibodies can be used to
produce 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.
Antibody fragments that recognize specific epitopes, e.g.,
extracellular epitopes, can be generated by techniques well known
in the art. Such fragments include Fab fragments produced by
proteolytic digestion, and Fab fragments generated by reducing
disulfide bridges. When used for immunotherapy, the monoclonal
antibodies, fragments thereof, or both may be unlabelled or labeled
with a therapeutic agent. These agents can be coupled directly or
indirectly to the monoclonal antibody by techniques well known in
the art, and include such agents as drugs, radioisotopes, lectins
and toxins.
[0183] The dosage ranges for the administration of monoclonal
antibodies are large enough to produce the desired effect, and will
vary with age, condition, weight, sex, age and the extent of the
condition to be treated, and can readily be determined by one
skilled in the art. Dosages can be about 0.1 mg/kg to about 2000
mg/kg. The monoclonal antibodies can be administered intravenously,
intraperitoneally, intramuscularly, and/or subcutaneously.
[0184] In certain embodiments of the invention, at least one
epitope encompassed by the antigenic peptide is a region of MG53, a
MG53 binding protein, and/or a MG53 receptor that is located on the
surface of the protein, e.g., a hydrophilic region. A
hydrophobicity analysis of the protein sequence will indicate which
regions of a polypeptide are particularly hydrophilic and,
therefore, are likely to encode surface residues useful for
targeting antibody production. As a means for targeting antibody
production, hydropathy plots showing regions of hydrophilicity and
hydrophobicity may be generated by any method well known in the
art, including, for example, the Kyte Doolittle or the Hopp Woods
methods, either with or without Fourier transformation. See, e.g.,
Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte
and Doolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated
herein by reference in their entirety. Antibodies that are specific
for one or more domains within an antigenic protein, or
derivatives, fragments, analogs or homologs thereof, are also
provided herein. A protein of the invention, or a derivative,
fragment, analog, homolog or ortholog thereof, may be utilized as
an immunogen in the generation of antibodies that
immunospecifically bind these protein components.
[0185] Human Antibodies
[0186] Fully human antibodies essentially relate to antibody
molecules in which the entire sequence of both the light chain and
the heavy chain, including the CDRs, arise from human genes. Such
antibodies are termed "human antibodies", or "fully human
antibodies" herein. Human monoclonal antibodies can be prepared by
the trioma technique; the human B-cell hybridoma technique (see
Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma
technique to produce human monoclonal antibodies (see Cole, et al.,
1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,
Inc., pp. 77-96). Human monoclonal antibodies may be utilized in
the practice of the present invention and may be produced by using
human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA
80: 2026-2030) or by transforming human B-cells with Epstein Barr
Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES
AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
[0187] In addition, human antibodies can also be produced using
additional techniques, including phage display libraries
(Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al.,
J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be
made by introducing human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in Marks et al.
(Bio/Technology, 10:779-783 (1992)); Lonberg et al. (Nature,
368:856-859 (1994)); Morrison (Nature, 368:812-13 (1994)); Fishwild
et al, (Nature Biotechnology, 14:845-51 (1996)); Neuberger (Nature
Biotechnology, 14:826 (1996)); and Lonberg and Huszar (Intern. Rev.
Immunol., 13:65-93 (1995)).
[0188] Human antibodies may additionally be produced using
transgenic nonhuman animals which are modified so as to produce
fully human antibodies rather than the animal's endogenous
antibodies in response to challenge by an antigen. The endogenous
genes encoding the heavy and light immunoglobulin chains in the
nonhuman host have been incapacitated, and active loci encoding
human heavy and light chain immunoglobulins are inserted into the
host's genome. The human genes are incorporated, for example, using
yeast artificial chromosomes containing the requisite human DNA
segments. An animal which provides all the desired modifications is
then obtained as progeny by crossbreeding intermediate transgenic
animals containing fewer than the full complement of the
modifications. The preferred embodiment of such a nonhuman animal
is a mouse, and is termed the Xenomouse.TM. as disclosed in PCT
publications WO 96/33735 and WO 96/34096.
[0189] Fab Fragments and Single Chain Antibodies
[0190] According to the invention, techniques can be adapted for
the production of single-chain antibodies specific to an antigenic
protein of the invention (see e.g., U.S. Pat. No. 4,946,778). In
addition, methods can be adapted for the construction of Fab
expression libraries (see e.g., Huse, et al., Science 246:1275-1281
(1989)) to allow rapid and effective identification of monoclonal
Fab fragments with the desired specificity for a protein or
derivatives, fragments, analogs or homologs thereof. Antibody
fragments that contain the idiotypes to a protein antigen may be
produced by techniques known in the art including, but not limited
to: (i) an F(ab')2 fragment produced by pepsin digestion of an
antibody molecule; (ii) an Fab fragment generated by reducing the
disulfide bridges of an F(ab')2 fragment; (iii) an Fab fragment
generated by the treatment of the antibody molecule with papain and
a reducing agent and (iv) Fv fragments.
[0191] Bispecific Antibodies
[0192] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for an antigenic protein of the invention. The
second binding target is any other antigen, and advantageously is a
cell-surface protein or receptor or receptor subunit. Methods for
making bispecific antibodies are known in the art. Traditionally,
the recombinant production of bispecific antibodies is based on the
co-expression of two immunoglobulin heavy-chain/light-chain pairs,
where the two heavy chains have different specificities (Milstein
and Cuello, Nature, 305:537-539 (1983)). Because of the random
assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of ten different
antibody molecules, of which only one has the correct bispecific
structure. Similar procedures are disclosed in WO 93/08829,
published May 13, 1993, and Traunecker et al., EMBO J.,
10:3655-3659 (1991).
[0193] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986); and Brennan et al., Science
229:81 (1985).
[0194] Additionally, Fab' fragments can be directly recovered from
E. coli and chemically coupled to form bispecific antibodies.
Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the
production of a fully humanized bispecific antibody F(ab')2
molecule. Each Fab' fragment was separately secreted from E. coli
and subjected to directed chemical coupling in vitro to form the
bispecific antibody. The bispecific antibody thus formed was able
to bind to cells overexpressing the ErbB2 receptor and normal human
T cells, as well as trigger the lytic activity of human cytotoxic
lymphocytes against human breast tumor targets.
[0195] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The "diabody" technology described by
Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)
has provided an alternative mechanism for making bispecific
antibody fragments. Another strategy for making bispecific antibody
fragments by the use of single-chain Fv (sFv) dimers has also been
reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For
example, trispecific antibodies can be prepared. Tutt et al., J.
Immunol. 147:60 (1991). Bispecific antibodies can also be used to
direct cytotoxic agents to cells which express a particular
antigen. These antibodies possess an antigen-binding arm and an arm
which binds a cytotoxic agent or a radionuclide chelator, such as
EOTUBE, DPTA, DOTA, or TETA.
[0196] Heteroconjugate Antibodies
[0197] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980), and for treatment of HIV infection (WO
91/00360; WO 92/200373; EP 03089). It is contemplated that the
antibodies can be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins can be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0198] Immunoconjugates
[0199] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a chemical agent, or a radioactive
isotope (i.e., a radioconjugate). Conjugates of the antibody and
cytotoxic agent are made using a variety of bifunctional
protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)
propionate (SPDP), iminothiolane (IT), bifunctional derivatives of
imidoesters (such as dimethyl adipimidate HCL), active esters (such
as disuccinimidyl suberate), aldehydes (such as glutareldehyde),
bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al.,
Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0200] Immunoliposomes
[0201] The antibodies disclosed herein can also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0202] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al .,
J. Biol. Chem. 257: 286-288 (1982) via a disulfide-interchange
reaction.
[0203] A therapeutically effective amount of an antibody of the
invention relates generally to the amount needed to achieve a
therapeutic objective. As noted above, this may be a binding
interaction between the antibody and its target antigen that, in
certain cases, interferes with the functioning of the target, and
in other cases, promotes a physiological response. The amount
required to be administered will furthermore depend on the binding
affinity of the antibody for its specific antigen, and will also
depend on the rate at which an administered antibody is depleted
from the free volume other subject to which it is administered.
Common ranges for therapeutically effective dosing of an antibody
or antibody fragment of the invention may be, by way of nonlimiting
example, from about 0.1 mg/kg body weight to about 500 mg/kg body
weight.-Common dosing frequencies may range, for example, from
twice daily to once a week.
[0204] Antibodies specifically binding a protein of the invention,
as well as other molecules identified by the screening assays
disclosed herein, can be administered for the treatment of various
disorders in the form of pharmaceutical compositions. Principles
and considerations involved in preparing such compositions, as well
as guidance in the choice of components are provided, for example,
in Remington: The Science And Practice Of Pharmacy 19th ed.
(Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.:
1995; Drug Absorption Enhancement: Concepts, Possibilities,
Limitations, And Trends, Harwood Academic Publishers, Langhorne,
Pa., 1994; and Peptide And Protein Drug Delivery (Advances In
Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York. The active
ingredients can also be entrapped in microcapsules prepared, for
example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacrylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles,
and nanocapsules) or in macroemulsions. The formulations to be used
for in vivo administration must be sterile. This is readily
accomplished by filtration through sterile filtration
membranes.
[0205] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0206] ELISA Assay
[0207] An agent for detecting an analyte protein is an antibody
capable of binding to an analyte protein, preferably an antibody
with a detectable label. Antibodies can be polyclonal, or more
preferably, monoclonal. An intact antibody, or a fragment thereof
(e.g., Fab or F(ab)2) can be used. The term "labeled", with regard
to the probe or antibody, is intended to encompass direct labeling
of the probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with another
reagent that is directly labeled. Examples of indirect labeling
include detection of a primary antibody using a
fluorescently-labeled secondary antibody and end-labeling of a DNA
probe with biotin such that it can be detected with
fluorescently-labeled streptavidin. The term "biological sample" is
intended to include tissues, cells and biological fluids isolated
from a subject, as well as tissues, cells and fluids present within
a subject. Included within the usage of the term "biological
sample", therefore, is blood and a fraction or component of blood
including blood serum, blood plasma, or lymph. That is, the
detection method of the invention can be used to detect an analyte
mRNA, protein, or genomic DNA in a biological sample in vitro as
well as in vivo. For example, in vitro techniques for detection of
an analyte mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of an analyte
protein include enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations, and immunofluorescence. In
vitro techniques for detection of an analyte genomic DNA include
Southern hybridizations. Procedures for conducting immunoassays are
described, for example in "ELISA: Theory and Practice: Methods in
Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press,
Totowa, N.J., 1995; "Immunoassay", E. Diamandis and T.
Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and
"Practice and Thory of Enzyme Immunoassays", P. Tijssen, Elsevier
Science Publishers, Amsterdam, 1985. Furthermore, in vivo
techniques for detection of an analyte protein include introducing
into a subject a labeled anti-an analyte protein antibody. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques intracavity, or transdermally, alone or
with effector cells.
[0208] Preparations for administration of the therapeutic of the
invention include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Aqueous
carriers include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Vehicles include
sodium chloride solution, Ringer's dextrose, dextrose and sodium
chloride, lactated Ringer's intravenous vehicles including fluid
and nutrient replenishers, electrolyte replenishers, and the like.
Preservatives and other additives may be added such as, for
example, antimicrobial agents, anti-oxidants, chelating agents and
inert gases and the like.
[0209] The compounds, nucleic acid molecules, polypeptides, and
antibodies (also referred to herein as "active compounds") of the
invention, and derivatives, fragments, analogs and homologs
thereof, can be incorporated into pharmaceutical compositions
suitable for administration. Such compositions typically comprise
the nucleic acid molecule, protein, or antibody and a
pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. Suitable
carriers are described in the most recent edition of Remington's
Pharmaceutical Sciences, a standard reference text in the field,
which is incorporated herein by reference. Preferred examples of
such carriers or diluents include, but are not limited to, water,
saline, finger's solutions, dextrose solution, and 5% human serum
albumin. Liposomes and non-aqueous vehicles such as fixed oils may
also be used. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0210] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical), transmucosal, intraperitoneal, and
rectal administration. Solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include
the following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfate; chelating
agents such as ethylenediaminetetraacetic acid (EDTA); buffers such
as acetates, citrates or phosphates, and agents for the adjustment
of tonicity such as sodium chloride or dextrose. The pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium
hydroxide. The parenteral preparation can be enclosed in ampoules,
disposable syringes or multiple dose vials made of glass or
plastic.
[0211] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor.TM.. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0212] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., the therapeutic complex of
the invention) in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, methods of preparation are vacuum drying and
freeze-drying that yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0213] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0214] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups, or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring, and sweetening
agents as appropriate.
[0215] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions may take the form of tablets
or lozenges formulated in conventional manner. For administration
by inhalation, the compounds for use according to the present
invention are conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebuliser, with the
use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethan-e, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of e.g. gelatin for use in
an inhaler or insufflator may be formulated containing a powder mix
of the compound and a suitable powder base such as lactose or
starch. The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions, or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing,
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use. The compounds may also be
formulated in rectal compositions such as suppositories or
retention enemas, e.g., containing conventional suppository bases
such as cocoa butter or other glycerides. In addition to the
formulations described previously, the compounds may also be
formulated as a depot preparation. Such long acting formulations
may be administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the compounds may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0216] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0217] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0218] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0219] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0220] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see, e.g., U.S. Pat. No.
5,328,470) or by stereotactic injection (see, e.g., Chen, et al.,
1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells that
produce the gene delivery system. The pharmaceutical compositions
can be included in a container, pack, or dispenser together with
instructions for administration.
[0221] A therapeutically effective dose refers to that amount of
the therapeutic sufficient to result in amelioration or delay of
symptoms. Toxicity and therapeutic efficacy of such compounds can
be determined by standard pharmaceutical procedures in cell
cultures or experimental animals, e.g., for determining the LD50
(the dose lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 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. Compounds that exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects. The data obtained from the
cell culture assays and animal studies can be used in formulating a
range of dosage for use in humans. The dosage of such compounds
lies preferably within a range of circulating concentrations that
include the ED50 with little or no toxicity. The dosage may vary
within this range depending upon the dosage form employed and the
route of administration utilized. For any compound used in the
method of the invention, the therapeutically effective dose can be
estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range that includes the IC50 (i.e., the concentration
of the test compound which achieves a half-maximal inhibition of
symptoms) as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans. Levels in
plasma may be measured, for example, by high performance liquid
chromatography.
[0222] Also disclosed according to the present invention is a kit
or system utilizing any one of the methods, selection strategies,
materials, or components described herein. Exemplary kits according
to the present disclosure will optionally, additionally include
instructions for performing methods or assays, packaging materials,
one or more containers which contain an assay, a device or system
components, or the like.
[0223] Additional objects and advantages of the present invention
will be appreciated by one of ordinary skill in the art in light of
the current description and examples of the preferred embodiments,
and are expressly included within the scope of the present
invention.
EXAMPLES
[0224] Discovery of MG53, a muscle specific TRIM family protein.
MG53 was isolated using a previously established an
immuno-proteomic approach that allows identification of novel
proteins involved in myogenesis, Ca.sup.2+ signaling and
maintenance of membrane integrity in striated muscle cells.
Briefly, this approach uses a monoclonal antibody library
containing .about.6500 clones that was generated from mice
immunized with triad-enriched membranes from rabbit skeletal
muscle. Antibodies of interest were selected based on the z-line
staining patterns of striated muscle sections observed under an
immunofluorescence microscope. The target-proteins were purified
through antibody-affinity column, and partial amino acid sequences
of the purified proteins were obtained. Based on the partial amino
acid sequence, the complete cDNA coding for the target gene was
isolated from a skeletal muscle cDNA library. Homologous gene
screening was then used to search for the presence of different
isoforms of the identified genes in other excitable tissues.
Finally, transgenic or knockout mouse models were generated to
study the in vivo physiological function of genes of interest.
[0225] Screening of this immuno-proteomic library for muscle
specific proteins led to the identification of an antigen
recognized by mAb5259 with a molecular size of 53 kilodaltons (kDa)
specifically with striated muscle tissues (FIG. 3B). The protein,
"MG53", was partially purified from rabbit skeletal muscle by a
mAb5259 immunoaffinity column and subjected to amino acid
sequencing. Skeletal muscle cDNA library screening and genomic
database searches identified the predicted amino acid sequences for
MG53 and the corresponding mg53 gene on the human 16p11.2 locus.
Northern blotting for the mg53 mRNA confirmed specific expression
with skeletal and cardiac muscle (FIG. 3C). Domain homology
analysis revealed that MG53 contains the prototypical tri-partite
motifs that include a Ring, B-box and Coiled-Coil (RBCC) moieties,
as well as a SPRY domain at the carboxyl-terminus (FIGS. 1, 2, and
3A). The SPRY domain is a conserved sequence first observed in the
ryanodine receptor Ca.sup.2+ release channel in the sarcoplasmic
reticulum of excitable cells. Of the approximately 60 TRIM family
members so far identified in various mammalian genomes, 15 members
carry a similar SPRY domain following the RBCC domain, and MG53
shows a conserved primary structure with these TRIM sub-family
proteins.
[0226] MG53 mediates vesicle trafficking in muscle cells. Although
there is no membrane-spanning segment or lipid-modification motif
in its primary structure, MG53 appears to be primarily restricted
to membrane structures in skeletal muscle. Immunohistochemical
analysis revealed specific labeling for MG53 in the sarcolemma
membrane and intracellular vesicles (FIG. 3D). Overexpression of
MG53 in the C2C12 myogenic cell line leads to dramatic
morphological changes. Cells transiently transfected with MG53 and
GFP displayed extensions of the plasma membrane with distinct
filapodia-like structures that were not present in cells expressing
GFP alone (FIG. 4A-D). Using a GFP-MG53 fusion construct, it was
found that MG53 is localized to both intracellular vesicles and the
plasma membrane of C2C12 myoblasts (FIG. 4B). Live cell
fluorescence imaging revealed dynamic intracellular trafficking and
fusion events in C2C12 cells overexpressing GFP-MG53. This GFP-MG53
mediated vesicle fusion at the cell surface membrane results in
budding of GFP-MG53 vesicles off the cell membrane (FIG. 4D). This
is confirmed by imaging of vesicle fusion events at the plasma
membrane using total internal reflection fluorescence (TIRF)
microscopy, which showed that vesicle fusion event are greatly
enhanced by co-expression of MG53 (data not shown). As a whole,
these experiments illustrate that endogenous MG53 is a
muscle-specific TRIM family protein that mediates trafficking of
intracellular vesicles to the sarcolemmal membrane.
[0227] MG53 is a muscle-specific protein that contains TRIM and
SPRY motifs. In previous studies we have established a monoclonal
antibody (mAb) library that targets proteins associated with the
triad junction in skeletal muscle. Screening of this
immuno-proteomic library for muscle specific proteins led to the
identification of an antigen named MG53 with a molecular size of 53
kilodaltons (kDa), which was recognized by mAb5259. MG53 was
partially purified from rabbit skeletal muscle by an immunoaffinity
column conjugated with mAb5259, and subjected to amino acid
sequencing. Based on the obtained partial amino acid sequences,
cDNAs encoding MG53 were isolated from rabbit and mouse skeletal
muscle libraries. Genomic library search identified the
corresponding MG53 gene on the human 16p11.2 locus. The predicted
amino acid sequences for MG53 in several species are shown in FIG.
1.
[0228] Domain homology analysis revealed that MG53 contains the
prototypical TRIM signature sequence of RBCC plus a SPRY domain at
the carboxyl-terminus, and thus belongs to the TRIM/RBCC family
(FIG. 1). Of the approximately 60 TRIM family members so far
identified in the mammalian genomes, 15 members carry a similar
SPRY domain following the RBCC domain, and MG53 shows a conserved
primary structure with these TRIM sub-family proteins (FIG. 2).
However, surprisingly and unexpectedly our studies indicate that
MG53 is the only TRIM family protein of those in FIG. 2 that
demonstrate membrane repair function.
[0229] Western blot assay confirms the muscle-specific expression
of MG53 in mouse tissues (FIG. 3B). Although there is no
membrane-spanning segment or lipid-modification motif in its
primary structure, MG53 appears to be primarily restricted to
membrane structures in skeletal muscle Immunohistochemical analysis
with mAb5259 showed specific labeling for MG53 in the sarcolemmal
and TT membranes in transverse sections of skeletal muscle fibers
(FIG. 3C). Moreover, transverse sections revealed localized
concentration of MG53 near the sarcolemmal membrane, with a broader
staining pattern than is typically observed for integral membrane
proteins of the sarcolemma. Thus, MG53 is a muscle specific TRIM
family protein that displays a unique subcellular distribution
pattern for a TRIM family protein.
[0230] Overexpression of MG53 produces filapodia-like structures in
both excitable and non-excitable cells. To elucidate the cell
biological function of MG53, mouse MG53 cDNA was expressed in C2C12
myogenic cells, as well as Chinese hamster ovary (CHO) cells. C2C12
cells at the myoblast stage do not express endogenous MG53 protein,
however differentiated C2C12 myotubes do express MG53. CHO cells
are non-excitable epithelial cells that contain no endogenous MG53
protein. As shown in FIG. 4A (left panel), transient transfection
of MG53 cDNA into C2C12 myoblasts or CHO cells produced the
expression of a recombinant protein of 53 kDa that could be
recognized by mAb5259. The molecular size of the recombinant
protein is identical to the endogenous MG53 present in both rabbit
and mouse muscles, thus confirming the identity of the isolated
cDNA clone as MG53. Co-transfection of cells with two plasmids
containing cDNAs that encode either EGFP or MG53 at a ratio of 10:1
provided a convenient method to identify transfected cells by
fluorescence microscopy. With confocal microscopic imaging, we
observed dramatic changes in morphology of cells transiently
transfected with MG53 (FIG. 4B). Specifically, extensions of the
cell surface membranes formed distinct filapodia-like structures in
both CHO cells and C2C12 myoblasts that transiently overexpress
MG53.
[0231] To further examine the MG53-induced changes in cell
morphology, two GFP-fusion constructs of MG53 were generated:
GFP-MG53 and MG53-GFP, with attachment of GFP to the amino-terminus
and carboxyl-terminus of MG53, respectively. Although both fusion
proteins can be expressed in CHO cells and C2C12 myoblasts (FIG.
4C, right panel), the subcellular distribution and functional
effects of GFP-MG53 and MG53-GFP were dramatically and surprisingly
different. Using confocal microscopy, it was found that GFP-MG53
fusion proteins were localized to both intracellular vesicles and
cell surface membranes in both CHO and C2C12 cells (FIG. 4C, left
panels). This result is consistent with immunostaining localization
of MG53 in skeletal muscle fibers (FIG. 3C), and suggests that MG53
participates in membrane trafficking events in muscle cells.
[0232] Unexpectedly, the distribution pattern of MG53-GFP fusion
protein was mostly cytosolic in both CHO and C2C12 cells (FIG. 4C,
right panels), which is in sharp contrast to the membrane-attached
distribution of GFP-MG53. In addition, the extensive filapodia-like
membrane extensions induced by overexpression of MG53 or GFP-MG53
were completely absent in cells transfected with MG53-GFP. Since
shielding the carboxyl-terminus of MG53 by fusion with GFP alters
the subcellular distribution of MG53, it is likely that the SPRY
motif at the carboxyl-terminal end of MG53 plays a role in
anchoring MG53 to the different membrane compartments and is
essential for MG53 function (see FIGS. 13 and 14).
[0233] Live cell fluorescence imaging identified dynamic
trafficking of intracellular vesicles, and active exocytotic fusion
and vesicle budding at the cell surface membrane, in cells
overexpressing GFP-MG53 (FIG. 4D). Close examination revealed the
occurrence of vesicle fusion events at the surface membrane (FIG.
4D, left panel). Budding of vesicles containing GFP-MG53 could be
clearly identified, as well as released extracellular vesicles
observed in the vicinity of transfected cells (FIG. 4D, right
panel).
[0234] Taken together, cell imaging studies suggest that MG53 can
localize to both intracellular vesicles and target to cell surface
membranes, and that it is a key mediator of membrane fusion and
vesicle budding.
[0235] MG53 mediates acute membrane repair in skeletal muscle
fibers following cellular injury. Vesicle fusion with the plasma
membrane is required for membrane repair and previous studies
indicate a role for dysferlin in maintenance of skeletal muscle
membrane integrity. Our findings indicate that MG53 is capable of
driving the trafficking of vesicles to the plasma membrane, perhaps
to mediate the repair process following membrane disruption. Acute
cellular injury generated by physical penetration of the plasma
membrane with a microelectrode leads to rapid recruitment of
GFP-MG53 vesicles toward the injury site (FIG. 12A). When more
severe damage that results in fracture of the cell occurs, the
repair site is densely labeled with GFP-MG53 (FIG. 12B). In
addition, this acute membrane repair also was observed in mature
C2C12 myotubes (see movies 2 and 3). This data indicates that
MG53-mediated vesicle trafficking play an active role in acute
repair of cell membrane.
[0236] To further define the physiological function of MG53 in
muscle membrane repair, a mouse model null for MG53 was generated
(FIGS. 9-11). The mg53-/- mice are viable up to 11 month of age
under unstressed conditions. In vivo stress tests revealed severe
defects in membrane repair function of the mg53-/- muscle. As shown
in FIG. 10C, membrane injury induced by down-hill running exercise
revealed severely compromised contractile function of the soleus
muscle from the mg53-/- mice. Without the strenuous exercise,
mg53-/- soleus muscles displayed some difficulty in recovery of
contractile function after ex vivo fatigue stimulation, compared
with the wild type (wt) controls (not shown). These differences can
be drastically exaggerated following exercise-induced damages at
8-10 month of age. Clearly, more severe damage could be found with
the mg53-/- muscle, where weaker and fluctuating contractile
function was observed in comparison with the wt muscle (FIG.
10D).
[0237] Injection of Evans blue dye into the intraperitoneal space
of mice directly monitors sarcolemmal membrane integrity after
down-hill exercise-induced muscle damage. As shown in FIG. 10E,
muscle fibers isolated from the mg53-/- mice showed significantly
more Evans blue staining than the wt muscle, revealing extensive
degree of exercise-induced muscle damage. This was confirmed by H/E
staining that illustrated increased dystrophy in the mg53-/- muscle
that was increased in aged mg53-/- mice compared to young mg53-/-
mice (FIG. 10A). Quantitative assay of total absorbance of Evans
blue extracted from muscle bundles provided direct support for the
increased muscle damage in the mg53-/- mice after down-hill running
(FIG. 10F).
[0238] Consistent with the role of MG53 in membrane repair,
elevated concentrations of MG53 was observed at the site of injury
with immunostaining of individual flexor digitorum brevis (FDB)
muscle fibers that were damaged during isolation (FIG. 11A). These
membrane patches would frequently co-localize with staining for
dysferlin. We directly evaluated the MG53-mediated membrane repair
function through measurement of FM-143 fluorescent dye entry after
laser-induced membrane damage to individual FDB muscle fibers. The
wt muscle fibers possessed intrinsic membrane repair function and
were fairly resistant to laser-induced damage of the sarcolemmal
membrane, as they displayed effective exclusion of the FM-143
fluorescent dye (FIG. 11B). Significant entry of FM-143 fluorescent
dye into the mg53-/- FDB muscle fibers could be observed following
laser-induced damage (FIG. 11C). The time-dependent accumulation of
FM-143 inside the FDB muscle fibers following laser damage of the
sarcolemmal membrane provides direct support for a defective
membrane repair function of the mg53-/- muscle (FIG. 11D).
[0239] Expression of MG53 is essential to maintain normal cardiac
membrane integrity. Defects in mg53-/- mice are not limited to
skeletal muscle fibers. During injection of Evans blue dye
.about.50% of the mg53-/- mice would die within 16 hours of
injection compared to none of the wild type animals injected.
Postmortem examination of mg53-/- hearts revealed extensive
labeling of cardiac muscle fibers with Evans blue, even in absence
of exercise stress (FIG. 9). We also found that exercise would
greatly exacerbate the extent of Evans blue staining in mg53-/-
hearts.
[0240] Role for MG53 in myotube formation during muscle
development. Membrane repair is only one of the cellular processes
that require dynamic trafficking of intracellular vesicles to allow
reorganization of cellular membranes. One such process in skeletal
muscle occurs during myogenesis. During the differentiation of
myoblasts into myotubes, the mononuclear myoblasts must fuse
together to form multinucleated myotubes. To directly examine the
role of MG53-mediated membrane fusion on the myogenesis of skeletal
muscle, a specific RNA interference probe was used to knockdown the
expression of endogenous MG53 in differentiating C2C12 myotubes. A
small hairpin (sh)RNA probe recognizing the nucleotide sequence
632-652 of the mouse MG53 cDNA suppressed greater than 80% of MG53
expression in cells transfected with shRNA-MG53, as compared with
cells transfected with a non-specific shRNA probe for a scrambled
version of the MG53 target sequence (FIG. 5A). Acute suppression of
MG53 resulted in a marked decrease in C2C12 myotube differentiation
(FIG. 5B). C2C12 myoblasts transfected with the shRNA-MG53 probe
formed significantly fewer myotubes at both day 5 and day 10 after
serum deprivation-induced differentiation (FIG. 5C). These results
suggest that normal expression of MG53 is necessary for the
differentiation of C2C12 myoblasts into myotubes.
[0241] Because caveolin-3 is developmentally regulated (FIG. 6A)
and can interact with MG53 (FIG. 6B), we tested whether
MG53-induced filapodia-like structure in C2C12 myoblasts could be
influenced by the overexpression of caveolin-3. As shown in FIG.
6D, concurrent overexpression of caveolin-3 and MG53 in either
C2C12 myoblasts lead to remarkable inhibition of the appearance of
filapodia-like structures associated with GFP-MG53 overexpression.
On average, C2C12 myoblasts transfected with caveolin-3 and
GFP-MG53 (in a ratio of 10:1) exhibited an 82.+-.6% reduction in
the appearance of filapodia-like structures, respectively (FIGS. 6E
and F). These results suggest that caveolin-3 represents one of the
molecular regulators of MG53-mediated membrane fusion events.
[0242] To further investigate the role of caveolin-3 in the
subcellular distribution of MG53 and the formation of
filapodia-like structures, a caveolin-3 shRNA plasmid (Table 1) was
constructed that includes an independent red fluorescence protein
expression cassette to provide a marker for shRNA transfected
cells. Western blot analysis shown in FIG. 7A reveals that the
shRNA-cav3 probe is highly efficient at suppressing the caveolin-3
expression in CHO cells transiently transfected with the caveolin-3
cDNA without affecting the expression of caveolin-1.
TABLE-US-00001 TABLE 1 Oligos for constructing the shRNA for MG53
and Caveolin-3. Plasmid Inserted oligos Scrambled shRNA for sense
5'-GTA CCT CGC CTG CCG TCC AAA GTT MG53 (SEQ ID NO. 18) GTA ATC AAG
AGT TAC AAC TTT GGA CGG CAG GCT TTT TGG AAA-3' antisense 5'-AGC TTT
TCC AAA AAG CCT GCC GTC (SEQ ID NO. 19) CAA AGT TGT AAC TCT TGA TTA
CAA CTT TGG ACG GCA GGC GAG-3' shRNA for MG53 sense 5'-GTA CCT CGA
GCT GTC AAG CCT GAA (SEQ ID NO. 20) CTC TTC AAG AGA GAG TT CAG GCT
TGA CAG CTC TTT TTG GAA A-3' antisense 5'-AGC TTT TCC AAA AAG AGC
TGT CAA (SEQ ID NO. 21) GCC TGA ACT CTC TCT TGA AGA GTT CAG GCT TGA
CAG CTC GAG-3' Scrambled shRNA for sense 5'-GAT CCG CGG AGA CAT AGC
CTG TAA Cav-3 (SEQ ID NO. 22) TTC AAG AGA TTA CAG GCT ATG TCT CCG
CTT TTT TAC CGG TG-3' antisense 5'-AAT TCA CCG GTA AAA AAG CGG AGA
(SEQ ID NO. 23) CAT AGC CTG TAA TCT CTT GAA TTA CAG GCT ATG TCT CCG
CG-3' shRNA for Cav-3 sense 5'-GAT CCG GAC ATT CAC TGC AAG GAG (SEQ
ID NO. 24) TTC AAG AGA CTC CTT GCA GTG AAT GTC CTT TTT TAC CGG
TG-3' antisense 5'-AAT TCA CCG GTA AAA AAG GAC ATT (SEQ ID NO. 25)
CAC TGC AAG GAG TCT CTT GAA CTC CTT GCA GTG AAT GTC CG-3'
[0243] While C2C12 myoblasts transfected with a non-specific shRNA
exhibit a normal differentiation pattern as shown by the abundant
red-fluorescent labeled myotubes in the left panel of FIG. 7B,
acute suppression of caveolin-3 could significantly inhibit the
differentiation of C2C12 myoblasts into myotubes (FIG. 7B, right
panel). On average, less than 10% of the shRNA-cav3 transfected
myoblasts marked by red-fluorescence could differentiate into
mature myotubes at day 6 after application of differentiation media
(FIG. 7C). This result is consistent with previous studies by other
investigators, which showed that the expression of caveolin-3 is
essential for differentiation of C2C12 myotubes.
[0244] Confocal microscopic imaging showed that transfection of
shRNA-cav3 into C2C12 myoblasts did not appear to affect the
subcellular distribution of GFP-MG53 expressed in these cells (FIG.
7D). In particular, the distinct pattern of vesicular distribution
of GFP-MG53 and filapodia-like membrane structures remained
unaffected by the transient transfection with either shRNA-cav3 or
the non-specific shRNA. This result is consistent with the lack of
expression of caveolin-3 in the myoblast stage of C2C12 cells.
[0245] Due to the essential nature of caveolin-3 in myotube
differentiation, the effect of methyl-.-cyclodextrin (M-.CD) on
C2C12 myoblasts overexpressing GFP -MG53 was tested to further
assay the functional impact of MG53-caveolin interaction on
membrane recycling. M-.CD can extract cholesterol from cell
membranes and has been widely used as an agent to disrupt caveolae
structures. As shown in FIG. 8A, myoblasts overexpressing GFP-MG53
exhibited spontaneous fusion of vesicles both intracellularly as
well as at the sarcolemmal membrane. These spontaneous fusion
events are slow and occur in the order of minutes. Following
treatment with M-.CD, exocytotic events become greatly enhanced
resulting in accelerated membrane fusion and massive budding of
membrane vesicles (FIG. 8B). These initial alterations are rapidly
induced, and extended incubation with M-.CD results in
solubilization of GFP-MG53 within the myoblast (FIG. 8C).
[0246] Caveolin-mediated internalization of membrane vesicles
likely play a regulatory role in restraining that excessive
exocyotic events generated by overexpression of MG53. Furthermore,
interaction of MG53 with caveolin is necessary to maintain
subcellular localization of MG53. This conclusion is supported by
results from additional experiments using mutant forms of
caveolin-3 (SEQ ID NO. 8).
[0247] Role of TRIM and SPRY motifs in MG53 function.
Structure/function assessment of the domains of MG53 (FIG. 13)
revealed a remarkable polarity of GFP fusion to MG53 in the
intracellular distribution of MG53. In particular, fusion of GFP to
the carboxyl-terminal end of MG53 alters the ability of MG53 to
partition to the vesicular compartment and to target to the
sarcolemmal membrane. To further test the function of the TRIM and
SPRY domains in facilitating the membrane-fusion function of MG53,
a series of deletion mutants coupled to GFP (FIG. 13A) were
generated.
[0248] To analyze the subcellular localization of these mutant
constructs of MG53, confocal microscopic imaging was applied to
C2C12 myoblasts following transient expression. As shown in FIG.
13B (right panels), GFP-TRIM or TRIM-GFP were predominantly
localized to intracellular vesicles without apparent labeling of
the sarcolemmal membrane. This result suggests that the SPRY
domain, which is absent from GFP-TRIM or TRIM-GFP, is necessary for
targeting of MG53 to the sarcolemmal membrane. The fact that
MG53-GFP exhibited a predominantly cytosolic distribution (FIG.
13B, left panel), further supports the role of SPRY in targeting
MG53 to the cell surface membrane.
[0249] Interestingly, although GFP-SPRY or SPRY-GFP displayed a
predominantly cytosolic pattern of distribution, they are clearly
excluded from intracellular vesicles (FIG. 13B, middle panels). The
cytosolic distribution pattern coupled with the exclusion of
localization at intracellular vesicles of GFP-SPRY and SPRY-GFP
likely reflects the role of TRIM. Presumably, the TRIM motif can
mediate the adherence of MG53 to intracellular vesicles (FIG. 13B,
right panels). The SPRY domain is insufficient to target to the
sarcolemma by itself, therefore the TRIM domain must be present in
tandem with the SPRY domain for proper trafficking of MG53 to the
sarcolemmal membrane. In addition, our co-immunoprecipitation data
shows that caveolin-3 interacts with the TRIM motif of MG53 (FIG.
13C). Thus, it is possible that the functional interaction between
MG53 and caveolin-3 may underlie some of the cellular factors
contributing to the diffuse pattern of GFP-SPRY and SPRY-GFP in
C2C12 myoblasts. Overall, the regulated distribution of MG53 to the
cell surface and intracellular compartments would likely result
from coordinated action between the TRIM and SPRY domains. This
requirement for both TRIM and SPRY for proper MG53 subcellular
localization also has apparent functional significance, as none of
these deletion mutants display the filapodia-like structures or the
robust vesicle budding events observed from overexpression of
full-length MG53.
[0250] MG53 can fully function in non-muscle cell types. Analysis
of MG53 function in myogenic C2C12 cells and in isolated skeletal
muscle fibers reveals an essential role for MG53 in vesicle
trafficking and membrane repair in striated muscle. Considering
that membrane repair is an essential to maintain cellular
homeostasis, it is likely that similar repair mechanisms in other
non-muscle cell types could use similar molecular machinery to
facilitate this process. To test this possibility, several of the
previous experiments conducted with C2C12 myogenic cells were
replicated with non-muscle Chinese hamster ovary (CHO) cells. In
these cells, a very similar phenotype to that seen in the C2C12
cells was found. First, GFP-MG53 could produce filapodia-like
protrusions of the plasma membrane and localize to both
intracellular vesicles and to the plasma membrane (FIGS. 6 and 14).
Second, MG53 deletion proteins behaved in an identical fashion to
that seen in C2C12 cells. Finally, caveolin-3 can also control the
activity of MG53 expressed in CHO cells (FIG. 14). As a result,
these studies indicate that MG53 acts through a conserved molecular
mechanism that is present in other cell types besides muscle.
[0251] Purification of recombinant MG53 and TAT-MG53. To supply
MG53 to the target cell to facilitate improved cellular
regeneration a cell penetrating peptide sequence derived from the
TAT gene in HIV is coupled with full-length MG53(TAT-MG53) and with
several MG53 deletion mutants (FIG. 15A). These fusion proteins can
be expressed in E. coli bacteria and effectively purified using
affinity chromatography (FIGS. 15B and 15C). We have previously
shown that the application of such fusion proteins to cell
monolayers results in effective translocation of recombinant
proteins into mammalian cells. Generation of these fusion proteins
should allow us to increase the amount of MG53 within target cells
so that we can resolve the therapeutic effects of MG53 on dermal
tissue.
[0252] Expression of recombinant MG53 can be performed in
eukaryotic or prokaryotic cells. FIG. 18 illustrates that
recombinant MG53 can be expressed in either eukaryotic or
prokaryotic systems. Briefly, recombinant MG53 is expressed in Sf9
cells as a fusion protein containing both a TAT peptide portion and
a six-histidine tag (6-HIS tag). This histidine tag can be used to
isolate and purify recombinant protein using filtration
chromatography techniques well known in the art. Panel (A) shows
the Coomassie blue stained gel of recombinant human MG53 protein
(arrow) fractions isolated from Sf9 cells with a Ni-NTA column.
Input=cell extract, FT=flow through, M=marker, E=elution number.
(B) Coomassie blue stained gel of recombinant human TAT-MG53
(arrow) isolated from Sf9 cells. The Coomassie blue stained gel in
(C) represents recombinant mouse TAT-MG53 (arrow) expressed and
isolated from E. coli.
[0253] Recombinant human TAT-MG53 can penetrate cells of different
origins. In order for MG53 to function it must be present
intracellularly. In order to demonstrate that recombinant MG53 can
be translocated across the cellular membrane in therapeutically
significant amounts HL-1 cardiomyocytes and 3T3 fibroblasts were
incubated with about 4 or 8 .mu.g/mL recombinant human TAT-MG53 for
15 minutes at 37.degree. C. (FIG. 17). The cells were washed three
times in a buffered salt solution and then lysed for western blot
analysis. Western blot shows that control cells (control) do not
contain endogenous MG53, however those incubated with TAT-MG53
contain ample intracellular TAT-MG53. Note that TAT-MG53 is
slightly larger than MG53 visualized from skeletal muscle extract
(muscle) due to the addition of the TAT cell penetrating peptide to
the protein. Multiple bands may be generated by intracellular
processing of the TAT-MG53 fusion protein. Therefore, in a
preferred embodiment of the MG53 polypeptide therapeutic, the
present invention comprises a recombinant polypeptide comprising a
TAT polypeptide portion and an MG53 polypeptide portion, wherein
the TAT and MG53 polypeptide portions are present in a single,
contiguous polypeptide chain.
[0254] Heterologous expression of MG53 in a human cell line results
in membrane repair in response to acute injury. FIG. 16
demonstrates that recombinant MG53 can be expressed in a
heterologous expression system and retain its ability to repair
cell membrane damage without the expression of additional proteins.
Specifically, MG53 was cloned into an expression vectors as a
fusion protein with red fluorescent protein (RFP). The fusion
protein was expressed in a human embryonic kidney cell line (HEK293
fibroblast cell line) and the cell's ability to repair membrane
damage was compared to cells expressing only RFP. Panel (a)
demonstrates that cell lines stably expressing an RFP (red
fluorescent protein) control protein show a cytosolic expression
pattern. However, in HEK293 cells expressing RFP only (FIG. 16A);
injury with a microelectrode results in no translocation of RFP to
the injury site (arrow). Some bleaching of RFP fluorescence occurs
from excessive entry of extracellular buffer (*). In contrast,
HEK293 cells that are stably expressing RFP-MG53 (c) show
localization to intracellular vesicles. Microelectrode injury of
HEK293 cells expressing RFP-MG53 (d) results in massive
translocation of MG53 to the injury site (arrow) in less than 90
seconds. This result demonstrates that recombinant MG53 can be
useful for repairing cellular and/or tissue damage in any cellular
environment. Although recombinant MG53 is able to repair injury to
cellular membranes when expressed in a heterologous system the
invention is not so limited. In certain embodiments, the invention
encompasses methods of co-expression of MG53 and caveolin-3 in
order to promote membrane repair in order to treat or prevent
tissue damage. In another embodiment, the present invention relates
to a therapeutic composition comprising a TAT-MG53 polypeptide and
a TAT-caveolin-3 polypeptide.
[0255] MG53 association with membranes and membrane repair depends
on interaction with phosphatidylserine. Lipid profiling (FIG. 19)
revealed that the purified recombinant MG53 could interact
specifically with phosphatidylserine (PS), lipids that
preferentially appear at the inner leaflet of the plasma membrane
and the cytoplasmic face of intracellular vesicles (FIG. 19A). If
this interaction allows MG53 to tether to intracellular membranes,
then vesicular accumulation following membrane disruption could be
monitored by the movement of Annexin-V, a protein known to interact
with PS. Using Annexin-V-GFP, we observed rapid labeling of
Annexin-V-GFP at the C2C12 myoblast injury site (FIG. 19B). The
accumulation of Annexin-V-GFP was accelerated by co-expression of
RFP-MG53, consistent with a role for MG53 in mediating the acute
membrane repair process. Live cell imaging demonstrated coordinated
movement of RFP-MG53 and Annexin-V-GFP toward the injury site.
[0256] Exemplary Methods
[0257] Identification and cloning of MG53--The preparation and
screening of a mAb library for microsomal proteins of rabbit
skeletal muscle were described previously (21). The preparation of
mAb5259 (IgG1 subclass) and immunoaffinity purification was carried
out as described previously (21). Purified MG53 was subjected to
amino acid sequence analysis and all sequences determined were
encoded in the rabbit MG53 cDNA (data not shown). Homology searches
in the databases found mouse and human MG53 using the rabbit
partial amino acid sequences. An exon region of the mouse MG53 gene
was amplified from mouse genomic DNA, and rabbit and mouse skeletal
muscle libraries were screened using the .sup.32P-labeled exon
fragment to yield full-length cDNAs.
[0258] Immunohistochemical and Immunostaining
analysis--Immunochemical analyses using mAb5259 were carried out as
described previously (21) Immunoelectron-microscopy using secondary
antibody conjugated with 15 nm gold particles was conduced as
described previously (17).
[0259] Cell culture--The C2C12 murine myoblast cell line used for
all studies was purchased from the American Type Culture Collection
(Manassas, Va.). Cells were grown in a humidified environment at
37.degree. C. and 5% CO.sub.2 in DMEM medium for C2C12 or Ham's F12
medium for CHO cells supplemented with 10% fetal bovine serum, 100
units/ml penicillin and 100 ug/ml streptomycin. In order to induce
myotube differentiation, C2C12 myoblasts were grown to confluence
and the medium was switched to DMEM containing 2% horse serum,
penicillin (100 U/ml), streptomycin (100 ng/ml). For transient
transfections, C2C12 myoblasts or CHO cells were plated at 70%
confluence in glass-bottom dishes. After 24 hours, cells were
transfected with plasmids described above using GeneJammer reagent
(Stratagene). Cells were visualized by live cell confocal imaging
at 24-48 hours after transfection or at times indicated for
individual experiments. In some experiments, C2C12 myoblasts were
allowed to differentiate into myotubes for the indicated time
before observation.
[0260] Plasmids construction--The full-length mouse MG53 cDNA and
associated truncation mutants were generated by PCR using the
primers described in supplemental table 1. For construction of
pCMS-MG53, after digestion by the appropriate restriction enzymes,
the PCR-amplified cDNA was inserted into pCMS-EGFP vector
(Invitrogen) at Nhe I/Xba I sites. For construct the GFP-MG53,
GFP-TRIM, GFP-SPRY, MG53-GFP, TRIM-GFP and SPRY-GFP, PCR products
were inserted into pEGFP-C1 at the XhoI/XbaI sites, or pEGFP-N1 at
the XhoI/KpnI sites.
[0261] Live cell imaging--To monitor intracellular trafficking of
GFP-MG53 either CHO or C2C12 cells were cultured in glass-bottom
dishes (Bioptechs Inc.) and transfected with the plasmids described
above. Fluorescence images (512.times.512) were captured at 3.18
s/frame using a BioRad 2100 Radiance laser scanning confocal
microscope with a 63.times.1.3NA oil immersion objective.
[0262] RNAi assay--The target sequence for shRNA knockdown of MG53
is at position 622-642 (GAG CTG TCA AGC CTG AAC TCT) in the mouse
MG53 cDNA. For caveolin-3, the target sequence is at position
363-380 (GAC ATT CAC TGC AAG GAG ATA). Complementary sense and
antisense oligonucleotides were synthesized. To construct the MG53
shRNA and control plasmids, annealed oligonucleotides were inserted
into psiRNA-hH1GFPzeo G2 (InvivoGene) at the Acc 65I/Hind III
restriction enzyme sites. For caveolin-3 shRNA and control
plasmids, annealed oligonucleotides were inserted into pRNAiDsRed
vector (BD Biosciences) at the EcoR I/BamH I restriction enzyme
sites. Each vector has as independent fluorescent protein
expression cassette (green or red) to act as markers of cell
transfection. All plasmids were confirmed by direct sequencing with
flanking primers and the down-regulation of MG53 and caveolin-3
protein expression was examined by Western blot analysis.
[0263] Western blot and Co-immunoprecipitation--Immunoblots were
using standard techniques. Briefly, C2C12 or CHO cells were
harvested and lysed with ice-cold modified RIPA buffer (150 mM
NaCl, 5 mM EDTA, 1% NP40, 20 mM Tris-HCl, pH 7.5) in the presence
of a cocktail of protease inhibitors (Sigma). 20 ng of total
protein were separated on a 4-12% SDS-polyacrylamide gel. A
standard protocol was used for co-immunoprecipitation studies of
MG53 and Caveolin-3. In brief, skeletal muscle tissue or C2C12
myotubes were lysed in 0.5 ml modified RIPA buffer. The whole cell
lysate (500 .mu.g) was incubated overnight with 5 .mu.g polyclonal
anti-MG53 (polyclonal antibody), or anti-caveolin-3 antibody (mAb).
As a negative control, 500 .mu.g whole cell lysate was incubated
with 5 .mu.g normal rabbit and mouse IgG and processed as described
above. The immune complexes were collected on protein G-Sepharose
beads by incubating for 2 hours and washed four times with RIPA
buffer.
[0264] It is understood that the detailed examples and embodiments
described herein are given by way of example for illustrative
purposes only, and are in no way considered to be limiting to the
invention. Various modifications or changes in light thereof will
be suggested to persons skilled in the art and are included within
the spirit and purview of this application and are considered
within the scope of the appended claims. For example, the relative
quantities of the ingredients may be varied to optimize the desired
effects, additional ingredients may be added, and/or similar
ingredients may be substituted for one or more of the ingredients
described. Additional advantageous features and functionalities
associated with the systems, methods, and processes of the present
invention will be apparent from the appended claims.
Sequence CWU 1
1
251477PRTHomo sapiensmisc_feature(1)..(477)Human MG53 Polypeptide
1Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu1 5
10 15Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His
Ser 20 25 30Phe Cys Arg Ala Cys Leu Gly Arg Val Ala Gly Glu Pro Ala
Ala Asp 35 40 45Gly Thr Val Leu Cys Pro Cys Cys Gln Ala Pro Thr Arg
Pro Gln Ala 50 55 60Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu
Gly Leu Ala Gln65 70 75 80Val Pro Gln Gly His Cys Glu Glu His Leu
Asp Pro Leu Ser Ile Tyr 85 90 95Cys Glu Gln Asp Arg Ala Leu Val Cys
Gly Val Cys Ala Ser Leu Gly 100 105 110Ser His Arg Gly His Arg Leu
Leu Pro Ala Ala Glu Ala His Ala Arg 115 120 125Leu Lys Thr Gln Leu
Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys 130 135 140Met Arg Lys
Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu145 150 155
160Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu
Asp Cys 180 185 190Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala
Leu Arg Arg Glu 195 200 205Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln
Leu Arg Gln Met Glu Lys 210 215 220Val Leu Glu Glu Val Ala Asp Lys
Pro Gln Thr Glu Phe Leu Met Lys225 230 235 240Tyr Cys Leu Val Thr
Ser Arg Leu Gln Lys Ile Leu Ala Glu Ser Pro 245 250 255Pro Pro Ala
Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp Asp Phe 260 265 270Lys
Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Leu 275 280
285Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala
Pro Pro305 310 315 320Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala
Val Ala Val Val Ala 325 330 335His Gln Gln Leu Ser Glu Gly Glu His
Tyr Trp Glu Val Asp Val Gly 340 345 350Asp Lys Pro Arg Trp Ala Leu
Gly Val Ile Ala Ala Glu Ala Pro Arg 355 360 365Arg Gly Arg Leu His
Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly 370 375 380Leu Arg Glu
Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro385 390 395
400Arg Ala Leu Arg Ser Pro Glu Arg Arg Pro Thr Arg Ile Gly Leu Tyr
405 410 415Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser
Asp Ala 420 425 430Asp Ala Leu Val Pro Leu Phe Ala Phe His Glu Arg
Leu Pro Arg Pro 435 440 445Val Tyr Pro Phe Phe Asp Val Cys Trp His
Asp Lys Gly Lys Asn Ala 450 455 460Gln Pro Leu Leu Leu Val Gly Pro
Glu Gly Ala Glu Ala465 470 47521434DNAHomo
sapiensmisc_feature(1)..(1434)Human MG53 cDNA 2atgtcggctg
cgcccggcct cctgcaccag gagctgtcct gcccgctgtg cctgcagctg 60ttcgacgcgc
ccgtgacagc cgagtgcggc cacagtttct gccgcgcctg cctaggccgc
120gtggccgggg agccggcggc ggatggcacc gttctctgcc cctgctgcca
ggcccccacg 180cggccgcagg cactcagcac caacctgcag ctggcgcgcc
tggtggaggg gctggcccag 240gtgccgcagg gccactgcga ggagcacctg
gacccgctga gcatctactg cgagcaggac 300cgcgcgctgg tgtgcggagt
gtgcgcctca ctcggctcgc accgcggtca tcgcctcctg 360cctgccgccg
aggcccacgc acgcctcaag acacagctgc cacagcagaa actgcagctg
420caggaggcat gcatgcgtaa ggagaagagt gtggctgtgc tggagcatca
gctggtggag 480gtggaggaga cagtgcgtca gttccggggg gccgtggggg
agcagctggg caagatgcgg 540gtgttcctgg ctgcactgga gggctccttg
gactgcgagg cagagcgtgt acggggtgag 600gcaggggtcg ccttgcgccg
ggagctgggg agcctgaact cttacctgga gcagctgcgg 660cagatggaga
aggtcctgga ggaggtggcg gacaagccgc agactgagtt cctcatgaaa
720tactgcctgg tgaccagcag gctgcagaag atcctggcag agtctccccc
acccgcccgt 780ctggacatcc agctgccaat tatctcagat gacttcaaat
tccaggtgtg gaggaagatg 840ttccgggctc tgatgccagc gctggaggag
ctgacctttg acccgagctc tgcgcacccg 900agcctggtgg tgtcttcctc
tggccgccgc gtggagtgct cggagcagaa ggcgccgccg 960gccggggagg
acccgcgcca gttcgacaag gcggtggcgg tggtggcgca ccagcagctc
1020tccgagggcg agcactactg ggaggtggat gttggcgaca agccgcgctg
ggcgctgggc 1080gtgatcgcgg ccgaggcccc ccgccgcggg cgcctgcacg
cggtgccctc gcagggcctg 1140tggctgctgg ggctgcgcga gggcaagatc
ctggaggcac acgtggaggc caaggagccg 1200cgcgctctgc gcagccccga
gaggcggccc acgcgcattg gcctttacct gagcttcggc 1260gacggcgtcc
tctccttcta cgatgccagc gacgccgacg cgctcgtgcc gctttttgcc
1320ttccacgagc gcctgcccag gcccgtgtac cccttcttcg acgtgtgctg
gcacgacaag 1380ggcaagaatg cccagccgct gctgctcgtg ggtcccgaag
gcgccgaggc ctga 14343477PRTMus musculusmisc_feature(1)..(477)Mouse
MG53 3Met Ser Ala Ala Pro Gly Leu Leu Arg Gln Glu Leu Ser Cys Pro
Leu1 5 10 15Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly
His Ser 20 25 30Phe Cys Arg Ala Cys Leu Ile Arg Val Ala Gly Glu Pro
Ala Ala Asp 35 40 45Gly Thr Val Ala Cys Pro Cys Cys Gln Ala Pro Thr
Arg Pro Gln Ala 50 55 60Leu Ser Thr Asn Leu Gln Leu Ser Arg Leu Val
Glu Gly Leu Ala Gln65 70 75 80Val Pro Gln Gly His Cys Glu Glu His
Leu Asp Pro Leu Ser Ile Tyr 85 90 95Cys Glu Gln Asp Arg Thr Leu Val
Cys Gly Val Cys Ala Ser Leu Gly 100 105 110Ser His Arg Gly His Arg
Leu Leu Pro Ala Ala Glu Ala Gln Ala Arg 115 120 125Leu Lys Thr Gln
Leu Pro Gln Gln Lys Met Gln Leu Gln Glu Ala Cys 130 135 140Met Arg
Lys Glu Lys Thr Val Ala Val Leu Glu His Gln Leu Val Glu145 150 155
160Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175Gly Lys Met Arg Met Phe Leu Ala Ala Leu Glu Ser Ser Leu
Asp Arg 180 185 190Glu Ala Glu Arg Val Arg Gly Asp Ala Gly Val Ala
Leu Arg Arg Glu 195 200 205Leu Ser Ser Leu Asn Ser Tyr Leu Glu Gln
Leu Arg Gln Met Glu Lys 210 215 220Val Leu Glu Glu Val Ala Asp Lys
Pro Gln Thr Glu Phe Leu Met Lys225 230 235 240Phe Cys Leu Val Thr
Ser Arg Leu Gln Lys Ile Leu Ser Glu Ser Pro 245 250 255Pro Pro Ala
Arg Leu Asp Ile Gln Leu Pro Val Ile Ser Asp Asp Phe 260 265 270Lys
Phe Gln Val Trp Lys Lys Met Phe Arg Ala Leu Met Pro Ala Leu 275 280
285Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Asp Gln Lys Ala
Pro Pro305 310 315 320Ala Gly Glu Asp Thr Arg Gln Phe Asp Lys Ala
Val Ala Val Val Ala 325 330 335Gln Gln Leu Leu Ser Gln Gly Glu His
Tyr Trp Glu Val Glu Val Gly 340 345 350Asp Lys Pro Arg Trp Ala Leu
Gly Val Met Ala Ala Asp Ala Ser Arg 355 360 365Arg Gly Arg Leu His
Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly 370 375 380Leu Arg Asp
Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro385 390 395
400Arg Ala Leu Arg Thr Pro Glu Arg Pro Pro Ala Arg Ile Gly Leu Tyr
405 410 415Leu Ser Phe Ala Asp Gly Val Leu Ala Phe Tyr Asp Ala Ser
Asn Pro 420 425 430Asp Val Leu Thr Pro Ile Phe Ser Phe His Glu Arg
Leu Pro Gly Pro 435 440 445Val Tyr Pro Ile Phe Asp Val Cys Trp His
Asp Lys Gly Lys Asn Ala 450 455 460Gln Pro Leu Leu Leu Val Gly Pro
Glu Gln Glu Gln Ala465 470 47541434DNAMus
musculusmisc_feature(1)..(1434)Mouse MG53 cDNA 4atgtcggctg
cacccggcct tctgcgtcag gaactgtcct gcccactgtg cttgcagctg 60ttcgatgcgc
cagtgacggc tgagtgtggc cacagtttct gccgtgcctg cctgatccgg
120gtggcagggg agcctgctgc ggacggcaca gttgcctgtc cctgttgtca
ggcacctaca 180cggccgcagg ctctaagcac taacctccag ttgtcacgcc
ttgtggaggg tttggcgcaa 240gtgccccaag gccactgcga ggaacacctg
gatccactga gcatctactg cgagcaggac 300cgcacacttg tgtgtggtgt
gtgtgcctcg ctcggttctc accgtggtca tcgtctcctg 360cctgccgctg
aagcccaagc acgcctcaag acacagcttc cacagcagaa gatgcagctg
420caggaggcat gcatgcgcaa ggagaagact gtagcggtgc tggagcatca
gctggtggag 480gtggaggaga cagtgcgcca gttccgggga gctgtcgggg
agcagctggg gaagatgcgg 540atgttcctgg ctgccctaga aagttctctg
gaccgtgaag cagaaagggt tcggggtgat 600gctggggttg ccttgcgtcg
ggagctgtca agcctgaact cttacctaga gcaactgagg 660cagatggaga
aggtgctgga ggaggtggct gacaagccac agacagaatt cctcatgaaa
720ttctgcctgg taaccagcag gctgcagaag atcctgtcag agtcaccacc
accggcaagg 780ctagatatcc agctgcctgt catctcagat gacttcaaat
tccaggtgtg gaagaagatg 840ttccgggctc tgatgccagc gctggaggaa
ctgacttttg accccagctc tgcgcacccg 900agcctggtgg tgtcctcctc
tggtcgccga gtggagtgct cagaccagaa ggcgccgcca 960gcgggagaag
acacgcgtca gttcgacaag gcagtagcgg tggtggcgca gcagctgctg
1020tcacagggcg agcactattg ggaggtggag gtgggcgaca aaccacgctg
ggccctggga 1080gtgatggcgg ctgacgcttc ccgccgtggc cggctgcacg
cggtgccctc acaggggctg 1140tggctgctgg gtctgcgcga tggcaagatc
ctggaggcgc acgtggaggc caaggagccg 1200cgggcactgc gcaccccaga
gaggcctccg gcgcgcattg gcctctacct aagcttcgca 1260gatggcgtcc
tggctttcta tgatgcgagc aaccccgacg tacttacgcc aatcttttct
1320ttccacgagc gtctgcccgg gccggtgtac cccatctttg acgtgtgctg
gcacgacaag 1380ggcaagaatg cccagcccct gctgcttgtg gggccggagc
aggaacaggc ctga 14345477PRTOryctolagus
cuniculusmisc_feature(1)..(477)Rabbit MG53 5Met Ser Ala Ala Pro Gly
Leu Leu His Gln Glu Leu Ser Cys Pro Leu1 5 10 15Cys Leu Gln Leu Phe
Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser 20 25 30Phe Cys Arg Ala
Cys Leu Ser Arg Val Ala Gly Glu Pro Ala Ala Asp 35 40 45Gly Thr Val
Asn Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala 50 55 60Leu Ser
Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln65 70 75
80Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95Cys Glu Gln Asp Arg Val Leu Val Cys Gly Val Cys Ala Ser Leu
Gly 100 105 110Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala
His Ser Arg 115 120 125Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln
Leu Gln Glu Ala Ser 130 135 140Met Arg Lys Glu Lys Ser Val Ala Val
Leu Glu His Gln Leu Thr Glu145 150 155 160Val Glu Glu Thr Val Arg
Gln Phe Arg Gly Ala Val Gly Glu Gln Leu 165 170 175Gly Lys Met Arg
Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg 180 185 190Glu Ala
Glu Arg Val Arg Ser Glu Ala Gly Val Ala Leu Arg Arg Glu 195 200
205Leu Gly Gly Leu His Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu
Met Lys225 230 235 240Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile
Leu Ala Glu Ser Pro 245 250 255Pro Pro Ala Arg Leu Asp Ile Gln Leu
Pro Ile Ile Ser Asp Asp Phe 260 265 270Lys Phe Gln Val Trp Arg Lys
Met Phe Arg Ala Leu Met Pro Ala Leu 275 280 285Glu Glu Leu Thr Phe
Asp Pro Ser Ser Ala His Pro Ser Leu Val Val 290 295 300Ser Pro Thr
Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro305 310 315
320Ala Gly Asp Asp Ala Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335Gln Gln Leu Leu Ser Asp Gly Glu His Tyr Trp Glu Val Glu
Val Gly 340 345 350Asp Lys Pro Arg Trp Ala Leu Gly Val Met Ala Ser
Glu Ala Ser Arg 355 360 365Arg Gly Arg Leu His Ala Val Pro Ser Gln
Gly Leu Trp Leu Leu Gly 370 375 380Leu Arg Asp Gly Lys Thr Leu Glu
Ala His Val Glu Ala Lys Glu Pro385 390 395 400Arg Ala Leu Arg Thr
Pro Glu Arg Arg Pro Thr Arg Leu Gly Leu Tyr 405 410 415Leu Ser Phe
Gly Asp Gly Val Leu Ala Phe Tyr Asp Ala Ser Asp Ala 420 425 430Asp
Ala Leu Glu Leu Leu Phe Ala Phe Arg Glu Arg Leu Pro Gly Pro 435 440
445Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
450 455 460Gln Pro Leu Leu Leu Val Gly Pro Asp Gly Gln Glu Ala465
470 47561434DNAOryctolagus cuniculusmisc_feature(1)..(1434)Rabbit
MG53 cDNA 6atgtcggccg cgcccggcct cctgcaccag gagctgtctt gcccgctgtg
cctgcagctg 60ttcgacgcgc ccgtgacagc cgagtgcggc cacagtttct gccgcgcctg
cctgagccgc 120gtggcggggg agccggcggc cgatggcacc gtgaactgcc
cgtgctgcca ggcgcccacg 180cggccgcagg cgctcagcac caacctgcag
ctggcgcgcc tggtggaggg gctggcgcag 240gtgccgcagg gccactgcga
ggagcacctg gacccgctga gcatctactg cgagcaggac 300cgcgttctcg
tgtgcggcgt gtgcgcctcg ctcggctcgc accgcggcca ccgcctgctg
360cccgccgccg aggcccactc gcgtctcaag acgcagctgc cccagcagaa
gctgcagctg 420caggaggcga gcatgcgcaa ggagaagagc gtggccgtgc
tggagcacca gctcacggag 480gtggaggaga cagtgcgtca gttccggggg
gcagtggggg agcagctggg caagatgcgg 540gtgttcctgg ccgccctgga
gggctccctg gaccgcgagg cagaacgtgt gcggagcgag 600gcgggggtgg
ccttgcggcg ggagctgggg ggcctccact cgtacctgga gcagctgcgg
660cagatggaga aggtgttgga ggaggtggct gacaagccac agaccgagtt
ccttatgaaa 720tattgcctgg tgaccagcag gctgcagaag atcctggcgg
agtcgccacc acctgctcgt 780ctggacatcc agctgcccat catttcagat
gacttcaaat tccaggtgtg gaggaagatg 840ttccgggctc tgatgccagc
gctggaggag ctgacctttg acccgagctc cgcgcacccg 900agcctcgtgg
tgtcacccac gggccgccga gtggagtgct cggagcagaa ggcgccgccc
960gccggggacg acgcgcgcca gttcgacaag gctgtggccg tggtggcgca
gcagctgctg 1020tccgacggcg agcactactg ggaggtggag gtgggcgaca
agccgcgctg ggcgctgggc 1080gtgatggcct ccgaggcgag ccgccgtggc
cggctgcacg ccgtgccctc acagggtttg 1140tggctgctgg ggctgcgcga
cggcaagacc ctggaggcgc acgtggaggc caaggagccg 1200cgcgcgctgc
gcaccccgga gcggcggccc acgcgcctcg gcctctacct cagcttcggc
1260gatggcgtgc tcgccttcta cgacgccagc gacgccgacg cgctcgagct
gctgtttgct 1320ttccgcgagc gcctgcccgg gcccgtgtac cccttcttcg
acgtgtgctg gcatgacaag 1380ggcaagaatg cgcagccgct gctgctcgtg
gggccggatg gccaggaggc ctga 14347477PRTHomo
sapiensMUTAGEN(29)..(29)C29L/C242A 7Met Ser Ala Ala Pro Gly Leu Leu
His Gln Glu Leu Ser Cys Pro Leu1 5 10 15Cys Leu Gln Leu Phe Asp Ala
Pro Val Thr Ala Glu Leu Gly His Ser 20 25 30Phe Cys Arg Ala Cys Leu
Gly Arg Val Ala Gly Glu Pro Ala Ala Asp 35 40 45Gly Thr Val Leu Cys
Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala 50 55 60Leu Ser Thr Asn
Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln65 70 75 80Val Pro
Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr 85 90 95Cys
Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys Ala Ser Leu Gly 100 105
110Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu
Ala Cys 130 135 140Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His
Gln Leu Val Glu145 150 155 160Val Glu Glu Thr Val Arg Gln Phe Arg
Gly Ala Val Gly Glu Gln Leu 165 170 175Gly Lys Met Arg Val Phe Leu
Ala Ala Leu Glu Gly Ser Leu Asp Cys 180 185 190Glu Ala Glu Arg Val
Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu 195 200 205Leu Gly Ser
Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys 210 215 220Val
Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys225 230
235 240Tyr Ala Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ser
Pro 245 250 255Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser
Asp Asp Phe 260 265 270Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala
Leu Met Pro Ala Leu
275 280 285Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu
Val Val 290 295 300Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Glu Gln
Lys Ala Pro Pro305 310 315 320Ala Gly Glu Asp Pro Arg Gln Phe Asp
Lys Ala Val Ala Val Val Ala 325 330 335His Gln Gln Leu Ser Glu Gly
Glu His Tyr Trp Glu Val Asp Val Gly 340 345 350Asp Lys Pro Arg Trp
Ala Leu Gly Val Ile Ala Ala Glu Ala Pro Arg 355 360 365Arg Gly Arg
Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly 370 375 380Leu
Arg Glu Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro385 390
395 400Arg Ala Leu Arg Ser Pro Glu Arg Arg Pro Thr Arg Ile Gly Leu
Tyr 405 410 415Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala
Ser Asp Ala 420 425 430Asp Ala Leu Val Pro Leu Phe Ala Phe His Glu
Arg Leu Pro Arg Pro 435 440 445Val Tyr Pro Phe Phe Asp Val Cys Trp
His Asp Lys Gly Lys Asn Ala 450 455 460Gln Pro Leu Leu Leu Val Gly
Pro Glu Gly Ala Glu Ala465 470 4758151PRTHomo
sapiensmisc_feature(1)..(151)P56539 CAV3_HUMAN CAVEOLIN-3 - HOMO
SAPIENS (HUMAN). 8Met Met Ala Glu Glu His Thr Asp Leu Glu Ala Gln
Ile Val Lys Asp1 5 10 15Ile His Cys Lys Glu Ile Asp Leu Val Asn Arg
Asp Pro Lys Asn Ile 20 25 30Asn Glu Asp Ile Val Lys Val Asp Phe Glu
Asp Val Ile Ala Glu Pro 35 40 45Val Gly Thr Tyr Ser Phe Asp Gly Val
Trp Lys Val Ser Tyr Thr Thr 50 55 60Phe Thr Val Ser Lys Tyr Trp Cys
Tyr Arg Leu Leu Ser Thr Leu Leu65 70 75 80Gly Val Pro Leu Ala Leu
Leu Trp Gly Phe Leu Phe Ala Cys Ile Ser 85 90 95Phe Cys His Ile Trp
Ala Val Val Pro Cys Ile Lys Ser Tyr Leu Ile 100 105 110Glu Ile Gln
Cys Ile Ser His Ile Tyr Ser Leu Cys Ile Arg Thr Phe 115 120 125Cys
Asn Pro Leu Phe Ala Ala Leu Gly Gln Val Cys Ser Ser Ile Lys 130 135
140Val Val Leu Arg Lys Glu Val145 1509477PRTDidelphis
sp.PEPTIDE(1)..(477)Opossum MG53 9Met Ser Gly Ala Pro Ala Leu Met
Gln Gly Met Tyr Gln Asp Leu Ser1 5 10 15Cys Pro Leu Cys Leu Lys Leu
Phe Asp Ala Pro Ile Thr Ala Glu Cys 20 25 30Gly His Ser Phe Cys Arg
Asn Cys Leu Leu Arg Leu Ala Pro Asp Pro 35 40 45Gln Ala Gly Thr Val
Leu Cys Pro Ser Cys Gln Ala Pro Thr Lys Pro 50 55 60Asp Gly Leu Asn
Thr Asn Gln Gln Leu Ala Arg Leu Val Glu Ser Leu65 70 75 80Ala Gln
Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser 85 90 95Val
Tyr Cys Glu Gln Asp Arg Ala Leu Ile Cys Gly Val Cys Ala Ser 100 105
110Leu Gly Lys His Arg Gly His Ser Val Val Thr Ala Ala Glu Ala His
115 120 125Gln Arg Met Lys Lys Gln Leu Pro Gln Gln Arg Leu Gln Leu
Gln Glu 130 135 140Ala Cys Met Arg Lys Glu Lys Thr Val Ala Leu Leu
Asp Arg Gln Leu145 150 155 160Ala Glu Val Glu Glu Thr Val Arg Gln
Phe Gln Arg Ala Val Gly Glu 165 170 175Gln Leu Gly Val Met Arg Ala
Phe Leu Ala Ala Leu Glu Ser Ser Leu 180 185 190Gly Lys Glu Ala Glu
Arg Val Thr Gly Glu Ala Gly Thr Ala Leu Lys 195 200 205Ala Glu Arg
Arg Ile Val Thr Ser Tyr Leu Asp Gln Leu Gln Gln Met 210 215 220Glu
Lys Val Leu Asp Glu Val Thr Asp Gln Pro Gln Thr Glu Phe Leu225 230
235 240Arg Lys Tyr Cys Leu Val Ile Ser Arg Leu Gln Lys Ile Leu Ala
Glu 245 250 255Ser Pro Pro Ala Ala Arg Leu Asp Ile Gln Leu Pro Ile
Ile Ser Asp 260 265 270Asp Phe Lys Phe Gln Val Trp Arg Lys Met Phe
Arg Ala Leu Met Pro 275 280 285Gly Met Glu Val Leu Thr Phe Asp Pro
Ala Ser Ala His Pro Ser Leu 290 295 300Leu Val Ser Pro Ser Gly Arg
Arg Val Glu Cys Val Glu Gln Lys Ala305 310 315 320Pro Pro Ala Gly
Asp Asp Pro Gln Gln Phe Asp Lys Ala Val Ala Leu 325 330 335Val Ala
Lys Gln Gln Leu Ser Glu Gly Glu His Tyr Trp Glu Val Glu 340 345
350Val Gly Asp Lys Pro Arg Trp Gly Leu Gly Leu Ile Ser Ala Asp Val
355 360 365Ser Arg Arg Gly Lys Leu His Pro Thr Pro Ser Gln Gly Phe
Trp Met 370 375 380Leu Gly Leu Arg Glu Gly Lys Val Tyr Glu Ala His
Val Glu Ser Lys385 390 395 400Glu Pro Lys Val Leu Lys Val Asp Gly
Arg Pro Ser Arg Ile Gly Leu 405 410 415Tyr Leu Ser Phe Arg Asp Gly
Val Leu Ser Phe Tyr Asp Ala Ser Asp 420 425 430Leu Asp Asn Leu Leu
Pro Leu Tyr Ala Phe His Glu Arg Leu Pro Gly 435 440 445Pro Val Tyr
Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn 450 455 460Ala
Gln Pro Leu Leu Leu Leu Gly Pro Asp Gly Glu Gln465 470
47510477PRTCanis sp.PEPTIDE(1)..(477)Dog MG53 10Met Ser Ala Ala Pro
Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu1 5 10 15Cys Leu Gln Leu
Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser 20 25 30Phe Cys Arg
Ala Cys Leu Ser Arg Val Ala Gly Glu Pro Ala Ala Asp 35 40 45Gly Thr
Val Pro Cys Pro Cys Cys Gln Ala Leu Thr Arg Pro Gln Ala 50 55 60Leu
Ser Thr Asn Gln Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln65 70 75
80Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95Cys Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys Ala Ser Leu
Gly 100 105 110Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala
His Ala Arg 115 120 125Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln
Leu Gln Glu Ala Cys 130 135 140Met Arg Lys Glu Lys Ser Val Ala Leu
Leu Glu His Gln Leu Met Glu145 150 155 160Val Glu Glu Met Val Arg
Gln Phe Arg Gly Ala Val Gly Glu Gln Leu 165 170 175Gly Lys Met Arg
Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg 180 185 190Glu Ala
Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu 195 200
205Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu
Met Lys225 230 235 240Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile
Leu Ala Glu Ser Pro 245 250 255Pro Pro Ala Arg Leu Asp Ile Gln Leu
Pro Val Ile Ser Asp Asp Phe 260 265 270Lys Phe Gln Val Trp Arg Lys
Met Phe Arg Ala Leu Met Pro Val Thr 275 280 285Lys Glu Leu Thr Phe
Asp Pro Ser Ser Ala His Pro Ser Leu Val Leu 290 295 300Ser Pro Ser
Gly Arg Arg Val Glu Cys Ser Asp Gln Lys Ala Pro Pro305 310 315
320Ala Gly Glu Asp Pro Cys Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335Gln Gln Val Leu Ser Asp Gly Glu His Tyr Trp Glu Val Gln
Val Gly 340 345 350Glu Lys Pro Arg Trp Ala Leu Gly Val Ile Ala Ala
Gln Ala Ser Arg 355 360 365Arg Gly Arg Leu His Ala Val Pro Ser Gln
Gly Leu Trp Leu Leu Gly 370 375 380Leu Arg Asp Gly Lys Ile Leu Glu
Ala His Val Glu Ala Lys Glu Pro385 390 395 400Arg Ala Leu Arg Thr
Pro Glu Arg Arg Pro Thr Arg Ile Gly Ile Tyr 405 410 415Leu Ser Phe
Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Pro 420 425 430Asp
Ala Leu Glu Leu Leu Phe Ala Phe His Glu Arg Leu Pro Gly Pro 435 440
445Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
450 455 460Gln Pro Leu Leu Leu Val Gly Pro Asp Gly Glu Glu Ala465
470 47511477PRTPan troglodytesPEPTIDE(1)..(477)Chimpanzee MG53
11Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu1
5 10 15Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His
Ser 20 25 30Phe Cys Arg Ala Cys Leu Gly Arg Val Ala Gly Glu Pro Ala
Ala Asp 35 40 45Gly Thr Val Leu Cys Pro Cys Cys Gln Ala Pro Thr Arg
Pro Gln Ala 50 55 60Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu
Gly Leu Ala Gln65 70 75 80Val Pro Gln Gly His Cys Glu Glu His Leu
Asp Pro Leu Ser Ile Tyr 85 90 95Cys Glu Gln Asp Arg Ala Leu Val Cys
Gly Val Cys Ala Ser Leu Gly 100 105 110Ser His Arg Gly His Arg Leu
Leu Pro Ala Ala Glu Ala His Ala Arg 115 120 125Leu Lys Thr Gln Leu
Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys 130 135 140Met Arg Lys
Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu145 150 155
160Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu
Asp Arg 180 185 190Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala
Leu Arg Arg Glu 195 200 205Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln
Leu Arg Gln Met Glu Lys 210 215 220Val Leu Glu Glu Val Ala Asp Lys
Pro Gln Thr Glu Phe Leu Met Lys225 230 235 240Tyr Cys Leu Val Thr
Ser Arg Leu Gln Lys Ile Leu Ala Glu Ser Pro 245 250 255Pro Pro Ala
Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp Asp Phe 260 265 270Lys
Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Leu 275 280
285Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala
Pro Pro305 310 315 320Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala
Val Ala Val Val Ala 325 330 335His Gln Gln Leu Ser Glu Gly Glu His
Tyr Trp Glu Val Asp Val Gly 340 345 350Asp Lys Pro Arg Trp Ala Leu
Gly Val Ile Ala Ala Glu Ala Pro Arg 355 360 365Arg Gly Arg Leu His
Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly 370 375 380Leu Arg Glu
Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro385 390 395
400Arg Ala Leu Arg Ser Pro Glu Arg Arg Pro Thr Arg Ile Gly Leu Tyr
405 410 415Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser
Asp Ala 420 425 430Asp Ala Leu Val Pro Leu Phe Ala Phe His Glu Arg
Leu Pro Arg Pro 435 440 445Val Tyr Pro Phe Phe Asp Val Cys Trp His
Asp Lys Gly Lys Asn Ala 450 455 460Gln Pro Leu Leu Leu Val Gly Pro
Glu Gly Ala Glu Ala465 470 47512477PRTMacaca
mulattaPEPTIDE(1)..(477)Rhesus Monkey MG53 12Met Ser Ala Ala Pro
Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu1 5 10 15Cys Leu Gln Leu
Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser 20 25 30Phe Cys Arg
Ala Cys Leu Gly Arg Val Ala Gly Glu Pro Ala Ala Asp 35 40 45Gly Thr
Val Leu Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala 50 55 60Leu
Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln65 70 75
80Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95Cys Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys Ala Ser Leu
Gly 100 105 110Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala
His Ala Arg 115 120 125Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln
Leu Gln Glu Ala Cys 130 135 140Met Arg Lys Glu Lys Ser Val Ala Val
Leu Glu His Gln Leu Val Glu145 150 155 160Val Glu Glu Thr Val Arg
Gln Phe Arg Gly Ala Val Gly Glu Gln Leu 165 170 175Gly Lys Met Arg
Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg 180 185 190Glu Ala
Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu 195 200
205Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu
Met Lys225 230 235 240Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile
Leu Ala Glu Ser Pro 245 250 255Pro Pro Ala Arg Leu Asp Ile Gln Leu
Pro Ile Ile Ser Asp Asp Phe 260 265 270Lys Phe Gln Val Trp Arg Lys
Met Phe Arg Ala Leu Met Pro Ala Leu 275 280 285Glu Glu Leu Thr Phe
Asp Pro Ser Ser Ala His Pro Ser Leu Val Val 290 295 300Ser Ser Ser
Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro305 310 315
320Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335His Gln Gln Leu Ser Glu Gly Glu His Tyr Trp Glu Val Glu
Val Gly 340 345 350Asp Lys Pro Arg Trp Ala Leu Gly Val Ile Ala Ala
Glu Gly Pro Arg 355 360 365Arg Gly Arg Leu His Ala Val Pro Ser Gln
Gly Leu Trp Leu Leu Gly 370 375 380Leu Arg Glu Gly Lys Ile Leu Glu
Ala His Val Glu Ala Lys Glu Pro385 390 395 400Arg Ala Leu Arg Ser
Pro Glu Arg Arg Pro Thr Arg Ile Gly Leu Tyr 405 410 415Leu Ser Phe
Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Ala 420 425 430Asp
Ala Leu Val Pro Leu Phe Ala Phe His Glu Arg Leu Pro Gly Pro 435 440
445Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ser
450 455 460Gln Pro Leu Leu Leu Val Gly Ser Glu Gly Ala Glu Ala465
470 47513482PRTBos sp.PEPTIDE(1)..(482)Bovine MG53 13Met Ser Ala
Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu1 5 10 15Cys Leu
Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser 20 25 30Phe
Cys Arg Ala Cys Leu Ser Arg Val Ala Gly Glu Pro Ala Ala Asp 35 40
45Gly Thr Val Leu Cys Pro Ser Cys Gln Ala Pro Thr Arg Pro Gln Ala
50 55 60Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala
Gln65 70 75 80Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu
Ser Ile Tyr 85 90 95Cys Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys
Ala Ser Leu Gly 100 105 110Ser His Arg Gly His Arg Leu Leu Pro Ala
Ala Glu Ala His Ala Arg 115 120 125Leu Lys Thr Gln Leu Pro Gln Gln
Lys Met Gln Leu Gln Glu Ala Cys 130 135 140Met Arg Lys Glu Lys Ser
Val Ala Leu Leu Glu His Gln Leu Leu Glu145 150 155 160Val Glu Glu
Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu 165 170 175Gly
Lys Met Arg Leu Phe Leu Ala Ala Leu Glu Gly
Ser Leu Asp Arg 180 185 190Glu Ala Glu Arg Val Arg Gly Glu Ala Gly
Val Ala Leu Arg Arg Glu 195 200 205Leu Gly Ser Leu Asn Ser Tyr Leu
Glu Gln Leu Arg Gln Met Glu Lys 210 215 220Val Leu Glu Glu Val Ala
Asp Lys Pro Gln Thr Glu Phe Leu Met Lys225 230 235 240Tyr Cys Leu
Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ser Pro 245 250 255Pro
Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp Asp Phe 260 265
270Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Arg
275 280 285Gln Glu Leu Thr Phe Asp Pro Ser Thr Ala His Pro Ser Leu
Val Leu 290 295 300Ser Asn Ser Gly Arg Cys Val Glu Cys Ser Glu Gln
Lys Ala Pro Pro305 310 315 320Ala Gly Glu Asp Pro Arg Gln Phe Asp
Lys Ala Val Ala Val Val Thr 325 330 335His Gln Leu Leu Ser Glu Gly
Glu His Tyr Trp Glu Val Glu Val Gly 340 345 350Asp Lys Pro Arg Trp
Ala Leu Gly Val Ile Gly Ala Gln Ala Gly Arg 355 360 365Arg Gly Arg
Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly 370 375 380Leu
Arg Asp Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro385 390
395 400Arg Ala Leu Arg Thr Pro Glu Arg Arg Pro Thr Arg Ile Gly Ile
Tyr 405 410 415Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala
Ser Asp Pro 420 425 430Asp Ala Leu Glu Leu Leu Phe Ala Phe His Glu
Arg Leu Pro Gly Pro 435 440 445Val Tyr Pro Phe Phe Asp Val Cys Trp
His Asp Lys Gly Lys Asn Ala 450 455 460Gln Pro Leu Leu Leu Val Gly
Pro Glu Val Ser Gly Gly Ser Gly Ser465 470 475 480Glu
Ala14477PRTRattus sp.PEPTIDE(1)..(477)Rat MG53 14Met Ser Thr Ala
Pro Gly Leu Leu Arg Gln Glu Leu Ser Cys Pro Leu1 5 10 15Cys Leu Gln
Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser 20 25 30Phe Cys
Arg Ala Cys Leu Ile Arg Val Ala Gly Glu Pro Ala Asp Asp 35 40 45Gly
Thr Val Ala Cys Pro Cys Cys Gln Ala Ser Thr Arg Pro Gln Ala 50 55
60Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln65
70 75 80Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile
Tyr 85 90 95Cys Glu Gln Asp Arg Thr Leu Val Cys Gly Val Cys Ala Ser
Leu Gly 100 105 110Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu
Ala His Ala Arg 115 120 125Leu Lys Thr Gln Leu Pro Gln Gln Lys Ala
Gln Leu Gln Glu Ala Cys 130 135 140Met Arg Lys Glu Lys Ser Val Ala
Val Leu Glu His Gln Leu Val Glu145 150 155 160Val Glu Glu Thr Val
Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu 165 170 175Gly Lys Met
Arg Met Phe Leu Ala Ala Leu Glu Ser Ser Leu Asp Arg 180 185 190Glu
Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu 195 200
205Leu Ser Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu
Met Lys225 230 235 240Phe Cys Leu Val Thr Ser Arg Leu Gln Lys Ile
Leu Ser Glu Ser Pro 245 250 255Pro Pro Ala Arg Leu Asp Ile Gln Leu
Pro Val Ile Ser Asp Asp Phe 260 265 270Lys Phe Gln Val Trp Lys Lys
Met Phe Arg Ala Leu Met Pro Glu Leu 275 280 285Glu Glu Leu Thr Phe
Asp Pro Ser Ser Ala His Pro Ser Leu Val Val 290 295 300Ser Ala Ser
Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro305 310 315
320Ala Gly Glu Asp Thr Cys Gln Phe Asp Lys Thr Val Ala Val Val Ala
325 330 335Lys Gln Leu Leu Ser Gln Gly Glu His Tyr Trp Glu Val Glu
Val Gly 340 345 350Asp Lys Pro Arg Trp Ala Leu Gly Val Met Ala Ala
Asp Ala Ser Arg 355 360 365Arg Gly Arg Leu His Ala Val Pro Ser Gln
Gly Leu Trp Leu Leu Gly 370 375 380Leu Arg Asp Gly Lys Ile Leu Glu
Ala His Val Glu Ala Lys Glu Pro385 390 395 400Arg Ala Leu Arg Thr
Pro Glu Arg Pro Pro Ala Arg Ile Gly Leu Tyr 405 410 415Leu Ser Phe
Ala Asp Gly Val Leu Thr Phe Tyr Asp Ala Ser Asn Thr 420 425 430Asp
Ala Leu Thr Pro Leu Phe Ser Phe His Glu Arg Leu Pro Gly Pro 435 440
445Val Tyr Pro Met Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ser
450 455 460Gln Pro Leu Leu Leu Val Gly Pro Asp Ser Glu Gln Ala465
470 47515477PRTXenopus laevisPEPTIDE(1)..(477)Xenopus laevis 15Met
Ser Thr Pro Gln Leu Met Gln Gly Met Gln Lys Asp Leu Thr Cys1 5 10
15Gln Leu Cys Leu Glu Leu Phe Arg Ala Pro Val Thr Pro Glu Cys Gly
20 25 30His Thr Phe Cys Gln Gly Cys Leu Thr Gly Val Pro Lys Asn Gln
Asp 35 40 45Gln Asn Gly Ser Thr Pro Cys Pro Thr Cys Gln Ser Pro Ser
Arg Pro 50 55 60Glu Thr Leu Gln Ile Asn Arg Gln Leu Glu His Leu Val
Gln Ser Phe65 70 75 80Lys Gln Val Pro Gln Gly His Cys Leu Glu His
Met Asp Pro Leu Ser 85 90 95Val Tyr Cys Glu Gln Asp Lys Glu Leu Ile
Cys Gly Val Cys Ala Ser 100 105 110Leu Gly Lys His Lys Gly His Asn
Ile Ile Thr Ala Ser Glu Ala Phe 115 120 125Ala Lys Leu Lys Arg Gln
Leu Pro Gln Gln Gln Val Ile Leu Gln Glu 130 135 140Ala Arg Leu Lys
Lys Glu Lys Thr Val Ala Val Leu Asp Arg Gln Val145 150 155 160Ala
Glu Val Gln Asp Thr Val Ser Arg Phe Lys Gly Asn Val Lys His 165 170
175Gln Leu Asn Ala Met Arg Ser Tyr Leu Asn Ile Met Glu Ala Ser Leu
180 185 190Gly Lys Glu Ala Asp Lys Ala Glu Ser Ala Ala Thr Glu Ala
Leu Leu 195 200 205Val Glu Arg Lys Thr Met Gly His Tyr Leu Asp Gln
Leu Arg Gln Met 210 215 220Glu Gly Val Leu Lys Asp Val Glu Gly Gln
Glu Gln Thr Glu Phe Leu225 230 235 240Arg Lys Tyr Cys Val Val Ala
Ala Arg Leu Asn Lys Ile Leu Ser Glu 245 250 255Ser Pro Pro Pro Gly
Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp 260 265 270Glu Phe Lys
Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro 275 280 285Ala
Leu Glu Asn Met Thr Phe Asp Pro Asp Thr Ala Gln Gln Tyr Leu 290 295
300Val Val Ser Ser Glu Gly Lys Ser Val Glu Cys Ala Asp Gln Lys
Gln305 310 315 320Ser Val Ser Asp Glu Pro Asn Arg Phe Asp Lys Ser
Asn Cys Leu Val 325 330 335Ser Lys Gln Ser Phe Thr Glu Gly Glu His
Tyr Trp Glu Val Ile Val 340 345 350Glu Asp Lys Pro Arg Trp Ala Leu
Gly Ile Ile Ser Glu Thr Ala Asn 355 360 365Arg Lys Gly Lys Leu His
Ala Thr Pro Ser Asn Gly Phe Trp Ile Ile 370 375 380Gly Cys Lys Glu
Gly Lys Val Tyr Glu Ala His Thr Glu Gln Lys Glu385 390 395 400Pro
Arg Val Leu Arg Val Glu Gly Arg Pro Glu Lys Ile Gly Val Tyr 405 410
415Leu Ser Phe Ser Asp Gly Val Val Ser Phe Phe Asp Ser Ser Asp Glu
420 425 430Asp Asn Leu Lys Leu Leu Tyr Thr Phe Asn Glu Arg Phe Ser
Gly Arg 435 440 445Leu His Pro Phe Phe Asp Val Cys Trp His Asp Lys
Gly Lys Asn Ser 450 455 460Gln Pro Leu Lys Ile Phe Tyr Pro Pro Ala
Glu Gln Leu465 470 47516477PRTXenopus sp.PEPTIDE(1)..(477)Xenopus
tropicalis MG53 16Met Ser Thr Pro Gln Leu Met Gln Gly Met Gln Lys
Asp Leu Thr Cys1 5 10 15Pro Leu Cys Leu Glu Leu Phe Arg Ala Pro Val
Thr Pro Glu Cys Gly 20 25 30His Thr Phe Cys Gln Gly Cys Leu Thr Gly
Ala Pro Lys Asn Gln Asp 35 40 45Gln Asn Gly Ser Thr Pro Cys Pro Thr
Cys Gln Thr Pro Ser Arg Pro 50 55 60Glu Thr Leu Gln Ile Asn Arg Gln
Leu Glu His Leu Val Gln Ser Phe65 70 75 80Lys Gln Val Pro Lys Gly
His Cys Leu Glu His Leu Asp Pro Leu Ser 85 90 95Val Tyr Cys Glu Gln
Asp Lys Glu Leu Ile Cys Gly Val Cys Ala Ser 100 105 110Leu Gly Lys
His Lys Gly His Asn Ile Ile Thr Ala Ala Glu Ala Tyr 115 120 125Ala
Lys Leu Lys Arg Gln Leu Pro Gln Gln Gln Val Ile Leu Gln Glu 130 135
140Ala Arg Leu Lys Lys Glu Lys Thr Val Ala Val Leu Asp Arg Gln
Val145 150 155 160Ala Glu Val Gln Asp Thr Val Ser Arg Phe Lys Gly
Asn Val Lys His 165 170 175Gln Leu Asn Ala Met Arg Ser Tyr Leu Ser
Ile Met Glu Ala Ser Leu 180 185 190Ser Lys Glu Ala Asp Asn Ala Glu
His Thr Ala Thr Glu Ala Leu Leu 195 200 205Val Glu Arg Lys Thr Met
Gly His Tyr Leu Asp Gln Leu Arg Gln Met 210 215 220Asp Gly Val Leu
Lys Asp Val Glu Ser Gln Glu Gln Thr Glu Phe Leu225 230 235 240Arg
Lys Tyr Cys Val Val Ala Ala Arg Leu Asn Lys Ile Leu Ala Glu 245 250
255Ser Pro Pro Pro Gly Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp
260 265 270Glu Phe Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu
Met Pro 275 280 285Ala Leu Glu Asn Leu Thr Phe Asp Pro Asp Thr Ala
Gln Gln Asn Leu 290 295 300Val Val Phe Ser Asp Gly Lys Ser Val Glu
Cys Ser Glu Gln Lys Gln305 310 315 320Ser Val Ser Asp Glu Pro Asn
Arg Phe Asp Lys Ser Asn Cys Leu Val 325 330 335Ser Lys Glu Ser Phe
Thr Glu Gly Glu His Tyr Trp Glu Val Leu Val 340 345 350Glu Asp Lys
Pro Arg Trp Ala Leu Gly Val Ile Ser Glu Thr Ala Asn 355 360 365Arg
Lys Gly Lys Leu His Ala Ser Pro Ser Asn Gly Phe Trp Leu Ile 370 375
380Gly Cys Lys Glu Gly Lys Val Tyr Glu Ala His Thr Glu Gln Lys
Glu385 390 395 400Pro Arg Val Leu Arg Val Glu Gly Arg Pro Glu Lys
Ile Gly Ile Tyr 405 410 415Leu Ser Phe Ser Asp Gly Val Val Ser Phe
Phe Asp Ser Ser Asp Glu 420 425 430Asp Asn Ile Lys Leu Leu Tyr Thr
Phe Asn Glu Arg Phe Ser Gly Arg 435 440 445Leu His Pro Phe Phe Asp
Val Cys Trp His Asp Lys Gly Lys Asn Ala 450 455 460Gln Pro Leu Lys
Ile Phe Tyr Pro Pro Ala Glu Gln Leu465 470 47517101PRTHuman
immunodeficiency virus type 1PEPTIDE(1)..(101)HIV-1 TAT protein
17Met Glu Pro Val Asp Pro Asn Leu Glu Pro Trp Lys His Pro Gly Ser1
5 10 15Gln Pro Pro Thr Ala Cys Ser Lys Cys Tyr Cys Lys Lys Cys Cys
Trp 20 25 30His Cys Gln Leu Cys Phe Leu Lys Lys Gly Leu Gly Ile Ser
Tyr Gly 35 40 45Arg Lys Lys Arg Lys His Arg Arg Gly Thr Pro Gln Ser
Ser Lys Asp 50 55 60His Gln Asn Pro Ile Pro Glu Gln Pro Leu Pro Ile
Ile Arg Gly Asn65 70 75 80Gln Thr Gly Pro Lys Glu Gln Lys Lys Thr
Val Ala Ser Lys Ala Glu 85 90 95Arg Asp Leu Cys Ala
1001866DNAArtificial SequenceChemically Synthesized 18gtacctcgcc
tgccgtccaa agttgtaatc aagagttaca actttggacg gcaggctttt 60tggaaa
661966DNAArtificial SequenceChemically Synthesized 19agcttttcca
aaaagcctgc cgtccaaagt tgtaactctt gattacaact ttggacggca 60ggcgag
662066DNAArtificial SequenceChemically Synthesized 20gtacctcgag
ctgtcaagcc tgaactcttc aagagagagt tcaggcttga cagctctttt 60tggaaa
662166DNAArtificial SequenceChemically Synthesized 21agcttttcca
aaaagagctg tcaagcctga actctctctt gaagagttca ggcttgacag 60ctcgag
662265DNAArtificial SequenceChemically Synthesized 22gatccgcgga
gacatagcct gtaattcaag agattacagg ctatgtctcc gcttttttac 60cggtg
652365DNAArtificial SequenceChemically Synthesized 23aattcaccgg
taaaaaagcg gagacatagc ctgtaatctc ttgaattaca ggctatgtct 60ccgcg
652465DNAArtificial SequenceChemically Synthesized 24gatccggaca
ttcactgcaa ggagttcaag agactccttg cagtgaatgt ccttttttac 60cggtg
652565DNAArtificial SequenceChemically Synthesized 25aattcaccgg
taaaaaagga cattcactgc aaggagtctc ttgaactcct tgcagtgaat 60gtccg
65
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