Note: Descriptions are shown in the official language in which they were submitted.
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PROTEINS RELATED TO NEURONAL REGENERATION
AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates generally to the treatment of neurological
injury and dysfunction associated with central nervous system trauma. In
particular,
the invention is directed to the identification of proteins which induce
neuronal
regeneration.
BACKGROUND OF THE INVENTION
The peripheral nervous system (PNS) comprises highly organized
groups of axon fibers or nerves external to the brain and spinal cord, such as
the
nerves in the limbs. In response to nerve damage, the peripheral nervous
system often
attempts to repair itself. While the return of lost functions is usually
incomplete,
generally the injured organism can adapt and function.
By contrast, damage to the central nervous system (CNS), comprising
the brain and spinal cord, is generally more serious, usually causing
permanent severe
disability or even death.
A number of conditions are known to affect both growth and
spontaneous regeneration in nerves, but the underlying mechanisms are not well
understood (Gibson et al., In Compered. Coretire. Educ. Pract. Yet., vol. 1 l,
pp., 1989,
938-945; and Daniloff et al., J. Cell Bio., 1986, 103:929-945). These
conditions
include the location of injury, the type of injury, the severity of injury,
and the age
and general health of the patient.
It has been reported that minor prior recoveries somehow prime the
nerve for greater recovery in secondary lesions, for example, recovery from an
earlier
compression injury.
There are no previous reports of an effective treatment for injuries to
neurons of the central nervous system, i. e. ; the brain and spinal cord (see,
M. Walker,
New Engl. J. Med., 1991, 324:1885-1887.
The lack of effective treatments for nervous system injuries may be
due to an insufficient understanding both of the formation of the nervous
system and
of its responses to injuries. Several attempts have been made to electrically
stimulate
injured
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nerves to try to cause regrowth; recovery was highly variable and inadequate
(see, B.
Sisken et al., Restorative Neurology and Neuroscience, 1990, 1:303-309; see
generally J. Daniloff et al., "The Molecular Bases of Nerve Regeneration," in
S.
Malhotra (ed.), Advances in Neural Science, vol. 2, 1993). The method that is
currently used most often to close gaps in severed nerves uses grafts of the
patient's
own sensory nerves, typically taken from the ankle; a minimal degree of
recovery and
permanent analgesia of the donor foot are the usual results.
Because an injured spinal cord has very limited ability to recover
spontaneously, and because the consequences of spinal cord injuries can be so
serious,
there is a particular need for an effective treatment of spinal cord injuries.
Paralytic
spinal cord injuries in the United States alone occur at the rate of about
10,000 per
year. Although the mortality rate is less than 10%, approximately 720
Americans per
million population are permanently disabled as a result of spinal cord
injuries. Most
of the injured are young people in the most productive stage of life.
Following injury to neuronal cells in the central nervous system, there
is often an abortive attempt by injured neural cells to generate new cellular
extensions
(dendrites and axons) in order to reestablish inter-neural contacts. In the
central
nervous system, these nerve sprouting and regeneration activities are often
modest
and only poorly sustained such that regeneration following stroke, trauma,
spinal cord
injury, etc., does not usually occur.
Thus, there is a need in the art for material and methods for treating
neuronal injury.
SUMMARY OF THE INVENTION
The present invention addresses this need. Applicants have
surprisingly discovered that a neuron-specific armadillo protein -- Neural
Plakophilin
Related Armadillo Protein (NPRAP) -- causes the development of numerous long,
cellular extensions, which are similar to axonal sprouting observed during
neuronal
regeneration and synapse formation.
One aspect of the invention is directed to a method of stimulating
growth of nerve cells, which method comprises contacting the nerve cells with
an
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hNPRAP having nerve growth stimulating activity in an amount effective to
cause
nerve cell growth.
In a specific embodiment related to a method of stimulating growth of
nerve cells, the method comprises contacting nerve cells with an hNPRAP
stimulating
agent in an amount sufficient to induce the expression of an hNPRAP and cause
nerve
cell growth.
A further related aspect of the invention is directed to a method of
stimulating neuronal regeneration in a mammal, which method comprises
administering to the mammal in need thereof an effective amount of an hNPRAP
or
an effective amount of an hNPRAP expression stimulating agent as set forth
above.
A further aspect of the invention is related to a pharmaceutical
composition comprising an hNPRAP having nerve growth stimulating activity, and
a
pharmaceutically acceptable carrier.
Yet another aspect of the invention is related to a pharmaceutical
1 S composition comprising an hNPRAP expression stimulating agent and a
pharmaceutically acceptable carrier.
In a specific embodiment, the invention provides a pharmaceutical
composition comprising an hNPRAP gene therapy vector, which vector comprises a
polynucleotid encoding hNPRAP and a promoter for expressing hNPRAP, and a
carrier. Naturally, such gene therarpy vectors are also part of the invention
as well.
A further aspect of the invention relates to a method for identifying
substances that modulate the expression of hNPRAP, which method comprises
contacting cultured cells that express hNPRAP with a test substance measuring
levels
of hNPRAP, as compared to a control in which the same cells that express
hNPRAP
are not contacted with the test substance, as an indication of modulatory
activity of
said test substance.
These and other aspects of the invention are disclosed more fully in the
accompanying detailed description.
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DETAILED DESCRIPTION
The human Neural Plakophilin Related Armadillo Protein ("hNPRAP")
(also described as GT24) consensus cDNA (SEQ ID N0:3) encodes a protein (SEQ
ID N0:4) of 1084 amino acid residues with a unique N-terminus, but with
homology
to proteins with armadillo (arm) repeat motifs at its C-terminus.
Applicants have now discovered that over-expression of hNPRAP, or
functional derivatives thereof containing one or more armadillo repeats,
causes the
development of numerous long, dendritic processes which typically terminate
upon
distantly located cells. These target cells need not necessarily be expressing
hNPRAP. The hNPRAP induced cellular extensions are highly similar to the
axonal
sprouting seen during neuronal regeneration and synapse formation.
Nucleotides 2920-2997 of the hNPRAP cDNA overlap the anonymous
microsatellite locus DSS478, therefore placing the hNPRAP gene on chromosome
SplS near the Cri-du-Chat deletion locus, a syndrome associated with
congenital
malformation and gross mental retardation. hNPRAP is described in detail in
copending commonly assigned U.S. Application Serial Nos. 08/888,077, filed
July 3,
1997 (PCT/CA97/00051), and 09/227,725, filed January 8, 1999 (PCT/CA99/00018),
both of which are incorporated herein by reference.
As described in U.S. Application Serial Nos. 08/888,077
(PCT/CA97/00051) and 09/227,725 (PCT/CA99/00018), hNPRAP is known to
interact with Presenilin I ("PS 1 ") and Presenilin II ("PS2") by direct
protein:protein
interaction studies. The domain of the PS 1 protein that interacts with hNPRAP
has
also been shown to interact with other proteins, such as armadillo repeat
proteins
p0071 and $-catenin.
On Northern blots, the hNPRAP gene is expressed as a range of
transcripts of 3.9 to S.0 kb in several regions of adult human brain, but is
expressed at
only very low levels in most non-neurologic tissues. Studies have shown that
PS 1
and hIVPRAP are both expressed in the same cell types and in
adjacent/contiguous
subcellular compartments.
In situ hybridization studies indicate that the transcriptional pattern of
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PS 1 and NPRAP overlap both in the brain of 4 month old mice, and in the
neural tube
and dorsal root ganglia of murine embryos. Both genes are expressed at high
levels in
dentate and hippocampal neurons, in scattered neocortical neurons, and in
cerebellar
Purkinje cells in adult mouse brain (Lee et al., J. Neurosci., 1996, 16:7513-
7525;
S Paffenholz and Franke, Differentiation, 1997, 61:293-304).
Immunocytochemical
studies show that PS 1 and hNPRAP have overlapping intracellular
distributions.
Thus, in non-confluent transfected cell cultures, hNPRAP has a predominantly
perinuclear cytoplasmic distribution contiguous with that of PS 1. In
contrast, in
confluent cells with abundant cell:cell contacts, hNPRAP is predominantly
located
near the cell membrane close to inter-cellular contact zones while PS 1
retains its
predominantly perinuclear distribution.
The invention is directed to the use of an hNPRAP to stimulate
neuronal regeneration and axon sprouting following a wide variety of insults
and
injuries. An "hNPRAP" is defined herein as a biologically active polypeptide
that
1 S contains a sequence of hNPRAP that mediate its nerve cell growth
stimulating
activity, e.g., the armadillo repeats. Thus, hNPRAP includes full-length
(naturally
occurring) hNPRAP, as well as biologically active analogues thereof. By
"analogues"
it is meant modifications such as point mutations, amino acid substitutions,
additions
or deletions, or other mammalian homologues, such as mouse (SEQ ID NO:S and
SEQ ID N0:6), which have similar activity to hNPRAP, the identification and
selection of which are well-known to those skilled in the art. In addition to
hNPRAP,
the use of recombinant proteins such as p120cas and chimeric proteins having
all or
parts of the C-terminal armadillo-like repeat and C-terminal unique sequences
of
hNPRAP may also be utilized in the practice of this invention. Analogues of
these
2S proteins which replicate the effects thereof may also be utilized in the
practice of this
invention.
In a first embodiment, the invention provides a method of stimulating
growth of nerve cells, comprising contacting nerve cells with an hNPRAP.
A second embodiment is directed to a method of stimulating growth of
nerve cells, comprising contacting nerve cells with an hNPRAP stimulating
agent in
an amount sufficient to induce the expression of hNPRAP. Such agents may
induce
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the expression of hNPRAP by positively binding to the hNPRAP gene to induce
expression, or may alter the interaction of hNPRAP with an inhibitor of hNPRAP
expression, e.g., by binding to the inhibitor itself or to hNPRAP such that
the inhibitor
no longer modulates the expression of hNPRAP.
Alternatively, the expression of hNPRAP may be induced by the use of
an appropriate viral vector system, or by the administration of recombinant
proteins,
biological molecules or small molecules which simulate or resemble either the
armadillo binding domain of the presenilins or the armadillo repeats of
hNPRAP.
Another embodiment is directed to a method for identifying substances that
simulate
or resemble (mimic) either the armadillo binding domain of the presenilins or
the
armadillo repeats of hNPRAP, and which substances cause neural growth.
Candidate
compounds which are shown to mimic either the armadillo binding domain of the
presenilins or the armadillo repeats of hNPRAP may be produced in
pharmaceutically
useful quantities for use in the treatment of neurological injury and
dysfunction
1 S associated with central nervous system trauma. Candidate compounds include
endogenous cellular components which interact with the presenilins in vivo and
which, therefore, provide new targets for pharmaceutical and therapeutic
interventions, as well as recombinant, synthetic and otherwise exogenous
compounds
which may have presenilin binding capacity and, therefore, may be candidates
for
pharmaceutical agents. Thus, in one procedure, cell lysates or tissue
homogenates
(e.g., human brain homogenates, lymphocyte lysates) may be screened for
proteins or
other compounds which bind to one of the normal or mutant presenilins.
Alternatively, any of a variety of exogenous compounds, both naturally
occurring
and/or synthetic (e.g., libraries of small molecules or peptides), may be
screened for
presenilin binding capacity. In each of these embodiments, an assay is
conducted to
detect binding between a presenilin component containing at least the
interacting
domain of a presenilin protein described herein and some other moiety.
As described in U.S. Application Serial No. 09/227,725, the presenilin
domain that interacts with PS-interacting proteins, such as armadillo repeat
proteins
hNPRAP, p0071 and $-catenin, has been identified as including or being
contained
in the sequence of amino acid residues from about 260 to about 409 of PS1 or
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corresponding residues from about 260 to about 390 in PS2. More preferably,
the
interacting domain contains or is contained in amino acid residues from about
372 to
about 399 of PS1 or corresponding residues from about 350 to about 380 in PS2.
The
amino acid sequences of wild-type human PS 1 and PS2 are shown in SEQ ID NO:1
and SEQ ID N0:2, respectively.
Binding may be detected by indirect functional measures reflecting the
functional consequences of the interaction (e.g., changes in intracellular
Ca2+, Na+,
KT, or GTP/GDP ratio, changes in apoptosis or microtubule associated protein
phosphorylation, changes in A$ peptide production or changes in the expression
of
other downstream genes which can be monitored by differential display, 2D gel
electrophoresis, differential hybridization, or SAGE methods) or by direct
measures
such as immunoprecipitation, the Biomolecular Interaction Assay (BIAcore) or
alteration of protein gel electrophoresis. The preferred methods involve
variations on
the following techniques: (1) direct extraction by affinity chromatography;
(2) co-
isolation of presenilin components and bound proteins or other compounds by
immunoprecipitation; (3) BIAcore analysis; and (4) the yeast two-hybrid
systems.
Other procedures include methods which detect abnormal processing of PS 1,
PS2,
APP, or proteins reacting with PSl, PS2, or APP (e.g., abnormal
phosphorylation,
glycosylation, glycation amidation or proteolytic cleavage) alterations in
presenilin
transcription, translation, and post-translational modification; alterations
in the
intracellular and extracellular trafficking of presenilin gene products; or
abnormal
intracellular localization of the presenilins.
The proteins or other compounds identified by these methods may then
be assayed for their ability to promote sprouting in axons of neuronal
cultures or
dendrite formation in non-neurological cells using morphometric analyses which
are
well-known to those skilled in the art of neuronal regeneration.
Alternatively, assays
for regeneration following sectioning of the optic nerve, spinal cord, etc. in
animals
may be performed. Such assays are well-known to those in the field of neuronal
regeneration.
The proteins or other compounds identified by these methods may be
purified and characterized by any of the standard methods known in the art.
Proteins
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may, for example, be purified and separated using electrophoretic (e.g., SDS-
PAGE,
2D PAGE) or chromatographic (e.g., HPLC) techniques and may then be
microsequenced. For proteins with a blocked N-terminus, cleavage (e.g., by
CNBr
and/or trypsin) of the particular binding protein is used to release peptide
fragments.
Further purification/characterization by HPLC and microsequencing and/or mass
spectrometry by conventional methods provides internal sequence data on such
blocked proteins. For non-protein compounds, standard organic chemical
analysis
techniques (e.g., IR, NMR and mass spectrometry; functional group analysis; X-
ray
crystallography) may be employed to determine their structure and identity.
These hNPRAPs, and compounds which activate hNPRAP, may be
employed in combination with a suitable pharmaceutical, physiologically
acceptable
carrier. Administration of hNPRAP of this invention can be through the
administration of hNPRAP peptides agonists or antagonists synthesized from
recombinant constructs of hNPRAP DNA or from peptide chemical synthesis (Woo,
et al., Protein Engineering, 1989, 3:29-37) or in the form of gene therapy
(Goldspiel
et al., Clin. Pharm., 1993, 12:488; Wu and Wu, Biotherapy, 1991, 3:87;
Mulligan,
Science, 1993, 260:926; Morgan and Anderson, Ann. Rev. Biochem., 1993, 62:191;
and, May TIBTECH, 1993, 11:155).
Generally, hNPRPA and/or activating agents) are administered as
pharmaceutical compositions comprising an effective amount of hNPRAP and/or
activating agents) in a pharmaceutical carrier. These reagents can be combined
for
therapeutic use with additional active or inert ingredients, e.g., in
conventional
pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants,
along
with physiologically innocuous stabilizers and excipients. A pharmaceutical
carrier
can be any compatible, non-toxic substance suitable for delivering the
compositions
of the invention to a patient.
The quantities of reagents necessary for effective therapy will depend
upon many different factors, including means of administration, target site,
physiological state of the patient, and other medicants administered. Thus,
treatment
dosages should be titrated to optimize safety and efficacy. Animal testing of
effective
doses for treatment of particular injuries will provide further predicative
indication of
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human dosage. Various considerations are described, e.g., in Gilman et al.
(eds.)
(1990) Goodman and Gilman's; The Pharmacological Bases of Therapeutics, 8th
Ed.,
Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed. ( 1990),
Mack
Publishing Co., Easton, PA. Methods for administration are discussed therein
and
below, e.g., for intravenous, intraperitoneal, or intramuscular
administration,
transfermal diffusion, and others. Pharmaceutically acceptable carriers
include water,
saline, buffers and other compounds described, e.g., in the Merck Index, Merck
&
Co., Rahway, New Jersey, and in Remington, supra. Slow release formulations,
or a
slow release apparatus, may be used for continuous administration.
Dosage range$ for hNPRAP and/or activating agents) would
ordinarily be expected to be in amounts lower than 1 mM concentrations,
typically
less than about 10 ~M concentrations, usually less than about 100 nM,
preferably less
than about 10 pM (picomolar), and most preferably less than about 1 fM
(femtomolar), with an appropriate carrier. Generally, treatment is initiated
with
I S smaller dosages which are less than the optimum dose of the compound.
Thereafter,
the dosage is increased by small increments until the optimum effect under the
circumstance is reached. Determination of the proper dosage and administration
regime for a particular situation is within the skill of the art.
Polypeptides and other compounds of the present invention which
activate or inhibit hNPRAP may be employed alone or in conjunction with other
compounds, such as therapeutic compounds. Once identified by the methods
described above, the candidate compounds may then be produced in quantities
sufficient for pharmaceutical .administration or testing (e.gJ., mg or mg or
greater
quantities), and formulated in a pharmaceutically acceptable carrier (see,
e.g.,
Remington's, supra).
Pharmaceutically acceptable carriers that may be used in these
pharmaceutical compositions include, but are not limited to, ion exchangers,
alumina,
aluminum stearate, lecithin, serum proteins, such as human serum albumin,
buffer
substances such as phosphates, glycine, sorbic acid, potassium sorbate,
partial
glyceride mixtures of saturated vegetable fatty acids, water, salts or
electrolytes, such
as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate,
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sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,
polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol, sodium
carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-
block
polymers, polyethylene glycol and wool fat.
5 The compositions of the present invention may be administered orally,
parenterally, by spray inhalation, topically, rectally, nasally, buccally,
vaginally or via
an implanted reservoir. The term "parenteral" as used herein includes
subcutaneous,
intravenous, intramuscular, intra-articular, intra-synovial, intrasternal,
intrathecal,
intrahepatic, intralesional and intracranial injection or infusion techniques.
10 Preferably, the compositions are administered orally, intraperitoneally or
intravenously.
Sterile injectable forms of the compositions of this invention may be
aqueous or oleaginous suspension. These suspensions may be formulated
according
to techniques known in the art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a sterile
injectable
solution or suspension in a non-toxic parenterally-acceptable diluent or
solvent, for
example as a solution in 1,3-butanediol. Among the acceptable vehicles and
solvents
that may be employed are water, Ringer's solution and isotonic sodium chloride
solution. In addition, sterile, fixed oils are conventionally employed as a
solvent or
suspending medium. For this purpose, any bland fixed oil may be employed
including
synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its
glyceride
derivatives are useful in the preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil, especially
in their
polyoxyethylated versions. These oil solutions or suspensions may also contain
a
long-chain alcohol diluent or dispersant.
The pharmaceutical compositions of this invention may be orally
administered in any orally acceptable dosage form including, but not limited
to,
capsules, tablets, aqueous suspensions or solutions. In the case of tablets
for oral use,
carriers which are commonly used include lactose and corn starch. Lubricating
agents, such as magnesium stearate, are also typically added. For oral
administration
in a capsule form, useful diluents include lactose and dried corn starch. When
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aqueous suspensions are required for oral use, the active ingredient is
combined with
emulsifying and suspending agents. If desired, certain sweetening, flavoring
or
coloring agents may also be added.
Alternatively, the pharmaceutical compositions of this invention may
be administered in the form of suppositories for rectal administration. These
can be
prepared by mixing the agent with a suitable non-irritating excipient which is
solid at
room temperature but liquid at rectal temperature and therefore will melt in
the
rectum to release the drug. Such materials include cocoa butter, beeswax and
polyethylene glycols.
The pharmaceutical compositions of this invention may also be
administered topically, especially when the target of treatment includes areas
or
organs readily accessible by topical application, including diseases of the
eye, the
skin, or the lower intestinal tract. Suitable topical formulations are readily
prepared
for each of these areas or organs.
Topical application for the lower intestinal tract can be effected in a
rectal suppository formulation (see above) or in a suitable enema formulation.
Topically-transdermal patches may also be used.
For topical applications, the pharmaceutical compositions may be
formulated in a suitable ointment containing the active component suspended or
dissolved in one or more carriers. Carriers for topical administration of the
compounds of this invention include, but are not limited to, mineral oil,
liquid
petrolatum, white petrolatum, ~ propylene glycol, polyoxyethylene,
polyoxypropylene
compound, emulsifying wax and water. Alternatively, the pharmaceutical
compositions can be formulated in a suitable lotion or cream containing the
active
components suspended or dissolved in one or more pharmaceutically acceptable
carriers. Suitable carriers include, but are not limited to, mineral oil,
sorbitan
monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-
octyldodecanol,
benzyl alcohol and water.
For ophthalmic use, the pharmaceutical compositions may be
formulated as micronized suspensions in isotonic, pH adjusted sterile saline,
or,
preferably., as solutions in isotonic, pH adjusted sterile saline, either with
our without
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a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic
uses,
the pharmaceutical compositions may be formulated in an ointment such as
petrolatum.
The pharmaceutical compositions of this invention may also be
administered by nasal aerosol or inhalation. Such compositions are prepared
according to techniques well-known in the art of pharmaceutical formulation
and may
be prepared as solutions in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability, fluorocarbons,
and/or
other conventional solubilizing or dispersing agents.
The methods and compositions of this invention may be used to treat
nerve damage caused by a wide variety of diseases or physical traumas. These
include, but are not limited to, Alzheimer's disease, Parkinson's disease,
amyotrophic
lateral sclerosis (ALS), multiple sclerosis, stroke and ischemia associated
with stroke,
neural paropathy, other neural degenerative diseases, motor neuron diseases,
sciatic
crush, peripheral neuropathy, particularly neuropathy associated with
diabetes, spinal
cord injuries and facial nerve crush.
The hNPRAP polynucleotides, polypeptides, agonists and antagonists
that are polypeptides may be employed in accordance with the present invention
by
expression of such polypeptides in treatment modalities often referred to as
"gene
therapy". Thus, for example, cells from a patient may be engineered with a
polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo. The
engineered cells can then be provided to a patient to be treated with the
polypeptide.
In this embodiment, cells may be engineered ex vivo, for example, by the use
of a
retroviral plasmid vector containing RNA encoding a polypeptide of the present
invention. Such methods are well-known in the art and their use in the present
invention will be apparent from the teachings herein.
Similarly, cells may be engineered in vivo for expression of a
polypeptide in vivo by procedures known in the art. For example, a
polynucleotide of
the invention may be engineered for expression in a replication defective
retroviral or
viral vector, as discussed above. The retroviral expression construct may then
be
isolated. A packaging cell is transduced with a retroviral plasmid vector
containing
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RNA encoding a polypeptide of the present invention, such that the packaging
cell
now produces infectious viral particles containing the gene of interest. These
viral
particles may be administered to a patient for engineering cells in vivo and
expression
of the polypeptide in vivo. These and other methods for administering a
polypeptide
of the present invention should be apparent to those skilled in the art from
the
teachings of the present invention.
Retroviruses or. viruses from which the plasmid vectors hereinabove-
mentioned may be derived include, but are not limited to, SimiForest Virus,
Lenti-
virus, Moloney Murine Leukemia Virus, Spleen Necrosis Virus, Rous Sarcoma
Virus,
Harvey Sarcoma Virus, Avian Leukosis Virus, Gibbon Ape Leukemia Virus, Human
Immunodeficiency Virus, Adenovirus, Myeloproliferative Sarcoma Virus, and
Mammary Tumor Virus. In a preferred embodiment, the retroviral plasmid vector
is
derived from Moloney Murine Leukemia Virus.
Such vectors will include one or more promoters for expressing the
polypeptide. Suitable promoters which may be employed include, but are not
limited
to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV)
promoter described in Miller et al., Biotechniques, 1989, 7:980-990. Cellular
promoters such as eukaryotic cellular promoters including, but not limited to,
the
histone, RNA polymerase III, and $-actin promoters, can also be used.
Additional
viral promoters which may be employed include, but are not limited to,
adenovirus
promoters such as the adenoviral major late promoter, thymidine kinase (TK)
promoters such as the Herpes Simplex thymidine kinase promoters; the
respiratory
syncytial virus (RSV) promoters; and B19 parvovirus promoters. The selection
of a
suitable promoter will be apparent to those skilled in the art from the
teachings
contained herein.
The nucleic acid sequence encoding the polypeptide of the present
invention may be placed under the control of an inducible promoter. Suitable
inducible promoters which may be employed include, but are not limited to, the
MMT
promoter, the metallothionein promoter; heat shock promoters; the albumin
promoter;
the ApoAI promoter; human globin promoters; viral thymidine kinase promoters;
and
human growth hormone promoters. The promoter may also be the native promoter
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which controls the gene encoding the polypeptide.
The retroviral plasmid vector is employed to transduce packaging cell
lines to form producer cell lines. Examples of packaging cells which may be
transfected include, but are not limited to, the PE501, PA317, .PSL-2, .omega.-
AM,
PA12, T19-14X, VT-19-17-H2, .omega.CRE, .omega.CRIP, GP+E-86,
GP+envAml2, and DAN cell lines as described in Miller, A., Human Gene Therapy,
1990, 1:5-14. The vector may be transduced into the packaging cells through
any
means known in the art. Such means include, but are not limited to,
electroporation,
the use of liposomes, and CaPOa precipitation. In one alternative, the
retroviral
plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and
then
administered to a host. The producer cell line will generate infectious
retroviral vector
particles, which include the nucleic acid sequences) encoding the
polypeptides. Such
retroviral vector particles may'then be employed to transduce eukaryotic
cells, either
in vitro or in vivo. The transduced eukaryotic cells will express the nucleic
acid
sequences) encoding the polypeptide. Eukaryotic cells which may be transduced
include, but are not limited to, embryonic stem cells, embryonic carcinoma
cells, as
well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,
keratinocytes,
endothelial cells, and bronchial epithelial cells.
Although the invention herein has been described with reference to
particular embodiments, it is to be understood that these embodiments are
merely
illustrative of the principles and applications of the present invention. It
is therefore
to be understood that numerous modifications may be made to the illustrative
embodiments and that other arrangements may be devised without departing from
the
spirit and scope of the present invention as defined by the appended claims.
Each patent, patent application, publication, and procedure disclosed in
this application is specifically incorporated by reference in its entirety.
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
1 /15
SEQUENCE LISTING
<110> The Governing Council of the University of Toronto
<120> Proteins Related to Neoronal
Regeneration and Uses Thereof
<130> 9267-59/PAR
<160> 6
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 467
<212> PRT
<213> human
<400> 1
Met Thr Glu Leu Pro Ala Pro Leu Ser Tyr Phe Gln Asn Ala Gln Met
1 5 10 15
Ser Glu Asp Asn His Leu Ser Asn Thr Val Arg Ser Gln Asn Asp Asn
20 25 30
Arg Glu Arg Gln Glu His Asn Asp Arg Arg Ser Leu Gly His Pro Glu
35 40 45
Pro Leu Ser Asn Gly Arg Pro Gln Gly Asn Ser Arg Gln Val Val Glu
50 55 60
Gln Asp Glu Glu Glu Asp Glu Glu Leu Thr Leu Lys Tyr Gly Ala Lys
65 70 75 80
His Val Ile Met Leu Phe Val Pro Val Thr Leu Cys Met Val Val Val
85 90 95
Val Ala Thr Ile Lys Ser Val Ser Phe Tyr Thr Arg Lys Asp Gly Gln
100 105 110
Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr Glu Thr Val Gly Gln Arg
115 120 125
Ala Leu His Ser Ile Leu Asn Ala Ala Ile Met Ile Ser Val Ile Val
130 135 140
Val Met Thr Ile Leu Leu Val Val Leu Tyr Lys Tyr Arg Cys Tyr Lys
145 150 155 160
Val Ile His Ala Trp Leu Ile Ile Ser Ser Leu Leu Leu Leu Phe Phe
165 170 175
Phe Ser Phe Ile Tyr Leu Gly Glu Val Phe Lys Thr Tyr Asn Val Ala
180 185 190
Val Asp Tyr Ile Thr Val Ala Leu Leu Ile Trp Asn Phe Gly Val Val
195 200 205
Gly Met Ile Ser Ile His Trp Lys Gly Pro Leu Arg Leu Gln Gln Ala
210 215 220
Tyr Leu Ile Met Ile Ser Ala Leu Met Ala Leu Val Phe Ile Lys Tyr
225 230 235 290
Leu Pro Glu Trp Thr Ala Trp Leu Ile Leu Ala Val Ile Ser Val Tyr
245 250 255
Asp Leu Val A1a Val Leu Cys Pro Lys Gly Pro Leu Arg Met Leu Val
260 265 270
Glu Thr Ala Gln Glu Arg Asn Glu Thr Leu Phe Pro Ala Leu Ile Tyr
275 280 285
SUBSTITUTE S~iE~T (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
2/15
Ser Ser Thr Met Val Trp Leu Val Asn Met Ala Glu Gly Asp Pro Glu
290 295 300
Ala Gln Arg Arg Val Ser Lys Asn Ser Lys Tyr Asn Ala Glu Ser Thr
305 310 315 320
Glu Arg Glu Ser Gln Asp Thr Val Ala Glu Asn Asp Asp Gly Gly Phe
325 330 335
Ser Glu Glu Trp Glu Ala Gln Arg Asp Ser His Leu Gly Pro His Arg
340 345 350
Ser Thr Pro Glu Ser Arg Ala Ala Val Gln Glu Leu Ser Ser Ser Ile
355 360 365
Leu Ala Gly Glu Asp Pro Glu Glu Arg Gly Val Lys Leu Gly Leu Gly
370 375 380
Asp Phe Ile Phe Tyr Ser Val Leu Val Gly Lys Ala Ser Ala Thr Ala
385 390 395 400
Ser Gly Asp Trp Asn Thr Thr Ile Ala Cys Phe Val Ala Ile Leu Ile
405 410 415
Gly Leu Cys Leu Thr Leu Leu Leu Leu Ala Ile Phe Lys Lys Ala Leu
420 425 430
Pro Ala Leu Pro Ile Ser Ile Thr Phe Gly Leu Val Phe Tyr Phe Ala
435 440 445
Thr Asp Tyr Leu Val Gln Pro Phe Met Asp Gln Leu Ala Phe His Gln
450 455 460
Phe Tyr Ile
465
<210> 2
<211> 448
<212> PRT
<213>. human
<400> 2
Met Leu Thr Phe Met Ala Ser Asp Ser Glu Glu Glu Val Cys Asp Glu
1 5 10 15
Arg Thr Ser Leu Met Ser Ala Glu Ser Pro Thr Pro Arg Ser Cys Gln
20 25 30
Glu Gly Arg Gln Gly Pro Glu Asp Gly Glu Asn Thr Ala Gln Trp Arg
35 40 45
Ser Gln Glu Asn Glu Glu Asp Gly Glu Glu Asp Pro Asp Arg Tyr Val
50 55 60
Cys Ser Gly Val Pro Gly Arg Pro Pro Gly Leu Glu Glu Glu Leu Thr
65 70 75 80
Leu Lys Tyr Gly Ala Lys His Val Ile Met Leu Phe Val Pro Val Thr
85 90 95
Leu Cys Met Ile Val Val Val Ala Thr Ile Lys Ser Val Arg Phe Tyr
100 105 110
Thr Glu Lys Asn Gly Gln Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr
115 120 125
Pro Ser Val Gly Gln Arg Leu Leu Asn Ser Val Leu Asn Thr Leu Ile
130 135 140
Met Ile Ser Val Ile Val Val Met Thr Ile Phe Leu Val Val Leu Tyr
145 150 155 160
Lys Tyr Arg Cys Tyr Lys Phe Ile His Gly Trp Leu Ile Met Ser Ser
165 170 175
Leu Met Leu Leu Phe Leu Phe Thr Tyr Ile Tyr Leu Gly Glu Val Leu
180 185 190
SUBSTITUTE SHEET (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
3/15
Lys Thr Tyr Asn Vai Ala Met Asp Tyr Pro Thr Leu Leu Leu Thr Val
195 200 205
Trp Asn Phe Gly Ala Val Gly Met Val Cys Ile His Trp Lys Gly Pro
210 215 220
Leu Val Leu Gln Gln Ala Tyr Leu Ile Met Ile Ser Ala Leu Met Ala
225 230 235 240
Leu Val Phe Ile Lys Tyr Leu Pro Glu Trp Ser Ala Trp Val Ile Leu
245 250 255
Gly Ala Ile Ser Val Tyr Asp Leu Val Ala Val Leu Cys Pro Lys Gly
260 265 270
Pro Leu Arg Met Leu Val Glu Thr Ala Gln Glu Arg Asn Glu Pro Ile
275 280 285
Phe Pro Ala Leu Ile Tyr Ser Ser Ala Met Val Trp Thr Val Gly Met
290 295 300
Ala Lys Leu Asp Pro Ser Ser Gln Gly Ala Leu Gln Leu Pro Tyr Asp
305 310 315 320
Pro Glu Met Glu Glu Asp Ser Tyr Asp Ser Phe Gly Glu Pro Ser Tyr
325 330 335
Pro Glu Val Phe Glu Pro Pro Leu Thr Gly Tyr Pro Gly Glu Glu Leu
340 345 350
Glu Glu Glu Glu Glu Arg Gly Val Lys Leu Gly Leu Gly Asp Phe Ile
355 360 365
Phe Tyr Ser Val Leu Val Gly Lys Ala Ala Ala Thr Gly Ser Gly Asp
370 375 380
Trp Asn Thr Thr Leu Ala Cys Phe Val Ala Ile Leu Ile Gly Leu Cys
385 390 395 400
Leu Thr Leu Leu Leu Leu Ala Val Phe Lys Lys Ala Leu Pro Ala Leu
405 410 415
Pro Ile Ser Ile Thr Phe Gly Leu Ile Phe Tyr Phe Ser Thr Asp Asn
420 425 930
Leu Val Arg Pro Phe Met Asp Thr Leu Ala Ser His Gln Leu Tyr Ile
435 440 445
<210> 3
<211> 4746
<212> DNA
<213> human
<400> 3
gccagcatcc cttgtcccgc ggccggctca gacaacaaaa gcggaagatg ctgcagttgg
gcaaggtcag gaccttgcct tgaaagccgg gcggcgccgc gcaacgcctc ttcccggact
120
gaggagctgt cgccggcgga gggtgcatgt ttgcgaggaa gccgccgggc gccgcgcctt
180
tgggagctat gcctgttcca gaccagcctt catcagcctc agagaagacg agttccctga
240
gccccggctt aaacacctcc aacggggatg gctctgaaac agaaaccacc tctgccatcc
300
tcgcctcagt caaagaacag gaattacagt ttgaaaggct gacccgagag ctggaggctg
360
aacggcagat cgtagccagc cagctggagc gatgcaagct cggatccgag actggcagca
420
tgagcagcat gagttcagca gaagagcagt ttcagtggca gtcacaagat ggtcaaaaag
480
SUSSTITUT~ SHEET (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
4/15
atatcgaaga tgagcttaca acaggtctcg agctggtgga ctcctgtatt aggtcactac
540
aggaatcagg aatacttgac ccacaggatt attctacagg tgaaaggccc agcctgctct
600
cccagagtgc acttcagctc aattccaaac ctgaagggtc tttccagtat ccggccagct
660
accatagcaa ccagaccctg gccctggggg aaaccacccc ttcacagctc ccggcccgag
720
gcacacaagc ccgagctacg ggccagagct tcagccaggg cacgaccagc cgcgccggcc
780
acctggcggg gcccgagccc gcgccgccgc cgccgccgcc gccgcgggag ccgttcgcgc
840
ccagcctggg cagcgccttc cacctgcccg acgcgccgcc cgccgccgcc gccgccgcgc
900
tctactactc cagctccacg ctgcccgcgc cgccgcgcgg gggctccccg ctggccgcgc
960
cccagggcgg ttcgcccacc aagctgcagc gcggcggctc ggcccccgag ggcgccacct
1020
acgccgcgcc gcgcggctcc tcgcccaagc agtcgcccag ccgcctggcc aagtcctaca
1080
gcaccagctc gcccatcaac atcgtcgtgt cctcggccgg cctgtccccg atccgcgtga
1140
cctcgccccc caccgtgcag tccaccatct cctcctcgcc catccaccag ctgagctcca
1200
ccatcggcac gtacgccacc ctgtcgccca ccaagcgcct ggtccacgcg tccgagcagt
1260
acagcaagca ctcgcaggag ctgtatgcca cggccaccct ccagaggccg ggcagcctgg
1320
cagctggttc ccgagcctca tacagcagcc agcatgggca cctgggccca gagttgcggg
1380
ccctgcagtc cccagaacac cacatagatc ccatctatga agaccgcgtc tatcagaagc
1440
cccctatgag gagtctcagc cagagccagg gggaccctct gccgccagca cacaccggca
1500
cctaccgcac gagcacagcc ccatcttccc ctggtgtcga ctccgtcccc ttgcagcgca
1560
caggcagcca gcacggccca cagaatgccg ccgcggccac cttccagagg gccagctatg
1620
ccgccggccc agcctccaat tacgcggacc cctaccgaca gctgcagtat tgtccctctg
1680
ttgagtctcc atacagcaaa tccggccctg ctctcccgcc tgaaggcacc ttggccaggt
1740
ccccgtccat tgatagcatt cagaaagatc ccagagaatt tggatggaga gacccggaac
1800
tgccggaagt gattcagatg ttgcagcacc agtttccctc ggtccagtct aacgcggcag
1860
cctacttgca acacctctgt tttggagaca acaaaattaa agccgagata aggagacaag
1920
gaggcatcca gctcctggtg gacctgttgg atcatcggat gaccgaagtc caccgtagtg
1980
cctgtggagc tctgagaaac ctggtgtatg ggaaggccaa cgatgataac aaaattgccc
2040
tgaaaaactg tggtggcatc ccagcactgg tgaggttact ccgcaagacg actgacctgg
2100
agatccggga gctggtcaca ggagtccttt ggaacctctc ctcatgcgat gcactcaaaa
2160
SUBSTITUTE SHEET (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
5/15
tgccaatcat ccaggatgcc ctagcagtac Lgaccaacgc ggtgattatc ccccactcag
2220
gctgggaaaa ttcgcctctt caggatgatc ggaaaataca gctgcattca tcacaggtgc
2280
tgcgtaacgc caccgggtgc ctaaggaatg ttagttcggc cggagaggag gcccgcagaa
2340
ggatgagaga gtgtgatggg cttacggatg ccttgctgta cgtgatccag tctgcgctgg
2400
ggagcagtga gatcgatagc aagaccgttg aaaactgtgt gtgcatttta aggaacctct
2460
cgtaccggct ggcggcagaa acgtctcagg gacagcacat gggcacggac gagctggacg
2520
ggctactctg tggcgaggcc aatggcaagg atgctgagag ctctgggtgc tggggcaaga
2580
agaagaagaa aaagaaatcc caagatcagt gggatggagt aggacctctt ccagactgtg
2640
ctgaaccacc aaaagggatc cagatgctgt ggcacccatc aatagtcaaa ccctacctca
2700
cactgctctc tgagtgctca aatccagaca cgctggaagg ggcggcaggc gccctgcaga
2760
acttggctgc agggagctgg aagtggtcag tatatatccg agccgctgtc cgaaaagaga
2820
aaggcctgcc catcctcgtg gagctgctcc gaatagacaa tgaccgtgtg gtgtgcgcgg
2880
tggccactgc gctgcggaac atggccttgg acgtcagaaa taaggagctc atcggcaaat
2940
acgccatgcg agacctagtc cacaggcttc caggagggaa caacagcaac aacactgcaa
3000
gcaaggccat gtcggatgac acagtgacag ctgtctgctg cacactgcac gaagtgatta
3060
ccaagaacat ggagaacgcc aaggccttac gggatgccgg tggcatcgag aagttggtcg
3120
gcatctccaa aagcaaagga gataaacact ctccaaaagt ggtcaaggct gcatctcagg
3180
tcctcaacag catgtggcag taccgagatc tgaggagtct ctacaaaaag gatggatggt
3240
cacaatacca ctttgtagcc tcgtcttcaa ccatcgagag ggaccggcaa aggccctact
3300
cctcctcccg cacgccctcc atctcccctg tgcgcgtgtc tcccaacaac cgctcagcaa
3360
gtgccccagc ttcacctcgg gaaatgatca gcctcaaaga aaggaaaaca gactacgagt
3420
gcaccggcag caacgccacc taccacggag ctaaaggcga acacacttcc aggaaagatg
3480
ccatgacagc tcaaaacact ggaatttcaa ctttgtatag gaattcttat ggtgcgcccg
3540
ctgaagacat caaacacaac caggtttcag cacagccagt cccacaggag cccagcagaa
3600
aagattacga gacctaccag ccatttcaga attccacaag aaattacgat gagtccttct
3660
tcgaggacca ggtccaccat cgccctcccg ccagcgagta caccatgcac ctgggtctca
3720
agtccaccgg caactacgtt gacttctact cagctgcccg tccctacagt gaactgaact
3780
atgaaacgag ccactacccg gcctcccccg actcctgggt gtgaggagca gggcacaggc
3840
SUBSTITUTE SHEET (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
6/15
gctccgggaa cagtgcatgt gcatgcatac cacaagacat ttctttctgt tttgtttttt
3900
tctcctgcaa atttagtttg ttaaagcctg ttccatagga aggctgtgat aaccagtaag
3960
gaaatattaa gagctatttt agaaagctaa atgaatcgca agttaacttg gaaatcagta
4020
gaaagctaaa gtgatcctaa atatgacagt gggcagcacc tttctagcgt gagctgtaaa
4080
gtaacgagaa gtgctttata ctgaacgtgg ttgatgggag gagagacgag gcattcgggc
9140
cggtggggcg taagggttat cgttaagcac aagacacaga atagtttaca cactgtgtgg
4200
gggacggctt ctcacgcttt gtttactctc ttcatccgtt gtgactctag gcttcaggtt
4260
gcattggggt tcctctgtac agcaagatgt ttcttgcctt ttgttaatgc attgttgtaa
4320
agtatttgat gtacattaca gattaaagaa gaaaagcgcg ttgtgtatat tacaccaatg
4380
ccgccgtgtt tcctcatcta tggttctaaa tattgcttca atttcaaact tttgaaagat
4440
gtatggattt ccagtttttc tttactttct cccagtatgt tttaacaaaa aaaaaaaaaa
4500
gcaggaaaaa aggaatattt agcagtattg ttcgttctga tatgtgaatt tgtttgtgac
4560
aactaaacaa ggcattcagc agtttctgac aattaacata catcattcca cactccttgt
4620
caacaaagtg ctttttcact gcctaaaatt ttagatgtag atatttgaaa tagatttttt
4680
catttatacc agttttcttt atgatgatac agtgttaaaa gaaaataaat tacaattgat
4740
ctgtca
4746
<210> 4
<211> 1225
<212> PRT
<213> human
<400> 4
Met Phe Ala Arg Lys Pro Pro Gly Ala Ala Pro Leu Gly Ala Met Pro
1 S 10 15
Val Pro Asp Gln Pro Ser Ser Ala Ser Glu Lys Thr Ser Ser Leu Ser
20 25 30
Pro Gly Leu Asn Thr Ser Asn Gly Asp Gly Ser Glu Thr Glu Thr Thr
35 40 45
Ser Ala Ile Leu Ala Ser Va1 Lys Glu Gln Glu Leu Gln Phe Glu Arg
50 55 60
Leu Thr Arg Glu Leu Glu Ala Glu Arg Gln Ile Val Ala Ser Gln Leu
65 70 75 80
Glu Arg Cys Lys Leu Gly Ser Glu Thr Gly Ser Met Ser Ser Met Ser
85 90 95
Ser Ala Glu Glu Gln Phe Gln Trp Gln Ser Gln Asp Gly Gln Lys Asp
100 105 110
Ile Glu Asp Glu Leu Thr Thr Gly Leu Glu Leu Val Asp Ser Cys Ile
115 120 125
SUBSTITUTE SHEET (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CAQO/00126
7/15
Arg Ser Leu Gln Giu Ser Gly Ile Leu Asp Pro Gln Asp Tyr 5er Thr
130 135 140
Gly Glu Arg Pro Ser Leu Leu Ser Gln Ser Ala Leu Gln Leu Asn Ser
145 150 155 160
Lys Pro Glu Gly Ser Phe Gln Tyr Pro Ala Ser Tyr His Ser Asn Gln
165 170 175
Thr Leu Ala Leu Gly Glu Thr Thr Pro Ser Gln Leu Pro Ala Arg Gly
180 185 190
Thr Gln Ala Arg Ala Thr Gly Gln Ser Phe Ser Gln Gly Thr Thr Ser
195 200 205
Arg Ala Gly His Leu Ala Gly Pro Glu Pro Ala Pro Pro Pro Pro Pro
210 215 220
Pro Pro Arg Glu Pro Phe Ala Pro Ser Leu Gly Ser Ala Phe His Leu
225 230 235 240
Pro Asp Ala Pro Pro Ala Ala Ala Ala Ala Ala Leu Tyr Tyr Ser Ser
245 250 255
Ser Thr Leu Pro Ala Pro Pro Arg Gly Gly Ser Pro Leu Ala Ala Pro
260 265 270
Gln Gly Gly Ser Pro Thr Lys Leu Gln Arg Gly Gly Ser Ala Pro Glu
275 280 285
Gly Ala Thr Tyr Ala Ala Pro Arg Gly Ser Ser Pro Lys Gln Ser Pro
290 295 300
Ser Arg Leu Ala Lys Ser Tyr Ser Thr Ser Ser Pro Ile Asn Ile Val
305 310 315 320
Val Ser Ser Ala Gly Leu Ser Pro Ile Arg Val Thr Ser Pro Pro Thr
325 330 335
Val Gln Ser Thr Ile Ser Ser Ser Pro Ile His Gln Leu Ser Ser Thr
340 345 350
Ile Gly Thr Tyr Ala Thr Leu Ser Pro Thr Lys Arg Leu Val His Ala
355 360 365
Ser Glu Gln Tyr Ser Lys His Ser Gln Glu Leu Tyr Ala Thr Ala Thr
370 375 380
Leu Gln Arg Pro Gly Ser Leu Ala Ala Gly Ser Arg Ala Ser Tyr Ser
385 390 395 400
Ser Gln His Gly His Leu Gly Pro Glu Leu Arg Ala Leu Gln Ser Pro
405 410 415
Glu His His Ile Asp Pro Ile Tyr Glu Asp Arg Val Tyr Gln Lys Pro
420 425 430
Pro Met Arg Ser Leu Ser Gln Ser Gln Gly Asp Pro Leu Pro Pro Ala
435 440 445
His Thr Gly Thr Tyr Arg Thr Ser Thr Ala Pro Ser Ser Pro Gly Val
450 455 460
Asp Ser Val Pro Leu Gln Arg Thr Gly Ser Gln His Gly Pro Gln Asn
465 470 475 480
Ala Ala Ala Ala Thr Phe Gln Arg Ala Ser Tyr Ala Ala Gly Pro Ala
485 490 495
Ser Asn Tyr Ala Asp Pro Tyr Arg Gln Leu Gln Tyr Cys Pro Ser Val
500 505 510
Glu Ser Pro Tyr Ser Lys Ser Gly Pro Ala Leu Pro Pro Glu Gly Thr
515 520 525
Leu Ala Arg Ser Pro Ser Ile Asp Ser Ile Gln Lys Asp Pro Arg Glu
530 535 590
Phe Gly Trp Arg Asp Pro Glu Leu Pro Glu Val Ile Gln Met Leu Gln
545 550 555 560
His Gln Phe Pro Ser Val Gln Ser Asn Ala Ala Ala Tyr Leu Gln His
565 570 575
SUBSTITUTE SHEET (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
8/15
Leu Cys Phe Gly Asp Asn Lys Ile Lys Aia Glu Ile Arg Arg Gln Gly
580 585 590
Gly Ile Gln Leu Leu Val Asp Leu Leu Asp His Arg Met Thr Glu Val
595 600 605
His Arg Ser Ala Cys Gly Ala Leu Arg Asn Leu Val Tyr Gly Lys Ala
610 615 620
Asn Asp Asp Asn Lys Ile Ala Leu Lys Asn Cys Gly Gly Ile Pro Ala
625 630 635 640
Leu Val Arg Leu Leu Arg Lys Thr Thr Asp Leu Glu Ile Arg Glu Leu
645 650 655
Val Thr Gly Val Leu Trp Asn Leu Ser Ser Cys Asp Ala Leu Lys Met
660 665 670
Pro Ile Ile Gln Asp Ala Leu Ala Val Leu Thr Asn Ala Val Ile Ile
675 680 685
Pro His Ser Gly Trp Glu Asn Ser Pro Leu Gln Asp Asp Arg Lys Ile
690 695 700
Gln Leu His Ser Ser Gln Val Leu Arg Asn Ala Thr Gly Cys Leu Arg
705 710 715 720
Asn Val Ser Ser Ala Gly Glu Glu Ala Arg Arg Arg Met Arg Glu Cys
725 730 735
Asp Gly Leu Thr Asp Ala Leu Leu Tyr Val Ile Gln Ser Ala Leu Gly
740 745 750
Ser Ser Glu Ile Asp Ser Lys Thr Val Glu Asn Cys Val Cys Ile Leu
755 760 765
Arg Asn Leu Ser Tyr Arg Leu Ala Ala Glu Thr Ser Gln Gly Gln His
770 775 780
Met Gly Thr Asp Glu Leu Asp Gly Leu Leu Cys Gly Glu Ala Asn Gly
785 790 795 800
Lys Asp Ala Glu Ser Ser Gly Cys Trp Gly Lys Lys Lys Lys Lys Lys
805 810 815
Lys Ser Gln Asp Gln Trp Asp Gly Val Gly Pro Leu Pro Asp Cys Ala
820 825 830
Glu Pro Pro Lys Gly Ile Gln Met Leu Trp His Pro Ser Ile Val Lys
835 840 845
Pro Tyr Leu Thr Leu Leu Ser Glu Cys Ser Asn Pro Asp Thr Leu Glu
850 855 860
Gly Ala Ala Gly Ala Leu Gln Asn Leu Ala Ala Gly Ser Trp Lys Trp
865 870 875 880
Ser Val Tyr Ile Arg Ala Ala Val Arg Lys Glu Lys Gly Leu Pro Ile
885 890 895
Leu Val Glu Leu Leu Arg Ile Asp Asn Asp Arg Val Val Cys Ala Val
900 905 910
Ala Thr Ala Leu Arg Asn Met Ala Leu Asp Val Arg Asn Lys Glu Leu
915 920 925
Ile Gly Lys Tyr Ala Met Arg Asp Leu Val His Arg Leu Pro Gly Gly
930 935 940
Asn Asn Ser Asn Asn Thr Ala Ser Lys Ala Met Ser Asp Asp Thr Val
945 950 955 960
Thr Ala Val Cys Cys Thr Leu His Glu Val Ile Thr Lys Asn Met Glu
965 970 975
Asn Ala Lys Ala Leu Arg Asp Ala Gly Gly Ile Glu Lys Leu Val Gly
980 985 990
Ile Ser Lys Ser Lys Gly Asp Lys His Ser Pro Lys Val Val Lys Ala
995 1000 1005
Ala Ser Gln Val Leu Asn Ser Met Trp Gln Tyr Arg Asp Leu Arg Ser
1010 1015 1020
SUBSTITUTE SNEET (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
9/15
Leu Tyr Lys Lys Asp Gly Trp Ser Gln Tyr His Phe Val Ala Ser Ser
1025 1030 1035 104
Ser Thr Ile Glu Arg Asp Arg Gln Arg Pro Tyr Ser Ser Ser Arg Thr
1045 1050 1055
Pro Ser Ile Ser Pro Val Arg Val Ser Pro Asn Asn Arg Ser Ala Ser
1060 1065 1070
Ala Pro Ala Ser Pro Arg Glu Met Ile Ser Leu Lys Glu Arg Lys Thr
1075 1080 1085
Asp Tyr Glu Cys Thr Gly Ser Asn Ala Thr Tyr His Gly Ala Lys Gly
1090 1095 1100
Glu His Thr Ser Arg Lys Asp Ala Met Thr Ala Gln Asn Thr Gly Ile
1105 1110 1115 112
Ser Thr Leu Tyr Arg Asn Ser Tyr Gly Ala Pro Ala Glu Asp Ile Lys
1125 1130 1135
His Asn Gln Val Ser Ala Gln Pro Val Pro Gln Glu Pro Ser Arg Lys
1140 1145 1150
Asp Tyr Glu Thr Tyr Gln Pro Phe Gln Asn Ser Thr Arg Asn Tyr Asp
1155 1160 1165
Glu Ser Phe Phe Glu Asp Gln Val His His Arg Pro Pro Ala Ser Glu
1170 1175 1180
Tyr Thr Met His Leu Gly Leu Lys Ser Thr Gly Asn Tyr Val Asp Phe
1185 1190 1195 120
Tyr Ser Ala Ala Arg Pro Tyr Ser Glu Leu Asn Tyr Glu Thr Ser His
1205 1210 1215
Tyr Pro Ala Ser Pro Asp Ser Trp Val
1220 1225
<210> 5
<211> 4998
<212> DNA
<213> mouse
<400> 5
aagcgccgga gccggccgcc gcggctgagc cggaggctga gctgcggcgc gcggcgggag
gagcctcgct ctcggcggcg gcggcggcgg cggcgacaca ggtggcgcgg gcggcgcgca
12
gggcgcagct cgagagcgct cggcgccggg cgccagggcg gcccaggctc gcgcccgcgg
180
cggcaaccgg ccgagcggag cggcgggcgc ggcggctcgg tagcccggcc cgagcccggg
240
gagccccgcg gaaccctgag catcccgcgg cgcccgccga gtcgggcagg gggcgctacg
300
ctcgccgcgc tcggaggggc ggccgggccg ggcgctgcgc actcgcgtcg ggagccgcct
360
ctcgcctgcc gcgctcgccc ctgctccccg ccagcatcac ttgtcccgcg gccgcgctcc
420
gacaacaaaa gcggaggatg ctgcagctgg gcaaggtcag gaccttgctc tgaagccggg
480
cggcggcgcg cacgcctttc ccccgactga ggagctgtct ttggcggcgg gtgcatgttc
540
gccaggaagc agtcgggcgc cgcgccgttc ggagctatgc ctgtcccaga ccagcctcca
600
tcagcctcag agaagaacag ctccttgagc ccaggcttaa acacctccaa tggtgatggc
660
SUBSTITUTE SHEET (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
10/15
tctgagacgg aaaccacctc tgctatcctt gcctccgtca aagaacagga attacagttt
720
gaaaggctga cccgagagct ggaggctgaa cgccagatcg tagccagcca gctggagcga
780
tgcaagcttg gctcggagac aggaagcatg agcagtatca gttcagcagg agagcagttt
840
cactggcaga cacaagatgg ccaaaaagat atcgaagatg aacttacaac gggccttgag
900
ctggtggact cctgtatccg ctctctgcag gagtcaggca ttctggaccc acaggattac
960
tccacaagtg aaaggcctag cctgctctcc cagagtgcac ttcagctcaa ttctaaacct
1020
gaagggtctt tccagtatcc ggccagctac catagcaacc agaccctggc cctgggtgac
1080
acagcccctt ctcagctccc agcacgcagc acgcaagccc gagctgccgg ccagagcttc
1140
agccagggca cgaccggccg cgcggggcac ctggcgggct ccgagcctgc gccaccgcct
1200
ccgcctccgc gggaaccgtt cgcgcccagc ctgggcagcg ccttccacct gcccgacgcg
1260
ccgcccgccg ccgcggcgct ctactactcc agctccacgc tgcccgcgcc gccgcgcggg
1320
ggctccccgc tgaccaccac gcagggcggc tcacccacca agctgcagcg cggaggctcg
1380
gcccccgagg gtgccgccta cgccgcgccg cgcggctcct cgcccaagca gtcgcccagc
1440
cgcctggcta agtcctacag caccagctcg cccatcaaca tcgtcgtgtc ctcggccggc
1500
ctgtccccga tccgcgtgac ctcgcccccc accgtgcagt ccaccatctc ctcttcgccc
1560
atccaccagc tgagctccac catcggcacc tacgccaccc tgtcgcccac caagcgcctg
1620
gtccacgcgt ctgagcagta cagcaagcat tcgcaggagc tgtatgccac cgccaccctc
1680
cagaggccgg gcagcctggc agctggatcc cgagcctcgt atagcagcca gcatgggcac
1740
ctggcccctg agctgcgggc cctgcagtcc ccagagcacc acatagaccc catctatgaa
1800
gaccgtgtct atcagaagcc ccctatgagg agtctcagcc agagccaggg ggatcctctg
1860
ccgccagcac ataccggcac cttccgcacg agcacagccc cgtcctcccc tggtgtcgac
1920
tccgtcccct tgcagcgcac aggcagccaa cacgggccac agaatgccgc cgcagccacc
1980
ttccagaggg ccagctatgc tgccggccca gcctccaact acgcagaccc ctaccgacag
2040
ctgcagtatt gtgcctccgt tgactctccg tacagcaaat ctggccctgc cctcccaccc
2100
gaaggcacct tggccagatc cccatccatc gacagcattc agaaagaccc cagggagttt
2160
ggatggagag acccggagct gcctgaagtg atacagatgt tacagcacca gttcccttca
2220
gtccagtcca atgctgcagc ttacctgcaa cacctctgtt ttggagacaa taaaattaag
2280
gcagagataa ggagacaagg agggatacag ctcctggtgg acctgctgga tcaccgaatg
2340
SUBSTITUTE SHEET (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CAQO/00126
11/15
acagaagtcc accgtagtgc ctgtggggct ctgaggaacc tggtgtatgg gaaggccaat
2400
gatgataaca aaatcgccct gaaaaactgt ggtggtatcc cagcgctggt gagactcctt
2460
cgcaagacca cagacctgga gatccgggag ctggtcacag gagtcctttg gaacctctca
2520
tcatgtgatg cactcaaaat gccaatcatc caggacgccc tggcagtgct gaccaatgcg
2580
gtgattatcc ctcactcggg ctgggagaat tcacctcttc aggatgatcg gaaaatacag
2640
ctgcattcat cacaggtgct gcgcaacgcc actgggtgcc taaggaatgt aagttcagct
2700
ggagaggagg cccgccgaag gatgcgggag tgtgatgggc tcacggatgc cttgctgtac
2760
gtgatccagt ctgcactggg gagcagtgag atcgatagca agaccgttga aaactgtgtg
2820
tgcatcttga ggaacctctc ctaccggcta gcagcagaaa cgtctcaggg acagcacatg
2880
ggcacagacg agctggacgg gctgctctgc ggggagacca acggcaaaga cacagagagt
2940
tctgggtgct ggggcaagaa gaagaagaaa aagaaatccc aggaccagtg ggatggagta
3000
ggacctcttc cagactgtgc agagccacca aaagggatcc agatgctgtg gcacccgtcc
3060
atagtcaaac cctacctcac actgctctct gagtgctcaa acccagacac gctggaaggg
3120
gcagcgggcg ccctgcagaa cttggctgca gggagctgga agggctgggc tgaggatgtg
3180
gcaggcatgg cgtatgccct acgttcactg ccagaggggg ctccctgcct gccacagtgg
3240
tccgtgtata tccgagctgc tgtccggaaa gagaaaggcc tgcccattct tgtggagctc
3300
ctccgaatag acaatgaccg tgtagtgtgt gcagtggcca cagcacttcg gaacatggcc
3360
ctcgatgtca gaaacaagga actcattggc aagtatgcca tgcgagacct ggtccaccgg
3420
cttcctggtg ggaacaacag caacaactcg gggagcaagg ccatgtcaga tgacaccgtg
3480
acggccgtgt gctgcaccct gcatgaagtg atcaccaaga acatggagaa tgccaaggcc
3540
ttacgggatg ctggtggcat cgagaagttg gtcggcatct ctaaaagcaa aggagacaag
3600
cactctccaa aggtggtcaa ggctgcttct caggtcctaa acagcatgtg gcagtatcgc
3660
gatctgagga gtctctacaa gaaggatgga tggtcacaat atcactttgt agcctcatct
3720
tcaaccatcg agagggatcg acaaaggccc tactcctcct cccgcacacc ctccatctct
3780
cccgtgcgtg tgtctcccaa caaccgctca gcaagtgccc cagcttcacc tcgggaaatg
3840
atcagcctca aagaaaggaa gacggactac gagtccgctg gcaacaacgc cacttaccac
3900
ggaactaaag gagaacacac ctccagaaaa gacaccatga cagctcaaaa cactggagtt
3960
tcaactttgt acaggaattc atacggtgcg cccgctgaag acatcaaaca gaaccaggtt
4020
SUBSTITUTE SHEET (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
12/15
tccacacagc ctgtccctca ggagcccagc aggaaagact acgagaccta ccagcccttt
4080
ccgaattcca cacgaaatta tgatgagtcc ttctttgagg accaggtcca ccaccgccct
4140
ccagccagcg agtacaccat gcacctgggc ctcaagtcca ctggcaacta tgtcgacttc
4200
tactctgcag cccgtcctta cagtgaactg aactatgaaa cgagccacta cccggcctcg
4260
cccgactcct gggtgtaagg agccaggaca cgaggcactc cggggacagt gcatgtgcat
4320
gcatacacca caggacattt tgtttctttt tttcttttct tttcttttgt tttttttttt
4380
ttttctttcc ctgcaaattt agtttgttaa agcctgttcc gtaggaaggc tgtgataacc
4490
aggaagaaat actcagagct attttagaaa gctaaaatga atcaagagtt aactgggaaa
4500
tcgataggaa gctaaacgca atcctaattg tgaccgcatt caacaccttt ctagtttgaa
4560
ctatagcatt ttgaaagtgc tttatagtcc ggtgaggctg aaggtaggag agaggagaca
4620
gtcagggtgg tgggcgtggt tatcgctaag cacaagacag actagtttac acactgtggg
4680
gacggcttct cacgctttgt ttactctctt catccgtgtg actctaggct tcaagttgca
4740
ttggggttcc tctgtacagc aagacgtctc ttgccttttg ttaatgcatt gttgtaaagt
4800
attcgatgta cattacagat taaagacgaa gagtgcattg tgtatattac accaatgcca
4860
ctgtgtttcc tcatcaatgg ttctaaatat tgcttcaatt tcaaactttt gaaagatgta
4920
tgggtttcca attttctttt tttttttctt tctcccagta tgttttaaca aaaaaggaaa
4980
aaaaaaacag gaaaaaaa
4998
<210> 6
<211> 1247
<212> PRT
<213> mouse
<400> 6
Met Phe Ala Arg Lys Gln Ser Gly Ala Ala Pro Phe Gly Ala Met Pro
1 5 10 15
Val Pro Asp Gln Pro Pro Ser Ala Ser Glu Lys Asn Ser Ser Leu Ser
20 25 30
Pro Gly Leu Asn Thr Ser Asn Gly Asp Gly Ser Glu Thr Glu Thr Thr
35 40 45
Ser Ala Ile Leu Ala Ser Val Lys Glu Gln Glu Leu Gln Phe Glu Arg
50 55 60
Leu Thr Arg Glu Leu Glu Ala Glu Arg Gln Ile Val Ala Ser Gln Leu
65 70 75 80
Glu Arg Cys Lys Leu Gly Ser Glu Thr Gly Ser Met Ser Ser Ile Ser
85 90 95
Ser Ala Gly Glu Gln Phe His Trp Gln Thr Gln Asp Gly Gln Lys Asp
100 105 110
SUBSTITUTE SHEET (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
13/15
Ile Glu Asp Glu Leu Thr Thr Gly Leu Glu Leu Val Asp Ser Cys Ile
115 120 125
Arg Ser Leu Gln Glu Ser Gly Ile Leu Asp Pro Gln Asp Tyr Ser Thr
130 135 140
Ser Glu Arg Pro Ser Leu Leu Ser Gln Ser Ala Leu Gln Leu Asn Ser
145 150 155 160
Lys Pro Glu Gly Ser Phe Gln Tyr Pro Ala Ser Tyr His Ser Asn Gln
165 170 175
Thr Leu Ala Leu Gly Asp Thr Ala Pro Ser Gln Leu Pro Ala Arg Ser
180 185 190
Thr Gln Ala Arg Ala Ala Gly Gln Ser Phe Ser Gln Gly Thr Thr Gly
195 200 205
Arg Ala Gly His Leu Ala Gly Ser Glu Pro Ala Pro Pro Pro Pro Pro
210 215 220
Pro Arg Glu Pro Phe Ala Pro Ser Leu Gly Ser Ala Phe His Leu Pro
225 230 235 240
Asp Ala Pro Pro Ala Ala Ala Ala Leu Tyr Tyr Ser Ser Ser Thr Leu
245 250 255
Pro Ala Pro Pro Arg Gly Gly Ser Pro Leu Thr Thr Thr Gln Gly Gly
260 265 270
Ser Pro Thr Lys Leu Gln Arg Gly Gly Ser Ala Pro Glu Gly Ala Ala
275 280 285
Tyr Ala Ala Pro Arg Gly Ser Ser Pro Lys Gln Ser Pro Ser Arg Leu
290 295 300
Ala Lys Ser Tyr Ser Thr Ser Ser Pro Ile Asn Ile Val Val Ser 5er
305 310 315 320
Ala Gly Leu Ser Pro Ile Arg Val Thr Ser Pro Pro Thr Val Gln Ser
325 330 335
Thr Ile Ser Ser Ser Pro Ile His Gln Leu Ser Ser Thr Ile Gly Thr
340 345 350
Tyr Ala Thr Leu Ser Pro Thr Lys Arg Leu Val His Ala Ser Glu Gln
355 360 365
Tyr Ser Lys His Ser Gln Glu Leu Tyr Ala Thr Ala Thr Leu Gln Arg
370 375 380
Pro Gly Ser Leu Ala Ala Gly Ser Arg Ala Ser Tyr Ser Ser Gln His
385 390 395 400
Gly His Leu Ala Pro Glu Leu Arg Ala Leu Gln Ser Pro Glu His His
405 410 415
Ile Asp Pro Ile Tyr Glu Asp Arg Val Tyr Gln Lys Pro Pro Met Arg
420 425 430
Ser Leu Ser Gln Ser Gln Gly Asp Pro Leu Pro Pro Ala His Thr Gly
435 440 445
Thr Phe Arg Thr Ser Thr Ala Pro Ser Ser Pro Gly Val Asp Ser Val
450 455 460
Pro Leu Gln Arg Thr Gly Ser Gln His Gly Pro Gln Asn Ala Ala Ala
465 470 475 480
Ala Thr Phe Gln Arg Ala 5er Tyr Ala Ala Gly Pro Ala Ser Asn Tyr
485 490 495
Ala Asp Pro Tyr Arg Gln Leu Gln Tyr Cys Ala Ser Val Asp Ser Pro
500 505 510
Tyr Ser Lys Ser Gly Pro Ala Leu Pro Pro Glu Gly Thr Leu Ala Arg
515 520 525
Ser Pro Ser Ile Asp Ser Ile Gln Lys Asp Pro Arg Glu Phe Gly Trp
530 535 540
Arg Asp Pro Glu Leu Pro Glu Val Ile Gln Met Leu Gln His Gln Phe
545 550 555 560
SUBSTITUTE SHiEET (RUL.E 28)
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
14/15
Pro Ser Val Gln Ser Asn Ala Ala Ala Tyr Leu Gln His Lea Cys Phe
565 570 575
Gly Asp Asn Lys Ile Lys Ala Glu Ile Arg Arg Gln Gly Gly Ile Gln
580 585 590
Leu Leu Val Asp Leu Leu Asp His Arg Met Thr Glu Val His Arg Ser
595 600 605
Ala Cys Gly Ala Leu Arg Asn Leu Val Tyr Gly Lys Ala Asn Asp Asp
610 615 620
Asn Lys Ile Ala Leu Lys Asn Cys Gly Gly Ile Pro Ala Leu Val Arg
625 630 635 640
Leu Leu Arg Lys Thr Thr Asp Leu Glu Ile Arg Glu Leu Val Thr Gly
645 650 655
Val Leu Trp Asn Leu Ser Ser Cys Asp Ala Leu Lys Met Pro Ile Ile
660 665 670
Gln Asp Ala Leu Ala Val Leu Thr Asn Ala Val Ile Ile Pro His Ser
675 680 685
Gly Trp Glu Asn Ser Pro Leu Gln Asp Asp Arg Lys Ile Gln Leu His
690 695 700
Ser Ser Gln Val Leu Arg Asn Ala Thr Gly Cys Leu Arg Asn Val Ser
705 710 715 720
Ser Ala Gly Glu Glu Ala Arg Arg Arg Met Arg Glu Cys Asp Gly Leu
725 730 735
Thr Asp Ala Leu Leu Tyr Val Ile Gln Ser Ala Leu Gly Ser Ser Glu
740 745 750
Ile Asp Ser Lys Thr Val Glu Asn Cys Val Cys Ile Leu Arg Asn Leu
755 760 765
Ser Tyr Arg Leu Ala Ala Glu Thr Ser Gln Gly Gln His Met Gly Thr
770 775 780
Asp Glu Leu Asp Gly Leu Leu Cys Gly Glu Thr Asn Gly Lys Asp Thr
785 790 795 800
Glu Ser Ser Gly Cys Trp Gly Lys Lys Lys Lys Lys Lys Lys Ser Gln
805 810 815
Asp Gln Trp Asp Gly Val Gly Pro Leu Pro Asp Cys Ala Glu Pro Pro
820 825 830
Lys Gly Ile Gln Met Leu Trp His Pro Ser Ile Val Lys Pro Tyr Leu
835 840 845
Thr Leu Leu Ser Glu Cys Ser Asn Pro Asp Thr Leu Glu Gly Ala Ala
850 855 860
Gly Ala Leu Gln Asn Leu Ala Ala Gly Ser Trp Lys Gly Trp Ala Glu
865 870 875 880
Asp Val Ala Gly Met Ala Tyr Ala Leu Arg Ser Leu Pro Glu Gly Ala
885 890 895
Pro Cys Leu Pro Gln Trp Ser Val Tyr Ile Arg Ala Ala Val Arg Lys
900 905 910
Glu Lys Gly Leu Pro Ile Leu Val Glu Leu Leu Arg Ile Asp Asn Asp
915 920 925
Arg Val Val Cys Ala Val Ala Thr Ala Leu Arg Asn Met Ala Leu Asp
930 935 940
Val Arg Asn Lys Glu Leu Ile Gly Lys Tyr Ala Met Arg Asp Leu Val
945 950 955 960
His Arg Leu Pro Gly Gly Asn Asn Ser Asn Asn Ser Gly Ser Lys Ala
965 970 975
Met Ser Asp Asp Thr Val Thr Ala Val Cys Cys Thr Leu His Glu Val
980 985 990
Ile Thr Lys Asn Met Glu Asn Ala Lys Ala Leu Arg Asp Ala Gly Gly
995 1000 1005
SUBSTITUTE Sh~EET (RULE 26)
CA 02361034 2001-08-02
WO 00/47615 PCT/CA00/00126
15/15
Ile Glu Lys Leu Val Gly Ile Ser Lys Ser Lys Gly Asp Lys His Ser
1010 1015 1020
Pro Lys Val Val Lys Ala Ala Ser Gln Val Leu Asn Ser Met Trp Gln
1025 1030 1035 104
Tyr Arg Asp Leu Arg Ser Leu Tyr Lys Lys Asp Gly Trp Ser Gln Tyr
1045 1050 1055
His Phe Val Ala Ser Ser Ser Thr Ile Glu Arg Asp Arg Gln Arg Pro
1060 1065 1070
Tyr Ser Ser Ser Arg Thr Pro Ser Ile Ser Pro Val Arg Val Ser Pro
1075 1080 1085
Asn Asn Arg Ser Ala Ser Ala Pro Ala Ser Pro Arg Glu Met Ile Ser
1090 1095 1100
Leu Lys Glu Arg Lys Thr Asp Tyr Glu Ser Ala Gly Asn Asn Ala Thr
1105 1110 1115 112
Tyr His Gly Thr Lys Gly Glu His Thr Ser Arg Lys Asp Thr Met Thr
1125 1130 1135
Ala Gln Asn Thr Gly Val Ser Thr Leu Tyr Arg Asn Ser Tyr Gly Ala
1140 1145 1150
Pro Ala Glu Asp Ile Lys Gln Asn Gln Val Ser Thr Gln Pro Val Pro
1155 1160 1165
Gln Glu Pro Ser Arg Lys Asp Tyr Glu Thr Tyr Gln Pro Phe Pro Asn
1170 1175 1180
Ser Thr Arg Asn Tyr Asp Glu Ser Phe Phe Glu Asp Gln Val His His
1185 1190 1195 120
Arg Pro Pro Ala Ser Glu Tyr Thr Met His Leu Gly Leu Lys Ser Thr
1205 1210 1215
Gly Asn Tyr Val Asp Phe Tyr Ser Ala Ala Arg Pro Tyr Ser Glu Leu
1220 1225 1230
Asn Tyr Glu Thr Ser His Tyr Pro Ala Ser Pro Asp Ser Trp Val
1235 1240 1245
SUBSTITUTE SHEET (RULE 26)
