U.S. patent application number 16/156578 was filed with the patent office on 2019-01-31 for biglycan variant polypeptides and methods of use.
The applicant listed for this patent is Brown Univerity, Tivorsan Pharmaceuticals, Inc.. Invention is credited to Justin Fallon, Elizabeth John.
Application Number | 20190031731 16/156578 |
Document ID | / |
Family ID | 50477853 |
Filed Date | 2019-01-31 |
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United States Patent
Application |
20190031731 |
Kind Code |
A1 |
Fallon; Justin ; et
al. |
January 31, 2019 |
BIGLYCAN VARIANT POLYPEPTIDES AND METHODS OF USE
Abstract
The invention provides compositions and methods for treating,
preventing, and diagnosing diseases or conditions associated with
an abnormal level or activity of biglycan; disorders associated
with an unstable cytoplasmic membrane, due, e.g., to an unstable
dystrophin associated protein complex (DAPC); disorders associated
with abnormal synapses or neuromuscular junctions, including those
resulting from an abnormal MuSK activation or acetylcholine
receptor (AChR) aggregation. Examples of diseases include
Amyotrophic Lateral Sclerosis (ALS), as well as muscular
dystrophies, such as Duchenne's Muscular Dystrophy, Becker's
Muscular Dystrophy, neuromuscular disorders and neurological
disorders.
Inventors: |
Fallon; Justin; (Providence,
RI) ; John; Elizabeth; (Richland, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tivorsan Pharmaceuticals, Inc.
Brown Univerity |
Saint Louis
Providence |
MO
RI |
US
US |
|
|
Family ID: |
50477853 |
Appl. No.: |
16/156578 |
Filed: |
October 10, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14050225 |
Oct 9, 2013 |
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16156578 |
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61712177 |
Oct 10, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/04 20180101; A61K
38/17 20130101; A61P 25/00 20180101; A61P 19/08 20180101; A61P
19/02 20180101; A61P 21/04 20180101; C07K 14/47 20130101; C07K
14/4725 20130101; G01N 33/6896 20130101; A61P 19/10 20180101; A61P
21/00 20180101; A61P 21/02 20180101; G01N 2400/00 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; G01N 33/68 20060101 G01N033/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grants
UO1NS064295 (NIH), R21AR055878 (NIAM) and HD023924 (DHHS) awarded
by the National Institutes of Health. The government has certain
rights in the invention.
Claims
1. A method for preventing or treating a disorder in a subject,
comprising, administering to the subject a composition comprising
an isolated biglycan variant polypeptide lacking glycosaminoglycan
side chains, wherein the variant polypeptide potentiates
agrin-induced AChR clustering.
2. The method of claim 1, wherein the variant polypeptide is the M
form of the polypeptide.
3. The method of claim 1, wherein the disorder is a muscular,
neuromuscular or neurological disorder.
4. The method of claim 3, wherein the disorder is associated with
an abnormal dystrophin-associated protein complex (DAPC).
5. The method of claim 3, wherein the disorder is characterized by
an abnormal neuromuscular junction or synapse in the subject.
6. The method of claim 3, wherein the disorder is muscular
dystrophy.
7. The method of claim 6, wherein the disorder is selected from the
group consisting of Duchenne's Muscular Dystrophy, Becker's
Muscular Dystrophy, Congenital Muscular Dystrophy, Lamb-girdle
Muscular Dystrophy, and mytonic dystrophy.
8. The method of claim 3, wherein the disorder is low bone mass,
osteoarthritis, or ectopic bone formation.
9. The method of claim 3, wherein the disorder is Amyotrophic
Lateral Sclerosis.
10. The method of claim 3, wherein the disorder is congestive heart
failure.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 14/050,225 filed Oct. 9, 2013, now pending;
which claims the benefit under 35 USC .sctn. 119(e) to U.S.
Application Ser. No. 61/712,177 filed Oct. 10, 2012, now expired.
The disclosure of each of the prior applications is considered part
of and is incorporated by reference in the disclosure of this
application.
INCORPORATION OF SEQUENCE LISTING
[0003] The material in the accompanying Sequence Listing is hereby
incorporated by reference into this application. The accompanying
sequence listing text file, named TIVOR1100-2_ST25.txt was created
on Oct. 9, 2018 and is 13 KB. The file can be assessed using
Microsoft Word on a computer that uses Windows OS.
BACKGROUND OF THE INVENTION
Field of the Invention
[0004] The present invention relates generally to proteoglycans,
and more specifically to biglycan variants and use of such variants
in the treatment of disease.
Background Information
[0005] Biglycan is a 37 kd extracellular matrix protein. In muscle,
biglycan exists in two glycoforms: a proteoglycan that bears two
glucosaminoglycan (GAG) side chains and a non-glycanated form that
lacks GAG chains. Both forms contain N-linked carbohydrates at two
sites. Previous work showed that in mdx mice the recombinant
non-glycanated version is effective in: 1) recruiting utrophin and
other DAPC components to the muscle membrane; 2) improving muscle
health; and 3) improving muscle function.
[0006] Biglycan is an extracellular matrix component of many parts
of the skeleton including bone, cartilage, tendon, teeth and
muscle. Biglycan is predominantly expressed as a proteoglycan, but
a mature form lacking GAG side chains (`nonglycanated`) has
recently been shown to have specific functions in muscle, synapses
and Wnt signaling in bone. The biglycan gene is on the X (and not
Y) chromosome and is dysregulated in Turner (XO) and Kleinfelter's
Syndromes (supernumery X) diseases, characterized by short and tall
stature respectively. Biglycan deficient mice have shorter bones as
well as lower bone mass (ostepenia/osteoporosis), another notable
feature observed in Turner Syndrome. The mechanisms underlying the
thinner and weaker bones produced without biglycan have been
studied in detail and point to the fact that biglycan modulates
multiple pathways critical to skeletal metabolism.
[0007] While biglycan is not needed for development of the
musculoskeletal system, it is required for the maintenance of its
integrity. In adult bone turnover is regulated by a fine balance
between bone formation by osteoblasts and bone resorption by
osteoclasts. In the absence of biglycan, there is decreased bone
formation due to defects in the maturation of osteogenic precursors
that form bone. Bone Morphogenic Protein 2/4 (BMP-2/4), a
well-known inducer of bone formation, is currently being used
therapeutically to aid bone repair. Bone-derived cells depleted of
biglycan have less BMP-2/4 binding and subsequently less osteogenic
differentiation. It is logical to conclude that biglycan could be a
prime candidate to enhance BMP-2/4 function in situations where it
is commonly used such as in bone regeneration and repair after
fracture or trauma.
[0008] Mice lacking biglycan also display pathologies typically
associated with skeletal aging. Specifically, by three months of
age, hallmark signs of osteoarthritis (OA) are evident in the
mutant mice, including fissures, cell clustering and loss of the
smooth articular cartilage surface on the joints. The OA is
detected in all weight bearing joints as well as in the
temporomandibular joint of the jaw. The effects of biglycan loss
are exacerbated by depletion of the related small leucine-rich
proteoglycan fibromodulin (Bgn-/0; Fmod-/- DKO). Molecular studies
point to the abnormal sequestration of the potent growth factor
TGF-beta in the combined absence of biglycan and fibromodulin
causing it to be "unleashed" and subsequently overactive. The
uncontrolled stimulation of TGF-beta in this context leads to
hyper-proliferation, premature differentiation of cartilage derived
cells, MMP induction and, ultimately, loss of the condyle tissue
integrity.
[0009] Biglycan can also control the fate of skeletal stem cells by
modulating the extracellular niche. This function was demonstrated
in ECM-rich tendon tissue that harbors a cell population with stem
cell features including clonogenicity, multipotency and
regenerative capabilities. The combined removal of biglycan and
fibromodulin caused tendon stem/progenitor cells to be
hypersensitive to BMP-2. Instead of differentiating into tendon,
these progenitors form multiple ectopic bones within the tendons
that affect the gait of the mice. Biglycan also controls other
factors critical to bone in addition to TGF-beta and BMP-2/4. In
humans, a mutation in the extracellular domain of the key Wnt
signaling molecule LRP-6 (R611C) causes elevated cholesterol and
osteopenia. Notably, exogenous application of non-glycanated
biglycan repaired the defective Wnt signaling in cells expressing
mutant LRP-6. Thus, biglycan could potentially ameliorate
pathologies caused by defective Wnt signaling. Taken together these
findings underscore the importance of biglycan in modulating
several key growth factor-mediated signaling pathways that regulate
skeletal tissue architecture and function.
[0010] Biglycan also plays a role in organizing membrane
architecture and function in muscle and at synapses. Muscle
membranes are highly specialized to transmit force, protect the
cell from contraction-induced damage and orchestrate signaling
pathways required for normal function. The dystrophin- and
utrophin-membrane glycoprotein complexes (DGC and UGC,
respectively) link the cytoskeleton to the extracellular matrix and
serve as a scaffold for signaling molecules in adult (DGC) and
immature (UGC) muscle. Biglycan binds to three shared components of
these complexes: the extracellular peripheral membrane protein
alpha-dystroglycan and the transmembrane proteins alpha- and
beta-sarcoglycan. Genetic studies show that biglycan regulates the
expression of utrophin, the two sarcoglycans and an intracellular
membrane-associated signaling complex comprised of dystrobrevin,
syntrophins and nNOS (neuronal nitric oxide synthase) in immature
muscle. Notably, dosing mice with recombinant non-glycanated
biglycan (rNG-BGN) can restore the expression of several of these
components to the membrane.
[0011] The role of biglycan in binding and regulating several
components of DGC and UGC, coupled with the ability to deliver
rNG-BGN systemically, suggested that biglycan could be a
therapeutic for Duchenne Muscular Dystrophy (DMD). DMD is the most
common form of muscular dystrophy and results from mutations in
dystrophin--a large intracellular protein that links the actin
cytoskeleton to the membrane and anchors the DGC. Notably, utrophin
upregulation can compensate for dystrophin loss in mouse models of
DMD (mdx; Davies). Systemically-delivered rNG-BGN recruits utrophin
to the membrane and improves muscle health and function in mdx
mice. The efficacy of the non-glycanated form (i.e., lacking GAG
side chains) in this therapeutic approach is most likely based on
two reasons. First, this form can be readily manufactured in a
homogeneous form. Second, the glyconated form of biglycan,
proteoglycan (PG), is proinflammatory, but the non-glycanated
(core) is not. A non-glycanated form of biglycan is currently in
preclinical development for DMD.
[0012] Biglycan is also important for synapse stabilization. In
biglycan-deficient mice, neuromuscular junctions form normally but
then they become unstable about three weeks after birth. The
mechanism of biglycan action at the synapses is likely to involve
MuSK, a receptor tyrosine kinase that is the master regulator of
synapse differentiation and maintenance. Biglycan binds to MuSK and
regulates its expression in vivo. Notably, synaptic loss is one of
the earliest abnormalities observed in almost all neurodegenerative
diseases, including ALS (amyotrophic lateral sclerosis) and SMA
(spinal muscular atrophy). Treatments that promote neuromuscular
junction stability could prolong function and potentially survival
in these devastating motor neuron diseases.
SUMMARY OF THE INVENTION
[0013] The invention is based on the discovery of a biglycan
variant termed T2-rhBGN (T2 recombinant human biglycan). In this
molecule the two sites of GAG addition have been mutated to
alanine. Enzymatic and carbohydrate analysis showed that this form
completely lacks GAG side chains. Stable clonal cell lines
expressing T2-rhBGN have been derived from CHO and HEK 293 hosts.
Data from three of these lines (CHO 57, CHO 171, HEK 293) are
included herein.
[0014] Biglycan plays important roles in the musculoskeletal
system. The fact that non-glycanated forms of biglycan are
effective in ameliorating muscle defects and that it can be
administered systemically makes it particularly amenable for tissue
and cell therapy. Taken together, it is reasonable to conclude that
biglycan holds promise as a novel therapeutic for numerous
musculoskeletal diseases including low bone mass, osteoarthritis,
ectopic bone formation and muscular dystrophy.
[0015] Accordingly, in one aspect, the present invention provides
an isolated biglycan variant polypeptide lacking glycosaminoglycan
(GAG) side chains. In various embodiments, the variant polypeptide
potentiates agrin-induced AChR clustering and is the M form of the
molecule. In one embodiment the variant polypeptide includes an
amino acid sequence which is at least about 90%, 95%, 96%, 97%, 98%
or 99% identical to SEQ ID NO: 1, or portion thereof, includes a
mutation at amino acid residue 42 or 47, or combination thereof. In
some embodiments, the mutation at amino acid residue 42 or 47 is
substitution with an alanine. In some embodiments, the variant
polypeptide includes an amino acid sequence as set forth in amino
acid residues 38-48 of SEQ ID NO: 1. In some embodiments, the
variant polypeptide includes an amino acid sequence as set forth in
amino acid residues 38-80 of SEQ ID NO: 1.
[0016] In another aspect, the invention provides an isolated
nucleic acid molecule encoding the variant polypeptide of the
present invention.
[0017] In another aspect, the invention provides an expression
cassette including the nucleic acid encoding the variant
polypeptide of the present invention.
[0018] In another aspect, the invention provides a vector including
the expression cassette of the present invention.
[0019] In another aspect, the invention provides a pharmaceutical
composition. In one embodiment, the pharmaceutical composition
includes the variant polypeptide of the present invention and a
pharmaceutically acceptable carrier. In another embodiment, the
pharmaceutical composition includes a nucleic acid molecule
encoding the variant polypeptide of the present invention and a
pharmaceutically acceptable carrier.
[0020] In yet another aspect, the invention provides a method of
preventing or treating a disorder in the subject. The method
includes administering to the subject a composition including the
variant polypeptide of the present invention or a pharmaceutical
composition including such a peptide as described herein.
[0021] In another aspect, the invention provides a method for
determining whether a subject has or is at risk of developing a
disorder. The method includes: a) determining a level or activity
of the variant polypeptide of the present invention in a sample
from the subject; and b) comparing the level or activity of the
variant polypeptide to that of a corresponding or known sample,
wherein an increase or decrease the level or activity of the
variant polypeptide as compared to that of the corresponding or
known sample is indicative of having or developing the disorder,
thereby determining whether the subject has or is at risk of
developing the disorder. In various embodiments, the method further
includes administering to the subject a composition including the
variant polypeptide of the present invention or pharmaceutical
composition as described herein.
[0022] In various embodiments, disorders include muscular,
neuromuscular and neurological disorders. Such disorders include
those associated with an abnormal dystrophin-associated protein
complex (DAPC) and those disorders that are characterized by an
abnormal neuromuscular junction or synapse in the subject. In some
embodiments, the disorder is muscular dystrophy, such as Duchenne's
Muscular Dystrophy, Becker's Muscular Dystrophy, Congenital
Muscular Dystrophy, Lamb-girdle Muscular Dystrophy, and mytonic
dystrophy, or the disorder is low bone mass, osteoarthritis, or
ectopic bone formation. In other embodiments, the disorder could be
other neurodegenerative disorders such as Amyotrophic Lateral
Sclerosis (ALS). In other embodiments, the disorder could be other
muscular disorders such as congestive heart failure.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1. Analytical Ultracentrifugation of HIC Pool Purified
from CHO Clone 57. HIC pool in 20 mM Tris, pH 8.0, 0.5M NaCl was
analyzed by AUC at 0.2, 0.6 and 2 mg/ml. The HIC pool resolved into
2 sedimentation peaks: M and D. Analysis of the sedimentation at
three concentrations showed that the dissociation constant for M-D
exchange in the HIC pool was .about.50 .mu.M.
[0024] FIGS. 2A-2B. Purification of M- and D-rhBGN by Size
Exclusion Chromatography. CHO clone 57 HIC pool in 20 mM Tris, pH
8.0, 0.5M NaCl was applied to a Superdex.TM. 200 column
equilibrated with 1.0M NaCl, 20 mM Tris, pH 8.0. FIG. 2A) First
SEC: HIC pool resolved into two discrete, partially overlapping
peaks that eluted at .about.18.7 (D) and 20.4 ml (M). FIG. 2B)
Fractions corresponding to M and D were re-analyzed on the size
exclusion column. The elution profiles (overlaid) show a clear
separation of the M and D forms and there was little exchange
between them.
[0025] FIGS. 3A-3B. Capillary Electrophoresis of M- and D-rhBGN. M-
and D-rhBGN were purified from CHO Clone 57 by size exclusion
chromatography. M and D were analyzed by capillary electrophoresis
using an Agilent Bioanalyzer 2100.TM. and Agilent Protein 80.TM.
assay kit. The apparent molecular weight and mobility of D-rhBGN
were greater than that of M-rhBGN, indicating differential
posttranslational modification of the two forms.
[0026] FIG. 4. N-linked Oligosaccharide Profiling Analysis of M-
and D-rhBGN. M- and D-rhBGN were purified from CHO Clone 57 by size
exclusion chromatography. N-glycans were cleaved from the samples,
purified and analyzed by MALDI/TOF-TOF. The percentages of each
glycan were calculated for each sample. M and D forms showed
varying amounts of complex-type glycans, branching and sialic
acid.
[0027] FIG. 5. Bioactivity of M- and D-rhBGN. M- and D-rhBGN were
purified from CHO Clone 171 by size exclusion chromatography and
tested for bioactivity using an AChR clustering assay. Note the
robust and broad dose-response observed with M-rhBGN and the
uniformly low activity at all concentrations of D-rhBGN.
[0028] FIG. 6. In vivo Efficacy of M-rhBGN. M-rhBGN was purified
from HEK-293 Clone 2 by size exclusion chromatography. Male mdx
mice were IP injected at P18 and P25 with the indicated doses of
M-rhBGN or vehicle. Diaphragm muscles were harvested at P32 and
myofibers with central nuclei were scored. M-rhBGN reduced the
percentage of myofibers with central nuclei (*p<0.05, 1-way
ANOVA analysis with post-hoc Dunnett's Multiple Comparison, n=7-10
animals/group). There was a statistically significant response
across a 20-fold range (0.5-10 mg/kg).
[0029] FIGS. 7A-7B. Near and Far UV CD of M- and D-rhBGN. FIG. 7A)
Near UV CD spectra of M-(black) and D-rhBGN (gray). There is a
difference between M and D, as seen in the expanded scale (right
panel), with D spectrum shifted upward. FIG. 7B) Far UV CD spectra
of M-(black) and D-rhBGN (gray). There is a difference in the
spectra of M and D, indicating the different forms have different
structures.
BRIEF DESCRIPTION OF THE INVENTION
[0030] The invention is based on the discovery of a biglycan
variant termed T2-rhBGN (T2 recombinant human biglycan) in which
sites of GAG addition have been mutated thereby eliminating
addition of GAG side chains to the variant. Such variants are
useful in various therapeutic and diagnostic application. As such,
in one aspect, the invention provides biglycan therapeutics for use
in maintaining the integrity of plasma cell membranes, in
particular, biglycan therapeutics which stabilize dystrophin
associated protein complexes (DAPC) in these membranes, thereby
preventing the disintegration of the membranes. The invention also
provides biglycan therapeutics which stimulate neuromuscular
junction formation, such as by stimulating postsynaptic membrane
differentiation, and more generally compounds which stimulate
synapse formation.
[0031] Preferred therapeutics of the invention have one or more
biological activities of biglycan, in addition to, or instead of,
being able to bind one or more components of the DAPC and/or MuSK.
For example, a therapeutic of the invention can stimulate
neuromuscular junction formation, in particular, postsynaptic
membrane differentiation, including inducing aggregation of AChRs
and/or stimulating or stimulating agrin-induced tyrosine
phorphorylation of MusK.
[0032] The therapeutics of the invention can be a protein or
derivative thereof, in particular a biglycan variant polypeptide as
described herein. The variant of the present invention has a
mutated sequence which eliminates addition of GAG side chains.
[0033] In a preferred embodiment, the variant of the invention is
the M form proteoglycan having a molecular weight from about 40 kDa
to about 44 kDa, as determined, e.g., by migration on an SDS
acrylamide gel. In the preferred embodiment, the "M form" is
generally considered monomeric, but is not required to be
monomeric. Fragments or portions of these proteoglycans are also
within the scope of the invention. While not wanting to be bound by
a particular theory it is believed that the D form of biglycan
negatively affects the M form (when in a mixture). In the preferred
embodiment, the "D form" is generally dimeric, but is not required
to be dimeric. It merely negatively affects the M form.
[0034] The proteoglycan of the invention is a member of the family
of small leucine-rich proteoglycans (SLRP), also referred to as
"nonaggreagating or small dermatan-sulfate proteoglycans because of
their inability to interact with hyaluronan, or because of their
type of glycosaminoglycans, respectively. SLRPs are organized into
three classes based on their protein and genomic organization. All
SLRPs are characterized by a central domain containing leucine rich
repeats (LRR) flanked at either side by small cysteine clusters.
The SLRPs are described, e.g., in Iozzo et al. (1998) Ann. Rev.
Biochem. 67:609, specifically incorporated herein by reference.
[0035] SLRP protein cores range from about 35-45 kD with one or two
GAG chains attached at the extreme N-terminus. The general
structure of the SLRP protein core consists of a tandem array of
6-10 leucine-rich repeats (LRR) flanked by domains with conserved,
disulfide-bonded cysteines. Depending upon the extent of
glycosylation and number of GAG chains, the native molecular weight
ranges from .about.100-250 kD. On the basis of their sequence
homology, Iozzo, supra, has proposed that SLRPs be grouped into
three classes consisting of: 1) biglycan and decorin; 2)
fibromodulin, lumican, keratocan, PREPLP, and osteoadherin; and 3)
epiphycan and osteoglycin. The most compelling feature of the SLRP
protein core are the LRRs. Such repeats (24aa each in the SLRPs)
mediate protein-protein interactions in a wide variety of
intracellular, transmembrane, and extracellular contexts (Kobe
& Deisenhofer, (1994) Trends Biochem. Sci. 19: 415-21). The
neurotrophin binding site on trkB, for example, is an LRR (Windisch
et al., (1995) Biochemistry 34: 11256-63). The repeats are thought
to have a general structure of an .alpha.-helix followed by
beta-sheet in an anti-parallel array, although sequence analysis
has suggested that this order might be reversed in the SLRPs
(Hocking et al., (1998) Matrix Biol. 17: 1-19). It is likely that
the conserved residues of each repeat dictate their secondary
structure, while the intervening amino acids determine specificity
of ligand binding.
[0036] Preferred SLRPs for use in the invention include Class I
SLRPs, such as biglycan. Nucleotide and amino acid sequences of
biglycan genes and proteins from various species are publically
available, such as in GenBank. For example, human biglycan can be
found under GenBank Accession No. J04599 (human hPGI encoding bone
small proteoglycan I (biglycan), described in Fisher et al. (1989)
J. Biol. Chem. 264: 4571) and M65154; cow biglycan can be found
under GenBank Accession No. L07953; rat biglycan can be found under
GenBank Accession No. U17834, mouse biglycan can be found under
GenBank Accession No. L20276 and X53928; ovis biglycan can be found
under GenBank Accession No. AF034842.
[0037] Typically, biglycan has two glycosaminoglycan (GAG) chains,
respectively. Their composition is tissue specific and can be
regulated at a number of levels (Hocking et al., (1998) Matrix Biol
17: 1-19). For example, the biglycan GAG from skin and cartilage is
predominantly dermatan sulfate, while biglycan synthesized in bone
is a chondroitin sulfate proteoglycan. Heparan sulfate side chains
have not been reported. Both the protein core and the cell type
contribute to the distinct glycosylation of these SLRPs.
[0038] However, as discussed herein, the proteoglycan of the
present invention has a mutated sequence which eliminates addition
of GAG side chains. Portions and fragments of the proteoglycans of
the invention are also within the scope of the invention. A portion
is typically at least five, 10, 15, or 20 amino acids long.
Preferred portions are those which are sufficient for exerting a
biological activity, such as interacting with a DAPC component.
Portions can comprise or consist of one or more specific domain of
a protein. Domains of biglycan include two cysteine-rich regions
(included in the N- and C-terminal 40-50 amino acids of mature
biglycan) and leucine-rich repeats (LRRs). The "LRR region" refers
to the region of biglycan containing the repeats, and consists
essentially of amino acids 81-314. Each individual repeat is
referred to herein as an "LRR." LRRs are believed to mediate
protein: protein interactions and may thus be sufficient for
stabilizing DAPCs and postsynaptic membranes.
[0039] A preferred biglycan of the invention consists of a portion
of biglycan that is capable of binding to a sarcoglycan. It has
been shown that the .alpha.-sarcoglycan binding domain of human
biglycan is located in the N-terminal domain of the mature biglycan
protein, i.e., amino acids 38-80, and more specifically, amino
acids 38-58 of SEQ ID NO: 1. The GAG chains are not necessary for
binding to .alpha.-sarcgoglycan. It has also been shown that the
C-terminal cysteine-rich domain mediates interaction with
.gamma.-sarcoglycan. Accordingly, preferred biglycans of the
invention include portions of biglycan consisting of the N-terminal
or the C-terminal cysteine-rich domain, i.e., amino acids 38-48,
38-80 and 315-368 of SEQ ID NO: 1. Combinations of certain domains
of biglycan are also within the scope of the invention.
[0040] Preferred fragments consist of at least about 30 amino
acids, at least about 40 amino acids, 50, 60, 70, 80, 90, 100, 150,
or 200 amino acids. Short portions of the proteoglycans of the
invention are termed "mini-proteoglycan of the invention." For
example, a biglycan core fragment of about 20, 30 or 40 amino acids
is referred to as a "mini-biglycan."
[0041] Human biglycan consists of 368 amino acids (SEQ ID NO: 1),
of which amino acids 1-19 constitute a signal peptide (GenBank
Accession No. NP001702 and Fisher et al., supra). Thus biglycan
without a signal peptide consists of amino acids 20-368 of SEQ ID
NO: 1. The mature biglycan protein consists of amino acids 38-368
of SEQ ID NO: 1, since amino acids 1-37, being a pre-propeptide,
are cleaved during processing. Amino acids 38-80 correspond to the
N-terminal cysteine-rich region. About amino acids 81-314
corresponds to the leucine rich repeat region, containing 10
repeats of about 24 or 23 amino acids.
[0042] A proteoglycan of the present invention is a variant
biglycan polypeptide having a mutated sequence with reference to
SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the variant
includes mutations at amino acid residues 42 and 47 (corresponding
to amino acid positions 5 and 10 of SEQ ID NO: 2) which eliminate
GAG side chains. In one embodiment, the variant has the amino acid
sequence of SEQ ID NO: 2, wherein either or both amino acids 5 and
10 are any amino acid other than serine (wild type). In one
embodiment, the variant has the amino acid sequence of SEQ ID NO: 3
in which amino acids 5 and 10 are alanine.
[0043] Preferred proteoglycans of the invention are encoded by
nucleotide sequences which are at least about 70%, preferably at
least about 80%, even more preferably at least about 85%, at least
about 90%, at least about 95%, at least about 98%, and even more
preferably at least about 99% identical to the nucleotide sequence
of an SLRP, e.g., biglycan, or portion thereof.
[0044] Preferred nucleic acids of the invention include those
encoding a polypeptide comprising an amino acid sequence which is
at least about 70%, preferably at least about 80%, even more
preferably at least about 85%, at least about 90%, at least about
95%, at least about 98%, and even more preferably at least about
99% identical to the nucleotide sequence of an SLRP, e.g.,
biglycan, or portion thereof. In one embodiment, the nucleic acid
encodes a polypeptide containing all or a fragment of SEQ ID NO:
1.
[0045] Methods for preparing compounds of the invention are well
known in the art. For a compound of the invention which is a
protein or a derivative thereof, the compound can be isolated from
a tissue or the compound can be recombinantly or synthetically
produced. Isolation of the protein from a tissue is described in
the Examples. The proteins or proteoglycans of the invention
isolated from tissue are preferably at least about 70%, preferably
at least about 80%, at least about 85%, at least about 90%, at
least about 95%, at least about 98% and most preferably, at least
about 99% pure. Accordingly, preferred compounds contain less than
about 1%, and even more preferably less than about 0.1% of material
from which the compound was extracted.
[0046] The protein of the invention can also be produced
recombinantly, according to methods well known in the art.
Typically, a gene encoding the protein is inserted into a plasmid
or vector, and the resulting construct is then transfected into
appropriate cells, in which the protein is then expressed, and from
which the protein is ultimately purified.
[0047] Accordingly, the present invention further pertains to
methods of producing the subject proteins. For example, a host cell
transfected with an expression vector encoding a protein of
interest can be cultured under appropriate conditions to allow
expression of the protein to occur. The protein may be secreted, by
inclusion of a secretion signal sequence, and isolated from a
mixture of cells and medium containing the protein. Alternatively,
the protein may be retained cytoplasmically and the cells
harvested, lysed and the protein isolated. A cell culture includes
host cells, media and other byproducts. Suitable media for cell
culture are well known in the art. The proteins can be isolated
from cell culture medium, host cells, or both using techniques
known in the art for purifying proteins, including ion-exchange
chromatography, gel filtration chromatography, ultrafiltration,
electrophoresis, and immunoaffinity purification with antibodies
specific for particular epitopes of the protein.
[0048] Thus, a coding sequence for a protein of the present
invention can be used to produce a recombinant form of the protein
via microbial or eukaryotic cellular processes. Ligating the
polynucleotide sequence into a gene construct, such as an
expression vector, and transforming or transfecting into hosts,
either eukaryotic (yeast, avian, insect or mammalian) or
prokaryotic (bacterial cells), are standard procedures.
[0049] Expression vehicles for production of a recombinant protein
include plasmids and other vectors. For instance, suitable vectors
for the expression of the instant fusion proteins include plasmids
of the types: pBR322-derived plasmids, pEMBL-derived plasmids,
pEX-derived plasmids, pBTac-derived plasmids and pUC-derived
plasmids for expression in prokaryotic cells, such as E. coli.
[0050] A number of vectors exist for the expression of recombinant
proteins in yeast. For instance, YEP24, YIPS, YEP51, YEP52, pYES2,
and YRP17 are cloning and expression vehicles useful in the
introduction of genetic constructs into S. cerevisiae (see, for
example, Broach et al., (1983) in Experimental Manipulation of Gene
Expression, ed. M. Inouye Academic Press, p. 83, incorporated by
reference herein). These vectors can replicate in E. coli due the
presence of the pBR322 ori, and in S. cerevisiae due to the
replication determinant of the yeast 2 micron plasmid. In addition,
drug resistance markers such as ampicillin can be used.
[0051] The protein can be produced either in eukaryotic cells,
e.g., mammalian cells, yeast cells, insect cell (baculovirus
system) or in prokaryotic cells. When biglycan is used in the
context of muscle cells, it is preferable to produce biglycan in
muscle cells, e.g., C2 muscle cells.
[0052] Alternatively, a protein core of a proteoglycan can be
produced in a prokaryote, which results in a protein without
glucose side chains.
[0053] Immortalized cell lines, e.g., muscle cell lines, such as
biglycan negative cell lines, can be obtained as described in Jat
et al., PNAS (1991) 88: 5096-100; Noble et al., (1992) Brain
Pathology 2: 39-46. In one embodiment, a H-2K.sup.b/tsA58
transgenic mouse is used. This mouse is a heterozygote harboring a
thermolabile immortalizing gene (the tsA58 mutant of SV40 large T
antigen) under the control of an interferon-inducible promoter
(this mouse is available at Charles River). When cells containing
this gene are cultured, they proliferate indefinitely at 33 C. in
the presence of interferon. However, when the temperature is raised
to 39 C. (at which temperature the tsA58 antigen is non-functional)
and interferon is removed, the cells cease dividing.
[0054] This method has been used for growing a wide variety of cell
types, including astrocytes, osteoclasts, trabecular network, and
colon epithelial cells (Chambers et al., (1993) PNAS 90: 5578-82;
Groves et al., (1993) Dev. Biol. 159: 87-104; Whitehead et al.,
(1993) PNAS 90: 587-91; Noble et al., (1995) Transgenic Res. 4:
215-25; Tamm et al., (1999) Invest. Ophtamol. Vis. Sci. 40:
1392-403. This technique is well suited for the production of
muscle cell lines. For example, in one study alone 65 separate
muscle cell lines were derived from animals ranging in age from
neonates to four weeks (Morgan et al., (1994) Dev. Biol. 162
486-98). These lines were maintained for upwards of 80 generations.
Remarkably, they not only formed myotubes when shifted to
non-permissive conditions in culture, but also formed muscle when
implanted into host mice. The H-2K.sup.b/tsA58 transgenic method
was also used by D. Glass and colleagues to produce a MuSK.sup.-/-
muscle cell line (Sugiyama et al., (1997) J. Cell Biol. 139:
181-91).
[0055] To produce conditionally immortalized cell lines, mice
having a specific mutation, e.g., a deficiency in biglycan or MuSK,
can be crossed with heterozygote H-2K.sup.b/tsA58 transgenic mice.
The crosses are straightforward since only one copy of the gene is
required for full activity. Muscle cells from neonatal animals can
then be plated out and grown under permissive conditions (33C with
interferon). Proliferating cells can then be cloned and samples
from each line shifted to the non-permissive temperature and tested
for their ability to form myotubes. Wild type; decorin.sup.-/-;
biglycan.sup.-/o; and decorin.sup.-/- biglycan.sup.-/o cell lines
are examples of cell lines which can be obtained using this
technique.
[0056] Although the preferred method for treating subjects with a
biglycan is by administration of the biglycan to the subject (based
at least on the efficiency of biglycan when added to cell
cultures), the proteoglycans of the invention can also be produced
in a subject, by gene therapy techniques. Thus, e.g., a subject can
receive an injection in a muscle (e.g., where the subject has a
muscle dystrophy) of a vector encoding a protein or proteoglycan of
the invention, such that the vector is capable of entering muscle
cells and being expressed therein. Alternatively, the vector can be
a viral vector, which is provided with the viral capsid and the
virus infects the cells, e.g., muscle cells and thereby deliver the
vector. Methods and vectors for gene therapy are well known in the
art. Illustrative methods are set forth below.
[0057] The preferred mammalian expression vectors contain both
prokaryotic sequences to facilitate the propagation of the vector
in bacteria, and one or more eukaryotic transcription units that
are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo,
pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7,
pko-neo and pHyg derived vectors are examples of mammalian
expression vectors suitable for transfection of eukaryotic cells.
Some of these vectors are modified with sequences from bacterial
plasmids, such as pBR322, to facilitate replication and drug
resistance selection in both prokaryotic and eukaryotic cells.
Alternatively, derivatives of viruses such as the bovine papilloma
virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205)
can be used for transient expression of proteins in eukaryotic
cells. Examples of other viral (including retroviral) expression
systems can be found below in the description of gene therapy
delivery systems. The various methods employed in the preparation
of the plasmids and transformation of host organisms are well known
in the art. For other suitable expression systems for both
prokaryotic and eukaryotic cells, as well as general recombinant
procedures, see Molecular Cloning: A Laboratory Manual, 2nd Ed.,
ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor
Laboratory Press, 1989) Chapters 16 and 17. In some instances, it
may be desirable to express the recombinant fusion proteins by the
use of a baculovirus expression system. Examples of such
baculovirus expression systems include pVL-derived vectors (such as
pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as
pAcUW1), and pBlueBac-derived vectors (such as the -gal containing
pBlueBac III).
[0058] In yet other embodiments, the subject expression constructs
are derived by insertion of the subject gene into viral vectors
including recombinant retroviruses, adenovirus, adeno-associated
virus, and herpes simplex virus-1, or recombinant bacterial or
eukaryotic plasmids. As described in greater detail below, such
embodiments of the subject expression constructs are specifically
contemplated for use in various in vivo and ex vivo gene therapy
protocols.
[0059] Retrovirus vectors and adeno-associated virus vectors are
generally understood to be the recombinant gene delivery system of
choice for the transfer of exogenous genes in vivo, particularly
into humans. These vectors provide efficient delivery of genes into
cells, and the transferred nucleic acids are stably integrated into
the chromosomal DNA of the host. A major prerequisite for the use
of retroviruses is to ensure the safety of their use, particularly
with regard to the possibility of the spread of wild-type virus in
the cell population. The development of specialized cell lines
(termed "packaging cells") which produce only replication-defective
retroviruses has increased the utility of retroviruses for gene
therapy, and defective retroviruses are well characterized for use
in gene transfer for gene therapy purposes (for a review see
Miller, A. D. (1990) Blood 76:271). Thus, recombinant retrovirus
can be constructed in which part of the retroviral coding sequence
(gag, pol, env) has been replaced by nucleic acid encoding a fusion
protein of the present invention rendering the retrovirus
replication defective. The replication defective retrovirus is then
packaged into virions which can be used to infect a target cell
through the use of a helper virus by standard techniques. Protocols
for producing recombinant retroviruses and for infecting cells in
vitro or in vivo with such viruses can be found in Current
Protocols in Molecular Biology, Ausubel, F. M. et al., (eds.)
Greene Publishing Associates, (1989), Sections 9.10-9.14 and other
standard laboratory manuals.
[0060] In clinical settings, the gene delivery systems can be
introduced into a patient by any of a number of methods, each of
which is familiar in the art. For instance, a pharmaceutical
preparation of the gene delivery system can be introduced
systemically, e.g., by intravenous injection, and specific
transduction of the construct in the target cells occurs
predominantly from specificity of transfection provided by the gene
delivery vehicle, cell-type or tissue-type expression due to the
transcriptional regulatory sequences controlling expression of the
gene, or a combination thereof. In other embodiments, initial
delivery of the recombinant gene is more limited with introduction
into the animal being quite localized. For example, the gene
delivery vehicle can be introduced by catheter (see U.S. Pat. No.
5,328,470) or by stereotactic injection (e.g., Chen et al., (1994)
PNAS USA 91: 3054-3057).
[0061] The gene encoding the proteoglycan of the invention can be
under the control of a constitutive, or inducible promoter. These
are well known in the art.
[0062] Methods for determining whether a compound has a biological
activity of a biglycan protein are described. A biological activity
of a biglycan protein is intended to refer to one or more of: the
ability to maintain the integrity of a plasma membrane; the ability
to stabilize DAPCs on plasma membranes; the ability to bind to one
or more components of DAPCs; e.g., binding to .alpha.-dystroglycan,
binding to a sarcoglycan component, such as .alpha.-sarcoglycan;
phosphorylation of .alpha.-sarcoglycan; binding to MuSK;
stimulating the formation of neuromuscular junctions, such as by
stimulating postsynaptic differentiation; stimulating AChR
aggregation; stimulation of MuSK phosphorylation and potentiation
of agrin-induced MuSK phosphorylation. Such methods can further be
adapted for screening libraries of compounds for identifying
compounds having one or more of the above-described activities.
[0063] Breakdown of cytoplasmic membranes, e.g., the presence of
"leaky membranes" can be determined by assays which measure the
release of creatine kinase or the absorption of Evans Blue dye, as
described, e.g., in Tinsley et al. (1996) Nature 384: 349 and
Straub et al. (1997) J. Cell Biol. 139: 375).
[0064] The compounds of the invention can also be tested in a
variety of animal models, in particular the mdx mice, which are
dystrophin negative.
Methods of Treatment
[0065] The invention provides therapeutic and prophylactic methods
of treatment of disorders including muscular, neuromuscular, and
neurological disorders. Therapeutic methods are intended to
eliminate or at least reduce at least one symptom of a disease or
disorder, and preferably cure the disease or disorder. Prophylactic
methods include those intended to prevent the appearance of a
disease or disorder, i.e., a method which is intended to combat the
appearance of the disease or disorder.
[0066] Furthermore, since DAPCs are also found on other cell types,
the invention also provides methods for treating diseases
associated with any abnormal DAPC. For example, DAPC are present in
the brain, and since, in addition, agrin has been found in senile
plaques in patients with Alzheimer's disease, neurological diseases
can also be treated or prevented according to the methods of the
invention. A further indication that neurological disorders can be
treated or prevented according to the methods described herein is
based on the observation that patients with muscular dystrophy
often also suffer from peripheral and central nervous system
disorder. Accordingly, about one third of patients with Duchenne
Muscular Dystrophy have a mental affliction, in particular, mental
retardation. Thus, dystrophin, and hence, DAPCs, are believed to
play a role in the nervous system.
[0067] Patients with Duchenne's Muscular Dystrophy also have
diaphragm problems, indicating a role for dystrophin, and possibly
DAPCs in diaphragms. Thus, therapeutics of the invention would also
find an application in disorders associated with diaphragm
abnormalities.
[0068] It should be noted that diseases that can be treated or
prevented include not only those in which biglycan is abnormal, but
more generally any disease or condition that is associated with a
defect that can be improved or cured by biglycan. In particular,
diseases that are characterized by a defect or an abnormality in
any component of the DAPC or component associated therewith,
thereby resulting, e.g., in an unstable plasma membrane, can be
treated or prevented according to the methods of the invention,
provided that the proteoglycan of the invention can at least
partially cure the defect resulting from the deficient component.
In particular, diseases that can be treated according to the method
of the invention include any disease associated with an unstable
DAPC, which can be rendered more stable by the presence of a
proteoglycan of the invention.
[0069] Exemplary Diseases and Disorders: Diseases or disorders that
are characterized by a destabilization or improper organization of
the plasma membrane of specific cell types include muscular
dystrophies (MDs), a group of genetic degenerative myopathies
characterized by weakness and muscle atrophy without nervous system
involvement. The three main types are pseudohypertrophic (Duchenne,
Becker), limb-girdle, and facioscapulohumeral. For example,
muscular dystrophies and muscular atrophies are characterized by a
breakdown of the muscle cell membrane, i.e., they are characterized
by leaky membranes, which are believed to result from a mutation in
a component of the DAPC, i.e., dystrophin. Mutations in the
sarcoglycans are also known to result in muscular dystrophies and
leaky membranes. Accordingly, the invention provides for methods
for treating or preventing diseases associated with mutations in
dystrophin and/or in sarcoglycans or other component of DAPCs, in
particular muscular dystrophies.
[0070] Dystrophin abnormalities are responsible for both the milder
Becker's Muscular Dystrophy (BMD) and the severe Duchenne's
Muscular Dystrophy (DMD). In BMD dystrophin is made, but it is
abnormal in either size and/or amount. The patient is mild to
moderately weak. In DMD no protein is made and the patient is
wheelchair-bound by age 13 and usually dies by age 20.
[0071] Another type of dystrophy that can be treated according to
the methods of the invention includes congenital muscular dystrophy
(CMD), a very disabling muscle disease of early clinical onset, is
the most frequent cause of severe neonatal hypotonia. Its
manifestations are noticed at birth or in the first months of life
and consist of muscle hypotonia, often associated with delayed
motor milestones, severe and early contractures and joint
deformities. Serum creatine kinase is raised, up to 30 times the
normal values, in the early stage of the disease, and then rapidly
decreases. The histological changes in the muscle biopsies consist
of large variation in the size of muscle fibers, a few necrotic and
regenerating fibers, marked increase in endomysial collagen tissue,
and no specific ultrastructural features. The diagnosis of CMD has
been based on the clinical picture and the morphological changes in
the muscle biopsy, but it cannot be made with certainty, as other
muscle disorders may present with similar clinico-pathological
features. Within the group of diseases classified as CMD, various
forms have been individualized. The two more common forms are the
occidental and the Japanese, the latter being associated with
severe mental disturbances, and usually referred to as Fukuyama
congenital muscular dystrophy (FCMD).
[0072] One form of congenital muscular dystrophy (CMD) has recently
been characterized as being caused by mutations in the laminin
alpha 2-chain gene. Laminin is a protein that associates with
DAPCs. Thus, the invention also provides methods for treating
diseases that are associated with abnormal molecules which normally
associate with DAPCs.
[0073] Other muscular dystrophies within the scope of the invention
include limb-girdle muscular dystrophy (LGMD), which represents a
clinically and genetically heterogeneous class of disorders. These
dystrophies are inherited as either autosomal dominant or recessive
traits. An autosomal dominant form, LGMD1A, was mapped to 5q31-q33
(Speer, M. C. et al., Am. J. Hum. Genet. 50:1211, 1992; Yamaoka, L.
Y. et al., Neuromusc. Disord. 4:471, 1994), while six genes
involved in the autosomal recessive forms were mapped to 15q15.1
(LGMD2A)(Beckmann, J. S. et al., C. R. Acad. Sci. Paris 312:141,
1991), 2p16-p13 (LGMD2B)(Bashir, R. et al., Hum. Mol. Genet. 3:455,
1994), 13q12 (LGMD2C)(Ben Othmane, K. et al., Nature Genet. 2:315,
1992; Azibi, K. et al., Hum. Mol. Genet. 2:1423, 1993),
17q12-q21.33 (LGMD2D)(Roberds, S. L. et al., Cell 78:625, 1994;
McNally, E. M., et. al., Proc. Nat. Acad. Sci. U.S.A. 91:9690,
1994), 4q12 (LG1MD2E)(Lim, L. E., et. al., Nat. Genet. 11:257,
1994; Bonnemann, C. G. et al. Nat. Genet. 11:266, 1995), and most
recently to 5q33-q34 (LGMD2F) (Passos-Bueno, M. R., et. al., Hum.
Mol. Genet. 5:815, 1996). Patients with LGMD2C, 2D and 2E have a
deficiency of components of the sarcoglycan complex resulting from
mutations in the genes encoding gamma-, alpha-, and
beta-sarcoglycan, respectively. The gene responsible for LGMD2A has
been identified as the muscle-specific calpain, whereas the genes
responsible for LGMD1A, 2B and 2F are still unknown.
[0074] Yet other types of muscular dystrophies that can be treated
according to the methods of the invention include Welander distal
myopathy (WDM), which is an autosomal dominant myopathy with
late-adult onset characterized by slow progression of distal muscle
weakness. The disorder is considered a model disease for hereditary
distal myopathies. The disease is linked to chromosome 2p13.
Another muscular dystrophy is Miyoshi myopathya, which is a distal
muscular dystrophy that is caused by mutations in the recently
cloned gene dysferlin, gene symbol DYSF (Weiler et al. (1999) Hum
Mol Genet. 8: 871-7). Yet other dystrophies include Hereditary
Distal Myopathy, Benign Congenital Hypotonia, Central Core disease,
Nemaline Myopathy, and Myotubular (centronuclear) myopathy.
[0075] Other diseases that can be treated or prevented according to
the methods of the invention include those characterized by tissue
atrophy, e.g., muscle atrophy, other than muscle atrophy resulting
from muscular dystrophies, provided that the atrophy is stopped or
slowed down upon treatment with a therapeutic of the invention.
Furthermore, the invention also provides methods for reversing
tissue atrophies, e.g., muscle atrophies.
[0076] Muscle atrophies can result from denervation (loss of
contact by the muscle with its nerve) due to nerve trauma;
degenerative, metabolic or inflammatory neuropathy (e.g.,
GuillianBarre syndrome), peripheral neuropathy, or damage to nerves
caused by environmental toxins or drugs. In another embodiment, the
muscle atrophy results from denervation due to a motor
neuronopathy. Such motor neuronopathies include, but are not
limited to: adult motor neuron disease, including Amyotrophic
Lateral Sclerosis (ALS or Lou Gehrig's disease); infantile and
juvenile spinal muscular atrophies, and autoimmune motor neuropathy
with multifocal conduction block. In another embodiment, the muscle
atrophy results from chronic disuse. Such disuse atrophy may stem
from conditions including, but not limited to: paralysis due to
stroke, spinal cord injury; skeletal immobilization due to trauma
(such as fracture, sprain or dislocation) or prolonged bed rest. In
yet another embodiment, the muscle atrophy results from metabolic
stress or nutritional insufficiency, including, but not limited to,
the cachexia of cancer and other chronic illnesses, fasting or
rhabdomyolysis, endocrine disorders such as, but not limited to,
disorders of the thyroid gland and diabetes.
[0077] Since muscle tissue atrophy and necrosis are often
accompanied by fibrosis of the affected tissue, the reversal or the
inhibition of atrophy or necrosis can also result in an inhibition
or reversal of fibrosis.
[0078] In addition, the therapeutics of the invention may be of use
in the treatment of acquired (toxic or inflammatory) myopathies.
Myopathies which occur as a consequence of an inflammatory disease
of muscle, include, but not limited to polymyositis and
dermatomyositis. Toxic myopathies may be due to agents, including,
but are not limited to adiodarone, chloroquine, clofibrate,
colchicine, doxorubicin, ethanol, hydroxychloroquine,
organophosphates, perihexyline, and vincristine.
[0079] Neuromuscular dystrophies within the scope of the invention
include myotonic dystrophy. Myotonic dystrophy (DM; or Steinert's
disease) is an autosomal dominant neuromuscular disease which is
the most common form of muscular dystrophy affecting adults. The
clinical picture in DM is well established but exceptionally
variable (Harper, P. S., Myotonic Dystrophy, 2nd ed., W. B.
Saunders Co., London, 1989). Although generally considered a
disease of muscle, with myotonia, progressive weakness and wasting,
DM is characterized by abnormalities in a variety of other systems.
DM patients often suffer from cardiac conduction defects, smooth
muscle involvement, hypersomnia, cataracts, abnormal glucose
response, and, in males, premature balding and testicular atrophy
(Harper, P. S., Myotonic Dystrophy, 2nd ed., W. B. Saunders Co.,
London, 1989). The mildest form, which is occasionally difficult to
diagnose, is seen in middle or old age and is characterized by
cataracts with little or no muscle involvement. The classical form,
showing myotonia and muscle weakness, most frequently has onset in
early adult life and in adolescence. The most severe form, which
occurs congenitally, is associated with generalized muscular
hypoplasia, mental retardation, and high neonatal mortality. This
disease and the gene affected is further described in U.S. Pat. No.
5,955,265.
[0080] Another neuromuscular disease is spinal muscular atrophy
("SMA"), which is the second most common neuromuscular disease in
children after Duchenne muscular dystrophy. SMA refers to a
debilitating neuromuscular disorder which primarily affects infants
and young children. This disorder is caused by degeneration of the
lower motor neurons, also known as the anterior horn cells of the
spinal cord. Normal lower motor neurons stimulate muscles to
contract. Neuronal degeneration reduces stimulation which causes
muscle tissue to atrophy (see, e.g., U.S. Pat. No. 5,882,868).
[0081] The above-described muscular dystrophies and myopathies are
skeletal muscle disorders. However, the invention also pertains to
disorders of smooth muscles, e.g., cardiac myopathies, including
hypertrophic cardiomyopathy, dilated cardiomyopathy and restrictive
cardiomyopathy. At least certain smooth muscles, e.g., cardiac
muscle, are rich in sarcoglycans. Mutations in sarcoglycans can
result in sarcolemmal instability at the myocardial level (see,
e.g., Melacini (1999) Muscle Nerve 22: 473). For example, animal
models in which a sarcoglycan is mutated show cardiac creatine
kinase elevation. In particular, it has been shown that
delta-sarcoglycan (Sgcd) null mice develop cardiomyopathy with
focal areas of necrosis as the histological hallmark in cardiac and
skeletal muscle. The animals also showed an absence of the
sarcoglycan-sarcospan (SG-SSPN) complex in skeletal and cardiac
membranes. Loss of vascular smooth muscle SG-SSPN complex was
associated with irregularities of the coronary vasculature. Thus,
disruption of the SG-SSPN complex in vascular smooth muscle
perturbs vascular function, which initiates cardiomyopathy and
exacerbates muscular dystrophy (Coral-Vazquez et al. (1999) Cell
98: 465).
[0082] Similarly to delta-sarcoglycan negative mice, mice lacking
.gamma.-sarcoglycan showed pronounced dystrophic muscle changes in
early life (Hack et al. (1998) J Cell Biol 142: 1279). By 20 wk of
age, these mice developed cardiomyopathy and died prematurely.
Furthermore, apoptotic myonuclei were abundant in skeletal muscle
lacking .gamma.-sarcoglycan, suggesting that programmed cell death
contributes to myofiber degeneration. Vital staining with Evans
blue dye revealed that muscle lacking .gamma.-sarcoglycan developed
membrane disruptions like those seen in dystrophin-deficient
muscle. It was also shown that the loss of gamma-sarcoglycan
produced secondary reduction of beta- and delta-sarcoglycan with
partial retention of .alpha.- and epsilon-sarcoglycan, indicating
that beta-, .gamma.-, and delta-sarcoglycan function as a unit.
Since the other components of the cytoplasmic membrane complex were
functional, the complex could be stabilized by the presence of a
therapeutic of the invention.
[0083] In addition to animal models, certain cardiomyopathies in
humans have been linked to mutations in dystrophin, dystroglycans
or sarcoglycans. For example, dystrophin has been identified as the
gene responsible for X-linked dilated cardiomyopathy (Towbin J. A.
(1998) Curr Opin Cell Biol 10: 131, and references therein). In
this case, the dystrophin gene contained a 5'-mutation which
results in cardiomyopathy without clinically-apparent skeletal
myopathy (Bies et al. (1997) J Mol Cell Cardiol 29: 3175.
[0084] Furthermore, cardiomyopathy was also found in subjects
having Duchenne's Muscular Dystrophy (associated with a mutated
dystrophin), or other types of muscular dystrophies, such as Limb
Girdle Muscular Dystrophy. For example, dilated cardiomyopathy was
present in one autosomal dominant case and in three advanced
autosomal recessive or sporadic patients, of whom two were found to
have alpha sarcoglycan deficiency. Two of these three patients and
three other cases showed ECG abnormalities known to be
characteristic of the dystrophinopathies. A strong association
between the absence of alpha sarcoglycan and the presence of
dilated cardiomyopathy was found. In six autosomal dominant cases
there were atrioventricular (AV) conduction disturbances,
increasing in severity with age and in concomitant presence of
muscle weakness. Pacemaker implantation was necessary in certain of
these patients (see van der Kooi (1998) Heart 79: 73).
[0085] Therapeutics of the invention can also be used to treat or
prevent cardiomyopathy, e.g., dilated cardiomyopathy, of viral
origin, e.g., resulting from an enterovirus infection, e.g., a
Coxsackievirus B3. It has been shown that purified Coxsackievirus
protease 2A cleaves dystrophin in vitro and during Coxsackievirus
infection of cultured myocytes and in infected mouse hearts,
leading to impaired dystrophin function (Badorff et al. (1999) Nat
Med 5: 320. Cleavage of dystrophin results in disruption of the
dystrophin-associated glycoproteins .alpha.-sarcoglycan and
beta-dystroglycan. Thus, cardiomyopathy could be prevented or
reversed by administration of a therapeutic of the invention to a
subject having been infected with a virus causing cardiomyopathy,
e.g., by disruption of dystrophin or a protein associated
therewith. Administration of the therapeutic could restabilize or
reorganize the cytoplasmic membrane of affected cardiac cells.
[0086] Thus, the therapeutics of the invention can also be used to
prevent or to treat smooth muscle disorders, such as cardiac
myopathies, and to stop atrophy and/or necrosis of cardiac smooth
muscle tissue. The treatment can also be used to promote survival
of myocytes. The therapeutics of the invention can also be used to
prevent or to treat heart failure, e.g., congestive heart failure.
The DGC is known to be involved in heart failure. As such
restoration of utrophin, nNOS and the sarcoglycans are expected to
be beneficial in treatment of heart failure. Further, therapeutics
of the invention are thought to help stabilize the DAPC in cardiac
myocytes. Additionally, normalization of heart weight in mdx mice
treated with biglycan has been observed.
[0087] Neurological disorders that can be treated according to the
methods of the invention include polymyositis, and neurogenic
disorders. Another neurological disease that can be treated is
Alzheimer's disease. Another neurological disease that can be
treated is Amyotrophic Lateral Sclerosis.
[0088] Other diseases that can be treated according to the methods
of the invention include those in which the proteoglycan of the
invention is present at abnormal levels, or has an abnormal
activity, relative to that in normal subjects. For example, a
disease or disorder could be caused by a lower level of biglycan,
resulting in, e.g., unstable cytoplasmic membranes. Alternatively,
a disease or disorder could result from an abnormally high level or
activity of biglycan, resulting in, e.g., overstimulation of MuSK
or over-aggregation of AChRs.
[0089] Yet other diseases or disorders that are within the scope of
the invention include those that are associated with an abnormal
interaction between a proteoglycan of the invention and another
molecule (other than those of the DAPC or MuSK), e.g., a complement
factor, such as C1q. For example, it has been shown that C1q
interacts with biglycan (Hocking et al. (1996) J. Biol. Chem. 271:
19571). It is also known that binding of C1q to cell surfaces
mediates a number of biological activities including enhancement of
phagocytosis and stimulation of superoxide production. Thus, since
biglycan binds to C1q, biglycan or another proteoglycan or core
thereof, of the invention could be used to inhibit the binding of
C1q to its receptor on cell surfaces to inhibit one or more of such
biological activities. In addition, compounds of the invention
which inhibit the interaction between C1q or other complement
component and a cell surface can also be used to inhibit complement
mediated necrosis of the cells and tissues containing such
cells.
[0090] Also within the scope of the invention are methods for
preventing or inhibiting infections of cells by microorganisms,
e.g., viruses. For example, it has been shown that dystroglycan is
a receptor via which certain microorganisms enter eukaryotic cells
(Science (1998) 282: 2079). Thus, by administrating to a subject a
therapeutic of the invention which occupies the site on
dystroglycan molecules to which the microorganism binds, entering
of the microorganism into the cell can be inhibited. This method
can be used, e.g., to prevent or inhibit Lassa Fever virus and
lymphocytic choriomeningitis virus (LCMV) infection, as well as
infection by other arenaviruses, including Oliveros, and Mobala.
Soluble alpha-dystroglycan was shown to block both LCMV and LFV
infection (Science (1998) 282: 2079).
[0091] In addition to cell cultures, e.g., established from
patients having, e.g., a muscular dystrophy, various animal models
can be used to select the most appropriate therapeutic for treating
a disease. In particular, to identify a therapeutic for use in
preventing or treating a muscular dystrophy or cardiomyophaty
associated with a mutated or absent DAPC component or, mice having
mutated versions of these proteins, or having null mutations in the
genes encoding these proteins, can be used. For example, mice
having a disrupted sarcoglycan, such as delta-sarcoglycan, can be
used. Such mice are described, e.g., Coral-Vazquez et al. (1999)
Cell 98: 465. Alternatively, mice deficient in dystrophin (mdx
mice), or in .alpha.- or .gamma.-sarcoglycans can be used. Such
mice have been described herein and in the literature. Additional
mice can be made according to known methods in the art. In an
illustrative embodiment to identify therapeutics, different
therapeutics are administered to delta-sarcoglycan null mice, and
the effect of the therapeutics are evaluated by studying cardiac
function. Another animal model that can be used for this purpose is
the cardiomyopathic hamster that does not express delta-sarcoglycan
due to a genomic deletion. This rat is an animal model for
autosomal recessive cardiomyopathy, and is further described in
Sakamoto et al. FEBS Lett 1999 (1999) 44: 124.
Effective Dose and Administration of Therapeutic Compositions
[0092] The above-described diseases or disorders can be treated or
ameliorated in a subject by administering to the subject a
pharmaceutically efficient amount of a bigylcan therapeutic of the
invention. Depending on whether the disease is caused by higher
levels or activity or by lower levels or activity of biglycan, an
agonist or an antagonist biglycan therapeutic is administered to a
subject having the disease. Although a person of skill in the art
will be able to predict which therapeutic to administer for
treating any of the diseases of the invention, tests can be
performed to determine the appropriate therapeutic to administer.
Such tests can use, e.g., animal models of the disease.
Alternatively, in cases where diseases are due to a mutation in,
e.g., biglycan, in vitro tests can be undertaken to determine the
effect of the mutation. This will allow the determination of what
type of therapeutic should be administered to a subject having this
type of mutation.
[0093] Another manner of administering a therapeutic of the
invention to a subject is by preparing cells expressing and
secreting the proteoglycan of interest, inserting the cells into a
matrix and administering this matrix to the subject at the desired
location. Thus, cells engineered in accordance with this invention
may also be encapsulated, e.g., using conventional biocompatible
materials and methods, prior to implantation into the host organism
or patient for the production of a therapeutic protein. See e.g.,
Hguyen et al, Tissue Implant Systems and Methods for Sustaining
viable High Cell Densities within a Host, U.S. Pat. No. 5,314,471
(Baxter International, Inc.); Uludag and Sefton, 1993, J Biomed.
Mater. Res. 27(10):1213-24 (HepG2 cells/hydroxyethyl
methacrylate-methyl methacrylate membranes); Chang et al, 1993, Hum
Gene Ther 4(4):433-40 (mouse Ltk-cells expressing
hGH/immunoprotective perm-selective alginate microcapsules; Reddy
et al, 1993, J Infect Dis 168(4):1082-3 (alginate); Tai and Sun,
1993, FASEB J 7(11):1061-9 (mouse fibroblasts expressing
hGH/alginate-poly-L-lysine-alginate membrane); Ao et al, 1995,
Transplantation Proc. 27(6):3349, 3350 (alginate); Rajotte et al,
1995, Transplantation Proc. 27(6):3389 (alginate); Lakey et al,
1995, Transplantation Proc. 27(6):3266 (alginate); Korbutt et al,
1995, Transplantation Proc. 27(6):3212 (alginate); Dorian et al,
U.S. Pat. No. 5,429,821 (alginate); Emerich et al, 1993, Exp Neurol
122(1):37-47 (polymer-encapsulated PC12 cells); Sagen et al, 1993,
J Neurosci 13(6):2415-23 (bovine chromaffin cells encapsulated in
semipermeable polymer membrane and implanted into rat spinal
subarachnoid space); Aebischer et al, 1994, Exp Neurol 126(2):151-8
(polymer-encapsulated rat PC12 cells implanted into monkeys; see
also Aebischer, WO 92/19595); Savelkoul et al, 1994, J Immunol
Methods 170(2):185-96 (encapsulated hybridomas producing
antibodies; encapsulated transfected cell lines expressing various
cytokines); Winn et al, 1994, PNAS USA 91(6):2324-8 (engineered BHK
cells expressing human nerve growth factor encapsulated in an
immunoisolation polymeric device and transplanted into rats);
Emerich et al, 1994, Prog Neuropsychopharmacol Biol Psychiatry
18(5):935-46 (polymer-encapsulated PC12 cells implanted into rats);
Kordower et al, 1994, PNAS USA 91(23):10898-902
(polymer-encapsulated engineered BHK cells expressing hNGF
implanted into monkeys) and Butler et al WO 95/04521 (encapsulated
device). The cells may then be introduced in encapsulated form into
an animal host, preferably a mammal and more preferably a human
subject in need thereof. Preferably the encapsulating material is
semipermeable, permitting release into the host of secreted
proteins produced by the encapsulated cells. In many embodiments
the semipermeable encapsulation renders the encapsulated cells
immunologically isolated from the host organism in which the
encapsulated cells are introduced. In those embodiments the cells
to be encapsulated may express one or more proteoglycans of the
host species and/or from viral proteins or proteins from species
other than the host species.
[0094] Alternatively, the therapeutic is a nucleic acid encoding
the core of a proteoglycan of the invention. Thus, a subject in
need thereof, may receive a dose of viral vector encoding the
protein of interest, which may be specifically targeted to a
specific tissue, e.g., a dystrophic tissue. The vector can be
administered in naked form, or it can be administered as a viral
particle (further described herein). For this purpose, various
techniques have been developed for modification of target tissue
and cells in vivo. A number of viral vectors have been developed,
such as described above, which allow for transfection and, in some
cases, integration of the virus into the host. See, for example,
Dubensky et al. (1984) Proc. Natl. Acad. Sci. USA 81, 7529-7533;
Kaneda et al., (1989) Science 243, 375-378; Hiebert et al. (1989)
Proc. Natl. Acad. Sci. USA 86, 3594-3598; Hatzoglu et al. (1990) J.
Biol. Chem. 265, 17285-17293 and Ferry, et al. (1991) Proc. Natl.
Acad. Sci. USA 88, 8377-8381. The vector may be administered by
injection, e.g., intravascularly or intramuscularly, inhalation, or
other parenteral mode. Non-viral delivery methods such as
administration of the DNA via complexes with liposomes or by
injection, catheter or biolistics may also be used.
[0095] In yet another embodiment, cells are obtained from a
subject, modified ex vivo, and introduced into the same or a
different subject. Additional methods of administration of the
therapeutic compounds are set forth below.
[0096] Toxicity: Toxicity and therapeutic efficacy of compounds of
the invention 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 Ed.sub.50 (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
LD.sub.50/ED.sub.50. Compounds which 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.
[0097] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. In particular, where the therapeutic is administered for
potentiating AChR aggregation, it is desirable to establish the
dose that will result in stimulation, if desired, or inhibition, if
desired. Tests can then be continued in medical tests. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 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
IC.sub.50 (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.
[0098] Pharmaceutical Compositions: Pharmaceutical compositions for
use in accordance with the present invention may be formulated in
conventional manner using one or more physiologically acceptable
carriers or excipients. Thus, the compounds and their
physiologically acceptable salts and solvates may be formulated for
administration by, for example, injection, inhalation or
insufflation (either through the mouth or the nose) or oral,
buccal, parenteral or rectal administration.
[0099] For such therapy, the compounds of the invention can be
formulated for a variety of loads of administration, including
systemic and topical or localized administration. Techniques and
formulations generally may be found in Remmington's Pharmaceutical
Sciences, Meade Publishing Co., Easton, Pa. For systemic
administration, injection is preferred, including intramuscular,
intravenous, intraperitoneal, and subcutaneous. For injection, the
compounds of the invention can be formulated in liquid solutions,
preferably in physiologically compatible buffers such as Hank's
solution or Ringer's solution. In addition, the compounds may be
formulated in solid form and redissolved or suspended immediately
prior to use. Lyophilized forms are also included.
[0100] 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., ationd 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.
[0101] 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, dichlorotetrafluoroethane, 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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 bile
salts and fusidic acid derivatives. In addition, detergents may be
used to facilitate permeation. Transmucosal administration may be
through nasal sprays or using suppositories. For topical
administration, the oligomers of the invention are formulated into
ointments, salves, gels, or creams as generally known in the art. A
wash solution can be used locally to treat an injury or
inflammation to accelerate healing.
[0106] In clinical settings, a gene delivery system for the
therapeutic gene encoding a proteoglycan of the invention can be
introduced into a patient by any of a number of methods, each of
which is familiar in the art. For instance, a pharmaceutical
preparation of the gene delivery system can be introduced
systemically, e.g., by intravenous injection, and specific
transduction of the protein in the target cells occurs
predominantly from specificity of transfection provided by the gene
delivery vehicle, cell-type or tissue-type expression due to the
transcriptional regulatory sequences controlling expression of the
receptor gene, or a combination thereof. In other embodiments,
initial delivery of the recombinant gene is more limited with
introduction into the animal being quite localized. For example,
the gene delivery vehicle can be introduced by catheter (see U.S.
Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen et al.
(1994) PNAS 91: 3054-3057). A gene encoding a proteoglycan of the
invention can be delivered in a gene therapy construct by
electroporation using techniques described, for example, by Dev et
al. ((1994) Cancer Treat Rev 20:105-115).
[0107] A preferred mode of delivering DNA to muscle cells include
using recombinant adeno-associated virus vectors, such as those
described in U.S. Pat. No. 5,858,351. Alternatively, genes have
been delivered to muscle by direct injection of plasmid DNA, such
as described by Wolff et al. (1990) Science 247:1465-1468; Acsadi
et al. (1991) Nature 352:815-818; Barr and Leiden (1991) Science
254:1507-1509. However, this mode of administration generally
results in sustained but generally low levels of expression. Low
but sustained expression levels are expected to be effective for
practicing the methods of the invention.
[0108] The pharmaceutical preparation of the gene therapy construct
or compound of the invention can consist essentially of the gene
delivery system in an acceptable diluent, or can comprise a slow
release matrix in which the gene delivery vehicle or compound is
imbedded. Alternatively, where the complete gene delivery system
can be produced intact from recombinant cells, e.g., retroviral
vectors, the pharmaceutical preparation can comprise one or more
cells which produce the gene delivery system.
[0109] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
Diagnostic Methods
[0110] Based at least on the observation that biglycan binds to at
least one component of DAPCs, protein complexes which are critical
for maintaining the integrity of plasma membranes, the invention
provides diagnostic methods for determining whether a subject has
or is likely to develop a disease or condition which is
characterized by, or associated with, plasma membrane instability,
in particular, abnormal or unstable DAPCs, such as muscular
dystrophies. Furthermore, it has been observed in an animal model
for muscular dystrophy, which lacks dystrophin, that the amount of
the proteoglycan biglycan is elevated, and thereby believed to be a
compensatory mechanism.
[0111] Furthermore, based at least on the observation that biglycan
binds to, and phosphorylates MuSK and potentiates agrin-induced
MuSK phosphorylation, and that biglycan stimulates agrin-mediated
AChR aggregation, the invention also provides diagnostic methods
for determining whether a subject has or is likely to develop a
disease or condition which is characterized by abnormal synapses or
neuromuscular junctions, e.g., neurological or neuromuscular
diseases.
[0112] Accordingly, the identification of abnormal levels or
activity of the proteoglycan of the invention in a subject would
indicate that the subject has, or is likely to develop a disease or
condition relating to abnormal or unstable DAPCs. Diseases can be
characterized by a high levels of proteoglycan of the invention,
e.g., if the cell compensates for the lack of another DAPC
component or molecule associating therewith, e.g., as seen in
dystrophin negative mice. Alternatively, a high level or activity
of proteoglycan of the invention can at least be part of the cause
of the disease.
[0113] In addition, an elevated level or activity of a proteoglycan
of the invention could be associated with, or be at least in part,
the cause of neurological or neuromuscular diseases, e.g., by
overstimulating AChR aggregation and/or activating MuSK.
[0114] Diseases are also likely to be caused or associated with a
lower level or activity of proteoglycan of the invention, which
may, e.g., cause DAPCs to be more unstable than those on cells of
subjects having a normal amount or activity of the proteoglycan of
the invention. Accordingly, a lower level or activity of the
proteoglycan of the invention in cells of a subject would result in
leaky membranes.
[0115] A lower level or activity of the proteoglycan of the
invention could also result in insufficient AChR aggregation and/or
insufficient MuSK activation, thereby resulting in abnormal
synapses or neuromuscular junctions. Such situations can thus
result in neurological or neuromuscular diseases, and result, e.g.,
in atrophy of tissues.
[0116] As used herein, the term "diagnostic assay" refers to the
specific use of the methods described herein to identify an
individual predisposed to a disease, such as a muscular disorder, a
neuromuscular disorder or a neurological disorder. Such diagnostic
assays are particularly useful as prenatal diagnostic assays, which
can be used to determine whether a fetus is predisposed to one or
more of these disorders. For prenatal diagnosis, for example, a
sample can be obtained by biopsy of muscle tissue from the fetus or
by biopsy of placenta from the pregnant mother.
[0117] In one embodiment, the method comprises determining the
level of, or the biological activity of a proteoglycan of the
invention relative to that in non-affected subjects, or determining
whether the proteoglycan or gene encoding it contains a mutation,
or abnormal glycan side chains.
[0118] A patient sample may be any cell, tissue, or body fluid but
is preferably muscle tissue, cerebrospinal fluid, blood, or a blood
fraction such as serum or plasma. As used herein, the term "sample"
refers to a specimen obtained from a subject, which can be a human
subject. In general, a tissue sample, which can be obtained, for
example, by biopsy of muscle or placenta of an individual suspected
of being predisposed to a disorder, is a suitable sample. In many
cases, it is useful to prepare the sample as a tissue section,
which can be examined by histologic analysis. Alternatively,
proteins or nucleic acids can be extracted from a sample and can be
examined using methods such as gel electrophoresis and appropriate
"blotting" methods, which are well known in the art and described
in detail below.
[0119] A sample can be obtained from a normal subject or from a
test subject, who is suspected of being predisposed to a disorder,
such as a muscular, neuromuscular or neurological disorder, and is
being examined for altered expression or localization of the
proteoglycan of the invention or altered expression of the mRNA
encoding the proteoglycan of the invention.
[0120] A sample obtained from a normal subject can be used as a
"control" sample, which is useful for comparison with a sample
obtained from a test subject. A control sample can be, for example,
a muscle sample or a placenta sample, which is obtained from an
age- and sex-matched individual who does not exhibit and is not
predisposed to a disorder, such as a muscular, neuromuscular, or
neurological disorder. A control sample exhibits a level of
expression and a pattern of expression of the proteoglycan of the
invention and a level of expression of the proteoglycan mRNA that
is characteristic of the human population in general and does not
significantly deviate from the normal levels of expression or
pattern of localization expected for a person in the population. It
is expected that, after a statistically significant number of
control samples have been examined, an amount of expression of the
proteoglycan of the invention per unit of a sample will be
determined to be normal for a control sample. As used herein, a
"normal" amount of proteoglycan of the invention in a control
sample means an amount that is within an expected range for a
person that is not predisposed to a disorder, e.g., a muscular,
neuromuscular, or neurological disorder.
[0121] Altered expression of the proteoglycan of the invention in a
sample obtained from a test subject can be identified qualitatively
by visually comparing, for example, photomicrographs of an
immunohistochemically stained control sample with the sample
obtained from the test subject. Alternatively, altered expression
of proteoglycan of the invention can be measured quantitatively
using, for example, densitometric analysis. Altered expression of
proteoglycan of the invention protein also can be determined using
methods of gel electrophoresis and, if desired, immunoblot
analysis. Such methods are well known in the art.
[0122] Methods for the determination of levels of dystrophin and
dystrophin-associated proteins are carried out by conventional
techniques. Such techniques are disclosed, for example, in U.S.
Pat. Nos. 5,187,063; 5,260,209; and 5,308,752, the disclosures of
which are incorporated herein by reference. International
Publication Number WO 89/06286 also discloses such conventional
techniques, as well as the nucleic sequence encoding
dystrophin.
[0123] Altered localization of the proteoglycan of the invention in
a sample also can be determined. As used herein, the term
"localization" refers to the pattern of deposition of the
proteoglycan of the invention in a sample. The localization of the
proteoglycan of the invention also can be determined qualitatively
or quantitatively. "Altered" localization refers to a pattern of
deposition of the proteoglycan of the invention in a sample that is
different from the pattern of localization observed in a control
sample.
[0124] The level of expression mRNA encoding the proteoglycan of
the invention can be determined and can be used to identify an
individual that is predisposed to a disorder, such as muscular,
neuromuscular, or neurological disorder. Methods for determining
the level of expression of proteoglycan mRNA in a sample are well
known in the art and include, for example, northern blot analysis,
which can be used to determine whether proteoglycan mRNA is
expressed at a normal level in a test sample. Northern blot
analysis also can be used to determine whether the proteoglycan
mRNA that is expressed in a cell is a full length transcript. For
example, an RNA sample obtained from a tissue sample can be
contacted with a nucleic acid probe that hybridizes to the mRNA
encoding the proteoglycan of the invention. One skilled in the art
would know that the probe can be a DNA or RNA probe and can be
prepared from a cDNA encoding the proteoglycan or can be
synthesized as an oligonucleotide. In addition, the skilled artisan
would recognize that such hybridization should be performed under
stringent conditions, which can be determined empirically (see, for
example, Sambrook et al., Molecular Cloning: A laboratory manual
(Cold Spring Harbor Laboratory Press 1989), which is incorporated
herein by reference). Methods for isolating intact total RNA and
poly A+ mRNA and for performing Northern blot analysis are well
known in the art (Sambrook et al., 1989).
[0125] A sensitive method of determining the level of expression of
mRNA encoding the proteoglycan of the invention in a sample is the
reverse transcriptase-polymerase chain reaction (RT-PCR), which is
well known in the art (see, for example, H. A. Erlich, PCR
Technology: Principles and applications for DNA amplification
(Stockton Press, 1989), which is incorporated herein by reference;
see chap. 8). The RT-PCR method is particularly useful for
examining a sample that fails to give a detectable signal by
northern blot analysis. Due to the amplification steps involved in
PCR analysis, a rare proteoglycan mRNA can be identified in a
sample.
[0126] Methods for determining levels of proteoglycan of the
invention can use, e.g., antibodies binding to the proteoglycan of
the invention. An antibody can be used in connection with a
conventional assay for the determination of levels of antigen in a
tissue of interest, e.g., muscle tissue. Any method which enables
the determination of protein levels present in muscle tissue based
on antibody binding is useful in connection with the present
invention. Preferred methods include Western blotting,
immunocytochemical analysis and enzyme-linked immunoadsorbent assay
(ELISA).
[0127] For assays which require solubilized extracellular matrix
(e.g., ELISA and Western blotting), the amount of muscle obtained
by biopsy should be sufficient to enable the extraction of the
proteoglycan of the invention in a quantity sufficient for
analysis. In an illustrative embodiment, the muscle tissue is
homogenized by mechanical disruption using an apparatus such as a
hand operated or motor driven glass homogenizer, a Waring blade
blender homogenizer, or an ultrasonic probe. Homogenization can be
carried out, for example, in a buffer having a pH of about 11 or
12, as further described in the Examples. The buffer can further
comprise protease inhibitors, e.g., 1 mM PMSF, 0.75 mM benzamidine,
1 mu g/ml aprotinin, 1 mu g/ml of leupeptin, 1 mu g/ml of pepstatin
A. The incubation is then carried out, e.g., on ice for 2 hr.
Following centrifugation, extracellular matrix solubilized in this
manner can then be processed by conventional methods for use, for
example, in Western blotting or ELISA analytical formats.
[0128] The solubilized extracellular matrix components, prepared as
described above can be analyzed by Western blotting by first
separating the components on a 3-12% SDS polyacrylamide gel
(Laemmli (1970) Nature 227, 680) followed by transfer to a solid
support, such as a nitrocellulose membrane, forming an exact
replica of the original protein separation but leaving the
transferred proteins accessible for further study. This solid
support bearing the transferred protein components is referred to
as an immunoblot. The detection of transferred proteins can be
accomplished by the use of general protein dyes such as Amido black
or Coomassie brilliant blue. Antibodies which are specific for the
proteoglycan of the invention can be labeled with a detectable
reporter group and used to stain the protein transferred to the
solid support. Alternatively, unlabeled antibodies specific for the
proteoglycan of the invention are incubated with an immunoblot
under conditions appropriate for binding. The specific binding of
these antibodies to the muscle tissue sample can be detected
through the use of labeled secondary antibodies by conventional
techniques.
[0129] The methods of the present invention can also be practiced
in an enzyme-linked immunoadsorbent assay (ELISA) format. In this
format, antibodies against the proteoglycan of the invention are
adsorbed to a solid support, in most cases a polystyrene microtiter
plate. After coating the support with antibody and washing, a
solubilized sample is added. Proteoglycan of the invention, if
present, will bind to the adsorbed antibodies. Next, a conjugate
that will also bind to the proteoglycan of interest is added. A
conjugates can be an antibody molecule which binds to the
proteoglycan of the invention, and to which an enzyme is covalently
bound. After addition of a chromogenic substrate for the enzyme,
the intensity of the colored reaction products generated will be
proportional to the amount of conjugated enzyme and thus indirectly
to the amount of bound proteoglycan of the invention. Since the
intensity of the developed color is proportional to the amount of
proteoglycan of the invention present, determination of the
intensity of the color produced by a standard series of
concentrations of proteoglycan of the invention will allow the
calculation of the amount of proteoglycan of the invention in an
unknown sample. Many variations of this assay exist as described in
Voller, A., Bidwell, D. E. and Bartlett, A., The Enzyme Linked
Immunoadsorbent Assay (ELISA): A guide with abstracts of microplate
applications, Dynatech Laboratories, Alexandria, Va. (1979).
[0130] Alternatively, tissue specimens (e.g., human biopsy samples)
can be tested for the presence of the components of the DAPC
complex by using monoclonal or polyclonal antibodies in an
immunohistochemical technique, such as the immunoperoxidase
staining procedure. In addition, immunofluorescent techniques can
be used to examine human tissue specimens. In a typical protocol,
slides containing cryostat sections of frozen, unfixed tissue
biopsy samples are air-dried and then incubated with an antibody
preparation against the proteoglycan (primary antibody) of the
invention in a humidified chamber at room temperature. The slides
are layered with a preparation of fluorescently labeled antibody
directed against the primary antibody. Labeled secondary antibodies
are also useful for detection. The staining pattern and intensities
within the sample can be determined by fluorescent light
microscopy.
[0131] Specific diseases or disorders, e.g., genetic diseases or
disorders, are associated with specific allelic variants of
polymorphic regions of certain genes, which do not necessarily
encode a mutated protein. Thus, the presence of a specific allelic
variant of a polymorphic region of a gene, such as a single
nucleotide polymorphism ("SNP"), in a subject can render the
subject susceptible to developing a specific disease or disorder.
Polymorphic regions in genes, e.g., a gene encoding a proteoglycan
of the invention, can be identified, by determining the nucleotide
sequence of genes in populations of individuals. If a polymorphic
region, e.g., SNP, is identified, then the link with a specific
disease can be determined by studying specific populations of
individuals, e.g., individuals which developed a specific disease,
such a muscular, neuromuscular, or neurological disease. A
polymorphic region can be located in any region of a gene, e.g.,
exons, in coding or non coding regions of exons, introns, and
promoter region.
[0132] It is likely that genes encoding proteoglycans of the
invention comprise polymorphic regions, specific alleles of which
may be associated with specific diseases or conditions or with an
increased likelihood of developing such diseases or conditions.
Thus, the invention provides methods for determining the identity
of the allele or allelic variant of a polymorphic region of a gene
encoding a proteoglycan of the invention in a subject, to thereby
determine whether the subject has or is at risk of developing a
disease or disorder associated with a specific allelic variant of a
polymorphic region.
[0133] In an exemplary embodiment, there is provided a nucleic acid
composition comprising a nucleic acid probe including a region of
nucleotide sequence which is capable of hybridizing to a sense or
anti sense sequence of a gene encoding a proteoglycan of the
invention or naturally occurring mutants thereof, or 5' or 3'
flanking sequences or intronic sequences naturally associated with
the subject proteoglycan genes or naturally occurring mutants
thereof. The nucleic acid of a cell is rendered accessible for
hybridization, the probe is contacted with the nucleic acid of the
sample, and the hybridization of the probe to the sample nucleic
acid is detected. Such techniques can be used to detect alterations
or allelic variants at either the genomic or mRNA level, including
deletions, substitutions, etc., as well as to determine mRNA
transcript levels.
[0134] The following examples are provided to further illustrate
the advantages and features of the present invention, but are not
intended to limit the scope of the invention. While they are
typical of those that might be used, other procedures,
methodologies, or techniques known to those skilled in the art may
alternatively be used.
Example 1
Production of T2-rhBGN
[0135] Stable CHO clones expressing T2-rhBGN produce a mixture of
two forms herein referred to as M and D or M-rhBGN and D-rhBGN.
[0136] T2-rhBGN was purified from conditioned media of CHO clone 57
and 171 and HEK 293 clone 2 by a combination of anion exchange and
hydrophobic interaction (HIC). Analytical ultracentrifugation (AUC)
of HIC pool purified from CHO Clone 57 resolved into two
sedimentation peaks: M with a sedimentation coefficient:
s.sub.20,w.apprxeq.2.88 and D with coefficient
s.sub.20,w.apprxeq.4.28 (FIG. 1). Analysis of the sedimentation at
three concentrations (0.2, 0.2, and 2 mg/ml) showed that the
dissociation constant for M-D exchange in the HIC pool was
.about.50 .mu.M. Experiments were performed at 20.degree. C., in a
Beckman Optima XL-I analytical ultracentrifuge. Spin Analytical
charcoal epon 12 mm double-sector centerpieces were used in an
eight-hole AN 50 Ti rotor at 45,000 rpm. Radial absorption scans
were measured at .lamda.=280 nm. Sedfit 12.44 was used for data
analysis, with a 68% confidence limit
(analyticalultracentrifugation.com). Sedimentation coefficients
were corrected to standard s.sub.20,w conditions using the
experimental buffer density and buffer viscosity as calculated with
SEDNTERP (sednterp.unh.edu). Buffer: 20 mM Tris, pH 8, 0.5M NaCl;
viscosity=0.01002 Poise; density=1.004989 g/mL. Partial specific
volume, v-bar=0.74413 mL/g calculated from amino acid composition
with Sednterp.
[0137] Further analysis of the HIC pool by size exclusion
chromatography (SEC) showed that the HIC pool resolved into two
distinguishable peaks that corresponded to M and D (FIG. 2). Clone
57 HIC pool in 20 mM Tris, pH 8.0, 0.5M NaCl was applied to a
Superdex 200 10/300 GL (GE Healthcare) equilibrated with 20 mM
Tris, pH 8.0, 1M NaCl. In the first SEC analysis (FIG. 1a),
T2-rhBGN resolved into two discrete, partially overlapping peaks
that eluted at .about.18.7 (D) and 20.4 ml (M). The fractions
corresponding to these two peaks were each re-run on the SEC
column. Highly discrete peaks were observed with the same elution
profiles (FIG. 1b). These analyses indicate that M and D can be
enriched by SEC and there is little exchange between the two
forms.
Example 2
M- and D-rhBGN are Chemically Distinct
[0138] The M and D forms purified from CHO clone 57 HIC pool over
two size exclusion columns were further analyzed by Capillary
Electrophoresis (FIG. 3) was conducted using an Agilent Bioanalyzer
2100 and Agilent Protein 80 assay kit under non-reducing conditions
according to manufacturer's protocols. The apparent molecular
weight and mobility of D-rhBGN (65.5 kd, 31.00, respectively) was
greater than that of M-rhBGN (60 kd, 30.44, respectively),
indicating differential posttranslational modification of the two
forms.
[0139] The M and D forms were characterized by N-linked
oligosaccaride profiling analysis (FIG. 4). Approximately 250 .mu.g
of M- and D-rhBGN were digested into glycopeptides with trypsin at
37.degree. C. overnight. The glycopeptides were purified with a C18
cartridge, eluted in sequential elutions of 20%, 40%, and 100%
isopropanol, combined, and dried. N-glycans were released by
enzymatic cleavage with PNGase F, and then purified of contaminants
with a C18 sep-pak cartridge. The carbohydrate fraction was eluted
with 5% acetic acid and dried by lyophilization. Released N-linked
oligosaccharides were permethylated based on a previously described
method (Anumula and Taylor, 1992). The glycans were dried with
nitrogen gas and profiled by MALDI-TOF analysis. MALDI/TOF-MS was
performed in the reflector positive ion mode using
.alpha.-dihyroxybenzoic acid (DHBA, 20 mg/mL solution in 50%
methanol:water) as a matrix. All spectra were obtained by using a
ABISciex 5800 MALDI/TOF-TOF. The percentages of each glycan were
calculated for each sample. M and D showed varying amounts of
complex-type glycans, branching and sialic acid.
Example 3
M-rhBGN is Highly Active in Bioassays
[0140] M-rhBGN purified from CHO clone 171 by two rounds of SEC was
analyzed (FIG. 5) by an in-vitro bioassay for biglycan activity
where the ability of biglycan to potentiate agrin-induced AChR
clustering in cultured H2K myotubes is measured based on the method
described in (Amenta et al., J. Neuroscience, 2012). M-rhBGN showed
a robust and broad dose-response. Activity was observed in a
30-fold range of >50% of maximal activity between 0.016-0.512
.mu.g/ml. (FIG. 5.)
Example 4
D-rhBGN Shows Low Bioactivity
[0141] The SEC fractions containing D-rhBGN were analyzed by the
bioassay describe above. As shown in FIG. 5, there was only low
activity (<25% of maximal) across the entire concentration range
tested (0.004-0.512 .mu.g/ml).
Example 5
Evaluation of In-Vivo Efficacy of the M-rhBGN
[0142] In vivo efficacy of M-rhBGN purified from HEK-293 Clone 2 by
size exclusion chromatography was evaluated in an in-vivo efficacy
model in mdx mice (FIG. 6). P18 male mdx mice were IP injected at
P18 and P25 with the indicated doses of M-rhBGN or vehicle only.
Diaphragms were harvested at P32 and flash frozen in liquid
nitrogen cooled isopentane. Frozen sections were prepared as
previously described (Mercado et. al., 2006) and H&E stained.
Sections were observed using a Nikon Eclipse E800 microscope and
montages of sections were acquired with NIS Elements. The
percentage of myofibers with centrally located nuclei were scored
by workers blind to experimental conditions. All of the myofibers
in a section from the mid-belly of the muscle were counted. M-rhBGN
reduced the percentage of myofibers with central nuclei
(*p<0.05, 1-way ANOVA analysis with post-hoc Dunnett's Multiple
Comparison, n=7-10 animals/group). There was a statistically
significant response across a 20-fold range (0.5-10 mg/kg).
Example 6
Evaluation of Structure of M- and D-rhBGN
[0143] The structure M- and D-rhBGN purified from CHO Clone 57 by
size exclusion chromatography was evaluated using circular
dichroism (CD)(FIG. 7). The 2 samples were analyzed by near and far
UV CD. CD measurements were carried out at room temperature using 2
cm cell for near UV CD and 0.02 cm cell for far UV CD. After
subtracting solvent spectrum (20 mM Tris, 0.5 M NaCl, pH 8.0), the
sample spectrum was converted to the mean residue ellipticity (CD
intensity per amino acid) using the protein concentration, the
path-length of the cell and the mean residue weight (112.2): note
that this calculation normalizes for the different protein
concentrations. The near and far UV CD spectra differ for M- and
D-rhBGN indicating they have different structures.
[0144] Although the invention has been described with reference to
the above example, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
41368PRTHomo sapiens 1Met Trp Pro Leu Trp Arg Leu Val Ser Leu Leu
Ala Leu Ser Gln Ala 1 5 10 15 Leu Pro Phe Glu Gln Arg Gly Phe Trp
Asp Phe Thr Leu Asp Asp Gly 20 25 30 Pro Phe Met Met Asn Asp Glu
Glu Ala Ser Gly Ala Asp Thr Ser Gly 35 40 45 Val Leu Asp Pro Asp
Ser Val Thr Pro Thr Tyr Ser Ala Met Cys Pro 50 55 60 Phe Gly Cys
His Cys His Leu Arg Val Val Gln Cys Ser Asp Leu Gly 65 70 75 80 Leu
Lys Ser Val Pro Lys Glu Ile Ser Pro Asp Thr Thr Leu Leu Asp 85 90
95 Leu Gln Asn Asn Asp Ile Ser Glu Leu Arg Lys Asp Asp Phe Lys Gly
100 105 110 Leu Gln His Leu Tyr Ala Leu Val Leu Val Asn Asn Lys Ile
Ser Lys 115 120 125 Ile His Glu Lys Ala Phe Ser Pro Leu Arg Lys Leu
Gln Lys Leu Tyr 130 135 140 Ile Ser Lys Asn His Leu Val Glu Ile Pro
Pro Asn Leu Pro Ser Ser 145 150 155 160 Leu Val Glu Leu Arg Ile His
Asp Asn Arg Ile Arg Lys Val Pro Lys 165 170 175 Gly Val Phe Ser Gly
Leu Arg Asn Met Asn Cys Ile Glu Met Gly Gly 180 185 190 Asn Pro Leu
Glu Asn Ser Gly Phe Glu Pro Gly Ala Phe Asp Gly Leu 195 200 205 Lys
Leu Asn Tyr Leu Arg Ile Ser Glu Ala Lys Leu Thr Gly Ile Pro 210 215
220 Lys Asp Leu Pro Glu Thr Leu Asn Glu Leu His Leu Asp His Asn Lys
225 230 235 240 Ile Gln Ala Ile Glu Leu Glu Asp Leu Leu Arg Tyr Ser
Lys Leu Tyr 245 250 255 Arg Leu Gly Leu Gly His Asn Gln Ile Arg Met
Ile Glu Asn Gly Ser 260 265 270 Leu Ser Phe Leu Pro Thr Leu Arg Glu
Leu His Leu Asp Asn Asn Lys 275 280 285 Leu Ala Arg Val Pro Ser Gly
Leu Pro Asp Leu Lys Leu Leu Gln Val 290 295 300 Val Tyr Leu His Ser
Asn Asn Ile Thr Lys Val Gly Val Asn Asp Phe 305 310 315 320 Cys Pro
Met Gly Phe Gly Val Lys Arg Ala Tyr Tyr Asn Gly Ile Ser 325 330 335
Leu Phe Asn Asn Pro Val Pro Tyr Trp Glu Val Gln Pro Ala Thr Phe 340
345 350 Arg Cys Val Thr Asp Arg Leu Ala Ile Gln Phe Gly Asn Tyr Lys
Lys 355 360 365 2331PRTArtificial SequenceSynthetic
Constructmisc_feature(5)..(5)Xaa can be any naturally occurring
amino acidmisc_feature(10)..(10)Xaa can be any naturally occurring
amino acid 2Asp Glu Glu Ala Xaa Gly Ala Asp Thr Xaa Gly Val Leu Asp
Pro Asp 1 5 10 15 Ser Val Thr Pro Thr Tyr Ser Ala Met Cys Pro Phe
Gly Cys His Cys 20 25 30 His Leu Arg Val Val Gln Cys Ser Asp Leu
Gly Leu Lys Ser Val Pro 35 40 45 Lys Glu Ile Ser Pro Asp Thr Thr
Leu Leu Asp Leu Gln Asn Asn Asp 50 55 60 Ile Ser Glu Leu Arg Lys
Asp Asp Phe Lys Gly Leu Gln His Leu Tyr 65 70 75 80 Ala Leu Val Leu
Val Asn Asn Lys Ile Ser Lys Ile His Glu Lys Ala 85 90 95 Phe Ser
Pro Leu Arg Lys Leu Gln Lys Leu Tyr Ile Ser Lys Asn His 100 105 110
Leu Val Glu Ile Pro Pro Asn Leu Pro Ser Ser Leu Val Glu Leu Arg 115
120 125 Ile His Asp Asn Arg Ile Arg Lys Val Pro Lys Gly Val Phe Ser
Gly 130 135 140 Leu Arg Asn Met Asn Cys Ile Glu Met Gly Gly Asn Pro
Leu Glu Asn 145 150 155 160 Ser Gly Phe Glu Pro Gly Ala Phe Asp Gly
Leu Lys Leu Asn Tyr Leu 165 170 175 Arg Ile Ser Glu Ala Lys Leu Thr
Gly Ile Pro Lys Asp Leu Pro Glu 180 185 190 Thr Leu Asn Glu Leu His
Leu Asp His Asn Lys Ile Gln Ala Ile Glu 195 200 205 Leu Glu Asp Leu
Leu Arg Tyr Ser Lys Leu Tyr Arg Leu Gly Leu Gly 210 215 220 His Asn
Gln Ile Arg Met Ile Glu Asn Gly Ser Leu Ser Phe Leu Pro 225 230 235
240 Thr Leu Arg Glu Leu His Leu Asp Asn Asn Lys Leu Ala Arg Val Pro
245 250 255 Ser Gly Leu Pro Asp Leu Lys Leu Leu Gln Val Val Tyr Leu
His Ser 260 265 270 Asn Asn Ile Thr Lys Val Gly Val Asn Asp Phe Cys
Pro Met Gly Phe 275 280 285 Gly Val Lys Arg Ala Tyr Tyr Asn Gly Ile
Ser Leu Phe Asn Asn Pro 290 295 300 Val Pro Tyr Trp Glu Val Gln Pro
Ala Thr Phe Arg Cys Val Thr Asp 305 310 315 320 Arg Leu Ala Ile Gln
Phe Gly Asn Tyr Lys Lys 325 330 3331PRTArtificial SequenceSynthetic
Construct 3Asp Glu Glu Ala Ala Gly Ala Asp Thr Ala Gly Val Leu Asp
Pro Asp 1 5 10 15 Ser Val Thr Pro Thr Tyr Ser Ala Met Cys Pro Phe
Gly Cys His Cys 20 25 30 His Leu Arg Val Val Gln Cys Ser Asp Leu
Gly Leu Lys Ser Val Pro 35 40 45 Lys Glu Ile Ser Pro Asp Thr Thr
Leu Leu Asp Leu Gln Asn Asn Asp 50 55 60 Ile Ser Glu Leu Arg Lys
Asp Asp Phe Lys Gly Leu Gln His Leu Tyr 65 70 75 80 Ala Leu Val Leu
Val Asn Asn Lys Ile Ser Lys Ile His Glu Lys Ala 85 90 95 Phe Ser
Pro Leu Arg Lys Leu Gln Lys Leu Tyr Ile Ser Lys Asn His 100 105 110
Leu Val Glu Ile Pro Pro Asn Leu Pro Ser Ser Leu Val Glu Leu Arg 115
120 125 Ile His Asp Asn Arg Ile Arg Lys Val Pro Lys Gly Val Phe Ser
Gly 130 135 140 Leu Arg Asn Met Asn Cys Ile Glu Met Gly Gly Asn Pro
Leu Glu Asn 145 150 155 160 Ser Gly Phe Glu Pro Gly Ala Phe Asp Gly
Leu Lys Leu Asn Tyr Leu 165 170 175 Arg Ile Ser Glu Ala Lys Leu Thr
Gly Ile Pro Lys Asp Leu Pro Glu 180 185 190 Thr Leu Asn Glu Leu His
Leu Asp His Asn Lys Ile Gln Ala Ile Glu 195 200 205 Leu Glu Asp Leu
Leu Arg Tyr Ser Lys Leu Tyr Arg Leu Gly Leu Gly 210 215 220 His Asn
Gln Ile Arg Met Ile Glu Asn Gly Ser Leu Ser Phe Leu Pro 225 230 235
240 Thr Leu Arg Glu Leu His Leu Asp Asn Asn Lys Leu Ala Arg Val Pro
245 250 255 Ser Gly Leu Pro Asp Leu Lys Leu Leu Gln Val Val Tyr Leu
His Ser 260 265 270 Asn Asn Ile Thr Lys Val Gly Val Asn Asp Phe Cys
Pro Met Gly Phe 275 280 285 Gly Val Lys Arg Ala Tyr Tyr Asn Gly Ile
Ser Leu Phe Asn Asn Pro 290 295 300 Val Pro Tyr Trp Glu Val Gln Pro
Ala Thr Phe Arg Cys Val Thr Asp 305 310 315 320 Arg Leu Ala Ile Gln
Phe Gly Asn Tyr Lys Lys 325 330 4368PRTHomo sapiens 4Met Trp Pro
Leu Trp Arg Leu Val Ser Leu Leu Ala Leu Ser Gln Ala 1 5 10 15 Leu
Pro Phe Glu Gln Arg Gly Phe Trp Asp Phe Thr Leu Asp Asp Gly 20 25
30 Pro Phe Met Met Asn Asp Glu Glu Ala Ala Gly Ala Asp Thr Ala Gly
35 40 45 Val Leu Asp Pro Asp Ser Val Thr Pro Thr Tyr Ser Ala Met
Cys Pro 50 55 60 Phe Gly Cys His Cys His Leu Arg Val Val Gln Cys
Ser Asp Leu Gly 65 70 75 80 Leu Lys Ser Val Pro Lys Glu Ile Ser Pro
Asp Thr Thr Leu Leu Asp 85 90 95 Leu Gln Asn Asn Asp Ile Ser Glu
Leu Arg Lys Asp Asp Phe Lys Gly 100 105 110 Leu Gln His Leu Tyr Ala
Leu Val Leu Val Asn Asn Lys Ile Ser Lys 115 120 125 Ile His Glu Lys
Ala Phe Ser Pro Leu Arg Lys Leu Gln Lys Leu Tyr 130 135 140 Ile Ser
Lys Asn His Leu Val Glu Ile Pro Pro Asn Leu Pro Ser Ser 145 150 155
160 Leu Val Glu Leu Arg Ile His Asp Asn Arg Ile Arg Lys Val Pro Lys
165 170 175 Gly Val Phe Ser Gly Leu Arg Asn Met Asn Cys Ile Glu Met
Gly Gly 180 185 190 Asn Pro Leu Glu Asn Ser Gly Phe Glu Pro Gly Ala
Phe Asp Gly Leu 195 200 205 Lys Leu Asn Tyr Leu Arg Ile Ser Glu Ala
Lys Leu Thr Gly Ile Pro 210 215 220 Lys Asp Leu Pro Glu Thr Leu Asn
Glu Leu His Leu Asp His Asn Lys 225 230 235 240 Ile Gln Ala Ile Glu
Leu Glu Asp Leu Leu Arg Tyr Ser Lys Leu Tyr 245 250 255 Arg Leu Gly
Leu Gly His Asn Gln Ile Arg Met Ile Glu Asn Gly Ser 260 265 270 Leu
Ser Phe Leu Pro Thr Leu Arg Glu Leu His Leu Asp Asn Asn Lys 275 280
285 Leu Ala Arg Val Pro Ser Gly Leu Pro Asp Leu Lys Leu Leu Gln Val
290 295 300 Val Tyr Leu His Ser Asn Asn Ile Thr Lys Val Gly Val Asn
Asp Phe 305 310 315 320 Cys Pro Met Gly Phe Gly Val Lys Arg Ala Tyr
Tyr Asn Gly Ile Ser 325 330 335 Leu Phe Asn Asn Pro Val Pro Tyr Trp
Glu Val Gln Pro Ala Thr Phe 340 345 350 Arg Cys Val Thr Asp Arg Leu
Ala Ile Gln Phe Gly Asn Tyr Lys Lys 355 360 365
* * * * *