CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
1
POLYNUCLEOTIDES AND POLYPEPTIDE SEQUENCES INVOLVED IN THE
PROCESS OF BONE REMODELING
FIELD OF THE INVENTION
This invention relates, in part. to unique and newly identified genetic
polynucleotides involved in the process of bone remodeling; variants and
derivatives of
the polynucleotides and corresponding polypeptides, uses of the
polynucleotides
polypeptides, variants and derivatives, methods and compositions for the
amelioration
of symptoms caused by bone remodeling disorders. including but not limited to
osteoporosis, osteopenia, osteomalacia. hyperparathyroidism, hypothyroidism,
hyperthyroidism, hypogonadism, thyrotoxicosis, systemic mastocytosis, adult
hypophosphatasia, hyperadrenocorticism, osteogenesis imperfecta, Paget's
disease,
Cushing's disease/syndrome, Turner syndrome, Gaucher disease, Ehlers-Danlos
syndrome, Marfan's syndrome, Menkes' syndrome, Fanconi's syndrome, multiple
myeloma, hypercalcemia, hypocalcemia, arthritides, periodontal disease,
rickets
(including vitamin D dependent, type I and II, and x-linked hypophosphatemic
rickets),
fibrogenesis imperfecta ossium, osteosclerotic disorders such as
pycnodysostosis and
damage caused by macrophage-mediated inflammatory processes.
In particular, this invention relates to polynucleotide expression profiles of
active
osteoclasts. the isolation and identification of polynucleotides.
polypeptides, variants
and derivatives involved in osteoclast activity, validation of the identified
polynucleotides for their potential as therapeutic targets and use of the
polynucleotides,
polypeptides. variants and derivatives for the amelioration of disease states
and
research purposes. as well as in diagnosis of disease states or in the
predisposition to
.. develop same,
BACKGROUND OF THE INVENTION
Bone is a dynamic connective tissue comprised of functionally distinct cell
populations required to support the structural, mechanical and biochemical
integrity of
.. bone and the human body's mineral homeostasis. The principal cell types
involved
include, osteoblasts responsible for bone formation and maintaining bone mass,
and
osteoclasts responsible for bone resorption. Osteoblasts and osteoclasts
function in a
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
2
dynamic process termed bone remodeling. The development and proliferation of
these
cells from their progenitors is governed by networks of growth factors and
cytokines
produced in the bone microenvironment as well as by systemic hormones. Bone
remodeling is ongoing throughout the lifetime of the individual and is
necessary for the
maintenance of healthy bone tissue and mineral homeostasis. The process
remains
largely in equilibrium and is governed by a complex interplay of systemic
hormones,
peptides and downstream signalling pathway proteins, local transcription
factors.
cytokines, growth factors and matrix remodeling genes.
Any interference or imbalance arising in the bone remodeling process can
produce skeletal disease, with the most common skeletal disorders
characterized by a
net decrease in bone mass. A primary cause of this reduction in bone mass is
an
increase in osteoclast number and/or activity, The most common of such
disease, and
perhaps the best known, is osteoporosis occurring particularly in women after
the onset
of menopause. In fact osteoporosis is the most significant underlying cause of
skeletal
fractures in late middle-aged and elderly women. While estrogen deficiency has
been
strongly implicated as a factor in postmenopausal osteoporosis. there is
longstanding
evidence that remodeling is a locally controlled process being that it takes
place in
discrete packets throughout the skeleton as first described by Frost over
forty years
ago (Frost H.M. 1964).
Since bone remodeling takes place in discrete packets, locally produced
hormones and enzymes may be more important than systemic hormones for the
initiation of bone resorption and the normal remodeling process. Such local
control is
mediated by osteoblasts and osteoclasts in the microenvironment in which they
operate. For example, osteoclasts attach to the bone matrix and form a
separate
compartment between themselves and the bone surface delimited by a sealing
zone
formed by a ring of actin surrounding the ruffled border, Multiple small
vesicles
transport enzymes toward the bone matrix and internalize partially digested
bone
matrix_ The microenvironment within the sealing zone is rich with the presence
of
lysosomal enzymes and is highly acidic compared to the normal physiological pH
of the
body, The ruffled border membrane also expresses RANK, the receptor for RANKL,
and macrophage-colony stimulating factor (M-CSF) receptor, both of which are
responsible for osteoclast differentiation, as well as the calcitonin receptor
capable of
rapidly inactivating the osteoclast (Baron. R. 2003).
CA 02785046 2013-02-08
3
In a complex pattern of inhibition and stimulation not yet fully understood,
growth hormone, insulin-like growth factor-1, the sex steroids, thyroid
hormone,
calciotrophic hormones such as PTH and prostaglandin E2, various cytokines,
such
as interleukin-1 beta, interleukin-6, and tumour necrosis factor-alpha, and
1,25-
dihydroxyvitamin D (calcitriol) act co-ordinately in the bone remodeling
process (Jilka
et al. 1992; Poli et al. 1994; Srivastava et al. 1998; de Vemejoul 1996).
Thus, it stands to reason that the unique local environments created by these
specialized cells is due to the expression of either unique genetic sequences
not
expressed in other tissues and/or splice variants of polynucleotides and
polypeptides
expressed in other tissues. The isolation and identification of
polynucleotides,
polypeptides and their variants and derivatives specific to osteoclast
activity will
permit a clearer understanding of the remodeling process and offer tissue
specific
therapeutic targets for the treatment of disease states related to bone
remodeling.
Many diseases linked to bone remodeling are poorly understood, generally
untreatable or treatable only to a limited extent. For example, osteoarthritis
is difficult
to treat as there is no cure and treatment focuses on relieving pain and
preventing
the affected joint from becoming deformed. Non-steroidal anti-inflammatory
drugs
(NSAIDs) are generally used to relieve pain.
Another example is osteoporosis where the only current medications
approved by the FDA for use in the United States are the anti-resorptive
agents that
prevent bone breakdown. Estrogen replacement therapy is one example of an anti-
resorptive agent. Others include alendronate (FosamaxTM- a biphosphonate anti-
resorptive), risedronate (ActonelTM - a bisphosphonate anti-resorptive),
raloxifene
(EvistaTM- selective estrogen receptor modulator (SERM)), calcitonin
(CalcimarTM- a
hormone), and parathyroid hormone/teriparatide (ForteoTM- a synthetic version
of the
human hormone, parathyroid hormone, which helps to regulate calcium
metabolism).
Bisphosphonates such as alendronate and risedronate bind permanently to
the surface of bone and interfere with osteoclast activity. This allows the
osteoblasts
to outpace the rate of resorption. The most common side effects are nausea,
abdominal pain and loose bowel movements. However, alendronate is reported to
also cause irritation and inflammation of the esophagus, and in some cases,
ulcers of
the esophagus. Risedronate is chemically different from alendronate and has
less
likelihood of causing esophagus irritation. However, certain foods, calcium,
iron
CA 02785046 2013-02-08
4
supplements, vitamins and minerals, or antacids containing calcium, magnesium,
or
aluminum can reduce the absorption of risedronate, thereby resulting in loss
of
effectiveness.
The most common side effect of Raloxifen and other SERMS (such as
Tamoxifen) are hot flashes. However, Raloxifene and other hormone replacement
therapies have been shown to increase the risk of blood clots, including deep
vein
thrombosis and pulmonary embolism, cardiovascular disease and cancer.
Calcitonin is not as effective in increasing bone density and strengthening
bone as estrogen and the other anti-resorptive agents. Common side effects of
either
injected or nasal spray calcitonin are nausea and flushing. Patients can
develop
nasal irritations, a runny nose, or nosebleeds. Injectable calcitonin can
cause local
skin redness at the site of injection, skin rash, and flushing.
A situation demonstrative of the link between several disorders or disease
states involving bone remodeling is that of the use of etidronate (DidronelTM)
first
approved by the FDA to treat Paget's disease. Paget's disease is a bone
disease
characterized by a disorderly and accelerated remodeling of the bone, leading
to
bone weakness and pain. DidronelTM has been used 'off-label' and in some
studies
shown to increase bone density in postmenopausal women with established
osteoporosis. It has also been found effective in preventing bone loss in
patients
requiring long-term steroid medications (such as Prednisone or Cortisone).
However,
high dose or continuous use of DidronelTM can cause another bone disease
called
osteomalacia. Like osteoporosis, osteomalacia can lead to weak bones with
increased risk of fractures. Because of osteomalacia concerns and lack of
enough
studies yet regarding reduction in the rate of bone fractures, the United
States FDA
has not approved DidronelTM for the treatment of osteoporosis.
Osteoporosis therapy has been largely focused on antiresorptive drugs that
reduce the rate of bone loss but emerging therapies show promise in increasing
bone
mineral density instead of merely maintaining it or slowing its deterioration.
The
osteoporosis early stage pipeline consists largely of drug candidates in new
therapeutic
classes, in particular cathepsin K inhibitors, osteoprotegerin and calcilytics
as well as
novel bisphosphonates. Some of these are examples where novel drugs exploiting
genomics programs are being developed based on a deeper understanding of bone
biology and have the potential to change the face of treatment of bone
disorders in the
CA 02785046 2013-02-08
long term.
There thus remains a need to better understand the bone remodeling process
and to provide new compositions that are useful for the diagnosis, prognosis,
treatment, prevention and evaluation of therapies for bone remodeling and
5 associated disorders. A method for analysing polynucleotide expression
patterns has
been developed and applied to identify polynucleotides, polypeptides, variants
and
derivatives specifically involved in bone remodeling.
The present invention seeks to meet these and other needs.
SUMMARY OF THE INVENTION
The present invention relates to polynucleotides comprising sequences
involved in the process of bone remodeling, the open reading frame of such
sequences, substantially identical sequences (e.g., variants (e.g., allelic
variant), non
human orthologs), substantially complementary sequences and fragments of any
one
of the above thereof.
The present invention relates to polypeptide comprising sequences involved
in the process of bone remodeling including biologically active analogs and
biologically active fragments thereof. The present invention also relates to
compositions that are useful for the diagnosis, prognosis, treatment,
prevention
and/or evaluation of therapies for bone remodeling and associated disorders.
In addition, the present invention relates to a method for analyzing
polynucleotide expression patterns, and applied in the identification of
polynucleotides, polypeptides, variants and derivatives specifically involved
in bone
remodeling.
The present invention relates to polynucleotide expression profiles of
osteoclasts, the isolation and identification of polynucleotides, their
corresponding
polypeptides, variants and derivatives involved in osteoclast activity,
validation of
these identified elements for their potential as therapeutic targets and use
of said
polynucleotides, polypeptides, variants and derivatives for the amelioration
of
disease states.
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
6
It is an object of the present invention to provide polynucleotides and/or
related
polypeptides that have been isolated and identified. More specifically, the
invention
provides (isolated or substantially purified) polynucleotides comprising or
consisting of
any one of SEQ. ID. NOs:1 to 33, SEQ ID NO.:85 or SEQ ID NO.:86 their coding
sequence (open reading frame) substantially identical sequence (e.g.,
variants,
orthologs (e.g., SEQ ID NO.:35)), substantially complementary sequences and
related
polypeptides comprising any one of SEQ ID NO.: 48-80 and polypeptides encoded
by
SEQ ID NO .85 or SEQ ID NO :86 which have been shown to be upregulated in a
highly specific fashion in osteoclasts. The present invention also relates to
polypeptide
analogs, variants (e.g., SEQ ID NO .81) and fragments thereof
NSEQ refers generally to polynucleotide sequences of the present invention
and includes for example, SEQ. ID. NOs:1 to 33, SEQ ID NO.:85 or SEQ ID NO.:86
whereas PSEQ refers generally to polypeptide sequences of the present
invention and
includes, for example. SEQ ID NO. :48 to 82 and polypeptides encoded by SEQ ID
NO.:85 or SEQ ID NO.:86. Of course it will be understood that NSEQ also
encompasses polynucleotide sequences which are designed or derived from SEQ.
ID.
NOs:1 to 33 SEQ ID NO.:85 or SEQ ID NO.:86 for example, their coding sequence,
complementary sequences. Non-limiting examples of such sequences are disclosed
herein (e.g. SEQ ID Nos 42-45).
As used herein the term -NSEQ" refers generally to polynucleotides sequences
comprising or consisting of any one of SEQ. ID. NOs:1 to 33, 85 or 86 (e.g..
an isolated
form) or comprising or consisting of a fragment of any one of SEQ ID NOs 1 to
33. 85
or 86. The term "NSEQ" more particularly refers to a polynucleotide sequence
comprising or consisting of a transcribed portion of any one of SEQ. ID. NOs.1
to 33.
85 or 86, which may be, for example, free of untranslated or untranslatable
portion(s)
(i.e., a coding portion of any one of SEQ ID Nos.: 1 to 33, 85 or 86). The
term "NSEQ"
additionally refers to a sequence substantially identical to any one of the
above and
more particularly substantially identical to polynucleotide sequence
comprising or
consisting of a transcribed portion of any one of SEQ. ID. Nos1 to 33, 85 or
86, which
may be, for example, free of untranslated or untranslatable portion(s). The
term
"NSEQ" additionally refers to a polynucleotide sequence region of any one of
SEQ. ID.
NOs:1 to 33, 85 or 86 which encodes or is able to encode a polypeptide. The
term
"NSEQ" also refers to a polynucleotide sequence able of encoding any one of
the
polypeptides described herein or a polypeptide fragment of any one of the
above
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
7
Finally, the term "NSEQ" also comprise a sequence substantially complementary
to
any one of the above.
The term "inhibitory NSEQ" generally refers to a sequence substantially
complementary to any one of SEQ. ID. Nos: 1 to 33, 85 or 86, substantially
complementary to a fragment of any one of SEQ. ID. Nos: 1 to 33, 85 or 86,
substantially complementary to a sequence substantially identical to SEQ. ID.
NOs:1 to
33, 85 or 86 and more particularly, substantially complementary to a
transcribed portion
of any one of SEQ. ID. NOs.1 to 33. 85 or 86 (e.g., which may be free of
unstranslated
or untranslatable portion) and which may have attenuating or even inhibitory
action
againts the transcription of a mRNA or against expression of a polypeptide
encoded by
a corresponding SEQ ID NOs..1 to 33, 85 or 86. Suitable "inhibitory NSEQ" may
have
for example and without limitation from about 10 to about 30 nucleotides. from
about 10
to about 25 nucleotides or from about 15 to about 20 nucleotides. As used
herein the
term "nucleotide" means deoxyribonucleotide or ribonucleotide. In an exemplary
embodiment, the use of nucleotide analogues is also encompassed in the present
invention.
The present invention relates in one aspect thereof to an isolated
polynucleotide
sequence having at least from about 80% to about 100% (e.g., 80%, 90%, 95%,
etc.)
sequence identity to a polynucleotide sequence selected from the group
consisting of
polynucleotides comprising (a) any one of a SEQ. ID. NOs.1 to 33 or SEQ ID NO
85 or
SEQ ID NO. :86, (b) an open reading frame of (a): (c) a full complement of (a)
or (b),
and ,(d) a fragment of any one of (a) to (c).
As used herein the term "unstranscribable region" may include for example. a
promoter region (or portion thereof), silencer region, enhancer region etc of
a
polynucleotide sequence.
As used herein the term "unstranslatable region" may include for example, an
initiator portion of a polynucleotide sequence (upstream of an initiator
codon, e.g.,
AUG), intronic regions, stop codon and/or region downstream of a stop codon
(including polyA tail, etc.).
Complements of the isolated polynucleotide sequence encompassed by the
present invention may be those, for example, which hybridize under high
stringency
conditions to any of the nucleotide sequences in (a), or (b). The high
stringency
conditions may comprise, for example, a hybridization reaction at 65 C in 5X
SSC, 5X
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
8
Denhardt's solution. 1% SDS, and 100 pg/ml denatured salmon sperm DNA
In accordance with the present invention, the polynucleotide sequence may be
used, for example, in the treatment of diseases or disorders involving bone
remodeling.
Fragments of polynucleotides may be used, for example, as probes for
determining the presence of the isolated polynucleotide (or its complement or
fragments thereof) in a sample, cell, tissue, etc. for experimental purposes
or for the
purpose of diagnostic of a diseases or disorders involving bone remodeling.
The present invention also relates to a combination comprising a plurality of
polynucleotides (substantially purified and/or isolated). The polynucleotides
may be
co-expressed with one or more genes known to be involved in bone remodeling.
Furthermore, the plurality of polynucleotides may be selected, for example,
from the
group consisting of a polynucleotide comprising (a) any one of SEQ. ID. NOs:1
to 33.
SEQ ID NO.:85 or SEQ ID NO.:86: (b) an open reading frame (a) (c) a
polynucleotide
sequence comprising or consisting of a transcribed portion of any one of SEQ.
ID
NOs:1 to 33, 85 or 86, which may be. for example, free of untranslated or
untranslatable portion(s) (d) a complementary sequence of any one of (a) to
(c); (e) a
sequence that hybridizes under high stringency conditions to any one of the
nucleotide
sequences of (a) to (d) and; (f) fragments of any one of (a) to (e).
The present invention further relates to a polynucleotide encoding any one of
the polypeptides described herein. In accordance with the present invention,
the
polynucleotide (RNA, DNA, etc.) may encode a polypeptide which may be selected
from the group consisting of any one of SEQ ID NO. :48 to 80. polypeptides
encoded by
SEQ ID NO.:85 or 86, analogs or fragments thereof (e.g., biologically active
fragments,
immunologically active fragments, etc.),
The present invention also relates to an isolated nucleic acid molecule
comprising the polynucleotides of the present invention, operatively linked to
a
nucleotide sequence encoding a heterologous polypeptide thereby encoding a
fusion
polypeptide.
The invention further relates to a polypeptide encoded by a polynucleotide of
SEQ. ID. NOs:1 to 33, SEQ ID NO.:85 or SEQ ID NO.:86 or more particularly from
the
open reading frame of any one of SEQ. ID. NOs.1 to 33, SEQ ID NO.:85 or SEQ ID
NO. :86, or a portion thereof. The invention also comprise the product of a
gene that is
co-expressed with one or more genes known to be involved in bone remodeling.
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
9
Isolated naturally occurring allelic variant are also encompassed by the
present
invention as well as synthetic variants (e.g., made by recombinant DNA
technology or
by chemical synthesis, etc.) such as biologically active variant which may
comprise one
or more amino acid substitutions (compared to a naturally occurring
polypeptide), such
as conservative or non conservative amino acid substitution.
The present invention, further provides a vector (mammalian, bacterial, viral,
etc.) comprising the polynucleotides described herein or fragments thereof,
such as an
expression vector. The vector may further comprise a nucleic acid sequence
which
may help in the regulation of expression of the polynucleotide and/or a
nucleotide
sequence encoding a tag (e.g., affinity tag; HA, GST, His etc.).
In accordance with the present invention, an expression vector may comprise.
for example. the following operatively linked elements:
a) a transcription promoter:
b) a polynucleotide segment (which may comprise an open reading
frame of any one of SEQ ID NOs :1-33. 85 or 86); and
c) a transcription terminator.
The invention also relates to an expression vector comprising a polynucleotide
described herein, a host cell transformed with the expression vector and a
method for
producing a polypeptide of the present invention.
The invention further relates to a vector comprising a polynucleotide or
polynucleotide fragment. Vectors which may comprise a sequence substantially
complementary to the polynucleotides of the present invention (e.g., siRNA,
shRNA)
are thus encompassed by the present invention. The vector may comprise
sequences
enabling transcription of the polynucleotide or polynucleotide fragment.
More particularly, the present invention therefore provides a cell which may
be
genetically engineered to contain and/or to express the polynucleotide
(including
complements and fragments) and/or polypeptides of the present invention The
cell
may be. for example. a mammalian cell, an insect cell, a bacteria cell. etc.
The present invention, therefore provides a host cell which may comprise a
vector as described herein. The cell may be, for example, a mammalian cell, an
insect
cell, a bacteria, etc. The cell may be able to express or expresses a
polypeptide
encoded by the polynucleotide described herein.
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
Methods of producing the polypeptides of the present invention encompassed
herewith includes for example, culturing the cell in conditions allowing the
transcription
of a gene or expression of the polypeptide. The polypeptide may be recovered,
for
example, from cell lysate or from the cell supernatant.
5 The invention
relates to the use of at least one polynucleotide comprising any
one of SEQ. ID. NOs:1 to 33, SEQ ID NO.:85 or SEQ ID NO :86 their coding
sequence.
substantially identical sequences, substantially complementary sequences or
fragments thereof on an array. The array may be used in a method for
diagnosing a
bone remodeling disease or disorder by hybridizing the array with a patient
sample
10 under conditions
to allow complex formation, detecting complex formation, and
comparing the amount of complex formation in the patient sample to that of
standards
for normal and diseased tissues wherein the complex formation in the patient
sample
indicates the presence of a bone remodeling disease or disorder. Of course,
the use of
a polynucleotide of the present invention in a diagnosis method is not
dependent
exclusively by way of a specific assay. The sequence or sequences may be used
in
conventionally used diagnosis methods known in the art.
The present invention also relates to a method of ameliorating bone remodeling
disease or disorder symptoms. or for inhibiting or delaying bone disease or
disorder.
the method may comprise: contacting a compound capable of specifically
inhibiting
activity or expression of a polynucleotide sequence described herein or a
polypeptide
described herein, in osteoclasts so that symptoms of the bone remodeling
disease or
disorder may be ameliorated, or the disease or disorder may be prevented,
delayed or
lowered.
The present invention further relates to a method for ameliorating bone
remodeling disease or disorder symptoms, or for inhibiting or delaying bone
disease or
disorder, the method may comprise: contacting a compound capable of
specifically
promoting activity or expression of a polynucleotide sequence described herein
or a
polypeptide described herein, in osteoclasts so that symptoms of the bone
remodeling
disease or disorder may be ameliorated, or the disease or disorder may be
prevented.
delayed or lowered.
The present invention also relates to a method of treating a condition in a
mammal characterized by a deficiency in, or need for, bone growth or
replacement
and/or an undesirable level of bone resorption, which method may comprise
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/0002 10
11
administering to a mammalian subject in need of such treatment an effective
amount of
a suitable compound described herein.
The present invention further relates to a method of using a polynucleotide
sequence described herein, a polypeptide described herein on an array and for
the use
of the array in a method for diagnosing a bone remodeling disease or disorder
by
hybridizing the array with a patient sample under conditions to allow complex
formation, detecting complex formation, and comparing the amount of complex
formation in the patient sample to that of standards for normal and diseased
tissues
wherein the complex formation in the patient sample may indicate the presence
of a
bone remodeling disease or disorder.
In accordance with the present invention, the polynucleotide sequence
described herein may be used for somatic cell gene therapy or for stem cell
gene
therapy.
The invention also relates to a pharmaceutical composition comprising a
polynucleotide described herein or a polypeptide encoded by the selected
polynucleotide or portion thereof and a suitable pharmaceutical carrier.
Additionally, the invention relates to products, compositions, processes and
methods that comprises a polynucleotide described herein, a polypeptide
encoded by
the polynucleotides a portion thereof. their variants or derivatives, for
research.
biological, clinical and therapeutic purposes.
The NSEQs and PSEQs may be used in diagnosis, prognosis. treatment.
prevention, and selection and evaluation of therapies for diseases and
disorders
involving bone remodeling including, but not limited to, osteoporosis,
osteopenia,
osteomalacia, hyperparathyroidism, hyperthyroidism, hyperthyroidism,
hypogonadism,
thyrotoxicosis, systemic mastocytosis, adult hypophosphatasia,
hyperadrenocorticism,
osteogenesis imperfecta, Paget's disease, Cushing's disease/syndrome, Tumer
syndrome, Gaucher disease, Ehlers-Danlos syndrome, Marfan's syndrome, Menkes'
syndrome, Fanconi's syndrome, multiple myeloma, hypercalcemia, hypocalcemia,
arthritides, periodontal disease. rickets (including vitamin D dependent, type
I and It.
and x-linked hypophosphatemic rickets), fibrogenesis imperfecta ossium,
osteosclerotic
disorders such as pycnodysostosis and damage caused by macrophage-mediated
inflammatory processes.
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
12
Use of NSEQ as a Screening Tool
The polynucleotides obtained by the present invention may be used to detect
and isolate expression products, for example, mRNA, complementary DNAs (cDNAs)
and proteins derived from or homologous to the NSEQs. In one embodiment the
expression of mRNAs homologous to the NSEQs of the present invention may be
detected. for example, by hybridization analysis. reverse transcription and in
vitro
nucleic acid amplification methods. Such procedures permit detection of mRNAs
in a
variety of tissue types or at different stages of development. The subject
nucleic acids
which are expressed in a tissue-specific or a developmental-stage-specific
manner are
useful as tissue-specific markers or for defining the developmental stage of a
sample of
cells or tissues that may define a particular disease state. One of skill in
the art may
readily adapt the NSEQs for these purposes.
Those skilled in the art will also recognize that the NSEQs, and its
expression
products such as cDNA nucleic acids and genomic DNA may be used to prepare
short
oligonucleotides sequences. For example, oligonucleotides having ten to twelve
nucleotides or more may be prepared which hybridize specifically to the
present
NSEQs and cDNAs and allow detection. identification and isolation of unique
nucleic
sequences by hybridization. Sequences of for example, at least 15-20
nucleotides may
be used and selected from regions that lack homology to other known sequences.
Sequences of 20 or more nucleotides that lack such homology show an increased
specificity toward the target sequence. Useful hybridization conditions for
probes and
primers are readily determinable by those of skill in the art. Stringent
hybridization
conditions encompassed herewith are those that may allow hybridization of
nucleic
acids that are greater than 90% homologous but which may prevent hybridization
of
nucleic acids that are less than 70% homologous. The specificity of a probe
may be
determined by whether it is made from a unique region, a regulatory region, or
from a
conserved motif. Both probe specificity and the stringency of diagnostic
hybridization or
amplification (maximal, high, intermediate, or low) reactions may be
determined
whether the probe identifies exactly complementary sequences, allelic
variants, or
related sequences. Probes designed to detect related sequences may have at
least
50% sequence identity to any of the selected polynucleotides.
It is to be understood herein that the NSEQs (substantially identical
sequences
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
13
and fragments thereof) may hybridize to a substantially complementary sequence
found in a test sample. Additionally, a sequence substantially complementary
to NSEQ
may bind a NSEQ found in a test sample.
Furthermore, a probe may be labelled by any procedure known in the art, for
example by incorporation of nucleotides linked to a "reporter molecule". A
"reporter
molecule", as used herein, may be a molecule that provides an analytically
identifiable
signal allowing detection of a hybridized probe. Detection may be either
qualitative or
quantitative. Commonly used reporter molecules include fluorophores, enzymes,
biotin,
chemiluminescent molecules, bioluminescent molecules, digoxigenin. avidin,
streptavidin or radioisotopes. Commonly used enzymes include horseradish
peroxidase. alkaline phosphatase. glucose oxidase and p-galactosidase. among
others. Enzymes may be conjugated to avidin or streptavidin for use with a
biotinylated
probe. Similarly, probes may be conjugated to avidin or streptavidin for use
with a
biotinylated enzyme. Incorporation of a reporter molecule into a DNA probe may
be by
any method known to the skilled artisan, for example by nick translation,
primer
extension, random oligo priming, by 3' or 5 end labeling or by other means. In
addition,
hybridization probes include the cloning of nucleic acid sequences into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro. The labelled
polynucleotide sequences may be used in Southern or northern analysis. dot
blot, or
other membrane-based technologies; in FOR technologies; and in micro arrays
utilizing
samples from subjects to detect altered expression. Oligonucleofides useful as
probes
for screening of samples by hybridization assays or as primers for
amplification may be
packaged into kits. Such kits may contain the probes or primers in a pre-
measured or
predetermined amount, as well as other suitably packaged reagents and
materials
needed for the particular hybridization or amplification protocol.ln another
embodiment.
the invention entails a substantially purified polypeptide encoded by the
polynucleotides of NSEQs, polypeptide analogs or polypeptide fragments
thereof. The
polypeptides whether in a premature, mature or fused form. may be isolated
from lysed
cells, or from the culture medium, and purified to the extent needed for the
intended
use. One of skill in the art may readily purify these proteins, polypeptides
and peptides
by any available procedure. For example, purification may be accomplished by
salt
fractionation, size exclusion chromatography, ion exchange chromatography,
reverse
phase chromatography, affinity chromatography and the like.
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
14
Use of NSEQ for Development of an Expression System
In order to express a biologically active polypeptide, NSEQ. or derivatives
thereof, may be inserted into an expression vector, i.e., a vector that
contains the
elements for transcriptional and translational control of the inserted coding
sequence in
a particular host. These elements may include regulatory sequences, such as
enhancers. constitutive and inducible promoters, and 5 and 3' un-translated
regions_
Methods that are well known to those skilled in the art may be used to
construct such
expression vectors. These methods include in vitro recombinant DNA techniques.
synthetic techniques, and in vivo genetic recombination_
A variety of expression vector/host cell systems known to those of skill in
the art
may be utilized to express NSEQ These include, but are not limited to.
microorganisms such as bacteria transformed with recombinant bacteriophage,
plasmid, or cosmid DNA expression vectors; yeast transformed with yeast
expression
vectors; insect cell systems infected with baculovirus vectors; plant cell
systems
transformed with viral or bacterial expression vectors; or animal cell
systems. For long-
term production of recombinant proteins in mammalian systems, stable
expression in
cell lines may be effected. For example, NSEQ may be transformed into cell
lines using
expression vectors that may contain viral origins of replication and/or
endogenous
expression elements and a selectable or visible marker gene on the same or on
a
separate vector. The invention is not to be limited by the vector or host cell
employed.
In general, host cells that contain NSEQ and that express a polypeptide
encoded by the NSEQ, or a portion thereof, may be identified by a variety of
procedures known to those of skill in the art. These procedures include, but
are not
limited to. DNA-DNA or DNA-RNA hybridizations. PCR amplification, and protein
bioassay or immunoassay techniques that include membrane, solution, or chip
based
technologies for the detection and/or quantification of nucleic acid or amino
acid
sequences. Immunological methods for detecting and measuring the expression of
polypeptides using either specific polyclonal or monoclonal antibodies are
known in the
art. Examples of such techniques include enzyme-linked immunosorbent assays
(ELISAs). radioimmunoassays (RIAs), and fluorescence activated cell sorting
(FACS),
Those of skill in the art may readily adapt these methodologies to the present
invention,
The present invention additionally relates to a bioassay for evaluating
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
compounds as potential antagonists of the polypeptide described herein, the
bioassay
may comprise:
a) culturing test cells in culture medium containing increasing
concentrations of at least one compound whose ability to inhibit the action of
a
5 polypeptide described herein is sought to be determined, wherein the test
cells may
contain a polynucleotide sequence described herein (for example, in a form
having
improved trans-activation transcription activity, relative to wild-type
polynucleotide, and
comprising a response element operatively linked to a reporter gene); and
thereafter
b) monitoring in the cells the level of expression of the product of
10 the reporter gene as a function of the concentration of the potential
antagonist
compound in the culture medium, thereby indicating the ability of the
potential
antagonist compound to inhibit activation of the polypeptide encoded by the
polynucleotide sequence described herein.
The present invention further relates to a bioassay for evaluating compounds
as
15 potential
agonists for a polypeptide encoded by the polynucleotide sequence
described herein, the bioassay may comprise:
a) culturing test cells in culture medium containing increasing
concentrations of at least one compound whose ability to promote the action of
the
polypeptide encoded by the polynucleotide sequence described herein is sought
to be
determined, wherein the test cells may contain a polynucleotide sequence
described
herein (for example, in a form having improved trans-activation transcription
activity,
relative to wild-type polynucleotide, and comprising a response element
operatively
linked to a reporter gene); and thereafter
b) monitoring in the cells the level of expression of the product of
the reporter gene as a function of the concentration of the potential agonist
compound
in the culture medium, thereby indicating the ability of the potential agonist
compound
to promote activation of a polypeptide encoded by the polynucleotide sequence
described herein.
Host cells transformed with NSEQ may be cultured under conditions for the
expression and recovery of the polypeptide from cell culture. The polypeptide
produced
by a transgenic cell may be secreted or retained intracellularly depending on
the
sequence and/or the vector used. As will be understood by those of skill in
the art.
expression vectors containing NSEQ may be designed to contain signal sequences
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
16
that direct secretion of the polypeptide through a prokaryotic or eukaryotic
cell
membrane. Due to the inherent degeneracy of the genetic code, other DNA
sequences
that encode substantially the same or a functionally equivalent amino acid
sequence
may be produced and used to express the polypeptide encoded by NSEQ. The
nucleotide sequences of the present invention may be engineered using methods
generally known in the art in order to alter the nucleotide sequences for a
variety of
purposes including, but not limited to, modification of the cloning,
processing, and/or
expression of the gene product. DNA shuffling by random fragmentation and FOR
reassembly of gene fragments and synthetic oligonucleotides may be used to
engineer
the nucleotide sequences. For example, oligonucleotide-mediated site-directed
mutagenesis may be used to introduce mutations that create new restriction
sites, alter
glycosylation patterns, change codon preference, produce splice variants, and
so forth.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the inserted sequences or to process the expressed polypeptide
in the
desired fashion. Such modifications of the polypeptide include, but are not
limited to.
acetylation, carboxylation. glycosylation. phosphorylation lipidation. and
acylation.
Post-translational processing, which cleaves a -prepro" form of the
polypeptide, may
also be used to specify protein targeting, folding, and/or activity Different
host cells that
have specific cellular machinery and characteristic mechanisms for post-
translational
activities (e.g.. CHO, HeLa, MDCK, HEK293, and W138) are available
commercially
and from the American Type Culture Collection (ATCC) and may be chosen to
ensure
the correct modification and processing of the expressed polypeptide.
Those of skill in the art will readily appreciate that natural, modified, or
recombinant nucleic acid sequences may be ligated to a heterologous sequence
resulting in translation of a fusion polypeptide containing heterologous
polypeptide
moieties in any of the aforementioned host systems. Such heterologous
polypeptide
moieties may facilitate purification of fusion polypeptides using commercially
available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase
(GST), maltose binding protein, thioredoxin, calmodulin binding peptide, 6-His
(His),
FLAG, c-myc. hemaglutinin (HA), and monoclonal antibody epitopes.
In yet a further aspect, the present invention relates to an isolated
polynucleotide which may comprise a nucleotide sequence encoding a fusion
protein,
the fusion protein may comprise a fusion partner fused to a peptide fragment
of a
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
17
protein encoded by, or a naturally occurring allelic variant polypeptide
encoded by, the
polynucleotide sequence described herein.
Those of skill in the art will also readily recognize that the nucleic acid
and
polypeptide sequences may be synthesized, in whole or in part. using chemical
or
enzymatic methods well known in the art. For example, peptide synthesis may be
performed using various solid-phase techniques and machines such as the ABI
431A
Peptide synthesizer (PE Biosystems) may be used to automate synthesis. If
desired.
the amino acid sequence may be altered during synthesis and/or combined with
sequences from other proteins to produce a variant protein.
Use of NSEQ as a Diagnostic Screening Tool
The skilled artisan will readily recognize that NSEQ may be used for
diagnostic
purposes to determine the absence, presence, or altered expression (i.e.
increased or
decreased compared to normal) of the gene. The polynucleotides may be at least
10
nucleotides long or at least 12 nucleotides long, or at least 15 nucleotides
long up to
any desired length and may comprise complementary RNA and DNA molecules,
branched nucleic acids. and/or peptide nucleic acids (PNAs). In one
alternative, the
polynucleotides may be used to detect and quantify gene expression in samples
in
which expression of NSEQ is correlated with disease. In another alternative,
NSEQ
may be used to detect genetic polymorphisms associated with a disease. These
polymorphisms may be detected in the transcript cDNA.
The invention provides for the use of at least one polynucleotide comprising
NSEQ (e.g., an open reading frame of NSEQ, a substantially complementary
sequence, a substantially identical sequence, and fragments thereof) on an
array and
for the use of that array in a method for diagnosing a bone remodeling disease
or
disorder by hybridizing the array with a patient sample under conditions to
allow
complex formation, detecting complex formation, and comparing the amount of
complex formation in the patient sample to that of standards for normal and
diseased
tissues wherein the complex formation in the patient sample indicates the
presence of
a bone remodeling disease or disorder.
In another embodiment, the present invention provides one or more
compartmentalized kits for detection of bone resorption disease states A first
kit may
have a receptacle containing at least one isolated probe Such a probe may be a
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
18
nucleic acid fragment which is present/absent in the genomic DNA of normal
cells but
which is absent/present in the genomic DNA of affected cells. Such a probe may
be
specific for a DNA site that is normally active/inactive but which may be
inactive/active
in certain cell types. Similarly, such a probe may be specific for a DNA site
that may be
abnormally expressed in certain cell types. Finally, such a probe may identify
a specific
DNA mutation. By specific for a DNA site is meant that the probe may be
capable of
hybridizing to the DNA sequence which is mutated, or may be capable of
hybridizing to
DNA sequences adjacent to the mutated DNA sequences. The probes provided in
the
present kits may have a covalently attached reporter molecule Probes and
reporter
molecules may be readily prepared as described above by those of skill in the
art.
Use of NSEQ as a Therapeutic
One of skill in the art will readily appreciate that the expression systems
and
assays discussed above may also be used to evaluate the efficacy of a
particular
therapeutic treatment regimen, in animal studies, in clinical trials, or to
monitor the
treatment of an individual subject. Once the presence of disease is
established and a
treatment protocol is initiated, hybridization or amplification assays may be
repeated on
a regular basis to determine if the level of expression in the patient begins
to
approximate the level observed in a healthy subject. The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several days to many years
In yet another aspect of the invention, an NSEQ, a portion thereof, or its
complement. may be used therapeutically for the purpose of expressing mRNA and
polypeptide, or conversely to block transcription or translation of the mRNA.
Expression
.. vectors may be constructed using elements from retroviruses, adenoviruses,
herpes or
vaccinia viruses, or bacterial plasmids, and the like. These vectors may be
used for
delivery of nucleotide sequences to a particular target organ, tissue, or cell
population.
Methods well known to those skilled in the art may be used to construct
vectors to
express nucleic acid sequences or their complements.
Alternatively, NSEQ, a portion thereof, or its complement, may be used for
somatic cell or stem cell gene therapy. Vectors may be introduced in vivo in
vitro, and
ex vivo. For ex vivo therapy, vectors are introduced into stem cells taken
from the
subject. and the resulting transgenic cells are clonally propagated for
autologous
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
19
transplant back into that same subject. Delivery of NSEQ by transfection,
liposome
injections, or polycationic amino polymers may be achieved using methods that
are
well known in the art. Additionally, endogenous NSEQ expression may be
inactivated
using homologous recombination methods that insert an inactive gene sequence
into
the coding region or other targeted region of NSEQ.
Depending on the specific goal to be achieved, vectors containing NSEQ may
be introduced into a cell or tissue to express a missing polypeptide or to
replace a non-
functional polypeptide. Of course, when one wishes to express PSEQ in a cell
or
tissue, one may use a NSEQ able to encode such PSEQ for that purpose or may
directly administer PSEQ to that cell or tissue.
On the other hand, when one wishes to attenuate or inhibit the expression of
PSEQ, one may use a NSEQ (e.g.. an inhibitory NSEQ) which is substantially
complementary to at least a portion of a NSEQ able to encode such PSEQ.
The expression of an inhibitory NSEQ may be done by cloning the inhibitory
NSEQ into a vector and introducing the vector into a cell to down-regulate the
expression of a polypeptide encoded by the target NSEQ.
Vectors containing NSEQ (e.g., including inhibitory NSEQ) may be transformed
into a cell or tissue to express a missing polypeptide or to replace a non-
functional
polypeptide. Similarly a vector constructed to express the complement of NSEQ
may
be transformed into a cell to down-regulate the over-expression of a
polypeptide
encoded by the polynucleotides of NSEQ, or a portion thereof. Complementary or
anti-
sense sequences may consist of an oligonucleotide derived from the
transcription
initiation site nucleotides between about positions -10 and +10 from the ATG
are
preferred. Similarly inhibition may be achieved using triple helix base
pairing
methodology. Triple helix pairing is useful because it causes inhibition of
the ability of
the double helix to open sufficiently for the binding of polymerases,
transcription
factors, or regulatory molecules. Recent therapeutic advances using triplex
DNA have
been described in the literature. (See, e.g., Gee et al. 1994)
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the
cleavage of mRNA and decrease the levels of particular mRNAs. such as those
comprising the polynucleotide sequences of the invention. Ribozymes may cleave
mRNA at specific cleavage sites. Alternatively, ribozymes may cleave mRNAs at
locations dictated by flanking regions that form complementary base pairs with
the
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
target mRNA. The construction and production of ribozymes is well known in the
art.
RNA molecules may be modified to increase intracellular stability and half-
life.
Possible modifications include, but are not limited to, the addition of
flanking sequences
at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'
0-methyl
5 .. rather than phosphodiester linkages within the backbone of the molecule.
Alternatively,
nontraditional bases such as inosine, queosine, and wybutosine, as well as
acetyl-,
methyl-. thio-, and similarly modified forms of adenine, cytidine, guanine,
thymine. and
uridine which are not as easily recognized by endogenous endonucleases, may be
included.
10 In addition to the active ingredients, a pharmaceutical composition may
contain
pharmaceutically acceptable carriers comprising excipients and auxiliaries
that facilitate
processing of the active compounds into preparations that may be used
pharmaceutically.
For any compound, the therapeutically effective dose may be estimated
initially
15 either in cell culture assays or in animal models such as mice, rats,
rabbits, dogs, or
pigs. An animal model may also be used to determine the concentration range
and
route of administration. Such information may then be used to determine useful
doses
and routes for administration in humans. These techniques are well known to
one
skilled in the art and a therapeutically effective dose refers to that amount
of active
20 ingredient that ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity
may be determined by standard pharmaceutical procedures in cell cultures or
with
experimental animals, such as by calculating and contrasting the ED50 (the
dose
therapeutically effective in 50% of the population) and L050 (the dose lethal
to 50% of
the population) statistics. Any of the therapeutic compositions described
above may be
applied to any subject in need of such therapy, including but not limited to
mammals
such as dogs, cats, cows, horses, rabbits, monkeys. and most preferably.
humans.
The pharmaceutical compositions utilized in this invention may be administered
by any number of routes including, but not limited to, oral, intravenous.
intramuscular,
intra-arterial, intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous,
intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
The term "Treatment" for purposes of this disclosure refers to both
therapeutic
treatment and prophylactic or preventative measures, wherein the object is to
prevent
or slow down (lessen) the targeted pathologic condition or disorder. Those in
need of
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
21
treatment include those already with the disorder as well as those prone to
have the
disorder or those in whom the disorder is to be prevented.
Use of NSEQ in General Research
The invention finally provides products, compositions, processes and methods
that utilize an NSEQ. their open reading frame, or a polypeptide encoded by
the
polynucleotides of NSEQ or their open reading frame, or a portion thereof,
their
variants, analogs, derivatives and fragments for research, biological,
clinical and
therapeutic purposes. For example, to identify splice variants, mutations, and
polymorphisms
NSEQ may be extended utilizing a partial nucleotide sequence and employing
various PCR-based methods known in the art to detect upstream sequences such
as
promoters and other regulatory elements. Additionally, one may use an XL-PCR
kit (PE
Biosystems, Foster City Calif.). nested primers, and commercially available
cDNA
libraries (Life Technologies, Rockville Md.) or genomic libraries (Clontech,
Palo Alto
Calif.) to extend the sequence.
The polynucleotides may also be used as targets in a micro-array. The micro-
array may be used to monitor the expression patterns of large numbers of genes
simultaneously and to identify splice variants, mutations, and polymorphisms.
.. Information derived from analyses of the expression patterns may be used to
determine gene function, to understand the genetic basis of a disease, to
diagnose a
disease, and to develop and monitor the activities of therapeutic agents used
to treat a
disease. Microarrays may also be used to detect genetic diversity, single
nucleotide
polymorphisms which may characterize a particular population, at the genomic
level.
In yet another embodiment. polynucleotides may be used to generate
hybridization probes useful in mapping the naturally occurring genomic
sequence
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome mapping techniques and genetic map data.
The present invention more particularly relates in one aspect thereof to a
method of representatively identifying an endogeneously differentially
expressed
sequence involved in osteoclast differentiation. The sequence may be, for
example.
differentially expressed in a differentiated osteoclast cell compared to an
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/0002 10
22
undifferentiated osteoclast precursor cell.
The method of the present invention may comprise:
a) separately providing total messenger RNA from (mature or
intermediately) differentiated human osteoclast cell and undifferentiated
human osteoclast precursor cell, the total messenger RNA may
comprise, for example, at least one endogeneously differentially
expressed sequence,
b) generating single-stranded cDNA from each messenger RNA of
differentiated human osteoclast cell and (e.g., randomly) tagging the 3'-
end of the single-stranded cDNA with a RNA polymerase promoter
sequence and a first sequence tag;
c) generating single-stranded cDNA from each messenger RNA of
undifferentiated human osteoclast precursor cell and (e.g., randomly)
tagging the 3'-end of the single-stranded cDNA with a RNA polymerase
promoter sequence and a second sequence tag;
d) separately generating partially
or completely double-stranded 5'-
tagged-DNA from each of b) and c), the double-stranded 5'-tagged-DNA
may thus comprise in a 5' to 3' direction, a double-stranded RNA
polymerase promoter, a first or second sequence tag and an
endogenously expressed sequence,
e) separately linearly amplifying a first and second tagged sense RNA from
each of d) with a RNA polymerase enzyme (which may be selected
based on the promoter used for tagging),
f) generating single-stranded complementary first or second tagged DNA
from one of e),
g) hybridizing the single-stranded complementary first or second tagged
DNA of f) with the other linearly amplified sense RNA of e).
h) recovering unhybridized RNA with the help of the first or second
sequence tag (for example by PCR or hybridization), and'
i) identifying (determining) the nucleotide sequence of unhybndized RNA.
Steps b) and/or c), may comprise generating a single copy of a single-stranded
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
23
cDNA.
The method may further comprise the step of comparatively determining the
presence of the identified endogeneously and differentially expressed sequence
in a
differentiated osteoclast cell relative to an undifferentiated osteoclast
precursor cell.
A sequence which is substantially absent (e.g., totally absent or present in
very
low quantity) from one of differentiated osteoclast cell or an
undifferentiated osteoclast
precursor cell and present in the other of differentiated osteoclast cell or
an
undifferentiated osteoclast precursor cell may therefore be selected.
The sequence thus selected may be a positive regulator of osteoclast
differentiation and therefore may represent an attractive target which may
advantageously be used to promote bone resorption or alternatively such target
may
be inhibited to lower or prevent bone resorption.
Alternatively, the sequence selected using the above method may be a negative
regulator of osteoclast differentiation and may therefore represent an
attractive target
which may advantageously be induced (e.g.. at the level of transcription,
translation,
activity etc.) or provided to a cell to lower or prevent bone resorption. Also
such
negative regulator may. upon its inhibition, serve as a target to promote bone
resorption.
In accordance with the present invention, the sequence may be further selected
based on a reduced or substantially absent expression in other normal tissue,
therefore
representing a candidate sequence specifically involved in osteoclast
differentiation
and bone remodeling.
The method may also further comprise a step of determining the complete
sequence of the nucleotide sequence and may also comprise determining the
coding
sequence of the nucleotide sequence.
The present invention also relates in a further aspect, to the isolated
endogeneously and differentially expressed sequence (polynucleotide and
polypeptide)
identified by the method of the present invention.
More particularly, the present invention encompasses a polynucleotide which
may comprise the identified polynucleotide sequence a polynucleotide which may
comprise the open reading frame of the identified polynucleotide sequence. a
polynucleotide which may comprise a nucleotide sequence substantially
identical to the
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
24
polynucleotide identified by the method of the present invention, a
polynucleotide which
may comprise a nucleotide sequence substantially complementary to the
polynucleotide identified by the method of the present invention, fragments
and splice
variant thereof, provided that the sequence does not consist in or comprise
SEQ ID
NO.:34.
In accordance with the present invention, the isolated endogeneously and
differentially expressed sequence of the present invention may be a complete
or partial
RNA molecule.
Isolated DNA molecule able to be transcribed into the RNA molecule of the
present invention are also encompassed herewith as well as vectors (including
expression vectors) comprising the such DNA or RNA molecule.
The present invention also relates to libraries comprising at least one
isolated
endogeneously and differentially expressed sequence identified herein (e.g..
partial or
complete RNA or DNA, substantially identical sequences or substantially
complementary sequences (e.g. probes) and fragments thereof (e.g..
oligonucleotides)).
In accordance with the present invention, the isolated endogeneously and
differentially expressed sequence may be selected, for example from the group
consisting of a polynucleotide which may consist in or comprise.
a) any one of SEQ ID NO.:1 to 33. SEQ ID NO..85 or SEQ ID NO.-86.
5) the open reading frame of any one of SEQ ID NO.i1 to 33. SEQ ID
NO.:85 or SEQ ID NO.:86,
c) a polynucleotide which may comprise a nucleotide sequence
substantially identical to a) or b), and;
ci) a polynucleotide which may comprise a nucleotide sequence
substantially comlpementary to any one of a) to c),
c) fragments of any one of a) to d)
In a further aspect the present invention relates to a polypeptide which may
be
encoded by the isolated endogeneously and differentially expressed sequence of
the
present invention.
In yet a further aspect the present invention relates to a polynucleotide able
to
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
encode a polypeptide of the present invention. Due to the degeneracy of the
genetic
code, it is to be understood herein that a multiplicity of polynucleotide
sequence may
encode the same polypeptide sequence and thus are encompassed by the present
invention.
5 Exemplary
polypeptides may comprise a sequence selected from the group
consisting of any one of SEQ ID NO.: 48 to 80, a polypeptide encoded by SEQ ID
NO.:85 or SEQ ID NO. .86
The present invention also relates to an isolated non-human ortholog
polynucleotide sequence (involved in bone remodeling), the open reading frame
of the
10 non-human
ortholog, substantially identical sequences, substantially complementary
sequences, fragments and splice variants thereof.
The present invention as well relates to an isolated polypeptide encoded by
the
non-human ortholog polynucleotide as well as biologically active analogs and
biologically active fragments thereof.
15 Exemplary
embodiments of non-human (e.g., mouse) ortholog polynucleotides
encompassed herewith include, for example, SEQ ID NO.35.
Exemplary embodiments of isolated polypeptide encoded by some non-human
orthologs identified herein include for example, a polypeptide such as SEQ ID
NO '82
The present invention also more particularly relates, in an additional aspect
20 thereof, to an
isolated polynucleotide which may be differentially expressed in
differentiated osteoclast cell compared to undifferentiated human osteoclast
precursor
cell.
The isolated polynucleotide may comprise a member selected from the group
consisting of:
25 a) a
polynucleotide which may comprise any one of SEQ ID NO. :1 to SEQ
ID NO.33, SEQ ID NO.:85 or SEQ ID NO.:86
b) a polynucleotide which may comprise the open reading frame of any
one of SEQ ID NO.:1 to SEQ ID NO.33, SEQ ID NO.:85 or SEQ ID
NO.:86;
c) a polynucleotide which may comprise a transcribed or transcribable
portion of any one of SEQ. ID. NOs:1 to SEQ ID NO.33, SEQ ID NO. 85
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
26
or SEQ ID NO.86, which may be. for example, free of untranslated or
untranslatable portion(s):
d) a polynucleotide which may comprise a translated or translatable portion
of any one of SEQ. ID. NOs:1 to SEQ ID NO.33, SEQ ID NO.:85 or SEQ
ID NO.:86 (e.g., coding portion),
e) a polynucleotide which may comprise a sequence substantially identical
(e.g., from about 50 to 100%, or about 60 to 100% or about 70 to 100%
or about 80 to 100% or about 85, 90, 95 to 100% identical over the
entire sequence or portion of sequences) to a), b) c) or d).
f) a polynucleotide which may comprise a sequence substantially
complementary (e.g.. from about 50 to 100% or about 60 to 1000/c or
about 70 to 100% or about 80 to 100% or about 85. 90. 95 to 100%
complementarity over the entire sequence or portion of sequences) to
a), 13). c) or d) and;
g) a fragment of any one of a) to f)
h) including polynucleotides which consist in the above.
Exemplary polynucleotides fragments of those listed above comprises
polynucleotides of at least 10 nucleic acids which may be substantially
complementary
to the nucleic acid sequence of any one of SEQ ID NO: 1 to 33, SEQ ID NO.:85
or
SEQ ID NO.:86, for example, fragments selected from the group consisting of
any one
of SEQ ID NO.: 42-45.
The present invention also relates to an isolated polynucleotide involved in
osteoclast differentiation, the isolated polynucleotide may be selected. for
example.
from the group consisting of;
a) a polynucleotide comprising any one of SEQ ID NO.: 1 to 33. SEQ ID
NO.:85 or SEQ ID NO.:86,
b) a polynucleotide comprising the open reading frame of any one of SEQ
ID NO.: 1 to 33, SEQ ID NO.:85 or SEQ ID NO.:86,
c) a polynucleotide which may comprise a transcribed or transcribable
portion of any one of SEQ. ID. NOsrl to SEQ ID NO.33, SEQ ID NO.:85
or SEQ ID NO.86, which may be, for example, free of untranslated or
untranslatable portion(s);
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
27
d) a polynucleotide which may comprise a translated or translatable portion
of any one of SEQ. ID. NOs:1 to SEQ ID NO 33, SEQ ID NO :85 or SEQ
ID NO.:86 (e.g., coding portion),
e) a polynucleotide substantially identical to a). b), c) or d),; and:
f) a sequence of at least 10 nucleic acids which may be substantially
complementary to the nucleic acid sequence of any one of SEQ ID
NO.:1 to 33, SEQ ID NO.:85 or SEQ ID NO.:86 or more particularly of
a), b), c) or d).
In accordance with the present invention the isolated polynucleotide may be
able to promote osteoclast differentiation (e.g., in a mammal or mammalian
cell
thereof), i.e, a positive regulator of osteoclast differenciation.
Further in accordance with the present invention, the isolated polynucleotide
may be able to inhibit, prevent or lower osteoclast differentiation (e.g in a
mammal or
mammalian cell thereof), t. e, a negative regulator of osteoclast
differenciation.
In yet a further aspect, the present invention relates to an isolated
polynucleotide which may be able to inhibit osteoclast differentiation (e.g..
in a mammal
or mammalian cell thereof). The polynucleotide may be selected, for example,
from the
group consisting of polynucleotides which may comprise a sequence of at least
10
nucleic acids which is complementary to the nucleic acid sequence of any one
of
NSEQ described herein.
Suitable polynucleotides include, for example, a polynucleotide having or
comprising those which are selected from the group consisting of SEQ ID NO. 42
to 45.
Suitable polynucleotides may be those which may be able to inhibit osteoclast
differentiation which has been induced by an inducer of osteoclast
differentiation such
as those listed herein.
In accordance with the present invention, the polynucleotide may be for
example, a RNA molecule, a DNA molecule, including those which are partial or
complete, single-stranded or double-stranded, hybrids, etc.
The present invention also relates to a vector (e.g., an expression vector)
comprising the polynucleotide of the present invention.
The present invention additionally relates in an aspect thereof to a library
of
polynucleotide sequences which may be differentially expressed in a
differentiated
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
28
osteoclast cell compared to an undifferentiated osteoclast precursor cell The
library
may comprise, for example. at least one member selected from the group
consisting of
a) a polynucleotide which may comprise any one of SEQ ID NO..1 to 33.
SEQ ID NO.:85 or SEQ ID NO.:86,
b) a polynucleotide which may comprise the open reading frame of any
one of SEQ ID NO.:1 to 33, SEQ ID NO.:85 or SEQ ID NO.:86,
c) a polynucleotide which may comprise a transcribed or transcribable
portion of any one of SEQ. ID. NOs:1 to SEQ ID NO.33. SEQ ID NO.:85
or SEQ ID NO.86, which may be, for example, free of untranslated or
untranslatable portion(s),
d) a polynucleotide which may comprise a translated or translatable portion
of any one of SEQ. ID. NOs:1 to SEQ ID NO 33. SEQ ID NO.:85 or SEQ
ID NO 86 (e.g., coding portion),
e) a polynucleotide which may comprise a sequence substantially identical
(e.g., from about 50 to 100%, or about 60 to 100% or about 70 to 1000/0
or about 80 to 100% or about 85, 90. 95 to 100% identical over the
entire sequence or portion of sequences) to a), b), c) or d):
f) a polynucleotide which may comprise a sequence substantially
complementary (e.g., from about 50 to 100%, or about 60 to 100% or
about 70 to 100% or about 80 to 100% or about 85, 90, 95 to 100%
complementarity over the entire sequence or portion of sequences) to
a), b), c) old) and;
g) a fragment of any one of a) to d).
The present invention also relates to an expression library which may comprise
a library of polynucleotides described herein. In accordance with the present
invention,
each of the polynucleotide may be contained within an expression vector.
Arrays and kits comprising a library of polynucleotide sequences (comprising
at
least one polynucleotide such as complementary sequences) of the present
invention
are also encompassed herewith
The present invention also provides in an additional aspect, a pharmaceutical
composition for inhibiting osteoclast differentiation (bone resorption and
bone
resorption related diseases or disorders), the pharmaceutical composition may
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/0002 10
29
comprise, for example;
a) an isolated polynucleotide as defined herein (e.g., able to inhibit
osteoclast differentiation) and:
b) a pharmaceutically acceptable carrier.
The present invention also provides in yet an additional aspect. a method for
inhibiting osteoclast differentiation (e.g., for inhibiting bone resorption or
for
ameliorating bone resorption) in a mammal (individual) in need thereof (or in
a
mammalian cell), the method may comprise administering an isolated
polynucleotide
(e.g., able to inhibit osteoclast differentiation) or a suitable
pharmaceutical composition
comprising such suitable polynucleotide.
In accordance with the present invention, the mammal in need may suffer, for
example and without limitation, from a condition selected from the group
consisting of
osteoporosis, osteopenia, osteomalacia, hyperparathyroidism, hyperthyroidism,
hypogonadism, thyrotoxicosis, systemic mastocytosis, adult hypophosphatasia,
hyperadrenocorticism, osteogenesis imperfecta. Paget's disease, Cushing's
disease/syndrome, Turner syndrome, Gaucher disease, Ehlers-Danlos syndrome,
Marfan's syndrome, Menkes' syndrome. Fanconi's syndrome. multiple myeloma.
hypercalcemia, hypocalcemia arthritides, periodontal disease. rickets
(includIng
vitamin D dependent, type I and II, and )(linked hypophosphatemic rickets)
fibrogenesis imperfecta ossium, osteosclerotic disorders such as
pycnodysostosis and
damage caused by macrophage-mediated inflammatory processes, etc.
In a further aspect, the present invention relates to the use of an isolated
polynucleotide (e.g., able to inhibit osteoclast differentiation) for the
preparation of a
medicament for the treatment of a bone resorption disease.
The present invention in another aspect thereof, provides a pharmaceutical
composition for promoting osteoclast differentiation in a mammal in need
thereof. The
pharmaceutical composition may comprise for example;
a. an isolated polynucleotide (e.g.. able to promote osteoclast
differentiation) and:
b. a pharmaceutically acceptable carrier.
The present invention also further provides a method for promoting osteoclast
differentiation in a mammal in need thereof (or in a mammalian cell), the
method may
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
comprise, for example, administering an isolated polynucleotide (e.g., able to
promote
osteoclast differentiation) or a suitable pharmaceutical composition as
described
above.
The present invention additionally relates to the use of an isolated
5 polynucleotide
(e.g.. able to promote osteoclast differentiation) for the preparation of a
medicament for the treatment of a disease associated with insufficient bone
resorption
(e.g., hyperostosis) or excessive bone growth.
The present invention also relates to the use of at least one polynucleotide
which may
be selected from the group consisting of:
10 a) a
polynucleotide comprising any one of SEQ ID NO.:1 to 33, SEQ ID
NO.:85 or SEQ ID NO .86.
b) a polynucleotide comprising the open reading frame of any one of SEQ
ID NO:1 to 33, SEQ ID NO.:85 or SEQ ID NO.86,
c) a polynucleotide which may comprise a transcribed or transcribable
15 portion of any one of SEQ. ID. NOs:1 to SEQ ID NO.33, SEQ ID NO.:85
or SEQ ID NO.86, which may be, for example. free of untranslated or
untranslatable portion(s);
d) a polynucleotide which may comprise a translated or translatable portion
of any one of SEQ. ID. NOs:1 to SEQ ID NO 33 SEQ ID NO :85 or SEQ
20 ID NO.:86 (e.g.. coding portion).
e) a polynucleotide comprising a sequence substantially identical (e.g.,
from about 50 to 100%, or about 60 to 100% or about 70 to 100% or
about 80 to 100% or about 85. 90, 95 to 100% identical over the entire
sequence or portion of sequences) to a), b), c) or d);
25 f) a
polynucleotide comprising a sequence substantially complementary
(e.g., from about 50 to 100`)/0, or about 60 to 100% or about 70 to 100%
or about 80 to 100% or about 85, 90, 95 to 100% complementarity over
the entire sequence or portion of sequences) to a), b), c) or d),
g) a fragment of any one of a) to f) and,
30 h) a library comprising any one of a) to g)
in the diagnosis of a condition related to bone remodeling (a bone disease).
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
31
Also encompassed by the present invention are kits for the diagnosis of a
condition related to bone remodeling. The kit may comprise a polynucleotide as
described herein.
The present invention also provides in an additional aspect, an isolated
polypeptide (polypeptide sequence) involved in osteoclast differentiation (in
a mammal
or a mammalian cell thereof). The
polypeptide may comprise (or consist in) a
sequence selected from the group consisting of.
a) any one of SEQ ID NO . 48 to 80,
b) a polypeptide able to be encoded and/or encoded by any one of
SEQ ID NO.:1 to 33, SEQ ID NO.:85 or SEQ ID NO.:86 (their
coding portion)
c) a biologically active fragment of any one of a) or b),
d) a biologically active analog of any one of a) or b).
In accordance with the present invention, the biologically active analog may
comprise, for example, at least one amino acid substitution (conservative or
non
conservative) compared to the original sequence. In accordance with the
present
invention, the analog may comprise, for example, at least one amino acid
substitution,
deletion or insertion in its amino acid sequence.
The substitution may be conservative or non-conservative The polypeptide
analog may be a biologically active analog or an immunogenic analog which may
comprise. for example at least one amino acid substitution (conservative or
non
conservative), for example, 1 to 5 1 to 10 1 to 15, 1 to 20. 1 to 50 etc
(including any
number there between) compared to the original sequence. An immunogenic analog
may comprise, for example, at least one amino acid substitution compared to
the
original sequence and may still be bound by an antibody specific for the
original
sequence.
In accordance with the present invention, a polypeptide fragment may comprise,
for example, at least 6 consecutive amino acids, at least 8 consecutive amino
acids or
more of an amino acid sequence described herein.
In yet a further aspect, the present invention provides a pharmaceutical
composition which may comprise, for example a polypeptide as described herein
and a
pharmaceutically acceptable carrier.
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
32
Methods for modulating osteoclast differentiation in a mammal in need thereof
(or in a mammalian cell) are also provided by the present invention, which
methods
may comprise administering an isolated polypeptide (e.g., able to promote
osteoclast
differentiation) or suitable pharmaceutical composition described herein.
In additional aspects, the present invention relates to the use of an isolated
polypeptide (e.g.. able to promote osteoclast differentiation) for the
preparation of a
medicament for the treatment of a disease associated with insufficient bone
resorption.
Methods for ameliorating bone resorption in an individual in need thereof are
also encompassed herewith, which method may comprise, for example
administering
an isolated polypeptide (e.g., able to inhibit osteoclast differentiation) or
suitable
pharmaceutical compositions which may comprise such polypeptide
In accordance with the present invention, the mammal may suffer, for example,
from a condition selected from the group consisting of osteoporosis,
osteopenia,
osteomalacia. hyperparathyroidism, hyperthyroidism, hypogonadism,
thyrotoxicosis.
systemic mastocytosis, adult hypophosphatasia, hyperadrenocorticism,
osteogenesis
imperfecta, Paget's disease, Cushing's disease/syndrome, Turner syndrome,
Gaucher
disease, Ehlers-Danlos syndrome, Marfan's syndrome, Menkes' syndrome,
Fanconi's
syndrome, multiple myeloma, hypercalcemia, hypocalcemia, arthritides,
periodontal
disease. rickets (including vitamin D dependent, type I and II. and x-linked
hypophosphatemic rickets), fibrogenesis imperfecta ossium, osteosclerotic
disorders
such as pycnodysostosis and damage caused by macrophage-mediated inflammatory
processes, etc.
In yet a further aspect, the present invention relates to the use of a
polypeptide
able to inhibit osteoclast differentiation in the preparation of a medicament
for the
treatment of a bone resorption disease in an individual in need thereof
The present invention also relates to a compound and the use of a compound
able to inhibit (e.g., in an osteoclast precursor cell) the activity or
expression of a
polypeptide which may be selected, for example, from the group consisting of
SEQ ID
NO.: 48 to 80 or a polypeptide encoded by SEQ ID NO :85 or SEQ ID NO.:86. in
the
preparation of a medicament for the treatment of a bone disease in an
individual in
need thereof.
In yet an additional aspect, the present invention relates to a method of
diagnosing a condition related to a bone resorption disorder or disease in an
individual
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000240
33
in need thereof. The method may comprise, for example, quantifying a
polynucleotide
described herein, such as, for example, polynucleotide selected from the group
consisting of those comprising or consisting of (a) SEQ ID NO. :1 to 33, SEQ
ID NO.:85
or SEQ ID NO.86, (b) a polynucleotide which may comprise the open reading
frame of
SEQ ID NO.: 1 to 33, SEQ ID NO.:85 or SEQ ID NO.:86, (c) a polynucleotide
which
may comprise a transcribed or transcribable portion of any one of SEQ. ID.
NOs:1 to
SEQ ID NO.33, SEQ ID NO.:85 or SEQ ID NO.:86 (d) a polynucleotide which may
comprise a translated or translatable portion of any one of SEQ. ID. NOs:1 to
SEQ ID
NO.33, SEQ ID NO.:85 or SEQ ID NO.:86; (e) substantially identical sequences
of any
one of (a) to (d): (f) substantially complementary sequences of any one of (a)
to (e), or
a polypeptide sequence which may be selected, for example, from the group
consisting
of SEQ ID NO.: 48 to 80 or a polypeptide encoded by SEQ ID NO.:85 or SEQ ID
NO.:86, and analogs thereof in a sample from the individual compared to a
standard or
normal value
The present invention also relates to an assay and method for identifying a
gene and/or protein involved in bone remodeling. The assay and method may
comprise silencing an endogenous gene of an osteoclast cell and providing the
cell
with a candidate gene (or protein). A candidate gene (or protein) positively
involved in
bone remodeling may be identified by its ability to complement the silenced
endogenous gene. For example, a
candidate gene involved in osteoclast
differentiation provided to a cell for which an endogenous gene has been
silenced. may
enable the cell to differentiate in the presence of an inducer such as, for
example,
RANKL.
The present invention further relates to a cell expressing an exogenous form
of
any one of the polypeptide (including variants, analogs etc.) or
polynucleotide of the
present invention (including substantially identical sequences substantially
complementary sequences, fragments, variants, orthologs etc).
In accordance with the present invention, the cell may be for example, a bone
cell. Also in accordance with the present invention, the cell may be an
osteoclast (at
any level of differentiation).
As used herein the term "exogenous form" is to be understood herein as a form
which is not naturally expressed by the cell in question.
In a further aspect, the present invention relates to an antibody (e.g..
isolated
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
34
antibody), or antigen-binding fragment thereof, that may specifically bind to
a protein or
polypeptide described herein The antibody
may be, for example a monoclonal
antibody, a polyclonal antibody an antibody generated using recombinant DNA
technologies. The antibody may originate for example, from a mouse, rat or any
other
mammal.
The antibody may also be a human antibody which may be obtained, for
example, from a transgenic non-human mammal capable of expressing human Ig
genes. The antibody may also be a humanised antibody which may comprise, for
example, one or more complementarity determining regions of non-human origin.
It
may also comprise a surface residue of a human antibody and/or framework
regions of
a human antibody. The antibody may also be a chimeric antibody which may
comprise, for example, variable domains of a non-human antibody and constant
domains of a human antibody.
Suitable antibodies may also include, for example, an antigen-binding
fragment.
an Fab fragment; an F(ab):, fragment, and Fv fragment, or a single-chain
antibody
comprising an antigen-binding fragment (e.g.. a single chain Fv)
The antibody of the present invention may be mutated and selected based on
an increased affinity and/or specificity for one of a polypeptide described
herein and/or
based on a reduced immunogenicity in a desired host.
The antibody may further comprise a detectable label attached thereto.
The present invention further relates to a method of producing antibodies able
to bind to one of a polypeptide, polypeptide fragments, or polypeptide analogs
described herein, the method may comprise:
a) immunizing a mammal (e.g., mouse, a transgenic mammal
capable of producing human Ig, etc.) with a suitable amount of a PSEQ
described herein including, for example, a polypeptide fragment
comprising at least 6 consecutive amino acids of a PSEQ:
b) collecting the serum from the mammal, and
c) isolating the polypeptide-specific antibodies from the serum of
the mammal
The method may further comprise the step of administering a second dose to
the animal.
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
The present invention also relates to a method of producing a hybridoma which
secretes an antibody that binds to a polypeptide described herein, the method
may
comprise.
a) immunizing a mammal (e.g.. mouse. a transgenic mammal
5 capable of producing human lg. etc.) with a suitable amount of a
PSEQ
thereof;
b) obtaining lymphoid cells from the immunized animal obtained
from (a);
c) fusing the lymphoid cells with an immortalizing cell to produce
10 hybrid cells; and
d) selecting hybrid cells which produce antibody that specifically
binds to a PSEQ thereof.
The present invention further relates to a method of producing an antibody
that
binds to one of the polypeptide described herein, the method may comprise:
15 a) synthesizing a library of antibodies (antigen binding
fragment) on
phage or ribosomes.
b) panning the library against a sample by bringing the phage
or
ribosomes into contact with a composition comprising a polypeptide or
polypeptide fragment described herein;
20 c) isolating phage which binds to the polypeptide or polypeptide
fragment, and;
d) obtaining an antibody from the phage or ribosomes.
The antibody of the present invention may thus be obtained, for example, from
a library (e.g., bacteriophage library) which may be prepared, for example, by
25 a) extracting cells which are responsible for production of
antibodies from a host mammal:
b) isolating RNA from the cells of (a):
C) reverse transcribing mRNA to produce cDNA
d) amplifying the cDNA using a (antibody-specific) primer,
and
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
36
e) inserting
the cDNA of (d) into a phage display vector or ribosome
display cassette such that antibodies are expressed on the
phage or ribosomes.
The host animal may be immunized with polypeptide and/or a polypeptide
.. fragment and/or analog described herein to induce an immune response prior
to
extracting the cells which are responsible for production of antibodies
The present invention also relates to a kit for specifically assaying a
polypeptide
described herein, the kit may comprise, for example, an antibody or antibody
fragment
capable of binding specifically to the polypeptide described herein.
The present invention further contemplates antibodies that may bind to PSEQ.
Suitable antibodies may bind to unique antigenic regions or epitopes in the
polypeptides, or a portion thereof. Epitopes and antigenic regions useful for
generating
antibodies may be found within the proteins, polypeptides or peptides by
procedures
available to one of skill in the art. For example, short, unique peptide
sequences may
be identified in the proteins and polypeptides that have little or no homology
to known
amino acid sequences. Preferably the region of a protein selected to act as a
peptide
epitope or antigen is not entirely hydrophobic: hydrophilic regions are
preferred
because those regions likely constitute surface epitopes rather than internal
regions of
the proteins and polypeptides. These surface epitopes are more readily
detected in
samples tested for the presence of the proteins and polypeptides Such
antibodies may
include, but are not limited to; polyclonal, monoclonal. chimeric, and single
chain
antibodies, Fab fragments and fragments produced by a Fab expression library.
The
production of antibodies is well known to one of skill in the art.
Peptides may be made by any procedure known to one of skill in the art, for
example, by using in vitro translation or chemical synthesis procedures. Short
peptides
which provide an antigenic epitope but which by themselves are too small to
induce an
immune response may be conjugated to a suitable carrier. Suitable carriers and
methods of linkage are well known in the art. Suitable carriers are typically
large
macromolecules such as proteins, polysaccharides and polymeric amino acids
Examples include serum albumins. keyhole limpet hemocyanin, ovalbumin,
polylysine
and the like. One of skill in the art may use available procedures and
coupling reagents
to link the desired peptide epitope to such a carrier. For example. coupling
reagents
may be used to form disulfide linkages or thioether linkages from the carrier
to the
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
37
peptide of interest. If the peptide lacks a disulfide group, one may be
provided by the
addition of a cysteine residue. Alternatively, coupling may be accomplished by
activation of carboxyl groups.
The minimum size of peptides useful for obtaining antigen specific antibodies
may vary widely. The minimum size must be sufficient to provide an antigenic
epitope
that is specific to the protein or polypeptide The maximum size is not
critical unless it is
desired to obtain antibodies to one particular epitope. For example, a large
polypeptide
may comprise multiple epitopes. one epitope being particularly useful and a
second
epitope being immunodominant. Typically, antigenic peptides selected from the
present
proteins and polypeptides will range from 5 to about 100 amino acids in
length. More
typically, however, such an antigenic peptide will be a maximum of about 50
amino
acids in length, and preferably a maximum of about 30 amino acids. It is
usually
desirable to select a sequence of about 6, 8, 10, 12 or 15 amino acids, up to
about 20
or 25 amino acids.
Amino acid sequences comprising useful epitopes may be identified in a
number of ways. For example, preparing a series of short peptides that taken
together
span the entire protein sequence may be used to screen the entire protein
sequence.
One of skill in the art may routinely test a few large polypeptides for the
presence of an
epitope showing a desired reactivity and also test progressively smaller and
overlapping fragments to identify a preferred epitope with the desired
specificity and
reactivity.
Antigenic polypeptides and peptides are useful for the production of
monoclonal
and polyclonal antibodies_ Antibodies to a polypeptide encoded by the
polynucleotides
of NSEQ, polypeptide analogs or portions thereof, may be generated using
methods
that are well known in the art. Such antibodies may include, but are not
limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments,
and
fragments produced by a Fab expression library. Neutralizing antibodies, such
as those
that inhibit dimer formation, are especially preferred for therapeutic use.
Monoclonal
antibodies may be prepared using any technique that provides for the
production of
antibody molecules by continuous cell lines in culture. These include, but are
not
limited to. the hybridoma, the human B-cell hybridoma, and the EBV-hybridoma
techniques. In addition, techniques developed for the production of chimeric
antibodies
may be used. Alternatively, techniques described for the production of single
chain
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
38
antibodies may be employed. Fabs that may contain specific binding sites for a
polypeptide encoded by the polynucleotides of NSEQ, or a portion thereof, may
also be
generated. Various immunoassays may be used to identify antibodies having the
desired specificity. Numerous protocols for competitive binding or
immunoradiometric
assays using either polyclonal or monoclonal antibodies with established
specificities
are well known in the art.
To obtain polyclonal antibodies, a selected animal may be immunized with a
protein or polypeptide. Serum from the animal may be collected and treated
according
to known procedures. Polyclonal antibodies to the protein or polypeptide of
interest
may then be purified by affinity chromatography. Techniques for producing
polyclonal
antisera are well known in the art.
Monoclonal antibodies (MAbs) may be made by one of several procedures
available to one of skill in the art, for example, by fusing antibody
producing cells with
immortalized cells and thereby making a hybridoma. The general methodology for
fusion of antibody producing B cells to an immortal cell line is well within
the province of
one skilled in the art. Another example is the generation of MAbs from mRNA
extracted
from bone marrow and spleen cells of immunized animals using combinatorial
antibody
library technology.
One drawback of MAbs derived from animals or from derived cell lines is that
although they may be administered to a patient for diagnostic or therapeutic
purposes,
they are often recognized as foreign antigens by the immune system and are
unsuitable for continued use. Antibodies that are not recognized as foreign
antigens by
the human immune system have greater potential for both diagnosis and
treatment
Methods for generating human and humanized antibodies are now well known in
the
art.
Chimeric antibodies may be constructed in which regions of a non-human MAb
are replaced by their human counterparts. A preferred chimeric antibody is one
that
has amino acid sequences that comprise one or more complementanty determining
regions (CDRs) of a non-human Mab that binds to a polypeptide encoded by the
polynucleotides of NSEQ, or a portion thereof, grafted to human framework
(FVV)
regions. Methods for producing such antibodies are well known in the art.
Amino acid
residues corresponding to CDRs and FVVs are known to one of average skill in
the art
A variety of methods have been developed to preserve or to enhance affinity
for
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
39
antigen of antibodies comprising grafted CDRs One way is to include in the
chimeric
antibody the foreign framework residues that influence the conformation of the
CDR
regions. A second way is to graft the foreign CDRs onto human variable domains
with
the closest homology to the foreign variable region. Thus, grafting of one or
more non-
human CDRs onto a human antibody may also involve the substitution of amino
acid
residues which are adjacent to a particular CDR sequence or which are not
contiguous
with the CDR sequence but which are packed against the CDR in the overall
antibody
variable domain structure and which affect the conformation of the CDR
Humanized
antibodies of the invention therefore include human antibodies which comprise
one or
more non-human CDRs as well as such antibodies in which additional
substitutions or
replacements have been made to preserve or enhance binding characteristics
Chimeric antibodies of the invention also include antibodies that have been
humanized by replacing surface-exposed residues to make the MAb appear human.
Because the internal packing of amino acid residues in the vicinity of the
antigen-
binding site remains unchanged, affinity is preserved. Substitution of surface-
exposed
residues of a polypeptide encoded by the polynucleotides of NSEQ (or a portion
thereof)-antibody according to the invention for the purpose of humanization
does not
mean substitution of CDR residues or adjacent residues that influence affinity
for a
polypeptide encoded by the polynucleotides of NSEQ, or a portion thereof.
Chimeric antibodies may also include antibodies where some or all non-human
constant domains have been replaced with human counterparts. This approach has
the
advantage that the antigen-binding site remains unaffected. However,
significant
amounts of non-human sequences may be present where variable domains are
derived entirely from non-human antibodies.
Antibodies of the invention include human antibodies (e.g . humanized) that
are
antibodies consisting essentially of human sequences. Human antibodies may be
obtained from phage display libraries wherein combinations of human heavy and
light
chain variable domains are displayed on the surface of filamentous phage.
Combinations of variable domains are typically displayed on filamentous phage
in the
form of Fab's or scFvs. The library may be screened for phage bearing
combinations of
variable domains having desired antigen-binding characteristics. Preferred
variable
domain combinations are characterized by high affinity for a polypeptide
encoded by
the polynucleotides of NSEQ, or a portion thereof. Preferred variable domain
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
combinations may also be characterized by high specificity for a polypeptide
encoded
by the polynucleotides of NSEQ, or a portion thereof. and little cross-
reactivity to other
related antigens. By screening from very large repertoires of antibody
fragments, (2-10
x 1010) a good diversity of high affinity Mabs may be isolated, with many
expected to
5 have sub-nanomolar affinities for a polypeptide encoded by the
polynucleotides of
NSEQ, or a portion thereof.
Alternatively, human antibodies may be obtained from transgenic animals into
which un-rearranged human Ig gene segments have been introduced and in which
the
endogenous mouse Ig genes have been inactivated. Preferred transgenic animals
10 contain very large contiguous Ig gene fragments that are over 1 Mb in
size but human
polypeptide-specific Mabs of moderate affinity may be raised from transgenic
animals
containing smaller gene loci. Transgenic animals capable of expressing only
human Ig
genes may also be used to raise polyclonal antiserum comprising antibodies
solely of
human origin.
15 Antibodies of
the invention may include those for which binding characteristics
have been improved by direct mutation or by methods of affinity maturation.
Affinity and
specificity may be modified or improved by mutating CDRs and screening for
antigen
binding sites having the desired characteristics. CDRs may be mutated in a
variety of
ways. One way is to randomize individual residues or combinations of residues
so that
20 in a population of otherwise identical antigen binding sites, all twenty
amino acids may
be found at particular positions. Alternatively, mutations may be induced over
a range
of CDR residues by error prone PCR methods. Phage display vectors containing
heavy
and light chain variable region gene may be propagated in mutator strains of
E. coll.
These methods of mutagenesis are illustrative of the many methods known to one
of
25 skill in the art.
Antibodies of the invention may include complete anti-polypeptide antibodies
as
well as antibody fragments and derivatives that comprise a binding site for a
polypeptide encoded by the polynucleotides of NSEQ, or a portion thereof
Derivatives
are macromolecules that comprise a binding site linked to a functional domain.
30 Functional domains may include, but are not limited to signalling
domains, toxins.
enzymes and cytokines.
The antibodies obtained by the means described herein may be useful for
detecting proteins, variant and derivative polypeptides in specific tissues or
in body
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
41
fluids. Moreover, detection of aberrantly expressed proteins or protein
fragments is
probative of a disease state. For example, expression of the present
polypeptides
encoded by the polynucleotides of NSEQ. or a portion thereof, may indicate
that the
protein is being expressed at an inappropriate rate or at an inappropriate
developmental stage. Hence, the present antibodies may be useful for detecting
diseases associated with protein expression from NSEQs disclosed herein.
A variety of protocols for measuring polypeptides, including ELISAs. RIAs, and
FACS, are well known in the art and provide a basis for diagnosing altered or
abnormal
levels of expression. Standard values for polypeptide expression are
established by
combining samples taken from healthy subjects, preferably human, with antibody
to the
polypeptide under conditions for complex formation. The amount of complex
formation
may be quantified by various methods, such as photometric means. Quantities of
polypeptide expressed in disease samples may be compared with standard values.
Deviation between standard and subject valuesmay establish the parameters for
diagnosing or monitoring disease.
Design of immunoassays is subject to a great deal of variation and a variety
of
these are known in the art. Immunoassays may use a monoclonal or polyclonal
antibody reagent that is directed against one epitope of the antigen being
assayed
Alternatively, a combination of monoclonal or polyclonal antibodies may be
used which
are directed against more than one epitope. Protocols may be based. for
example,
upon competition where one may use competitive drug screening assays in which
neutralizing antibodies capable of binding a polypeptide encoded by the
polynucleotides of NSEQ, or a portion thereof, specifically compete with a
test
compound for binding the polypeptide. Alternatively one may use, direct
antigen-
antibody reactions or sandwich type assays and protocols may. for example,
make use
of solid supports or immunoprecipitation. Furthermore, antibodies may be
labelled with
a reporter molecule for easy detection. Assays that amplify the signal from a
bound
reagent are also known. Examples include immunoassays that utilize avidin and
biotin
or which utilize enzyme-labelled antibody or antigen conjugates, such as ELISA
assays.
Kits suitable for immunodiagnosis and containing the appropriate labelled
reagents include antibodies directed against the polypeptide protein epitopes
or
antigenic regions, packaged appropriately with the remaining reagents and
materials
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
42
required for the conduct of the assay. as well as a suitable set of assay
instructions.
The present invention therefore provides a kit for specifically assaying a
polypeptide described herein, the kit may comprise, for example, an antibody
or
antibody fragment capable of binding specifically to the polypeptide described
herein.
In accordance with the present invention, the kit may be a diagnostic kit,
which
may comprise,
a) one or more antibodies described herein; and
b) a detection reagent which may comprise a reporter group
In accordance with the present invention, the antibodies may be immobilized on
a solid support. The detection reagent may comprise, for example, an anti-
immunoglobulin, protein G. protein A or lectin etc. The reporter group may be
selected,
without limitation, from the group consisting of radioisotopes, fluorescent
groups,
luminescent groups, enzymes, biotin and dye particles.
In an additional aspect, the present invention provides a method for
identifying an
inhibitory compound (inhibitor, antagonist) which may be able to impair the
function
(activity) or expression of a polypeptide described herein, such as, for
example. those
which may be selected from the group consisting of SEQ ID NO ; 48 to 80 or a
polypeptide encoded by SEQ ID NO 85 or SEQ ID NO '86. and analogs thereof. The
method may comprise contacting the polypeptide or a cell expressing the
polypeptide
with a candidate compound and measuring the function (activity) or expression
of the
polypeptide. A reduction in the function or activity of the polypeptide
(compared to the
absence of the candidate compound) may positively identify a suitable
inhibitory
compound.
In accordance with the present invention; the impaired function or activity
may
be associated with a reduced ability of the polypeptide to promote osteoclast
differentiation, such as osteoclast differentiation induced by an inducer
described
herein or known in the art.
In accordance with the present invention the cell may not naturally
(endogenously) express (polypeptide may substantially be unexpressed in a
cell) the
polypeptide or analog or alternatively, the expression of a naturally
expressed
polypeptide analog may be repressed.
For example, suitable method of screening for an inhibitor of SEQ ID NO 1,
CA 02785046 2012-08-02
WO 2007/093042 PC T/CA 200
7/000 2 10
43
may comprise repressing the expression of the mouse ortholog SEQ ID NO.:35 in
a
mouse osteoclast cell and evaluating differentiation of the osteoclast cell
comprising
SEQ ID NO. :1 in the presence or absence of a candidate inhibitor and for
example, an
inducer of osteoclast differentiation (e.g.. RANKLY
The present invention also provides a method for identifying an inhibitory
compound (inhibitor, antagonist) able to impair the function (activity) or
expression of a
polypeptide such as, for example SEQ ID NO 1 or SEQ ID NO .2 The method may
comprise, for example, contacting the (isolated) polypeptide or a cell
expressing the
polypeptide with a candidate compound and measuring the function (activity) or
expression of the polypeptide. A reduction in the function or activity of the
polypeptide
(compared to the absence of the candidate compound) may thus positively
identify a
suitable inhibitory compound.
In accordance with the present invention, the impaired function or activity
may
be associated, for example, with a reduced ability of the polypeptide to
inhibit or
promote osteoclast differentiation.
The cell used to carry the screening test may not naturally (endogenously)
express the polypeptide or analogs, or alternatively the expression of a
naturally
expressed polypeptide analog may be repressed.
The present invention also relates to a method of identifying a positive or a
negative regulator of osteoclast differentiation_ The method may comprise
for
example. performing a knockdown effect as described herein. The method may
more
particularly comprise a) providing an osteoclast cell with a compound (e g
siRNA) able
to specifically inhibit a target sequence (e.g.. a polynucleotide or
polypeptide as
described herein), b) inducing differentiation (e.g., with an inducer such as.
for
example, RANKL) and c) determining the level of differentiation of the
osteoclast cell
(e.g., measuring the number of differentiated cells, their rate of
differentiation, specific
marker of differentiation etc).
Upon inhibition of a positive regulator, the levels of osteoclast
differentiation will
appear lowered. Upon inhibition of a negative regulator, the level of
osteoclast
differentiation will apear increased.
Another method of identifying a positive or a negative regulator of osteoclast
differentiation is to a) provide a cell with one of a target sequence
described herein
(polypeptide or polynucleotide able to express a polypeptide) b) to induce
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
44
differentiation (e.g., with an inducer such as, for example, RANKL) and c) to
determine
the level of differentiation of the osteoclast cell (e.g., measuring the
number of
differentiated cells, their rate of differentiation, specific marker of
differentiation etc).
A cell provided with a positive regulator of osteoclast differentiation may
have
an increased level of differentiation. A cell provided with a negative
regulator of
osteoclast differentiation may have a decreased level of differentiation.
The present invention aslo provides a method of identifying a compound
capable of interfering with osteoclast differentiation, the method may
comprise
contacting a cell including therein a non-endogenous polynucleotide sequence
comprising any one of SEQ ID NO.:1 to 33. 85 or 86 (a coding portion) and
quantifying
(e.g. the number of) differentiated osteoclasts. A reduction in osteoclast
differentiation
in the presence of the compound in comparison to the absence of the compound
may
be indicative of an antagonist of osteoclast differentiation, while an
increase in
osteoclast differentiation in the presence of the compound in comparison to
the
absence of the compound may be indicative of an agonist of osteoclast
differentiation.
In accordance with the present invention, the cell may also comprise an
endogenous form of a polynucleotide.
As used herein the term "endogenous" means a substance that naturally
originates from within an organism, tissue or cell. The term
"endogenous
polynucleotide" refers to a chromosomal form of a polynucleotide or RNA
version
(hnRNA. mRNA) produced by the chromosal form of the polynucleotide The term
"endogenous polypeptide- refers to the form of the protein encoded by an -
endogenous
polynucleotide".
As used herein the term "non-endogenous" or -exogenous" is used in
opposition to "endogenous" in that the substance is provided from an external
source
although it may be introduced within the cell. The term "non-endogenous
polynucleotide" refers to a synthetic polynucleotide introduced within the
cell and
include for example and without limitation, a vector comprising a sequence of
interest,
a synthetic mRNA, an oligonucleotide comprising a NSEQ etc. The term "non-
endogenous polypeptide" refers to the form of the protein encoded by an "non-
endogenous polynucleotide",
The present invention also relate to a method of identifying a compound
capable of interfering with osteoclast differentiation, the method may
comprise
CA 02785046 2013-02-08
contacting a cell including therein a non-endogenous polypeptide sequence
comprising any one of SEQ ID NO.: 48 to 80 and quantifying (e.g. the number
of)
differentiated osteoclasts. A reduction in osteoclast differentiation in the
presence of
the compound in comparison to the absence of the compound may be indicative of
5 an antagonist of osteoclast differentiation while an increase in osteoclast
differentiation in the presence of the compound in comparison to the absence
of the
compound may be indicative of an agonist of osteoclast differentiation.
As used herein the term "sequence identity" relates to (consecutive)
nucleotides of a nucleotide sequence which with reference to an original
nucleotide
10 sequence. The identity may be compared over a region or over the
total sequence of
a nucleic acid sequence.
Thus, "identity" may be compared, for example, over a region of 3, 4, 5, 10,
19, 20 nucleotides or more (and any number there between). It is to be
understood
herein that gaps of non-identical nucleotides may be found between identical
nucleic
15 acids. For
example, a polynucleotide may have 100% identity with another
polynucleotide over a portion thereof. However, when the entire sequence of
both
polynucleotides is compared, the two polynucleotides may have 50% of their
overall
(total) sequence identical to one another.
Polynucleotides of the present invention or portion thereof having from about
20 50 to about 100%, or about 60 to about 100% or about 70 to about
100% or about 80
to about 100% or about 85%, about 90%, about 95% to about 100% sequence
identity with an original polynucleotide are encompassed herewith. It is known
by
those of skill in the art, that a polynucleotide having from about 50% to 100%
identity
may function (e.g., anneal to a substantially complementary sequence) in a
manner
25 similar to an original polynucleotide and therefore may be used in
replacement of an
original polynucleotide. For example a polynucleotide (a nucleic acid
sequence) may
comprise or have from about 50% to 100% identity with an original
polynucleotide
over a defined region and may still work as efficiently or sufficiently to
achieve the
present invention.
30
Percent identity may be determined, for example, with an algorithm GAP,
BESTFIT', or FASTA in the Wisconsin Genetics Software Package Release 7.0,
using default gap weights.
As used herein the terms " sequence complementarity" refers to (consecutive)
nucleotides of a nucleotide sequence which are complementary to a reference
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/1)00210
46
(original) nucleotide sequence The complementarity may be compared over a
region
or over the total sequence of a nucleic acid sequence.
Polynucleotides of the present invention or portion thereof having from about
50
to about 100%, or about 60 to about 100% or about 70 to about 100% or about 80
to
about 100% or about 85%, about 90%, about 95% to about 100% sequence
complementarity with an original polynucleotide are thus encompassed herewith.
It is
known by those of skill in the art, that an polynucleotide having from about
50% to
100% complementarity with an original sequence may anneal to that sequence in
a
manner sufficient to carry out the present invention (e.g., inhibit expression
of the
original polynucleotide).
An "analogue" is to be understood herein as a molecule having a biological
activity and chemical structure similar to that of a polypeptide described
herein. An
"analogue" may have sequence similarity with that of an original sequence or a
portion
of an original sequence and may also have a modification of its structure as
discussed
herein. For example, an "analogue" may have at least 90 A) sequence
similarity with
an original sequence or a portion of an original sequence. An "analogue" may
also
have, for example: at least 70 A) or even 50 A sequence similarity (or less,
i.e.. at least
40%) with an original sequence or a portion of an original sequence
Also, an "analogue" with reference to a polypeptide may have, for example, at
least 50 % sequence similarity to an original sequence with a combination of
one or
more modification in a backbone or side-chain of an amino acid, or an addition
of a
group or another molecule, etc.
"Polynucleotide" generally refers to any polyribonucleotide or polydeoxyribo-
nucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA.
"Polynucleotides" include, without limitation single- and double-stranded DNA,
DNA
that is a mixture of single- and double-stranded regions, single- and double-
stranded
RNA, and RNA that is a mixture of single- and double-stranded regions hybrid
molecules comprising DNA and RNA that may be single-stranded or more typically
double-stranded or a mixture of single- and double-stranded regions In
addition,
"polynucleotide" refers to triple-stranded regions comprising RNA or DNA or
both RNA
and DNA. The term polynucleotide also includes DNAs or RNAs containing one or
more modified bases and DNAs or RNAs with backbones modified for stability or
for
other reasons. "Modified" bases include, for example, tritylated bases and
unusual
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
47
bases such as inosine. A variety of modifications may be made to DNA and RNA.
thus
"polynucleotide" embraces chemically. enzymatically or metabolically modified
forms of
polynucleotides as typically found in nature, as well as the chemical forms of
DNA and
RNA characteristic of viruses and cells. 'Polynucleotide" includes but is not
limited to
linear and end-closed molecules. "Polynucleotide" also embraces relatively
short
polynucleotides, often referred to as oligonucleotides.
"Polypeptides" refers to any peptide or protein comprising two or more amino
acids joined to each other by peptide bonds or modified peptide bonds (i.e.,
peptide
isosteres). "Polypeptide" refers to both short chains, commonly referred as
peptides,
oligopeptides or oligomers, and to longer chains generally referred to as
proteins. As
described above, polypeptides may contain amino acids other than the 20 gene-
encoded amino acids.
As used herein the term "polypeptide analog" relates to mutants, variants,
chimeras. fusions, deletions, additions and any other type of modifications
made
relative to a given polypeptide.
As used herein the term "biologically active" refers to a variant or fragment
which
retains some or all of the biological activity of the natural polypeptide,
i.e., to be able to
promote or inhibit osteoclast differentiation. Polypeptides or fragments of
the present
invention may also include "immunologically active" polypeptides or fragments.
"Immunologically active polypeptides or fragments may be useful for
immunization
purposes (e.g. in the generation of antibodies).
Thus. biologically active polypeptides in the form of the original
polypeptides,
fragments (modified or not), analogues (modified or not), derivatives
(modified or not).
homologues, (modified or not) of the polypeptides described herein are
encompassed
by the present invention.
Therefore, any polypeptide having a modification compared to an original
polypeptide which does not destroy significantly a desired biological activity
is
encompassed herein It is well known in the art, that a number of modifications
may be
made to the polypeptides of the present invention without deleteriously
affecting their
biological activity. These modifications may, on the other hand, keep or
increase the
biological activity of the original polypeptide or may optimize one or more of
the
particularity (e.g. stability, bioavailability, etc.) of the polypeptides of
the present
invention which, in some instance might be desirable. Polypeptides of the
present
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
48
invention may comprise for example, those containing amino acid sequences
modified
either by natural processes, such as posttranslational processing, or by
chemical
modification techniques which are known in the art. Modifications may occur
anywhere
in a polypeptide including the polypeptide backbone the amino acid side-chains
and
the amino- or carboxy-terminus. It will be appreciated that the same type
of
modification may be present in the same or varying degrees at several sites in
a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. It is to
be understood herein that more than one modification to the polypeptides
described
herein are encompassed by the present invention to the extent that the
biological
.. activity is similar to the original (parent) polypeptide.
As discussed above, polypeptide modification may comprise, for example,
amino acid insertion (i.e., addition), deletion and substitution (i.e.,
replacement), either
conservative or non-conservative (e.g., D-amino acids, desamino acids) in the
polypeptide sequence where such changes do not substantially alter the overall
biological activity of the polypeptide.
Example of substitutions may be those, which are conservative (i.e., wherein a
residue is replaced by another of the same general type or group) or when
wanted
non-conservative (i.e., wherein a residue is replaced by an amino acid of
another type)
In addition a non-naturally occurring amino acid may substitute for a
naturally
occurring amino acid (i.e., non-naturally occurring conservative amino acid
substitution
or a non-naturally occurring non-conservative amino acid substitution).
As is understood, naturally occurring amino acids may be sub-classified as
acidic, basic, neutral and polar, or neutral and non-polar. Furthermore, three
of the
encoded amino acids are aromatic. It may be of use that encoded polypeptides
differing from the determined polypeptide of the present invention contain
substituted
codons for amino acids, which are from the same type or group as that of the
amino
acid to be replaced. Thus, in some cases, the basic amino acids Lys, Arg and
His may
be interchangeable; the acidic amino acids Asp and Glu may be interchangeable,
the
neutral polar amino acids Ser, Thr, Cys, Gln. and Asn may be interchangeable:
the
non-polar aliphatic amino acids Gly, Ala, Val. Ile, and Leu are
interchangeable but
because of size Gly and Ala are more closely related and Val, Ile and Leu are
more
closely related to each other. and the aromatic amino acids Phe. Trp and Tyr
may be
interchangeable_
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
49
It should be further noted that if the polypeptides are made synthetically,
substitutions by amino acids, which are not naturally encoded by DNA (non-
naturally
occurring or unnatural amino acid) may also be made.
A non-naturally occurring amino acid is to be understood herein as an amino
acid which is not naturally produced or found in a mammal A non-naturally
occurring
amino acid comprises a D-amino acid, an amino acid having an acetylaminomethyl
group attached to a sulfur atom of a cysteine. a pegylated amino acid. etc.
The
inclusion of a non-naturally occurring amino acid in a defined polypeptide
sequence will
therefore generate a derivative of the original polypeptide. Non-naturally
occurring
amino acids (residues) include also the omega amino acids of the formula
NH2(CH2),COOH wherein n is 2-6, neutral nonpolar amino acids, such as
sarcosine, t-
butyl alanine, t-butyl glycine, N-methyl isoleucine, norleucine, etc.
Phenylglycine may
substitute for Trp. Tyr or Phe; citrulline and methionine sulfoxide are
neutral nonpolar.
cysteic acid is acidic, and ornithine is basic. Proline may be substituted
with
hydroxyproline and retain the conformation conferring properties.
It is known in the art that analogues may be generated by substitutional
mutagenesis and retain the biological activity of the polypeptides of the
present
invention. These analogues have at least one amino acid residue in the protein
molecule removed and a different residue inserted in its place. For example,
one site
of interest for substitutional mutagenesis may include but are not restricted
to sites
identified as the active site(s). or immunological site(s) Other sites of
interest may be
those, for example, in which particular residues obtained from various species
are
identical. These
positions may be important for biological activity Examples of
substitutions identified as "conservative substitutions" are shown in Table A.
If such
substitutions result in a change not desired, then other type of
substitutions.
denominated -exemplary substitutions" in Table A, or as further described
herein in
reference to amino acid classes, are introduced and the products screened.
In some cases it may be of interest to modify the biological activity of a
polypeptide by amino acid substitution, insertion, or deletion. For
example.
modification of a polypeptide may result in an increase in the polypeptide's
biological
activity, may modulate its toxicity, may result in changes in bioavailability
or in stability.
or may modulate its immunological activity or immunological identity
Substantial
modifications in function or immunological identity are accomplished by
selecting
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
substitutions that differ significantly in their effect on maintaining (a) the
structure of the
polypeptide backbone in the area of the substitution, for example, as a sheet
or helical
conformation. (b) the charge or hydrophobicity of the molecule at the target
site, or (c)
the bulk of the side chain. Naturally occurring residues are divided into
groups based
5 on common side chain properties:
(1) hydrophobic: norleucine. methionine (Met), Alanine (Ala), Valine (Val),
Leucine
(Leu), lsoleucine (Ile)
(2) neutral hydrophilic: Cysteine (Cys), Serine (Ser). Threonine (Thr)
(3) acidic: Aspartic acid (Asp), Glutamic acid (Glu)
10 (4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His). Lysine
(Lys)
Arginine (Arg)
(5) residues that influence chain orientation: Glycine (Gly), Proline (Pro);
and
aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe)
Non-conservative substitutions will entail exchanging a member of one of these
15 classes for another.
TABLE A. Examplary amino acid substitution
Original residue Exemplary substitution = Conservative
substitution
Ala (A) Val. Leu, Ile Val
Arg (R) Lys. Gin, Asn Lys
Asn (N) Gin. His, Lys. Arg Gin
Asp (D) Glu IGlu
Cys (C) Ser Ser
Gln (Q) Asn Asn
Glu (E) Asp Asp
Gly (G) Pro Pro
His (H) Asn, Gin, Lys, Arg Arg
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
51
!Original residue 'Exemplary substitution r Conservative
substitution
Ile (I) Leu, Val, Met. Ala, Phe, Leu
norleucine
Leu (L) Norleucine, Ile, Val, Met, Ile
Ala, Phe
Lys (K) Arg, Gin, Asn Arg
Met (M) Leu, Phe. Ile Leu
=
Phe (F) . Leu, Val. Ile, Ala Leu
Pro (P) Gly Gly
Ser (S) = Thr Thr
Thr (T) Ser f Ser
Tip (W) Tyr ; Tyr
Tyr (Y) Trp, Phe, Thr, Ser Phe
Val (V) Ile, Leu, Met, Phe, Ala, Leu
norleucine
It is to be understood herein; that if a "range" or "group" of substances
(e.g.
amino acids). substituents" or the like is mentioned or if other types of a
particular
characteristic (e.g. temperature. pressure. chemical structure, time, etc) is
mentioned,
the present invention relates to and explicitly incorporates herein each and
every
specific member and combination of sub-ranges or sub-groups therein whatsoever
Thus, any specified range or group is to be understood as a shorthand way of
referring
to each and every member of a range or group individually as well as each and
every
possible sub-ranges or sub-groups encompassed therein; and similarly with
respect to
any sub-ranges or sub-groups therein. Thus, for example, with respect to a
percentage
(c)/0) of identity of from about 80 to 100%, it is to be understood as
specifically
incorporating herein each and every individual %, as well as sub-range, such
as for
example 80%, 81%, 84.78%. 93%, 99% etc.; and similarly with respect to other
parameters such as, concentrations, elements, etc.
CA 02785046 2013-02-08
52
It is in particular to be understood herein that the methods of the present
invention each include each and every individual steps described thereby as
well as
those defined as positively including particular steps or excluding particular
steps or a
combination thereof; for example an exclusionary definition for a method of
the
present invention, may read as follows: "provided that said polynucleotide
does not
comprise or consist in SEQ ID NO.:34 or the open reading frame of SEQ ID
NO.:34"
or "provided that said polypeptide does not comprise or consist in SEQ ID
NO.:82" or
"provided that said polynucleotide fragment or said polypeptide fragment is
less than
X unit (e.g., nucleotides or amino acids) long or more than X unit (e.g.,
nucleotides or
amino acids) long".
Other objects, features, advantages, and aspects of the present invention will
become apparent to those skilled in the art from the following description.
BRIEF DESCRIPTION OF THE DRAWINGS
In the appended drawings:
For each of Figures 1 to 34 and 38-39 macroarrays were prepared using RAMP
amplified RNA from human precursor cells (A-F 1), and differentiated
intermediate (A-F
2 ¨ 3) and mature osteoclasts for four human donors (A-F 4), and 30 different
normal
human tissues (adrenal (A5), liver (B5), lung (C5), ovary (D5), skeletal
muscle (E5),
heart (F5), cervix (G5), thyroid (H5), breast (A6), placenta (B6), adrenal
cortex (C6),
kidney (D6), vena cava (E6), fallopian tube (F6), pancreas (G6), testicle
(H6), jejunum
(A7), aorta (B7), esophagus (C7), prostate (D7), stomach (E7), spleen (F7),
ileum (G7),
trachea (A8), brain (B8), colon (C8), thymus (D8), small intestine (E8),
bladder (F8) and
duodenum (G8)). The STAR dsDNA clone representing the respective SEQ ID NOs.
was labeled with 32P and hybridized to the macroarray. The probe labeling
reaction was
also spiked with a dsDNA sequence for Arabidopsis, which hybridizes to the
same
sequence spotted on the macroarray (M) in order to serve as a control for the
labeling
reaction. Quantitation of the hybridization signal at each spot was performed
using a
CA 02785046 2013-02-08
53
STORM 820 PhosphorimagerTM and the lmageQuantTM TL software (Amersham
Biosciences, Piscataway, NJ). A log2 value representing the average of the
signals for
the precursors (A-F 1) was used as the baseline and was subtracted from the
log2
value obtained for each of the remaining samples in order to determine their
relative
abundancies compared to the precursors and plotted as a bar graph (right
panel).
Fig. 1 is a picture of the macroarray hybridization results and quantitation
of the
signal intensities showing the differential expression data for STAR selected
osteoclast-specific human SEQ. ID. NO. 1. The hybridization results obtained
confirms
its upregulation in all of the human osteoclast samples with generally higher
expression
in the more mature osteoclasts (A-F 2 ¨ 4) compared to the precursors (A-F 1)
and little
or no expression in all or most normal tissues (A-H 5 ¨ 6 and A-G 7 ¨ 8);
Fig. 2 is a picture of the macroarray hybridization results and quantitation
of the
signal intensities showing the differential expression data for STAR selected
osteoclast-specific human SEQ. ID. NO. 2. The hybridization results obtained
confirms
its upregulation in all of the human osteoclast samples with generally higher
expression
in the more mature osteoclasts (A-F 2 ¨4) compared to the precursors (A-F 1)
and little
or no expression in all or most normal tissues (A-H 5 ¨ 6 and A-G 7 ¨ 8);
Fig. 3 is a picture of the macroarray hybridization results and quantitation
of the
signal intensities showing the differential expression data for STAR selected
osteoclast-specific human SEQ. ID. NO. 3. The hybridization results obtained
confirms
its upregulation in all of the human osteoclast samples with generally higher
expression
in the more mature osteoclasts (A-F 2 ¨ 4) compared to the precursors (A-F 1)
and little
or no expression in all or most normal tissues (A-H 5 ¨ 6 and A-G 7 ¨ 8);
Fig. 4 is a picture of the macroarray hybridization results and quantitation
of the
signal intensities showing the differential expression data for STAR selected
osteoclast-specific human SEQ. ID. NO. 4. The hybridization results obtained
confirms
its upregulation in all of the human osteoclast samples with generally higher
expression
in the more mature osteoclasts (A-F 2 ¨4) compared to the precursors (A-F 1)
and little
or no expression in all or most normal tissues (A-H 5 ¨ 6 and A-G 7 ¨ 8);
Fig. 5 is a picture of the macroarray hybridization results and quantitation
of the
signal intensities showing the differential expression data for STAR selected
osteoclast-specific human SEQ. ID. NO. 5. The hybridization results obtained
confirms
its upregulation in all of the human osteoclast samples with generally higher
expression
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
54
in the more mature osteoclasts (A-F 2 - 4) compared to the precursors (A-F 1)
and little
or no expression in all or most normal tissues (A-H 5 - 6 and A-G 7 - 8):
Fig. 6 is a picture of the macroarray hybridization results and quantitation
of the
signal intensities showing the differential expression data for STAR selected
.. osteoclast-specific human SEQ ID NO. 6 The hybridization results obtained
confirms
its upregulation in all of the human osteoclast samples with generally higher
expression
in the more mature osteoclasts (A-F 2 - 4) compared to the precursors (A-F 1)
and little
or no expression in all or most normal tissues (A-H 5 - 6 and A-G 7 -
Fig. 7 is a picture of the macroarray hybridization results and quantitation
of the
signal intensities showing the differential expression data for STAR selected
osteoclast-specific human SEQ. ID. NO. 7. The hybridization results obtained
confirms
its upregulation in all of the human osteoclast samples with generally higher
expression
in the more mature osteoclasts (A-F 2 - 4) compared to the precursors (A-F 1)
and little
or no expression in all or most normal tissues (A-H 5 - 6 and A-G 7 - 8):
Fig. 8 is a picture of the macroarray hybridization results and quantitation
of the
signal intensities showing the differential expression data for STAR selected
osteoclast-specific human SEQ. ID. NO 8. The hybridization results obtained
confirms
its upregulation in all of the human osteoclast samples with generally higher
expression
in the more mature osteoclasts (A-F 2 - 4) compared to the precursors (A-F 1)
and little
or no expression in all or most normal tissues (A-H 5 - 6 and A-G 7 - 8):
Fig. 9 is a picture of the macroarray hybridization results and quantitation
of the
signal intensities showing the differential expression data for STAR selected
osteoclast-specific human SEQ. ID. NO. 9. The hybridization results obtained
confirms
its upregulation in all of the human osteoclast samples with generally higher
expression
in the more mature osteoclasts (A-F 2 - 4) compared to the precursors (A-F 1)
and little
or no expression in all or most normal tissues (A-H 5 - 6 and A-G 7 - 8):
Fig. 10 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO, 10. The hybridization results obtained
.. confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 - 6 and A-
G 7 -8).
Fig. 11 is a picture of the macroarray hybridization results and quantitation
of
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 11. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
5 1) and little or no expression in all or most normal tissues (A-H 5 -6
and A-G 7 - 8):
Fig. 12 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 12. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
10 expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 - 6 and A-
G 7 - 8.:
Fig. 13 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 13. The hybridization results obtained
15 confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 -6 and AG
7 - 8).
Fig. 14 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
20 osteoclast-specific human SEQ. ID, NO. 14. The hybridization results
obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 - 6 and A-
G 7 8):
Fig. 15 is a picture of the macroarray hybridization results and quantitation
of
25 the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 15. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 -4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 - 6 and A-
G 7 - 8):
30 Fig. 16 is a
picture of the macroarray hybridization results and quantitation of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 16 The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher,
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
56
expression in the more mature osteoclasts (A-F 2 ¨ 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 ¨ 6 and A-
G 7 ¨8);
Fig. 17 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 17. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 ¨ 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 ¨ 6 and A-
G 7 ¨ 8..
Fig 18 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 18. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 ¨ 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 ¨ 6 and A-
G 7 ¨ 8):
Fig. 19 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 19. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 ¨ 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 ¨ 6 and A-
G 7 ¨ 8):
Fig. 20 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ ID, NO. 20. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 ¨ 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 ¨ 6 and A-
G 7 ¨ 8):
Fig. 21 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 21. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 ¨ 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 ¨ 6 and A-
G 7 ¨ 8):
Fig. 22 is a picture of the macroarray hybridization results and quantitation
of
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
57
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 22. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 -6 and A-G
7 - 8);
Fig. 23 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 23. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 -6 and A-G
7 - 8):
Fig 24 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 24. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 - 6 and A-
G 7 - 8);
Fig. 25 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 25. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 - 6 and A-
G 7 - 8);
Fig. 26 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 26. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 -4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 - 6 and A-
G 7 -8):
Fig. 27 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID, NO. 27. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
58
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 -6 and A-G
7 - 8);
Fig. 28 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 28. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 - 6 and A-
G 7 - 8);
Fig. 29 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 29. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 - 6 and A-
G 7 - 8).
Fig. 30 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 30. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 -6 and A-G
7 - 8);
Fig. 31 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO, 31. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 - 6 and A-
G 7 - 8):
Fig. 32 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 32. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 - 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 - 6 and A-
G 7 -8).
Fig. 33 is a picture of the macroarray hybridization results and quantitation
of
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
59
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 33. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 ¨ 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 ¨ 6 and A-
G 7 ¨ 8);
Fig. 34 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 34. The hybridization results obtained
confirms its upregulation in all of the human osteoclast samples with
generally higher
expression in the more mature osteoclasts (A-F 2 ¨ 4) compared to the
precursors (A-F
1) and little or no expression in all or most normal tissues (A-H 5 ¨ 6 and A-
G 7 ¨ 8),
Fig. 35 is a picture showing the knockdown effects on osteoclastogenesis by
attenuating the endogenous expression of SEQ. ID. NO. 1 (A80326) and SEQ. ID.
NO.
2 (AB0369) using shRNA. A significant decrease in the number of multinucleated
osteoclasts was observed from precursor cells infected with the AB0326 shRNA
(Figure 35A; bottom panel) and AB0369 shRNA (Figure 1B, bottom panel) compared
to
those with the lacZ shRNA (Figure 35A and B, top panels). These results
clearly
indicated that expression of the gene encoding SEQ. ID. NO. 1 (AB0326) and
SEQ. ID,
NO. 2 (AB0369) are required for osteoclast differentiation;
Fig. 36 is a picture showing the knockdown effects on osteoclastogenesis of
the
mouse orthologue for AB0326 (SEQ. ID. NO. 35) in the RAW 264.7 model using
shRNA-0326.2 (SEQ. ID. NO. 45). The RAW-0326.2 cell line produced
significantly
less osteoclasts (Figure 36; bottom panel) compared to the cell line
containing the
scrambled shRNA (Figure 36; top panel). This result, coupled with that
obtained in the
human osteoclast precursor cells using the lentiviral shRNA delivery system
demonstrate that in both human and mouse, AB0326 gene product is clearly
required
for osteoclastogenesis;
Fig. 37 is a picture showing the results of a functional complementation assay
for SEQ. ID. NO. 1 (AB0326) in RAW-0326.2 cells to screen for inhibitors of
osteoclastogenesis. The RAW-0326.2 cells transfected with the empty pd2 vector
are
unable to form osteoclasts in the presence of RANK ligand (center panel)
indicating
that the mouse AB0326 shRNA is still capable of silencing the AB0326 gene
expression in these cells. Conversely, the cells transfected with the cDNA for
the
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
human AB0326 (pd2-hAB0326) are rescued and thus, differentiate more
efficiently into
osteoclasts in response to RANK ligand (right panel). Wild-type RAW 264.7
cells
containing the empty vector (pd2) did not adversly affect the formation of
osteoclasts in
the presence of RANK ligand (left panel) ruling out an effect due to pd2 Thus,
this
5 complementation assay can be used to screen for inhibitors of the human
AB0326
polypeptide;
Fig. 38 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential Expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 85. Macroarrays were prepared using
RAMP
10 amplified RNA from human precursor cells (A-F 1), and differentiated
intermediate and
mature osteoclasts for four human donors (A-F 2 - 4), and 30 different normal
human
tissues (adrenal. liver, lung, ovary, skeletal muscle, heart, cervix, thyroid.
breast,
placenta, adrenal cortex, kidney, vena cava, fallopian tube. pancreas,
testicle, jejunum,
aorta, esophagus, prostate. stomach, spleen, ileum, trachea, brain, colon,
thymus.
15 small intestine, bladder and duodenum (A-H 5 - 6 and A-G 7 - 8)). The
STAR clone
representing SEQ. ID. NO. 85 was labeled with 32P and hybridized to the
macroarray
The hybridization results obtained confirms its upregulation in all of the
human
osteoclast samples with generally higher expression in the more mature
osteoclasts (A-
F 2 - 4) compared to the precursors (Al - Fl) and little or no expression in
all or most
20 normal tissues (A-H 5 - 6 and A-G 7 - 8), and,
Fig. 39 is a picture of the macroarray hybridization results and quantitation
of
the signal intensities showing the differential Expression data for STAR
selected
osteoclast-specific human SEQ. ID. NO. 86. Macroarrays were prepared using
RAMP
amplified RNA from human precursor cells (A-F 1). and differentiated
intermediate and
25 mature osteoclasts for four human donors (A-F 2 - 4), and 30 different
normal human
tissues (adrenal, liver, lung, ovary, skeletal muscle, heart, cervix, thyroid,
breast,
placenta, adrenal cortex, kidney, vena cava, fallopian tube, pancreas,
testicle. jejunum,
aorta, esophagus, prostate, stomach, spleen, ileum, trachea, brain, colon,
thymus.
small intestine, bladder and duodenum (A-H 5 - 6 and A-G 7 - 8)). The STAR
clone
30 representing SEQ. ID NO. 86 was labeled with 32P and hybridized to the
macroarray
The hybridization results obtained confirms its upregulation in all of the
human
osteoclast samples with generally higher expression in the more mature
osteoclasts (A-
F 2 -4) compared to the precursors (Al - Fl) and little or no expression in
all or most
normal tissues (A-H 5 - 6 and A-G 7 - 8).
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
61
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The applicant employed a carefully planned strategy to identify and isolate
genetic sequences involved in osteoclastogenesis and bone remodeling. The
process
involved the following steps: 1) preparation of highly representative cDNA
libraries
using mRNA isolated from precursors and differentiated intermediate and mature
osteoclasts of human origin; 2) isolation of sequences upregulated during
osteoclastogenesis: 3) identification and characterization of upregulated
sequences: 4)
selection of upregulated sequences for tissue specificity; and 5)
determination of
knock-down effects on osteoclastogenesis. The results discussed in this
disclosure
demonstrate the advantage of targeting osteoclast genes that are specific to
this
differentiated cell type and provide a more efficient screening method when
studying
the genetic basis of diseases and disorders. Genes that are known to have a
role in
other areas of biology have been shown to play a critical role in
osteoclastogenesis and
osteoclast function. Genes that are known but have not had a role assigned to
them
until the present disclosure have also been isolated and shown to have a
critical role in
osteoclastogenesis and osteoclast function. Finally, novel genes have been
identified
and play a role, however, applicant reserves their disclosure until further
study has
been completed:
The present invention is illustrated in further details below in a non-
limiting
fashion.
A- Material and Methods
Commercially available reagents referred to in the present disclosure were
used
according to supplier's instructions unless otherwise indicated. Throughout
the present
disclosure certain starting materials were prepared as follows:
B - Preparation of osteoclast differentiated cells
The RAW 264.7 (RAW) osteoclast precursor cell line and human precursor cells
(peripheral blood mononuclear cells or CD34+ progenitors) are well known in
the art as
murine and human models of osteoclastogenesis. These murine and human
osteoclasts are therefore excellent sources of materials for isolating and
characterizing
CA 02785046 2012-08-02
62
genes specialized for osteoclast function.
Human primary osteoclasts were differentiated from G-CSF-mobilized peripheral
blood mononuclear cells (Cambrex, East Rutherford, NJ) as described by the
supplier in
the presence of 35 ng/ml M-CSF and 100 ng/ml RANK ligand. Multinucleated TRAP-
staining osteoclasts were visible by 11-14 days. Osteoclasts were also derived
from
human osteoclasts precursor cells (CD34+ progenitors) (Cambrex, East
Rutherford, NJ)
and cultured as described by the supplier. In the latter case, osteoclasts
were obtained
after 7 days.
RAW cells were purchased from American Type Culture Collection and
maintained in high glucose DMEM containing 10% fetal bovine serum and
antibiotics.
The cells were sub-cultured bi-weekly to a maximum of 10-12 passages. For
osteoclast
differentiation experiments, RAW cells were seeded in 96-well plates at a
density of 4 x
103 cells/well and allowed to plate for 24h. Differentiation was induced in
high glucose
DMEM, 10% charcoal-treated foetal bovine serum (Hyclone, Logan, UT), 0.05%
BSA,
antibiotics, 10 ng/ml macrophage colony stimulating factor (M-CSF), and 100
ng/ml
receptor activator of NF-kB (RANK) ligand. The plates were re-fed on day 3 and
osteoclasts were clearly visible by day 4. Typically, the cells were stained
for tartrate-
resistant acid phosphatase (TRAP) on day 4 or 5 unless otherwise indicated.
For TRAP
staining, the cells were washed with PBS and fixed in 10% formaldehyde for 1
h. After
two PBS washes, the cells were rendered lightly permeable in 0.2% TritonTm X-
100 in
PBS for 5 min before washing in PBS. Staining was conducted at 37 C for 20-25
min in
0.01% Naphtol AS-MX phosphate, 0.06% Fast Red Violet, 50 mM sodium tartrate,
100
mM sodium acetate, pH 5.2. Cells were visualized microscopically.
C- Method of Isolating Differentially Expressed mRNA
Key to the discovery of differentially expressed sequences unique to
osteoclasts
is the use of the applicant's patented STAR technology (Subtractive
Transcription-based
Amplification of mRNA; U.S. Patent No. 5,712,127 Malek et al., issued on
January 27,
1998). In this procedure, mRNA isolated from intermediate and mature
osteoclasts is
used to prepare "tester RNA", which is hybridized to complementary single-
stranded
"driver DNA" prepared from osteoclast precursor mRNA and only the un-
hybridized
"tester RNA" is recovered, and used to create cloned cDNA libraries, termed
"subtracted
libraries". Thus, the "subtracted libraries" are enriched for
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
63
differentially expressed sequences inclusive of rare and novel mRNAs often
missed by
micro-array hybridization analysis. These rare and novel mRNA are thought to
be
representative of important gene targets for the development of better
diagnostic and
therapeutic strategies.
The clones contained in the enriched "subtracted libraries' are identified by
DNA sequence analysis and their potential function assessed by acquiring
information
available in public databases (NCB' and GeneCard). The non-redundant clones
are
then used to prepare DNA micro-arrays, which are used to quantify their
relative
differential expression patterns by hybridization to fluorescent cDNA probes.
Two
classes of cDNA probes may be used those which are generated from either RNA
transcripts prepared from the same subtracted libraries (subtracted probes) or
from
mRNA isolated from different osteoclast samples (standard probes). The use of
subtracted probes provides increased sensitivity for detecting the low
abundance
mRNA sequences that are preserved and enriched by STAR. Furthermore, the
specificity of the differentially expressed sequences to osteoclast is
measured by
hybridizing radio-labeled probes prepared from each selected sequence to
macroarrays containing RNA from different osteoclast samples and different
normal
human tissues. Additionally, Northern blot analysis is performed so as to
confirm the
presence of one or more specific mRNA species in the osteoclast samples.
Following
this, the full-length cDNAs representative of the mRNA species and/or spliced
variants
are cloned in E. coli DH10B.
A major challenge in gene expression profiling is the limited quantities of
RNA
available for molecular analysis The amount of RNA isolated from many
osteoclast
samples or human specimens (needle aspiration, laser capture micro-dissection
(LCM)
samples and transfected cultured cells) is often insufficient for preparing:
1)
conventional tester and driver materials for STAR. 2) standard cDNA probes for
DNA
micro-array analysis; 3) RNA macroarrays for testing the specificity of
expression 4)
Northern blots and; 5) full-length cDNA clones for further biological
validation and
characterization etc. Thus, the applicant has developed a proprietary
technology called
RAMP (RNA Amplification Procedure) (U.S. Patent Application No. 11/000,958
published under No. US 2005/0153333A1 on July 14. 2005 and entitled "Selective
Terminal Tagging of Nucleic Acids"), which linearly amplifies the mRNA
contained in
total RNA samples yielding microgram quantities of amplified RNA sufficient
for the
various analytical applications. The RAMP RNA produced is largely full-length
mRNA-
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
64
like sequences as a result of the proprietary method for adding a terminal
sequence tag
to the 3'-ends of single-stranded cDNA molecules, for use in linear
transcription
amplification. Greater than 99.5% of the sequences amplified in RAMP reactions
show
<2-fold variability and thus, RAMP provides unbiased RNA samples in quantities
sufficient to enable the discovery of the unique mRNA sequences involved in
osteoclastogenesis.
D- Preparation of Human Osteoclasts Subtracted Library
Two human primary precursor cells from two different donors (Cambrex. East
Rutherford, NJ), and the corresponding intermediate (day 3 and day 7) and
mature
(days 11-14) osteoclasts were prepared as described above Isolation of
cellular RNA
followed by mRNA purification from each was performed using standard methods
(Qiagen. Mississauga, ON). Following the teachings of Malek et al (U S Patent
No
5,712.127), 2 pg of poly A+ mRNA from each sample were used to prepare highly
representative (>2x106 CFU) cDNA libraries in specialized plasmid vectors
necessary
for preparing tester and driver materials. In each case, first-strand cDNA was
synthesized using an oligo dT" primer with 3' locking nucleotides (e.g., A, G
or C) and
containing a Not I recognition site. Next, second-strand cDNA synthesis was
performed
according to the manufacturer's procedure for double-stranded cDNA synthesis
(Invitrogen, Burlington, ON) and the resulting double-stranded cDNA ligated to
linkers
containing an Asc I recognition site (New England Biolabs, Pickering, ON). The
double-stranded cDNAs were then digested with Asc I and Not I restriction
enzymes
(New England Biolabs, Pickering, ON), purified from the excess linkers using
the cDNA
fractionation column from Invitrogen (Burlington, ON) as specified by the
manufacturer
and each ligated into specialized plasmid vectors - p14 (SEQ. ID. NO 36) and
p17+
(SEQ ID. NO:37) used for preparing tester and driver materials respectively
Thereafter, the ligated cDNAs were transformed into E. coli DH10B resulting in
the
desired cDNA libraries (RAW 264.7-precursor-p14, RAW 264 7-precursor-p17+, RAW
264.7-osteoclasts-p14 and RAW 264.7-osteoclasts-p17+). The plasmid DNA pool
for
each cDNA library was purified and a 2-pg aliquot of each linearized with Not
I
restriction enzyme. In vitro transcription of the Not I digested p14 and p17+
plasmid
libraries was then performed with T7 RNA polymerase and sp6 RNA polymerase
respectively (Ambion, Austin, TX).
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
Next, in order to prepare 3-represented tester and driver libraries, a 10-pg
aliquot of each of the in vitro synthesized RNA was converted to double-
stranded
cDNA by performing first-strand cDNA synthesis as described above followed by
primer-directed (primer OGS 77 for p14 (SEQ. ID. NO:40) and primer OGS 302 for
5 p17+ (SEQ. ID N0,41)) second-strand DNA synthesis using Advantage-2 Taq
polymerase (BD Biosciences Clontech, Mississauga, ON). The sequences
corresponding to OGS 77 and OGS 302 were introduced into the in vitro
synthesized
RNA by way of the specialized vectors used for preparing the cDNA libraries.
Thereafter, 6x 1-pg aliquots of each double-stranded cDNA was digested
individually
10 with one of the following 4-base recognition restriction enzymes Rsa I.
Sau3A1, Mse I,
Msp 1, MinP1 I and Bsh 12361 (MBI Fermentas, Burlington, ON), yielding up to
six
possible 3'-fragments for each RNA species contained in the cDNA library.
Following
digestion, the restriction enzymes were inactivated with phenol and the set of
six
reactions pooled. The restriction enzymes sites were then blunted with T4 DNA
15 polymerase and ligated to linkers containing an Asc 1 recognition site.
Each linker-
adapted pooled DNA sample was digested with Asc I and Not I restriction
enzymes.
desalted and ligated to specialized plasmid vectors, p14 and p17 (p17 plasmid
vector is
similar to the p17+ plasmid vector except for the sequence corresponding to
SEQ. ID.
N0,41), and transformed into E. coli DH10B. The plasmid DNA pool for each p14
and
20 p17 3'-represented library was purified (Qiagen, Mississauga, ON) and a
2-rig aliquot
of each digested with Not 1 restriction enzyme. and transcribed in vitro with
either T7
RNA polymerase or sp6 RNA polymerase (Ambion, Austin. TX). The resulting p14
3'-
represented RNA was used directly as "tester RNA" whereas, the p17 3'-
represented
RNA was used to synthesize first-strand cDNA as described above, which then
served
25 as "driver DNA". Each "driver DNA" reaction was treated with RNase A and
RNase H to
remove the RNA, phenol extracted and desalted before use.
The following 3'-represented libraries were prepared:
Tester 1 (donor 1 ¨ day 3) - human intermediate osteoclast-3' in p14
Tester 2 (donor 1 ¨ day 7 - human intermediate osteoclast)-3' in p14
30 Tester 3 (donor 1 ¨ day 11 - human mature osteoclast)-I in p14
Tester 4 (donor 2 ¨ day 3 - human intermediate osteoclast)-3 in p14
Tester 5 (donor 2 ¨ day 7 - human intermediate osteoclast)-3' in p14
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
66
Tester 6 (donor 2 ¨ day 13 - human mature osteoclast)-3' in p14
Driver 1 (donor 1 ¨ day 3) - human precursor-3' in p17
Driver 2 (donor 2¨ day 3) - human precursor-3' in p17
The tester RNA samples were subtracted following the teachings of U.S patent
No. 5.712,127 with the corresponding driver DNA in a ratio of 1:100 for either
1-or 2-
rounds following the teachings of Malek et at. (U.S. Patent No. 5,712,127).
Additionally,
control reactions containing tester RNA and no driver DNA, and tester RNA plus
driver
DNA but no RNase H were prepared. The tester RNA remaining in each reaction
after
subtraction was converted to double-stranded DNA, and a volume of 5% removed
and
amplified in a standard PCR reaction for 30-cycles for analytical purposes.
The
remaining 95% of only the driver plus RNase H subtracted samples were
amplified for
4-cycles in PCR, digested with Asc I and Not I restriction enzymes, and one
half ligated
into the pCATRMAN (SEQ. ID. NO:38) plasmid vector and the other half, into the
p20
(SEQ. ID. NO:39) plasmid vector. The ligated materials were transformed into
E. coli
DH1OB and individual clones contained in the pCATRMAN libraries were picked
for
further analysis (DNA sequencing and hybridization) whereas, clones contained
in
each p20 library were pooled for use as subtracted probes. Each 4-cycles
amplified
cloned subtracted library contained between 25.000 and 40.000 colonies
The following cloned subtracted libraries were prepared:
SL90 - tester 1 (day 3 osteoclast) minus driver 1 (precursor) (1-round) in
pCATRMAN,
SL91 - tester 2 (day 7 osteoclast) minus driver 1 (precursor) (1-round) in
pCATRMAN:
5L92-tester 3 (day 11 osteoclast) minus driver 1 (precursor) (1-round) in
pCATRMAN:
SL108-tester 1 (day 3 osteoclast) minus driver 1 (precursor) (2-rounds) in
pCATRMAN.
SL109-tester 2 (day 7 osteoclast) minus driver 1 (precursor) (2-rounds) in
pCATRMAN:
SL110-tester3 (day 11 osteoclast) minus driver 1(precursor) (2-rounds) in
pCATRMAN:
SL93 -tester 4 (day 3 osteoclast) minus driver 2 (precursor) (1-round) in
pCATRMAN,
SL94 - tester 5 (day 7 osteoclast) minus driver 2 (precursor) (1-round) in
pCATRMAN,
SL95-tester 6 (day 13 osteoclast) minus driver 2 (precursor) (1-round) in
pCATRMAN,
SL87 - tester 4 (day 3 osteoclast) minus driver 2 (precursor) (2-rounds) in
pCATRMAN
SL88 - tester 5 (day 7 osteoclast) minus driver 2 (precursor) (2-rounds) in
pCATRMAN:
CA 02785046 2012-08-02
67
SL89- tester 6 (day 11 osteoclast) minus driver 2 (precursor) (2-rounds) in
pCATRMAN
A 5-pL aliquot of the 30-cycles PCR amplified subtracted materials described
above
were visualized on a 1.5% agarose gel containing ethidium bromide and then
transferred
to HybondTM N+ (Amersham Biosciences, Piscataway, NJ) nylon membrane for
Southern blot analysis. Using radiolabeled probes specific to the CTSK
(cathepsin K;
NM_000396.2) gene, which is known to be upregulated in osteoclasts, and GAPDH
(glyceraldehyde-3-phosphate dehydrogenase; M32599.1), which is a non-
differentially
expressed house-keeping gene, it was evident that there was subtraction of
GAPDH but
not CTSK. Based on these results, it was anticipated that the subtracted
libraries would
.. be enriched for differentially expressed upregulated sequences.
E - Sequence identification and annotation of clones contained in the
subtracted
libraries:
A total of 6,912 individual colonies contained in the pCATRMAN subtracted
libraries (5L87-95 and SL108-110) described above were randomly picked using a
Qbot
(Genetix Inc., Boston, MA) into 60 pL of autoclaved water. Then, 42 pL of each
was
used in a 100-pL standard PCR reaction containing oligonucleotide primers, OGS
1 and
OGS 142 and amplified for 40-cycles (94 C for 10 minutes, 40x (94 C for 40
seconds,
55 C for 30 seconds and 72 C for 2 minutes) followed by 72 C for 7 minutes)
in 96-
wells microtitre plates using HotStartTM Taq polymerase (Qiagen, Mississauga,
ON).
The completed PCR reactions were desalted using the 96-well filter plates
(Corning) and
the amplicons recovered in 100 pL 10mM Tris (pH 8.0). A 5-pL aliquot of each
PCR
reaction was visualized on a 1.5% agarose gel containing ethidium bromide and
only
those reactions containing a single amplified product were selected for DNA
sequence
analysis using standard DNA sequencing performed on an ABI 3100 instrument
(Applied
Biosystems, Foster City, CA). Each DNA sequence obtained was given a Sequence
Identification Number and entered into a database for subsequent tracking and
annotation.
Each sequence was selected for BLAST analysis of public databases (e.g.
NCBI). Absent from these sequences were the standard housekeeping genes
(GAPDH,
actin, most ribosomal proteins etc.), which was a good indication that the
subtracted
library was depleted of at least the relatively abundant non-differentially
expressed
sequences.
CA 02785046 2012-08-02
W02007/093042
PCT/CA2007/000210
68
Once sequencing and annotation of the selected clones were completed. the
next step involved identifying those sequences that were actually upregulated
in
osteoclasts compared to precursors.
F - Hybridization analysis for identifying upregulated sequences
The PCR amplicons representing the annotated sequences from the
pCATRMAN libraries described above were used to prepare DNA microarrays The
purified PCR amplicons contained in 70 pL of the PCR reactions prepared in the
previous section was lyophilized and each reconstituted in 20 pL of spotting
solution
comprising 3xSSC and 0.1% sarkosyl. DNA micro-arrays of each amplicon in
triplicate
were then prepared using CMT-GAP2 slides (Corning, Corning, NY) and the GMS
417
spotter (Affymetrix, Santa Clara, CA).
The DNA micro-arrays were then hybridized with either standard or subtracted
cy3 and cy5 labelled cDNA probes as recommended by the supplier (Amersham
Biosciences, Piscataway, NJ). The standard cDNA probes were synthesized using
RAMP amplified RNA prepared from the different human osteoclast samples and
the
corresponding precursors. It is well known to the skilled artisan that
standard cDNA
probes only provide limited sensitivity of detection and consequently low
abundance
sequences contained in the cDNA probes are usually missed. Thus, the
hybridization
analysis was also performed using cy3 and cy5 labelled subtracted cDNA probes
prepared from subtracted libraries representing the different tester and
driver materials.
These subtracted libraries may be enriched for low abundance sequences as a
result
of following the teachings of Malek et al., and therefore. may provide
increased
detection sensitivity.
All hybridization reactions were performed using the dye-swap procedure as
recommended by the supplier (Amersham Biosciences, Piscataway, NJ) and
approximately 500 putatively differentially expressed upregulated (>2-fold)
sequences
were selected for further analysis.
G - Determining osteoclast specificity of the differentially expressed
sequences
identified:
The differentially expressed sequences identified in Section F for the
different
CA 02785046 2012-08-02
69
human osteoclast subtracted libraries were tested for osteoclast specificity
by
hybridization to nylon membrane-based macroarrays. The macroarrays were
prepared
using RAMP amplified RNA from human precursors and osteoclasts (intermediate
and
mature) of six independent experiments from 4 different donors (3 males and 1
female),
and 30 normal human tissues (adrenal, liver, lung, ovary, skeletal muscle,
heart, cervix,
thyroid, breast, placenta, adrenal cortex, kidney, vena cava, fallopian tube,
pancreas,
testicle, jejunum, aorta, esophagus, prostate, stomach, spleen, ileum,
trachea, brain,
colon, thymus, small intestine, bladder and duodenum) purchased commercially
(Ambion, Austin, TX). Because of the limited quantities of mRNA available for
many of
these samples, it was necessary to first amplify the mRNA using the RAMP
methodology. Each amplified RNA sample was reconstituted to a final
concentration of
250 ng/pL in 3xSSC and 0.1% sarkosyl in a 96-well microtitre plate and 1 pL
spotted
onto HybondTM N+ nylon membranes using the specialized MULTI-PRINTTm apparatus
(VP Scientific, San Diego, CA), air dried and UV-cross linked. A total of 400
different
sequences selected from 5L87-95 and SL108-110 were individually radiolabeled
with a-
32P-dCTP using the random priming procedure recommended by the supplier
(Amersham, Piscataway, NJ) and used as probes on the macroarrays.
Hybridization and
washing steps were performed following standard procedures well known to those
skilled in the art.
Of the 500 sequences tested, approximately 85% were found to be upregulated in
all of
the osteoclast RNA samples that were used to prepare the macroarrays. However,
many
of these sequences were also readily detected in a majority of the different
normal
human tissues. Based on these results, those sequences that appeared to be
associated with experimental variability and those that were detected in many
of the
other human tissues at significantly elevated levels were eliminated.
Consequently, only
sequences, which appeared to be upregulated and highly osteoclast-specific,
were
selected for biological validation studies. Included in this set of 35 genes
were 4 (SEQ.
ID. NOs. 30-33) where there was a significant upregulation in mature
osteoclasts
compared to most normal tissues but because the expression of these genes were
30 overall lower in the precursor cells, they appeared to be elevated in
the normal tissues
after quantitation Figure 30-33; bar graph). However, their expression in the
normal
tissues was still relatively lower than that of the mature osteoclasts. Thus,
these genes
may still be important regulators in osteoclastogenesis and bone resorption
and were
therefore selected for biological validation. This subset
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
of 35 sequences does not included genes also identified such as, CTSK, TRAP,
MMP9. CST3 and CKB amongst others since these were previously reported in the
literature to be upregulated in osteoclasts. The macroarray data for CST3
(SEQ. ID.
NO. 34) is included to exemplify the hybridization pattern and specificity of
a gene that
5 is already known to be a key regulator of the osteoclast resorption
process. One gene
(ANKH; SEQ. ID. NO. 17) was included in the subset of 35 genes although it was
previously reported in the database (NCB! - Gene) to play a role in bone
mineralization.
However, the observed bone phenotype resulting from mutations in the ANKH gene
was not specifically linked to its upregulation in osteoclasts. Thus our data
suggests the
10 important role for ANKH may be associated with osteoclast activity
during bone
remodeling.
Fig. 1-33, 38 and 39 show the macroarray patterns and quantitation of the
hybridization signals of the osteoclasts and normal human tissues relative to
precursor
cells for the 35 sequences selected for biological validation. Amongst the 35
selected
15 sequences were 24 genes with functional annotation 9 genes with no
functional
annotation and 2 novel sequences (genomic hits). The identification of gene
products
involved in regulating osteoclast differentiation and function has thus led to
the
discovery of novel targets for the development of new and specific therapies
of disease
states characterized by abnormal bone remodeling. Representative sequences
20 summarized in Table 1 are presented below and corresponding sequences
are
illustrated in Table 5.
SEQ. ID. NO:1:
SEQ. ID. Na1 (Table 5) corresponds to a previously identified gene that
25 encodes a hypothetical protein. L00284266 with an unknown function (see
Table 1).
We have demonstrated that this gene is markedly upregulated in intermediate
and
mature osteoclast compared to precursor cells and other normal human tissues
(Figure
1), which have not been previously reported. Thus, it is believed that this
gene may be
required for osteoclastogenesis and/or bone remodeling.
30 SEQ. ID. NO:2:
SEQ. ID. NO:2 (Table 5) corresponds to a previously identified gene that
encodes a predicted open reading frame, C6orf82 with an unknown function (see
Table
1). We have demonstrated that this gene is markedly upregulated in
intermediate and
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
71
mature osteoclast compared to precursor cells and other normal human tissues
(Figure
2), which have not been previously reported. At least 5 transcript variants of
this gene
coding for 3 protein isoforms has been identified so far (NCBI). Thus, it is
believed that
this gene may be required for osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:3:
SEQ. ID. NO:3 (Table 5) corresponds to a previously identified gene that
encodes a hypothetical protein, L0C133308 with an unknown function (see Table
1)
but may be involved in the process of pH regulation. We have demonstrated that
this
gene is markedly upregulated in intermediate and mature osteoclast compared to
precursor cells and other normal human tissues (Figure 3), which have not been
previously reported. Thus, it is believed that this gene may be required for
osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:4:
SEQ. ID. NO:4 (Table 5) corresponds to a previously identified gene that
encodes a hypothetical protein. L0C116211 with an unknown function (see Table
1)
We have demonstrated that this gene is markedly upregulated in intermediate
and
mature osteoclast compared to precursor cells and other normal human tissues
(Figure
4), which have not been previously reported. Thus, it is implified that this
gene may be
required for osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:5
SEQ. ID. NO:5 (Table 5) corresponds to a previously identified gene that
encodes a predicted protein, L0C151194 (similar to hepatocellular carcinoma-
associated antigen HCA557b). with unknown function (see Table 1). We have
demonstrated that this gene is markedly upregulated in intermediate and mature
osteoclast compared to precursor cells and other normal human tissues (Figure
5),
which have not been previously reported. Thus, it is believed that this gene
may be
required for osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:6:
SEQ. ID. NO:6 (Table 5) corresponds to a previously identified gene that
encodes a protein, chemokine (C-X-C motif) ligand 5 (CXCL5), which is an
inflammatory chemokine that belongs to the CXC chemokine family
(see Table 1). We have demonstrated that this gene is significantly
upregulated in
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
72
mature osteoclast compared to precursor cells and other normal human tissues
(Figure
6), which have not been previously reported. Thus, it is believed that this
gene may be
required for osteoclastogenesis and/or bone remodeling
SEQ. ID. NO:7:
SEQ. ID. NO:7(Table 5) corresponds to a previously identified gene that
encodes a protein, ATPase, H+ transporting. lysosomal accessory protein 2
(ATP6AP2), which is associated with adenosine triphosphatases (ATPases).
Proton-
translocating ATPases have fundamental roles in energy conservation, secondary
active transport, acidification of intracellular compartments, and cellular pH
homeostasis (see Table 1). We have demonstrated that this gene is markedly
upregulated in mature osteoclast compared to precursor cells and other normal
human
tissues (Figure 7), which have not been previously reported. Thus, it is
believed that
this gene may be required for osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:8
SEQ. ID. NO:8 (Table 5) corresponds to a previously identified gene that
encodes a protein, ubiquitin-specific protease 12-like 1 (USP12). which is
associated
with ubiquitin-dependent protein catabolism (see Table 1) We have demonstrated
that
this gene is markedly upregulated in intermediate and mature osteoclast
compared to
precursor cells and other normal human tissues (Figure 8), which have not been
previously reported. Thus, it is believed that this gene may be required for
osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:9
SEQ. ID. NO:9 (Table 5) corresponds to a previously identified gene that
encodes a protein, Ubiquitin-conjugating enzyme E2E 1 (UBC4/5 homolog. yeast)
(UBE2E1), which is associated with ubiquitin-dependent protein catabolism (see
Table
1). So far, there are 2 transcript variants and protein isoforms reported for
this gene
We have demonstrated that this gene is significantly upregulated in mature
osteoclast
compared to precursor cells and other normal human tissues (Figure 9). which
have
not been previously reported. Thus. it is believed that this gene may be
required for
osteoclastogenesis and/or bone remodeling
SEQ. ID. NO:10
SEQ. ID. NO.10 (Table 5) corresponds to a previously identified gene that
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
73
encodes a protein, Emopamil binding protein-like (EBPL), which may have
cholestenol
delta-isomerase activity (see Table 1). We have demonstrated that this gene is
markedly upregulated in intermediate and mature osteoclast compared to
precursor
cells and other normal human tissues (Figure 10), which have not been
previously
reported Thus, it is believed that this gene may be required for
osteoclastogenesis
and/or bone remodeling.
SEQ. ID. NO:11
SEQ. ID. NO:11 (Table 5) corresponds to a previously identified gene that
encodes a protein, development and differentiation enhancing factor 1 (DDEF1),
which
may be involved in cell motility and adhesion (see Table 1). We have
demonstrated
that this gene is markedly upregulated in intermediate and mature osteoclast
compared
to precursor cells and other normal human tissues (Figure 11), which have not
been
previously reported. Thus, it is believed that this gene may be required for
osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:12
SEQ. ID. NO 12 (Table 5) corresponds to a previously identified gene that
encodes a protein. member 7 of the SLAM family (SLAM7), which may have
receptor
activity and involved in cell adhesion but still not fully characterized (see
Table 1). We
have demonstrated that this gene is markedly upregulated in mature osteoclast
compared to precursor cells and other normal human tissues (Figure 12), which
have
not been previously reported. Thus, it is believed that this gene may be
required for
osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:13
SEQ. ID. NO:13 (Table 5) corresponds to a previously identified gene that
encodes a protein, Ubiquitin-conjugating enzyme E2E 3 (UBC4/5 homolog. yeast)
(UBE2E3), which is associated with ubiquitin-dependent protein catabolism (see
Table
1). There are 2 transcript variants documented so far, which code for the same
protein
isofrom. We have demonstrated that this gene is markedly upregulated in mature
osteoclast compared to precursor cells and other normal human tissues (Figure
1),
which have not been previously reported. Thus, it is believed that this gene
may be
required for osteoclastogenesis and/or bone remodeling.
CA 02785046 2012-08-02
PCT/CA2007/0002 10
WO 2007/093042
74
SEQ. ID. NO:14
SEQ. ID. NO:14 (Table 5) corresponds to a previously identified gene that
encodes a protein, Galanin (GAL), which is associated with neuropeptide
hormone
activity (see Table 1). We have demonstrated that this gene is markedly
upregulated in
intermediate and mature osteoclast compared to precursor cells and other
normal
human tissues except for colon (Figure 14) which have not been previously
reported.
Thus, it is believed that this gene may be required for osteoclastogenesis
and/or bone
remodeling.
SEQ. ID. NO:15
SEQ. ID. NO:15 (Table 5) corresponds to a previously identified gene that
encodes a protein, Cytokine-like nuclear factor n-pac (N-PAC), which may have
oxireductase activity (see Table 1). We have demonstrated that this gene is
markedly
upregulated in intermediate and mature osteoclast compared to precursor cells
and
other normal human tissues (Figure 15), which have not been previously
reported.
However, some overexpression of this gene but still way below that of mature
osteoclasts were seen in heart, fallopian tube, spleen and cervix. Thus, it is
believed
that this gene may be required for osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:16
SEQ. ID. NO:16 (Table 5) corresponds to a previously identified gene that
encodes a protein. Integrin alpha X (antigen CD11C (p150), alpha polypeptide)
(ITGAX), which is involved in cell adhesion and ion binding (see Table 1) We
have
demonstrated that this gene is markedly upregulated in intermediate and mature
osteoclast compared to precursor cells and other normal human tissues (Figure
16),
which have not been previously reported. Minimal expression but much lower
than
mature osteoclasts is observed for this gene in adrenal, lung and spleen
amongst the
normal tissues. Thus, it is believed that this gene may be required for
osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:17
SEQ. ID. NO:17 (Table 5) corresponds to a previously identified gene that
encodes a protein, Ankylosis. progressive homolog (mouse) (ANKH), which is
involved
in regulating pyrophosphate levels, suggested as a possible mechanism
regulating
tissue calcification (see Table 1). We have demonstrated that this gene is
markedly
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
upregulated in intermediate and mature osteoclast compared to precursor cells
and
other normal human tissues (Figure 17), which have not been previously
reported.
However, this gene has been reported to be involved in bone mineralization but
without
evidence of its upregulation in osteoclasts (Malkin et al., 2005). Thus, it is
believed that
5 this gene may be required for osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:18
SEQ. ID. NO:18 (Table 5) corresponds to a previously identified gene that
encodes a protein, ATPase, H+ transporting. lysosomal 70kD. V1 subunit A.
which is
involved in hydrogen-transporting ATPase activity, rotational mechanism (see
Table 1).
10 We have demonstrated that this gene is markedly upregulated in mature
osteoclast
compared to precursor cells and other normal human tissues (Figure 18). which
have
not been previously reported. Thus, it is believed that this gene may be
required for
osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:19
15 SEQ. ID. NO:19 (Table 5) corresponds to a previously identified gene
that
encodes a predicted open reading frame coding for protein, FLJ10874
(chromosome 1
open reading frame 75), which has no known function (see Table 1). We have
demonstrated that this gene is significantly upregulated in mature osteoclast
compared
to precursor cells and other normal human tissues (Figure 19), which have not
been
20 previously reported. Thus, it is believed that this gene may be required
for
osteoclastogenesis and/or bone remodeling
SEQ. ID. NO:20
SEQ. ID. NO 20 (Table 5) corresponds to a previously identified gene that
encodes a protein: Integrin beta 1 binding protein 1 (ITGB1BP1). which has an
25 important role during integrin-dependent cell adhesion (see Table 1).
Two transcript
variants and protein isoforms for this gene has been isolated. We have
demonstrated
that this gene is significantly upregulated in mature osteoclast compared to
precursor
cells and other normal human tissues (Figure 20), which have not been
previously
reported. Thus, it is believed that this gene may be required for
osteoclastogenesis
30 and/or bone remodeling.
SEQ. ID. NO:21
SEQ. ID. NO:21 (Table 5) corresponds to a previously identified gene that
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
76
encodes a protein, Thioredoxin-like 5 (TXNL5), which has no known function
(see
Table 1). We have demonstrated that this gene is significantly upregulated in
intermediate and mature osteoclast compared to precursor cells and other
normal
human tissues with the exception of esophagus (Figure 21), which have not been
previously reported. Thus, it is believed that this gene may be required for
osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:22
SEQ. ID. NO:22 (Table 5) corresponds to a previously identified gene that
encodes a protein. C-type lectin domain family 4, member E (CLECSF9), which
has no
known specific function (see Table 1). Members of this family share a common
protein
fold and have diverse functions, such as cell adhesion, cell-cell signaling
glycoprotein
turnover and roles in inflammation and immune response. We have demonstrated
that
this gene is significantly upregulated in mature osteoclast compared to
precursor cells
and other normal human tissues with the exception of lung and spleen (Figure
22).
which have not been previously reported. At this point, we cannot rule out
cross
hybridization to family members in lung and spleen. Thus, it is believed that
this gene
may be required for osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:23
SEQ. ID. NO 23 (Table 5) corresponds to a previously identified gene that
encodes a protein, RAB33A, member RAS oncogene family (RAB33A). which has
GTPase activity (see Table 1). We have demonstrated that this gene is
significantly
upregulated in intermediate and mature osteoclast compared to precursor cells
and
other normal human tissues with the exception of brain (Figure 23), which have
not
been previously reported Thus, it is believed that this gene may be required
for
osteoclastogenesis and/or bone remodeling
SEQ. ID. NO:24
SEQ. ID. NO:24 (Table 5) corresponds to a previously identified gene that
encodes a protein, Down syndrome critical region gene 1 (DSCR1), which
interacts
with calcineurin A and inhibits calcineurin-dependent signaling pathways,
possibly
affecting central nervous system development (see Table 1). There are 3
transcript
variants and protein isofroms isolated so far. We have demonstrated that this
gene is
markedly upregulated in intermediate and mature osteoclast compared to
precursor
cells and other normal human tissues (Figure 24), which have not been
previously
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
77
reported, Thus, it is believed that this gene may be required for
osteoclastogenesis
and/or bone remodeling.
SEQ. ID. NO:25
SEQ. ID. NO:25 (Table 5) corresponds to a previously identified gene that
encodes a protein, SNARE protein Ykt6 (YKT6), which is one of the SNARE
recognition molecules implicated in vesicular transport between secretory
compartments (see Table 1). We have demonstrated that this gene is
significantly
upregulated in mature osteoclast compared to precursor cells and other normal
human
tissues (Figure 25), which have not been previously reported. Thus, it is
believed that
this gene may be required for osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:26
SEQ ID. NO:26 (Table 5) corresponds to a previously identified gene that
encodes a protein, Actinin, alpha 1 (ACTN1). which is cytoskeletal. and
involved in
actin binding and adhesion (see Table 1). We have demonstrated that this gene
is
significantly upregulated in intermediate and mature osteoclast compared to
precursor
cells and other normal human tissues (Figure 26), which have not been
previously
reported. Thus, it is believed that this gene may be required for
osteoclastogenesis
and/or bone remodeling.
SEQ. ID. NO:27
SEQ. ID. NO:27 (Table 5) corresponds to a previously identified gene that
encodes a protein, CIpX caseinolytic peptidase X homolog (E. coli) (CLPX),
which may
be involved in protein turnover (see Table 1). We have demonstrated that this
gene is
significantly upregulated in intermediate and mature osteoclast compared to
precursor
cells and other normal human tissues (Figure 27), which have not been
previously
reported. Thus, it is believed that this gene may be required for
osteoclastogenesis
and/or bone remodeling.
SEQ. ID. NO:28
SEQ. ID. NO:28 (Table 5) corresponds to a previously identified gene that
encodes a protein, Carbonic anhydrase II (CA2), which has carbonate
dehydratase
activity (see Table 1). Defects in this enzyme are associated with
osteopetrosis and
renal tubular acidosis (McMahon et al., 2001) and have been shown to be
upregulated
in mature osteoclasts under induced acidic pH conditions (Biskobing and Fan,
2000)
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
78
We have demonstrated that this gene is markedly upregulated in intermediate
and
mature osteoclast compared to precursor cells independent of induced acidic pH
conditions and other normal human tissues (Figure 28), which have not been
previously reported. However, elevated expression of this gene was also
observed in
colon and stomach but still significantly below the levels of mature
osteoclasts. Thus, it
is believed that this gene may be required for osteoclastogenesis and/or bone
remodeling.
SEQ. ID. NO:29
SEQ. ID. NO:29 (Table 5) corresponds to a previously identified gene that
encodes a protein, Sorting nexin 10 (SNX10), whose function has not been
determined
(see Table 1). We have demonstrated that this gene is markedly upregulated in
mature
osteoclast compared to precursor cells and most normal human tissues (Figure
29).
which have not been previously reported However, elevated expression of this
gene
was also observed in liver, brain, lung, adrenal cortex, kidney and spleen but
still
significantly below the levels of mature osteoclasts. Thus, it is believed
that this gene
may be required for osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:30
SEQ. ID. NO:30 (Table 5) corresponds to a previously identified gene that
encodes a protein. Tudor domain containing 3 (TDRD3), whose function has not
been
determined but may be involved in nucleic acid binding (see Table 1). We have
demonstrated that this gene is markedly upregulated in mature osteoclast
compared to
precursor cells and most normal human tissues (Figure 30), which have not been
previously reported. However, above baseline expression of this gene was
observed in
the normal human tissues because of a lower than normal precursor level but it
was
still significantly below the levels of mature osteoclasts, Thus, this gene
was still
selected. Thus, it is believed that this gene may be required for
osteoclastogenesis
and/or bone remodeling.
SEQ. ID. NO:31
SEQ ID NO 31 (Table 5) corresponds to a previously identified gene that
encodes a protein. Selenoprotein P. plasma. 1 (SEPP1), which has been
implicated as
an oxidant defense in the extracellular space and in the transport of selenium
(see
Table 1). This gene encodes a selenoprotein that contains multiple
selenocysteines.
Selenocysteine is encoded by the usual stop codon UGA. The unususal amino
acids
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
79
are indicated as 'Li' in the amino acid sequence in SEQ. ID. NO:78 (Table 5)
or by Xaa
in the sequence listing. We have demonstrated that this gene is markedly
upregulated
in intermediate and mature osteoclast compared to precursor cells and most
normal
human tissues (Figure 31), which have not been previously reported However,
above
baseline expression of this gene was observed in the normal human tissues
because
of a lower than normal precursor level but it was still significantly below
the levels of
mature osteoclasts. Thus, this gene was still selected. Thus, it is believed
that this
gene may be required for osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:32
SEQ. ID. NO:32 (Table 5) corresponds to a previously identified gene that
encodes a hypothetical protein, KIAA0040. which has no known function (see
Table 1).
We have demonstrated that this gene is markedly upregulated in intermediate
and
mature osteoclast compared to precursor cells and most normal human tissues
(Figure
32). which have not been previously reported. However, above baseline
expression of
this gene was observed in the normal human tissues because of a lower than
normal
precursor level but it was still significantly below the levels of mature
osteoclasts. Thus
this gene was still selected. Thus, it is believed that this gene may be
required for
osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:33
SEQ. ID. NO:33 (Table 5) corresponds to a previously identified gene that
encodes a protein. Dipeptidylpeptidase 4 (CD26, adenosine deaminase complexing
protein 2) (DPP4), which is an intrinsic membrane glycoprotein and a serine
exopeptidase that cleaves X-proline dipeptides from the N-terminus of
polypeptides
(see Table 1). We have demonstrated that this gene is markedly upregulated in
,
intermediate and mature osteoclast compared to precursor cells and most normal
human tissues (Figure 33), which have not been previously reported. However,
above
baseline expression of this gene was observed in the normal human tissues
except for
placenta, lung, ovary, kidney, prostate and small intestine because of a lower
than
normal precursor level but it was still significantly below the levels of
mature
osteoclasts. Thus, this gene was still selected. Thus, it is believed that
this gene may
be required for osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:34:
SEQ ID. NO:34 (Table 5) corresponds to a previously identified gene that
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
encodes a protein, cystatin C precursor, with members of the cystatin family
known to
be inhibitor of cysteine proteases (see Table 1). We have demonstrated that
this gene
is markedly upregulated in intermediate and mature osteoclast compared to
precursor
cells and other normal human tissues (Figure 34). which have not been
previously
5 .. reported. However, it is well documented that cystatin C plays a critical
role in inhibiting
bone resorption due to osteoclasts (Brage et al . 2005) Thus, the
hybridization profile
for this gene is an excellent example of highly upregulated and specific
sequences
related to osteoclasts.
SEQ. ID. NO:85
10 SEQ. ID. NO:85 (Table 5) encodes an unknown protein found on chromosome
1 (clone RP11-344F13), which contains a novel gene (see Table 1). We have
demonstrated that this gene is markedly upregulated in intermediate and mature
osteoclast compared to precursor cells and other normal human tissues (Figure
38),
which have not been previously reported. Thus, it is implified that this gene
may be
15 required for osteoclastogenesis and/or bone remodeling.
SEQ. ID. NO:86
SEQ. ID. NO 86 (Table 5) encodes no known protein. Unknown gene with matching
Est sequence in the data base corresponding to BQ182670 isolated from an
osteoarthritic cartilage sample (see Table 1). We have demonstrated that this
gene is
20 significantly upregulated in intermediate and mature osteoclast compared
to precursor
cells and other normal human tissues (Figure 39). which have not been
previously
reported. Thus, it is implified that this gene may be required for
osteoclastogenesis
and/or bone remodeling.
25 .. H- Cloning of full-length cDNAs of selected sequences from osteoclast
mRNA:
It was necessary to obtain full-length cDNA sequences in order to perform
functional studies of the expressed proteins. Spliced variants are
increasingly being
implicated in tissue specific functions and as such, it is important to work
with cDNA
clones from the system under study. Applicant also recognizes that spliced
variants
30 may not always be involved. Thus. the applicant's approach has been to
isolate the
relevant full-length cDNA sequences directly from osteoclasts in order to
identify
variants and their potential role with respect to specificity
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
81
Coding cDNA clones were isolated using both a 5'-RACE strategy (Invitrogen,
Burlington, ON) and a standard two-primer gene specific approach in PCR. The
5'-
RACE strategy used cDNA prepared from cap-selected osteoclast RNA and/or RAMP
amplified osteoclast RNA. For amplification using gene specific primers,
either cDNA
prepared from RAMP RNA or total RNA was used. All cDNAs were synthesized
following standard reverse transcription procedures (Invitrogen, Burlington.
ON) The
cDNA sequences obtained were cloned in E.coli DH10B and the nucleotide
sequences
for multiple clones determined. Thereafter, the cDNA sequences for each set
were
aligned and the open reading frame(s) (ORF) identified using standard software
(e.g.
ORF Finder-NCBI). Table 2 shows the concensus sequence of the cDNA clones for
the
coding region for SEQ. ID. NO.1 (SEQ. ID. NO. 83) and SEQ. ID. NO.2 (SEQ. ID.
NO
84) obtained from a human osteoclast sample, which were identical to that of
the
published sequences corresponding to Accession# NM_213602 and NM_001014433
(NCBI), respectively.
I- RNA Interference Studies
RNA interference is a recently discovered gene regulation mechanism that
involves the sequence-specific decrease in a gene's expression by targeting
the mRNA
for degradation and although originally described in plants, it has been
discovered
across many animal kingdoms from protozoans and invertebrates to higher
eukaryotes
(reviewed in Agrawal et al., 2003). In physiological settings, the mechanism
of RNA
interference is triggered by the presence of double-stranded RNA molecules
that are
cleaved by an RNAse III-like protein active in cells, called Dicer, which
releases the 21-
23 bp siRNAs. The siRNA, in a homology-driven manner, complexes into a RNA-
protein amalgamation termed RISC (RNA-induced silencing complex) in the
presence
of mRNA to cause degradation resulting in attenuation of that mRNA's
expression
(Agrawal et al.. 2003).
Current approaches to studying the function of genes, such as gene knockout
mice and dominant negatives, are often inefficient, and generally expensive,
and time-
consuming. RNA interference is proving to be a method of choice for the
analysis of a
large number of genes in a quick and relatively inexpensive manner. Although
transfection of synthetic siRNAs is an efficient method, the effects are often
transient at
best (Hannon G J., 2002), Delivery of plasmids expressing short hairpin RNAs
by
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
82
stable transfection has been successful in allowing for the analysis of RNA
interference
in longer-term studies (Brummelkamp et al., 2002; Elbashir et al., 2001). In
addition,
more recent advances have permitted the expression of siRNA molecules, in the
form
of short hairpin RNAs, in primary human cells using viral delivery methods
such as
lentivirus (Lee et al., 2004; Rubinson et at. 2003).
J- Determination of knockdown effects on osteoclastogenesis
In order to develop a screening method for the human candidate genes. RNA
interference was adapted to deliver shRNAs into human osteoclast precursor
cells so
that the expression of the candidate genes could be attenuated This approach
would
then allow osteoclast differentiation to be carried out in cells containing
decreased
expression of these genes to determine their requirement, if any, in this
process
To this end, a commercial lentiviral shRNA delivery system (Invitrogen.
Burlington, ON) was utilized to introduce specific shRNAs into human
osteoclast
precursor cells. The techniques used were as described by the manufacturer
unless
otherwise stated. In this example, the results obtained for two of the
candidate genes,
SEQ. ID. NO. 1 (AB0326) and SEQ. ID. NO. 2 (AB0369) tested so far, are
presented.
The proteins encoded by both of these two genes have no known function. The
shRNA
sequences used to specifically target SEQ. ID. NO. 1 and SEQ. ID. NO. 2 were 5-
CAGG000AGGAGTCCAATT-3' (SEQ. ID. NO. 42) and 5'-
TCCCGTCTTTGGGTCAAAA-3' (SEQ. ID. NO. 43) respectively. Briefly, a template for
the expression of the shRNA was cloned into the lentiviral expression vector
and co-
transfected in 293FT cells with expression vectors for the viral structural
proteins After
two days supernatants containing the lentivirus were collected and stored at
¨80 C
Human osteoclast precursors purchased from Cambrex (East Rutherford NJ) were
seeded in 24-well plates and cultured in complete medium containing macrophage-
colony stimulating factor and allowed to adhere for three days. After washing
with PBS,
the cells were infected with 20 MOls (multiplicity of infection) of either
lentiviral particles
containing a shRNA specific for the bacterial lacZ gene as a control (lacZ
shRNA) or
SEQ. ID. NO. 1 (A80326 shRNA) or SEQ. ID. NO. 2 (AB0369 shRNA). After 24h, the
infected cells were treated with same medium containing 100 ng/ml RANK ligand
for 5
¨ 8 days to allow for differentiation of osteoclast from precursor cells.
Mature
osteoclasts were fixed with formaldehyde and stained for TRAP expression as
follows
CA 02785046 2012-08-02
83
the cells were washed with PBS and fixed in 10% formaldehyde for 1h. After two
PBS
washes, the cells were lightly permeabilized in 0.2% Tritonna X-100 in PBS for
5 min
before washing in PBS. Staining was conducted at 37 C for 20 ¨ 25 min in 0.01%
Naphtol AS-MX phosphate, 0.06% Fast Red Violet, 50 mM sodium tartrate, 100 mM
sodium acetate, pH 5.2. The stained cells were visualized by light microscopy
and
photographed (magnification: 40X). A significant decrease in the number of
multinucleated osteoclasts was observed from precursor cells infected with the
AB0326
shRNA (Figure 35A; bottom panel) and AB0369 shRNA (Figure 35B; bottom panel)
compared to those with the lacZ shRNA (Figure 35A and B; top panels).
Therefore, in
both cases, the respective lentiviral shRNA (SEQ. ID. NOs. 42 and 43,
respectively)
(Table 4) perturbed osteoclastogenesis. These results clearly indicated that
expression
of the gene encoding SEQ. ID. NO. 1 (AB0326) and SEQ. ID. NO. 2 (AB0369) are
required for osteoclast differentiation.
Similar experimentations to those described above are carried out for other
sequences (SEQ ID NO.3 to SEQ ID NO.:33, SEQ ID NO.:85 or SEQ ID NO.:86).
K- Biological validation of the mouse orthologue for AB0326 (SEQ. ID. NO. 35)
in
osteoclastogenesis using the RAW 264.7 model
As a means of developing a drug screening assay for the discovery of
therapeutic molecules capable of attenuating human osteoclasts differentation
and
activity using the targets identified, it was necessary to turn to another
osteoclast
differentiation model. The RAW 264.7 (RAW) osteoclast precursor cell line is
well known
in the art as a murine model of osteoclastogenesis. However, due to the
difficulty in
transiently transfecting RAW cells, stable transfection was used as an
approach where
.. shRNA are expressed in the RAW cells constitutively. This permitted long
term studies
such as osteoclast differentiation to be carried out in the presence of
specific shRNAs
specific to the mouse orthologues of the human targets identified.
RAW cells were purchased from American Type Culture Collection (Manassass,
VA) and maintained in high glucose DMEM containing 10% fetal bovine serum and
antibiotics. The cells were sub-cultured bi-weekly to a maximum of 10-12
passages. For
osteoclast differentiation experiments, RAW cells were seeded in 96-well
plates at a
density of 4 x 103 cells/well and allowed to plate for 24h. Differentiation
was induced in
CA 02785046 2012-08-02
84
high glucose DMEM, 10% charcoal-treated foetal bovine serum (obtained from
Hyclone,
Logan, UT), 0.05% BSA, antibiotics, 10 ng/ml macrophage colony stimulating
factor (M-
CSF), and 100 ng/ml RANK ligand. The plates were re-fed on day 3 and
osteoclasts
were clearly visible by day 4. Typically, the cells were stained for TRAP on
day 4 or 5
unless otherwise indicated.
To incorporate the shRNA-expression cassettes into the RAW cell chromosomes,
the pSilencer 2.0 plasmid (SEQ. ID. NO. 47) was purchased from Ambion (Austin,
TX)
and sequence-specific oligonucleotides were ligated as recommended by the
manufacturer. Two shRNA expression plasmids were designed and the sequences
used
for attenuating the mouse ortholog of AB0326 (SEQ. ID. NO. 35) gene expression
were
5'-GCGCCGCGGATCGTCAACA-3' (SEQ. ID. NO. 44) and 5'-
ACACGTGCACGGCGGCCAA-3' (SEQ. ID. NO. 45). A plasmid supplied by Ambion
containing a scrambled shRNA sequence with no known homology to any mammalian
gene was also included as a negative control in these experiments. RAW cells
were
seeded in 6-well plates at a density of 5 x 105 cells/well and transfected
with 1 pg of
each plasmid using FugeneTM6 (Roche, Laval, QC) as described in the protocol.
After
selection of stable transfectants in medium containing 2 pg/ml puromycin, the
cell lines
were expanded and tested in the presence of RANK ligand for
osteoclastogenesis.
The stably transfected cell lines were designated RAW-0326.1, RAW-0326.2 and
RAW-ctl. In 96-well plates in triplicate, 4 000 cells/well were seeded and
treated with 100
ng/ml RANK ligand. After 4 days, osteoclasts were stained for TRAP expression
and
visualized by light microscopy (magnification was 40X and 100X as depicted in
the left
and right panels, respectively).
The representative results for the RAW-0326.2 line is shown in Figure 36. The
RAW-
0326.2 cell line produced significantly less osteoclasts (Figure 36; bottom
panel)
compared to the cell line containing the scrambled shRNA (Figure 36; top
panel). The
RAW-0326.1 cell line also showed attenuation of the mouse ortholog of AB0326
but not
as pronounced (data not shown). Therefore, as observed for SEQ ID NO. :42 and
43,
siRNAs to the mouse orthologue (SEQ. ID. NOs. 44 and 45) (Table 4) appear to
phenotypically perturb osteoclast differentiation in the mouse model as well.
These
results, coupled with that obtained in the human osteoclast precursor cells
using the
lentiviral shRNA delivery system (section J), demonstrate that in both human
and
mouse, AB0326 gene product is clearly required for osteoclastogenesis.
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
L- A functional complementation assay for SEQ. ID. NO. 1 (AB0326) in RAW 264.6
cells to screen for inhibitors of osteoclasto genesis
To establish a screening assay based on SEQ. ID. NO. 1 (AB0326) to find small
5 molecules capable of attenuating osteoclast differentiation, the cDNA
encoding human
A80326 was introduced into the RAW-0326.2 cell line. Thus, if the human AB0326
plays an identical functional role as the mouse orthologue in RAW 264.7 cells,
it should
restore the osteoclastogenesis capabilities of the RAW-0326.2 cell line.
To accomplish this task, the RAW-0326.2 cell line was transfected with an
10 eukaryotic expression vector encoding the full length cDNA for human
AB0326, termed
pd2-hAB0326. This expression vector (pd2, SEQ. ID. NO. 47) was modified from a
commercial vector, pd2-EGFP-N1 (Clontech. Mountain View, CA) where the EGFP
gene was replaced by the full length coding sequence of the human AB0326 cDNA
The AB0326 gene expression was driven by a strong CMV promoter Stable
15 transfectants were selected using the antibiotic, G418. This resulted in
a RAW-0326.2
cell line that expressed the human AB0326 gene product in which, the mouse
orthologue of AB0326 was silenced. As a control. RAW-0326.2 cells were
transfected
with the pd2 empty vector, which should not complement the AB0326 shRNA
activity.
Also, the pd2 empty vector was transfected into RAW 264.7 cells to serve as a
further
20 .. control. After selection of stable pools of cells, 4 000 cells/well were
seeded in 96-well
plates and treated for 4 days with 100 ng/ml RANK ligand. Following fixation
with
formaldehyde, the cells were stained for TRAP, an osteoclast-specific marker
gene. As
shown in Figure 37, the RAW-0326.2 cells transfected with the empty pd2 vector
are
still unable to form osteoclasts in the presence of RANK ligand (center panel)
indicating
25 .. that the mouse AB0326 shRNA is still capable of silencing the AB0326
gene
expression in these cells_ Conversely, the cells transfected with human AB0326
(pd2-
hAB0326) are rescued and thus, differentiate into more osteoclasts in response
to
RANK ligand (right panel). RAW 264.7 cells containing the empty vector (pd2)
did not
adversly affect the formation of osteoclasts in the presence of RANK ligand
(left panel)
30 .. These results confirm that the mouse and human orthologues of AB0326 are
functionally conserved in osteoclast differentiation
This particular type of cell-based assay can now serve as the basis for
screening compounds capable of binding to and inhibiting the function of human
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
86
AB0326. A compound library could be applied to this 'rescued' cell line in
order to
identify molecules (small molecule drugs, peptides, or antibodies) capable of
inhibiting
AB0326. Any reduction in osteoclast differentiation measured by a reduction in
the
expression of TRAP would be indicative of a decrease in human AB0326 activity.
This
assay is applicable to any gene required for proper osteoclast differentiation
in RAW
cells. A complementation assay can be developed for any human gene and used as
the basis for drug screening.
Similar experimentation to those described above are carried out for other
sequences (SEQ ID NO.3 to SEQ ID NO.:33 or SEQ ID NO.:85 or SEQ ID NO .86)
This type of assay may be used to screen for molecules capable of increasing
or
decreasing (e.g., inhibiting) the activity or expression of NSEQ or PSEQ.
In the NSEQs of the present invention, their methods, compositions, uses, its,
assays or else, the polynucleotide may either individually or in group
(collectively) more
particularly be (or may comprise or consist in) either;
a translatable portion of either SEQ ID NO.:1, of SEQ ID NO.:2, of SEQ ID
NO.:3, of SEQ ID NO.:4, of SEQ ID NO.:5, of SEQ ID NO.:6, of SEQ ID NO.:7, of
SEQ
ID NO.:8, of SEQ ID NO.:9, of SEQ ID NO .10, of SEQ ID NO 11, of SEQ ID NO 12.
of SEQ ID NO.:13, of SEQ ID NO.:14, of SEQ ID NO :15, of SEQ ID NO.:16. of SEQ
ID NO 17 of SEQ ID NO 18, of SEQ ID NO :19, of SEQ ID NO 20. of SEQ ID
NO :21, of SEQ ID NO.:22, of SEQ ID NO .23. of SEQ ID NO ;24 of SEQ ID NO '25.
of
SEQ ID NO :26. of SEQ ID NO. 27. of SEQ ID NO 28, of SEQ ID NO 29, of SEQ ID
NO.30, of SEQ ID NO. 31, of SEQ ID NO -32. of SEQ ID NO '33. of SEQ ID NO .85
or
of SEQ ID NO. :86;
sequence substantially identical to a translatable portion of SEQ ID NO..1, of
SEQ ID NO.:2, of SEQ ID NO :3, of SEQ ID NO.:4, of SEQ ID NO 5, of SEQ ID NO
6.
of SEQ ID NO.:7, of SEQ ID NO.:8, of SEQ ID NO.:9, of SEQ ID NO.:10, of SEQ ID
NO.:11, of SEQ ID NO.:12, of SEQ ID NO.13, of SEQ ID NO.:14, of SEQ ID NO. 15.
of SEQ ID NO.:16, of SEQ ID NO.:17, of SEQ ID NO.:18. of SEQ ID NO.:19, of SEQ
ID
NO :20, of SEQ ID NO.:21, of SEQ ID NO.:22, of SEQ ID NO. 23, of SEQ ID
NO.:24, of
SEQ ID NO :25, of SEQ ID NO.:26, of SEQ ID NO :27, of SEQ ID NO -28, of SEQ ID
NO :29, of SEQ ID NO :30. of SEQ ID NO. 31, of SEQ ID NO 32, of SEQ ID NO 33.
of SEQ ID NO :85 or of SEQ ID NO '86:
a sequence substantially complementary to a translatable portion of SEQ ID
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
87
NO.:1, a fragment of a transcribable portion of SEQ ID NO. :1 of SEQ ID NO.
:2, of SEQ
ID NO.:3, of SEQ ID NO. 4, of SEQ ID NO.:5, of SEQ ID NO.:6, of SEQ ID NO 7,
of
SEQ ID NO.:8, of SEQ ID NO.:9, of SEQ ID NO.:10, of SEQ ID NO.:11, of SEQ ID
NO.:12, of SEQ ID NO.:13, of SEQ ID NO.:14, of SEQ ID NO.:15, of SEQ ID NO.
16.
of SEQ ID NO.17, of SEQ ID NO 18, of SEQ ID NO.:19, of SEQ ID NO.20. of SEQ ID
NO.21, of SEQ ID NO.:22, of SEQ ID NO. 23. of SEQ ID NO.:24, of SEQ ID NO 25,
of
SEQ ID NO 26, of SEQ ID NO.27, of SEQ ID NO.:28, of SEQ ID NO.:29, of SEQ ID
NO.:30, of SEQ ID NO 31, of SEQ ID NO..32, of SEQ ID Na.33, of SEQ ID NO .85
or
of SEQ ID NO.:86:
a fragment of a sequence substantially identical to a translatable portion of
SEQ
ID NO 1, of SEQ ID NO 2, of SEQ ID NO.:3, of SEQ ID NO 4, of SEQ ID NO. 5. of
SEQ ID NO.:6. of SEQ ID NO :7, of SEQ ID NO 8, of SEQ ID NO 9. of SEQ ID
NO.:10. of SEQ ID NO.:11, of SEQ ID NO.:12. of SEQ ID NO.:13, of SEQ ID NO.
14,
of SEQ ID NO.:15, of SEQ ID NO.:16, of SEQ ID NO.:17, of SEQ ID NO.:18, of SEQ
ID
NO.:19. of SEQ ID NO 20. of SEQ ID NO :21, of SEQ ID NO.:22, of SEQ ID NO 23,
of
SEQ ID NO.:24. of SEQ ID NO :25, of SEQ ID NO.:26, of SEQ ID NO.:27. of SEQ ID
NO.:28, of SEQ ID NO.:29, of SEQ ID NO.:30. of SEQ ID NO.:31. of SEQ ID NO
:32,
of SEQ ID NO :33, of SEQ ID NO.:85 or of SEQ ID NO.:86;
a fragment of a sequence substantially complementary to a translatable portion
of SEQ ID NO.:1, of SEQ ID NO. 2. of SEQ ID NO.:3, of SEQ ID NO .-4, of SEQ ID
NO 5, of SEQ ID NO.:6, of SEQ ID NO. 7, of SEQ ID NO .8. of SEQ ID NO .9. of
SEQ
ID NO .10. of SEQ ID NO.:11. of SEQ ID NO.:12, of SEQ ID NO. 13. of SEQ ID
NO.14, of SEQ ID NO .15. of SEQ ID NO. 16 of SEQ ID NO 17. of SEQ ID NO -18.
of
SEQ ID NO.:19, of SEQ ID NO :20. of SEQ ID NO :21, of SEQ ID NO .22 of SEQ ID
NO.:23. of SEQ ID NO.:24, of SEQ ID NO.:25, of SEQ ID NO.26. of SEQ ID NO .27.
of
SEQ ID NO.:28, of SEQ ID NO.:29, of SEQ ID NO 30, of SEQ ID NO.:31. of SEQ ID
NO :32, of SEQ ID NO.:33, of SEQ ID NO.:85 or of SEQ ID NO :86;
or a library comprising any of the above_
In the PSEQs of the present invention, their methods, compositions, uses, kits
assays, or else, the polypeptide may either individually or in group
(collectively) more
particularly be (or may comprise or consist in) either ;
SEQ ID NO.:48, SEQ ID NO.:49, SEQ ID NO.150, SEQ ID NO. 51, SEQ ID
NO.:52, SEQ ID NO :53, SEQ ID NO.:54, SEQ ID NO.:55, SEQ ID NO.:56. SEQ ID
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
88
NO. 57, SEQ ID NO :58, SEQ ID NO.:59, SEQ ID NO.:60, SEQ ID NO :61, SEQ ID
NO.:62, SEQ ID NO.:63, SEQ ID NO.:64, SEQ ID NO.:65, SEQ ID NO:66, SEQ ID
NO.:67, SEQ ID NO.:68, SEQ ID NO :69, SEQ ID NO.:70, SEQ ID NO.:71, SEQ ID
NO.:72, SEQ ID NO.:73, SEQ ID NO.74, SEQ ID NO.:75 SEQ ID NO.:76, SEQ ID
NO '77, SEQ ID NO.:78, SEQ ID NO.:79 or SEQ ID NO.:80;
a fragment of SEQ ID NO.:48, SEQ ID NO :49. SEQ ID NO .50, SEQ ID
NO.:51, SEQ ID NO.:52, SEQ ID NO.53, SEQ ID NO.:54, SEQ ID NO.:55, SEQ ID
NO.56, SEQ ID NO .57. SEQ ID NO :58, SEQ ID NO.:59, SEQ ID NO 60, SEQ ID
NO :61, SEQ ID NO.62, SEQ ID NO 63, SEQ ID NO.:64. SEQ ID NO 65, SEQ ID
NO..66. SEQ ID NO.:67, SEQ ID NO. 68, SEQ ID NO 69, SEQ ID NO.:70. SEQ ID
NO 71 SEQ ID NO.:72, SEQ ID NO .73. SEQ ID NO .74, SEQ ID NO.:75 SEQ ID
NO.:76, SEQ ID NO.77, SEQ ID NO.:78, SEQ ID NO.:79 or SEQ ID NO. 80;
or a biologically active analog, variant or a non-human hortologue of SEQ ID
NO.:48, SEQ ID NO.:49, SEQ ID NO.:50, SEQ ID NO .51, SEQ ID NO :52, SEQ ID
.. NO :53, SEQ ID NO.:54, SEQ ID NO.:55, SEQ ID NO.:56, SEQ ID NO. 57, SEQ ID
NO.:58, SEQ ID NO.:59, SEQ ID NO.:60, SEQ ID NO.:61, SEQ ID NO.:62, SEQ ID
NO.:63, SEQ ID NO.:64, SEQ ID NO.:65, SEQ ID NO.:66, SEQ ID NO.:67, SEQ ID
NO. 68, SEQ ID NO.:69, SEQ ID NO.70, SEQ ID NO.:71, SEQ ID NO.:72, SEQ ID
NO.:73, SEQ ID NO.:74, SEQ ID NO.:75 SEQ ID NO.76, SEQ ID NO.77. SEQ ID
NO.:78, SEQ ID NO.:79 or SEQ ID NO. 80.
One of skill in the art will readily recognize that orthologues for all
mammals
maybe identified and verified using well-established techniques in the art,
and that this
disclosure is in no way limited to one mammal The term ''mammal(s)" for
purposes of
this disclosure refers to any animal classified as a mammal, including humans,
.. domestic and farm animals, and zoo, sports, or pet animals, such as dogs
cats, cattle.
horses, sheep. pigs. goats, rabbits, etc. Preferably. the mammal is human.
The sequences in the experiments discussed above are representative of the
NSEQ being claimed and in no way limit the scope of the invention. The
disclosure of
the roles of the NSEQs in osteoclastogenesis and osteoclast function satisfies
a need
in the art to better understand the bone remodeling process, providing new
compositions that are useful for the diagnosis, prognosis, treatment,
prevention and
evaluation of therapies for bone remodeling and associated disorders.
The art of genetic manipulation, molecular biology and pharmaceutical target
CA 02785046 2013-02-08
89
development have advanced considerably in the last two decades. It will be
readily apparent to those skilled in the art that newly identified functions
for genetic
sequences and corresponding protein sequences allows those sequences, variants
and derivatives to be used directly or indirectly in real world applications
for the
development of research tools, diagnostic tools, therapies and treatments for
disorders or disease states in which the genetic sequences have been
implicated.
TABLE 1 ¨ Differentially expressed sequences found in osteoclasts.
Nucleotide NCB! Accession ORF Function
Sequence
Unigene Number Nucleotide
No. Positions/
#/Gene
Symbol/Gen Polypeptide
e ID sequence No.
SEQ ID NO. Hs.287692 NM 213602 150-1136 hypothetical
protein
1 L0C284266;
/ CD33L3 encoding SEQ
membrane associated
ID NO.:48
/ 284266 function unknown
SEQ ID NO. Hs.520070 NM 001014 104-700 chromosome 6 open
2 433¨ reading frame 82
/ C6orf82
membrane
/51596
associated
with
encoding SEQ unknown function
ID NO.: 49
SEQ ID NO. Hs.546482 NM 178833 633-2246 hypothetical
protein
3 L0C133308 possibly
/ L00133308 encoding SEQ
involved in regulation
ID NO.: 50
/133308 of pH
SEQ ID NO. Hs.135997 NM_138461 112-741 transmembrane 4 L
4 six family member 19
/ LOC116211 encoding SEQ
ID NO.: 51 ; function unknown
/ 116211
SEQ ID NO. Hs.558655 NM_145280 172-82 hypothetical
protein
5 L0C151194
/ LOC151194 encoding SEQ
ID NO.: 52
/ 151194
SEQ ID NO. Hs.89714 NM_002994 119-463 chemokine (C-
X-C
6 motif) ligand
5
/ CXCL5 encoding SEQ
ID NO.: 53 precursor; chemokine
CA 02785046 2012-08-02
WO 2007/093042 PCT/CA2007/000210
Nucleotide NCB! Accession ORF Function
Sequence Unigene Number Nucleotide
No. Positions/
Gene
Symbol/Gen Polypeptide
e ID sequence No.
/ 6374 activity
SEQ ID NO. Hs.495960 NM_005765 103-1155 ATPase, H+
7 transporting,
/ ATP6AP2 encoding SEQ
ID NO 54 lysosomal accessory
.:
/10159 protein 2;receptor
activity
SEQ ID NO. Hs.42400 NM_182488 259-1371 ubiquitin-specific
8 protease 12-like 1;
/ USP12
cysteine-type
/ 219333 encoding SEQ endopeptidase activity
ID NO.: 55
SEQ ID NO Hs 164853 NM 003341 175-756 ubiquitin-conjugating
9 enzyme E2E 1
/ UBE2E1
isoform 1. ligase
/ 7324 encoding SEQ activity
ID NO.: 56
SEQ ID NO. Hs.433278 NM_032565 53-673 emopamil binding
10 related protein.
/ EBPL
de1ta8-delta7 integral
/ 84650 encoding SEQ to membrane
ID NO.: 57
SEQ ID NO. Hs.106015 NM_018482 29-3418 development and
11 differentiation
/ DDEF1
enhancing factor 1.
/ 50807 encoding SEQ membrane
ID NO.: 58
SEQ ID NO. Hs.517265 NM 021181 16-1023 SLAM family member
12 receptor activity
/ SLAMF7
/ 57823 encoding SEQ
ID NO.. 59
SEQ ID NO Hs.470804 NM 006357 385-1008 ubiquitin-conjugating
13 enzyme E2E 3,
/ UBE2E3
ligase activity
/ 10477 encoding SEQ
ID NO.: 60
SEQ ID NO. Hs.278959 NM_015973 177-548 galanin preproprotein:
14 neuropeptide
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
91
Nucleotide NCB! Accession ORF Function
Sequence
Unigene Number Nucleotide
No. Positions/
#/Gene
Symbol/Gen Polypeptide
e ID sequence No.
/ GAL hormone activity
/51083 encoding SEQ
ID NO.: 61
SEQ ID NO. NM_032569 NM_032569 19-1680 cytokine-like nuclear
15 factor n-pac: 3-
/ N-PAC
hydroxyisobutyrate
/ 84656 encoding SEQ dehydrogenase-like
ID NO.: 62
SEQ ID NO. Hs.248472 NM 000887 68-3559 integrin alpha X
16 precursor. cell-matrix
/ ITGAX
adhesion
/ 3687 encodingSEQ
ID NO 63
SEQ ID NO. Hs.156727 NM_054027 321=1799 ankylosis. progressive
17 homolog. regulation of
/ ANKH
bone mineralization
/ 1827 encoding SEQ
ID NO.: 64
SEQ ID NO. Hs.477155 NM 001690 67-1920 ATPase. H+
18 transporting.
/ ATP6V1A
lysosomal 70kD, V1
/ 523 encoding SEQ subunit A. isoform 1
ID NO.: 65
: proton transport.
hydrolase activity
SEQ ID NO. Hs.445386 NM_018252 139-1191 hypothetical protein
19 L0055248
/ FLJ10874
/55248 encoding SEQ
ID NO.: 66
SEQ ID NO. Hs 467662 NM 004763 170-772 integrin cytoplasmic
20 domain-associated
/ ITGB1BP1 encoding SEQ
ID NO.: 67 protein 1
/9270
: cell adhesion
SEQ ID NO. Hs.408236 NM 032731 77-448 thioredoxin-like 5
21
/ TXNL5 function unknown
/84817 F encoding SEQ
ID NO: 68
L. ____________________________________________________________
_1
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
92
Nucleotide NCB! Accession ORF Function
Sequence Number Nucleotide
Unigene
No. Positions/
#/Gene
Symbol/Gen Polypeptide
e ID sequence No.
SEQ ID NO. Hs.236516 NM 014358 152-811 C-type lectin.
22 superfamily member 9
/ CLECSF9 encoding SEQ
ID NO.: 69 = ;integral to membrane
/ 26253
SEQ ID NO I Hs.56294 NM 004794 265-978 ; Ras-related protein
23 ' Rab-33A; small
/ RAB33A
GTPase mediated
/ 9363 encoding SEQ signal transduction
ID NO: 70
SEQ ID NO. Hs.282326 NM 004414 73-831 calcipressin 1 isoform
24 a; interacts with
/ DSCR1
calcineurin A and
/1827 encoding SEQ inhibits calcineurin-
ID NO.: 71 dependent signaling
pathways
SEQ ID NO. Hs.520794 NM 006555 158-754 SNARE protein Ykt6:
25 vesicular transport
/ YKT6
between secretory
/ 10652 encoding SEQ compartments
ID NO.: 72
SEQ ID NO. Hs.509765 NM_001102 I 184-2862 alpha-actinin 1:
26 structural constituent
/ ACTN1 of cytoskeleton.
/ 87 encoding SEQ calcium ion binding
ID NO: 73
SEQ ID NO. Hs.113823 NM 006660 73-1974 CIpX caseinolytic
27 protease X homolog;
/ CLPX
/ 10845 encoding SEQ energy-dependent
ID NO.: 74 regulator of
proteolysis
SEQ ID NO. Hs.155097 NM_000067 66-848 carbonic anhydrase
28 carbonate
/ CA2 encoding SEQ
ID NO.: 75 dehydratase activity
/ 760
SEQ ID NO. Hs 520714 NM 013322 216-821 sorting nexin 10
29
/ SNX10 encoding SEQ ; function unknown
ID NO.: 76
/29887
SEQ ID NO. Hs.525061 I NM 030794 258-2213 tudor domain
30 I ¨
containing 3: nucleic
/ TDRD3 encoding SEQ
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
93
Nucleotide NCB! Accession ORF Function
Sequence Unigene Number Nucleotide
No. Positions/
#/Gene
Symbol/Gen Polypeptide
e ID sequence No.
/ 81550 ID NO.: 77 acid binding
SEQ ID NO. Hs.275775 NM 005410 101-1246 selenoprotein P
31
/ SEPP1 encoding SEQ : extracellular space
' / 6414 ID NO 78 implicated in defense I
SEQ ID NO. Hs.518138 NM 014656 1 921-1382 1 KIAA0040 novel
32 protein
/ KIAA0040 encoding SEQ
/9674 ID NO.: 79
SEQ ID NO. Hs.368912 NM 001935 562-2862 dipeptidylpeptidase
33 IV:
/ DPP4 encoding SEQ activi aminopeptidase
ty
ID NO.: 80
/1803
SEQ ID NO. Hs.304682 NM 000099 76-516 cysteine protease
34 i
/CST3 encoding SEQ inhibitor activity
/1471 ID NO.: 81
SEQ ID NO. None AL357873 Novel novel
/none
/none
SEQ ID NO. AL645465 novel J novel
86
/BQ182670
TABLE 2 ¨ Shows the concensus sequences for SEQ. ID. NO. 1 and SEQ. ID. NO. 2
cloned from a mature human osteoclast sample.
Sequence ORF Polypeptide
Identification Nucleotide sequence No.
Positions
SEQ ID NO. 83 1-987 SEQ ID NO. 48
SEQ ID NO. 84 I 1-471 SEQ ID NO. 49
CA 02785046 2012-08-02
WO 2007/093042 PCT/CA2007/000210
94
TABLE 3 -List of mouse orthologue for AB0326
Sequence NCB! Accession ORF Nucleotide
Polypeptide
Identification Unigene Number Positions sequence
Cluster No.
SEQ ID None XM_884636 122-1102/ SEQ ID 1
1
NO. 35 /L00620235 1 similar to neural cell NO .82
620235 adhesion molecule 1
/
= 2/unknown function
TABLE 4 - list of additional sequences identification of plasmids and shRNA
oligonucleotides
Sequence name Description
Identification
SEQ. ID. NO. 36 = p14 Vector for STAR
SEQ. ID. NO. 37 p17+ Vector for STAR
SEQ. ID. NO. 38 pCATRMAN Vector for STAR
SEQ. ID. NO. 39 p20 Vector for STAR
1 SEQ. ID. NO. 40 OGS 77 Primer used for STAR
p14 vector
SEQ. ID. NO 41 OGS 302 Primer used for STAR
p17+ vector
SEQ. ID. NO: 42 human 0326.1 siRNA sequence for
SEQ. ID. NO. 1
SEQ. ID. NO: 43 Human 0369.1 shRNA sequence for
SEQ. ID. NO. 2
SEQ. ID. NO: 44 mouse 0326.1 shRNA sequence for
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
Sequence name Description
Identification
SEQ. ID. NO. 35
SEQ. ID. NO: 45 mouse 0326.2 shRNA sequence for
SEQ ID NO. 35
SEQ. ID. NO: 46 pSilencer2.0 vector
SEQ. ID. NO: 47 pd2 vector
0
TABLE 5 -
=
NucleotideSequence
ORFs
(5'-3')
^SEQIDNO.:1
SEQIDNO.:48
TCCGCCTCCCGCAGAGCCCACAGGGACCTGCAGATCTGAGTGCCCTGCCCACCCCCGCCCGCCTTCCTTCCCCCACCAC
GCCTGGGA MEKSIWLLACLAWVLPTGSFVRT
GGGCCCTCACTGGGGAGGTGGCCGAGAACGGGTCTGGCCTGGGGTGTTCAGATGCTCACAGCATGGAAAAGTCCATCTG
GCTGCTGG KIDTTENLLNTEVHSSPAQRWSM
CCTCCTTGCCGTGCGTTCTCCCGACAGGCTCATTTGTGAGAACTAAAATAGATACTACCGAGAACTTGCTCAACACAGA
GGTGCACA QVPPEVSAEAGDAAVLPCTFTHt,
GCTCGCCAGCGCAGCGCTGGTCCATGCAGGTGCCACCCGAGGTGAGCGCGCAGGCAGGCGACGCGGCAGTGCTGCCCTC
CACCTTCA HRHYDCPLTAIWRAGEPYAGPQV 0
CGCACCCGCACCGCCACTACGACGGGCCGCTGACGGCCATCTGGCGCGCGGGCGAGCCCTATGCGGGCCCGCAGGTGTT
CCGCTGCG FRCAAARGSELCQTALSLHGRFR
CTGCGCCGCGGGGCACCGAGCTCTGCCAGACGOCGCTGAGCCTGCACGGCCGCTTCCGCCTGCTGGGCAACCCGCGCCG
CAACCACC LLGNPRRNDLSLRVERLALADDR 0
N.)
CO TcTcGcTGcGcGTcGAGCGccTCGCCCTGGCTGAcGACCGcCGCTACTTcTGccGceTcGAGTTcc-
CcGGceAcGTCCATGAceGcT RYFCRVEFAGDVHDRYESRHGVR
ACGAGAGCCGCCACGGCGTCCGCCTGCACGTGACAGCCGCGCCGCGGATCGTCAACATCTCCGTCCTGCCCAGTCCGGC
TCACGCCT LHVTAAPRIVNISVLPSPAHAFR 01
0
TCCGCGCGCTCTGCACTGCCGAAGGGGAGCCGCCGCCCGCCCTCCCCTGGTCCGGCCCGGCCCTGCGCAACAGCTTGGC
AGCCCTGC ALCTAEGEPPPALAWSGPALGNS
GGACCCCGCGTGAGGGTCACGGCCACCTAGTGACCGCCGAACTGCCCCCACTGACCCATGACGGCCGCTACACGTGTAC
GGCCGCCA LAAVRSPREGHGHLVTAELPALT
rn
ACAGCCTGGGCCGCTCCGAGGCCAGCGTCTACCTCTTCCGCTTCCATGGCGCCAGCGCCGCCICGACGGTCGCCCTCCT
GCTCGGCG hDGRYTCTAANSLGRSEASVYLF N.)
0
(1)
CTCTCGGCTTCAAGGCGCTGCTGCTGCTCGGGGTCCTGGCCGCCCGCGCTGCCCGCCGCCGCCCAGAGCATCTGGACAC
CCCGGACA RFRGASGASTVALLLGALOFKAL
CCCCACCACGGTCCCAGGCCCAGGAGTCCAATTATGAAAATTTGAGCCAGATGAACCCCCGGAGCCCACCAGCCACCAT
GTGCTCAC LLLGVLAARAARRRPERLDTPDT N.)
CGTGAGGAGTCC C TCAC C CAC CAA CAT C CATTTCACCACTGTAAAGAACAAA GGC CAGT GC GAGG
C TTGGC T GGCACAGC CAGT C CT PPRS QAQESNYENLS QMNP RS P P 0
GGTTCTCCGGCACCTTGGCAGCCCC CAGCTGGGTGGCT C C TCC CCT GOT CAAGGT CAAGAC C CTGC T
CAAGGAGGCT CATCTGGC C T ATMCS P
7J CCTATGTGGACAACCATTTCGGAGCTC C C TGATATTTTTGCCAGCAT TTCG TAAATGTGCATAC GT
CTGTGTGTGTGTGTGTGTGTG 0
N.)
AGAGAGAGAGACAGAGAG TACAC GCA TTAGCT TGAGCGTGAAAC TTC CAGAAATG TT CC CTTGCC
CTTT CTTAC CTAGAACAC CTGC
TATAGTAAAGCAGACAGGAAACTGTT
a)
"0
-4
=
t.4
SEQIDNO . :2
SEQIDNO : 4 9
ACGGAAACGGGCGTGCCATTTCCGCGCACGTCTG CAGATGCGGTAGTCGATTGGT CAAGTCTCCCATGGCTCCTC
CTTCATCAGGAG MIGS GLAGSGGAGG PS S TVTWCA
GTGGGCAAACCGCGCCATGATAGGCTCGGGATTGGCTGGCTCTGGAGGCGCAGGTGGTCCTTCTTCTACTGTCACATGG
TGCGCGCT LFSNHVAATQASLLLS FVWMPAL
GTTTTCTAATCACGTGC-
CTGCCACCCAGGCCTCTCTGCTCCTGTCTTTTGTTTGGATOCCGGCGCTGCTGCCTGTGGCCTCCCGCCT L
PVASRLLLL PRVLLTMASGS PP
TTTGTTGCTACCCC GP.GTCTTGCTGACCATGGCCTCTGGAAGCCCTCCGACC CAG CC CTCGCCGGCCTC GC-
ATTCCGGCTCTGGCTA TQ PS PASDSG SGYVPGSVSAAFV
CGTTCCGGGCTCGGTCTCTGCAGCCTTTGTTACTTGCCCCAACGAGAAGGTCGCCAAGGAGATCGCcAGC-
GCCGTGGTGGAGAAGCG TCPNEKVAKEIARAVVEICRLP_AC
CCTAGCAGCCTGCGTCAACCTCATCCCTcAGATTACATCCATCTATGAGTGGAAAGGGAAC-
ATCGAGGAAGACAGTGAGGTGCTGAT VNL 'POI TS /YEWICGKIEEDS EV
GATGATTAAAACCCA.P.AGTTCCTTGGTCCCAGCTTTGACAGATTTTGTTCGTTCTGTGCACCCTTACGAAGTGGCCG
AGGTAP.TTGC LMM IKTQS S LvPALTD FVPsvHp
ATTGC CTGTG
cAACP_GGGGAACTTTCCGTACCTGCAGTGGGTGCGCCAGGTCACAGAGTCAGTTTCTGACTCTATCACAGTC CTGC
C yEvAEVIALPVEQGNFPYLQWVE
ATGATGAGCCCTGTTCCTGCTCATCATGAAGATCCCCGCGATACTTCAACGCCTTCTGACTTCCAGGTGATGACTGGGC
CCCCAATA QvTESVSDS ITVLP
AATC CC GT CTTTGGGTCTCT CTG CCAAAAAAAAAAAAAAA
0
co
co
co
rn
n.)
Cl)
0
rn
n.)
rn
co
7J
n.)
N.)
Z71
t.)
0
SEQIDNO . :3
SEQIDNO . : 5 0
CGGTGTcTCGTCATCTCCGGGAAGACTCGGCGCCTGGGTCCG CG
CTCTcTGGGTAAGcTTICCGGGAAGCTTTCCCGGGAGCTCGCT MGDEDKRI TYED SEP S TGMNYTP
GGTCCTGGCCCCAGPAGCCTGCGGACCCGCCCAGGGAGGATAAGC.AGCTGAAAGACCGCGCGGTGcCGcTccGAGGCC
CCGGGACGT S MHQEAQEETIIMIGAGIDANE PT
GGGCCCATGGTCGGCCTGGCGCCAC CTTTCCGGGGGAAGCCACGCGCACGAGGCATCGCACG
CGGCTCTGCACCCGCGCCGCCGGAC EGS ILLKSSEKKLQETPTEANHV
CTGAAACCCGGCGGAGGGCACACGGGGCTGCCGCTecGGG C CC
CGGACCAACCCATGCTTACTCCGGAGCCTGTA.CCGGCGC CGACG QRLRQMLAC PPTIGLLDRVI TNVT
GGICGGACCTCCCTGCGCGGIGTCGCCCAGCGGG'ITCGTGCGAAAGGCOGGG CCGACTACACGCGG
TGCCGCGCCCTGAGAC:CGT TT I IVLLWAVMS ITGSECLPGGNL
ATCTGCAGTCAACGCAGCCTGCCGGCTCAGCCTGGGAAGATGCGCGAATCGGGAACCCCAGAGCGCGGTGGCTAGACCG
GGCTCCGC EGI I I LPYCAIIGGKLLGLIKLP
CGCCTCCCCCACAGCCCCITTCCTAATCGTTCAGACGGAGCCTGGICGAGTTCGCCGGAG-
ACTGCCAGATCTCGTTCCTCTTCCCTG TL P PL PSLLGMLLAGFL/RNI PV
TGTCATCTTCTTAATTATAAATAATGGGGGATGAAGATAAAAGAATTACATATGAAGATTCAGAACCATCcACAGc-
AATGAATTACA INDNVQIKHKWS S S LES IALS I I
CGCCCTCCATGCATCAAGAAGCACAGGAGGAGACAGTTATGAAGCTCAAAGGTATAC-
ATGCAAATGAACCAACAGAAGGAAGTATTC LATRAGLGLDSICALKKLKGVCVRL
TTTTGAAAAGCAGTGP_AAAAP-
kGCTACAAGAP_ACACCAACTGAAGCAAATCACGTACP.AAGACTGAGACAAATGCTGGCTTGCCCTC SMG PC
/VEACTSALLAHYLL GLP
CACATGGTTTACTGGACAGGGTCATAACAAATG T TACCATCATTGTTCTT
CTGTGGGCTGTAGTTTGGTCAATTACTGGCAGTGAAT WQCAIGF I LG1TVL GAVS PAVVVPSM
cn GTCTTCCTGGAGGAAACCTATTTC-
GAATTATAATCCTATTCTATTGTGCCATCATTGGTGGTAAACTTTIGGGGCTTATTAAGTTAC
LLLQGGGYGVEKGVPTLLMAAGS
CTAcATTGCCTCCACTGCGTTCTCTTCTTGGCATGCTGCTTGCAGGGTTTCTaATCAGAAATATCCCAGTCATCAACGA
TAATGTGC FDDILAITGENTCLGIAESTGST
cn AGATCAAG CACAAGTGGTCTICCTCTITGAGAAGCATAGCCCTGTCTATCATTC TGGTTCGTC-
CTGGCCTTC-GTCTC-GATTCAAAGG VENVLRGVLEVVIGVATGSVLGF
CO
CO
CC CTGAAGAAGTTAAAGGGCGTTTGTGTAAGACTGrccATGGGTCCCTGTATTGTC-
GAGGCGTGCACATCTGCTCTTCTTGCCCATT F QYFP SRDQD KINCKRTFLVLG
171 CTGCTGGGTTTAC CATGGCAAIGGGGATTTATACTGGGTTTTGITTTAGGTGcT.GTATC T
CCAGCTGTTGTGc-TGCCTTcAATGc LSVLAvESSvHFGFPGSGGLCTL 0
--1 TCCTTTTGcAGGGAGGAGGCTATGGTGTTGAGAAGGGTG
TCCCAACCTTGCTCATSGCAGCTGGCAGCTTCGATGACATTCTGGCCA VMP.FLAGMGvirsEKAEVEKIIAV
fli
TcAcToGcTTcAAcAcATGeTTGGGCATAGCCTTTTCCACAGGCTCTACTGICTTTAATGTCCTCAGAGGAGTTTIGGA
GGTGGTAA AwDI FULL FGL IGAEVS IAS LE n.)
Cl) TTGGTGroccAAcTGGATemrrcTTGGATTTTTcATTcAGTAcTTTc
CAAGCCGTGACC_AGGAcAAACTTGTGTGTAAGAGAAcAT PETVGLCVATVCIAVLIRILTTF co
TCCTTGTGTTGGGGTTGTCTGTGCTAG CTGTGTTCAGCAGTGTGCATTTTGGTTTCC
CTGGATCAGGAGGACTGTGCACGTTGCTCA LMVCFAG PNLKEKI F S PAWLPK
Ill TGGCTTTC CTTGC_P_GGCATGGGATGGACCAGCGAAAAGGcAGAG G
TTGAAAAGATAATTGCAGTTGCCTGGGACATTITT CAGCC CC ATVQAAIGSVALDTP_RS HGEKQL 0
TTCTTITTSGACTAATTGGAGCAGAGGTATCTATTGCATCTCTCAGACCAGAAACIGTAGGCCTTIGTGTTGCCACCGT
AGGCATTG EDYGMDVLTVAELS IL ITAPIGS co
CAGTATTGATACGAATTTTGACTACATTTCTGATGGTGTGTTTTGCTGGTITTAACTTAAAAGAAAAGATATTTATTTC
TTTTGcAT LLIGLLGpELLQKVERQNKDEEV 0
GGCTTCCAAAGGCCACAGTTCAGGCTGCAATAGGATCTGTGGCTTTGGACACAGCAAGGICACATGGAGAGAAACAATT
AGAGGACT QGETSvQV n.)
rn
ATGGAATGGATGTGTTGAcAGTGecATTTTTeTccATccTcATcAcAccccuakTTGGAAGTerccrTATTGGTTTAcT
GGGccccA.
GGCTTCTGCAGAAAGTTGAACATCAAAATAAAGATGAAGAAGTTCAAGGAGAGACTTcTGTGCAAGTTTAGAc-
GTGAAAAGAGAGAG
cr)
TGCTGAACATAATGITTAGAAAGCTGCTACTTTTTTCAAGATGCATATTGAAATP.TGTP.ATGITTAAGCTTAAAATG
TAATAGAACC
AP.P.AGTGTAGCTOTTTCTITAAACAGCATTTTTAGCCCTTGCTCTTTCCATGTGGGTGGTAATGATTCTATATCCCC
AAAAAAAAAA
AAAAAAAAAAA
'1.;w
SEQIDNO.:4
SEQIDNO.:51
GACAACCTTCAGGTCCAGCCCTGGAGCMGAGGAGTGGAGCCCCACTCTGAAGACGCAGCCTITCTCCAGGTTCTGTCTC
TCCCATT MVSSPCTPASSRTCSRILGLSLG
CTGATTCTTGACACCAGATGCAGGATGGTGICCTCTCCCTGCACGCCGGDAAGCTCACGCACTTGCTCCCGTATCCTGW
ACTGAGC TAALFAAGANIZALLLPNWDVTYL
L.4
CTTGGGACTGCAGCCCTGTTTGCTGCTGGGGCaAACGTGGCACTCCTCCTTCCTAACTGGGATGTCACCTACCTGTTGA
GGGGCCTC LRGLLGRHAMLGTGLWGGGLMVL
4-
CTTGCCAGGCATGCCATGCTGGGAACTGGGCTCTGGGGAGGAGGCCTCATGGTACTCACTGCAGCTATCCTCATCTCCT
TGATGGGC TAAILISLMGWRYGDFSKSGLCR
TGGAGATACGGCTGCTTCAGTAAGAGTGCGCTOTGTCGAAGCGTGCTTACTGCTCTOTTGTCAGGTGGCCTGGCTTTAC
TTGGAGCC SVLTALLSGGLALLGALICFVTS
CTGATTTGCTTTGTCACTTCTGGAGTTGCTCTGAAAGATCGTCCTTTTTGCATGTTTGATGTTTCATCCTTCAATCAGA
CACAAGCT GVALKDGPFCMFDVSSFNQTQAW
TGGAAATATGGTTACCCATTCAAAGACCTGCATAGTAGGAATTATCTGTATGACCGTTCGCTCTGGAACTCCGTCTSCC
TGGAGCCC KYGYPFKDLHSRNYLYDRSLWNS
TCTGCAGCTGTTGTCTGGCACGTGTCCCTCTTCTCCGCCCTTCTGTGCATCAGCCTGCTCCAGCTTCTCCTGGTGGTCG
TTCATGTC VCLEPSAAVVWHVSLFSALLCIS
ATCAACAGCCITCTGGGCCTTTTCTGCAGCCITTGCGAGAAGTGACAGGCAGAACCTTCACTTGCAAGCATGGGTGTTT
TCATCATC LLQLLLVVVHVINSLLGLFCSLC
GGCTGTCTTGAATCCTTTCTACAAGGAGTGGGTTCAGGCCDTCTGTGGTTAAAGACTGTATCCATGCTGTGCTCAAGGA
GGAACTGG EX
CAAATGCTGAATATTCTQCAGAAGAAATGCCTCAGCTTACAAAACATTTATCAGAAAACATTAAAGATAAATTAAAAGG
TAATCATG 0
cn GTGAPIAAAAAASLAP.W...A.
n.)
CCI
Cl)
(13
(1)
rn
171
co
n.)
rn
cr)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
100
H a H 0 n Z .-1 > +
a ,y1 E.HCP.IxIftii-i
g>g NaJanqp
xo.-lopcDogg
a cz 4 0 8 Ee.
O 0 a. >1-INH,-
H OH H (r) ui 14 u >
rsi
It) E. H :.> ,..1 v..1 1-4 (01 III
- E.0( 05,,E.>.,1> 41
= CP1HcnCD4101--IFil-1
2 cnt
n 11, H PC A
H Pk.....¶7:4 C.DZ CA
i'i),' cc',4 'A,'-. 8 g
Cl) Z ,. > F. E. 1- i-1 pc 1....
HUH LA H El H [-J' (A r. r, ', 8 g 6,
6 0 g `E?. H El P. ti: I1 ) H 0 0 .E-.1: EC1 8 g 6 Pc
E.0<a0UP0aH0E.E.E.Hu0000
0 0 U 0 0 / a U 0 0 a a 4 CD y CD CD y 0 a
UUCDOr Hu E.0a0HuHouUu
0 cDa E.E.000HHHE.E.4 E. 0
u 0 a C. u u 0 E--, E. 0 0 u U 0 E. uu u 0
u Kt auchuF, a cD0E-+KCua H Hu00
a UUKI:E.E.Ucia a a UE.PagE.0
q0c9Hua 00 cDuug c..) Hu a cDUE.
LI 6 8 `,' U rEr Ec.) -.' t 6 E: , g g .,i. `,'c 8
HoHgour...,HHHHHu4H000
,...) C., UuE.HHE.HuHu0HOuUHU a
0 0 u V U U H E., a E. 0 E. 4 E. a H u H U U
uUE.E.0UUuLD000U,H0a40Hu
UOHHOH ,,: HUE.< KC 0, E-. E. E. U / CD t.
8 ug E6 [; 8 ,E=' E,-' 'E, E6' EY. 1 'E*4 El 6 ' C-D) El .
A .e.E.,,CPCOLDUE.C.)UE.,FICJE. CDOE.
UCDUE.E.E. E. E. a a U a a E. u U H 0
i
E,-' 6 8 6 c,' 8 'e, i-_,) r;, ,,== E.-: E. .., '4
',; F. SE5. 8 ri
H 0 E. 0 a a U H a a H F. k.-, ,_, 0 E ,.) CD
ti:
E-. gr, 0 PC CD PC CD E-. CD A E-. U E. PC rt: U E. pl: U U
E-. 0 E. u 0 u 1. u cD, E. u a E. E. E. E-+
O E. E. u E. u E. a a a H 0 U F. t,=4 E. t). H S-2 8
g -,) -,"3 g ,`: ,E;.' g ill ,`-g) 6 8 g 6' r, g 8 g 0
gc98`gr,606'''''',.E.' (-) H PK [-I
E-1 Pf: CD 1-+ 0 E. Kg 0 6 ,r4 (x"C) 8 ,4 Ll r, g g 4
u E. CD CD, E. H A E. E. E. E. 0 a g 0 cD
Ii: CD a 0 CD 0 CD a CD a HUHU E. a u c..4 0 ,.
E-, 0 u 0 CD U r, E. E. E. a E-, PC F4 E. 0 E. A H 0
P. 'c'5 ,C-)p3EY+ -4' 8 r4 i,' ,:õ.5' ,.`3 `-'
,,.,:cH V-1.8 8 ri
gouHo HuPc7F-. auat.1,,t..90
u0E.E.OuUE.E.HucDUE. g CD(D a
U a E.00 H HuHE-4 a HUO 0 ci0 0
6 gl E( '2, Et , +Ea' r+ FY , Er'C P. U E. g 8 E.4
., 00,,,,,,,,,,,E,,, H,/ g ,,,, ,6:
O g 0 CD H H F. 0.4 0 4 E. 0 c) U CD 6 !.. ts
1
O 0 aULDE.0<0E.OHE.E. K4 r .
CDUOVE.CDCDC3U,rcrCr HE'F"1.(1EC,
ECj (i EC. CD Li' LE-2. (Cni C:D 0 8 E1 8 `E?, EC,' [-_,' g 0
g0000g0g000ogggggiu..., 5
a CD ED H a < E. u H 4 E, 1-. F. PC CD E. A (9 A U
0 ,, 0 0 0 c..D H ,.- y y ,5 o y o E. F. .9 ,(i..., (0)
0 It H a 0 a H It 0 U a H U 0 H H H H
U 0 E. 0 E. H a a E-; a H E. H A U E. El El (8
a UHUIDE. aug a E.H auEE-:,,u b 4
r7Y)E.g.F-.) 8 6 2i, li:', d ' E,-' ..-), Eg N E--. 6 8 0 (C59
CD E. C.9 U C9 CD U U CD U E. HE. E.g
O00 V E. pa ()i: Ha <Oa u 0
C) F,C U a U E. 0 E-+ E. / u < U E. 4 1 4 E-4 E.
OuggUE.c.DOuE. uuuE.HE.Huuu
uHugUrg0Hu 4 0E+Huuqua y,
HaU00a 0a HO,P4 gcr,,..,U,,E` ,,,L F. tc3 ',41,
CD F. PI: A U r_.: ,(0.1 r!). id ,i: 6 t-; 6 E. A E. CD 1
8 r:, ,T4 8 (c)7 CD U U P4 It H U 0 Kg H 0 Kg a U H It
6r8b4EP.66.6,11 (1`6'El E, :: r, ,tc. r.)' 5
-,) 666`,Z6t-if-,' 6 n [-,'F-t)E,-,' ';'4U88 R
/
HUHOU004E.a, t._ E. F-. c cic.DE-+UOPC..)
CI u 4 < 1-, CI 4 U Pt C.) E. E. CD E. U H PC CD C.), ,,,q E.
0 E-19 E-.) .)q 8 '6 8 8 `,-);; ,`,-) r. ,r;1; El El 8 ,` '6
fr4
O HU 0a H F-4 0 A 1.9 CD I. ID H U El
C.) ,.., 8 6
In UUOU CD 1, a OU0a aHOHOA t.
- ii E. 0 C..) 0 E. E. a -a 0 E- , u a u WE-. E, c 0
, ,, rõ r. ,,. Ey,
= E= E, E. H HO E. a Pc gg HOC) U E. U H
O 000 a Oa 00HuHOOF.C2S9,0p, y,
= 0 ci. o 0 E. 0 E. g HE. E.C.TD E, 4 t-
. ,-4. µc)., '7'6
fZI 0 a E.aUHE.H aHOE. 0 uu0
H OUUUCDOCDE.E, g q g ri r, 8 ,) ,-; ,,,t,
0 ,:4 0 0 0 A: H H E-1 U E-1 r.1, U f -I 4 E, 0 E,.., 0
E4 UCDUE-IC.DgUACDPCPCgE.
C) UCDO<OUPCE.C.JUE.UPgi-.H AHUULD
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
101
j. CI H
c.1) H 0
cri cl. m r=1
rt. < fir ci) H
o cn <
rn ?Ho. H
L2 (tt OEsEs
E
t14 E. M
O 0 0
n cn a u oi ,a,
H g g > a+ g
01 g g U 0 al
al 0 g r14 H la,
cn a a < m
utno4E.W4E, E. 0 F. E. g4 of4E-,<HP<UHPE.0PU
UH,u4rouPPE,HoOppochouuouuE.,4
<OWEJ PCDHUDCJUA<E1 <o<opo4PE.PKE
pi.; E. H u CJHUE.E.E.UPHE.A0C)<CDOHUPE. 4 E.1
0 E. U E. A H u U KE < H g E. P 4 4 < E-, u o 4 < Kg E. P ,, Es Es
E. H E A P 0 0 U E. E. A PA 4 A
U= POHUPHOPPHUAHE. UCDOCDE.K4CDOE.HP0
PUAOUIDUAOF. 4 E-I < F.0 UPOOPOHHAACDE.
E.00<E-..A,E.E.E.<4F.E.E.AUAUE-.00H L5UP 4 E.
U 0 F. u o 0 H 0 H H U 0 0 U Ui E. A p E. u
UUFUHACJOOHE.E.00CDAOUU4 UU oHu
OE. puoo 0 4 HOP OCJAPPOL)<U 0 HOLD E-4 E.E. ,
UPCDC)AAAE.C.?E.A AE.<uc.DAHUCA<AH 4H0
/ AUPCDAUF.E.0,UHLDHE.,<UHAUC..5 HCDA E-.0E-,
E-- UUCDOACD , HUCDE--.UE-.c1.<PCDC.) 4 Kg < 4 E.F.
UPE.A0A00E.0 UHAP CDHOOPAAE. E.F.E.
POUE-luAH<A 0,-4PU OPE. .4 OHAE. APE.
UCDOA E-.F.CDA.<0 E-.E.ULDE--.PCDOE-.F.E.A NH<
E.u0uHu4 044 UAHHUE. .< E.E.E.040E.
U U 0 CD U E. U 0 A 0 El 0 E. E. PA E. U < < CD CD 0 E. 0 0 E. U E.
0 U 0 U E. 0 0 E. u E-1 4,4 rc U a, E. F. A E. A E. El U o
E. 4 4 u
U H u E. E. A E. A U U A E. E. 0 4 E. U U < A U E. U < < E-, o E.
0 0 0 E . H U E. A. 0 A a. E.-, re, E-, U o H P E. 0 E. 0 c..) E. A < E. CD
H E. E. 0 E. () E. E. A n. 0 [-, < ril 0 r4., E. 0 Kg < 0 E-. E-, c..) A E. E.
<
u 0 U E-4 U r.) F. U 0 E--. < u E-, E. 0 H, E. E. U E, 0 E. q E-. E. E.
F.
H U U CD U E. F. 0 P E. A A U A: A A < < < CD U A U F. E. F. A
rUE.UPC-DA(DAE-.CJOHULDUC9HHHOU E.AOU
0 UPHUHE.AF.CDPAHCDCDPHE.PH AHCDP
00000 HHOAH E--.C9HU KE o 4 < H 10E.0=KqE;'
U(DHIDA/HUACD E-, E-, U: Es E.P.A<OA< AAPAL)
E-(C) U H gl.: A 0 H t. A A 0 A u u a, H 0 u H 0 A 4a. 0
00000 PA AAHE. P o o 4, u u < 4 0 POPE.E.HE-.
U U U E. A F. L3 P E. U E. A E. g E. U C)
E. 0 A 0 0 gc ,:: 0 EA E--.
POO AuLIPHUHAAE.HE.00H<AACDHACDUHA
u u 4 4 CJO OPAUUAUPCJHOOPPOUHA F. HC)
O U c.) E. E. P H E. 0 C.) F. E.
E. C.) 0 H 4 E. -A H E. U E. E. CD U E-.
UOUOAAHUF.HPHETUCDP-40E1OHE. F.0
H < 0 < 0 U H 0 H F. U E. CD A 1= U CD CD CD H H E. U A E.
HUUUE.CDOAAAPA.A 0,01ACDE. HUE. P0
U U H E. F. U E. a; A F. A H A
E. 4., E. U E. F. A 0 E. U El H 0 E.
E. E. 4 A U A E. E. U E. C) E. P A A C.) CD U 0 E. U .< 0 E. 0
ALDUCE.CJOHO E. APPC.5.ZE.r(000 AU 000E.
A E. U E. 00HU0 0 HHHHU40H.g0444 C.5 E. E. A
1 ci U U E. A E. 0 H 0 E. < CD C) A A Cl c.) 4
4; H E. U E. E-, E-, E-i
U.U0 H00UH.c004,<0-444H 0 U CD P E. Hug()
E. H o H E. P U E. .< .< 0 -. F. <
E. 0 r< H < .< 0 E. P Kg E. E. KE <
uu0 /E-,opuucn ,r, E-IHUOPPUUE-
.U.APUHA,
U u A. u E-. c-, o, U 4 H 4 E-
, 4 U 0 .4 0 H 0 4 H 0 E. U E.
H 1.9 U U HPAE.E.C.DPg04a:
UAE.F.HPAACDE.
AACJUOCD < H 4 E. 5 ,,,.,õF, . 00H.40HU0E.E-.
000E.04.00CDP E.E.PH A Ag<CDOE.E1AF.
O E. A A a, 0 1-. E. A r< E. 4 E. U E. c.) U u A A
P E. 0 E.
0 A u U A A < E. E, 0 0 E. c) C. 0 < U < P A A F. H A U
U E. cn H cn 4 El A A CD F. A u E. 0 E. 0
Ci H 0 P E-' U , E' ,4
E. c) U E. 0 E. 0 U E. E. E. E. a, 0 A A A 0 E. 0 0 H U E. E. H E-,
O<.<00HOUUE-,,<00H H<Kg aPKEEnHuo/pp
ocio<ug< 1,..õ,,, A U F. U o 0 CJ E. E. r
HUH0-40 A PHE-.UUAHHA < CJAOPPE. Es Es
o 4 u o 4 H 0 A (-) E-, < P < re, o 4 < < 0 u H 0 0 E-, 0 gti
< o o < 0 u 4 H < o PHAHAE.A AUCDOHUA cl
u F. E. A, A A A 0 U 0 E. H F, C.) Ai A A 0 U A CD F. F. U 0 A
A E. c..) U A C.) 0 0 E. U C) E. CD A HUE-,0H0HU <40
UU000 < <HO HUE.0
HE.......:E.0E.C.7.<0 < A 1.E.
UE.E.UE. 0000 HEUHE.E. Ch ,cr H E. H 0 P r CJ CJ E.
O 0 CJ A U 0 A E. H < 8 E, 0 ,
Cl E. H E. E. g E. 0 0 4 P
,<HOUU0<00:E. H H t, 0 E., E-, 4 .I, E. E. E. A E. E-, E. E.
Pc tiE-,OUACDE.H0 4H04E.E.UE.U C.5 CDPAUH AU,
1
(5 CD U A P a, 0 0 E-, E-1 P.< pc F. A E. 0 H < CD CD CD E. 4 4 El
OULDOUCJOAD E. 4 En < E-, 0 < 4 0 < 0 F4 .'4 E-4 1. 0 0
4 Oes<UK:0H0.4E.KCE.E.C7HU HUUL7HHE-.U0.<
i
0",0U0 U,..,<-.< <0<HHE.H <HE.P0H0 < U F.
CJU(DAA UAHOUgHE--.UP A UCDUE.HE.HPU
F,C0E.HHouopppH4 E-.E.E.E.P PH<PHO HE-.E.
ti7 UU0H00,<HOHHH ACJHUHP UUL)PUE. H < H
=== OPOHOHAHHUHOOPAPH,OUAUF440E-10.< Of-'0 U E. 0 E. 0 0 E. CJ E-. t.9 C-. < <
..< E. E. U U 0 U E. 0 E. < H < 4
o < 0 E-I E. 0 0 E. C.) 0 0 E.
g E. E , E. 4 4 P C.J P 5 F. U A s4 H 0
Z A U 0 F. PA CI 0 A E. P <E.OH<E-. < E-, u < c) H 0 F. E. E.
IC) CDUHE.A<HUE-100A E4 E.U0E.OPU000 H E. 4 <
H < < U 0 0 0 U E. P A H A r 4 u c) E, 4 0 E. E-, F. 0 a. E. A H F.
0 000000HUAA10E.E.,<HHACJA E. 0 E-4 a El 4
Cs) 0 C.) EJ 0 E. E. 0 CJ F. A 0 CJ H H A E. 0 U Q 0 E. E--. 0 <
E. E.
Cs) POCDPOHOHoU OHAL5 ACJAULDOPOHOHA E.
V OUUPCDP4POE.00404000PHOPOHAE.APA:
SUBSTITUTE SHEET (RULE 26)
SEQIDNO . : 7
SEQ IDNO . : 5 4 ),4
CTGGACGAGTCCGAGCGCGTCACCTCCTCACGCTCCGCCTGTCGCGGGTGTCccGcCGGCCCGTTCCGTGTCGCCCCGC
AGTGCTGC MAVFVVLLALVAGVLGNEFS ILK
GGCCGCCGCGGCACCATGGCTGTGTTTSTCGTGCTCCTGGCGTTGGIGGCGGGTGITTTGGGGAACGAGTTTAGTATAT
TAAAATCA SPGSVVFRNGNWPIPGERIPDVA
CCAGGGTCTGTTGTTTTCCGAAATC-GAAATTGGCCTATAC CAGGAGAGCGGATO CC.AGACGTGGC TC-
CATTGTCCATGGGCTTCT DT ALSMGFSVKEDLSWPGLAVGNLF
GTGAAAGAAGACCTTTCTIGGCCAGGACICGCAGIGGGTAACCTGTTTCATCGTCGTCGGGCTACCC-
TCATGGTGATGGTGAAGGGA 1-1RPRATVMVMVKGVNKLALPPGS
GTGAACAAACTGGCTCTACC CC
CAGGCAGTGTCATTTCGTACCCTTTGGAGAATGCAGTTCCTTITAGTCTTGACAGTGTTSCAAAT VIS
YPLENAVPFSLDS VANS INS
TCCATTCACTCCTTATTTTCTGAGGAAACTCCTGTTSTTTTGCAGTTGGCTCCCAGTGAGGAP.AGAGTGTATATGGTA
GOGAAGGGA LES EETPVVL QLADS EERVYMVG
AACTCAGTGITTGAAGACCTTTCAGTCP_CCTTGCGCCAGCTCCGTAATCGCCTGTTTCAAGAAAACTCTGTTCTGAGT
TCACTCCCC KANSVFEDLSVTLRQLRNRLFQE
CTCAATTCTCTGAGTAGGAACAATGAAGTTGACCTGCTCTITCTTTCTGAACTGCAAGTGCTACATGP.TP.TTTCAAG
CTTGCTGTCT NSVLS S LPLNS LS RNNEVDLLFL
CGTCATAAGCATCTAGCCAAGGATCATTCTCCTGATTTATATTCACTGGAGCTGGCAGGTTTGGATGAAATTGGGAAGC
GTTATGGG SELQVLHDISSLLSRH.KHLAKDH
GAAGACTCTGAACAATTCAGAGATGCTTCTAAGATCCTTGTTGACGCTCTGCTLAAAGTTTGCAGATGACATGTAGAGT
CTTTATGGT SPDLYSLELAGLDEIGK-RYGEDS
cn
GGGAATGCAGTGGTAGAGTTAGTCACTGTCAAGTCATTTGACACCTGCCTCATT.AGGAAGACAAGGACTATCCTTGAG
GCAAAACAA EQFRDASKILVDALQKFADDMYS
GCGAAGAACCCAGCAASTCCCTATAACCTTGCATATAAGTATAATITTGAATATTCCOTGGTTITCAACATGGTACTTT
GGATA.ATG LYGGNAVVELVTVKSFDTSLIP.K
CO
AT CGCCTTGGCGTTGGCTGTGATTATCACCTCTTACAATATTTC-GAACATG GATCCTSGATATC-
ATAGCATCATTTATAGGATGACA TRTILEAKQAKN PAS PYNLAYKY
7=- 1 AAC CAGAAGATTCGAATGGATTGAATGTTACCIGTG CCAGAATTAGAAAAGGGGGTTGG2-
'2=ATTGGCTGTTTTGTTAAA.ATATATCT NFEYSVVFNMVLWIMIALALAVI
T T T AG TGTGCTTT AAA G TAG AT A G T AT AC T TTA CAT T TA TAAAAAAAAAT CAP.A
TTTT GTT CTT TA TTTTG T GTG TGC CTGT GA T G T I TS YNIWNMD PGYDS I IYRMTNQ
TTTTCTAGAGTGAATTATAGTATTGACGTGAATCCCACTGTGGTATAGATTCCATAATATC.:CTTGAATATTATGATA
TAGGCATTTA KIRYLD ND
cn
ATAACATTGATTTCATTCTGITTAATGAATTTGGAP.ATATGCACTGAAAGAP.ATGTAAPAC.ATTTAGAATAGCTCG
TGTTATGGAAA 0
AAAGTGCACTGAATTTATTAGACAAACTTACGAATG CTTAACTTCTTTACACAGCATAGGTGAAAATCATAT
TTGGGCTATTGTATA
CTATGAACAATTTGTAAATGTCTTAATTTGATGTAAATAACTCTGAAACAAGAGAAAA'
GGTTTTTAACTTAGAGTAGCCCTAAAATA
TGGATGTGCTTATATAATCGCTTAGTTTTGSAACTGTATCTGAGTAACAGAGGACAGCTOTTTTTTAACCCTCTTCTGC
AAGTTIGT co
TGACCTACATGGGCTAATATGGATACTAAAAATACTACATTGATCTAAGAAGAAACTAGCCTTGTGGAGTATATAGATG
CTTTTCAT
TATACACACAAAAATcCcTGAGGGAcATTTTGAGGCATGRATATAAAAcATTTTTATTTCAGTAAcrrrrcceccTGTG
TAAGTTAc
TATGGTTTGTGSTACAACTTCATTCTATAGAATATTAAGTGGAAGTGGGTGAATTCTACITTTTATGTTGGAGTGGACC
AATGTCTA
TCAAGAGTGACAAATAAAGTTP_ATGATGATTCCAAAAAAAAAA
;74
SEQIDNO : 8
SEQIDNO : 55
AGCGGGGCAGCGGCTGCGCCCTGCGCCGGGGCGGP.GCCGGGGGCGGGCCGGCGGCCC-
GCAGGCGGGGGCTGGGGCCCGAGGCCGGGA ME ILMTvS KFAS I CTMGANASAL '76
GTGCCTGAGCGCCGGCGGCGACGACGGCAGCGGCGGCCCAGCGGGCTCGGTGGTTGGGTCCGCGGCGGCTCGGGGTCCG
CCCGCGGG EKE IGPEQFFVNEHYFGLVNEGN
CTGCGGTGCGAGCGGGCGGCCCGGCTCCCCTCCTCCCCCGCCCGCCGCCGCCGCTGTGATTGGGTGC-
AAGATGGCGCTGGCCGGATG TCYCNSVLQALYECRPFREKVLA
GAAAT CC TAATGACAG T CTC CAAATTCGCCTC CA Tc TGTACcATGGG c G ccAATGCTTCC
GCATTAGAGAAAGAGATTGG TC CAGAA YKSQPRKKESI.LTCLADLEHs TA
CAGTTTCCGGTCAATGAGCACTATTTTGGATTAGTCAATTTTGGGAATACCTGCTACTGCAATTCAGTTCTTCAAGCAC
TTTATTTT TQKKKVGVIP PKKFITRLRKENE
TGTCGTCCATTTCGGGAAAAAGTTCTTGCGTATAAGAGTCAACCTAGGAAAAAGGAC-
AGCCTTCTTACATGCTTAGCAGATCTCTTC LFDNYMQQDAHEFLNYLLNT IAD
C_ATAGCATAGCCACTCAGAAGAAAAAGGTTGGAGTAATACCCCCTAAGAAGTTCATCACAAGATTACGGAAAGAAAAT
GAGCTTTTT ILQEERKQEKQNGRDENGNIDNE
GACAACTACATGCAACAAGATGCCCATGAATTCTTAAATTACCTA.CTAAATACAATTGCTGATATTTTACAAGAAGAG
AGAAAGCAG !INNS TPDPTWVDEIFQGTLTNET
GAAAAACAAAATGGTCGTTTACCTAATGGTAATATTGATAATGAAAATAATP.ACAGCACACCAGACCCAACGTGGGTT
GATGAGATT RCLTCETISSICDEDFLDLSVDVE 0
TTTCAGGGAACATTAACTAATGAAACCAGATGTCTTACTTGTGAAACTATAAGCAGCP_AAGATGAAGATTTTTTAGAC
CTTTCTGTT QNTS ITHCLRGF SNTETLC SEYK
co
GACGTGGAACAAAATACATCAATTACTCACTGCTTAAGGGGTTTCAGCAACACAGAAACTCTGTGCAGTGAATACAAGT
ATTACTGT YYCEECRSKQEP_HKRMKvKKLPM
CO
GAAGAGTGTCGCAGCAAACAGGAAGcACACAA_ACGGATGAAAGTTAAAAAACTGCCCATGATTCTAGCTCTACACCTG
AAGAGATTT ILALHLK.RFKYMDQUIRYTKLSY n.)
.4AATA.TATGGATCAACTTCATcGATATACAAAACTCTCTTACCGGGTAGTTTTTCCTTTAGP_ACTTCGTCTGTTTA
ACACTTCAGGT RVVEPLELRLENTSc-DATNEDRm co
(xi
GATGCCACCAATCCAGAcAGAATGTACGACCTTGTTGCTGTTGTGGTTCACTGTGGAAGTGGTCCcAATCGAGGCcATT
ATATTGCA YDLVAVVVECGSGENRGH.YIATV
ATAGTTAAGAGTCATGATTTTTGGTTGTTGTTTGATGACGACATTGTAGAAAAAATAGATGCACAAGCTATTGAAGAAT
TCTACGGG KSEDEVLLFDDDIVEKIDAQATE cD
TTGACATCAGATATCTCAAAGAACTCTGAGTCTGGTTACATCCTTTTCTATCAGTCTCGGGACTGAGAGGGAACCGTGA
TGAP_GAGA EFYGLTS D Is KNS Es G Y ILFYQS
n.)
CACTTTCTGCCTCATTTCTTCTCTGGTTATTTTGGAAAGGATCAAGCACTGATTTTTCAAGAAAAGAGAAATGCAGGAA
GCTCAGGG RD 0
co
GGCAGTAGCACACTTTGCACACGATAP.AGCAAAGACGATGGATTGACAAGCCCTTCCGATCATGGTAGTTGATTTATT
TGCTCAGGT
n.)
ATCATGCTGTCTC-
TACAGTTCCATACAACAAGGAGGTGAAATCAGAGATACCAGCTCCTCTTTTAAAACAGCCTTCCAGTcATTGGC
ACGCATTTTCTCTTTATTAATTGCACCAATAATGCTTTGAATTCCTTGGGGGTGCAGTAGAAAGAATCGGAATCTGTGC
CGTATTGA
co
TAAGGAGATGP.TGTTGAACACACTGCATAAATTTGCCTGGTTCAGTATGTATAGAAGCATATTCAGTGGTCTTTTCAA
GAGTAAACC
o
7J
AGAAATACTTTTGGGCCCAACACTTGCAGTTGCCTTCCTGATGTAAAAACTAACATGCTAGATAATCCAGTGTCGGGAA
GACAAAGA n.)
TGITTTGCTTCTCTGAAGAAGCTTATAATAATATACAGTATATGTATATGTAGGGAGCAATTGGTCAAAAGTGGCTITT
TGTTTCCC
CAAGGGGAAAGACTC-
GCTTTGTAATTATAATTTTTTCCTTATTTATTTTACTTAAAACTGGTAGAGTCTAAGTATTATATGAAGTGC
n.)
CCATGATTCTGTCAGTAAATTTGAACATATTTTTATTAGTTAATGTCAGTTTP.AGTTGTCCTTTTGTTTGTTTCTATT
TTTAAGGTG
cr)
AATTTTAATTTCTATCTGAAATCAGTTAAGATACCTTGAGP.AAAACTGCAGTGAGAGGAGATAAATATCCTTTTTCAG
GAGGAACTG
ATATCTCTGGCTAAATATTTGTCCTTTTATTATGGTTTCTAAATCAGTTA.TTTTcTTCAGCTTTAATTTCATAAAATT
AAAAAACTA
TTTTAAAAAAAAAAAP_AAAA.A.AAAAAAAAAAAAAAAAAAAA
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
104
rti 0) A E=1 cx= 2 04 A
2 In P. 0 p.. TH F. 0 cI) 2 õ-) .4 a=
cil P. c), fi, CI 0 Pc I U c4 0 0 F. 2 H
cnIccq.C9P..c.JIn :1 c 8 (utri
Hx...,,_1 (1. H u
uc H E. Pi 0 (c) 2 yoox&-,x 0 0
co 2 R cn P. 01 0 . U 0 VI 2 0 re, 2 2 0
2 a , 4 (X .0 .-.1 H 7. Pi P. 1
Ln E. Ey :3 1.4 2 E. in
= P N >1 ,1-1 '', u, ,'-' == gc 0 P. < . X 0 P.,
0 P Of I-1 (21 U H En S. 0 NI 1=4 ,..1 2 E. ?-. (.1
2 In 0. 1-4 2 0 PI E. 0
0
H CI 2 0 cc. I-I KC 0 E. (-c 41 f,,r cl cn p. 4 u u
01
PI c() Ey cn a( C.) E-4 VI go , (.., 0 0 pr=C Lxt H . 54 0
(I) x E. 0 0 f2 3 PC P4 0
UHUU<OHOHO(..91c4E-.< 0 0 E. H 4 H < E. 0 1
c..) C.) C.) (.4 U 0 E. E. rl 0 U 0 0 u u U < E.
C9C9UOUUr4c4HuccU cl,E.0 E.UuE.C9yOUH
cg U U 0 H E. IJ F. cr C.) E. 0 .4 / 0 0
U E. U U 0 E. UE-; E. E . a; .0 U E. 4 E. 0
8 0 0 0 0 0 0 0
c4oduguE.1 E. A
0c9<HOCIE.c4E.E.U0E-.< 0 000c4FuU C.E.
0 0 (I, (11.9 4 c..) U E. u .4 C. H E. F. U 0 u (.4 E. (4
U u E. u g a ei Et E-, E.
UOU<HPUUE.HUUE.F. .4
0 0 1 0 U 0 0 E. cg u
I
CDCD AP-A.440000
E. 0 4 U C.) CJ H 00E-(440HUE. E.
0 y clEIcEJU4(E.E. f-,U 00E+04 (.401H0
C) C) E.04(44Ou .41(.9E-(UE. C) c..9 (9 U U < E. 0
0 0 / 0 UE. E. 4 E. < u E. 4 U 0 U F.
A 0 A U U 0 E. U E. U U < 0 F. 4 U U 4. 1
0 E' U '. U c4 < HACDHAUE.CDA CD 0 0 H UUU
U 0 H < E. 4 CD E. g F. U E. g
gcla E. uuout,oHouopog 00E.E. UCJE.HE.
U 0 0 < H U E. 4 0 A E. 0 A E. C.) 0 A CD U u F. u 0 U E. u E...
0
<C9C.).4.40EØ4U9c400E. ggE, u ED gE.E.CDHUE(..)000 , 1.--, V E.
UE.1 < .4 0
.4014,q8 1-
(10E,40u4u 0uu04004 4
. 0 0 E.E-, pc g < 0 E. 0 E-, F4 F4 C,D C., &4
4F. P4OH<E.E.E.U<UCDPCOU 0 0 0 E. < 0 CJ E. C.) E.
0 E
O E. C.) cc. E-I E. 0 0 4, 0 <4 u E. < < 0 U
E. 0 F. c4 U .4 E. 0 E. 0 4
.400ua,U4(.94,004uHuH4 U E. E. U E. Kg F. E. /
C9 E. At E . ., ..0 U El a' Ei ral 0 u E-, u 0 a' 0 c.) H 4 0 U
(.7 (...) E-.
c4UUCDCDU HHHE. 4, C.) E--, C.!, V 44 C) U E. A, E. U
0 E. U 0
O0cJuE.F. CDCJA00E.E. E.H04 (9 0 u U < E. 0 E. (4 E. /
U 0 < 0 < 4 E. CJ 0 4'1 0 0 (-) 0 E. C.)
F. E. E. F.
0 0 gr cr: E-' E-' F. U .4 0 U f-, 0 0 U E-== E. 0 0 0 a:
Kr. u UUUUHOH E. e. 0 4 E. C. F, 4,
0 = A 0 U E. U 0 E. EJE. A A C.) 0 CD A c9
Ci U 4; I---. C.) 0 E. 0 0 E.
O A H U C..) A U U 0 0 CD E . CD 0 E. f-, A
0 0 41. C.) E-4 t i 0 A E. E.
E.0,00AUACJACD,7=0E. 4 -,ugt, g.ou 0 u < 0 4 .0 0
C.) 4 E. E. A 0 0 0 E. PC U PC 0 0 1 u 0 U 0 cc C.) i
H 4 0 U E. < H E. A A 0 E. E. E. F. (4 E-. 4 E.
U 4 U E. cl: U F. U 0 0 E. 4E. E. 4
E. U C. U V A ugo,E,E,E,Ur-c (..) (3 (1) u 0 0 E. < U
E.
r,
UUCCE.OH04 a. r¶..7 UE.CDA a' oo o 0 E. E. E. F. 0 0 (..)
O < E. U 0 E. E. < 4 U 0 U .4 U F. 4 <
(..) U H,4 0E. E. 0 4 4.
c-i' EE--: c.-h' cd t7i µ,'µIzi 0 8 P-, i-', 6' ti g 40E.c400F.C../HcJE.
0 E. E. E. 0 E. E. A E. U A
E. u F. < F4 HA CDE.AHE.0P0 =OUUACDUHUH 4 H
HCJU 0(....E.000HC..).4 F, < C.) E-, 00 <PC HOE--
1q00 H
O 0 U ti U E. U A E. U E. (..9 0 E. 0 (J 0
U 0 E. A E. 0 re,
00C1HE.CD
CJ 0 (..) U E-4 E. E. < 0 cc < 0 U .4 c4 5 U, (..) (.... F. u < F. < r.
0 H
(.900 0.40u A 0E.64 ACDCDHA 000 HHUOHA E.
E. A E. 0 CD 0 g U E-40HU0E.E-, E. 00000001H<H
CJ Ou1 H E. E. cl<H0,404CJE HUF.C90H4 CJE.0
U H E. 0 E. F. F. E. < 0 c..) 0 E.+ 0 U E. 0 0
(-A _ E. E. E.
F. C.) U U A E. 0 / E. A A 4 E. 0I. CD E. 1
HAOUHE.HE-.C.JAH 000E.H
O C) U 0 0 E. CD H U 0 H 0 CD A E.
000H0000 i1
HO 0 A A A E. gc_10E-,E-104E-. 4 .4 000E.c..9C)(.9E-.00
UOUOUCJ U .4 0 4 E. E. c...1 E. E. Cd E.D E. A 0 E-= 0 0 A H
CD U CD E. 0 E. A U E. E. 4 u 0 (4 0 0 OUCDFAUHE.u4H
E. 0 A A E. E. A H rc E--, 0 0 E. E. F. E. E. HOUHOOHO .4
Lc U < U U E. 0 E. 0 H
O0004 r-, r-, u o o g E, ED 0 0 [-i U 0 E.E.
< 0 0 < c4 0 ct E. E. E. E. U u H 0 E. U
cJ E. (J U C.) CA
E. U 0 U E. H E. U U 0 0 A H CJ A CD A OU0HE.044 4 E. E-;
A C.) CD F. C-E CD A E--, A 0 0 E. H H U H A 0 CD (-= A 0 E. E. A CD E. 0
HO 000000
E. U 0 A A H H E. H 0 E. A A E--. Cll. A 0 H E. U E. E. A C.) C.D E.
ci E-. U E. cg U C.) < 0 F. E.
E.C_DH 4UCDHE.C.E040 E.04, 4 u0ouououe4ug
oge-1 E. El =I' 4 E. H U E. E. E. OH HO
FI, on
0 U E. 0 0 .24 E. U 0 cl.)
c..) 0 0 rt; U 0 U U 0 0 u E. A E. E. 0 < 0 0 C.) E. H u C.) < F. H
U .4 U 0 C.) E. E. U cg < E. E. U 4 U E. 0
E. E. H 4 H E. PC < < E.
O C.) cg 4.444E-,0000 4000 cp uu<E1 4 gal 0 U A El
cr) E.0000HE.00A A A, 00E.E. H HOUU 004 E. A
== E-=4E.C.DAOCJUAUCJH CDE.H0 == UHE-=0 ACDE.E.0
= A U A A E. H Ei H 0 E.E. E. E. 0 U E. =
0 0 0 H 0 C.) A U LI E.
I
O u 4_ , 0 E.CDCDHOOU 0
ra,U0
2 U u 0 0 E. u cc 4 Li E. < u E. < .4 c..5 0 2 F. 0 C.) 5 E. E. U 0 ,....4 cJ
<
CI OU04000<<ciE.E.E.4U0E. n E.E.0 c9u<HE.E.<
H A r=D E. g A E. A a, a. E. [-,un; 4 4 4 ct H U E. 0 , E. 0 Li .4 U 0 (.9
01 a, guougg [-,uug E= A cc 0 U E. 0 0 c..9 0 U U E. 04 u F. 0 <
PI 0 U E. <, E. 0 0 u U H H E. cc U 0 E. U in E. 0 0 0 H E. A E. A E. 0
cn 0 0 4 A A 0 A E. A E. KC A H A E. < H cr) E. 0 u E. 0 F. U 0 E. H cl
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
105
f:2)WE.Wc..20nXzE.Clg0Nr:4000(nr.i0Xrpfleipciult4f).
tnig,-100NIAMOH0+xc4Xx ',4 q ril r:4 .-IP.MOP
0 Ers 0
_10in,-j'.41-10,,-1+4(t)t.410h-10cocnci.ami.-100+(a.,
E. 34"A'vf1,-, El Z,.4u)8:',',"-teat-.4`jl`j1 2 ri c',.., ,9RE'T'IN2
,q,-19,P,INN`08, R .6,, 22588 r, 0 'F-4 V2. E' Ef 11 (8,
2 rx, 22,E-3,8,gHo
8 0 6 R '61 'A }t: M Q r=1 ',I.
0 c'itEi',P2, >.n1-1,-',.-.2-1'4).
. 2 [-TO tc70('-+Ip141-1-iE.Or-134f..41.-1',31.541R mchOMA .. M
,f4 rnw_ wr-,g 4-4o141;YaCiat-IZE40u) 4ftrqln , xa
- (44 M4-1aZ0zXOrrz4,4awor.404-404 fr, u) 4 .00Hca,c:
16 ,1 u '' 9 8' Er", 0 8 r7, 3 0' 0 8 8 8 g " " OZU z '-
caE-4r4ZXE-4_,-104.1
carrl >1.9Hq0100qwwW1004E.Eactioqcnor4UP4 xo
Q Ft to; 'EYx?c,-,Nwpatt,lt:Im uw441-44-14-404nui-4E-
,ZmE.04:4
H cO>0 <>124f4r4pE.r=15114 E.-,PCHcnE-, E..10,0E.40
Ri gHE.z4c0X44MX0E.0(.9u0E-.c=?N0,0 E.PN'00,-44n
cn ZOE, .111E-,0,-1P4001q>, 4y. x E-4 r.,1 x g
r4C/),--14.71-uco0OH
,. ,0 , ,0 , ,µE. ,. _E-. H C. E". E-. < < L.7 0 E-. < 4 (.7 < < E-. E.-
. Ed, U Ep 0 <
µ-.'s-',.',."0(-)<<E-,(900E-.E...C.DE-,<PCE-1L90Ktggiuu0u
E-40HE-4000uE.E-Igu0E-.KtuKtuc(!),,i 8 te4 E., 8 v,,,,5,E,,,,,,,c;
4 U 0 E-, E. .1,' E. E. E. 4 < E. 4 E-4 0 0 A
Pc <F,E,U1.135UE.C9u44H0E.C90000(11.964UuU0UU
000FF(90t.00,00UUHF0 f...70E.0E-,<CJUE,<U,U0
g Ed,. VD 4H A 0 CJ U < 0 El CJ
0 444 0E-40E-4040 U y U
KG U 0 E. U E9 ill 8) 8) 8 Pc --,' 8 6
l 84 A 8 8) El EE-: 8 6 LI 0 05 8 .E-: 8 8,
/
HE.V.) E.,:t 400,4;ugE-4KL41-.0H0 E--,440().(-)(puE.-.
u r-, < u u 0 4 4g0uE-.E-,ououuy UU0E.0 UUKGKG
010000E-, Pc t--,u00E--44CJHUE.<CE, OUOU 0E-4y0u
00E--40E-igg00g,,t0g ugH4E-4E-4U 4UggE-.0uugu
H g c.h 0 0 0 u E-4 HOUgUKt cp gt g g OHO 4, t: 0 CJ C-c 0 p E. 4
/
F4'8vE=',E,-,:iP,u-,18g,Y, 8858800riEti,F4888El 8!
0E. HUO (J,PI,E, ,,,rt <00A
4000E0 E-400uuo0
KC0004 4.KtE-10uHugHu0F40E.E.
6uKtuuHE, a,
0 U g atO U U H 4 4 4 Kt u u u u E-, 4 E. E. 0 E. o<oFu4
6 6 8 e, 6 o 8 8 8 ti .4 0 il E', 0 8 il 8 r, 0 8 8 8 8 8 8 1..., 6 `, 1-2 6
i
uuP,<()E-,4E-,<.,..Pue,E-:E-10E-,<E-,E,F, KGE.E-40g0KtU0
H4,400E-.0000400E/uuue,u0uuE-,000KtE--,Kta040
000H04000E,404<u(940E.<<UE.4E.OUHOUU<
LJUUHE.0444<gE.uuu0gE--,04u4.uogguoV)u,E,
FC3c9u()(90UHE.4u4E.(9u0H44U0y4a401.0 ED4U
Eg 8 6 (94E-.F40uu4004u41.4Fr
0E-,p.-4040Sup
0E-4K4E-, a , g G U E. Kt .24 C. E. U U KG E 1 ,, 0 U t; , - , re -, 4,
0 Es : ,,
EH.,q0cla;HHE.UF04004uE.000 4E.0 y ty-fl
Hu<40 r=0E--,E,c9E-
tU.QUE.KGE.,,GOOF,a,05s2u<s...p
<0<4E-, F444(94H<F(94()UFFC3UF4U(6cE-JcEl ._-,.t.6
(.'6rõ 88808885 KV: 8
P.,(E_:88',C0 ricicrZgc'50Hu4EDF
U44UU44(9FuE.404/ 4UUUUE.44E.4U4UUU,64
EIE-400,4000400gE.0 0,tE-44E-4uuE-40',E-,uuuKtu u
uuV-IE.,,44,tuE-4F,400E-.c.,00.E-40E-4000044uuu0,FE-1,,
E-. 5 4E.040E.u4/E.E.04E.UUU4E-
1H,14 tu:z8 (E,,, 8 ,3 6
c., y u E-. c..) (.1 o 9 u 4 E. 4 0 E-, 4 U 4 0 Li
4 u o, E. _ _ . _ a
(9(9u0F4c5 ,a, f-_,, CJ El U H H E-I H'4 4; gC
E.H44UU_FLec2,c- j_.
,t-r'66 .(Cj5' 6 r . . 26 '7. L/ pE ' 0 4" 5 E-, E-, ',-), (-0. f -''.2
E--: .9, t [-i 16' 8 6 8 V: 6' E. L ' ', N i i
uuKt E-4gt-.QC.),,rt./-.,.1 E-.t-,< t-,L",<t-
.U(....CDC.90EJE-.4;<
CJUE-. UE.E--.<1=C<'Ut.)E. 6E-40E-
400q000Ktc.68.6Er.-:,
H<U0E.H<ACJAHKGcl.G.G0HUHU,HOUU
00 E. r. E., 0 < U 0 g U U E. 0 Ci 0 44U4UuHu4 OFE.40
U u 0 F-,. F F 4 H 4 U 0 E. E. C.) F 4 U U 4 4 4
0400E.P 0u0E-,04 A H C.7 H gt E. < 0 v H CI 8 8, V.: `;', (ei 8 -
?e,
400E10E. 00F E.(90, 4 4yuuugE-.0 u ' t.-:,(6,0,-_,;
1L-Igl 6 ,(2,.(,' ,F,TEd rl(c-3'66'.=6c,-i 0 8 6
6 6 6 0 < E, õ 6 õ
1
EJ Ei) 0 8 E-, E.-4, 0 ,E-' ,4
E-, 0 '-' 0 F.,5 ,',),E`,6)8',,-E',1'6(E-2,6'u'UVii-2,6
c<'88`48888808g8,600q60 '6 P, i' tO , P., 'El '6 , V:686'8
O E-, E-, E-, E. H 1 0 E. KG E. <0.E,E.LJAUUHCAEc.; 8
8 [ -,- , i -2, 0 ,c _P4 * EL;
Kt 4 c..) 0 E. E. 0 CJ H A 0 Ci H 0 U E. 0 .
C9U0HOU OH UErE..0L,IHOKGE, .Cdtr,,,C(4 6 < 8 8 1
HHE.KGUUU U0 pc H CJ 4 F F F E. 4 0 u u
4E80004 4 0 0 0 0 HOUE.E.HOUst E. ,66re:2,, 6
U00E.UU0E.00<U<H4 F OF FOHUUU0
f.CD[49(,-)t 6 P-, 6 '6' V, 8 8 8 8 8 6 8 8 ,` 8 1 0 8 8 8 8 V46
6 8 8 8 8 't. ,<' ` 8, pci 8' 0 ri 8
8",µ": õ ',;!, E-: 8 8 Et: 0 1 8 8 E-., ,La.: El
1
E-, E-, 0 0 4 E-, , 00E-
.....0U0UUOC.70080E.U055U
UUUE.HOE.E.UE. 4 0 0 ''4 HO U E" V; , : 6 r, P. , 'El r j < 0 ! F(-0.1 8 (.)
il
OH
E.., 0 H E. KG U U 5G U E. .5q E. .,G 0 C..) Et , , , . , _ , , ,, ,,,,
,õ u , , . u
0040u0004g000-0$0t-,KtuguouucLjr,c, ,,,,, , d ,,, 0 t, 7 c' , 0,
uE.c.,HUE.40E.E.OU,E--,c+E.,),<LJO<Hci, ,4E,,HH0,,1
,t0,5E.,:,:c U 8 uu40.; ,RiK4OHHH51.0 rj,,cuu
OFKt.EØ4, 0 0 nguKcuy,7.c. u<0uu0ogg u
,u,,,SE
HUOHUU04: <<E.0UUU<E,E.HKGE.00E-.00 a. -... ,
0 E-. 0 ,..q U EG 0 U E. U P 4: C.7 ogguuE.4 H E. C...) E. U E. ,,q 0 q U CJ U
Ed
:-, 2 0 8 El 0 ,".9 8 Eta, 0 R '.4) E' E4) .c) 6 --''') E. E..- g c'' 5) 6
:%) 6 6 6 'd H Pt (Ej.E ' ri
, - , u u u < u k . -., c i E . uouH0yE-4E-4
0goc./ut.-4E3c)Fir 8 EE:,,
- E. g 4 E-, g E, 00H<Uu<guyu,o4uu< , i,
= E.y340E-, .eE.4E--
,UE.D0004,04 uE-40uE-,u 4
2 i--,' 8 < 6 6 8 ,V: 6 r 8 `' ,,-Eil r.: EG' L., Et: il El 8 'd ',("C
Et: F,;c' 8 i-1 6 `,!) 6 6
al 0uu0u0000 E-40u0,<E.cp<E, y U0 8 8 8 ,E.4, EL,) ,,
UAUE.OHU-5GOUUOU ,
'a EY. P P. E1 ,,ri r 0 ,E.-. ',=._4, .i.
. ,, ,, ODE. ....' 1.D E. 0 E. 0 U E. 0 0 0 r
1
hi) 868 08E,:-'c 8E-.4'c,`,.?.0(''S,`:488ElEltli--.D 8ev, 6UE18888
1
SUBSTITUTE SHEET (RULE 26)
(9Z 37112:1)133HS ainiasans
.3n nignn,3 4,. 2 0 r3 .3 n . non.3n5.63oHnnoonHon
Hn " Ho 5 0 n 0 25 '.--1 .1 ?-1 ?'-3' 'An n n OH H 3 4F3' 2,'23 8 'n-3
r.31 loon.3.3.343HOHH, c).3,3
r3S,5.a.3 Hann 9,00
,o, n .--3 Hnnrn).o,-3,-.3-30 - a5.1-3 no..3oo4.r
13p2.-3
nH 9 >> r flgnoaann.30).(1,3 ononn.3.3
Pg.), 1 >9-) !:-.1
Peen'5"),151-)g:,!'3r,-.3)229,53-19:,,,c)51.,
0 .-3 H )... >.. 0 ,-3 r) r) .-3 p 0
'n, 0 H H 0 .-3 H H H n .3 .3 .3 a .3 n n 0 0 0
O.3.3 9,0.3PcHS..300.3HH>000.--3(39,X,
nnnononnnn,Ron, S,4-3.30n0H GI .-3
I n S,HrI0H
O n n ona..-30.3nnons,9
n.3:1.00. P 4 H H . / ''... 3 3. 3, H n o.3 3. ,i, 0 H a
,3>
= 3= br nG) 2o.'"
9' 9-' 'An 8rc) Pr, On 9,-6) R nn ',-1 9
c) C) ,o.-306.1ann..3o>+-3 .3c,-).3.-3a.g.3nnor
Han H.3n.3 :,µ05..00rn.-30,-
n5.nna.3.3.3aannong
a 0 P H P 0 3, annr
r3npr,.30,nnotor,.35Ø0 .3
S.. .3 n 9. CI 0
4-)I1 .-.3 ,-)RnnooRn4-1),pon,Rnn
E
;-3 H H 05.0 0-3 OH) .3.30Hr r r . - .-
- . i .3 00nROR:,,
,:,,-, p o.... > 0 >c, H .3 .--3 .-
.3 0 H P H 0 0 0 3, 0 0 H . 0 P 3... 0 > 50. 0
O G) ... 0 0 n H n ).. o r .3 0 .3 H H
0 n n r 5- .-3 .3 .3 ..-A :0 n > p > >
'c)5' rIn'vC. H,3 '(1)9"',931')?-
'3*H1:-V'V-)','-',92,99`)88)`'')
5 9 C1)9.1.8 ,(23 1--3 'p .-3 ; H 1123 H .3 n n '
, (C) a P o .3 P, .3 rri 1 1
/ (IR (-)RhH.-3no ..on .3 Ø,p,o,-3h)o.pr,,,I 0.30,0>a
O n H:nanng.35.
.3H.30n:, HH.-3.-30000.-3.30nnn
= nHnonr .3n,3 a.) (30.-3000.-300.3nno.3;>0:P H n .-3 on
H H H 3, =-= 0 1.. H 3, RR,-3 .00o00,AR 4-,,,,,,- o,-3(-)0 ,
Rni-an,-,, Ho.3s , - 3 n > r3r3 :3, P '.-1:3=.0? .3p,,annon
.3 H nan.3.3nnpHH c.(-,,t-3a.onn
Ho.30 ,0n
1
.3 r .3 .-3 .ona,-3,,,- H P H H 3., ?.., 3, H 1-3 g n
0 n r n o n ).., 0 H
.L.oHn a or :vr. a naHH a n > .-3 .-3 H H n :-3 .3npna,30naa
aran,-3nneln,,,,- H H P ,,o.3,-;)..L..,ornnHan0s=,,grn
Hp .3.3a or '),r o or HnHHnHgor .3 4-)na., :,.pan nn
1-3 3
1
H .3 0 n n n g a. n r rn Haa5. 0 a> r ,-.3 , a o nnon
.-3 9. .3r n.3?.noHngr r)Hn',-, r nr H n no o,-30.3,0nn
'. 33 an H. -3 Hc- ) 51 aC") 5.'D. Ha Ha 2 Pp cl 2 Ha Hn Hn r .' . ' P '0
P. i -, o'-3 H na ) .(; ) Hn an .' 'a nn na '.( ) (-) >0
I
C) , , .,. .4 1-3,3 Ho p, ,1 . , ,, ., - , or ) p, , -34 .) no > ,. 3 2 2 r,
r. ) i 2 c). 3 2 kJ . p : 1 .0 ou 1 r, n: i , , ,,.v rn 5 2 n 5 .0 2
$.. .3 .3,Rn04-14-)onRono> :,,,-304,4-)noonqiinop0,-.2nn4-)
;J. a a n n 0 o > n H H .+ A3 o . n H o H a o n n .-
3 r-=-)
3Q,-D6)),,,
1
H .-3 .3 H ,-3n1=,pp, r,n.,1
.36.),-3 ,(-3.-3,-3-,n,-3.3nnant0:1:,0000
ir4 >0 n H 1 ?in ,3)::. ,,3 >.:_3 6-1 2 4)$ 61.3,.3a 8 cl .'l. ;,,..3 1,-13 Ho
n,-3 HP 2 kr) 2 6):i. ._3=P 2,,.,>4-),.., rn
o.aa.-3.3 4,01-3, .3 H H H r. ,A n a .3
.3 n .3 nonnon
ono nR>R,R00(,,,,,,-36),-31,r,,),,a.-3n9oRn nPn,-3):,..-32
p , , ,-, ,,-, a ,-, .3 H H H H ra ,r, o . 0 n .3 r
;1, ,, N-, n > H n n 5. n a a
na 9 .-3.10PP5,PP Hon G-,,nnon.3 HPHOIPHOH00
Ha HH Ha 0 P= , c-)' Pn ' - 'n 1 9 RH '$3 Q nn ).3) n'''' '-'0 H Ho '''a >. -
3 '30 2 a >6 ) P Qn >r ) 2 8 2 2
1-3 a H P ,1 0 n 3' H a n n n n o 1-3 r n > ,A n .3 IA a .3 n > o H n n .3 n
tv Hong3'c)5.3' .3 S, .3 S.' .3n>n.3.grnanHo o on> ri-I
HaHHHHono.iagoa.nnHHo ' p p .3 I-3 H t--3 G :,>.. ,.,-, a,
n R .3 H n a H .4 H o o
n H H r H r R ?., o R 0 o 0 n n R n H
b . 3 0 O.30.3 R p.:,,' 0 R ,?,,,, 1 .1 ,,-_33 ,..,6 p, ;-.i ,,-,3
(?)...0)00.-3 0 > 0 .3 0 0
,, PA 08 '0, 9,. , >1 0,93 1-3 .03 1 H nn na 0.3 ,_.3,,
HH,Ao ,A:P ,..-3 Ho :1-3 CE1 ??-3:,,, 4( -1, , . ,'4.- )' r).-'3 P3n (;),93
>2 n)r)
1
C) n,-3 ,-3 0 y, .3 n H S.. H 0 H 9, .3 n () H n .3 , H 0 0 P. > n.in
3,
H PnE.3.3.-3,-3Hnao.,-3,-3,3 n a r 04-)Pn,--3,-34.),non
Ivna,3 00a.-1,-3> ,ronHon.3 nSln, pnprInn annpv-
.3a, R,-)43' n,-3,,,- ,,,nn(i) nn5nR,R,.-30
Rc-1
0 0 4-)
0 P. .-3 n n .3 ,, .3 .3 0 p, pr. Ho H 0 .-3 0 H 3, 0 H I 3 3. nr
3,H.3(--)00n ,-3:i,0401-30,-3,P,3,..30
.3pFinano 3'3,nr-1
a n .3 ,--3 0 0 0 0 0 0 0 r- .3 ,--3> . 3 H H 0 n o H ,r, > 0 ,-3 .-3 n a .3 o
n n ,--3
0,-3,-3,3,--30.-3.3a3'.3aHHog0:va,0n3'a.oaa3'0.ona
'-a3 G3S . .- 3.-3 1 Hn Ha ra 1 . 'c- ) ' -/ G - ) an Hn . 1 H, 3 n> Q. - 3 2
' - 30 n P., r3S . .. D ' ' 30 1 H9 ,- 9 P. 5-' ' ' . G )9 , 8 C'n
3. 0 H .-3 33' H H 0 n n n ,-3 :1, ..3 3. 3')' 0 H 0 0 0 P. C))' H
0 P 3, n
HHHn.3.30.3nr H5.0Hagp.3 0.3,--1nRn,R4-)04-)on
-3().-3() H H 3, ..3.3n.3 nran.loani,1-3
.0,:vnn.--onn, .3 n
cn Z hi HK) .ti 0 H
...I hi 0 X .11 t. k. HO
U H 01 730.-c..i.t.tx300t.0,ou3r3uu3
0 (C) 73733rI73O1:-.73cr33,ho .-3 En N
73H0t+-3,o07,,Ln<0.zit.-. kilo()
H 0 Z P, C 0 0 0 (r) >, ,o ,31 a 41 '-33, W
trl H H cn H .11 H 0 L---. ( H 73
L--. r3 t-. En
0 '1 0 r=) t. 0 RI ft, Cn 0 N tr, C.) Cl)
En
OHNUNUNO 9=1 .3 Z .3 ),1 .-C En
tt1 DJ z 0 En t. hi H H Lo icp y 0 id 4-)
ki
73 oxZt-ipoot-itimat-,.311-10
HD-Jo*cit_-.Utri-I=.0 Cf. U-3 r. 73 30
X C x t", Lw. 730 (-'0 rii-V kl 0 Erl,r( .10
ED H W H C:1 ci It 71 En H En ,u
0 In Z ti
('3) C)'!) ,1:1 0, P.-. CA hi ti) 03) 73
H030I H'-!) ti0 r.f), t.tazH
0H ,t1 Z ti 'A (7) apttiNtIroc
cn M 0 VI C ,cr t.-1 tzr H .3 Id rn x to it,
og,cr3, ' ',, TiN 'R, .t. ,:i ,C-Z,,, -,1,,, ,
, , ? , ,, 2, i : , 73 -21,_3 ',1õ3 Fr ,
H tr.1 P.. NJ ICI 3, V, X X W N ..1 ,ci qi ati .3
L-. ti../ L-. 9. 0 0 'CI ,d H L-", t.i Z rd
.t7 En 43
901-
zi-ocooitooz thix
01z000IL00ZV3/13d
ZO-80-Z10Z 91OS8LZO VD
AGTTCTTTTTCCCCCAGAGGGGAAAGTTATGTTcTGCAAATAGTGTGTGTCTTATTTTACTGTTGAACAGCAATTGCTA
TTTATTTT
TTTATTGCCTAGAACTTCAACATGTTGTATAGGAATCCTGTAGTGCCACTAGTTAAATGCcC-
AATTCTCATCTGGATGTTACCATeA
AACATCAGTACACTTGTCATTTCACATGTGTTTAATGTGACAGTTTTTCAGTACTGTATGTGTTAATTTCTACTTTTTT
TAATATTT
AAAATTGCTTTTAAATAAACATATTCTCAGTTGATCCC
c.4
4-
0
n.)
CO
co
cp
0
n.)
rn
0
cnni
n.)
co
n.)
rn
0)
SEQIDNO.:22
SEQIDNO.:59
CITCCAGAGAGCAATATGGCTGGITCCCCAACATGCCTCACCCTCATCTATATCCTTTGGCAGCTCACAGGOTCAGCAG
CCTCTGGA MAG5PTCLTLIYILWQLTGSAA5 71-
CCCGTGAAAGAGCTGGICGGITCCGTIGGTGGGGCCGTGACTTICCCCCTGAAGICCAAAGTAAAGCAAGITCACTCTA
TTGTCTGG GPVKELVGSVGGAVTFPLKSKVK
w
ACCTTCAACACAACCCCTCTTGTCACCATACAGCCAGAAGGGGGCACTATCATAGTGACCCAAAATCGTAATAGGGAGA
GAGTAGAC QVDSIVWTENTTPLVTIQPEGGT
TICCCAGAIGGAGGCTACTCCCTGAAGCTCAGCAAACTGAAGAAGAATGACTCAGGGATCTACTATGTGGGOATATACA
GCTCATCA IIVTQNRNRERVDFPDGGYSLKL
CTCCAGCAGCCCTCCACCCAGGAGTACGTGCTGCATGTCTACGAGCACCTGTCAAAGCCTAAAGICACCATGGGTCTGC
AGAGCAAT SKLKENDSGIYYVGIYSSSLQQP
AAGAATGGCACCTGTGTGACCAATCTGACATGCTGCATGGAACATGGGGAAGAGGATGTGATTIATACCTOGAAGGCCC
IGGGGCAA SIQEYVTEVY7HL&KPKVTMGLQ
OCAGCCAATGAGTCCOATAATOGGTCCATCCTCCCCATCTCCTGGAGATGGGGAGAAAGTGATATGACCTTCATCTGCG
TTGCCAGG SNKNGTCVTNLTCCMEHGEEDVI
AACCCTGTCAGCAGAAACTTCTCAAGCCCCATCCTTGCCAGGAAGCTCTGTOAAGGTGCTGCTGATGACCCAGATTCCT
CCATGGTC YTWKALGOAANESHNOSILPISW
CTCCTGIGTCTCCTGTIGGIGCCCCTCCTGCTCACTCTCTTIGTACTGGGGCTATTTCTTIGGITICTGAAGAGAGAGA
GACAAGAA RWGESDMTFICVARNPVSRNSS
GAGTACATTGAAGAGAAGAAGAGAGIGGACATTIGICGGGAAACTCCTAACATATGCCCCCATTCTGGAGAGAACACAG
AGTACCAC PILARKLCEGAADDPDSSMVLLC
ACAATCCCTCACACTAATAGAACAATCCTAAAGGAAGATCCAGCAAATACGGTTTACTCCACTGIGGAAATACCGAAAA
AGATGGAA LLINPLLL5LFVLGLFLWFLKRE 0
AATCCCCACTCACTGCTCACGATGCCAGACACACCAAGGCTATTTGCCTATGAGAATOTTATCTAGACAGCAGTOCACT
CCCCTAAG RQBEYIEEKERVDICREIPNICP
TCTCTGCTCAAAAAARAAACAATICTCGCCCCAAAGAAAACAATCAGAAGAATTCACTGAITTGACTAGAAACATCAAG
GAAGAATG HSGENTEYDTIPHTNRTILKEDP 0
CO
cn
AAGAAcGTTGAcTTTTTTccAGGATAAATTATcTcTGATGcTTcTTTAGATTTAAGAGTTcATAATTccATccAcTGcT
GAGAAATc ANTIryszwEiplacmENplisLLTm
TCCTCAAACCCAGAAOGTTTAATCACTICATCCCAAAAATGGGATTGTGAATGTCAGCAAACCATAAAAAAAGTGCTTA
GAAGTATT PDTPRLFAYENVI
-71
CCTATAGAAATGTAAATGCAACOTCACACATATTAATGACAGCCTGTTGTATTAATGATGGCTCCAGGTCAGTGTCTGG
AGTTTCAT CD
CO
TCCATCCCAGGGCTTGGAIGTAAGGATTATACCAAGAGTCTIGCTACCAGGAGGGCAAGAAGACCAAAACAGACAGACA
AGTCCAGC
fli
AGAAGCAGATGCACCTGACAAAANIGGAIGTATTAATTGGCTCTATAAACTATGTGCCCAGCACTATGCTGAGCTTACA
CTAATTGG
0
Cn
TCAGACGTGCTGTCTGCCCICATGAAATTGGCTCCAAATGAATGAACTACTTTCATGAGCAGTTGTAGCAGGCCTGACC
ACAGATIC
CCAGAGGGCCAGGIGTOGATCCACAGGACTTGAAGGICAAAGTTCACAAAGATGAAGAATCAGGGTAGCTCACCATGTT
TGGCAGAT
rn
ACTATAATGGAGACACAGAAGTGTGCATGGCCCAAGGACAAGGACCTCCAGCCAGGCTTCATTTAIGCACTTGTCCTGC
AAAAGAAA
AGTCTAGGTTTTAAGGCTGTGCCAGAACCCATCCCAATAAAGAGACCGAGTCTGAAGTCACATTGTAAATCTAGTGTP.
GGAGACTTG CO
7J
GAGTcAGGcAGTGAGAcTGGTGGGGCACGGGGGGCAGTGGGTACTTGTAAACCTTTAARGATGGTTAATTCATTcAATA
GATATTTA
n.)
TTAAGAACCTATGCGGCCCGGCATGGIGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCAAGGTGGGTGGGTCATC
TGAGGICA
rn
GGAGTTCAAGACCAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAGATACAAAAATTTGCTGAGCGTGGTOGTG
TOCACCTO
TAATCCCAGCTACTCGAGAGGCCAAGGCATGAGAATCGCTTGAACCTGGGAGGTGGAGGTTGCAGTGAGCTGAGATGGC
ACCACTGC
ACTCC'GCCTAGGCAACGAGAGCAAAACTCCAATACAAACAAACAAACAAACACCTGTGCTAGGTCAGTCTGGCACGTA
AGATGAAC
ATCCC
ACCAACACAGAGCTCACOATCTCTTATACTTAAGTGAAAAACATGGGGAAGGGGAAAGGGGAATGGCTGCTTITGATAT
GT
TCCCTOACACATATCTTGAATOGAGACCTCCCTACCAAGTGATOAAAGTOTTGAAAAACTTAATAACAAATGCTTOTTG
GGCAAGAA
TGGGATTGAGGATTATCTTCTCTCAGAAAGGCATTGTGAAGGAATTGAGCCAGATCTCTCTCCCTACTGCAAAACCCTA
TIGTAGTA
AAAAAGTCTTCTTTACTATCTTAATAAAACAGATATTGTGAGATTCAAAAAAAAAAAAAAAA
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
109
< Or X H Z En ,-,
A 00HACJHcn ccl 0 Z H
CONHZ.IZE.ON
0 camea4H.car, a , cog z n
fa E1E < W, W. Ei, o. KrOxH,.-1
ri) r4 En Kt: PI fr, 2 ril 2 .1 44
0
qD
.= a a, E. a, a. fl, Q .1 = .1NMAC=1
= (El Co M c). la, M .1 .4 = a x a, c4 a4
u)
0 w a, to n 0 a, u fa 0
Z
n
H A a, w H E. n o ci) a H OZZXP.Q
0 z q E. DI u) cn C. .1 H 0 rc..DZMC.1
41 0 Z Z =< Z 0 cc) .1 0( 41 3 A .4 0
ul
O000H0H<UN<DHU<0E. u < < < E-, 4 E.
CJUUCDuU0E.
0 0 C) 0 UE-10,04E-EU0E.
U 0 00E-.4
0000/U/E-./ ..eUN Er4U 0<u<4U4F-.
U<E..)00(!),,E.E.L) UOU E. E. uUU0Kc0E.F.
00000,-CE-1000(..)<0
C.)0004.0/0,<E-,40E-.P FEE, A, i= C-.) U 0 < C.) 0 E.
0C)H<V9aE.
NUCJOCDC)<U0NE.,<HE.0 4 0 <00U ()KEE.
0 0 0 4 4 KCD0CJKCE..<00UH0 U<CDE. c.JHrg
0 0 0 0 U U N a CJ < 0UHNOUH0,
0 0 U a E-( C.) U
(JOU u(.9 0 C.) E. U N < U 0 E. 0 0 E. C.) <
ULDUE.UUE.
00000C.JHUCJE.<0E.C.DCD<H 0 0 0 D < E. E.
O004000
000 CD H CD E. H E. E. 0-1 < u 0 < 4oug4
UU0000E.0
U 0 E. uogouoo-04 E. U 4 E. E. C.) 0U0<0E-
.0E.
UUOciu<<EDU 4 E. c) Pg 0 U <
U U E. H a 0 E. E. a C.) KC E-.000,<E-,
U Cr E. E. 0 U E-, 0 CDCDU<CDHOH
U U CJ H 0 a,
OUUOUUUNKt
CJUE.U0aU a; u 4. ,--C E.CDUE.CDua a. E, U CD 5 o u f-, gl,
O 0 0 .4 u 4 E--+ 4 04 0 u 4 E, 0 CJ CD E. 0
C.9 u CD 0 C.) CJ 1 el
0000c.JaNUCJOC.5<<<<<e) E. HOHHHU E.
0 U U < E.-, 0
UE.U00(.9,<<WV/<<04c_) E. C., 0 < E. 0 E. 0 0 E.
our,uogoor401, Q U E. E. 0 0 E. E. 00H00UE-EP
(.9,i
i CD U H U 0 F U D
O U U CJ a a, CD E. U E. U E. E. 4 F CJ 0
HUUCU0Ha
U H<H CD E.UKC<KC E. 4UU H E-, U<EJ
UUu/< U00/
< / U ci 04V4o4U.CFu u 4 L h (..) F .5
U 4C1Uu4u4,u E-4 E. cD a E. E. < 000aa E.
0 F.
000000Hu
OUOUCIOUU
O C.) < U < < 0 < < E. U u < 0 E. U U E.
U 0 CJ U U E. 0
O E. 0 H 0 0 < H H U 0 < o < < o < <
0000 E.) 0 F.
ED E. U U U E. pr, 0 KC CD 0 0 H E.
OUU000E.E.
E 0 u4DourcuouE,d, f,u
0000
i 000E,
O E, u E-1 u 4 U 4 0 4 U / / E. E. cl
OUU0cHU
O 0 C.) E. 0 0 E.) E. E. / uuUC.)01 UHE.
0 U U CJ E. C.) 0 0 E.
O U U E. < U E-1 0 E. U U < F. 0 E. 4 F.
E. 0 F, 0 E. 8
, õ E-. 0 E - - , c _1 F 4 H E. c.,), 0 E. E. 0
E , U ()C./UE.(700U
CD U C) C.9 a U a, D E.E. .a. E. U E., U U .5 E. E-, u
u0r400,-4040E,E, 040 100,0-4 0U00,<<UH0
9 ,c u 0 u u E-, .i. 4 EJ E.) U 4 E. 0 CJ 4 L. in 0 V U E. KC
0UU<00UKO0N00DE4 4 0
yoE-.4,400444P,440 of-,,,S 000E-.0o4
U E. 0 u < E. E. ti 0 0 0 E. 0 U E. E. E. 0 u CD 0 C.) H 1) U <
U u 0 < E. < c..9 0 H0000U000
z-,0E,00E-4441
Ci 0 Ci 0 E-4 < < 0 E-, E. CD U KC C.) 0 0 <EJE-.0U
/ 00H-4E-.000E.
UOU<UE.E. a
CDHUCDCDOU 0
c..) U E. E. < H U 5.
CJ = < U E. 4 0 U U < u H E. E. 0 U U U U CD 0 E. u
Ou<N0a0DE.C.DHCJUNE. ,a, 0 o 0-,4u00E,00u
04 0 0 4 <u<a1H,4000U0U0a4 0 0000 E.0 E-. 0
OurJuKeuE-.0E-.0U<H<HE.E.0 000,<0E.u40
0000000HE.
uU00 8 4 4 H 0
ONUCJNOUNOUNC)H00_. 4 6 Uc..)<E. OOP
Ci <
DuUNUNaHH0000E.HH KC 0 U C) E. C., CD 8
0 U U 0 E. 0 C.) a, 0 U) 0 E. D U 0 CD E. 000 H H E. ,1
E. E. U E. U U E. C) 0 4: 0 0 .g 0 0 A: F,
,1 000 00F4 ,E, u (3 4 El 4 < UE-40 0 0
00 < 0 <0< U 004; < 00 (JO< E.< H U000UH<0E-,
U U U C.) 0 0 CD .<
= 0= UC.)0CJUUUC700KCU<<EHE.CD =
0 OHOOUH<U0E.HyHOUHE.F. 0 000 E.U-KHE.
2 uu 0E--,(JE-00¶Jogt, E.00E.E.H Z U(1)0 U,40E-,4
q 0 0 0 U 0 0 U < < E. < 0 E--, H .< 4 E. n of:Jo 04)0p,
H F<HUU< UOUCJOHE.HUHU H UUE, 00UE.E-,
0 UOUUOU HE.H044HUOUU 01 4U<O0UE, < 0
C.1 404000 444H0004E-t-,0 w 0rf:00E,0u40
En 00u000 0 H E. E. 0 CD U H 0 < < cr) U 0 < KC < E. CI E. E.
SUBSTITUTE SHEET (RULE 26)
SEQIDNO.:15
SEQIDNO.:62
CGGTGGTTGGGTGGTAAGATGGCGGCTGTGAGTCTGCGGCTCGGCGACTTGGTGT.GOGGGAAACTCGGCCGATATCCT
CCTTGGCCA MAAVSLRLGDLVWGKLGRYPPWP
GGAAAGATTGTTAATCCACCAAAGGACTTGAAGAAACCTCGCGGAAAGAAATGCTTCTTTGTGAAATTTTTTGGAACAG
AAGATCAT GKIVNPPKDLKKPRGKKCFFVKF
GCCTGGATCAAAGTGGAACAGCTGAAGCCATATCATGCTCATAAAGAGGAAATGATAAAAATTAACAAGGGTAAACGAT
TCCAGCAA FGTEDHAWIKVEQLKETHAHKEE
GCGGTAGATGCTGTCGAAGAGTTCCTCAGGAGAGCCAAAGGGAAAGACCAGACGTCATCCCACAATTCTTCTGATGACA
AGAATCGA MIKINKGKRFQQAVDAVEEFLRR
CGTAATTCCAGTGAGGAGAGAAGTAGGCCAAACTCAGGTGATGAGAAGCGCAAACTTAGCCTGTCTGAAGGGAAGGTGA
AGAAGAAC AKGKDQTSSHNSSDDRNRRNSSE
ATGGGAGAAGGAAAGAAGAGGGTGTCTTCAGGCTCTTCAGAGAGAGGCTCCAAATCCCCTCTGAAAAGAGCCCAAGAGC
AAAGTCCC ER'SRPNSGDEKRKLSLSEGKVKK
CGGAAGCGGGGTCGOCCCCCAPAGGATGAGAAGGATCTCACCATCCCGGAGTCTAGTACCGTGAAGGGGATGATGGCCG
GACCGATG NMGEGKKRVSSGSSERGSKSPLK
GCCGCGTTTAAATGGCAGCCAACCGCAAGCGAGCCTGTTAAAGATGCAGATCCTCATTTCCATCATTTCCTGCTAAGCC
AAACAGAG RAQEQSPRKRGRPPKDEKDLTIP
AAGCCAGCTGTCTGTTACCAGGCAATCACGAAGAAGTTGAAAATATGTGAAGAGGAAACTGGCTCCACCTCCATCCAGG
CAGCTGAC ESSTVKGMMAGPMAAFKWQPTAS
AGCACAGCCGTGAATGGCAGCATCACACCCACAGAMAAAAGATAGGATTTTTGGGCCTTGGTCTCATGGGAAGTGGAAT
CGTCTCC EPVXDADPHEHRFLLSQTEKPAV
AACTTGCTAAAAATGGGTCACACAGTGACTGTCTGGAACCGCACTGCAGAGAAATGTGATTTGTTCATCCAGGAGGGGG
CCCGTCTG CYQAITKKLKTCEEETGSTSIQA 0
cn
GGAAGAACCCCCGCTGAAGTCGTCTCAACCTGCGACATCACTTTCGCCTGCGIGTCGGATCCCAAGGCGGCCAAGGACC
TGGTGCTG ADSTAVNGSTTPTDKKIGFLGLG
GGCCCCAGTGGTGTGCTGCAAGGGATCCGCCCTGGGAAGTGCTACGTGGACATGTCAACAGTGGACGCTGACACCGTCA
CTGAGCTG LMGSGIVSNLLKMGHTVTVWURT
n.)
CO
GCCCAGGTGATTGTGTCCAGGGGGGGGCGCTTTCTGGAAGCCCCCGTCTCAGGGAATCAGCAGCTGTCTAATGACGGGA
TGTTGGTG AEKCDLFIQEGARLGRTPAEVVS
cn co
ATCTTAGCGGCTGGAGACAGGGGCTTATATGAGGACTGCAGCAGCTGCTTCCAGGCGATGGGGAAGACCTCCTTCTTCC
TAGGTGAA TCDITFACVSDPKAAKDLVLGPS
-71
GTGGGCAATGCAGCCAAGATGATGCTGATCGTGAACATGGTCCAAGGGAGCTTCATGGCCACTATTGCCGAGGGGCTGA
CCCTGGCC GVLQGIRPGKCYVDMSTVDADTV
CAGGTGACAGGCCAGTCCCAGCAGACACTCTTGGACATCCTCAATCAGGGACAGTTGGCCAGCATCTTCCTGGACCAGA
AGTGCCAA TELAQVIVSRGGRFLEAPVSGNQ
AATATCCTGCAAGGAAACTTTAAGCCTGATTTCTACCTGAAATACATTCAGAAGGATCTCCGCTTAGCCATTGCGCTGG
GTGATGCG QLSNDGMLVILAAGDRGLYEDCS n.)
Cl)
GTCAACCATCCGACTCCCATGGCAGCTGCAGCAAATGAGGTGTACRAAAGAGCCAAGGCGCTGGACCAGTCCGACAACG
ATATGTCC SCFQAMGKTSPFLGEVGNAAKMM
GCCGTGTACCGAGCCTACATACACTAAGCTGTCCACACCCCGCCCTCACCCCTCCAATCCCCCCTCTGACCCCCTCTTC
CTCACATG LIVNMVQGSFMATIAEGLTLAQV n.)
GGGTCGGGGGCCTGGGAGTTCATTCTGGACCAGCCCACCTATCTCCATTTCCTTTTATACAGACTTTGAGACTTGCCAT
CAGCACAG TGQSQQTLLDILNQGQLASIFLD
CACACAGCAGCACCCTTCCCCTGAGGCCGGTGGGGAGGGGACAAGTGTCAGCAGGATTGGCGTGTGGGAAAGCTCTTGA
GCTGGGCA QKCONILQGNEKPDPYLKYIQKD
CTGGCCCCCCGGACGAGGTGGCTGTGTGTTCACACACACACACACACACACACACACACACACACACAGGCTCTCGcCC
CAGGATAG LRLAIALGDAVNHPTPMAAAANE
n.)
AAGCTGCCCAGAAACTGOTGCCTGGCTTTTTTTCTTCCGAGCTTGTCTTATCTCAAAcCCCTTCCAGTCAAGGAACTAG
AATCAGCA VYKRAKALDQSDNDMSAVYRAYI
r¨
ACGAGAGTTGGAAGCCTTCCCACAGCTTCCCCCAGAGCGAAGAGGcTGTAGTCATGTCCCCATCCCCCACTGGATTCCC
TACAAGGA H
=
GAGGCCTTGGGCCCAGATGAGCCAGTACAGACTCCAGACAGAGGGGCCCTTGGGGCCCTCCAACCTCAGGTGATGAGCT
GAGAAAGA
cn
TGTTCACGTCTAAGCGTCCAGTGTGCACCCAGCGCTCCATAGACGCCTTTGTGAACTGAAil_AGAGACTGGCAGAGTC
CCGAGAAGAT
GGGGCCCTGGCTTTCCAGGGAGTGCAGCAAGCAGCCGGCCTGCAGGTGAGOATGGAGGCCCGGCCCTCACCGCCTCGAA
GCCATGCC
CCAGATGCCACTGCCACAGCGGGCGCTCGCTCCTCCCTAGGCTOTTTTAGTATTTGGATTTGCATTCCATCCCTTGGGA
GGGAGTCC
TCAGGGCCACTAGTGATGAGCCAAGAGGAGTGGGGGTTGGGGGCGCTCCTTTCTGTTTCCGTTAGGCCACAGACTCTTC
ACCTGGCT
CTGAAGAGCCACTCTTACCTCGGTCCCCTCCCAGTGGTCCCACCTTCTCCACCCTGCCCTGCCAAGTCCCCTOCATGCC
CACCGCTC
"''r'ATCCTCPCTCrTrTCrCTCTTCCTCCCGTGGAGACAGTATTTCTTTCTGTCTGTCCCTTTGGCCCAGACCCAGC
CTGACCAACG
0
ATGAGCATTTCTTAGGCTCAGCTCTTGATACGGAAACGAGTGTCTTCACTCCAGCCAGCATCATGGTCTTCGGTGCTTC
CCGGGCCC
GGGGTCTGTCGGGAGGGAAGAGAACTGGGCCTGACCTACCTGAACTGACTGGCCCTCCGAGGTGGGTCTGGGACATCCT
AGAC-GCCC
TACATTTGTCCTTGGATAGGGGACCGGGGGGGGCTTGGAATGTTGCAAAAAAAAAGTTACCCAAGGGATGTCAGTTTTT
TATCCCTC
TGCATGGGTTGGATTTTCCAAAATCATAATTTGCAGAAGGAAGGCCAGCATTTACGATGCAATP_TGTAATTATATATA
GGGTGC-CCA c.4
CACTAGGGCGGGGTCCTTCCCCC CTACACAGCTTTG GC CCCTTTCAGAGATTAGAAACTGGGTTAGAGGATTG
CAGAAGACGAG TGG
GGGGAGGGCAGGCAAGATGCCTGTCGGGTTTTTAGCACAGTTCATTTCACTGGGATTTTGAAGCATTTCTGTCTGAACA
CAAGCCTG
TTCTP_GTCCTGGCGGPACP_CACTGGGGGTGGGGGCGGGGCAAC-
ATGCGGTAATGAAACCGGTTAGTCAATTTTGTCTTAATATTGTT
GACAATTGTGTAAAGTTCCTTTTTATGAATATTTCTGTTTP.AGCTATTTCACCTTTCTTTTGAAATCCTTCCCTTTTA
AGGAGAAAA
TGTGACACTTGTGAAAPAGCTTC-
TP.AGAAAGCCCCTCCCTTTTTTTCTTTAAACCTTTAAATGACAAATCTAGGTAATTAAGGTTGT
GAATTTTTATTTTTGCTTTGTTTTTAATGAACATTTGTCTTTCAGAATAGGATTGTGTGATAATGTTTAAATGGCAAAA
ACAMACA
TGATTTTGTGCAATTAACAAAGCTACTGCAAGAAAAATAAAACACTTCTTGGTAACACAAAAAAAP-
AAAAAAAAAAA.k 0
cn
0
n.)
CO
co
cn
-71
n.)
r r
n.)
rn
co
n.)
rn
ct)
1,4
CA 02785046 2012-08-02
PCT/C A2007/000210
WO 2007/093042
1 1 2
C) 0 4E44 cna a FAH 4 4 ca OH
E.FFC>..U(Drtu)0 H>Og
CYcnW.lOrr.-.4E3 a In t-1 r4 ft, 0 L-10,Cr:1>.-41X
>. -4 4. ot ri) 04 al E....4a cn A w a EH .. H r...
ti, x > pa. 4 01 1-1 0, a PI
0,4 > E.C.Da al -t AHHaHaa HS,,,CaE,ZHE, W
En
0a0 CD
Hanar.µ,EHE1>p4nE.Ocnr44cnop%arx.-fataa.-1
0 a. 4E1,01-10r., A =1AXE-,x0,--,vraomp c4 4ucn
o c.r1 E-4 41 0 E-, aHr=.0 tae,0 HP-.20)01-,00 ..
r..10)0lHata0
E.C1011.CJaaa01,4.,S.a H ChHa S.H0>a,00a0 a aHN
2 '' 1 S ,, '8, 'C4 P-, PI l'F'4 r3"'
'.' 2 cl ECD' 'al .2.1 f;4' 2 g 1 . PI '''
0 0(aqn L, >4 >4 Fifil00(/)1:4>='
E, KC H 4 0 a CY a gq>Nr.,r.arr,cnr:d ci¶..H1-1 ara
r.,-) Hvj )-1 o Cl) U W I HO) C.DC.DOHC,,,4 4 0,510H a
?00,01-101:)
VD 0 PI4 W I-I PI 0 01 CP g rZ4 r,u)MOOmar EnZa$1,00NI--1,4
14 01 a 01 0 a a 4 .-1 4 a a Cl) CD X CD ci> H
CD fLi a a 4 2 H
= a a Ed E. E. a E. 0 4 a S-t x 0 ul .. po 0 E. S. A
actli...
0 C., KC U 4 Z 01 r a vl 0 Kt CDPF.1.-1 OH 00)
Z P4040) r44,,ia QHF4ox F. .4 pc.DcDMCD4aWaEn
A
H Hi:i' EY,1 P44EU.3. 2 rti (I V, `,_.1 r,'n4 F61, CO 'I? EC
5. 8 F c) 1 2 O. 21 _
0, g 4.1 ci, ci, 0, u, 0, ,-4 H D., 0 . . ,..., cy
z 0 cn p En a n
44 E.r.1 >,,10> CP t-I 01HHaF-
1,0,fr,aZ0Howaa?Haa awn;
Ca Ca F. cti 0 0 U a H 0) a 4 .- n w x En a Z t4 0 ua >, 1=1 ci ..> 0. 4 0
u) Cl)
_
00 CD HUHHU H4U4CDUE.,4 000CDUOUUHHHE.PCD
00004a: E. E. HOHUOUCJu 0000 CDKCH HOE,E, F. 4 E.
4HHULJUOU<CDP,HOUUOU0OUCDUUCJHUUCJUKt
000 CDE,Pt 40 OHaHauu0a0 E.,4 HUHOCDU 4 (Duo
000000o4UUaa0u0a0<uuua0augtE.044
outpououu uu4E.oucDUUU0E.000,11UH 4 4U0
000E.F4 400 4004 4 4F.c.JOUHE.C9cDE.40 0-' C) UCJE,
0E,UeU0.40:0C.JHUE-t4000.040u4.44u4u
uu u0(.94r..1(9Uu.440o0u040Huociouougu
u4_. CJ g E, CD E, 4 A A U F. a:
F. 4 E, u H E. F. a: E, H U 4 E, 0 4
gOuE.404 4 00 Fl,c7CJO0C.DE-.401,UcJuUcJU HOC)
00 CJOUOCDU UU OCDUCJOgCJOUE.CDOUE.UHUU0
6
0 F. 1, 4 a: E. E. EH 0 EH
H E., u, < EH U H. a. 4 E. 4:g0U00I:t.
0Ha00E-100Uu<H a uOuuH,EHUHHaa0,<Hu
H. 0 H a EH a EH g E-
iHOUCJUUE-.E.UOCJOE.W0E.UuC,
0 0 0 H Q 0 CJ 4 .4 4 H U 4 U U C., 4 0 U E. KC F. 0 0 0 E. H E, U C.,
4U044 A 00 .40,E,000H 40 HOE.DUCJUUU0 0 E.E.H
OCJE,004,40a,AE,E.T_DHC)u000F.CDC.DHUUHUUUE,
HHUHOU0.4 PC HE' OH HE.E.CJUE-tHOOVJU
040
.4E-.0000,4CD UAL CDOHCDF.F.000000H4 HO HOC)
CJU KFCUUCJOU C.,04 H4 4HuuE.0E.00000uuu.40
F. U U U CD C_D E. E. E. 4 E. H a, CD 0 E. U U C.D CD U 4 g o g5 uEH<E,
0 0 a 0 H EH 0 E a a EH E-, 0 0 0 a EH H. 0 0 a 000 C,,0 HOC)
g 4 PC 0 0 0 u EHUDaUUHOUUE_IHoUUEHEH 00000
HUH< F4 AHU U0O<HUHEHOEHHOUCDE,HU 4F. 004
u44000HHOOCDOE,HUOHOPUHOHUg 0 CJ (DOC?
EHOqouou0 CJE-.E.OUCDUE,UOUUCJCD 0 OA 0 0 uuy
E'e(JP:.:4;EHUaEHre.uHauF,a0.4()EDUE.a, PH Ftgu
00 .10C-Jua,E, 400CDE.F.F.0 UUU<HUOU00.4 U00
CJ cD a U U 0 0 U U 1.-. 0 0 U U E. OUuH, 0 CJ U E. (...! CD U E. I, E.-.
4: g dU4CDE,4 CJUCDPOUU4 U00 Kt E,00,1,4 0 U
00 CJut-44.---
,0,4004F4HANCDOCDUOFPCUE,CJ g U
HO CJOLDu0,40u E. E-,UUCDUC.Dc.70000CDUU 0 04
0K4UE,HUHLJUHE. 0E-.40U0CDa:41.204F.CDCD
5 (9 5
F. CD U CD 0 E. 4 a' 4 4 -4 H a: E. 0 CD 0 cD H 0 U 0 a a H a U E.,.. Hgg
g:g 6 UOUCDCA UUFFC E.U00004 CJOUOuHU 0 0000
El
U u Ft, U Ft u 4 E. U E. 0 0 0 ,...) U
CD CJ g 0 E, U U Ci U E. a E.-, g: g0ug06 E-.00E.4400Uu0E.ouE.4uUu0u
u 4 0 u 0 0 4 E. E, a: 0 0 U U C., g U E. U H
U C-, E., C... CD u
ug cDHUUUHUE--tet HUOH 4 HUHOC.DUHOUE. H CDE-.0
HO OUVUOE, KCKCE...a 40HUE.HUCDOOKCUOU H 0.40
[JO E.UUUHE,UCJUCJOUOU C.DUK4 0004E-, 0 U00
O H CD E. 0 E. E. .. F. E. CD a, U
H KC a; 4 6oci0E,H.,HE,06,H0
E-, 8000E, Huuya0CDUH ouc..900auuaa0
0 u CJUOUUCDHU5 0 H, H U u C9 0 u u U u u u 0 c_, E-,E;
1
6 6' ',;` 8) ,') 8 E-' t,"0 6 8 Ec; '4 8
6 6 (,-p) ,tt' 6 8 6 8 t-i it, 6 ., 8
HO 0UCJE.DUUg U000HyuUCDUOCDNE.H. c..),p(õJ 5 p
OH H U U Kt CD CJ 4 H 4 U 0 4 c, 0 4 4 0 CD
N 0 E, V. ,I. 1 Y 71.3 ,,...; L-6,
OHOyOuCJHU Kg Ug 0 rz.,00 E.CDOUOUgg ,4, V
4 E.CDCJOUU CJOUUCJ E-I 0 0 U C.) U 0 U 0 0,<U0uHuuu
O00,<EHuuEHEHu0U0HaL)Ha0H4EHE-<:HHHV 1 5
O 0 Hoc) 5 E.E-,c)0 c. HouougH04uU4HUHu004 E-1
A:guE.0Uu0OugUoHuu0E.uuuguHuuu 40'u
OH u0E.Horc guuugt.gu .4E.00E,H1D1HOE-.0
u CJF.E.UUHUU0 CDUOLDUCDCJUHo_40E.uu uouu
0H0 C., 0 0 V 0 U F. 4 CD 1, u F. 0 E. U u u UE-,C9IJU C.,HOU
0U4044HE.444u4,E,CJUOUCJHE-,ggg00 0 0 g 4
OE.a000440004 guE,04000000H000HOE.
C-; E. U HUU00 E, 0 E, 0 C./ CJ 0 0 0 V E, U U 0 OUEHHOUHL)
aUUOUUHEHEHO<UU0HEHOuE, r' OH H HO 0 rc ,I HO
gg0CE,gergU0g0H0CD5U0000HUgggUE-.5, Fa, U E--,
UK,HHHUCJUE,E.HgEHEHEJUEDHHauurJUUUuOUC9
0 u 4 U U U. 0 u 0 0 U U E. 4 4 E, U 0 U u U U CD 4 4 a 0 a a EH
E-,oHauUH E-1 E, U 0 E.!) E-, 0 g CD g 0 0 g CJ gg g a c.9 o cp g Pc u
UgOUUUg E-, HUHEHUHUEDUUEHOUu0uUgC70Hrg
U E, Kg 0 0 0 C.1 E, U 0 Ft E. E. U E. H F, E-, 4 H 0 F. E. CJ E. 0 H. g 0
0
E, E-, 0 g .-4 0 E. E-, 0 0 g g g U 54 U 0 4 0 u 4 u E..) E-, 0 0 0 0 4 0
Huouuou000uu0guuguH00000c-,00E-'06
u 0 F. g U E. 0 4 E. 0 CJ a: 4 U E, 0 A F. 0 -4 H 4 0 4 u K4 EHEH0
E, 0 (I) E-, a U 0 u E, 0 U 0 a a EH a 0 U H -a a 0 0 u o a ci E-, 0 0
Uri:0 00 u k c.) 0 E-, u E. 0 u ci 0 u ULDUC104000Uou
00HS,UHaCJE-HaEH/aEHUaUE-,040 HE.E.E.UUE.0
440o4ciouE.F4 0E,gUt..70H0g 00CJUgUOU
UCJUHE,CJUCJUgU 00E,OC.700000-10000UHOU
U H 0 CD c..) H E. 0 0 U E. F. 4 E-, U E. 4 E. 0 0 C., 0 CD H KC 0 E. E. E.
4
to E. E. 4E. E. E. KC Ks: 4 1, 0 E, A 0 U 0 KG C., H CD U 0 E. U CD CD H
U U U
H 0 0 CJO 0 Ut..10 000,gC9E,E.J00 CJOE-, 40 QUO UCJUOC.)
O H 4 4 u 0 E. u E. U 4 4 F. U 0 0 E. 4 0 5 0 (9 E. u E. E-I 5 U E. U
= H0000HUUUCDCDCDUOHCDKCO4PCOCDCJg0-IUF.CHUH
0 HUgucpuu 5 UE,C1g4UC.9r E,
UKCCõDOULDOUCJOEJOU
O UHOgHHHUgg ...COE, OUOHOE,gUE,E,CJUE,
C) V VOE'VUE,g00-g u 5 5 ,t 5 5 c. E.,40U4OUCDHE.
H g VE--.UUg0C9UUHOUUE,00U0HUOHOU gE, 000
CO HE,E,HUOPH H E-.KCC1E-10000gE,E,UgE.(7OUgU
PI 0 0 0, U H 0 0 U E. F. H 1-. E. E. ..< E. 0 0 CJ U 0 0 U 0 g g g
Ii) g gC-DUOUHU, 0E,f, E.) . 0 gu 00gg 4 E-, E,0000u
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
113
41 4, P) C!) H c,i H HE-'> cr) fl, in < ri, 0 ora,
ozoit_1õ1õ,,,120.-g.a,41a> H >, pk: cr) g
.' ', 4 9 ti F4 1:4 c`-=1 tia cr;' 6 gi '8
0 0 H E-, R. H 0 H Op cr) rg 0 u) H H CD
.-1E-, Urr.1,--1)-1C10140zHENEAHM.-1Z
CDH,1E1 caxpc9r.,,,g1 cl.,irni=11<
0 C. ZZ 44 .4 tg .1E-.1., a, ,--1 0 4. c=1
.-D.L.-.P4,0g EnE.c/22>HE-. <<UZE, ,-)P1
õ-,--'1,4a,C7HD4H<grx-, WHOOF-(H.-1%
=tr),-1(1HH.-Ice,x(00100H<MNE-.,-IZL4
H 1 c.) .-1 H 0 U fa, H, Cl >.,n õ,,,E, 0 0) 1,)
1-1.H Ho)?-,OHHa, Cl) Cl 0)0 4c. 0) g I-4 >-, P.
VI U 'Kg CI 4.4) X r-41 p to > U A cal > frG ul 0 P,,
0. :. 4. .f.go.,ixoa> c,r)cd7
µ24,-.1a. Ch rtiCDH E-, ',-.1 > Cl Cl) z.9 x 1:1,
5,1134,111,0F,401,4r4E. H H (1)Ilt[1.1> H H C.1E-.
1 E-1,3,4u)4 gC 0.-Ivn?-.uir., a; ,cy H G4D
01,404 u) U)E.4 u) ..> C.) > OH LD E-,
02 E,E,., >-,CDE..,4X1-t00u)00 a >0
Ptr.,44..u) p4HH H .altno+Hr=4,-10.,E-4
E,H.E-,-,H a-.0-,0 HHHH:1)cni-11-.10,a-,01
1
CiL)C)<<CDHUE-,<HuquHrrHEJOUOC),<
4 4 4 0 4 E-,UH<<C)<<UNg .4 UE. E. H K:0
0000 OUUUCIUHUgg UH E-.."4,4:ETIU 0 0
E. E-.C5g a a E.,<UUUUE-,C7CDUUCJOg0
< E-, < U 0 < E-. C) E. U g U C) E. 4, E-,
E-, E. 0 0 0.1; E.
C9 40 UOUOULDUCJE,<E,<U0CDOCICJH
g < a: g U E-, CD E-t U e, u 0 H E--, E-, C.) E-, pG E-, H 0 0 ()
uguUUCJOCDCJUE-,HCD<HE-,E,<CD0H,0E-,
U 0 ,4 r- USD(.9 Cl C.) E. r.4 U, 0 d E. E-. 0 0 < 0 0 E. ci
C.) < g 0 < H < 0 U 0 chd KC UH 0 < 4 E-, H 0 H cD
000uUcho,CH<CJE,u<a:E-,QUCDHgc..) U
C)HULDUCJOgUITgc E-. JUCDHOgHguC) u
00 < H UUH c.) 0 ...4 0 H,C.) E-, c9 PK 00 /:
O u 0 E-, U U < 0 ,g= < 0 N (--, U a' U
u F-, C.) 4, E.
U U U U U E. uUque0Hduut_!,F. of.) 0. U
O 0 0 ,,. E-, < U < a, E-, < 0 E, c.) E-,
E, u 0 0 0 0 H
O < a: < 0 < < 0 g H 0 < IJ < H C.) E-, af, 0 Ei H <
f-, 0 C.) E-+ C9 CD 0 U U 0 E-, 0 E-, H H U H E, 0 E-, a, c..) u
CDE-1(1,<<H,E-,<E,CDOC.9rchuOU<c)0(7
uchg CD<H,HuH<C7E, C.JCJHOE-,CD<<H
C.) H. E. 0 0 E-, 0 U C.) C) C9 H H. U E-, tg H
g 0 E-, <
E-,<E-,E.E.<<CJE.CD00HOE-,E,HuE-,000CD
E4b000UCD<O0U<U<HUU0CDE-10<g
O 00 E-,HOHOE.000E.HUHE.U0 4E.
E. ti E. 4 a H E. E. gi E. U H 0 UPg 4 CIE.,404
'400 U 0 .1 CJ g 4 0 U U 0 C.) E-, .< 0 E-, U 0 0 H gli
U < EP U, 0 r... 0 a, U a. U U U < E. u U U C) Ci 0 ry a.
HOU< E-, a q .4 .4 000HP0udUc9KC,Q.0
O<OH K4u.c2 0H d0dUHE.u<KCHE.0<
U0 4 00uH0 004dE.P00CDUH0E.0
4 E. gg 0 0 0 g4 u 4 4 U U 0 HE-..= 0 E.. E. gC E-. < 0
00E-,<E-.000<HUE,OHH000<<UHE,
O E-, CJ CJ 0 0 0 0 0 0 0
0 0 g H. F. U E, 0 0 U U grt ,
,
.4 H, E-, 4 E-if,d H < H. U E-, H < E. C.) Q CD U H U 0 Li
A. CJOU00000CJ<UHuHOCDE-,U000
u0H0E-.00H0<c9000000<CJOUg
E. H 0 E-. C.) E-, H 0 HP .4; ..4 0 H E-, U E+ a; 0 c7 CD 0
=
K4 u ri ,=4 CDHCD<CJOUCJEAHHE-,C)Uc9g 4 H
00gUat<CDHE.uuUu<0HHOOLIC9HH
dUE.000d000<uH0u<U0Hu gCH
000 0 0 E-, u U 0 < 0 0 0 H. E-, 0 C) < U HOF
O 4dH gCuE.HuUc9 Kt uHE-,HOUHHOHai
E-1 0 a; 0 0 E-1 Ei 0 rr g L9 E-, 4 U E. OUHUE.C.5<HKI,
ugC0E.Ug4u 0 g4Uu H dd V< 00a: H> .4
t9 r." E-4 g -,,4 CJ U C) C.) E-4 / U g CD H 0 U U (,.7 0 R , U R. ;
,d0Ougc c9E-tH4 4 <, .9Hc9,<C),<UCDHE,
t..) 0 0 0 0 d U 0 0 U E. 0 E. E. () a4 0 U CD E. E.
UHU 0 HUU HUE. Hr-C H U ....¶9E.H E. H U 0
HOC) ruuci H0000E.4.:0E-10 E. UHE.0
UU ti OUUC90..t Ø...400410<gF-, 0 0 U 0 E-
,
0 E7, E. < u E-, ,.., < () C.) C..) c...1 c...)
C.9 0 C...) U < Pq 0 u E. 0
4uu0 (900 gcuOUUHUHE.K4UUuE.Kr HE-,
0HC9 004U U) Ho uuHud c9E.u0E.DdHu
cDug H I--Ø<<c)E-.C.)CJE--,E,<<UU<UOUOE,
HOC!) HC0004CDUOHUHOO<HuHE-,uHF,
O(000 E--,UE-, 0 .1: C9 H E. H C9 U 0 0 U rt., U E. C./ 4, 0
CI8E.KUE.0 HU HE, HOE. HE-000U 0 H ,:l .4 u
6 U000,4E-4C)<U<HH000CD<OLDUCD<0
CD 0 C.7 < U 0 0 CD a, CD c) E-, 0 0 H U 0 0 < ,,C H .4
O U < rcg E. d 0 H H 1-, < < U C.) 0 0 U g 0 c7 g 0 E--, <
OUU00,<UH,<5-.00E,c9<010g0E-,C),<E,
O00E-, 0 1.00 < E.0<(..E.E. OCIa, U 0 0 OH u
HUE-'
0 0 0: E-, E-, E, E. 0 0 U U E. U E. E. a u (.9 u a
E-, E-. E.E.0E. d E.u0 00 u dc1C9 HUH 04:E.
U uu (.9 U H 0 0 H E.HuHu uold (JP <Hu0E-.
O E. U gC E. u 0 < u 0 / U U E--, CJ 0 0 E-, H u H, ch u <
<CD<C9HHOL)00 UULDUHE.OUHOCDO.
CDUC)<C100C)00 000HOUHOPUE0
C,94UUrE,CD<OCJ E-.00(.1HC..)0CDC)00
0 OH El Hu 0000 U 0 g E-, U E-. C) 0 C.9 L. g 1
E-, E-, 0 0 C.) 0 0 0 E. 0 E. E. c) E. E--, u .4 E-,
0 Uci H
E-.F<HHCDOE-.<P6Hg<UOUUOLDOU U
O E, g H. < E. E.-, c7 u a' ' U 0 0 U 0 CD U CD g H, U E-, E-,
r<C)0(1c)000<cichHouutiguOUUUHO
ucJC)V9cJ C9C9CDOU<H,C7<HHIUU 4 HUOHO
,< g U<OHOH O CJ UHHUUE-,HOOH F.
E . 1
U U UH,HU,OC)0<c)E--,E.F.C)<<000H
O00 E. E. E. U 0 U 0 H 0 0 U 0 Ca E. U U 4 E, U E-,
(9 (9 F:4 Li C9 0 0 0 0 < g 0 0 U < g wHuu c..1 U E. u
u 0 o ci F,UHE-,E,C)C7H<UCJOHUUE-.0,4,<H,
0 E-, (.7 < U u b E-, E.0 E. E. 0 V. 0 U 0 E. 4 d u H 0
L'.'oc . H E. C.J E-.<HUClUg UC)C9H<c)c)E.E-.E,
uuc..)<0<000.< 0(0C)<HUU.C7gUg<
<H04E1 004E. 0F:400000U gCuc90E.
0HUrr UUNKC000E.0004E.E.Hu0KCE.<
E. u E. ciuuuuu U 0 E. u H d 0 E. < E. E. H 0 KC gc __ ......
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
1 1 4
H> g a z g a a E. 0 H> a 4 E. a a ria g g cl
a g X u >-, cx)Xr4E4HE.E.HzryHI-1.-Jgri
0 04HZI.,[4Hu)0E.
E.-, P, H 4 u H U) cnurg 1-10 a
P. E. or., = 4 r.=.X0 4 X 4 01:4 r E. a E.-, re a g P,
E 14 H EZ H Z 4:) cD C.) til HP 1,--10X
H 0 x g =?-t re, a a>. p, 0 S.. .-1 E--, a n
ri,z a g H (..9>ull-tEnz 0HP, Ul 4 r=-=
.-1 CI1 ,-Icil 400 q gX a Hat:404
43 -,KCY,enE.C.9X.44H cPCIEE).0P,<L1)
X04.4ZX>.E.H41.Hul >fr.,C..1-ir.. HON
Z
0 E-1D3.-. > HZ .4E.E.P. 04E4
Z ,-100rK?aloax 5 ., ,r.,,_,,., ,,..õ...,
0 El, 0 a -4 a rig>. gclgi-IX?1,E,E-tg
E. g H. UN pirnr=101-,HE-, 1.4 CI) .94X040.4X
C,
N z.DEri fl ti';'-, g, , 41 E'-.1 'El gi
U)
O 4 U E. 0 Eh 0 g U 0 E. E-, 0 E. E. 0 Ei 0 0 0 0 4 E. 4 D 0 0 U 4 C.)
1
HOUU.HHUO U 00H0UUE.004400 H044H4 HOP
0 0 0 4 E. 0 E. 0 4 a: 0 U P L.) E. 4 E. E. 0 CD E-1 E. E. 0 44 0 44 4 ES
E. HO < < U 0 Ogg 0.4 C.) E. 0 U CD 4 Ft4
E.0E-t0U0U0 HUUHE.EJUOU0004 E-,q444 (Duo
C90 u U 0 HUHHO OPOUPH OC.9E-.E.CDE. 00000 4 u 4
HUU 4 0 0 E. 4 0 Ft 4 0 Fl 0. E9 4 4.4E-10 O04f.0 UHU
E-,0u0OUD0UU0UuHO0 ED ouU00i-,400E, "-,0
E. UUHOUCDUH<H0004 KCEDULtugE.K4u044 OPH
EJ 0 0 U 0 U 0 E. 0 U C.) 44 CD kJ ,...0 0 <4 0 44 0 0 E. 4 H E. U 0 0 E. U
HUU0 0, E. 4 0 E. E. U L!) E-, EJO<OCJUHOPOUCJ U 4 HUH
< E.) 1. U CD (_) 4 E. E-, 0 E. 0 E-I ,4 E. E. E. 0 U 0 E. E.
U CJ C., C.,
0 D0004 000guEtu044404E.E-,00000)E.K4 000
0= 00UHUE. <OH HPH<HHUE.<44HPUUEJLI0UU
0 CD C.) 0 E. 4 0 0 0 E. 0 < U 0 KC, E. a; U0OuHu0 HOE. < HO
0 UcJUU UOCDHE.0CDUOUU UU,44CJE.C.)<CEIHO OU
CJ C.) CD C. UE-, E. E. 0 E. U 4 u 4 E. E. E-, 0 g, 0 0 4 u
(9 E. <
EJOGE.0E.0004441: 044U 0 0000E-.0 44U PE, 00
UUHIOCJOHHUU 000 44 0004E.DuE,<0 OF
E. 00 <HP440000E01 U E.E.00HD<U0E. OE.
E.Ou UH4UULD4,4UHO U4000E.EnOUE--,< <E.<
E.,...40(Doc9E-.00,4 U404_940E. UHOLDHC/CDHC) UgU
E.< 0H444040E-t0E-,u00 4 E.PH440E-.0 0E.0 01)E.
1-' '":0000000E-.0gE.U0L-, 4 0000E. e, g 01) g C.) r r, E.
E.00E,11,E-.E. C. C II E-. OUE-,Ouuu 0000000HE. < .1: E ' E.
E. 0 E. < 0 a', U 0 E. E. kJ < 0 04 HU U04 0 UE-,U<EA Cf.. C.) 4 1
OC)(..)H000 444H CD<HOULDU 000 4400C)OUC) (JO
E. E-. 0 E.J E. E. E. 44 U E. E , 0 E. al E. H E. E. U Cl 44 E. < 4 U 0 E. r"
H
O 00 g H oo,,,,c9c9 0 0 E-t Et 0 0 E. y 0 0 E-, Et U 4 0 (.7 E, 0 4 E.
O0000CJOHOF,E.H04g000004044.,E.00 E. pc E.
EtuU0KCUUKCHE.E.4E-tUUr H H U KC El EJ440E. 4 00P
CJ 000 0E.044r)(J,CDUC)4 HOCJHOE.E.OHUOHHE.
UCJ0000 U c..1, 44Kcoc_Do 0
E.00E.FitiUU/0 OFF,
uuuuE,KC Et 4 u04 E-tguuuE.K4 .,,=
F1040 0 U0E-1
E. 0 0 0 < F. a: 0 U 0 0 < rr .1G U, E. E-, U 0 < < E. 0 4 4: < C.' 0
0 4U000 HUHUOUUCJ5,C) OUHOE.44E.C..4 0 OP a;
0000.000E-.0Cuq(DE-,0400H00ggcDE-,00Hugga,
0 CD C-) 4 0 CD 4 a: 0 0 0 0 0 U < < H g E. g 0 U(DUE-,H0 OUE.
O <OU at 00004 o0E-,E.ODUUU00E.0000U OrCU
O 0 0 U H E. 0 4 E. E. E. E. E. 0 CJ 0 E. U0444 0 E. 4 CD EJ C.) 0 0
L) 4 (-, C) g 0 4 E. 44 <U40 4E-, 4 00E200(.00g E-,<F, 4
0E.E.0000(J00 UCE.E.OUPOC..)00 4CUUCJ CD CIE. gy CD
CJUCD at at 0KCKCF,E.4K4uE.0HOUPPOU40 44, ciC) CD cit E-.
KJ CD E-. u c9 cr9 P H C9 E. (.9 0 (9
E4 E. C.) 0 0 < < E. E. U ,4 4 U 0 <
0 HUHU04C<E.H CD00E.000E-ty00u4KCK? (-tug cIt u
OUE.0u400u4E-,youE-tHE.H <PEAUE.tJH E.CDEJ gl.
Ur.)004E-1.4000 HUUCJO<HU <VJO4C.)44 E. HOP
O 0000004E-14UUE.U0 0HU 0 EJ4C)4< E. U4g
0 0 0 0 EH 0 E. E-c (,) C, C7 0 < E. E. u E. E. 4,
CJ E. E. 4 0 a; kJ E.
0 E. 0 E. < 4 E.E.E.E.04E.U00(70<0 E-, o E-. o 4 U 0 4
19<0000 HouciE.04UE.E.Jc/OHE.UCD< 4r0H
000 F' 0C-)040 H HqE-lu tE.E.Pu HoO4HHE-.Oo 0
uou0(90.400.4u4.5044HOE-,4 0E.HOE.E-, E-, E.cD E.
OCJUE-.0 Out...cid, 04 dur....HHUEDOVEJP:HOUCJE-.00
O CD 0 0 Et E. ,4 c9 u u E-
, c_9 (D CI g 0 E. c., u g E. E-, U E. E. E. CD U U
UE-.(9 E. UU.-4 <OH <4 <044, HOA,40 V< KC a V J 0 0 0 0 0
0 I-Ø40cDO Ho o (Doc.9 gouH5E.ouu uociutial
'(-)t-,0c-,E,E-,a:E-,EJ U440(9(.0 ,0<<F,HUHU00UU
000 at ugUE.U000q4 guE,40 <04LTE-,000 u
C-- , < F,E L,F UU00,,UE.,..0uOuQ)up g UE. 1-,4 HHHU
E-, 0 U 0 E. 4 E. U 4 0 E-, u u E. U U. E. -4 4 u u 0 0 4 u o 0 c...)
OE.U400E-.CDE.E. 4U<HC.)40 44004000 <E. 0 4C at U
C.) E. U U CD U 19 a: C.) rt U CI V E. V CJ 4 0 0 u 0 CJ E. u u E. C) 0 <
< (..., c..9 o E. E. E. E. E. 0 E. E. U f. .4 E. < E. E. 4 H E. E. a; LI U
0 0 U
<<CDOE.CDCD,CUOU<CD<<U0UOPOHE. at OUCJUE.E.
O0 4U Ci40 0000000Pgd<00000 OCJCJUHU,
5,0CD OPUE.E.E. HE.E.E. ci4UUCJOH44CDUE-10 OVE.E.
i U Et uu &4 0uUH O c., U40 4 4 4.9 HO 00o4 CJEJ 4
E.
00
O 4U HOE-.0 40 0 UUE.000HOOPO < F.c CJE. 0400
<000CD<CD0UPUdE-.E. , E.E.EHE-14,40,4 c.-30 OUP 4
U000000 40000 40 OE, 4uHOE.E. uE.E.(DPU
HOUQuO0U0 ut...Dug 4 HUOU,e-,<<E. Hu4HE.E-,
O40UE.,4 E. <0E-144E.UgUQE-,<Ou DE,HUU 0000
HUUE-,400U4 E.OUUEDOCOUHE44040UH '4(70 H
N CJOU 000 E.E.,4
HOE.HEDUOE-,000040PHUOU<U
,-, 0,4 04E-,4 <HO 00E-,U<PHHOUUUUH 4tJE. at <,,'I, 0
000004 :.E.,,t,40400E.4E.1-,u0UHE.E.UU U4 <
= 000 4UU UtDuU004HuU04E.HOuUE.4 DE9g,
O 000 OUE-,E.U,<0.4HUocDgcc.9E-.8o0o(Deo uc94 o
Z 4cDC)ouc.9 c94E-1H4E-.4oHocpu c., QE.E.44H0c9
O Uat000>000ciL c!..Ho
E. C. 0 C. C.) C. C9 E-, 0 H. CD Li E-, 0 cl .4
H 0 0 U CD C.) HU El< .:, , 04 C.) U C., E. E. U E.
H E. 0 ,,4 0 E.4 0000
o? Ho U UE-10E-,04 <0,,,,'
UOU 0 0,404 4 4 0E.E.0 ("DUE,
E4 <0 0 UUE.uHu u ot.30<pu
4U ouciugu0u 0000
en 4 F,0 E, 4 0400 4 H4 Ou
4CHOE.HUg44 4U0 00E-,E-,
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
115
__________________________________________________ -
UHU<4E.4044HE.E. E-, E, U
H. PUL)04:4HOE/UHE.000
U 0 H H E. E.
OHE.E.UUE. 0 E. 0 H U 0 H U
CDUU4E-, OH 00E-.4UUHE.
440UOLDOE. 4HOHOU 0 4'
U ci 0 4 CD U U 4 U
0 E. E. 4 F. 0
0 U
vc.94 .5 E-.ouq.c 000E-,
/
55E-.00.4uc.5 E. 4 0 U
4
uF.0t/HE-,40 0 UHOOU4
0 E. OH 4 H 4 pc 0 OH 40404,
I
E-. 0 0 0 4 0 U, 4 KC
L)E.U0E.E4.4HU 04 46<4.0
C.) U 4
U u o E., y o
0 0 0 4 U 4 U 0 4' 0 0 0 E-4 0 0 0 KC
4 E.F.F.H0E-.04H000044
E.U4V.I.E.E.00)0H<UHOO
E-.04 000E-, F., .4E-,E-,Huu05
500 0E-,E,40000E-,00,50
UHUu 4E.E.0001.4 0(30WH E, H 0 g, F. Ul
4, 0 u u
1
.5 E.F.UPUE.U4E. H 4, 4 4: CD EJ
U ,,,,. 0 0 A: 0
OUL)E.E.E.000<0 H4E-.0,-C
E.040E-14HUKCHHO HOHUU
U 4L)04E.OUE.40F,H E, E. U U
HUH0 E-,L(q4;HugE, 4 g4U
ypcc..,E-.E-,01,00,.1:
1
H2404 H 0000H<OHU
HIUU
UFO() HgE,U0H00UUHU
WUHHOH
OHU u E-. E, U 0 0 4 Ci 4 0 U 4
E. H F. 0 E. H 0 0 .4 U
U 4 u 0 U 0 gc E. 0 L.) F. F4 4 ..= E. U
C/E. OH HE.L)40 E.00
E.E.H4E. UQu4E-.0EUHHE.40
HO00 E. L) u U
E--.H00 UgH4UK40E,
HUH 4 H E. P KC
H ,..c ,,c t-, ,,c E-, E-, c9
0= ,,,cfCgtogc F. 0 4 H U E. U 0 4 0 4
E. 0 H 0 0 E. 4 0 pc E. H E. E, 0 0 0 0
HHHU,4,4r1tH00E-,4 H0E-IU
CJUU4U 4 rd E-1 CD C.) Fl: U, E, E-1 C5 U
,ouc.., py00
HE-,04(7E-, 0004
40g4g E-.E-,E-,E,Ur0c,)0
4.E-IHE,a, Kr UOUE-,0 04c.7E,
COUHO CSUKCE,C9E, H0H0
EA 40HOUE.44 00E. 0c1)04
E. HE-.0E, H40E-.
U 4 U 4 H, 4., H 0 C C < E. E . E . I< CC!
E, H E-, U E, < E-1 H 0 H U U E.-, UUHH
H 0 E-+ 0 0 4 0 0 H E, CD
r..7 U ,r4 0 H H 4 4 0 4 0
E. u 4 0 ,c L)
E. 4 H. U U 4 0 F. 0 1 CD
C '4 U 0 U 0
HHU0440HCDHU
g HHH 4 E. 4 E. -,40UHUH40
0 4 F . u L9
UF,<E,H0E.C3F.00uH0E,44
E, E--, q E. u 4 E. C.) 4 C!)u 0 4 0 4 E.) 0 4 E. U E. 0 0 4 0
0 0 E. U H 4 P E-.
E. H C.) E. 0 0 E. 4 HO
U E. 4 4 0 E. g H 4 E. E-. 0 5 u u u
C.) U E. u .4 0 Ci44000,4uUKCE,
4*O.U.UU4E.01E.U04C)E..4
E. u 0 ,
401/H4HE.
UuC)40444.40E.E.H.E. 4 u 1-, 1
SUBSTITUTE SHEET (RULE 26)
SEQIDNO :18
SEQIDNO . : GS 1,0
GACAGCCTCTGGGICCTCGGTCGGTACAGTCTCTGCACCTCGCGCCCCAGCAGGTAAACTAACATTATGGATTTTTCCA
AGCTACCC MDFS KLPKILDEDKESTEGYVEG
AAAATACTCG.ATGAAGATAAAGAAAGCACATTIGGTTATGTGCATGGGGTCTCAGGACCTGTGGTTACA.GCCTGTGA
CATGGCGGGT VSGPVVTACDMAGAAMYELVRVG
1,4
GCAGCCATGTATGAGCTGGTGAGA.GTGGGCCACAGCGAATTGGTTGGAGAGATTATTCGATTGGAGGGTGACATGC-
CTACTATTCAG HS ELVGE I IRLEGDMATIQVYEE
GTGTATCAAGAAACTICTGGIGIGTCTGTIGGAGATCCTG'TACTTCC-
CACTGGTAPACCCCICTCTGTAGAGCTIGGICCTGGCATT TS GVSVGD PVLRTGKPLSVELGP
ATGGGAG CCATT TTTGATGGTATT CAAAGAC C TTTG TC GGATATCAGCAGT CAGAC C CAAAG
CATCTA CATCC CCAGAGGAGTAP.AC G TMGAIFDGIQRPLSD IS SQT QS
GTGICTGCTCTTAGCAGAGATATCAAATGGGACTITACACCTIGCAAAAACCTACGGGTTGGTAGTCATATCACTGGCG
GAGACATT IYIPRGVNVSALS RD IKWD FTPC
TATGGAATTGICAGTGAGAACTCGCTTATCAAACACP_A_AATCATGTTACCCCCACGAAACAGAGGAACTGTAACTTA
CAn'TGCTCCA KNLRVGSH I TGGD IYGIVS ENS L
CCTGGGAATTATGATP_CCTCTGATGTTGTCTTGGAGCTIGAATTTGAAGGIGT.A_A_A_GGAGAAGTTCACCATGGT
GCAAGTATGGCCT IKHKIML PPRNRGPVTYTAPPGN
GTACGTCAAGTTCGAC CTGTCACTGAGAAGCTGC CAGCCP_ATCATCCTCTGTTGACTGG
CCAGAGAGTCCTTGATG CCCTTTTTCCG YDTSDVVLELEFEGVKEKFTMVQ
TGTGICCAGGGAGGAACTP_CTGCTATCCCTGGAGCCITTGGCTGIGGAAAGACAGTGATATCACAGTCTCTATCCAAG
TATTCTAAC VWPVRQVRPVTE.KLPANHPLLTG
cn
0
=
AGTGATGTAATCATCTATGTAC-
GATGTGGTGAAAGAGGAAATGAGATGICTGAAGTCCTCCGGGACTICCCAGAGCTCACAAT.GGAG
QRVLDALFPCVQGGTTAI PGAFG n.)
CO
GTTGATGGTAAGGTAGAGTCAP.TTATGAAGAGGACAGCTTTGGTAGCCAATACCTCCAATATGCCTGTTGCTGCTAGA
GAAGCCTCT CGKTVIS QS LS KYSNSDVT IYVG
co
(I)
ATTTATACTGGAATCACACTGICAGAGTACTICCGTGACATGGGCTATCATGTCAGTATGATGGCTGACTCTACCTCTA
GATGGGCT CGERGNEMSEVLRD FPELTMEVD 01
GAGGCCCTTAGAGAAATCTCTG GTCGTTTAGCTGAAATGCC TGCAGATAGTGGAT ATCCAG
CCIATCTIGGTGCCCGTCTGGCCT CG GKVES IMKRIALvANTSNmPVAA
-71
TITTATGP_ACGAGCAGGCAGGGTGAAATGTCTIGGAAATCCTGAAAGAGAAGGGAGTGTCAGCATTGTP.C-
GAGCAGTTICTCCACCT HEASIYTGI TLSEYFRDMGYHVS
-
GGIGGTGATT'ITTCTGATCCAGTTACATCTGCCACTCTIGGTATCGTTCAGGTGITCTGOGC3CTTAGATAAGAAACT
AGCTCAACGT MMADSTSRWAEALREISGRLAEM n.)
AAGCATTTC CCCTCTGT CAATTGGC TCATCAG CTACAG CAAG TATATGCG TGCC TTGGATG
P_ATACTATGACAAA CACTTCACAGAG PADS GYPAYLGARLAS FYERAGR
cn
=
TTCG TTC C TCTGAGGA
CGAAAGCTAAGGAAATTCTG CAGGAAGAAGAAGAC CTGGCAGAAA TTGTACAGCT TGTGGGAAAGG CTTCT
VKCLGNPEREGSvS IVGAvS PPG n.)
TIGGCAGAAACAGATAAAATCACTCTGGAGGTAGCAAAACTTATCAAAGATGATTTCCTACAACAAAATGC-
ATATACTCCTTATGAC GDFSD PVTSATLGIVQVFWGLDK
co
¨1
AGGTTCTGCCCATTCTACAAGACAGTAGGGATGCTGICC_AACATGATTGCATTTTATGATATGGCTCGTP.GAGCTGT
TGAAACCACT KLAQRKTIFPSVNWLISYSKYMRA
GCCCAGAGTGACAATAAAAT CACATGGTCCATTATTCGTGAGCACATGGGAC-ACATC
CTCTATAAACTTTCCTCCATGAAATTCAAG LDEYYDKHFTEFVPLRTKAICEIL
n.)
GATCCACTGAAAGATGGTGAGGCAAAGATCAAAAGCGACTATGCACAACTTCTTGAAGACATGCAGAATG
CATTCCGTAG CCTTGAA QE EEDLAE VQLVGKAS LAETDK
r"
GATTP_GAAGCCITGAAGATTACP.ACTGTGATTTCCTITTCCTCAGCAAGCTCCIATGTGTATATTTTCCTGAATTTC
TCATCTCAAA ITLEVAKL I KDDFLQQNGYT PYD
CCCITTGCTICITTATTGTGCAGCTTTGAGACTAGTGCCTATGTGTGTTATTTGITTCCcTOTTTTITTGGTAGGICTT
ATATAAAA RFC P FYKTVGMLSNMIAFYDMAR
cr)
CAAACATTC CTT TGTTCTAGTGTTGTGAAG GGC CT CCCICTT CCTT
TATCTGAAGT GG TGAATATAGTAAATATACATTCTGGT TAC RAVETTAQSDNKITWS I IRE HMG
ACTACTGTAAACTIGTATGTAGGGTGATGACCCTCTTIGTCCTAGGTGTACCCITTCCICATCTCTATTXL.ATTGTAA
A.CAGGACTA DILYKLSSMKFKD PLKDGEAKIK
CTGCATGTACTCTCTTTGCAGTGAATTIGGAAIGGAAGGCCAGGTTTCTATP.ACTTTTGAACAGGTACTITGTGAAAT
GACTCAATT SDYAQLLEDmQNAFRs LED
TCTATTGTGGTAAGCTCATTGGCAGCTTAGCATTTTGCAP.AGGAATTGCTTTGCAGGAAATATTTAATTITCAAAAAC
ATAATGATT
PATGTTCCAATTATGCATCACTTC
CCCCAGTATAAATCAGGAATGTTTGTGAGAAACCATTGGGAACTATACTCTTTTTATTTTTAT
TTITTATTTITTTTATTATTTITTITTIGGGGA_CGGAGTGICCCTCTTGTTGCCCAGGCTGGAGTGCAATGGCGTGAT
CTTGGCTCA
.75
t7.-J
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
117
,
EJ 0 0 4, u E. H Er.
8 [5 8 41 0 g il 8 [5 u 8
8 8 6' il 8'
0 8 El ri [5 '.; f5 1 1 0 8 6 r r 5 P, f-J1.1 E. E::
0 Eg-44 OH
CJEDg4Pgra: CD E.HC9Lio.E.0 C.74000H
El ii [5 P El [5 UHEH5 0 F.'
0 0 1 El CJ 0 E. L9 F. C9 E. Eq E. E.4 H C7 g4 CJ
KJ H
8 8 0 f5 8 L7 g g .c) [; I?: Et4 F13 g 8 f5 P. il r
i HUHry
E4 El, U 8 tl
0 CJ C., ,4 o c!.! u, u o o c-, -'
..
',;.,
El 8 0 2 6' 8 2 E= 6 g 0 0 0 E. Eg' g E: [5 H El
HUH
HHKDKJHULDg4C7 g/LJCDOC9 g ,gc o E. E--.
8 8 8 0 ,t fl 2 6' 6 '
8,-, [5 0 82 `.0 EY,
8 El [5 [5 il N 8 E. F.' EH F7' El El El P. El 0 8 U 8 U
`-' ' " E' C.) 2. El E-4 El Eg EL1 C?) 5 r.,. r, (9tr...
(ii,..! (6. (,,i v_..
V El u c) E. F E4 rg 0 U
UH
U (.7 E.
UH 0 E. E. EA CD Pc E, CJ 611 g 0 0 F. F, F.
U 6 0
IJ gE.E.E.UHC9gL7 gc .0 p o . 5
b ii 8u r; V' g c[i r4' ri E ([ 3E, r5 P, '[:.5 3-, !'-' 1
000 ,0000.4 i -,¶.E--.E-.-0¶-,u,cHuL)
U g `d 6'. ,E 0 0 HHH0 !i 8 0 El
_ Ei
Eluur E-'1uf81.ur,E'E(2, oci -,7 ' CJ 0 u (7 K../ H E. E. 0 F.
C-)
V, 0 cA i E r fl ,'-Z E, 0 'i 1 El fl 8 E( 0
0 H u C.) CJ (9U U F, E. 0 E. 0 CD 0 H U E. H E.
E. H H E. 0 EA g g g EA C7 cn rc H O. ., ,H,. P EL1
E.-, El g E. H 04 gli i. li Pi g...,
gS2C1 EHgC7HHHH,Uoiti Hti
gHg4HP_P
,E1, ii E-.' P 8 P. E E.'; t', 8 r r, v, 8, ' ;!,c'
kJ V, ri 0 E.
8 8 2 il t5
g/ E. E.
E' " '' t-' 8 5 'r. F4 V., E-2 P. r ..4 6' 8 1
1
8" " " E'. 1 cõ 0 8 8 0 E5 2 El 0 1 E5 E5 g ';-4' ;5. El E.
., 0 8., .2 õ Eõ E.
HEC), Et.., ,F"-,
__ ,
tj,.! CH, " r,4. [5 1 [5 i4 EE3 r ',c. g g U 'El r, r 4 g
,, , ,-, r, ti 0 0 CD KC E4 rg Ch rg 0 F. Cp 0 Ch E" F.
ii
KJ E.
F?-:, ,t...;
b`'E----:Eirs,n_i8,`,-4-'E--:.nr-90E100E-.01-50
$)
R 8, [5 t; EJEc.3.
/
4) ,e, u g g !-. Ch CJ gi CJ CJ tJ C) C..) u E. 19 H
C7 E4 CD 0 H E, E. 0 CD U E. E. 0 g F. U E6. 1 ,(.1,1 .4
El 8 2 EI [5 .' 1 8 . EE--,' fl 8 8 ',;.!. `E-4 u 4
'
SUBSTITUTE SHEET (RULE 26)
SEQIDNO . :19
SEQIDNO . 166 0
1,0
ACGCCTGGTCTCTGGGACGCCCCTCCGGACCCGTTTCGCCTCGCGGAGCCGGTAGGTCCAGC3TGCAGCGGCCGCAGTG
CTGCGTCCG MIRQERS TS YQELS EELVQVVES
TGCGCCGCGGGCTGGGGCGGTCTCAGGTGTGCCGPAGCTCTGGTCAGTGCCATGATCCGGCAGGAGCGCTCCP_CATCC
TACCAGGAG SELADEQDKETVRVQGPGILPGL
C TGAGTGAGGAGTTGGTC CAGGTGGTTGAGAGCTCAGAG CTGGCAGACGAG
CAGGACAAGGAGACGGTCAGAGTCCAAGG TCCGGGT DSESASSS IRF S KACLICNVESVL
ATCTTACCAGGCCTGGACAGCGAGTCCGCCTCCAGCAGCATCGGCTICAGCAAGGCCTGCCTGAAGAACGTCTTCTCGG
TCCTACTC LIFTYLLLMAVAVELVYRTITDF
AT.CTTCATCTACCTGCTGCTCATGGCTGTGGCCGTCTTCCTGGTCTACCGGACCATCAC.AGACTTTCGTGAGAAACT
CAAGCACC CT . REKLKHPVMSVSYICEVDRYDAPG
GTCATGTCTGTGTCTTACAAGGAAGTGGATCC-CTATGA.TGCCCCAGGTATTGCCTTGTACC
CCGGTCAGGCCCAGTTGCTCAGCTGT IALYPGQAQLLSCKHHYEVTPPL
AAGCACCATTACGAGGTCATTCCTCCTCTGACAAGCCCTGGCCAC-
CCGGGTGACATGAATTGCACCACCCAGAGGATCAACTACACG TS PGQPGDMNCTTQRINYTDPFS
GACCCCTTCTCCAATCAGACTGTGAAATCTGCCCTGATTGTCCAGGGGCCCCGGGAAGTGAAAAAGCGGGAGCTGG'TC
TTCCTCCAG NQTVKSALIVQGPREVKKRFLVF
TTCCGCCTGAACAAGAGTAGTGAGGACTTCAGCGCCATTGATTAGCTCCTCTTCTCTTCTTTCCAGGAGTTCCTGCAAA
GCCCAAAC LQFRLNKS SED FSAIDYLLFS S F
AGGGTAGGCTTCATGCAGGCCTGTGAGAGTGCCTGTTCCAGCTGGAAGTTCTCTGGGGGCTTCCGCACCTGGGTCAAGA
TGTCACTG QEFLQS PNRVGFMQACESACSSW
GTAAAGACCAAGGAGGAGGATGGGCGGGAAGCAGTGGAGTTCCGGCAGGAGACAAGTGTGGTTAACTACATTGACCAGA
GGCCAGCT KFSGGERTWVKMSLVICTICEEDGR 0
GCCAAAA.AAAGTGCTCA_ATTGTTTTTTGTGGTCTTTGAATGGAAAGATCCTTTCATCCAGAAAGTCCAAGATATAGT
CACTGCC_AAT EAVEFRQETSVVNYIDQRPAAKK
cn
CC TTGGAACACAAT TGCTGTT CTCTGTGG CG C CTTCTTGG CAT TATTTAAAG CAGCAGAGTTTG C
CAAAC TGAGTATAAAATC- GATG SAQL F FVVFEWKD P F I QKVQDIV0
CO
ATCAAAATTAGAAAGAGATACCTTAPAAGAAG.AGGTCAGGCAACGP.GCCACATAAGCTGAAGTCACCTCGCGTTGTT
TAGAGAACTC- TANPWNTIALLCGAFLALFKAAE n.)
cn
co
TCCACATCAATGGGAGCTGTCATCACTTCCACTTTGTAP-
ACGGAGCTATCAACAATCCTGTACTCACTTGAAGAAATGGGGCCTTGC MIMS I KWMI KIRKRYLKRRGQA
tri
7:1
TGGGAGGAACAGCATGTAAAACTGGAACTTCTAACCCCGTCCCAPAAGAGGCGGTGTAGAGCCTAATAGAAGAGACTAA
TGGATAAA TSHIS op
CCTACAAGTTATTTAAATATTTAAATTATTAATAAACTTTTTAAAGAGCTGGCCAATGACTTTTGAATAGGGTTTGTAG
AAGATG CC
TTTCTTCCTGTTTGGTTCATTGTATTGTATTAGGTTAAGCTCTACTAGGGTAATC-
PAGGCTCTACTTTTCACTTTTTAAAAGTGGAC n.)
cn
AAAAGAGTGTGATITTc7rmccAAAAATTccrGAGTATcAAGAcGTGcAGGTcATGcrrTGGAGccTATccAcTGTAcA
cAATGG
CAAAACCGTATGACTTTGGCATCATCTG CCATTGATG
TCCAGCCTCTGACATGCTCTTTGATTTGTTAAATGTTAAATGAGACTTTA
n.)
AGGCTACTAGAAACTAGTAATTAAGTTTCTTAATGGACTGAGTAGCCACCTACTTGTCCGGCTAGAATGTTTGTTGATG
TATGAGTT
0
TAGATTAACACTCAAAAGCACTAGGACAGATGTACATAGAAGGTGCCTACTCATTGTATTTTGATGATTTCATTAACAG
GTAAATAA co
AAGTTAATACAAAAGGAACGAGTGTGACAATATGAATATCTGCTCAATCATCGGGCACAATTACTTTCATTTGGTGACT
TCCAAGGA
CAAAAAGGTAGTATGAGTCTGGACTCCCAAGATGGATCTAACTCTCAAGGTATGTTCTAACTGCTTCCAGGGAAGGGTT
TGTTAGGC
ATGGCAACTGATGGCAGGTGTCCAGAAAGAGTGACCTGGTGTCCCCGAGGAAGCTGGGTTPACTCTTTACTGTGTCCAC
AAAACTAC
CCATCATATGAGGAAGGGGTATACGCAGTGTGACCCTCAAAAAG
CTTTTAGCCTAGCCTTTGACAGAAATGAGTATGCATTAAAAAA
=
cr)
AAGTCTATTTTTCACATTAAGGTTCTAAAAATTGTTTCCAGAGTTTTAAATTATTTATGTGCCTGTTGCTTCAAAGAGG
ACTTGGTA
GCATTTCCTAAATTTTGTAATCTGGCTTCCGATAATCCAAAGGGP.ATAACTCAAATGTATGAATAGGCATTTTAAATG
GGAAGPAAC
TGTTTTTTGGATGAATGATTAAAAGTGAACTGTATAAAG
-4
SEOIDNO : 2 0
SEQIDNO . 67
GCGGACGTGGGCAGGAGGGCTGGAAAAGCCGGCGCTGGAGCGGGAACGGGAGTAGCTGCCTGGGCGCCAAAGGCCGCGG
CACTCCCA MFRKGKKRHSSSSSQSSEISTKS
CGCGGACCCCGAAGTC
CGCAACCCGGGGATGGGCCCGCGGCTGCGAGGGGATCTTCTCTGGATCAAGCAATGGTGGTGAAAAATG TT
KSVDSSLGGLSRSSTVASLDTDS
TCGCAAGGGCAAAAAACGACACAGTAGTAGCAGTTCCCAAAGTAGCGAAATCAGTACTAAGAGCAAGTCTGTGGATTCT
AGCCTTGG TKS S GQSNNNSDTCAS FRI KYVG
GGGTCTTTCACGATCCAGCACTGTGGCCAGCCICGACACAGATTCCACCAAAAGCTCAGGACAAAGCAACAATAATTCA
GALACCTG AIEKLKLSGKGLEGPLDLINYI
TGCAGAATTTCGAATAAAATATGTTGGTGCCATTGAGAAACTGAAACTCTCCGAGGGAAAP.GGCCTTGAAGGGCCATT
AGACCTGAT DvAQQDGKLPFVPPEEEFIMGVS
AAATTATATAGACGTTGCCCAGCP_AGATSGMAGTTGCCTITTGTTCCTCCGGAGGAAGA_a_TTTATTATGGGAGTTT
CCA.2-.' GTATGG KYGIKVSTSDQYDVLERIGILLYLI
CATAAAAGTATCAACATCAGATCAATATGAIGTTTTGCACAGGC.ATGCTCTCTACTTAATAATCCGGATGGTGTGTTA
CGP.TGACGG IPMVCYDDGLGAGKSLLALKTTD
TCTGGGGGCGGGAAAAAGCTTACTGGCTCTGAAGACCACAGATGCAP_CCAATGAGGAATAC.11_GCCTGTGGGTTTA
TCP_GTCCAACAG ASNEEYSLIWY. QCNSLEQAQAIC
CCTGGAACA.AGCACAAGCCATTTGCAAGGTTTTATCCACCGCTTTTGACTCTGTATTAACATCTGAGAAACCCTGAAT
CCTGCAATC KVLSTAFDSVLTSEKP
AAGTAGAAGTCAACTTCATCTGAAAGTTCAGCTGTTTTCAAACTGCAATGCTGAAATGTTATGCAAATAATGAAGTTAT
CCCTTGCT
CTAGATITTCTGAAGAAAATGGATTGTOTAAAATGCTGATCATTTGTTTATTAAAATGTGTCCTATTACACAGTGAGTT
PACTCTCA 0
ATGAAGTCATCTATITTCTGGGCTACTTCATTTC-
TCITTTTCAACTTCTAATAAGCTTAACCTAAGTGTCP_CGAAGACGAGA
cn
TGTCACAGAGGTCCACTCAGTGACAAACACACACTGAP.GGCCTGAGGGAAGACTGAGGACATGGGCTCAGTGGTGGCT
TCCCAGTCA
N.)
CO TGGTATCACTGGCATGGACCTCTGTCCGGCAGAGGTC-
TGGACTGGP_GACCAGGATTCATGCTGGTCTGGAACAATGACATTGCCAAC
cn
co
TTAAGACACACAAAGCAGATTTTCAGAAGTGTCTGGTCAP.GATAP_CATGCIGGCCAACCACAATTCCTAGAGTTAII
GAGAACCTTAA tri
AAGATTACCGCTCATGCTAAAAGTATGTAAAGATCCCATGTACAGTATGATAGTGTACTTTTITTAAAGGACTC-
TC.AATATACAAAA
CT T TAAAGATTAPAAACATTAAAAATAA.AARAA
rrl
N.)
0
cn
SEQIDNO.:21
SEQIDNO.:68 N.)
CCTCGCCCCGCCTACGCGGGAAC CC.AACCGCGGCGACCGGACGTGCACTCCTCCAGTAGCGOCTGCACGT
CCTGCAATGGC CCGCTA MARYEEVSVSGFEEFHRAVEQIIN
co
TGAGGAGGTGAGCGTGTCCGGCTTCGAGGAGTTCCACCGGGCCGTGGAACAGCACAATGGCAAGACCATTTTCGCCTAC
TITAcGGG GKTIFAYFTGSKDAGGKSWCPDC
=
TTCTAAGGACGCCG-
GGGCGAAA_AGCTCGTSCCCCGACTGCGTC-CAGGCTGP.ACCAGTCGTACGAGAGGGGCTGRAGCACATTP-GTGA
VQAEPVVREGLKIIISEGCVFIYC N.)
AGGATGTGTGTTCATCTACTCCCAAGTAGGAGAAAAGCCTTATTGGAAAGATCCAAATAATGACTTCAGAAAAAACTTG
AAAGTAAC QVGEKPYWKDPNNDFRKNLKVTA
rn
AGCAGTGCCTACACTACTTAAGTATGGAACACCTCAAAAACTGGTAGAATCTGAGTGTCTTCAGGCCAACCTOGTGGAA
ATGTTGTT VPTLLKYGTPQIGIATESECLQ.ANL
cr)
CTCTGAAGATTAAGATTTTAGGATGGCAATCATgTCTTGATSTCCTGATTTGTTCTAGTATCAATAP_ACTGTATACTT
GCTTTGAAT VEMLFS ED
TCATGTTAGCAATAAATCATGTTAAP_AP_P_ACTGGCATGTGTCTAAACAATAGAGTGCTATTAAMTGCCCATGP.AC
CTTTAGTTTGC
CTGTAATACATGGATATTTTTAAGATATAP
kGAAGTCTTCAGAAATAGCAGTAAAGGCTCAAAGGAACGTGATTCTTGAAGGTGACG
GTAATACCTA.A.AAACTCCTAAAGGTGCAGAGC
r)
___________________________________ _
______________________________________________________
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
120
a E., >4 >4 4 tee en pao z
14' 2 6 [:4, ,t1 EF, Fi
'tin' ,Z' [I 8" CE!: 6 f=1
' ix, E-0 tx4 P-I Cil Eir o 0
UHIZC.1U fe.1M
0 or 4 u
F Cil 0 P4 Cf) 4 F F 4
al (.1/-14./0/-14e0tZ cc)
0
P ,, .` '
c3 CO rar 0 H 41 0 ... ...,
Z
0
H
or co>crrucni-raNzH
E.)
440E-.04E.E.rPCJ004U4U0H4E. 4 U
00E-ruEruo4 UHHOHUCJOHE.0440H
4E-.00H000 UE.E.C.DHHOC.DE.40E.U<C)
CD C..) u 4 H /4 0 U E. U E. 4 /4 C./ E. E-. Ei E. U E. 4 4 E. H
OPCDCDP0H4 4 404 rt. C-, (..) 4 4UHUE.C.DHE-e
4 0 Er Kg U o 4 u 4 u 4 o o o Er u U F K4 F E.
8 E-, 2: /,Yt r g g 8 8 6 8 0... * 00 8 2 r 04 8 4E); 4444 E
04 u u u c.9 o E-r 4 u 4 ,r, 4 E--.00U0 u U, 4 4
--r) -..r, g cE?, p, t 8 4 '6 8 ( 9=5 r, 'd 6 i:: 8 EU (0) (,- )4 8 [Ell
UP EreE.C./ 4CDUCDE-1 .e,CD O Ce 44,HUCDUH u E.
4 U El 0 C., 4 4 H 4 H E. 0 4 C9 E. H 4 H C, C.) el. 4
C/E.H HE.CJC540 E.C..1.0 4 r, E. C.) CDUUU
ti El r.i 8482 ,1 4, v6P r, EY. b" 'E5. .1 8Ø184V-.1-1 1
E. u 0 4 , 0 0 0 u Er , 4 ci 4 Er E. Er 4 or Er 0 0
.,0,
U.OgFUUF 4 E. (9 -4 4 4 E.HPHUHH/4/4E-14
Ig6c,..),4H/E,c9CJE.U. UU. C) 4 E.H4E.HU0 4H
C1E.CJE.1E.V. 8 Et, 2 f,-:: 8 6 c,,-49r-18ggreE/:: 8 8 8
,'¶ 8 8 8 8 2 0. '6 5 c,' Ft: 8' 0 6 8 8 8 Et: 8 El '' ',.-4
Et: * E,"El '6 r-: 8 8 8 8 '6 cE.--.9 8 8 [--: 8' 2 .), " 2 8 V. , c jr- :
Ec : .1 8 ii: E;;; ._,;) ,v ,,E.. [11 0 _,,., U 0 E-, 0 0 E-. E- , H E-/
E. i...,,1 t-j,
,4 0 0 E. E. U U 4 E. 0 r.E.--/) 1 Eli (C-.).1 (5 E-.) Er-
/) E. 0. * 2 ..=; E.
.er, 4 H CD -4 Ce KJ U. E. CD, A. 0 0 E. C., C.) U E. E. ,-,q CD C..1 H 0
0 0 (-) 4 4 4 KI, Kg E4 0 0 U F F. 0 4 04 E. U U E.
H40 CDUC.D. Pr F H44E.UUHHHE-.4E.E.Ch
.4) Ell 8 Pi 6 .,L3 , ,&' LI 2 t; - L ) ,
5 gu pu f=-,.F3: 9.() r Et: g: .), EE:: r_: i:i ,e,' ,,:,),
1
O00 H0E-e4 .44.')FUOF t-'0,-,P 40H0E.UH
E. 4 E. CD U 0 0 0 0 H E. E. CD (9 E. 4 U 4 Er E. 0 . 0 E. 0 4
f'- .) /L) '6 :4, 8 8, 8 8 8 0, 4';
.,,,,(1 n''. 8 44) fl 8 8 :2 8 ,Ejii E,="g` L'D t i
H00E/U040040E. ll , C.J E.U00,,H g4CIE.0
4 C.1 0 4 E-/ E. 0 4 E. el, 4 E. 4 CD E. U 0 4 0 o Er E. 4 0 4
Ej, el..! F.) E.:C' il 8 8 V. 6 4 8 8
El 8 b? 8 8 8 8 6 8 8 8
6 E -. E. 0 U C ./ E . CD 0 H 4 4 e5 0 E . E . 0 , r_ ." 1
[7 : !,. , [_q, ,E7gi r,
8, 8 rE--.) 2 'E-4, $ 8' u. i-4 r6 8' 4 8, 8 o E. E. u R ED u .
I
* u 111 8 8. 'C.' 8 u 8 8 8 8 V-.4 -,' 8 8 (c)D E;t' id CEi E4'
ELIE ECDPSFE4'E 6680,868E,A;.8i--1288E.
1--,' 0 8 'E--: 8, 8' .-', 8 8 ''. r-: 8
',-3 '' 8 8 8 t.-: ,c.-Z f.. Ec-,' E,;'4 ,1 6
8 'El u t".' * 6 8 6 * -.' r. u Eg C,4) ,Ei 8 8 2 8 8 8
,,c ., 0 E., 0 0 /4 U E. CD U 4 CD 4 U E. U 0 E. U E. KC 0 H H
r E -.) E4 ,?4 E- .- : t5 E: : . .7 i [ 7 '6 8 8 8 2 Et
6 8 8 flt' -.91 01
E-' CDHU,-, 000.cCJE-.4E.E.U0E.E.E.CDULeHE.
I
CD 4 0 0 H E. El La (.1 A -4 0 Er p. sd. o 4 ce.-15 u 6 6, c-.-! ,,,,, ,,E,9
E=;-c`
HCDH00ETE-..De44H Liu 44 õ.c-.1. . _
' 0 4 C./ E. 4 E. CD E. 0 4 E. E. E. E. U 6 t, r..., 6
re, ,t--,,. t., ,._.,.,
/ it'u EE -,/ t' H E. F EE-4 1(Ch ECY') E )/ 8 [1 EC ', tin H)
(E 2, CE '8 E'
4 F.9 /L,-11 8 tl: 8 8 u 8 8 8 6 EE---'. 6 '0' 8
il E-. 2 V-.' 4 r, 8
O440 4. CD CD CD E. H E. E. , c[....),
EE.3 E. E,o.-, E., ,c..-4, 0 Ec-,, Ec-jr Ec:., cc!,
o 0 E. 0 0
4 0 0 H 0 4 C. V, Fi ,f:) 8 6 E. 0 (.7) H rõ
ci: El 0 0 ., 4
' 0 , 6' ' 2 8H 81 ' - '8 . 4 .1 . 8 .-.4 ! -, 3 1E - ' r.
7 EC- :( 9 r ,E ) 8H UC 9 ' -. . I .0 6H X * - ' I : 1H SH H8 .*E I
u 0 u E. E. Ci C.) 0 El 4 E. H 0 4 E. I ET, C..1 0 C.1 4 0
E-e E.
EJ 0 4 0 4 u 4 Er 4 0 ,E,. E.' CJE1HHE.Et,.(2
f -I 2 ,F,- Et: V-: 8 -,' 1 w &: 8 4
8 u El u r: 6 8 8) 2 E-1 E. n
C4 5 y,, ku k" E,:4' -). B E 8 E;r:., ii 8 Ec-_,"E-4 Eg 8 E,--
: b' 8 V ', 6' `E-_,)
CV ,-, c-4, ...= %, CJ U E. 4 E. 4 4 CD 41040E-ruE-1 E. F 4
(5 0 004UE.004CJE.E.40 0040U 4H4E.
. E-14H4HOU4E-144 4H00_4440HUE.00
O CJCD4UU
c)C.)Q00E.E.CDHpUL)C.)4E-, 4pOrEc,
Z
O P4 '6 c; 8 6 P-, 8 6 i--: 8
8' 8 r. 8 ',J. ',=-'t `,4 8, 8 8 8 r: 1-_.= .A
H
a 8 0 6 8 8 8 * `,,) 6 b' 8 E' Vc V: Et: 8 8 Cf.) [7 r7 'E4.: 6 Et: '6 2
E4 `E--,) 1 Pi 8 4 8 8 6 8 8 ,,.. ,E.t 6 8 `..": 2 8 8 8 8 8 r, 2
O _
SUBSTITUTE SHEET (RULE 26)
0
SEQIDNO 23
SEQIDNO : 7 0 1,4
CCTCCTCTCCCTGG CTTTTGTGTTGGTGCCTCCGAGCTGCAAGGAGGGTGCGCTGGAGGAGGAGGAGGGG
GGCCCGGAGTGAGAGGC MAQP ILGHGS LQPASAAGLAS LE
ACCCCCTTCACGCGCGCGCGCGCACAcGGTGCCGGCGCACGCACACACGGGCGGACACACACACACGCGCGCACACACA
CACGCACA LDS S LDQvVQTRIFKI I VIGDSN
GAGCTCGCTCGCCTCGAGCGCACGAACGTGGACGTTCTCTTTGTGTGGAGCCCTCAAGGGC-
GGTTGGGGCCCCGGTTCGGTCCGGGG VGKTCLTFRFCGGTFPD KTEAT I 1,4
GAGATGGCGCAGCCCATCCTGGGccATGGGAGcCTGcAcCcCGCCTCGGccGCTGGCCTGGCGTCCCTGGAGCTcGAcT
CGTCGcTG GVDFREKTvEIEGERTKvQviNDT
GACCAGTACGTGCAGATTCGCATCTTCAAAATAATCGTGATTGGGGACTCCAACGTGGGCA.AGACCTGCCTC-
ACCTTCCGCTTCTGC AC-QERFRKSMvERyyRNvRAvvF
GGGGGTACCTTCCCAGACAAGACTGAAGCCACCATCGGCGTGGACTTCAGGGAGAAGACCGTGGAAATCGAGGGCGAGA
AGATCAAG VYDVTKMTS FTNLKMWTQECNGH
GTTCAGGTGTGGGACACAGCAGGTCAGGAACGTTTCCGCAAAAGCATGDTCGAC-
CATTACTACCGCAACGTACATGCCGTGGTCTTC AVPPLVPKVLVGNKCDLREQIQV
GTCTATGACGTCACCAAGATGACATCTTTCACCAACCTCAAAATGTGGATCCAAGAATGCAATGGGCATGCTGTGCCCC
CACTAGTC PSNLALKFADAHNMLLFETSAKD
CCCAAAGTGCTTGTGGGCAACAAGTGTGACTTGAGGGAACAGATCCAGGTGCCCTCCAACTTAGCCCTGAAATTTGCTG
ATGCCCAC PKESQNVESIFMCLAcRLKAQKS 0
AACATGCTCTTGTTTGAGACATCGGCCAAGGACCCCAAAGAGAGCCAGAACGTGGAGTCC-ATTTTCATGTGCTTC-
GCTTGCCGATTG LLYRDAERQQGKVQKLEFPQRAN
cn
AAGGCCCAGAAATCCCTGCTGTATCGTGATGCTGAGAGGCAGCAGGGGAAGGTGCAGAPACTGGAGTTCCCACAGGAAG
CTAACAGT S KTS C PC
n.)
AAAACTTCCTGTCCTTGTTGAAACCAAACGAT.ATAAATACAAGATAAATTATCACTGGAGTTTTTTCTTTCCCTTTTT
TCTGTGcCT
CO GCATAATGCTGACACCTGCTTGTTTCCATAC_P.AATTGATATCAAAATAAAATTTGTATAGATT
.AAAAAAA co
cn
¨4
rn
0
Cl)
rn
0
rn
co
0
n.)
r¨
k.4
SEQIDNO . : 2 4
SEQIDNO . :71
GGAGCGCGTGAGGCTCCGGCGCGCAAGCCCGGAGCAGCCCGCTGGGGCGcAcAGGGTCGCGcGGGCGCGGGGATC-
GAGGACGGCGTG NEDGVAGPQLGAAAEAARAP...EAR
GCCGGTCCCCAGCT.
CGGGGCCGCGGCGGAGGCGGCGGAGGCGGCCGAGGCGCGAGCGCGGCCCGGGGTGACGCTGcGGCC CTTCGCG
ARPGVTLAP FA PLSGAAEADEGG
CC
CCTCTCGGGGGCGGCCGAGGCGGACGAGGGCGGCGGCGACTGGAGCTTCATTGACIGCGAGATGGAGGAGGTGGACCTG
CRGGAC GEMS FIDCENEEVDLQDLPSATI
CTGCCCAGCGCCACCATCGCCTGTCACCIGGACCCGCGCGTGITCGTGGACGGCCTGTGCCGGGCCAAATTTGAGTCCC
TCTITAGG ACHTZPRVFVDGLCRAKFESI,FR
ACGTATGACAP.GGACATCACCTITCAGTATITTAAGAGCTTCAA.ACGAGTCACAATAAACTICAGCAACCCCITCTC
CGCAGCAGAT TYDKDITFQYFKS FKRVRINFSN
GCCAGGCTCCAGCTOCATAAGACTGAGITTCTGGGAAAGGAAATCAAGTTATATTITGCTCAGACCTTACACATAGGAA
GCTCACAC PFSAADARLQL.HKTEFT,GICEMICI,
CIGGCTCCGCCAAATCCAGACAAGCAGITTCTGATCTOCCCTCCCGCCTCTCCC-CCAGTGGGATGGAAACA_kC-
TGGAAGATGCGACC YFAQTLH GS S HLAP PNPD KQFL
CCAGTCATAAACTATGATCTCTTATATGCCATCTCCAAGCTGGGGCCAGGGGAAAAGTATGAATTGCACGCAGCGACTG
ACACCACT IS PPAS PPVGWKQVEDATPVINY
CCCAGCGTGC-
TGGTCCATGTAIGTGAGAGTGATCAAGAGAAGGAGGAAGAAGAGGAAATGGAAAGANTGAGGAGACCTAAGCCAAAA
DLLYAI 5 KLGPCEICZELHAATDT
ATTATCCAGACCAGGAGGCCGGAGTACACGCCGATCCACCTCAGCTGAA.CTGGCACGCGACGAGGACGCATTCCAAAT
CATACTCAC TPSVVW11.7CESDQEKEEEEEMER
GGGAGGAATCTT
TTACTGTGGAGGIGCCIGGTCACGACTTCTICGGAGGIGGCAGCCGAGATCGGGGIGGCAGRAATQCCAGTICAT
MRRPKPKI/QTRRPF,YT.PIIILS
0
GTTGCTCAGAAGAGAATCAAGGCCGTGICCCCTTGTTCTAATGCTGCACACCAGTTACTGITCATGGCACCCGGGAATG
ACTTGGGC
CAATCACTGAGTTIGTGGTGATCGCACAAGGACATTTGGGACTGICTTGAGA.AAACAGATP.ATGATAGTGITTTGTA
CTIGTTCTIT
TCTGGIAGGTTCTGICTGIGCCAACGGCAGGITGATCAGTGAGCTCAGGAGAGAGCTTCCTGITTCTAAGTGGCCTGCA
GGGGCCAC n.)
CO
TCICTACTGGTAGGAAGAGGTACCACAGGAAGCCGCCTAGTGCAGAGAGGTTGTGAA.P.ACAGCAGCAATGCAATGIG
GAAATTGTAG El) CO
=ITC CTI'T
CTICCCTCATGITCTCATGITTGTGCATGTATATTACTGATTTACAAGP_CTAACCITIGTTCGTATATAAAGTTACA
tri
CCGTTGTTGTT'ITACATCTITTGGGAAGCCAGGAAAGCGTTIGGAAAACGTATCACCTTICCCAGATTCTCGGATTCT
CGACTCTTT
GCAACAGCACTTGCTTGCGGAACTCTTCCTGGA.ATGCATTCACTCAGC.ATCCCCAACCGTGCAACGTGTAACTTGTG
CTITTGCAAA
M
AGAAGTIGATCTGAPIATTCCTCTSTAGAATTTAGCTTATACAATTCAGAGAATAGCAGTTTCACTGCCAACTTTTAGT
GGGTGAGA.A n.)
ATITTAGTTTAGGTGTTTC-
GGATCGGACCTCAGTTTCTGTTGTTTCTTTTATGIGGTGGTTTCTATACATGAATCATAGCCAAAAAC
TTITTIGGAAACTGTTGGTTGAGATAGTTGGTTCTTTTACCCCACGAAGACATCAAGATACACTTGTAAATAAAGCTGA
TAGCATAT n.)
FT1
ATTCATACC7GTTGTACACTTGGGTGAAAAGTATGGCAGTGGGAGACT.AAGATGTATTAACCTACCTC-
TGAATCATATGTTCTAGGA
AA.A.GCTGTTCCCATGTCTAACAGGACTTGAATTCAAAGCATGTCAAGIGGATAGTAGATCTGTGGCGATATGAGAGG
GATGCAGTGC .. co
CTITCCCCATTCATICCIGATGGAATTGTTATACTAGGTTAACATTTGTAATTTITTTCTAGTTGTAATGTGTATGICT
GGTAAATA .. n.)
C GGTATTATATTTTGGCCTTACAATACCGTAAC_.kATTITTC-
TCATTITGAAATACTTAATGCCAAGTAACAATGCATGCTTIGGAAAT
r-
m
TTGGAAGATGOTTTTATTCTTTGAGPAGCAAATATGTTTGCATTARATGCTTTGATTGTTCATATCAAGAAATTGATTG
AACGTTCT
CAAACCCTGTTTACGGTACTTGGTAAGAGGGAGCCGGTTIGGGAGAGACCATTGCATCGCTGICCAAGTGTTTCTTGTT
AAGTGCTT
*-7-;
TTAAACTGGAGAGGCTAACCTCAAAATATITTIITTP_A-
CTGCATTCTATAATAAATOGGCACAGTATGCTCCTTACAGAAAAAAAAA
A A AW IA ARA AAAAAAP..21_ AAA
;==
0
SEQIDNO . :25
SE Q IDNO . :72 14
...,
GATTGCGAGCCAGGAGGAGGAAGCC GGCGGTGGC CCCGTCAGCAGCCGGCTGCTGAGAGGCCGGTAGGCGGCC-
GCGGTCCCGAGGGG
--,
CGGCGGCCGCGCTOCTCCCTGAGAACGC-GTCC CGCAGCTGGGCAGGCGGGCGOCCTGAGC-
GCGCGGAGCCATGAAGCTGTACAG CC T MKS, YS LSVIZYKGEAKVIILLKAAY 'a
CAGCGTCCTCTACAAAGGCGAGGCCAAGGTGGTGCTGCTCAAAGCCGCATACGATGTGTCTTCCTICAGCTITTTC
DVS S FS FFQRSSVQEFMTFTSQLCAGAGATCCAG Co4
IVERSSKGTRASVKEQDYLCHVY
COTTCAGGAATTCATGACCTTCACGAGTCAACIGP_TTOTGGAGCGCTCATCGAAAGGCACTAGAGCTTCTGTCAAAGA
ACA.AGACTA 4=
TCTGTGcc
P.CGTCTACGICCGGAATGATAGTCTIGCAGGTOTGGICATTGCTGACAATGAATACCCATCCCGGGIGGCCTITACCT
T VRNDSLAGVVIADNEYPSRVAFT
GCTGGAGAAGGTACTAGATGANITCTCCAAGCAAGICGACAGGATAGACTGGCCAGTAGGATCCCCIGCTACAATCCAT
TACCcAGC LLEKVLD E FS KQVDRIDWPVGSP
CCTGGATGGTCACCTCAGTAGATACCAGAACCCACGAGAAGCTGATCCCATGACTAAAGTGCAGGCCGA_ACTAGATC-
AGP_CCAAAAT ATIHYPALDGELSRYQNPREADP
MTKVQAELDETKIILHNTMESLL
CATTCTGCACAACACCATGGAC-
TCTCTGTTAGAGCGAGGTGAGAAGCTAGATGACTTGGTOTCCAAATCCGAGGTGCTGGGAACACA
GTCTAAAGCCTICTATAA.b_ACTGCCCGSAAACAAAACTCATGCTGTGCCATCATOTGATGC2-,'
GCCTGCCAGAGGCCCAATGCTGGAA ERCEKLDDLVSKSEVLGTQSKAF
TCGCAC CAT CATTCACATCAGAACTGCAGCCCCTGGA.AAAGAAGAGACAGCCATAC-
ACGAGGAGCr=AGAGTGGGGGCAGACTCGC CA YKTARXQNS CCAIM
TTTITATITTGAAGTTCCTOCGAGAAATGGATGGTGGAAGGGTGGCC-
AATGTTCAAATTCATATGTGTGGTAGTGATTCTTGGAAAG 0
AATTTGAGGTCCCCAAAGGIGTATTITTOGGCAAATGAAACCATAAACTCCGACTGGCTTCTGTAGATGCCAAAGGCCT
CTITTTCA
Cl)
GCTAACCCTOGGAAGGCTCTGIGGGAGGGAGGTCGGAGCCAGCTGTTICTCCATCITTGGTATATCTITGGATCTTATT
TGTACATT o
C
tv
CO
AATGATATTAACACTCCAGTGGGGGGTC-
GGGAGTCCCTGATGCTAGGGCTGGGGIGOGTGGAGTTTGAAGACTCTTGGGAAAGCCTC ...1
Cl) TCC
TGGGGCCACTGTTGGGGGIGGGAGTGAGCCCACCACAGAGGCCACAGGCAGGCCCCCACTTCAGGCCCAAGGC
CTGGGGCGGGG .. _ co
-1 01
-4
GOAACAGTCACTGOGTCTCAGATTCTGAGACTGTTOTTTAGCTTACCTTICTGCTAGGATIGC-
CTICCCGCAGAGGGCAGGGCCCAT (V
.)
0
G
o.
C
CCTAAGCAGCTTCCAAGTCCCACAAAGGTGGCTTGTGGGAGGATITGGRAGGAGCTGCATTGIGGGCGC-
GGAGTGIGTGGGTTGGGT cn
¨I
TCGTACCAGCAAGTAGACTAGGAACTGAGCCCAGGAA.AGGGGGATGTITTCCTGGTGTTTGGATGGTCAGCTOGGAGT
GICCATCAT
M
n.)
CAGGGGAAGATCAAACACAGGTGCACTCAGCTGCCCAGGGCCTCTOGGACACTTGCCTTGACTTGCAACTTGCCTTGAA
CATCACGA 0
Cl)
1-,
2
TCARAGCAGCAGGIGCTGIGGTCTCTCAUATTGATITTTATTTGACTCTGTGGCTCTAAGACTGCCITGA.A.CCGCCT
GAGGCCTAT tv
M
GCATcTGAACAAGTGGGTCTCTCCCTTGAGC_ACCAGGAGTGGOTGCCAGCCGGCCCCGAGGATTCCCAGCACCCCACC
TATGGTCIT I
o
M
-1
GCCAGCATAGGCTTGCTAGTTCCTTCTTGOTCAGAGGTAGCTGCAGAOGGGGGAGGCCAAGGOTTTGGTCTAAGCTGTG
CCCTGCCA co
I
CCTGGCAGGAGGCCCACTCACTGCCCAAGTCATGGCAACAGGCTGGAGCAGCCCAGGAGATGGGCCTP_AAATGTTCTC
GATCCCTTG o
X c
GGTCCTAGTGTTATGTTCCAGTCTGCCCACCTGTGCTCAGGATGCAGCCCTGGGATCCAGCACCCATGGAAGCTTCTGC
TGGGATGG tv
r
TGICACCTATGGGITTTGAACC_AGTGIGGTATGGICCTTGGGAGCTCTGCTCTGAGCTTGCCACACTGCTGAGAGCAC
CCACTOTCC
rn
TGACCAGAGICTCAGTGGTCCTGACCCCCAATGTGGGCAGGGGCTGGGCAGGAGGGTGGGGICTGCTGTGGGTTCAGAG
GACTCCAC
ry
cn
CTCCTGGCTGGTTTACCTGCTGCTGCCCATTTT CTCTGGGTACTCCEGGCCAGAGGAC
TTTAGCCTACCCCTGAAGAG CCTGTCCAT
.....
GTCATTTTCCTACTGCCATAGATACCCTAAGCCCAGGOCCCCTTG.AGGCCCAGACTCAOCCTGCCCACTGGTOCCGGA
GACGGAGTO
GAGTGGGCCTGGATCCGAGGGATGCTACCTCTCCCTTTC
CCACTTGAGGACCCTGGOGAGAGATGGGGGCGGGGAAAATGGAGGTAT
GAATTTGOGGTAAGAGGAAGTGAGATCTCCGCTTGCAGGTCAGCCCCTGCCTTGCAGGGCC-GC-
CTGGCTTGACTCAGGCCCTGTGAG *10
n
ATAGAGGGCCCAGCCCAGCCCCACCCACAGATCCCCTGCTCCIGTTGIGTTCTGTTGTAAAICATTTGGCGAGACTCTA
TTTTAG TA
-i
ACTGCTGCCTAACTTCCCTr2TGTTCTATTTGAGAGGCGCCTGTCTGGATAAAGTTGICTTGAAATTTCAAAAAAAPAA
-AAAAAP.A n
- -
_______________________________________________________________________________
__________
k=J
--A
0
0
0
kJ
...6
=
(9z 9-mH) 133HS 3iniusens
anan9n0o4392,nn9n.30nnon9n9009,annnn in
2,99,0n0 9.- CI H 0n09-9.3n9,009nnn02,9,02,no9,0 oi
na0onn02.002,n0.30(30annn.3>nopaponan o
. (.70oP ,.c)onaa.35,A.an,b,n6-)a)...- anaPP:'06)apna H
0 .--3n0.39(30.3n.--399.-309.3909-n0an.3n9innna o
an0nnnhnonnroonann=00nno.--3n9,annn.--3
9.30.30nnnnnn nn9-na9-02,nn2,9,-36)o6).3 ,,c.) o
paanr).,,,>a,,o, aap>6)6) a 0 0 0 a.. na a> a.3o6) =
nnnnnoci)oonanoonnnnon, narann =
a .- a anan2,02,9029,- 0 H 9.-nny,n "D, 0 0 0 n H 0 P= n n NJ
al
OH annanonanoo.3nonn00,3nHpn00.3n
-,06).,->c.,a,-3n.-36).-.3nacinnnaanolopn.-6,n
H b, x..po. 0 0 4) a. .4 Is Is a ,-, 4, n Is a 0 0 0 Is H 0 Is 0 Is 0 ),,,
--3nn 0 r) a n a a n o a .p.. a a 0 o o n n n a a n a a a a a n a a
2,.--3nn2.nnann.-390.00.30009,909,-n09-onnn0
n.-3n2,009,009.3.3n996)9,n0n 99,099 02,92,n9
nonnonnnfrInna.-3.30a0.309.00.3n90n0o0no
9, 2, OH 9. 9 n 9 n a 9 a n o n 2. .3 0 n ). 0 =., 6) oaaonaan
a ona ananon000nnaonnnob.nnnonno0nn
O0/000.3no2,906)6).-3000.39,n0ao0n9:0n2,0nn
9n02,90000>ono2,n0 ,.--3tapnann.-3 a> :0,- a4),.,nn
C) 0 y, o 0 o 0 0 0 .3 n 0 0 y, n n 6) n 6) o o n .3 n 6) 6) n a Is a Is a
onenonnn09,9,annnon9n.30)-30n509, H 0 H
D. 0 D. 0 H D. :D., 0 > .3nn6)e32,0.3 H n a n a a a a .-3 a ,r, 0 4) n
nr-32.0JJnonnannnonnanoonn .3 .3 n o n o a a n a
naoc)6),a0non2,92,99=0nnn09.- nan9pns.nno
.3 =,n)=,,,PP6-ipn,-3.-3no6)10'bnnaaa>nan;0.-anca
a6).-3 anao .- anon.3.-05,A0n6)n6)n0onnn9a09
C) 2. n n - '0 0 9 0 a n n o a 0 0 o .3 2. 0 y, J, 0 0 0 n 'p 9 0 n 0 a
oo,_3000y,,n9-*ppn.3aoap.-3ar)n
n :r. n n o anan .3 o a n n a a .3 a as n .3 o a a a a o n n .p.
naaann>nna6-19-nopon9n-309,-loon9 90
a nan:p.p .,.2,0n 91-30.32,09.9.9(3.300n0nno 0
nnanonaaanan, 0410000000).-3000ann0
06-)00:,.>a=,4.> anra).,nonnooann>nno>xi-n
1,
.:naanonn2,0,--3 nn0002,09,- ,-3)=. .-3.-
3.3..- an
annnnnnnanno naaannaa oonoononna
n=,(-)5.D=,anannnon,..6.1a>,..a,n):,,an.-iann
Onan09,-399.19.9.36)nano>apnPaoa:roao6)6)
C) - 0 Is 0 n r) 0 4-) 0 0 0 0 a n c) o a 0 a n a a Isb 00063.3.3a
no9n9002,00 -9na.--3onn2,9no9,anb, F-1.-34-).-3,-3,An
C) Is a .3 n H .3 Is > S). ),, > 0
P 0 1-3 ,-3 a , , , r ) ) 0 Is 15p a a 13 a Is 6) 0
=,.-3nonnvonapannoomnonnnoonoanna
n>2,0 n9 ar06.).too.onnoaaanann.bbabb.-ib
1 nn.ana r) > Jr,(33-nn 99 ..-36-)p> a aonnagb 0 I-)
n:0(,).-3annoa ,=,-6)nn 000nnoonno>n nna ,n
ant0(30:0.30oD,,na>o 6) nanbnaanna):,.3non
)nonp,..-3 .,nn6),--3.3 ,. .oc) b.a:l..-,on.-
3no-3()15,A6)
C) Is n n H 0 0 a 0 0 n n a n n 0 D. 0 0 0 .3 n 0 0 0 n n n n 0 0 0
no2,00a9-9n9n909nonan99,09,0n99.3nn90
99,32,(39,906).-3.3.3-3a)%.-1.-3na.-3>n,--3annon
anaanoannaannananonannaannbnpba
C)06)6) o n .0(300n2,00n0o0o2,a2,a3ann.-305,:'000y.0
*1 .v 0 0 4") rt 1,. nonnna,--30.3nno.332,10 up.3n9-on
ann.-302, Jr,nn9nn000(-3(30.3 9 nannn.30
=,ap,--3 6-1.3 aHnf-3,,-=,,,a,,PPaP) .3 a a a
.+- 6,6)6)
annpnn 0 ra 6) annonnan n n H n 0 C) .4 0 n 2, o n c-3 0
-39n 2-,n:02,nnn>onn9np63.-3.---39,0 00(3'002.9
:00n -,.-30-3.-3:0;0.-in>'.0pnap,0n;pna,.0,-3,-3ob'paa
nonp 0 .3 .3 nnoonnoonaannn00000n o anon
aaaacia),,a.-3)=onaa.36-)anaaaroa96-3
009 n 0 09,n92. a 0 .-3 a annn .-.3 .3 >, a a > a n H 0 0 P= ,,,
1 C) 9 n nn00n.30n.lon.3n2,nr3002,0nnn o (73'30
9.0n0n0n9o2,9ann02,009n2,0aa9-2,no G/n
On06-32,02.09.3-3 :09nn2.0J,,209,02,09- 0 a n x, n
nnnnan 0H0r00000000D.00H00000 0 0
oannaano> naapon ,- oPnnno - no2,9n0o
noaaPaa>41 >>5,,pnaa0 6-)c-Y.3PnP a> apnana
C) n a n a 0 0 0 r.,) 9no99nn a n a 0 0 0 0 0 0 (2, G) 0 :?=, 0 r) n
npa>c)06).nap->ana:,.-3.31-3nbab,annnynnr)
a)..aa:r9>'pt-3.-36)Pan.-3.-3n a 6).36,n)d .,.,a o, ,nn>>can
aaononao>nanpanannoa 0 (739- annn9on9n
2,909,2,nonnrn.06)aannnn:woon'ivbb 6-) > nno
.-3.3.36) a x, c) ;w 4-) > H' ..- on:00.3Paana
b.-3.3),a ann
on>noonan 0000:voaoaoaanannnononn
onatapnnov.-loano0naG)044.3,-Ionana),,
0 H )-3 a -,a>6--..,6) .3 0 0 0 :v H (-,, P' tap ana6....3>a
06) a a a a 0 0 6) 0 0 H 0 0 0 0 0 0 0 0 H 0 n GI 0 0 >pr 0 0 0 r)
O cl, a (-,) n n > n a a n ono>. 2,n2,0n9onnaaay,nnn
00no9.--3:0nHnana:t. - P ,,,:l.,,--3n.-3P>oantn,..--3p n
na=pannnnanna a:Nana/Dana =,..3.-)nonnnanP
aoa,aa:onnanb)Japo.na oona.3 ono ,,a6". , 0 0 0
> 0 .-3 Han'pa.3>nrinannal-3.3.--3n02.02,99.32.nn
nOn0nn00.3non90nnno6)na9J.9 ,-3 =,Øi-annan
- ________________________________________________________
Al 1.1 r z x pi CA 10 tn H 0 0 0 r a '4 tol p r<<
V 0 0 a on a < p 0630 r tri 21 r tyi z ,t, pi pi H ti < ,-ti tn V' Y...)
Cn .3 .ti .. H
tn H Pi 0 0 ..3 to to o ).. o zt x
z x 'to, toi :b' Pi H PI t. a to > a b
to to o x ..., r ar
rltitoiko<arNNtxtro.-3 tpit-tzto z
0 En 1-C t=i z to ..zt 41 Pi a . 0 Col En 70 XP P.HZ
tr1 õrt,, 0 0
C b toi tri Pi 0 H tC 70 )-4 hri N ,,i Pi i,
,-.3Hxf--iN < azx .1,,v] tri'xi 63.-4z63 .<xpqr
,-1 H 0 0 0) ED, M A htl H )0 1-4 trs n r n a to a H Pi co to
Pi to -4
H.41-1PdPotong .,--3, W CO toPNni orator ..1-ico6)xxl-Eit.18
,,,
61 <H H t-' to .--. 2, pi H li apoiroirtotri H <ro 63<663HatoitoN-4
ri n) H X
).) ).< H 0 +el H tn ,ro 0 D. < En H 0 Pi H 0 H =
0 ,r.-.3 ow..1 10
H . :.,q c=16-), Hi 0 tn En 64 6') 0 X 4 tn PI .< 0 0 0
H tg. H hi
'4 ;-3 ,o 9 ad hi i--. ,o rd n o I-I C,J < 'n 0 Pri I.<
0 0, 0 H < PD D:, t=1
Pi :4 x ,o a .- tr, a H )0 H P. tu En X ..n .3 to ),,,- ql G)
10 t, tn t-f t=i
<.4Hor a or, tIN =Z H b .3 7d1-101 1=1 t-I,vP;) Ai HcNn
ti
V) .3 t7 ,-3 > HHH )--4). H7J1-10C).ti
tianx.oior zz
z z o r r H Ea toi r n H r lo xt z to .- z rol o H z p .3 N X Cfl tl
0 1.i riPit-taa= .,=at-.ti-.Norz .3
7elt,00ct. r .1-tt.i.-ir x
r-Ixtqtiapitri x?.,colotaNxt4 o,xPz1 En 0 D=, 0 H ND 0 moor 0
0H0011:100<trIXH z Pi t.i w
-17 I-
Z1-060/LOOZ OA%
01Z000/L00ZVJ/Icl
ZO-80-Z10Z 9VOS8LZO VD
CA 02785046 2012-08-02
WO 2907/993942 PCT/CA2007/000210
125
[1,1 P `),
0 .4 H 01 a a,
g 1.71 H Q E'1, 2 HP4>-.00:rEo HEXION01-10M,-4>a) ;110
1.,..UEnE.H.a,a1 U/9.E.1.4...x0r1.,rot-I r:14
Q N
rCli col Evl 6' 'Eli Z' 2 N
Walt...WED.-4H '-
N 0 ;.. `j)" N '3 1 5 2 q Di 2J`-',
,n)g; .: ,(:), c,4 ,1 iu r' ., c' p 8 6
2
8 P. 2' ill 8" nF.. .7._
Cil E 2 3 P r.... 6 8
P 9 R RI
E4
ai
I E. HUUUHUU
U E..) E. < el 8 6 El 8 8 8 El 8 8 ..e 6 8 6 6 8 6 8 b' '6 8 6
8 8 L') 6' U E. t", E-, E-. U 0
g H u 0 F Er H F. i6 U(.UU ' 8 ,V, '6 6 8 b' .e)
t.", t.; E,-e' 6
UUU4 H00
U(.90,0 0 E.) E. 0 H u
E.
U U04:0 (<; rl. E. 1-' E.E' 2
4E-ic.2u C., .1: () C.) . 0 0 CJE.U0E.cieUt. H0000
0 HI V F i) (E 2, t - .7 , uE.0E.NUU0<E-.HbrdHOS,C?E-j'!'j
U U..Ale.,..1;"E-.'3; 8 .`' 0 0 6" E-.00E.HH
V. EC.) EET 1 rY' H 8 ' 8 8 E... 8." ..,- 8 6 E. E. 0 < < 0:
< H E-, < HO
U 0 < 0 < 0 < ,t.4 80,`,-e'',-
,i'ElaltLe) 6,t10.68,1Te:,,1 E--).
E.... 0 E. < E. E. 0 0 E. < E. 0 u 0 E. 0 0 (-. 8 8 6 8 '1 l E 4 .()=
;..0 5 = g," '6 8 8 8 0 8 0 0 < 0 0 U.) . Rp -..) 6. 6 -; rõ , ,
,. ,,, ,., 1 F. E. 0 0 E. U 0 < < U <
.-4) 8 8 8,te'888 El086168818 '.',-,'8.'6""..;
0HUUUHE.
U E. " " EE-_.,..,=6..E6.NE1E6-3,8,- ,
.9t-f_i
. = C.> E. E. U H E.-, UU= HU= O ., E. ' , , , u E- .
0 U < U 0 0 0 0 <
U 0 E. 0 0 < a' 0 < (11 E. < 0 gi, UUHHUUHHHHA
8 b. 8 8 . `,1i 8 ,, p4 u, [-_! u
0 E. A r; t-,¶0"6 0 i'-', b ;11
. 0 E
El.) 6 EE.. 8 ECD. (S. F.:0Ha,UU00,4,0 rt, U 1
E-7 EiC1 P. Er , E. -. -4' ) 000 8 8 f,-"q '' 8 8 r-, 8
8' '' L?
E. < E. 1,4 E-, U (..) = 8 866E86 EU' E. U H. 8El0888-itiu8
u u CI E-, < H p (.9 (D U E. E-. q u F. CD E-, 0 E. 0 E. 0 g C.) F4 p
k_,,, 8
8 8 8 8 u 0 E.-. u 0 E.- L=e u,:c ,H El _=),
F,' ,c? EP., `- =Z E.
888,08 t_') EY180.i.708.0E.00000E-,<OUUE.90,<
U u U U E. E. E. y g ei E-4 E,r-t. E,'5, Cp t1,4 0.
4' 8 8 Pe 8 n (V '1.
U r..) 0 0 [.. E. C.:.
U . 8 r-% 8 ,t,l, 'd 6 P4 ', * (E?, 8 P A F, ,$) (E2,
r(-4)
U c... g ., E.
E. 0 0 E. 0 E. u 886V-D,rl.`-'8H6,6",te' 68$'6",e88
u U E. < < E. 0
E.U0H0E--,< 86:80186808,e8i-,8 Elt;86886
E.E.UHU<E. 0H000,0u0U00UU0OHL.90 cp g (.../
u cl C.) r, C 7 = U CI
O 0 H E. E. H (9 (9 "E'ElE16%-lEt;ii8
66[1 6 ",,C, t;il `1 :t
8 8, 8 8 ,'el 8 R i.i-) !4._: Eq'4 s'-.J.) 0 < < 0
H U U 0 0 0 4 0
L.)L.... U Pa; L-, t. 0 E.. <PH .o.q c E.E.E.000 = 0,
E. E. E. H E. H
i-j (.;rj, 6 8 2 8 6 8 8 WV ))0U 8 .18U 6 '0" 0 ',',
u E-, u E,E.E.F.
<UU<HU0 E:). Hb' El 8 r4 0 6 6V661868`8E,,[483,
E. u E. U 0 0 E.
00<<E.E.0 8 6 1E-4 8 8 8 8 ,Ee" ("- 8 `,= 6 6 u 8 8 ''c 8
E. : r 4
< u E.E. u E. g H 0H E. <PUUK.) E.C..)
HHO00E. 0 HU
OCJOUL)E.0 0 0 0 0 0 r.-) E. u F=C 0 F. u c.D o o .1. g u u E. Kr q
8 8 8 t -,' 6 8 i s..). c? EC-; t P :<( 5 , .5E- ' 0 EP, 59 ((i 'k' 'd
b' i"-: gi 8 8 8 8 q 8 ',i
u 0 C.1 E. U E.
UE. 0e. FA:U L--,-. 8 El ,E,-" 8 'Et
O 0 0 0 E.
H0000HH 00<< HARI. E,:,-. (i.) tE....% E, .6. ,E,-if, E-
-,, cc.-= 8 (,-_,., '.g.
00UUUU Ft: E.' ti CD el r2) (t=. CD E, c.)
E. g 0 (.3 cD 0 E. E-, H EA
O u y < U E. U ''' u . ,I= p= c '
0000<00 0 cDUE.,<U0
6 HD 8 g t 6 "i-9 6 t.>" 8p
0 u E. E. 0 H U E. u ,i, E--. < E.
< E. H < E.., 0 0 A
8 8 8 8 8 8 ocu<000[900000000UOU g
u 0,
OU 8 8 8 1 8 El r. h9 ri E,E, 8
0E.E.
O= 0U 8 8 0 8' (,: E;e" ,e 6
`,'e 8' 8 8 `,e) 8 ?-.. 8 E;e1 V: 6
c., E--. g (9 g E-.
CJOCUHE.
E,E,,e0E.-,, 8' 8 8 6, 8 6 8 8 6 8 ., 6 r," ri 8 0 8 8" 6)
6
) tj 8 r." 8 8 " < 1 " < ,9 " 6 5 tl 8 1 8 6 . 8 8 8
8
O000HHH s '4" uPu H K.
...)z) <U00000
8 8 8 re ' 8 EC',
I `'. b' 8 8 8 8 8 8 8 '
U u u L) H H 6
(Du 8 6_, '6 QPI: 0 r 8 6" 6 `c-,) 8 CE.-.7 6 8 6 6 8 El u 8
8E, .)CE_, .
000 PPE. H OHHOOUE.0< 8 E.- p., i. (F_D 0
H U U E. 0 U 0 E. (W.' 04i:0 0
(C) 880PeEl88 rii
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
WO 2007/093042 PCT/CA2007/000210
126
H X a H >.;> z z
H g a o w ul w p4 ;=4 (:)1 rK al z a
a x 0 cn
g ,-1
n....õNp
gt=21E--,owz a, a al
1:14 T4 H p ral Z A z ,-1 X rc. E. E. a Z
E. a r4 f4 En n, z 1-1 0 En 0 in pi ,--1 fri z
,-.4 z a Cn C/3 0 E. Z 0 0 Si 0 fi= cn Er] p
HOO N Ln ri-4X01X>Ent.DEn>
01 rri > x 1.-
a OHH R - pHarlac_9>Ø-1
,-1 X C9 q 0, q = 0 a 4 4 ,-.1 W X E, }-1 C4
E. P E. En ..4 A 0
X 4 X Z 0 Z
C.1 En f4 a, H 1:4 n 01 >-, 0 4 a H pi X E4 cn
CI Ex. 14 a >4 fat H XrdcncnzMar.n..1.-10g
a q r4 0
En ac q P-, 0 01 :4 E4 a al Ea a g A 0 > La z
t4 cn 0 09 PE4 la 0 H
4: 0 Z E4 GI crE
U E. E. E. E. CD4HE,<<OU40004H440
U 4 c9UONHHHUgUHFH
H u ED U E. 0 u CD u 0 u u E. 0 E.
E. 4 E. H g OHHHUUN H4E.HEDHHOE-,
H Ft CD 0 E.
H u 0 0 g E-.0000(90H0c.DE. CDE.OHE.<
u H H a; g OUP NOHuOugOEHUE.gH0
E-I= ggUE. E. u o 0 U 0 u 0 A U F. .6 EH 0 CD 4
UOHHH
CDOE,E.H u 0 a, H u U E. g U E.
U U H U H U U U u 0 E. 0 0
c...1 E. u 0 0 0 0 g 404 E-1 E.
4 U 4 H HOUF4UH4E.CDOE.OHEAU 0E.
E. E. 0 5 C9HUUgH uguE.HE-ogy E. at
coq 4 4
t g (-1 g F. 0 t..9 C.) 0 F00
0 U 0 H 0 H 0 g u g g
0 E. 0 E. g UUOUUNgE-.0 UHHHEHHOU
E. g U E. A g E. g 4 g: ED g (.1) C.) CD E. u 0 g g
E.
0 0 0 ci E. tH E, u U A d H Eg E-, E-,
E.9 E. u ri,
E-, E, 0 0 OuucDCDCDOcJa,a,004 ,,;0
U 0 U E. E. A E.E.UULDH40H
0 0 U fa: Kr: HOUE.E.E.E.E.00 E--.
u U H A H g U H .,
1= ": U 0= 0 UU4E.OUOUUpt0E-,
UPC 00uUUE-,HH44 V. E. E. U
E.../ U g UQUAUOUE.U00A H4HE.
gc9C9UF1,000:000gH ,AE-,ci
UHUU 00A gq0c...90,,,,IgHHAa.OH E.
E.HgEHU U A U 0 U g g A U 4 0 E. g 4
u g u H Ft OUE. 6,ANg ONUCJO,AE. E. H
U U g 0 g 0 Cy E. 4
c.910U/gE-.AgUONON
4 U E. H 0 E.HHOOE. Augu0E.OggH 4
E. UE-,4p,4E-,4 UHAEHEHAHNE.C.../0
u 4E.400 HE-,u40<0 N E. 4
UgHg A 0000 E, E.H0gc..90E-.00
Ougul. 0 0 U E. u u E. 0 E. E. EH H
HUH0U1Uc1HHH
UUHOUUH
U Ci g o c.)
c..) E. E. 0 < HNO0UuHUHAC9E.ga,C9 0 H
CDU4,HE-, HUHO
u E. E. E. E.
UOCDE,E. c90E-,0C9UOE.OAHHc_9gHHOH
uHEHHE, cic_90AgOc9000uUE-.0HE-4r4
0 A u E-, 0
0 A u al 0 o E. 0 g u H U H H 0 g E.
gg0u0 000 0000E-100HgE.E.0 gE-,
c.9 u g g H C./000AEHLYUc9u0E-, g E. g
OHuHU
g g E. 0 a; a.,E.Auu 0HguuNgUE-.00H
000 H. A 0 E. H u
E.U05HgEDUE.00d0g00
= g 0 A g 000H(9000,ACDHHgE.A,Agg
000 U E. EH E. 0 u A C.7 E-, N U E-, E-, U pc
q E. A A u OguggE.E.HH0HUogUE-,gH
AH0H0 U0E-.019HOU0U0,110U0gHg
0 E. E. E. 1 U U u 0 E. E. (.9 g E-
. H U
CD E.', CD ED H OUU,AE.g U01..90Hgg0 4 4 E-,
E. E. 1.9 g A UUUOU0 HgUOUNOE, E. 4 4
HHOH u g u E. La; 0 0 g u 0 E. H E. E. E. H 0 E. 1
0 g H u 0 E. g A 0 H U E. Pt 0E-, < I
.=-1 E-, E-4 ED Ci C., E. H (.7 U 0 a; H c) E. 0 0
E. H E. I
co
OHUHU ru OOHUUUHOHOH ac I
E. U U 0 0 = = g g E-, 0 0 E. U U U g A E. H 0 A g U
U
U u 0 H EH
0 044E-104CDHa,<H004<0UU
Z. UUAL9E-.4.0000.guHH.a;HE.g
g H 0 U c_i 0
u.__,UUUUU0c9acCJEDOUH U
H
H 0 0 c.., g 0
g 0 g g g g r4 OuguHuguU0 7gE.c-)0C.91-.0
cn
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
1 27
Cr4 S4 i,..1 E al E. I4 CD
PC CI 0 CI (11
H = re) D/ 0 4 cn RI '04
U CJ Op
U) D4 a .4 4 cil C11
Z 0 Z C4 DI CD E./
E, n4 2 0 E. Z ch a
X !-I LE. .1 CI X cn C4
> H DI .4
U) C) ,
ID
r...
ts1 , F4 6 0, tc A E
= c,1 c) . cl, C) cn H H C.;
C> HK,..1[401.4C10
Z 0 >. iZ rs) 4 4 rs1 2
A 0 DI Kr)
H
a ca.U01.1:4tagcn
rsi rs, Fs, tr) ',>
U)
_ ___________________________________________________
Uuu5C) 40EDUU E. E.E.HUE.E.H05E,E.H.45HE.0 f .4
L) E. I. .g cb c) kDi.-. 5HOHHOUHE.H05H1.55E.E.E.E.H
u u e.) E. 5 0 1-, 4
L) C..7 (.7 50000E.4,0Upcur.)0.4000HHOH5H5E.5
r.J U E.E.)044 rJ CJ H C.) / a' H H
6 0 CI E. U g .4 E. CD U
C.) EH E. E. E. t) 4 , 4 0 .4 E. / E. U 4 E-, EA EA
E. E. 0 C9 .4 E. 4 U
0 HOH0000,4505E.E.OH<H050.4H5.4E-1555 1,
UUE-.0045HUE.54.E.5H0H5,400-5555HH, u a
U)0 0 H E, 5 5 u .4 E. 4, 4 E. C) CD E. 0 a 0 4 E. L) CD E. CD E. a 4F-.
E. C) .4 .4 C) 0 CD C.) cD c) C) E. C) a A E.4 U E. CD .4
c) .4 E. E. C)
0 0 4 U 0 0 5 LI E. H (J 0 a: E. E. E. u E. U 5
0 CJ 5 0 E. E. E. H
4. 0 I. 0 E. E. CJ H 5 E. 5 1. E. E. H U 4 E, t.)
(.7 f4 E. u H 5 0 E. 5
0 U 0 rt E. 0 E. E. 0 u u E. E. EA F, 5 (9 0 u H 5 0 E. H U rai L.7 E. E.
H'-)a,
00 H 5. 0 0 .4 0 5 E. / 0 0 0 5 F4 g 0 E, H H (.3 5 E. 5 H U ei E.
O 0 5 H E. 0 CD<Ha H0r<cDUE.HaV.,<H4a 4 u H 4 4
UUAU 5 .4 4. ED0 4 0 u u E.C.> 5. r.) 0 u C.) < u 0 H < E. a
CD CD 0 C) 4 U 0 Pk C.) E. 4 E. 0 E. U FQ E. 4 0 E. C) 5 E, CJ
= 0.1
O C) 4,, E. ,4 0 0 E. CD
C.) 0 / a 0 E, A E. E. U 0 CC 4 U A 0, E. U 0, 9
O (i l'1,4 6t U U H 0 E. 5. ,4 4 .5
g Fl. 0 E./ CI 9 Ply A ec 5. U 5
0 CD CD
CD 0 0 4 KC CD 1, 0 E. ,4 i-, E-, (7
CD C) 4 0 0 t9 U E. E. CD CD A U E. () .4 F. E. E. E. E. A 0 E. E. E.
E.
0 (.) ,4 t-i E. E. U E. E. C.) E. C9 E. E. E. CJ E. CD KC E-,
4 E, 4 E.
E U 4
0 cD to 0 0 cD E.1 E4 E. 4 .4 E. E. E. u gq CJ 0 44 0 E. E. C) EJ C..)
Ch. E. CI cc
O Ch 0 E. CD U U ED c) PC CJ 0
E. F. E. 4 E. C9 0 4 () E. CD 4 4 V. 4
CD .e
C) 0 4 E. 5 0 E. . E. 0 0 U E. (.4 54 E. a C) .4 H H 5 E. 5,
U 0 0 E, 4U C) 4 EH C) C) Lh 4 0 C) H U u 0 5 (I E, t) 0 0 E. 4 CD E.
CD U E. C) 0 0 u U .4 H E. E. 0 E. CD E, .4 E, E. E. t) E. g4 U E. E. (9
a
g U E. g F, g g Kt 0 E. U
cJ H E. E. H E. 1 a / E. H .4 F. CD 5 E. 5
U) E. E. ED U () c) U E. 4 51 E. / 4 u 4 E. E4 E.) 4 E. 1 EH 4 9
0 0 0 E, CD () F. E. E. g V H C/ KC E. F. E. ,4 C9 0 E. .5
CD FL
CD 04 E. E4 0 CD E. E. t9 E. E. C.) 0 CD 0 1, E. E. E. C9 tJ
,..c E. 0
CD 4 .4 4: E. t) L) .4 .4 .4 U Cl) 0 4 .4 E-, L.) t) Kc E4 .4 1 4 C.)
C..) 4 E.
0 gl, 0 IJ 0 E, U 44.4 Ch Pt El 0 C.) .4 C) 0C C9 E. ch 0 E. (9 (D
0 (D
CD L) a 4: E. g 0 0 <
, t, 0 a E. ch 4 Cl) 0 I, 4 F. .0 E. I, .4 E. a
E. E. t) E. t. 4 0 E. A
i
(9 .µ4 (9 E. 0 U 0,4 .4 a. 0 0 FC CD CI E. E. A
E'l H 0 0 E. E. t) E. E. A
KC 0 H U E, KC Cl) A U Cl) E. 0 U 0 .4 04 4-4 E--, Ei 4 0 ,4 (.1
F. E, .4 E. E. 4
CI E. 0 E. E. rC 4 0 E, E+ U .4 E, eC 0 CD P4 E. .4 C) E. E. E., 0
,, E. C) E. E.
CD LD CD F. gC CD H ft El U U E. (.3 PC Cl) .4 (J E., E, E4 4 Ch 0 e---
, A pc () 4-, 4,
c) 04 E. K: 5 E. g rg. g4 u 4 E. U c) E-4 f. E-, E' E. H E. H CD a H E. .4 U 5
4
CD 0 0 u CD 4 0 d Cl) H L. E. U E. CD E. E. H g4 E. a < E. t) u E. E. E. E. 0
0,
0 F. U 0 ED a u E. .4 4 C., .4 E. a U E4 E, E.-, F. Ft El Ft 9 E. CJ F. U
..4 E.
O E. 0 E, ,4 ll.0 E4 0 E.
0 E. E. E. .4 .4 0 .4 4 E. CD .4 Ch f,.9 ,.-oq E. E. E. E. 1-4
ED 0 E. u E. CJ E, E, E. CD i, E. (..) .4 .4 0 u U .4 C.9 E4 E. a E. 4 U Ft
El
HU0H5E,H um:4 5.UHE.E.05.40 -5E.ciE. E-I U E. E, Cl U E.
CD CD F. E. E. E. 0 E, CD c) u F. a t, 0 E. 0 E. E., PC .4 t) 0 .4 .4 .4 E. H
E. E.
U Ci CD C) '' E". C,. U .4 CD .4 C 0 E., .0 I, .4 gr, .4 CD gl 4.) H.4 H 0
44 E.) 4
,4 CD 04 Cl) F., E-. ,4 E. 0 E. H, 4 4 CD E4 CD FN PC CD 44 H-4 E. E. HO 0 HO
E.. ci E. c9 4, F. E. E. .4 C) o,' C. a () a E. .4 a E. 4 0 Cl) () V E. < E.
Cl El E.
() (9 4 CD .4 C9 E. CD E. E. 5 CD CI C9 gC U E. Ch E. CD g4 E. U U U 40044
CD C./ 0 4 (7, f. E. 5 4 C./ E. H. a 4 C) 0 4 .4 CD g4 u (9 E. 0 E. L9 C) .4
.4 0
c) U E. eD Pi, CJ U 4 0 0 ,-. ,,,t: a 1-, E., E. 44 () (.9 0 E. 4 g g th PIC
E. 0 u 4
C.) 0 E. CD .4 0 .0 E, E+ .0 PC E, .4 (.7, g4 .4 0 F. .4 C. E. E. C) 0 cC 6c
.4 pc c3 Ch
Ch (di g ...q u) < 0 C.) 1 C) E. E. E. a E. E. H E. U A E. E. E. C) FL E. Ch
cC .0 c)
0 CS K., g T., L9 g g Kt E.-, 0 PC CI U PC
Pt E. a .4 E. F. c) a 4 .4 E--, t) g
(D () C'D '4 , (' 0 E. Ci E. 0 E. U .4 E. U 44, .4 E. E. .4 g .4
E. E. U c.)
Ch E., C).4 E. 9 4, 04 E. E. E. C/ C) () E.
.4 a E-1 E-, 1,-, CD H.4 0 E. E. CD F.
c) 0 0 u cn A4 u U)'1 F. .4 E. c1 .4 .4 u E. E-1 E. u E. g 1-, E. ,.4 0 .4 E.
CI
Ch U) C) Ea E-4 E. (9 0 a CJ E. < FL A E. 4 4 CJ Ch E, 0 E. E.
E. U < E.
CJ U 0 E. 0 C) a 0 E. c) 0 E. E. 5 4 E. E. 4 E. E. E. a E.
CD 0 E. 4;
Cl) t) CD 4 E. F. 0 0 th / 0 H c9 LA E. 4 (9 E. F. E. U E. E. E.
E. L) 0 a
01 0 C .4 C-9 CJ 4C C El E. a 0 E. () 0 F. E. E.
0 U CJ E. HC9 cC +C gC
N CD 0 0 CD 0 U EA LI C) U 4 Ch c9 E. 4 E. .4 ft E4 r4
c) 4 E. CJ E. E.
C) 0 CD .4 E, U U E. 0 r4 .4 E. E. CD / 5 .4 E. CD CD E. 0 E. 04 u < LD
= CD (h .4 E. .4 E. c) I. E. .4 E. C5 .4 E, E.
E. u E. C.) E. E. E. C.) E. rg E. rJ E.
6 C) 0 0 0 CJ H E. E. 0 U).4 El 5 0 CD u CD E. u E. gf E. E.
E. H.4 .4.4
Z CD U 0 C) 1 0 C9 0 CD E. 0 .4 U E. .4 0 F. .4 .4 0 .4 0 E.
E. CJ 0 E.
Cl ,, 0 C.) E. 01 ii E. pc a .4 E. 0 .4 0 0 C.) E4
E. E. E, E. 4 E. CJ 1 CD
H C) 0
0 .4 c) 5 0 4 E. E4 Ch C.) , E. C) 0
E. 0 C5 CD .4 .0 al 0 01 E. a E.
ril 0 0 E, 0 E, cf 0 u p< 5 v4 E. 5 E. eD c) 5 g4 E. f. 4 U 0 04 rC 4 E.
L., FL c) _
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 20071093042
128
, g N al > 01 hi t-q 0 a, __ p4 44 0 44 (0) al 01 tn R EA Z 1,4 ti3 74 4 fii
CY 7-
U.) 0 Er, (-4 4 z 51 CD CD, 0 fl, g 0) E, r4 CY 0
014 Z pg, 4 74
0) I-. rl .4 2 r,4 H H 4 HO 2 6 c,2 .r: w P 4 P N ,.1 ',-?,
f4 P
Z 0 7
04.p.4c5r)0.0y04,-i uenci.-107E-.44y3 eip ,g0:441[01(30
ff, gi m m gl g 1
g (n P, FA e4 c, c7 O4 0 1 4,2 '1
E. 4.1 C)0 P1 CD 4 .3 C4 g ph4 w pl c.) .4 0
V4 i-, a. i-4 74 7. 4 04
',1 1,:,1 '2 2 P R 6. ,Y, ','e: ..'," '4 g r, t=,,1
F:,,: '("l ,?,' 8:
.. (, E. E. K HZ OH g3 ,. ,-1 i9 5 oa u in pt C7 P. E.
HO 8
6
al en 0) 0 > z a, 0 c4 01 G-I c. C.! La P. ta Pi Z P4
C) (:YE--vrgp1;,4, xa. P ':', VI Co CA CI CO olxAzxgg
1-4 til (4
E. .4 ;4 0) 0 z 44 P.; (9 r' 2 01 ?4
ka e4 Ch Pi CD Pi C7 era En VA p4 X .t ,1 y, _
CC i 11 t
cn X Vi 0 .4 cy 0 4c Z A LU Fl 1-4 ,4 0 Z ?A 0 0 Ei a. pl fil E. 01
__________________________________________________ _
0 (5 CT t9 c9 c) gS g 4 CD CT, ci g4 CD El g
0 E. E4 U 0 U E. ,. .4 0 E. u (4 (5 4 gt c) U .4
44 P4 kJ c) 0 t5 44
E4 c--) Ke, L, CD CI C/ 0 (.., 0 .1, 0 C-4 0 0, 0 0 A CD c.) 0 E. ti K4 0 U g
g
0 t; g 3 59 r, 8 t; ,i): ,'-s' g 6 ES N E4 ';(1, `4 V, 8 8 4 ES
( i94 p4 0 D il ri 8 il 0, H =,,c H 0 0 0 9 c) ci u C) 4 E.
E4 (9 u g u (9 H .4 (9 PC U CD, U fa: E4 Al, Ali 0 AC)
, [1 '6 t"), E7,e' q [:', ',q1 ','cli F, EE i?, 1
El N [--: 8 t-j ,1 8 8) E ,11
(3 (7 0 A Ch C9 (D Cõ) CI Ch Ch. C.) (D gl 0 rC ,C,.?, Ci p, () E4 C)
4 ..-,
[5 ,T. E1 8 '6 U , () 8 ,V 'A N' ', ';;4! ES ES El El fl'. V, El i4 H q 6
E'
6 8 ES 8 0 ES E3 ". 8 E4 6 El El N 8 1-4 8 El 3 t4 C? f-J'
9 P, cry t51 ,, ,,_3. 1 tl t-D. * .1 6 8 8 8 8 õ N '4
f5 6 '4,)!.
,, ,.-- ,- ,-.. ,,, ,--. ,-, ,c, E. (9 E. E. E. 4 .t E4 4 4 E.
44 44 44 E. A, (J E--+ El
C5 CD u E, 04 E, 0 FF (.7 u E. 12 1 pg., U r( 0 r( 4 ,. 0 L.) p g p (9, E.,
0 0, E" 8 0 ;l E4 8 8 ' 4 'C I'S 0 il 8 E?, E4 0 6 '' 8 ES 8 ES Ea
'A g 1 t-r-i' * 0 8 1 8 t.:: cfl (VII 0. P.0 L55 (,-4. cl 8 6
',..:-ii i--: E
,
E, E. 0, C) 0: 0 E. E-, Ch 0: LJ 4. 0 rr 0: 0 0 E, rc 0 (4 E. ,4 E, (2) ,,, E.
,,, ot
8 0 8 i-9' 8 r.;i " gi 8 8 ',:' (C.)) Eg--,C g: ,-,c' g:9 8 :-T)
E:1 2 fC' 1--:
g .,1, !-.4' 'icli 4'-' 'g E4 8 g t,'. Li 8 ti; E.)4 t--_,µ g, 8 L9 E; g !;
[:: t; 0 (1, ,E,t' ,.
u E. E. E. 0 E-, .4 &I E-, k-, 4 (D (9 F4 () re 0 E, ES ,4 A '.1/, 0 (h E, E.
0 -.... Al
'E.1 .( g r. El ,V1 8 8 I'D' E: 8 8 g g trj 1 g 8 8 )- ',,',.
t; 8 2, El 14 21
i(9 E.) 0 I-, Li CJ U Ch E. 0 u CD Pk E. iii t,) Cu. .4 E-,--4. ,6 ,õõ:
:õ9, 0 Eg 8
l 8 `4": El E5 E,'t 4 r) ,`µ,, 1 5 1, H 0 E. U t9 Z1,9
0 ti p cn
1
8 E u V,' P. ',', 8 8 8 El ,,, 0 '6' ,., 1-4 N ES 4 8
[l 8 El
* Up (4, -'4 [4 8 'E1 E:' d E: 8 E, il t2, 0 8 8 0, ES
8 0 El
1
CD E. u g E. U E. py (9 (D o< E. ci E-, C) 44 H g g g 0
44 E,
E--, H 0 co E, E. 0 ... g .4 0 Ch r.) H El H c5 E. E. .t AI; 0
E. V E. ri 0
F6 r-, (1 gi H Eg- 0 E9, 8 E,-,' 8 u ',,- '2,. 8 -
_1 s 1 v, il
õ õ õ õ ,, õ õ ,, 4 õ E. 44 c) E. t.) Ch 0 0 CT
04 E--. Ei (9 0, g
(9.(D " EC71 g"1 " ACH 8 '4" 6 :). ",,i1 6 t; ,":- 6 U t;
6 [5 tD r,
i
g'; L9 8 6 6 0 0 gC f-f 4 0 g E. (9 C) g
0 U E. 0 c5 EA 19 g
gc Ef gC 0, F, E-, H E. 0 E. E4 4 0
E. g E. E, c, g (9 H CJ cD gs (9 (9 E.
2 8 (,-:4 5 El Eg ,'-' 8 6 -!, ?) E4 E4 N E,, g El
0, t; 'A ES 'i g
ES 11 8 8 Eõ ',,'-'4 El 0 E: 6 El 6 (., El 8 El E,
0 El 6 El ',,-.4 i4 0 6
6 il E4 tri 0 ',,- E;-, F, I?, [?, g N N 0 El N P, ',.-
NI 8 8 il; g E4 El
H HLDHHHHU<UHH Ca H 0 Huu.Ku4p.[-,tciusrt..-2,b6
,
1.-.:4
kJ (9 PE, F, E. A CD 4 0 xl, 4 0 U, ,4 ..' 4c 0 Oc E-, E.? c7 0 E. 1 c) f,
c) gs
,t- 0 / cl 8 8 6 0, 8 8 V 6 '4 F.: E., 8 El 4 1 N 8 8 i f5
E_,E, 4..E.r.,,,,g(90_0004000, g 0 g d, g 0
H 0,,
1
il
8 E4 V. El 4 g 8 8 8 E4 V, 8 4 8 El u g 0 8 E, El 8 8 ',,i
0 f" 6 8 ES 1 8 g E4 8 8 6 () 6 6 CD E6 6 t; c=-ii i.) U
[..)) 8 E. n C) Eci'i ,1 'a 8 6 E, ES (4i ES ES g, ES 8 ES I:: 8
E. 0 E, 0 CD g4 0 E. 0 gt u c)
u g 0 E. ps ch. 8 c7 g, cõD, c..3 c_.õ) (y, t5 8
E. E. 0 E. (J E, E. F. F, r g y t: El g t.,. E, 6. u 40 4
..-..3 c,
E. CJ E. C.) F. E. E, V CD U N., A 4
U E. E. 4 04 E. U 0 0 0 E., KC a, E. 4 4 .,µ ,E1 0 t,i tl tt..-; cl
Ec,
tr:!õ i...1 '4! ri 8. 6 8 8 6 El 8 66 ES 8 ES 2(i ', (i g E.
E.
ts r. C., ,4 a LI l..! H 04 4 U U u Kt g4 .4 44 0 41, 0 A pc gs (9 E. E. 4 E.
2 8 Ig." t'D t--1 ,'=.4 8 H EE:" tej 6 5 8 R ",-4 -*)' El
E-,' 0 0, 4 8 8 Et: 8 0 -2.
.. E.-. CD E. (J CI CJ CD 0 E., I-4 f4 ,4 g4 c, 0 () E-, cl () ,4 0 k-, E. pt
c? E. c) cD E.
1 H g(6 _ õ ,. õ. , õ,_
91 E, U.C) El (9 E-f CJ 4 E, 0 C.) ,S4 E. E. 44 . y.D
t:,., t::: .02 ci-õ, , _ .s.? r.-õ,
C) kJ E. E. 44 E. cD E4 CD 0 CS ki 5s 5s 5s s9 il t7., ,a ri,
Lc...), D ;..f;. ,,_._ 7,1!
1-4 L) g W (2) LI (--1 () E-' 4004 Pi 8' 8 8 d E. 'A 0 1 E. 0 CJ= 0 H
C5
O
1 V. g E.'.!).!
SUBSTITUTE SHEET (RULE 26)
SEQIDNO . : 3 1 S
EQIDNO . : 7 8
ATAAATATCAGAGTGTGCTGCTGTGGCTTTGTGGAGCTGCC.AGAGTAAAGCAAAGAGAAAGGAAGCAGGCCCGTTGGA
AGTGGTTGT NWRSLGLALALCLLPSGGTESQD
GACAAOCCCAGCAATGTGGAGAAGCCIGGGGCTTGCCCTGGCTCTCTGTCTCCTCCCATCGGGAGGAACAGAGAGCCAG
GACCAAAG QS S LCKQEPAWS I RDQD PlvILNSN =.c
CTCCTTATGTAAGCAACCCCCAGCCTGGAGCATAAGAGATCAAGATTCPATGCTAAACTCCAATGGTTCAGTGACTGTG
GTTGCTCT GSVTVVALLQASITYLCILQASKI,
TCTTCAAGCCAGCTGATACCTGTGCATACTGCAGGCATCTAAATTAGAAGACCTGCGAGTAA.A.ACTGAAGAAAGP.A
GGATATTCIAA EDLRVKLICKEGYSNISYIVVNHQ
TATTTCT TATAT TO TTG TTAAT CATCAAGGAATCTC TTCT CGAT TAAAATA CA CACATC
TT1tAGAATAAGGTTTCAGAGCATATT CC GT S SELKYTELENICVSEHIPVYQ
TGTTTATCP.ACAAGAAGAAAACCAAPICAGATGTCTGGACTCTITTARATC-
GAP..GCAAAGATGACTTCOTCATATATGATAGATGTGG QEENQTDVWTLLNGSKDDFLIYD
CCGTOTTGTATATCATCTTGGTTTC-
CCTTITTCCTTCCTAACTTICCCATATGTAGAAGP.AGCCATTAAGATTGCTTACTGTGAP.AA
RCGRLVYHLGLPFSFLTFPYVEE
GAAAIGTGGP.AACTGCTCTCTCACGACTCTCAAAGATGP.AGACTITTGTAAACG
TGTATCTTTGGCTACTGTGGATAAAACAGTTGA AIKIAYCF,KKCGNCSLTTLKDED
AACTCCATCGCCTCATTACCATCATGAGCATCATCACAATCATG'GACATCAGCACCTTGGCAGCAGTGAGCTTICAGA
GAATC.AGCA PCKRVSLATVDKTVETPSEHY1111
ACCAGGAGCACCAAATGCTCCTACTCATCCTGCTCCTCCAGGCCITCATCACCACCATAAGCACAAGGGTCAGCATAGG
CAGGGICA EHBHNHGFIQHLGSSELSENQQPG C)
CCCAGAGAACCGAGATATGOCAGCAAGTGAAGATTTACAAGATTTACAAAAGAAGCTCTGTCGAAAGAGATGTATA_kA
TCAATTACT APNAPTH PAP PGLEHHHEEKGQH
cn
CTGIA.AATTGCCCAOP_GATTCAGAGTTGGCTCCTAGGAGCTGATGCTGCCATTc.;TCGP.CATCTGATATTTGAAA
AAACAGGGTCTGC RQGHPENRDMPASEDLQDLQICKL
AATCACTTGACAGTGTAAAGAA.A.A.ocTOcCATCTTTATGTAGCTGACAGGGACTTCGGGCAGAGGAGAACATARCT
GAATCTTGTCA CRKRCINQLLCKLPTDSELAPRS N.)
03
GTGACGTTTGCCTCCAGCTGCCTGACAAATAAGTCAGCAGCTTATACCCACAGAAGCCAGIGCCAGTTGACGCTGAAAG
AATCAGGC UCCHCRHLIFEKTGSAITUQCKE co
cn
A.kkAAP.GTGAGAATGACCTTCAAACTAAATATTTAAi% ATAGGACATP_CTCCCCAATTTAC-T
CTAGACACAATTICATTTCCAGCATT NLPSLCS UQG LRAEENITE S CQU
TTTATAAACTACCAAATTAGTGAACCAWATAGAAATTAGATTTGTGCAAACATGGAGAAATCTACTGAATTGGCTTCCA
GATTTT EL PPAALIQI S QQL IPTEASAS tiR CO
Az...kTITTATGTCATAGAAATATTGACTCAAACCATATTTTTTATGAIGGAGCF_ACTGAAAGGTGATTGCAGCTIT
TGGTTAATATGT ITKNQAKKUELTPSN
Ill
N.)
CITTTITTTTCTTTITCCAGTGTTCTATTTGCTTTAATGAGAATAGAPACGTAPACTATGACCTAGGGGITTCTGTTGG
ATAATTAG
Cl)
CAGTTTAGAATGGAGGAAGAACAACAAAGACATGOTTTCCATTITTTICTTTAOTTATCTCTCAAAACAATATTACTTT
GTOTTTTc N.)
PATCTTCTACITITAACTAATAAAATAAGTGGATTTTGTATTTTAAGATCCAGAAATAoTTAACACGTGAATATTTTGC
TAAAAAAG
CATATATAACTATTTTAAATATCCATTTATCTTTIGTATATCTAAGACTCATCCTGATTITTACTATCACACATGAATA
AAGCCTIT co
GTATCTTTCTTTCTCTAATGTTGTATCATACTCTTCTAAAACTTGAGTGGCTGTCTTAAAAGATATAAGGGGAAAGATA
ATATTGTC
TGTCTCTATATTGCTTAGTAAGTATTTCCATAGTCP_ATGATGGITTAATAGGTP.P.P_CCAAACCCTATAAACCTGA
CCTCCTTTATGG N.)
TTAATACTATTAAGCAAGAATGCAGTACAGAATTGGATACAGTACGGATTTGICCAAATAla_ATTCAATAAPAACCTT
AAAGCTGAAA
AAA.PAP
4A.A.A.AAAAAAAAAAAPLAWAAAAAAAAAMAAAAAAAAAAWMAP.P_AAAPAP_a_ARARAAAAAPAAAA
C)
-17!
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
130
U M: CO
EC; CI, mii,
P H Li H ED 11 11
µ81 R FL-). c4''. j C)) 1
0 E. fr. ii, P. ,H g r=
.--.
r4c4C.7200
c; 1 14 P., .1 f... 8,
z
n
En H . En H P.
i, ccs , _ia ,i ,.1 , .), ) g 2r ,,
2:
u U 4 C.) ci U 0 U, a 4), 0
0 (.9 U ,4 u4 0 0 AT, A 0 0 a E. CD
CD CD 0 a 0 E. A ED H 0 H 4, 0 H 0 a EDE..) F. a a U 0E. a H
E.DaEDE.DE.ArCED ciaNgaE.HuUEDED aNaaE.AE-1
(90ag UUCDON 000euu0NrCEDgEDF.¾(900HHU
0004 r E-.00UH4e,0,E.0HauguHE-,aHqqag
EiNaUUEDgE.HHE.JurcaCiUHOONOaraCiaaau
U E. 0 CI, U 0 U U EJ I. a ED a E. F. a U a E. 0 F. U a E. E.-. E-. 0.1. E.
H
U F. 0 a Ei E. 0 CD E. C. E. 0 u ED CD u U a 0 0 0 E. a 0 a a ED E. 0
8o 01. 1---, 0., 3,q tici ,4ch 8E. ,c,r):Ci (E2,0 Ei, E6,E-. cti r6(.7 3. .
c Ei .,,,, . - , 8E7, 8 ,,_,,,,, Et E,5:E. E.b, ctiDN ELIE. au ,,,;.a EC:
ci 0 U a U 0 a a a 0 a E.1 E. a AUHHUHUUAH
[...E-.E.,400Uu0E-.0uUcDciUUgOtEDE.rCHE--.H.H.E.NU
u H g U E. 0 U U a 0 r1: g 0 0 a C.> E. CD E. E. 0 c..) 0 u u .=:q ,. U
H
ci U F. H. a V H E. ...4 0 0
a a a 0 E. U a E. 0 F.
UH0CDUOU,HEDaa g 4 0 NH AA 0 A EDUrc AgONE.Ha
H. F. a E. 0 A A F. E. CD C.; cD U, E.... CD A U U g q g E. E. H ED F.., H 0
F. F.
UgUU00-achagUEDaE.gHaciclug00E-'00 a
U 0 E. 0 E. U a E. CDUE.E.0<0000,< <UCD<CD E. a E.
rCaUOUE.iy0CDONUagE.c.DE-10 HIE-IgUaa0 , g
U 0 U g 0 0 e-.. g g E. V U a F. g E-, CD E. U E. * ED 6 0 U ci
CD
1
6 E, E-. U
o o ,.: u 6 6 6 6 6 r, ,P4 `.,-- 8 8 E,-; r, c.i
CC; (..,0 8 6 q 6 8
0,0, El g 0 U U 5
U, ci U a; E. 0 E. E. A a, E-. AgAHuHr4U4,0E--,[y, ce, EE2, ,.,., EC-% t.,
E(.3
,.., 0) u":" go tC).E. EC) EY< ECU r4a, - '54 ''. - A 80 t - -15 ' Ed Ef 5A
6E - µ Ec:j C9E - , 0) 8 0, 05 Hu uE, DO S. EY. 6 5
q u E . q o (5 0 u c5 r4 E... 0 N g a E.) Ci C./ ,, A E-1 * 0 E8 E-. Ea, r,
Ey, < E4
C)
- - 5 .0 c' '- )0 Pi rd At ' cD, N P. , ' ' - - 4, P. , 6" ) E i j- d ' 0_ , '
- " )), c (6 0, u?' 8, " "(3 0Ã ' '6' 8 8 u4 F-; gE'
E.CDHOgau*.gOON ''' , YV9C-"--"-)t'5,-c) 6 '4 cr_,' i-.)., b'
ri '6' Er_; r, 8 S 6 u 1:: ,E,7,1 u E9 `,J,2, 8 8 0 ri CE'l E.' El. 0 < U U a;
E E. F.
UU.,40:Na CDFE-, Er-,_,Vi.r.. cD<CDEUE.CDCDUytEicC--.CD,Api,El:
re..1) (.;- ccj7 E9 ..j.) t-; 6 u P. o ,V: '0' -)' ';5 E6 E i-
-.' 'E,' 8 `-',.,=c 8 `,-t' 0 A 1 '6; 0 (.6
u .. u, gr, o u E-4, i--. gc U 0 C.J C..! 0 CD gt CD o,
u E--, ,.., E-, C.) < CD g, .-5 0 ,4
O ,.,. a t-j 0 ,l, '. Q Ki. fr. 0 rt. E. ri
E. ET. a 0 u q E-, H GC u y * .E4;
' .4 8 `,- c' c, c. ) . i ; EY, ,V, cc:9 Ec?, b) i-- ..) ,..-.)D 8 8 1-.' 8 5
8 6 f_-.; ..',' 5 o 8 8 ..4
C!) ,4 CD 0 C. F-, E-, 0 A U U U U E. E-, a E. U El a 0 Ei 0 -a E. H c; a o F.
g U CD a U E. u ED 0 r4; u F. C.; <4 E. a; CJ H A < 0 E. U a 0 U u 0 < C.,
U CD < U 0 E. 0 E. a U 0 0 CD U U E. g E. CD 4. f. F A CD E. CD U < < E.
CDHACJEE<HAEC.,<E-,a:p4ue. HuciE.ciE.E.F.E.JauH
E. ci 0 H E... U 0 0 ED g g Ey a u H U 0 a CD A 0 0 E. U * (El a Ed
FD 8C . D E,8 --)j,,4 114 '-''F'4 c.). rA 8 84 g 80 L"5:_) F.7 tE:'90; EE 1H .
H E 1 ---- j ' 6E - , 8E - , ', V- , Q '' E - ', r. i r (4 r. ;- .
CDONOutiE-.0gOgagE.F.agu-a0000E.PF!ED,HHC),1
O C. c.) r-, u u E-, u .< A 0 0 CJ CD 0 < U < a FI: < U E.E.00ua L,-,' '11
i
E 0 (...) 0 CD E. CJ 0 0 0 C. 0 U E 0 u g 0 F, c" u ,rq ,a E-, u E. 0 E. au
OCJAUCDHCJE.01EgFU,CD,CD<F<CCD.CIFEC,D,,E.10, GC E0 N Al
8 `,' , 8, 5: 84 `E. 3 8 8 o o 4, '4 6 5: , E--: Egl [7.1. .?C E. F4 8 F4 E.,
,,, : o o ,s1 5,
0 E. 0 0 0 4 0, 0 g a U U < ,..., < E. U C9 0 4. < E . F. E. f. E. a: < a,, a.
ug,4E-,u5,ouo,cuuu,..,4,,,q aHUHaE.E.E.Hociuci
H 0 CD E. H u U 0 A ED ED Ei ED Ei E. E. a 0 E. 0 F. E. ED H 0 A H H F. CD
U A E. CDUNE.HC/F<FEE.C.9<H<EUEUCD A E. A Cc/CD ,a.
0 CD U U 0 0 a UUAU CD u E--. 0 LI UNHU AU a E. E. u, 1.... UN
O 0 0 a a r...D Pc 0 I.) E..D < H. < 0 H g.. H g U 0 U H a H 0 0 ri. :,;,,
t. EL:
Ha .: E, ,O, E. 0 E.gauCDE-..0ci,HEDHuNuE-.HHEiu ,ug,
CJ v 0 U U CD < U U U a a; C--, E. a E. F. y 0 C.) E. a y A
A H ET. E-, El
O00<cJVD0000,E440.< 0<ECJ<< gyE,E0,80(E-1_, l(D)t-_,,,
46 _ .> , ,I. C-. F-' H8 aU 8 '' t". ; L5 E -,Ed L') ( ), = El E5 v", . ; " 1E
. UU (E . 40 EC la . " jd 6.0 5E - , 1 Hc 9 *H 0, : E - ', 4 u' 4 ' r
UOUOUNE-400E. 400UUHHE-100044 4 UNEDa
U A E. E. cD H. a CD ED U
CD a E. E. H a N ED E. H. 0 _ I-4, c-2 u F. p. El
CDUUF0<0000EC.1<UUCHEE 0E-..E-IA!,JEFSJI.
C., CD FC 44 :viE-,04uE,-grE-.0400,4 0(.7,.k 0 t'. a, N t' u
gc9000 piaaHUcigHUEDHEJOgaH (ED. 8 p. Ev8 ,F,.
Ci CD a CJ CD E-, C., 0 Cl E. 0 0 tJ c..., 0 F C-1 E. E 0 A U ,
EN a 0 C.D 0 ,4 -...4 o o ., u u ,,, a: H 0 g U E-. 0 a
E. q -.¶.. 0 E. H. E. E-_, H. H
,-,1 HuHga -agE-rauci HEE.C.0U FFC-JU .50_. ,-,
;,
0A0,000A001r-, A AAF. u u u E....uHaH 0 gli
=
a 00 F-1 P, `,.-.Z 8 E' 8 u 8 c.) 6 ',-_), (-
'4 `,-)) ,E -. 6 8 6 V, : 8 Vc E. EE-2.
2 CDU 00cDOUrCHO UN. t--, 0000E. <U<E0C-,y Ec
OHOHUg., ,4uoyqc).1-,,tugug-,a,.._ C-cl < c...?, L'.. ra ,C,D, ..-), y. y
o9 0 0 .. .9 A E-,,..0-..:Q1 CDHA,5. UV
1-2 ,..2 ,:...; L., -.., =3 õy4 ,E.,:, E, r,,,,, c, ,E1,. ,,,,,.
0 E--, A 0 A cJC/Cia:FE-. E-,, N ,, '41-"i'''''''' .<F E-;
PI U Ei 0 ED E. 0 H a U U 0 . u E. u H 0 u 0 H E. CD E. 1-. U 0
0 U ED E---. 0 ED H ,= A C.) N 0 U H g (9 0 E-. a E. g U g 0 0 CD 0 a a E-
. 0
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093942
131
()(..qggc-,u(..,E--.rigHF-toHggr-,gp.¶Jg
H <U0HUHOE.E,u&-gHaHugHR.0E-
E. H g U F. E. < E. CD 4 U 0 E.
C.9 1 < 4 4
u u .4 u Q.)t, E..) 4 a 0 E. EE. OUUCDOU<C9CDHUU U
E.C9H. <UU
0040E-..4U<dULDC9 L.)/auL9a 6 4
<OUHUrc 4 ciE.a:4E-,0E.0 UE.04, 0
U E. H c) C.) H C..) au,uouaHa H CD 4 CD CJ E.
U40E.C900(JaUU4C9H4f.0 0 000
4 H H g H 4 (7 U 04 H LI 0 U U U H'( ,4 a: 0 4 E.
U a, HC) < < < E. E-4 44 U < E. U UHU 1.
CD E. U 0 0 0 U H 0 E. CD L9 U F. H a.E.a:U CD L9 U
HOUC-904UUCJ<< aLpuHHa L.) ouga
0 0
HuugH E. E. 4 U U u 0 E-. E. u E. 4 E. U
U 000 c,CJ44000U E. <HU4 OU 4u
U < E. 0 .< 0 E. E. E. E. 4 < U E. U 4 0 4 Ui E. 0
CJO<CJOU40000044 4044400 U4
< E. E. < K4E. dup.40uouud HOHH
ChUU HQUCJE.HU < C9UU a E.I.C9C90 a
00C) UOUUE.U<HCJ0404 C.),c4 1C)
0 CJ E. 0 ci C., E. C.) U C., C.) CJ E. c..) 04 U E. u
a
HoHauuoHluuouaHoaugaH a
ou 0 U U E. 0 u < 0 E. g C.`J E. 0 4 0 U U E. ....., F.
a, 42 u 0 a; u E. -04 0 UHU < 0
HHE.E-10C.DdCJUJOE-.0 ()C./ CDC/HOLD
H F. U 0 4 (.1 0 E. < E. 0 C9 U 0 g 4 4 U U E. E. E.
H 4 E. U 0 0 ac...),ouHE,uu U)-' E. U U 4 4
.4 CJ 0 (.9 E. 0 CD E. U V a: E. < g 4 E. E. H 0 ED E.
Ud4HUUE. U0HUE.0 U<UU00 g
ouuuHaa uat-AuvougE.ogHoa
C9E.CIPUUUCDULDUUL944U0C90<40
0 0 4 4 0 E. U < U 0 E. .4 0 0 0 U < 4, 0 H
E. 4 < 0 44 44 0 0 E. 4 U 44 44 4 0 4 C.) U U 0 E.
O (I 0 UUOHUU<UU0u0 E. 4UE.<0
OUrCJOHu 4 a,(JC7aLTH,ua 4 0
CD D, E. E. U 4 < 0 0 E. 0 a, 0 E. < E. C) U E.
4 a HUCDUCJ<E.HE.4)a; E.cil.ry<ci(D4.,
OE.OcJE.OHre.4Ur 0/4 00H0E.
0444000 <004 04 <E.000<
E.H00H4E. UCDHU 00 HUH E. <
CDE.4E.Uc9E.C9E.HUUHUD HUH4E-,.0
E.HUgU(90E-.40E.L9U<UHUUUD k.) E.
U I-. U < H < 4 4 U C.5. U H U 0 r4 0 E. E. V E. E. F.
0 4 4 0 E. E. C) 0 Ci a, E. C) H 0 0 E. U U C.!) 4 E. 0
0 H CD 4 U U H 0 U U E. 4 H. U CD U E. C) 0 < E. E. U
O d I, CJUE.C.) OU 404 <ULDHUUHC,CDUE.
4E.E.AU<<gt-.<000E-.4E-140<HE.<c)
E,dr,40(.9uuE,4[-.F.Hu0E.E-,-.4 C.? a, 0 U C.)
HUCJE.E.E.CDUE. E-4 44 U C901-.1.04,04 4 4
E4 0 U 4 u 4 4 rd E--, E. U qa E. 0 U U EJ U H E.
UHUUCDHUL10 HCJ aCDE-10U<HUOCJ
'CU U < 4 4 4 4 E. _ , E. E. u CD H E. E.
HO 'CU 04 0 chuUt.H0 r,,t,t / 0 E.
E. UF<U<UULDHU aoua aua u E.
C.) HU ONE.HEJ E.4 E. E--, CD < 0 CD ,...0 E-. E. 0
0 E. .-r_ , u 0 c) El u C!) E. c..) U 4 E-. E , U E. a; H 0 E. LD 0
gE4uU4HaLDOE.UUCDE.PHOUH40.<
U E-. E. 0 U 0 a E. CD E. I. 0 0 U U C9 < 4 U U < < ,
U4UHU406.00U<CDOPHHU040H<
E. 4 < 0 F,C 1. E. 0 E. UHU C) 'C HE. CD 4 F. E. E. 0 H
u H U E. U 0 E. gga E. U 0 U 1. E. E. CD E. E. t, E. H
CJ g U u H u E. 0 FL Pc E. E. u 0 H 4 4 F. u (.1 E. 0
UUUOU4U00,a.E.E.44HH 00U 4UE.
0E.E.000UE.<E,HUL)'0U0H HU ciU c.)
E. au', E. 4 E. .4 u u u C.) E. E. E. 04 E. U 4 g 4 E.
c_) 0.4 uu00004u OH HHU E. E. u H U g 0 0 H
U0C7H<HOUUCJWUCDE. E-i g E-4 PHUO E.
<HOLJUUE.Urd 4 <1. 0 HO HiJC7E.0
0004UU0E.E. 0E.U4UHO V.)H0 '04 E.
E.E. U 0 0 4 U 4 4 u E. F. d 4 H E. 0 E-. a 4 0
UUE.C.)E.U04 E.E.E.00uU uHHOE.E.
40UOUKE.E.C.)4E.E.0000 4.4U C.)< 004
E.C.J040400000UHOHE.gouuuaH04
O4UH<H4U04 0U440E.4 HUOHO (1)U
O U U < E. 0 4 E. U 0 U < u < 0 E. H 0 E, 0
F.E.r<U4OULAUHUOLDOCJ .,.. aucauE-.
E.g<HUOUH c9CJE.00C7H E. E. < HE.
Ci c.D E. 0 0 4 0 0 E. < U CD 4 E. 0 C.9 1 a E. U 6
O < 0 E. U 0 E. H 4: CD U E. F. E. rd 0 ' E. a CD 0 pq
E. u U E. 0040U <UUUU4L9E.<0 E. U PIS E.
U a' C.7 E. H E. U CD E. U E. CJ E. U U 1
H. 4 0 C.) 5 E.
C9 d E. 0 U 0U PUL)E.E.gur, HU< 4
OE.U4H(JE.u4UHE.UUU UHOE-4<.
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
132
E-, 0Z0HOC4H,p,IXO>.>.:OH>-,tncoZzi-10>aZcoE. tn
H,.1>,c)11-1).4>a.riz>0000 egclizaHoolmuHw 2CD
H H H0 C.+ pr4EntAlrE.NHel0, 0 H CI) 0 (f) E. 0 0 Q4 H VI ,
a-, En a 4.1 0 E. 0 H rAC E. 0 E, H a, D. >. 0 0 Z 0 cn H
E.Fin >OHIZ}A 4a.rif4>44[4,--Ig .,-4x3,41.1H
,E40 t:2to,
>gu2tnn pH 4 74 14 ,..1 0 a. < . CA a 44 EA, E4 M
74
1 a 0 X 4 U a 0 41 4 U x
0 3 z w co 3 RI z '41 a 0 3 z r4 H H z ix CD AEG E. e4 < 2 En En DJ,
5,a>. cn E4 E. H E. rAl a, 4 Cgt+ HOP-10Z 5,--1XCY cr-1
OH (11C,.->-' 00a),,CCnC.Dr4 >0.Zcon a c-1 coo
q 4 >4 e, La -., ZHHHHHHO [4 a 4 u rAl
H a .,4, 0 0 > 0
C
,0 00Srilr-,H .04H HCak-,Z0141440NE-40U1-1 r.,-,g
>.w>4.4w
..-1E.,-.1>co3g I-1 En>H0p>Z0E400.ale4 OHEn> PI
= t9pg ,-4t0tAl Az
,,ACEnf.a. z tnala..-la-.40Ene4000,4E.
0 4 P, H CD X c4 12 41 0 E. g4 e4 (5 E. E.
FA.1 U a, (7 4 [,, 0 C4 CD Ei e4
Z
H ,.azz,41?-+H (.9010V,ZHHHENci) R
till>, E.0>HUO
H P-
O F E q ril ,A4 =,=4 0
0
Ha,a4Ht).74E.En.-A,P,EnU PlEntina,HEacn0a EnE.N>r4
cn X cl, c4 >, En 4 a. al E. E.E., E. > 1.0 ...I CI) H x,-
1C211...cr)>001gc.9 4r-Dr=1
E4 U 0 U U 0 0 < E. E. EA H r) U U g g E. 0 E. 0 E, Ch 0 F, a: < 0 U
O UUUUUUN. g A5 0 <EDUON g g ti i ,5 H
<NOLO<
HE-EL/0000U HO 0,40000000,C 4.0,400E.
uulAcDOHNE, <0 CD OUCDHE. g HUOUUUHO
H
UHOUCDUC)C) HUHOUHE.C.90g HH g E.KCUggE.E.KC
U 0 u CD .C..9 U 0 f=C CD g E. 0 0 U U U E. g 0 0 g C.) U N E. 4, 41 CD
C.)
Ch 0 CD U U CD H U g E. El g 0 f-, (9 E. < U t. E. 0 El 0 Er C.) a; U
U
HOHKCHHOgg(JUUUUHUUCJUggHUHguE.L.DC)g
U KC U U U u o H. Li E. CSOHHUHHHUHH U CI 0 a; a; .4 000
UUHCJgE.E.E.0,,HHHCDC.9gUOKCHE.E.C.90HE.Ua H .-1: E.
U 0 U 0 g H 0 C) KC U C.) 0 0 U g H g g g CD g E. g U Cj HHH
C.)<,CU HUCD g U gE.E.E.E.CDC)00(9g0 C.) 000 g U
l'OUC)E-',40,1A0,400,400,4F.E. E.H.NOHOE.c....CU
0 0 CD U H CD H g4 0 gt 0 C.) 0 4 C.) g g C., E-4 E.0 B 6 E. 0 rC
CDC)u0PCJ , F CDE.HgE.C.Dgc.11c9 Hga; E. PHU H CD
HUQE.C.DC9uUHUKCUCDE.E.E.HHgUE.HCDE-4u0 U E-,u
OUPH4
.E.Duc.)40E-,HE4E-,c)c)E.E..1 0 4 U a. a4 E4 4 04;
u g< C) 0 gc u c.) U U E. E. g g U CD E. H U C.) E.-, 4 U U 0
,..c 4 (E-,0
upc(DE.H.0,4E.H(.9E,U<NH.CDUTDUE.. UH0E.CDU00 4,
0 F-.00 UU0 0 < ,A4 < E. 0 C.) U H E. g 0 E-, < E. 0 E. U E, E. E.
H 0 CD A4 a: U 0 a: E, (D u 0 < 0 E. 0 E. 0 At E. E. 0 0 U t) 4 (9 4 0
HIDUUCJUQUg4H0 OuHUHUH E.H.400 HgCLDFu.,C
E.K4 UU E.O K4Pc .4 H0040 .4 H .4 0 E. H g
C9 U U g H U
1
0 E.
CDHE.C9 CJUU0 00 gHHHHOHHE.HUE.H UggE.E.0
HHUH CD U> E. E. E. CD CD E. U u F. UE.HU H r,4 F E. CD U> 4 0 0
0 E, U r4 C) 0 F. (..) AC E. 0 a: Ch a: E. H < N 0 0 ,E4 U 0 4: E, F F4 0 E. 0
PHE.CDCDUC)C9C)HE. Ug4HE.HUgac F, E4 < 0 QJ E-, E-) 0 0 0 < ,
O E. U CD 0 C., E. A4 E. .45.4KCUUUHHAHHOgCDUC),<CDU g
H KCOCD UPPUgUa,UUCJUE.4 g OH 4E--,0E.HE.C.9E.c.9 C)
CD 00C-)E.U0 gOgUgt.)0E. E. E.5 0 0 < 00.4E.5005C9U
E. H U U U E. 0 E. <4 E. 0 C) g4 E. CJ 0 0 CD E, 0 E-, 0 t, (.7 E.55 E. CJ
Lir - D ,q: Q CD C9 g Ci C.) 0 g a; E. 0 g õ, F Eg4 E.. 0 u E.C.)
g a, 0 E.
/
OUC)0C9tig0Hggn.HgE. CDHOUg UOggUga 0
OH UUOCDO E.E.V OUPCJI ,gUlDgQ C9g0C)C),g F4 E-
O CD C) t..) C.) U 0 a; E.
g H g F, a, E. E. E. 0 0 C) UHUH < ,E4 0 CD 0
H00000E-.0g4PC0d F00r40E.r4 550E400E. E.00D
C9OUUr4UCDF 000d clE.E.CDOCIUgE.E.E.E4.4E-
,E.cD 0
O CD H C.) u ,4 0 e< 0 <
KC Ci E. E. C.) E. U CD g H g g4 .--4 g4 U 0 E. U E.
U CJ C) U U Ch 0 0 E. 0 E.
C.) U 0 4 E. E. ED E. U H E. g E. 0 0 E. E. <
OgCJE.CJO a g gOUE. 4 ( ( 4 H H CDPE.HHUHUO<HaC,A4U
o PC ti (i < < (3 Q 0 E--, 5 E-, ,4 g 0 U E. AC U ED C) < < g4 0 <C CD .4 0
,4
u0u0EØ45.5 E.a..400u0.4H,4000NON5uLDE-,
UHNO,C(900,AcUULDE.c.),414E.UP,A4,4 E-4 q r4, E-= H ,14 g 0 H
CDCJE.CDE.Ha;CDE. E. AA: 0 E. AC U .A.c u < 0 a. a. 0 a, pc H 0 0 E. 0
HOHUN0,1.0HE4H .4 0 u .c. E. 5 5 0 0 d, <0E.C.)0gUU
E.HULDE. (9cD-UH,040 H -,A4UOHON,E4a4E,CDEDH<Uclac
O 0 U U U E.,. E. t., E. ac, A4 PqE-,E-
104,0Pc E, H QH CDOUE.a;
500E-004 E.000 ,00000.404u0u 5.4000
u5CDUO<CDUUKCH<HH g HE.E.c9H CD g C_9 U H E---, U 4: 0 4:
0 0 U U U C., L., 4G ==C C_J HHHAH U C.) E--. E-1 1...) /..0 < E. u 0 0
UHU
E. OLD (-) 0 C9 '4 4 F,F,Hor.DE.000514 OU00,4E.00
E4E4U00g00g5U0g. E.H a; E - -, 0 0 0 (-) < 4 A E U H CD CD CD u tl',
0 E. 0 0 E4 E-, gr. . U CD U (.9 0 E. H Ell 14. U H
H U Kr.
KCO00000Q E.-. HO 4 H. 0 < (9, 0 ,A4 0 ,C < HH HO 0 0 ED
9 c., E. F.
U CD U U C!) 0 0 Q E. E. H (.9 U a, E-4 CO
H U c..) E., 0 H U E. H U 0
E. < 0 c..) U C) U E. g E. E. H 0 Kt: u U 0 H H E. 0 E . < 0 E. E. c.)
E.
E4 CD CD <4 U El 4 ci 4 4 4 < H c9 0 < 6 g < (9 AU 4 C7 E. 0
E. E4 ,4 U 0 0 cD E. 0 0 0 C) 0 U 0 U U C.) g E. U 0 CD HUH HA
CD CD CD C, H 0 ." E..., E. CD E. H 4 4 ,,, H E. a; H 4 g E. CD E.
C.) 0 6, CJ
O U U U U U C.9 ch U cD E.
U (5(5 a, a' C) U g CD :. E. 0 E. C7 4 U CD E,
CD 4 U UU 4 CD CD 4 E. 00 E. E. F. 0, CD O. CD i. <4 4 E-'
E' El E-' C.JU
E. 0 E. U 0 0 g H CD < A A g U E-4 U 0 4, c5 -. U 4 E. 4 C.) '4 4 4
U U f4 0 4 U CD CD <4 H U u .4 C. u () E. 4 E. C.) E. a, .:4 E. c9
0 E. 0
0 E-4 kJ U U F. C) CJ E. 4 C7 CD al g H a; (9 0 () U> (9
H> (9 4 4 (9 E.
E. (7 0 U c.9 u C) U g 4 0 0 G.4 P 5 < E. 4 0 .4 c) 5 E. g E. E. E. a. C5 a:
(D E. U ,24 E. < E. U E. CD 0 (
CD U r4 .4 u E4 4 0 c; E. 5 4
CD c9 4 0 cD c9 0 F.
Hc) 0 c.) (..) f.9 E. 0 E. F. E.. 0 ,c c) g U 0 .=C U 0 CD
E, E, 4..) E. <4 E, <
O (-, E' gUHUE. U U A 0
OUUcl HuF, 0 Co C, 4 E--, g4 H E. CD 5
O CD 0 E. CD g CD g 0 CD E. C.) CD U E. E-, 0 E.
.,.4 H 0.0 E. CD C5 D ET' 1
U CA F. U 0 <C 0 C.: E. H
E, F E. E. E. H E., E-, 0 CJ Pi, E. 0 U g H P,.. E. U.
.4 E-, 0 (,) H 0 E4 0 A5 U E. Z <4 F. CI U E. CD
C'J <4 H 5 E--. E. ,5 5
1 1
0 cJ U 0 U CJ 0 < 4 () (7 E-, 0 A E. 0 KC u E. 0 C) E. <C U E. CJ
KCC.100C..)00u OH E.E--gCaa,H, 4 E, g4()U0(40E,419
OC)A000UE, E-,c)E-.4.4c)cDuE,H5u0 5
0 E. E. t.c.)0
45.0)00055 E.E.HHHOUtDgH g HOE.,.¶,c) H F.E.E.
ocE.c),,CUchgcuAg4HtQg4,4g4H-pcUE.Fr.gr_Du H K4 H .-Cc7 cD
ni UCDQUChg 0E.E.E.HguE.E.E.K400CDEAu,c40 H F H U00
ol 000c)Uul.gE.C7a,004 E. 0 pc < E. 0 < U U < (el ED 04 Fs U
O CJ 0 U 0 U t.) 0 C.) 0
PC E. CD a, E. E. U a, CJ E. El 0 E. U E. < a4 0 0
E. E. U 0 U 0 E. U g4 E. U g E. H E, 0 C.) Pc 5 5 E-4 H 0 4 a, u Q c.) 0 u
0. UUUUUHUgHHE.HHrCH ggo<0 Hg HUg Ug 0 0 E.
Z < E. (9 () 0 E. E. C., E. U E. E. 1 E. E. HUH OH .4 4 OH U E. U U
LD ,4
0 uoucDpc,E-, E-4,c:E.,CE. HA OH E.-1-gc..95.00CDE.C.) gOE.0 E.
H HO HE.CAUCDC-9.4E-,.4 (94 00E-,u.40 uf.E.50E40
0) EICDOU0 EJE.E.PHUHa/U g4r44Cic.) E.004 Kt; ,C E. P
HEH gOE. E.E.Ug ga; pC <CDOUC)C9C) 9 CiUgUCDE.C)
CS U U 0 0 U CD U U < g 0 0 0 0 C.9 CJ N .=4 g4 N 0 gg U 4, t.D
g C)
SUBSTITUTE SHEET (RULE 26)
AGAAATTCAACAGTCATGAGCAGAGCTGAAAATTTTAAACAAGTTGAGTACCTCCTTATTCATGGAACAGCAGATGATA
ACGTTCAC RAENFKQVEYLLIHGTADDNVHF
TTTCAGCAGTCAGCTCAGATCTCCAAAGCCCTGGTCGATGTTGGAGTGGATTTCCAGGCAATGTGGTATACTGATGAAG
ACCATGGA QQSAQISKALVDVGVDFQAMWYT
ATAGCTAGCAGCACAGCACACCAACATATATATACCCACATGAGCCACTTCATAAAACAATGTTTCTCTTTACCTTAGC
ACCTCAAA DEDHGIASSTAHQHIYTHMSHFI
ATACCATGCCATTTAAAGCTTATTAAAACTCATTTTTGTTTTCATTATCTCAAAACTGCACTGTCAAGATGATGATGAT
CTTTAAAA KQCFSLP
TACACACTCAAATCAAGAAACTTAAGGTTACCTTTGTTCCCAAATTTCATACCTATCATCTTAAGTAGGGACTTCTGTC
TTCACAAC
AGATTATTACCTTACAGAAGTTTGAATTATCCGGTCGGGTTTTATTGTTTAAAATCATTTCTGCATCAGCTGCTGAAAC
AACAAATA
GGAATTGTTTTTATGGAGGCTTTGCATAGATTCCCTGAGCAGGATTTTAATCTTTTTCTAArTGGAcTGGTTGAAATGT
TGTTCTC'r
TCTTTAAAGGGATGGCAAGATGTGGGCAGTGATGTCACTAGGGCAGGGACAGGATAAGAGGGATTAGGGAGAGAAGATA
GCAGGGCA
TGGCTGGGAACCCAAGICCAAGCATACCAACACGAGCAGGCTACTGTCAGCTCCCCTCGGAGAAGAGCTGTTCACAGCC
AGACTGGC
ACAGTTTTCTGAGAAAGACTATTCAAACAGTCTCAGGAAATCAAATATGCAAAGCACTGACTTCTAAGTAAAACCACAG
CAGTTGAA 0
AAGACTCCAAAGAAATGTAAGGGAAACTGCCAGCAACGCAGGCCCCCAGGTGCCAGTTATGGCTATAGGTGCTACAAAA
ACACAGCA P
cn
AGGGTGATGGGAAAGCATTGTAAATGTGCTTTTAAAAAAAAATACTGATGTTCCTAGTGAAAGAGGCAGCTTGAAACTG
AGATGTGA 0
rs,
CO
ACACATCAGCTTGCCCTGTTAAAAGATGAAAATATTTGTATCACAAATCTTAACTTGAAGGAGTCCTTGCATCAATTTT
TCTTATTT
cATTrcTTTGAGTGTcTTAATTAAAAGAATATTTTAAcTTccTTGGAcTcATTTTAAAAAATGGAAcATAApATAcAAT
eTTATGTA
TTATTATTcccATTcTAcATAcTATGGAATTTcTcccAGTcATTTRATAApaGTGccTTcATTTTTavAGALAAAAAAA
AAAAAA 0
rs,
cn SEQIDNO.:34 SEQIDNO.:81Ill
0
0
CGCAGCGGGTCCTCTCTATCTAGCTCCAGCCTCTCGCCTGCGCCCCACTCCCCGCGTCCCGCGTCCTAGCCGACCATGG
CCGGGCCC MAGPLRAPLLLLAILAVALAVSP
CTGCGCGCCCCGCTGCTCCTGCTGGCCATCCTGGCCGTGGCCCTGOCCGTGAGCCCCGCGGCCGGCTCCAGTCCCGGCA
AGCCGCCG AAGSSPGKPPRLVGGPMDASVEE 0
rs,
CGCCTGGTGGGAGGCCCCATGGACGCCAGCGTGGAGGAGGAGGGTGTGCGGCGTGCACTGGACTTTGCCGTCGGCGAGT
ACAACAAA EGVRRALDFAVGEYNKASNDMYH
GCCAGCAACGACATGTACCACAGCCGCGCGCTGCAGGTGGTGCGCGCCCGCAAGCAGATCGTAGCTGGGGTGAACTACT
TCTTGGAC SRALQVVRARKQIVAGVNYFLDV
GTGGAGCTGGGCCGAACCACGTGTACCAAGACCCAGCCCAACTTGGACAACTGCCCCTTCCATGACCAGCCACATCTGA
AAAGGAAA ELGRTTCTKTQPNLDNCPFHDQP
cs)
GCATTCTGCTCTTTCCAGATCTACGCTGTGCCTTGGCAGGGCACAATGACCTTGTCGAAATCCACCTGTCAGGACGCCT
AGGGGTCT HLKRKAFCSFQIYAVPWQGTNTL
GTACCGGGCTGGCCTGTGCCTATCACCTCTTATGCACACCTCCCACCCCCTGTATTCCCACCCCTGGACTGGTGGCCCC
TGCCTTGG SKSTCQDA
GGAAGGTCTCCCCATGTGCCTGCACCAGGAGACAGACAGAGAAGGCAGCAGGCGGCCTTTGTTGCTCAGCAAGGGGCTC
TGCCCTCC
CTCCTTCCTTCTTGCTTCTCATAGCCCCGGTGTGCGGTGCATACACCCCCACCTCCTGCAATAAAATAGTAGCATCGGC
AAAAAAAA
pokkAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
17.1
SEQIDNO.:35
SEQIDNO.:82
CCCAGCGGCCCTGCAGACTTGGCACAGAGCACACCCACCTGCCTTTGTCACAGCACACTAAGAAGGTTCTCTGTGGTGA
CCAGGCTG MEGSLQLLACLACVLQMGSLVKT (.=
GGTAGAGGGCTGCTGGGTCTGCAGGCGTCAGAGCATGGAGGGGTCCCTCCAACTCCTGGCCTGCTTGGCCTGTGTGCTC
CAGATGGG RRDASGDLLNTEAHSAPAQRWSM
ATCCCTTGTGAAAACTAGAAGAGACGCTTCGGGGGATCTGCTCAACACAGAGGCGCACAGTGCCCCGGCGCAGCGCTGG
TCCATGCA QVPAEVNAEAGDAAVLPCTFTHP
GGTGCCCGCGGAGGTGAACGCGGAGGCTGGCGACGCGGCGGTGCTGCCCTGCACCTTCACGCACCCGCACCGCCACTAC
GACGGGCC HRHYDGPLTAIWRSGEPYAGPQV
GCTGACGGCCATCTGGCGCTCGGGCGAGCCGTACGCOGGCCCGCAGGTGTTCCGCTGCACCGCGGCGCCGGGCAGCGAG
CTGTGCCA FRCTAAPGSELCQTALSLHGRFR
GACGGCGCTGAGCCTGCACGGCCGCTTCCGCCTGCTGGGCAACCCGCGCCGCAACGACCTGTCCCTGCGCGTCGAGCGC
CTCGCCCT LLGNPRRNDLSLRVERLALADSG
GGCGGACAGCGGCCGCTACTTCTGCCGCGTGGAGTTCACCGGCGACGCCCACGATCGCTATGAGAGTCGCCATGGGGTC
CGTCMCG RYFCRVEFTGDAHDRYESRHGVR.
CGTGACTGCTGCGCCGCGGATCGTCAACATCTCGGTGCTGCCGGGCCCCGCGCACGCCTTCCGCGCGCTCTGCACCGCC
GAGGGGGA LRVTAAPRIVNISVLPGPAHAFR
GCCCCCGCCCGCCCTCGCCTGGTCGGGTCCCGCCCCAGGCAACAGCTCCGCTGCCCTGCAGGGCCAGGGTCACGGCTAC
CAGGTGAC ALCTAEGEPPPALAWSGPAPGNS 0
CGCCGAGTTGCCCGCGCTGACCCGCGACGGCCGCTACACGTGCACGGCGGCCAATAGCCTGGGCCGCGCCGAGGCCAGC
GTCTACCT SAALQGQGHGYQVTAELPALTRD
GTTCCGCTTCCACGGCGCCCCCGGAACCTCGACCCTAGCGCTCCTGCTGGGCGCGCTGGGCCTCAAGGCCTTGCTGCTG
CTTGGCAT GRYTCTAANSLGRAEASVYLERE
0
TCTGGGAGCGCGTGCCACCCGACGCCGACTAGATCACCTGGTCCCCCAGGACACCCCTCCACGTGCGGACCAGGACACT
TCACCTAT RGAPGTSTLALLLGAILLKALLL N.)
CO CTGGGGCTGAGCTGAAGAAATAGAAGATCTGAAAGACCTGCATAAACTCCAACGCTAG
LGILGARATRRRLDHLVPOTPP
cn
RADQDTSPIWGSAEEIEDLIOLH
0
KLQR
N.)
0
0
co
0
n.)
.00
t.)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093942
135
___________________________________________________ ,
___________________________________________________ ,
8 8 ,E,' El El 8 ,' 8 8 8 (5C) 2 6 El 8 24 El El 4 4 El 8
Et: 6 4 6 L
1
E. 0 c9 .4 E. A4 C) E. gg (7 CI L9 0 H c) c) c) g (9 E, E.
(5 0 y E, E. 4
E. E. .4 () (7 E., 0 (7 .., r4 0 p (7 4 (5.4 CD r4 U U .0 C) (50 g4 y a C)
CD a
.8 P. Et. r) E4 8 8 ,',,', 6 8 8 El 8 8 8 ;1 8 Ll Et.' 6 8 tl 8
8 8 4 ,'-4' 8 ti,
E.4 0 4 0 (7 0 E-, .4 H 0 0 E. 0 E4 E., CY 0 .4 E. 0 E. CY H E, gg H.4 0
E,'- El 8 '6''' 8 8 8 El El 8 E3 El
2 6 8 El 8 " 6 El El 6 2 8 2 8
1
0 E. Cy E. E. 0 0 U c.) Cc 0 .4 0 LD .4 .4 ,1 0 E. t) 6. (9 4) E. Ed= Ei t-
I gq CD E.
0 .4 .4 EA (5 g C.) (7 C) K1: E, L3 E. CY H E. C9 E. C) .4 0 (7 E. (7 E.
El E. a 0 u
E. C.) E. E. E. L7 0 CD 0 0 EA E. .4 (9 E. E., c9 ,r, u u t4 (7 (9 (9 E,
E. c) c..?
8 El 5 8 8 E4 2 8 8 8 El 2 2 6 ," 8 El 8 8 0 8 S () 2 "..1 F-,' E-ii 8 8'
g E. 4 E, EA 0 < .4 E. Cy AII S4 E. E.
(9 E. E. c9 E. r. AcJgL9U E. 0
c) ED c, (9 E. E. ,4 y : .1; Ei E. .4 L7 y c) E-4 .4 (9 u 4 fg E. ci .4 E4
E, 4 EJ CY
,i34 (5' 6 1 6 8 g 8 Cj U 8 61 .4( u,5 (g9c I; Eci; r.
6 U i% 0 t; o [1 o 8, tl
0 (D E, E. CD E. C) (7 I. < L9 E. ., L3 0 o.C,
,,t I) E. CD I. (9 E. c9 E, CY CD .0 C.3 E.
6 El 8 E, 8 El E1 0 " 8 8 8 8 8 2 6 E4 ,`,4) 8 6 EI [5 2 El 6
[5 El g) F. 8
El il 1 E4' 8 El 8 El f4 El ',- 6 LS 6 El '8 ',-
'4 8 4 6 8 ': 6 El 6 E.- g [5 f
2 4 8 E--,,' E P. 6 6' ';',1 Z5 6 8 El f4 El 8 1 '-i 8, 8 i4 8 8 6 '<' 8
'4. '.' 68
0 E. U CD U 0 (9 U CD U U ,,,: t. E. .1, E7 (9 .4 E.
u H f', 0 b; 0 .-4 n.: E4
8 8 F' E f. 8 .-_' E4 6 El 8 f4 2: S 8 6 g 8 8 El E':-
8 El 8 El u H t-.4' P. t;
'6 EC-1 P. U ': 8 'il' 8 8 8 8 8 F:.' il t.
(-:, 8 '6'i?, i P4 6 El 6 8 8 8
< 6 u E. c9 C) U 4 = 0 E. E. F. .4
EA EA .1, E. < r4 (7 E. .4 EA 0 0 ,q CD ge si, (7
[1 t7 8 F.% E-61 8 P. g; Ed 8 8 E, ,HUH 8 EE.--1 4 8 6 E--
,' 6 -?. Z5 6 Z'3 8 '.'4 8
.4 U C9 CD E. E. E, C) c9 C) 04 Ci (J c,C FC E. E. E. C) .e L7 C9 Al C) E, i1
U CD gg (9 .., C9
E-2. i-l; Cl E4 8 8 6 8 6 u '2 8 r3 8 2 6' 6' 6 6
[2, [1 6 gll Eil c' P .',4! 6
E., _ k4 4 4 El! CD E.
B 8 6 El P-: 8 2 8 El El 8 '4 El El El 6 8 t1 El 6 g fl 8' 8 EY. r, 8
0 (2 E. 0 0 .4 E.) (7 Cy 0 L3 L) .., E, 0 0 E.
.4 (5 0 _ , u I. .4 .4 U Ot .() 0 0 L9
F. (-4 u .4 u u U E. E. U CJ 0 LI H .4 c) L) gq ID E. E. (J (9 CD a < < < --_,
(...! C), 0
i'!.'5 r, p."r, p.r,õ ,<L) ti 6 El 8 8 0 8 8 2 6 j
8 2 'i 8 El 8 2 4 8 8 6 '6 8
.õ ., u c7 (9 E. 0 E4 E. p; E. E. ,4 .4 u L9 4 u U C7 L3 E.
(-4 (.4 (5
E. (J c9 0 0 L9 0 E. (9 L9 E, E. E-, 0 0 0 U oe C3 C.) 0 E. C3 U .4 (7 E. 0 4
C.) .4 ,4
U L9 o Cy C.) 0 0 Li CD .4 E. 0 C7 4. ,' 0 EA E. H 0 .4 E. E. t7 EA .4 .4 0 H
0 0 ()
HUH U E. U CD C9 .4 El E.7 0 cl CI L9 E. T.? E. y U y E. < p (q Ei 8 E, 8 ..g.
?.-2 !I,
1
E., 4 8 CY cc?, C) Ei.J, CJUnUOUy CD y y 5 1;-. QD U c,,. 0 ko. i..; .3 r_.
0 ,....; c(-:11 Lc...,
E4 < u 2 CD il CY -.3 up fr"... up _-.4 2 f--! E.% S E.
0 E. ,- E. 0 c9 C. c)
CD 0 ,C u H Pc C9 u u CD ci u E, Ll 0 Pt; tC4 . E. (9 U C9 !dc H
E. Li
E. 0 0 C7 (7 C) C) E4 ' E. CJ 0 U 0 4 4 Ft E, U E, 0 ,C CI E. 0 0 6 s_,,. ,õ,
g il
2. 6 6 g g El 0 t--: ,' 8 ri!
6 8 8 8, El 6 6 8 El 8 (;.i El 8 () 8 ',' 3 6 ,,
0 , E. C3 0 ,,C E. c9 4 E. H E. 0 0 ,4 .4 ,E-A S.? S2 p S2 g ,H ,, ,S.%
..,' rci! ,J 8 Fcd:
(,, FC f= E. E-. C3 E, (3 U 0 a: E. 4 0 U 5 E. u C2 .1; V ,,, c. H " " ..., a
t_: 6, (J 0
P. El 6 8 S 8, 8 8 ' 8 6 El 4 (3 Vi E4-4' g EE1 8 8 El 2 (9 ..' 0
0 =....
U Cy Cy .4 El cy u E, .4 c9 CD L9 .4 L) .4 c) c9 E. C.) 0 U E.
F. L7 ,rC () C) C.) 0
rt 0 0 CD H ,,t L3 L3 E., E. E. E. 0 CD e E. xl: 0 C9
0 Ei U 0 c../ gg E. y CD UP. c9
f3 El 8 8 8 8' 6 8 Et-2 8 8 8 0 6
El 0 8 E3 il L 6 II,' g 8 8 8 6
1 8 8 F. 8 El 8 8 El EL El 8 8 4 8 El 2: 6 8 2 i- El r g t; 8 El 8 8
E, 6 4 " El t5 8 4 8 6 8 8 4 8 8 EY E. 8 0 t7 E. 8 8 8 ,, 6 i:: 8
6' 8 P. E.' 8 8 8 0 8 2 8 8 8 E., '..- 8 il El 2 8 1 8 2 2 2 11 6 t; P.
' E"' 8 ,'C E3 2 8 8 2 8 E-3 6 8 8 6
,'' 8 E-4' '6 E5 g E.-4' ,11 El 8 4 8 8 6 E--. (2 C9
O C) _ g (.1 () CD EA (7 (7 (7 C,
q CJ C5 P Kg (.9 g4 cp u Kc E. (9 A C./ 0 4 0 c9 C..9
0 CD 0 ,4 (9 E. E. c9 cy g4 u E-, gt, Ft (50 ,4 (7 6g CY 04 c7 Cy U HUH , cp
Kg p p. ,.,..1
0 .Ei'-c' !c.-.!! " ,-4, (;. Pu --.F! '' " -.)..), '',6. Li Y." 8 0
8 V, 8 6' g P. g g (4
,, ,_, ....,,,,,_,,_. E- ,,,, (il ,,. ,..., ,v li V., g
Pd, e,C U E. U E. L.) C) C.) C9 CJ E, CJ a
,,, [5 8 El El 12. 8 El E. a E.-4' t.-). El 4. E-, El 4 8
',,' 8 ,g:. El El 8 t5 El
,.., E., 0 < E4 '41 4 E. 0 0 (9 0 C9 0 E. gg 0 I) I) C9 El I) __ CD .r...p p
c__, p --._. J. s.p E ! I.--!
.: 8 8 8 El 0 8 El 8 8 8 8 8 6 8, ';-4' 6 6 2 E 8 8 8 8 8 8
(.,. ;!.' -!,
0 CY 0 E. 0 CD E-4 0 t4 K4 CD CY E. CY E. E. CP E. 64 4 C.) [-", E. E.
L7 E. 0 E. E. 0 C9
CY E.
E g g il ill i,-4' ,D4 V. 6. 8 il 8 8 t; (35 !- F. ("6 Eg-4' (el Z;
ti 6 Eg t5 El CD E'
" E. 0 CD E' '.' CI 09 u (9 c9 t. E. 4,
F. CD ., 0 c) E. E. c9 U 0 E. C) 4 U F. 0 L.?
g Er.. t5. Eg cj '6 [7'9 t5 t5 8 8 0 8 ; 8 8 f:: ill [-p: 8
P. 8 f5 2 ?- 4 2
,,õ ", 060E-.6uu<ugug ..õ.E.,(2.1E-. 4 CDUCDC.100E.E. .0 vi
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
136
4 C7 UUUUOUUUHUQ
U 0
C.) CD 04 H g H 0 HE. A
U, E-.UUCD<U000U0 00CJE.E.OUHU U
< 0 g0E-'0E--'0UciatUactU< ,E.C9H<OUOU0C7E.
U U at E. U C7 E. 0 U U E. H 0 < A H 0 0 EA El at rz4 H
U H
0 cl C.JHUHOV.hat0H0C7(7< <UE.CJ<E.UHHE.E,
U CJ<<HE. UE. (9HE.dUCIC7E.UUU<C7HE.0 u
U 0 aCUPHOHL70 <C)(70<04 ogq. uu,,cgc...qu<
u
H 8 8 (Ei 1 .2 ('-.; 8 8 8 3 8 8 fl El P, 8 E9 :2 E LI: 3 8 r, 8 8 --:
i E. C., 0 U 0 f-, 0 gq K: 0 0 0 H E-I E-+ U 0 H 0 at 0 E. 0 F.5 E. d
E. I.
U UE.<0,E.CØ0d0U000L)UHdCh 0,<C7 UUHUU
C7 0 UU<NOUUUUU<U0 4 A .4 0 H
E...) H 0 A' ..., 0 E. H
0 A 00H<Chat CJOU<NUHUE.H0HU<C10a4 E-, g
g u <HUal,F,(30000E.E.FtC7HHC7dLJUd E-, ...1 A 0 0 U
0 0 E. 0 0 E. U E. 0 U 0 C) 0 C) E. 0 0 at 0 E. 0 E. U 0 < U U U, C7
(Fl!)
4 U
A .4 HHE.A.4 E.HA U.4..GEØ4.4E.E.OHNOHE. U
E-, E. rtE.UU<Upt 0d HOE.E.00E.E.<<UUChatH E.
0 H OH0000000 U0L7do. 6i9oPuotn E.U000
(... A; 4 0 0 0 E. 0 E. 0 0 E-. 4 0 H a, 1 < U E. (7 H 0 H (9 E.
(.9 H (70HHOE.<H<UUE.0000.< CJUUrd0d0E.<
E. U E.E.<U0ChacH(9<(.7000E.<0 UatUUE.Uat E.4 CD
0: ei CDUat.E.HOE.OHCJUE.Hat Oat E.HE.(70<<C9H<E,
0 0 0 0 u H < 0 I. U 0 E. C7 U d 0 0 C) CJ Cl rt at U H U (.7 < 0 U
d E. at HUUUU < E, E. < E-,0HuE.o<ECJC7UucJH A, Et t.
U H E-, 0 0 < E. N U 0 < U 0 U E. 0 U E. E. U E. 0 U < 0 < <
4 U
O H E.0,00<U0000U<U< NEI
FC7f4HUE,<UU
E-4 a, E. E. < U H U < g E. U U 0 CJ E, C..1 U E. N U E. 0 E. U
1.9 U .4 n. 0 U E. 0 U E, <00 HO<C7(90E.UUE.< E. C_7 E. U U
U U OUNE-,010U0UUUUUU E.01HAEØ40U0 A
U 0 UUC)<U <0.-I<U00W0E.< H H u U 0 E. E. E. U
at 0 <C70CJED UU<OE-,<H<HHat E.< -gq c.9 E-, e,
E.,qc5
E. c.) HOE.00HUCha,H(J<LJHUHLDH g4 ID pc H e4 cõ, ri: 41 H
Ecu UU
?, H 0 H 0 0 E. (9 U C.9 < 0 H E. E. 0 E. H E. U E. 4... E-. 0
0
0 0 E. E. HO 0 UrcUU < <E-.E.E-, U < 0 0< U E. U
g4c)of--,ugot., dUdE.U(DE.E.C9HE.at H E.Hgq_. E.
0 0 CI E.E.E.CIUUUCJOUat000<00UUNHUHU
E, 0 HUOU.< HH0U0HU<OZDUP4 HOULDCDH; H a.
(2., U U(7000 OUUUE.HULJE.E.HatUE.E.U< A. E. U
E-, H. cp 0 E. 0 0 E. E., ,4 Fr CD (...1 N 4 < E--+ L7 U 0 E. 0 Ch < c9 H
0 U U
0 d AF40UCJAUCh.4 UUCJUeHOWN A 00,ACJE-.4 U
O 0 0 C.)00 0UUHHUU0OH <UU ONHU 00,...
00 UE.<<OD<U<HUU<ON(.9 U0000NuCA
(FU 1-i H < < UUUUUUCFUU
HO E. U 0 0 0 d g U CD CD H CD H E-+ 4 E. E. A 0
A 0 4.qAHAZDOH00E-,E.HUEJ00 CiEJOHE.J.....<0E.
E. 4 0 A 4 U < U 0 0 U 0 < E-, 0 0 4 4 U H E-. E-. U < E-. H 0 E. < <
0 0 E. U 0 u 0 W ,r H C.7 U U C.) oc E-, CD F. L) CJ C.) H gq Ci 0 H 0
U 0 0 H 0:qt,UUL)1.)UKC U 00 U OUC1 Ea 000 0 U 0. g u
r_i Ch .t (70H<U0E. E. E-, U 0 < U 0 gqou ,-quu,4 U 0 0 U H
,t 0 U d E-1000<0U<U0.<0<4,00 HH0E.<0H<E.
OH' U UUU<OO<U<E.<0000UUHUU< Kg AHOU0
0 E. 0 0000000HOHLJOL10.4.4E-40H0FAOHOUA
0 E. ,i= 0 < U 0 C9 E. L. U 0 E.
a( U < U U U U E. CJ CJ U E. U < U U CJ
A U ,= U000.4 at E. 0 < N E. E.
C7 (7 d HUOU00E-, < < 0 E.
O -..,q H 4!0oE-,ouHUU0
6tHCJOU HUOatOrt HHOUat
F4 Pc 0 D 0 e. g: F-4 pg C) u CS C.) 0 U 4 t. < E. E. < H 0 u cp 0. E.
0 0
0 U 0 U 0: ,..., E. NAOHE-.0
AUCJC/HUE..40E-,..,00.110A A
E-, 0 0 4 0 0 E-, U U 0 E. .4 0
< U 0 < Q 4 0 0 HU,G0 U a. H, E. 0
U 0 H 0 0 U E.
< 0 E. (E-.) EC-7' 8 3 8 Ec2 '6 'il4 ti
t-D' 8' 8 2 N it', ',." 8 8 -'. E. A H. . H
H E. 0 4 0 0
04!C7U0C74!HE.C7UUOUU0UHUUHE.0
Ch U a' E-I U < C_7 U E. 0 0 H H H 0 U
< 0 E. kJ H < U 0 0 E. H d H
H 0 d (7 0 EJ 0 u CJ U CJ H CJ H E.
E. E. 0 H at ac 0 0 < at 0 H 0 U
E. 0 H E. H HO 0-F-'
,ri:ogyugovHOK4,o,--¶--, HOU [J
g4 K4 E. EA < CJ U 0 E. 8 E. U 0 U 0
H < C.) U E. E. 0 U (9 H E-, 0 0 U
0 Ci a=C OUU0a,
2.< E. U < u 0 u H 4 Ei u r..)
4 0 A 4 0 Ft g gC
U HO ,C Ci U U ' U 0 E-. E. C.) u
C./ E. g u 0 ,: E-4 0 H 4 E. U E. 0 E. E.
0 A 0 0 0 < E-, A 0 A 0 0 A E., U 0
U E-. 0 4 I. A U 0 H A A A E.
.FU C) 0 0 U U U C7H00 00000E. ..4 CD
< 0 0 d E. 0 at < U
CD Clg g 0 E.-, U H E-, 0 U g UN g g LD 0 g CD g 1 E.pc
0 C-10 HUH
g CD 0, N U CD H C..) uHue,i,uu,-5. ouHH ust: 4000
000 r", E.U<NR00001UUUKOU UH E0E-100cy
E. E. 0 == 00.4E-, UUE--..1,4.40000 < U E. CJ
E. U u c..) q
A E. E. = A 0 q E-, 0 CD E. a, a, E. E. < H d
E. < < 0 at 0 E. 0 <
0 +, U 0 U U fat H E. E. E. H d d U 4. 0 E. E.
I. < PC .4 E.H 4 (7 <
8 u '' z 0 ,..c EØ4 L--, 000000E-
..40uU0E.L.A00U0u
EJ E.-. C:1 0 0 A U 0 E. -4 0 E. U U 0
U 0 A 0 E. H A A 0 0 0 0 4..) 0 E.
U 0 0 H
H4!UNNE.4!UUUOUH00H4!p4u04!uoutpc..)u
O U E. 0 E-. 01 0 0. 5--, 0 H
0 ,< 0 U H. U E.. E. 0 H < .4 0 0 H H. H 0 E,
E. 0 .4 r4 [--. E. o 0 c4 E--, E-, 0 U
E. A 0 U E. 0 0 H 0 E. H C., E. E-, < H U
< 0 0 cri E. E-, E. <
U f-, .4 .4 C. 0 0 HUUE.J.:G4,4HurcuchuuE.<
, __ -
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
137
E. 4 0 U U 0 0 040E.U0H00400000440E.C.)04u
U U, 4 0 0 U E. U u 0 al E. E. 4 Er. C.7 E. U E. 0 4 E. U CD U U U Ff ,..0
E.
< C9 4 4 F.S.u04000E.00E.4E.0HOU 4 0 4
H 1 c.) 4 0 0 0 4 U 0 (Cl U 0 c.) 4 U 4 H H 4 H 0 4 E. 0- H. A
C.)
0 4 Ci E. U 1A HOC) 0 0 E. U 0 0 E. E. 0 H 0 E.
Cif. uUCJO H E. C.7 U H a. 0 C.) C-, CD 44 0 (Cl 0 E. U U H A E. o
Er, U
El %E-41 8
i u040u0Uuuc)OuPUE.4E.E.)4000
4 0 Pi, g r , C 9 Pc r4 C ) C 2 (Cl CD OH4 E.H.C.300E.HE.
4 E. 0 E. U 3 U 0 4 U U 4 0 H E. E. 4 H. H 4 H 4 F.7 E. 4 E. 0
E., 4 U . E. 0 ( E. 0 E. U 0 4 H C.) 4 U H
0 E. 0 E. A E.7 F. 4 U (41
0, E. 4 U u F.-,00E.Ou0,40HE.E.OE.HE. Hut. U
4000000 E. E. E. U 0 C.) 4 U U 4 4 E-, E. E. U .4 0 0 4 U E.
0 4 U < c!) < U r..) 0 0 CD ft U A H U C.9 E-t E-1 0 E. E 4 H E. H 4 0 4
E. H P.< 0 E. E. 0
E. E. 4 0 U 4 0 .:Dq ill) E74 ti9 8 8 (i-_,) '65 (.1,, 8 8
'(5 EY. 8 8 t; 6' i--', t--,'
4 0 U C.) EJ E. H u ,..i E. H E. 0 Er. E-.,. C.) 4 g
E,
E. 4 E. El 0 4 U 4 E.) E. E. 4 rc 0 U E. u 4 E. 0 U 0 E. U y ro 0 E, 0 Li
1E1(400Ft 0 0 gt U 0 4 C i 0 U E.) 4 E. 0 0 E. 4 0 c.) 4 E.) I. 0
OUE.004 rt:HOUE.E.U000H4UU OHHU00
EA 4 u u A C.} 0 0 : C . - l',',q E.) 4 H. U (..) 4 (Cl El 0 4 A U00019HUO
0 4 0 0 E. C.7 u OU414C;Uu0u(7040 1.5 A A u A q E. A
w g E, A CD C9 C.? E.044000Ht..9E-.1.4E.E. 40404 a,C..)0
C..) 0 F', 0 E. r4 U ,,4 0 0 0 E.--.00E-,PE-,0000
UU0E.0040
r.q E-, U 0 0 -KC 4 0 ,,t 0 0 0 c.) .r E. 0 0 4 4 0 E. E. E. u 4 E. E. 0 E.
4
C) H H H 0 H E. 4UV004H0uuuLD HOHUCJUE.4U0H
4 E. 4 .4 0 0 E. 0 0 CJ U ci 0 4 t..) 0, u U Q.) U 0 4 Er. 0 c...D 0 0 U 4,
4:
0 .4 E. 0 U U a: Q.) E. 0 E. E. H U 0 4 CJOgc0(.744,<CDU<U000
U E. E. 4 0 0 E-, 0 0 4 0 (-) r- LI C.D rc U 4: < C.) C.9 4 H E. 0 E. 4 0
F. 4
O4400HU 40CD4U4E.60UUOUUE.uc..)44H0u
O C.) E. 4 E. 4 0 HEDUUE.C9H C9U044 4HUE40 uHuu
O 4 E. 0 E. 0 u 4 E. 4 0 E. 4 E. u 0 4' 4 E. U E. E. 4 4 u
E. u 0 E. 0
AHAHOAH E. H E. u E.) E. 4 u g u U U 0 E. U C.) U E. U a, 0 U
4 4 U C) E. U E. 0 4 E, 0 4 E. E. E. 0 CD < / f--, u 0 u 0 0 H 4 4 0
E. 0 Ci E. cD 4 CJ 40UHuu04E.UOu E.00404E.H0U
..¶7/00C1E. U I. < U U U u c...q u 4 E. 4 H E. 4 H 0 U 0 0 E. u
ga; U U 0 CD U E.<<CDNE-, 9, U UUE-UP<C9 E. < U 0 4, 0 4
0 0 r...) C.) C..)0U0E.4U C.)040400E.0 .40 00,HE,
0 40 000 4 0 CJ ' <UHE.E.NUC E-,<CDC...DCDUUOU<
0 CD < < 4: CD 0 U U 0 F E. 0 F. U CD r..) 0 4 4 E. 4 0 0 E. E, 4 E. 4
/E. 4 a. u E. CD CJ 0 a: 0 C.) 0 0 4 E. E. 0 C l 0 CD 0 C.) 0 C.) E. U C.)
E. E.
e. E-, 0 E. 4 U 4 4 C., E. 0 0 E. E. E. 0 U a: 0 E. U E. 4 U 0 0 E. E.4
UCJE-.040 E. a, U U U C.) E. 0 E. H E. E-, (9 E. 4 4 0 0 4 4 0 E. 0
E.E.0U<F, E. E. 4 9, ,4 0 < C.) U < U U E. U gE CD < E. U E. CJ 0
4, U 0 0 0 CD CD ,4 E. U u E-, U CD U CD E. < U 0 F E. CD U 0 E. E. CD 0
<FUUUF 0 H 4 4 E. 0 4 0 0 C9 0 E. 4 E. U H E. 0 U 4 E. C.9 E. U
C) 0 4 4 0 0 0 V . 4 0 E. 4 U 4 0 0 0 E. 4 H 0 U 4 t7 4 4 U 4 4
o E, u u u El E. E. 4 C.) 0 E-, H Ei UUHAUCD<HCIE.,<FUE.EDE.
U E. E-, F. 0 ED 4 E. 0 4 U 4 C.9 0 4 E. U 0 U E. 0 4 E. 4 L., 0 H 4 4 4
0 4 0 A A o 0 .4 HUUE.00000U0H4,040U4H044
CD E. U 0 U U 0 40E.HuuluH440t....404010or...)Hu
H F < H F 0 0 0 .4 U C--, c-, r..7 C.) Li Ft, u H H C.), 0 4 4 0
0 0 E.
10C)00H0 E.C.90000, UU040C) 4 C.) E. E. 0 C. C.) 0
CDUCDU<C9 OUU01-, ,:4 400004 4UHEJHUUU
00UUH40 4 E. U 0 H 4; E. E. 1,4 E. 4 0 E. AgoguE, 9
:=,.n t-, E. u ,,c ,,, o U U E. E-, EA E. , a: U < CD E-, E. E. <
< E. H 4 U
U u 0 E. 0 u 0 0 Ci 0 4H0 0000H4Ouu OH 400uu
8 ',-'t 6' x): 8 tj i 0 ,x, E. , u Er, (..J U 0 U 0 4, 0 E. E-. 4
.4 0 0 0 0 U U
0E,E.4UUUOUE.UCDE.44004:0E-,UOu
4 E. C.) 0 U H U C.)00E.04 4 0 U H U E. F 0 E. < < U CD E. H E. (5
44 F 0 < 0 E. CD < 0HHOU4H400H00E.C9E.E-,C) E. H 4 I.
0 H 0 C.) 0 E. 0 C., 0 U E. 4 4 E. 0 0 E. 0 U U 4 Ft E. 0 4 0 0 L.) 0 E.
0 6q u o g (9 E-, 4 UUUOU0C9HE.4E-40 OUE.E.OUUHU
< U CJ U ti r4 U 4 4 0 4404 < U 0 4 E. < < E. 0 E-. E. E.
E. 0 E.
4
CJOUE.0CD<E, < 0 U < < < CO 8 o (E-4 8 ,r5 8 8 `-, 8 '.,? Et?, 8 EY,
,E' E(1, 8 (C-P0' SI
rDE-.00E-400 E. E. 0 4 uguE,c9c..9(.94 upugE.-,uE.C-, f-,
E. E. U 0 U U E. 0 < E.. 44 U H r E. F 4 r..) 0 0 E. Li 0 U 4 0 H E. 0
E. gC E. u 4 0 E. F. 4 c.. E. 0 0 4uo(94c.924clouguE)0A
u 4 u H EJ E. E. 0 E--µ E. E. U U 0 a; CI CO E. E. ii u 0
U H E. 4 0 E. 0
0H4HOCIU 0 et O 0 0 U 0 U U E. 0 E. 0 E. E. u E. C.) U 0 U
4HOEJE.E.0 oru,SUOUE-.4E-.00E-.'0E-.E.u04E.OE-,u
0 4 0 u .4 H 0 E. 4 4 E-.04000E.F.E.00E.040E.0 E-.4
H H U CI 0 4 4 E. OHE.E. A 0400000UUE,4000H A 40
0 8000004 U E.E.0 UUOU40C94.440HuE.0 4u
0 U 0 C./ e= E-, 0 4 Ft r.., 4 0 CD U 4 E.
U Fi 0 E. E. 0 H CJ 4 0 0 E. 0
0 CJ.40UHU co CJUE-4000C90E.E.40E.HUAU(..)4H A 4 0
El E. E. 0 4o4(9 rn H U U El CD CI C.9 CJ CD U E. 0 0 4 0 E. 0 E. [..) 0 u
u
U E-4 E - . U 4 4 0 0 = = 0004,004'E.FE.U.:4:0<<FUCJ u u c_q o
HEØ4H0t1 = 4C.)44E.E.14U44E.U44HHOE.H0E.E.
E.H00040 0 u K.7 E-, E, 0.4 u 4
u E. 4C.,CJUE.40U4 < Hurl;
E.E.E9EHE.Uu Z UU4u40 r0E.E.00E.E.44UU04H
O004C.)E.H0 A 00u0000 U(9044,400HCIU0E.00
U U 4 E. E. 4 E. E. H E. 0 E. 0 E. 0 0 E. 4 0 E. 4 EJ U 4 0 E. 0 E, 0 E.
0 E. E. 0 0 CJ ,4 u a F. E. 0 E-. 4 E. A V U E. U 0 Ct) 0 4. U C.) C.) g 0
< 0
4E-. 40E000 ta k4000a.E.040000E-.=40 0400E+04
u 0 4 0 E. 0 U E. 0 H u E. 0 E. 0 E. U U E. E. 4 L) ,4 E. E. E. 0 0 4 4 0 F.
SUBSTITUTE SHEET (RULE 26)
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
138
.. __________________________________________________
r..) 0 4 H U C.) Ci a, b) E. U a CD E. tD 0 u a a E. CD
H a 0 0
A U E. E. a 0 0 F. (--)aE. E-,q0E.00t.)E.E-i0 A .5
aE.00HE-.
ac-DaaCJOE,u CD a E. 0 a 0 a 0 a a, U E.-, (JAE, U
u CD C.) E. a E. a 0 0 CJ E. I U 0 0 E. C.) U U a 0
Ci 0 A E. 0 E. CD tJ a , F. 0 H CD CD a, U 0 0 a 0 a 0 / U U a U
E. U u 0 a 0 0 E. F. LI 0 0 C-3 F. E.
a,
a a gl. U U E. CJ H H C)0000LJ U0CJHUE.CDF. E-.C.)HC)
0 H. 0 0 E. 0 A U CD0E.E.0a0CDUE.aE-.001.0E.E.U0aE,
0 a cA CJ 0 CI E---. H 0 a H E. 0 a ft 0 V ID E. E. T. ti 0 E. 0 ,a 0 E. 0
HaCJ Cit5C.D0 _____________________________ 0 0 CD a E--. E. a 0 Ft
CD CJ 0 0 0 0 U E-. a 0 u t j E ,
00CD C..1 /you 1. 0 U u u 0 0 4 u 0 4
KC 4 C9 P U P 0
Ug,A0 OU0 1 E-, 0 H 0 A u 0 u g z. C.) E.-, o I-, E. 9 H. U a 0 0
E. u 0 a A a: 0 E. E. E. E. 0 0 0 CD 0
H. F. A 0 H E. C) a 0U a E.
0 E. a 0 F. ch E.0HCJHOU000LJE-.0040E-
,0E-.0cD
I
U < A 1-, 0 < U 0 < E. E. P C.) ft a ch a, a E. U C.) U
U 0 u E. 0 CD 000 a a a E. E. U 4.4 E-, U 4 < E-, E-,
0 H. E.
U E. E. cD U H. I--.HUE.E.H AcJ
UH<OU0 HachUaa
(1,_ , u q 0 Ht_DHHOChE.Hrt auu
a E. CJ Ci C.) E. 0 Chc-JE.HuCJuUL)
00(Daaa0CDE.000
aU0a4C2CDU PHaaE.C9HUCh A H 40E-,Kcul 4 uE., 0
E. 0 E. 0 0 0 t. E-. E-.0-aa0HaEla 0HCJ000a uCiCift
0 U E. E. E. 0 0 a CJE.aa0chaE.C.Da0000Ha0 Ugtue
HuaCJAaCh C7 <000H0HOHL/CJE,H<CJAHHHOO<
.40HUOUCJC) CDE.a0a0E.C.)0E.0UKC00UuCia 4 u E-,
0 F.E.4 H E. 0 0 a 0C-A 0,< 0H E, aPIDEACJE.CD p 0
.000
UaOuth0H0 00E. E.HUclaCJOUHEDUE. a E-, (..) E-i 0
E. a 0 u 0 C.) a CD, (!) E. V a 0 0 0 U CD U Kt: ti a
E. E---. a E. 0 .4 E.
H a, 0 0 0 U E-. a 0 000HaUHluaaH00C9UE. A 001) F-' F-'
0 a a E-. E. U a a 0 E.(DE-.00E-
.a,CDUHCha000E.c.JCJHa'
a000E-.0000 ,E.E.
Cha000CJCJ0000E.0aHal.C3
E.
0 0 El H 1=4 a 0 0 a a <UP U<Alr4U0000E-4<.4 H H U r.)
0 0 A 0/6C 0 U H H UyO<UU H, 0 a a E. E. a a E. a
a CD
E. Kg E. C.) 0 0 E. CI U H U 0 0 H CJ 0 E.
C) a, 0 E. C.) E. 0 E. a 0 ...4 H
a a. E. 0 Ft CD E. 0 .4000E-, 0 0 H 0 0 CD a 0 E. E. E. 0 E. E. H
CJ E.
0 0 E. 0 U 0 E. 0 cJ U 0 E. E. E. E. E. U 0 0 a u c) rt 4 E. E. E. U a 0 0
arDaCJ0,40,Ha E. E. a 0 0 0 0 0 a 0 at E. 0 0 E. E. 0 E. E. a H E.
0 a 0 U 0 0 a, U a U C) a E-, 0 < u ci U U 0 0 A 0 0 0 < 0 0 U U H
HCJOUH0C9,AH CI C9 V E. .4 a, H E. 0 0 ID L. 0 Ft 0 0 U a E. 0 0 CD
E. ,..q.Nuugq,c0 U E, U U 0 < 0 0 CJ u H E-, U 0 E, E-, H a C.) P E. 0
0 a a E. H 0 0 U CD a; 0 0 a; U H E, < a CD C) E. C.) a E. 0 CI 0 E. C.) 0
a
F-. '1. a C) CD .0 0 uucDocuuuu AE,00E-
.4 00 A
U H.E.0cDCJUE. E.aE.E.chuuE.E.u00C9 E-
,4uu 0 E-' Hu
0 E. a E. 0 a 0 E. E-.<0001CJ r4E-4(JuK40E,0g4 u 00,0
C.; aUHUE.E.O auE.E.U.a achCJU)0000a0 ChEt aU
U 0 E. a E. 0 0 c.) Kc ath0a0a, CJOC,DE.CDE.Hat-.E.
aC)ach
a a E. 0 U Er E. CD a 00CDCJaCh0E-.0cDE.HH uucDua u 0 0E,
E. 0 a 0 0 a E. C9 E. H0E-4000000aE.00a-4,0E.F.E.CiaH
E. E. E. 0 0 0 a a E. Ei E. 0 a U U c3 0 a H. 0 U U 0 a E. 0 H ci C.) CJ E.
C.)0a00000a a,00000000a0000000 E.CD0C.D
E.C9a0000,E.Ch PCU0E.00E.H.E.00a00,400 aChua
4V)uachuE.c.) 0 E. H E. ch a 0 0 a u 0 a 0 rt < 0 0 H H CD H
!.2 H E. 0 p50.-40 H U 0 U E. 0 4 CJ A H < 0 U U 0 0 0 f.-4 Li
0 a a
CD CJ E. EA CJ A A U 0 CD 0 (.70554uuE-,(DHOOUC544A<HUE-, 49 54
,,.,4 E-, 0 C..) < ,,,C T.!) H L.) < ,r4 U H a: H U 0 a a H 0 E. E. a A CJ
E-,
a E. EA 0 U CD a 0 a uach(JrcHE-,00HaUatJur.JUE.ci(JyH
U E. E. ch E. H 0 0 u 0 u 0 0 E. a E. CD A E. E. E. 0 0 a A E. C.) 0 U SJ
CD
F. CD tJ 0 a U E. E. 0 1DE..100HuUcDaH000 HUChacha
U Cr) U a E. F. a; H H 0 H ,,4c.),¶,v()U0c.)K4E.A
H E. a H. C;; u
F. 0 H F. 0 0 (..) .4 U UEJ '0g4A(.7E,HclUUUHUH<OH<U
U < H U E, 0 CJ 0 444E-tuutiC--, 4 0 u0 AU UcDE-.0-
ach
,r0U 0E-.0C)E. 0 a u a E--, a U U F. 1--
. , E. E. 0 0 H < 0 0 0 u
U E-, Ci C.) u 0 o U 0 H U 0 U u H L-, U H U 0 U 0 < H A (.9 0 E-,
.4 U U ,=4 (D 4!) U f--, A ,:4 A Er E. a 0 0 0 0 a E. H. CD C.) U 0 U 0 CD
U E.
r. ' 6 P. 8 6 0 F. C9 U E, 0 CD E.
E., E-. CD 0 C 0 H. ci E. a U 0
HUUacJtJUUHUE.E.E.E.c7F.aaChcha
0 E. a a F. a 0 aucJaE.E.a0achauciaUUH0acD.CF.
0 E-+UE-, UUUch F. U 0 0 a E. C) 0 H C, CD U 0 E. a u C.) E. E. 0 0
0
O 0 E. / E. a E. 0
1
0 H H 0 H g H 0 at 0 0 0 0 El PCH0F.E.01-)44
0 AU 0 a a 0 E-t a u 0 0 CJ H. a, CJ <000Fla H EJ U < 0 al
O HA E-, 0 H U gq E.000E. CDHE-
.0C.)0F.a0C-DaHIDHaE.
Cl E, Ei ft U H 0 a; 000F E.ft U 4 00< E.-
.uuu H04E-, 4(...0
H E, a E. ft 0 U a u U a a 0 U 0 E. 0 CD 0
0 CD 0 0 H a CD g 0 0, a
H E. a ch U 0 0 U 0 art0HE.E.E.00 ur-
140y0c0 04 0
CJ C., 0 < 0 a 0 F` C., 0 (5 C H (-) U P U 0 C,C.) 0E.E.0a
am, 4
E. E. E. E. 40 E-. [-, 0 0 C.) E-, 0 r.. . .9 ( 7 CD u u 4 0 u K4
Ki (J E-, K. E.
E. a E. H a CD t) 0 FtHO C.)0lJE, 4u0c.90 .< Kcc..)E ,
u
O 0 0 E. E. U E. cn U Ch 0 u C.) ch E. E, E.
0, E -. ..: u I -. 4 9
0 CD E. 4 E- . U C...) m E. CJ Fg 8 H. H. E-,-. E. a
CJ a 0 H E. H <
E. a U a 0 - 0 0 U
EEc,,,t-i,c3., 6 6 6 ';'4 t-A 8 0 Et (i (-i 8, t8J P . 0õ' op' E t ;- , ' '
, ,H1 6' N 1
E9 (.., 8 8' rl 8 '(', -,) (E.% -'. t.," 8 6 i-',
r.j4 [ ; 8 El 14 8 ti: 6 'El E. u
I
o= puo,,,,,,,E,., u H HUUU E. < 4 E . 0 p E. -, c / i -_,4 ,6 t . ; g
f_c, ).,
Ha rg E,....00 K. 4 0. E. E-E r4: E-1 v400000E-4E.
E. 0 u E. E. E. 0 E. E. v-a H 0 U U Pe, a a CD a a 0 0 E. a H 0 H E.
a E. a H a 0: 0 0 H cri E. U E. 0 a u ch a u0 00uu 40 0 to E-. E-,
SUBSTITUTE SHEET (RULE 26)
GTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAA
GCGGTTAG
CTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAAT
TCTCTTAC
TGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGA
CCGAGTTG
CTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCT
TCGGGGCG
AAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCT
TTTACTTT
CACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGA
ATACTCAT
ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAG
AAAAATAA
ACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACC
TATAAAAA
TAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAG
ACGGTCAC
AGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGG
CTTAACTA
TGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATOCGTAAGGAGAAAATAC
CGCATCAG
GCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCG
AAAGGGGG
0
ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG
0
CSEQIDNO.:40
CO
Cl) AATTCTAATACGACTCACTATAGGGAGACGAGAGCACCTGGATAGGTT
0
SEQIDNO.:41
CAD IA
CD
GCCTGCACCAACAGTMACA
Cl) SEQIDNO.:42
171 CAGGCCCAGGAGTCCAATT
0
rn
0
SEQIDNO.:43
0
7J
TCCCGTCTTTGGGTCANAA
r¨
SEQIDNO.:44
17.! GCGCCGCGGATCGTCAACA
SEQIDNO.:45
ACACGTGCACGGCGGC CAA
ra
0
SEQIDNO : 4 6
TCGCGCGTTTCGGIGATGAtGGTGAAAACCTCTGACACATGCAGCTCCCC-
GAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGA
GCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGICGGGGCTGGCTTAACTATGCGGCATCAGAGCAGAT
TGTACTGA
GP.GIGCACCATATGCGGIGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCCCATCAGGCGCCATTCGCCATTCA
GGCTGCGCA
ACTGTTGGGAAGGGCGATCGGTGCGGGCCICTTCGCTATTACGCCAGCTGGCGAPAGGGGGATGTGCTGCAAGGCGATT
AAGTTGGG
TAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTITCCAAAP.AACTACCGTTGTTA
TAGGTGTCT
CTTGAACACCTATA.n.CAMCGTAGTGGATCCCGCGTCCITTCCACAAGATATATILAACCLA_AGAAATCGAAATACT
TTCAAGTTACG
GTAAGCATATGATAGTCCATTITAAAACATAATTTTP_AAACTGCAPACTACCCAAGAAATTATTACTTTCTACGTCP-
CGTATTTTGT
ACTA.ATATCITTGIGTTTACAGTCAR_ATTAATTCTAAITATCTCTCTAACAGCCTTGTATCGTATATGCAAATATGA
AGGAATCATG
GGAAATAGGCCCTCTICCTGCCCGACCTTGGCGCGCGCTCGGCGCGCGGTCACGCTCCGTCACGTGGTGCGTTTTGCCT
GCGCGTCT
TTCCACTGGGGAATTCATGCTTCTCCTCCCTTTAGTGAGGGTAATTCTCTCTCTCTCCCTATAGTGAGTCGTATTAATT
CCTTCTCT
TCTATAGIGTCACCTAAATCGTTGCAATTCGTAATCATGTCATACCIGTTTCCTGTGTGAAATIGTTATCCGCTCACAA
TTCCACAC
cn
.RAcATAccheccGep.Accth,AAGTGTAAAeccTeGeGTGccTAATGAGTGAccTAAcTcAcATTAATTGcGTTecG
crcAcmccc
GCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTG
GGCGCTCT 0
C1:1
TCCGCTTCCTCGCTCACICACTCGCTGCGCTCGGTCGTICGGCTGCGGCGAGCGGTATCAGCTCACTCP_AAGGCGGI-
zkATACGGTTA n.)
cn co
TC CACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGC
C.AGCAAAAGGCCAGGP.ACCGTAAAP.AGGCCGCGTTGCTG
-71
GCGTTTITCCATAGGCTCCGCCCCCCTGACGP.GCATCACARAAATCGACGCTCAAGICAGAGOTGGCGAAACCCGACA
GGACTATAA
0
AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCG
CCTTTCTC
CCTTCGGGAP,GCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG
GGCTGTOTG n.)
cn
CACGAACCCCCCGTTCAGCCCGACCGCMCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGP-
CTTATCGCCZ- 0
CTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTP
_ACTACGGC n.)
TACACTAGRAGAP.CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTICGGAWAGAGTTGGTAGCTCTTGATC
CGGCAAA .. 0
AAAACCACCGCTGGTAGCGGIGGTTTTITTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGA.AGAT
CCITTGATC co
TTTICTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATITTGGTCATGAGATTATCAAAAAGGATCT
TCACCTAG
n.)
C ATCCTTITAAATT
'AAAAgTGAAGTTTTAAATCAATCTAAAGTATATATGAGTAARCTTGGICTGACAGTTACCAATGCTTPLATCAGT
r-
GAGGCACCTATCTCAGCGATCTGTCTATTTCGTICATCCATAGTTGCCTGACTCCCCGTCGTGTAGATPACTACGATAC
GGGAGGGC
TTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCT CAC CGGCTC
CAGATTTATCAGCAATAAACCAGCCAGCCGGA
2)
AGGGCCGAGCGCAGAAGIGGTCCTGCAP6CTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGPAGCTAGAGTA
AGTAGTTCG
CCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCAT
TCAGCTCC
GGTTCCCAAcGATcAAGGCGAGTTAcATGATcCCCcATGTTGTCcAAAA;U,GCGGTTAC-
CTCCTTCGGTCCTCCGATCGTTGTCAGA
AGTAAGTTGGCCGCAGTGTTATCACTcATeeTTATGGCAGCACTGCATAATTcTcTTAcT=ATGccATccGTAAGATGc
TTTTCT
GraAcTcGTGAGTACTrP
4CCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAAT
ACCGCGCCACATAGCAGAACTTMAA.AGTGCTCATCATTGGAAAACGTTCTTCGC-GGCGA;a-
A.ACTCTCAAGGATCTTP.CCGCTGTTG
-
CA 02785046 2012-08-02
PCT/CA2007/000210
WO 2007/093042
141
_________________________________________________ i
gur...)...quouout,opociur.
u 0 U 0; U 0 CI T.9 U U U 0 U U U EJ E. 0 E.
I54UUUUU
E?, tCjD 8 g EY, il b' ill 8 8 8 '6 8 8 ti 8
0 E. .4 0 < H UUUUUUUUUO
E.E.urJuU04.0H<E.HUQU0,0
FI E-..4UDEDE.OchouUou5440
5400 UE...4E-louour.7(.70uu0E-.
UUHUUUUU g 5
<4 E- , Ci E-, (DE.uUtD0c.DuchUuu00, -,
0 UU0000UUHrc<
HHU < H, 0 U 0 CJ U E.
u u-, 0000 0 u u u
c2<UUUH 4:0c.D0c9UUFC< H
O u U E. U E. CD U ci U E. U < 0 0 <
HE--,HUrCE-, <04:0U0E-,HU UE-,
UHHHHUUUUUUUUUU
E. E. UUHUUUHUHUOHU
HE-.0u0HE-,<EDUr_DU<POU Hrg
PHOO<HP<UOuyCJUHUE.00;
0 CJ 0 E. CJ CJ CJ EJ 0 U 0 U E. 0 E.
U 0 E. < 0 H 0 0 0 C) 0 u E. H H
UUUUUUUUUUUOU
U= EUUUUHUUUHU
H= E, CDUUugu0,000cJEJOHuc.,
HUE.,<U0CDHUE3UUraciUCJ E. 4 4
(JE-, i E--.E.E-.4u540oViuvuE.uE,
.4u 0, ,,,C0uE.HU .0ciu<00 E.
0 u E. U u 0 cJ 00000g E-, t-,
E. < CD 0 E. U CD 0 < U U (9 t. 4.
U 0 g U U0E-14:0u0cDCJUu<HFU
E.E.E.00UHUOU(Dua, V (.7 C.!) C9 H U
g E. E-i C.9 g U E-, U U a, E. 0 u E. E. CJ E. 0 a
U0E-.<<HuUUUuuLEULDUE.E.
O H < 0 4, U CD rC U E., E, U F,C CJ 0 FE.
4 E. 0 0 E. 0 CD C.J 0 0 0 0 4 E. CD 4 CI 0
U 040E.4E.uourf uouuu44
E-, H 0 E. < OUNOUHU a, U cd 04 4. 4.
E.C<UrCUOUEDuouU0,-CFUE-,E.
uchrc UHU<UU0000(DUUEJHEJ
E. = C.) 0 U 0 UOUH ED U E. C) U)
OUHU<UO<UUCDUUCJOE.4HA
E. g E. 0 c-D C-) g U .: C- , U CJ E--, E. C., F, Q 0 C.>
kJ 0 F4 0 < E. H U 0 < 0 E. CD El C.) U U E. 0
0,14 UHUUUUHUUUUHH
4 0 < 0 0 0 4 U 0 ,_< CJ V CD U 0 U 4 0 0
UCDCJUO< f"UUC-1 CDC 7 0 ouout.54
u u 54 u 4 u (.7 u u u u u u u a; 4 E, H CJ
CJ 4 H CJ U CJ < LI U 0 CDOE.04044E,
44 <FOLUU<UCD<CDUOUrc pc H
UE.004000000CDUCDU4HUH
O ,uq o u u f. E-, u 4 4 E. UHUU<.CDE.
E. 4 U U U E. 0 CD CD 0 E.54E.7uouE., E.
O 0 0 U HUU CD U 0 U 0 0 U E. 4 H
UCD<QUUHUHU<<CJOH044E,
E. 0 CD 0 E. gl, U C., 0 u 0 0 cD 0 0 < 0 0 ,4C
U4H Hup,c0<UULDEJCDUcDcD0E.c.,
< < 4(70E-.4E.704uuElugE744.7,
urpCDUC90000CDUCJO <0 .5
U HO rCuHre,c_DUcicDcauurr<
u U0UuuciPE-, E. E. H 0 E. g E. 4 ,7(,1
E-,E.OU E.U000< HUCDOUEIE.0
0 0 E. U 0 U 0 CD Ca C_D 0 CD 0 U U E. < 1 E.
E.E.7E-.54 0 CJ 0 U U 0 0 0 0 < < 0 U U. CD
CDCIHHE.UOU<24HUE.00,4 al u
E . CJ V 0 V C,7 0 CJ ,... 0 0 0 C..) U 0 0 u E-,
4 0 E.C1E.UOU
O E. H 4 4 H V kJ CJ0u000uucDUH
O 0 U u U E.C.DUOUCJUOU< E-, E-, u
E. ouuuuu4 c940uuuUouE. C.)
1
H C) C) 000 Oa UPCOUCD,CCJUUHU
O KCHHOU<UCDUUCDOUCDU<4 0
µCUULDCDUUU<UCDE, 4,<HOCD04 4 CD
U0E. F,000E.404000 004 4
U 49 <4 OHOHOUVIDOOUth< E. U 0
E-, < E. CD 0 < CJ < U E., g E. UUOU 4 0
41
.-4E-., g .K4 0 U 0 01=4 O OgUUUUg
0 0 E, 0 UN.. UOUUUU- gUOUU E-, U
1=4 0 .4 0 U E-, CJE,00,<UUCDE.4UCJ4 E.
SUBSTITUTE SHEET (RULE 26)
SEQIDNO , :47
TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTA
AATGGCCC
GCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTC.AATAATGACGTATGTTCCCATAGTAACGCCAATAGGGAC
TITCCATTG
ACGTCAATGGGIGGAGTATTTACGGTMACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA
TTGACGT
CAATGACGGTAAATGGCCCGCCTGGCATTATGcCCAGTACATGAcCTTATGGGAcTTTccTACTTGGCAGTACATCTAC
GTATTAGT
CATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCA
AGTCTCCA
CCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCA
TTGACGCA
AATGCGCGGTAGGCGTGTACGC-TGGGAGGTCTATATAAGCAGAGCTC-
GTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGACTCA
GATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGACGGTACcGcc-
GGCCCGGGATCcACCGGGGCCGCGACTCTAGATCATAATCAGC
CATACCACATTTGTAGAGGTTTTAcTTGCTTTAAAMACCTCCCAcACCTCCCCCTGAACCTGAAACATAAAATGAATGC
AATTGTT
GTTGT TAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCAT CACAAATT
TCACAAATAAAGCATTT TTTTCACTG
cn
CATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGCGTAAATTGTAAGCGTTLATATTTTGTTAAAATTC
GCGTTAAA
03
ITTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAP.AATCCCTTATAAATCAAAAGAATAGACCGA
GATAGGGTT
(/)
GAGTGTTGTTCCAGTTTGGAACAAGAGTC
cACTATTAAAGAAcGTGGACTccAACGTCAAAGGGcGAAAAACCGTCTATCAGGGcGA
0
:71
TGGCCCACTACGTGAACCATCACCCTAATCAAGTTTITTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAA
GGGAGcCC
co
CCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGG
GCGCTGGC
AAGTGTAGCGGTCACGCTGCGCGTAAcCACCAcACCcGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACT
TTTCGGGG 0
Cr)
APATGTGCGCGGAACCCCTATTTGTTTATTTTTCTI4AATAcATTCAAATATGTATCCGCTCATGAGACAATAACCCTG
ATAAATGCT
TCAATAATATTGAAAAAGGAAGAGTCCTGAGGCC-GPAAC-
APiccAGCTGTGGARTGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGC
n.)
rn
TCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAIATTAGTCAGCRACCAGGTGTGGAAAGTCcCcAGGcTCCCCA
GCAGGCAGA 0
AGTATGcAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCcCGCCCCTAACTCCGC
CCAGTTCC n.)
GCCCATT CT C CGCCCCATGGCTGACTAATTT TT TTTATTTATG CAGAGGCCGAGGCCGCCT CGGCCT
CTGAGCTATTCCAGAAGTAG
TGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAGATCGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAA
CAAGATGG co
r-
ATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACP.P_CAGACAATCGGCTGCT
CTGATGCCGC
CGTGTTCCGGCTGTCAGCGCAGGGC- CGCCCGGTTCTT TT TGTC AAGACCGA CCTGTCCGGTGC
CCTGAATGAACTGCAAGACGAGGC n.)
cr)
AGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGP-
AGGGACTGGCTGCT
A T TGGGCGAAGTG C CGGGGCAGGATCT CCTGT CATCT CA CCTTGC TCCT
GCCGAGAAAGTATCCATCATGGCTGAT G CAATGCGGCG
GCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGARACATCGCATCGAGCGAGCACGTACTCGGATG
GAAGCCGG
TC TTGTCGATCAGGATGATC TGGACGAAGAGC AT CAGGGGC TCGCGCCAGC C GP.AC TGTTCGC
CAGGCTCAAGGCGAGCATGCCCGA
CGGCGAGGATCTCGTCGTGACCCATGGC GATGCCTGCTT G CCGAATATCATGGTGGAAAATGGCCGCTTT
TCTGGAT T CAT CGACTG
TGGCCGGCTGGGTGTGGCC-
GACCGCTATCAGGACATAGCGTTGCcTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGA
ccecrrcorccrccrrrAce-
GTATcGccGcTcccGATTcGcAGcccArrcGccTTcTATceccrrctrGAccAorrcrTcreAcceGG
ACT CTGGGGTTCap
4ATOAC_'CGACCAAGCC.',ACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCIATGAAAGGTTG
k=.)
CA 02785046 2012-08-02
WO 2007/093042 PCT/CA2007/000210
143
0
.11.,õc, flc 0
0)0E--.4a a pc 00 0 0 a.
(34; U 0 rn a. P4 ri 4 0 0 .Z
O fa4 tal a+ U) 1-1 PI U) CD 4 F.I 4
O ui 0 o=C [..1 p4 ,-1 p4 ,-4
a+En
FS al ic) U .-1 H 0 cn al
co
a. U a, F.
H = = pt 0 51 E. En CI KS H a. [4 PC crl ri)
rd = ,-1 ,-1 < ,-.1 0 2 a I:4 C.,.1 P4 E. < 41
U0 ,-IZCIlfaci 1..¶,) P. 0 a. 0 0 0
HZ VI > 0 pc a > -4 al cn F. cn 0 4 a.
4) f:) H 0 a 0 En
OH EnE-. P.,>.<ZUr.(4000U
O0 V,P0..XU0F., Ug0 0 aZ
'c) Crl c4 H ri4 E-I
H ul
0 E. U u 0 0 g 0 0 0 F. 0 U H 0 0 0 U U U U 0
OUgU00
(.7 0 0 (.9 0 0 U 'H < (J c. 0 00 00000000000
OHUE-.0<<OHOE.E-.<0 0 U C.) 0 0 0 0 F. 0 cJ 0
000000u 4 (.9 0 u
0 u 0 U 0 E. 0 0 a: <0000<UUOUU
E.0:0UUUOUUUU
U<PCOPUFE <0000 HU C)U<HOKCOUOOPC
00000000HE-.<000 al 00HUPC 0 PC g 0 PC
E-c 0 0 CJ U 0 U 0 U C I 0
KCUOUE.U.PCOU04E-.00 g<00000E-.Cic.)0
0 < 0 0 < E-. C) 0 E. 4 0 0 0 < 0 0 0 C.) 0 0 H. 0 0 0 KC
U U 0 El 0 P <000000PCUPO
00000000000:
0 <000400 0 0 PC rOOP<PCDC_DO0
OHE.0100E-.00 HUH U 0 U U 0 U 0 E. 0 0
H<<OH C.,,<<0;d0<0 , 0 0 < g 0 U U < 0 0 -
E. pc u 0 0 U H 0 0 u PC KC00E-.0 Ouci0r4
C.) 0 U u 0 U 0 0 0 < < 0 u U 00 0 (,) 0 0 U 0 CJ 0 0
EJ U 0 0 0 < 0 < uH01-.0000000
Fo0r3FigHU 0 <PS 0 .= a Fl 0 H EJ C) CJ c) u E. 0 E-.
0ggc0u0E-Tr_DE,0(.9E-40r-+ a. 0 U 0 0 c.7 C.) 4 LI U f-,
0 < 0 0 0 E. C.) a: 0 < H 0 0 C) El pc 0 0 1
O <KC PC g HE-.4:Hour.Dc.) goggurDc9clur.9
=
0g000 E-, OUO<E-.00 0 U U 0 u u u 0 U 0
FigUU0 HH,OU0.:< g4 E. H000400000
i 00000000E-100
H010000:0000E.
E.E.C.10 E.E.1.0000U H u U U KC 000E-.001
4,100 0000C)C.) 0 0 HU FE 0 0 KCC)guut,
C./ < CJ (.1 E-c U 00000000 <000000000E,
E--.U00UHC)(3000 a: UPPCJUE-cUP<CDPE
CJ 0 < U P U 0 0 0 0 0 0 P 0 P OUKCOOCJOHO Pr
H0H0<0<0000 0 g E, 01.7 0 0 0 0 0 4 0 06
4 U 4 0 4 4 U E-cPCJUPU 0 KCO<H<KCE-10 HU
OKS0000000<< Uu chuucJC)UOUO=uF.
00UKCE.<FSE-.<<c) uu <0(700000000
CJ P U P C.J U t.7 Ci 0 0 U 0t-.0000040E-.<
0 P 0 u 0 u al 0 0
U F. E-4 c.) E. 0 0 El guur.90g 0 0 U 0 U 0 < U U 0 0
O (..) (..) C.7 g U C.) 0 0 0 E-. 0 u a; E--. i--.
(.., g 00 HFS
0000000 -pc El00H UE-.00c.)000c,nou
(J 0UU < H<FS 400 <00 0000000000
FS 0 FS Pc HOUUHUrOU E. C') d g 0 E. 0 0: < E. U
U0U00H0U0F. 0000 00000000000
CJUOP gug FEUOPEPOP Huu000E-.00C90
F. y00 0 0 H 0 0 HOPC04000004
000FE00<0P00
C., Pc Ci 0 CJ 0 FE 0 g 0 pc: Fc 0 0 u ci cJ u u 0 u 0 U
HOOUHP c FCE--c< H <0 E. 0 < 0 0 HP40 PC 0 u
FSOOHHE, UU0U0HE. 00000000C) H
O < 0 E. E-.0 0000000 0000U0UH0 0
E-c pc rc r_l g EJOUHUKC (DUE-. UU<O000 0000
<00 g 0 . P 1 0 U 0 0 PC P E-c 0 0 U U U CJ CJ 0 0 H V
CDOUPCE,E, <P0E-cOCJO 0 PC 0 00U 0000 FE
0 r., (..) U E. E. ,.= 0 c.J < 0 U C9 H E.000H000E.00
H U E. U < H U KC 0 < 0 4 E-. PC E-. 0 0 0 U FS C7 CJ 0 0 U
ugou w ch CD 0 C) E--. 0 U g U 0000000U0UPE PC
0 0 < rS H 0 H < < 0 0 g 0Ug00<000E-100
0 P 0 0 < < 0 0 E-.
U= PEPUPE-cCJUUO<OOP UCIOPCJUUOUE.CJO
U C.) P 0 0 < 0 P < H U 0000E-.,<H0gUUU
Og C_h CJ U 0 E-c E-= E-c C.) 0 0 Pc 0 0 g 4000E.C)<0.t 0
O 00c)<<C)C)<KC r-1 PE P H 0 CJ U C) 0 CJ 0 0 0
C.) U PC
FEFEOCJE-c<OOLIPFEOPP PpCHOFEOUP00PC 0
0 0 CD V FE P Pc 0 < E. E-c 0 E-c P CJOHH00000C)0E-c
0 0 CJ FE E-c Pc 0 E-c P 0 < < KC H. 000000000000
0 4 0 0 < 0 0 0 0 4 0 o 0 u F.E.00<< 0:00HUO
U g 0 0 E. U < < U P < PC 0 0 0 0 4 EJ 0:0000CJUH
c_.) 0 < U < U < g c...q gopc HU 0000000<000 0
00 pc 00E.E. E-. C..) 0 E-1 0 0 0 0 g C.) P 0 g 0 0 U E--. 0 P
H I U 0, E. E. 00011CJOUH 00E+0 000 CDFSL)FS <
HKCE-.E-.C) uchgg goo() ug UOUU00E-.000
HuuH0HUH0E.< U
0 E. E. ugogr.90c9E-, F=4 <00000HE-.00<
C.) PUE--cc.JOOFE <OOP H 0000000.00000
H PUO0P0PUCJOE.P rn 0<PPC000000E-1 0
c'500POFEUEJOPPCPEUP co H<0000 g0C/0
<E.PPrE0E-cPPU0C-IgU == 000000 L ..) 0 u U P <
i 00 H00E-.00U< <000 = E. H U < 0 0 < g E. < U
C...7 H E. E. < F. E. E. 0 E. 0 E 0 0 (J 0 U < 0 U FE E-c 0 0 Li
0<0P0UPPC/0UC)00 Z OgUUU0UU004
E-c E. 0 F4 0 P 00 0-4 E-.0(9E-1E-. .zi 0000 0E-100UKS u
00000000.4E.<< [Jo H 0P000000CJFE00
O000< F.H.E.000< ou 0 00000 000004 C)
O000E--.<00,40c)HFSH al H<0CJE-+E-.E-.E.000 0
cf) g 0, CD 0 U E. 0 U 0 E. 0 <
SUBSTITUTE SHEET (RULE 26)
=
Co4
SEQIDNO :84
Identical to
ATGCCGGCGCTGCTGCC TGTGGCC TCCCGCC TTTTGTTGC TACCCCGAGT CT TGCTGAC
CATGGCCTCTGGAAGCC C TCCGACC CAG SEQIDNO . : 4 9
CCCTCGCCGGCCTCGGATT CCGGCTC TGGC TACGTTCCGGGCTCGGTC TC TGCAGCCTTTGTTAC
TTGCCCCAACGAGAAG GT CGCC MIGSGLAGSGGAGGPSSTVTWCA
AAGGAGATCGCCAGGG C CGTGGT GGAGAAGCGCCTAGCAG CCTGCGTCAAC C TCATC C
CTCAGATTACATC CATC TAT GAGTGGAAA LFSNHVAATQAS LLLS FVWMPAL
GGGAAGATCGAGGAAGACAGTGAGGTGCTGATGATGATTAAAAC CCAAAGTTC C TTGGT CCCAGC
TTTGACAGAT TTT GT T CGTTC T L PVASRLLLLPRVLLTMASGSPP
GTGCACCCTTACGAAGTGGC CGAGGTAATTGCATTGCCTGTGGAACAGGGGAACTTTC CG TACC TGCAGTGGG
T GC GCCAGGTCACA TOPS PASDS GS GYVPGSVSAAFV
co GAGTCAGTTTCTGACT C TAT CACAGTCCTG C CATGA
TCPNEKVAKE IARAVVEKRLAAC 0
VNL I PQ I TS IYEWKGK TEED S EV
CO
LMMIKTQS S LVPAL TD FVRSVHP 0
cr)
YEVAEVIALPVE Q GNI" P YLQWVR
QVTESVS DS I TVL P
0
SEQ ID NO. 85:
.A
rn
CATGTGCCAACATGCAGGTTTG CTCATATNTATAC TT T TGCCATGTTGG T GTGC T GCACCCATTAACT
CGTCATTTAGCAT TAGGTA
TAT T T CTTAATGCTAT C C CTCC CCCCTCCCTC CAC C CCACAACAGTCCC CGC TGGTGTGTGAT G
TT CCCAAAT T TTTT TT T TC TCAT
rTl rrl
CANCATTATCNCTAAACAACATTGAATGAAACAACATTGAGGAT
CTGCTATATTTGAAAATAAAAATATAACTAAAAATAATACAAA
TTTTAAAAATACAGTG TAACAA CTATTTACATAGAATTTACATTGTATTAGGTATTGNANGTAAT CTAGAG TT
GAT TTAAAGGAGGG
GNGT C CAAACTTTTGGC TT CCCTGGGCCACAC TGGAANAANAATTGTC TTGGGCTACCCATAAAATACAC
TAACAATAGC TGATAAC
GA
SEQ ID NO. 86
GC TGATT TACAGAGTTT CCT CCTTATAATAT TCAAATG T
CCATTTTCAATAACAGCAACAAACTACAAAGAAACAGGAAAG TAT GG T
CTACTCACAGA
"t1
=
=
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
145
REFERENCES:
PATENTS:
U.S. Patent No. 5,712,127 Malek et al.. January 27, 1998
U.S. Patent No. 6,498,024, Malek et al.. December 24, 2002
U.S. Patent Application No. 11/000,958 field on December 2, 2003 published
under
No. US 2005/0153333A1 on July 14, 2005 and entitled 'Selective Terminal
Tagging of
Nucleic Acids'
US Patent 6.617 434 Duffy, September 9, 2003
US Patent 6,451,555 Duffy. September 17, 2002
OTHER REFERENCES:
1. Frost H.M.,
1964 Dymanics of Bone Remodeling. In: Bone Biodynamics. Little
and Brown, Boston, MA, USA pp 315:
2. Baron, R., Anatomy and Biology of Bone Matrix and Cellular Elements, In:
Primer
on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, Fifth
Edition 2003. American Society for Bone and Mineral Research, Washington DC.
pp 1-8,
3. Jilka, R. L. et at,, "Increased Osteoclast Development After Esgtrogen
Loss:
Mediation by Interleukin-6", Science 257: 88-91 (1992)
4. Poli. V et al., "Interleukin-6 deficient mice are protected from bone
loss caused by
estrogen depletion", EMBO J 13 1189-1196 (1994).
5. Srivastava, S et al.. "Estrogen Blocks M-CSF Gene Expression and
Osteoclast
Formation by Regulating Phosphorylation of Egr-1 and Its Interaction with Sp-
1", J
Olin Invest 102: 1850-1859 (1998).
6. de Vernejoul, M. C., "Dynamics of Bone Remodeling: Biochemical and
Pathophysiological Basis", Eur J Olin Chem Olin Biochem 34: 729-734 (1996).
7. Netzel-Arnett. S., J. D. Hooper, et al. (2003). "Membrane anchored
serine
CA 02785046 2012-08-02
WO 2007/093042 PC
T/CA2007/000210
146
proteases: a rapidly expanding group of cell surface proteolytic enzymes with
potential roles in cancer." Cancer Metastasis Rev 22(2-3): 237-58.
8. Shan, J.
L. Yuan, et al. (2002). "TSP50, a possible protease in human testes, is
activated in breast cancer epithelial cells."CancerRes 62(1): 290-4.
9. Yuan, L.. J. Shan, et al. (1999). "Isolation of a novel gene, TSP50, by
a
hypomethylated DNA fragment in human breast cancer." Cancer Res 59(13):
3215-21.
Nishi, T. and M. Forgac (2002). "The vacuolar (H+)-ATPases--nature's most
versatile proton pumps." Nat Rev Mol Cell Biol 3(2): 94-103.
10 11. Nishi,
T . S. Kawasaki-Nishi, et al (2003) "Expression and function of the mouse
V-ATPase d subunit isoforms." J Biol Chem 278(47) 46396-402
12. Morello,
R., L. Tonachini, et al. (1999). "cDNA cloning, characterization and
chromosome mapping of Crtap encoding the mouse cartilage associated protein."
Matrix Biol 18(3): 319-24.
13. Tonachini, L., R. Morello, et al. (1999). "cDNA cloning,
characterization and
chromosome mapping of the gene encoding human cartilage associated protein
(CRTAP)." Cytogenet Cell Genet 87(3-4): 191-4.
14. Kawai, J.,
A. Shinagawa, et al. (2001). "Functional annotation of a full-length
mouse cDNA collection." Nature 409(6821): 685-90.
15. Strausberg, R. L.. E. A. Feingold. et al. (2002). "Generation and
initial analysis of
more than 15,000 full-length human and mouse cDNA sequences." Proc Natl
Acad Sci U S A 99(26): 16899-903.
16. Janssen.
E.. M. Zhu, et al (2003). "LAB- a new membrane-associated adaptor
molecule in B cell activation." Nat lmmunol 4(2): 117-23
17. Kawaida, R.. T. Ohtsuka, et al. (2003). "Jun dimerization protein 2
(JDP2), a
member of the AP-1 family of transcription factor, mediates osteoclast
differentiation induced by RANKL." J Exp Med 197(8): 1029-35.
18. Agrawal, N., P. V. Dasaradhi, et al. (2003). "RNA interference: biology,
mechanism, and applications." Microbiol Mol Biol Rev 67(4): 657-85.
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
147
19. Hannon, G. J. (2002). "RNA interference." Nature 418(6894)= 244-51
20. Brummelkamp, T. R. R Bernards. et al. (2002). "A system for stable
expression
of short interfering RNAs in mammalian cells." Science 296(5567): 550-3
21. Elbashir, et al. (2001). "Duplexes of 21-nucleotide RNAs mediate RNA
interference in cultured mammalian cells." Nature 411(6836): 494-8.
22. Lee, J. S. Z. Hmama, et al. (2004). "Stable gene silencing in human
monocytic
cell lines using lentiviral-delivered small interference RNA. Silencing of the
p110alpha isoform of phosphoinositide 3-kinase reveals differential regulation
of
adherence induced by 1alpha,25-dihydroxycholecalciferol and bacterial
lipopolysaccharide." J Biol Chem 279(10): 9379-88.
23. Rubinson, D. A. C. P. Dillon, et al. (2003). "A lentivirus-based system
to
functionally silence genes in primary mammalian cells, stem cells and
transgenic
mice by RNA interference." Nat Genet 33(3)= 401-6.
24. Boyle. W J W S. Simonet, et al (2003). "Osteoclast differentiation and
activation " Nature 423(6937). 337-42
25. Gee et al. In: Huber and Carr (1994) Molecular and Immunologic
Approaches.
Futura Publishing Co., Mt. Kisco N.Y., pp. 163-177.
26. Smith, A. N. F. Jouret, et al. (2005). "Vacuolar H+-ATPase d2 subunit:
molecular
characterization, developmental regulation. and localization to specialized
proton
pumps in kidney and bone." J Am Soc Nephrol 16(5): 1245-56
27. Smith, A. N.. J. Skaug, et al. (2000). "Mutations in ATP6N1B, encoding
a new
kidney vacuolar proton pump 116-kD subunit, cause recessive distal renal
tubular
acidosis with preserved hearing." Nat Genet 26(1): 71-5.
28. Stehberger, P. A., N. Schulz, et al. (2003). "Localization and
regulation of the
ATP6V0A4 (a4) vacuolar H+-ATPase subunit defective in an inherited form of
distal renal tubular acidosis." J Am Soc Nephrol 14(12)- 3027-38.
29. Malkin I, Dahm S. Suk A Kobyliansky E. Toliat M. Ruf N. Livshits C.
Nurnberg P
Association of ANKH gene polymorphisms with radiographic hand bone size and
geometry in a Chuvasha population. Bone. 2005 Feb;36(2).365-73
CA 02785046 2012-08-02
WO 2007/093042
PCT/CA2007/000210
148
30 McMahon C,
VVill A, Hu P, Shah GN, Sly WS, Smith OR Bone marrow
transplantation corrects osteopetrosis in the carbonic anhydrase II deficiency
syndrome. Blood. 2001 Apr 1:97(7).1947-50.
31. Biskobing DM, Fan D. Acid pH increases carbonic anhydrase II and
calcitonin
receptor mRNA expression in mature osteoclasts. Calcif Tissue Int 2000
Aug:67(2).178-83.
32. Brage M, Abrahamson M. Lindstrom V, Grubb A, Lerner UH. Different
cysteine
proteinases involved in bone resorption and osteoclast formation. Calcif
Tissue
Int. 2005 Jun;76(6):439-47. Epub 2005 May 19.
