U.S. patent application number 16/197434 was filed with the patent office on 2019-03-14 for methods and assays for treating subjects with shank3 deletion, mutation or reduced expression.
This patent application is currently assigned to ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI. The applicant listed for this patent is ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI. Invention is credited to Joseph D. Buxbaum, Ozlem Gunal, Takeshi Sakurai.
Application Number | 20190076553 16/197434 |
Document ID | / |
Family ID | 44992247 |
Filed Date | 2019-03-14 |
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United States Patent
Application |
20190076553 |
Kind Code |
A1 |
Buxbaum; Joseph D. ; et
al. |
March 14, 2019 |
METHODS AND ASSAYS FOR TREATING SUBJECTS WITH SHANK3 DELETION,
MUTATION OR REDUCED EXPRESSION
Abstract
Methods and assays are disclosed for treating subjects with
22q13 deletion syndrome or SHANK3 deletion or duplication, mutation
or reduced expression, where the methods comprise administering to
the subject insulin-like growth factor 1 (IGF-1), IGF-1-derived
peptide or analog, growth hormone, an AMPAkine, a compound that
directly or indirectly enhances glutamate neurotransmission,
including by inhibiting inhibitory (most typically GABA)
transmission, or an agent that activates the growth hormone
receptor or the insulin-like growth factor 1 (IGF-1) receptor, or a
downstream signaling pathway thereof.
Inventors: |
Buxbaum; Joseph D.; (New
York, NY) ; Sakurai; Takeshi; (New York, NY) ;
Gunal; Ozlem; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI |
New York |
NY |
US |
|
|
Assignee: |
ICAHN SCHOOL OF MEDICINE AT MOUNT
SINAI
New York
NY
|
Family ID: |
44992247 |
Appl. No.: |
16/197434 |
Filed: |
November 21, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14192924 |
Feb 28, 2014 |
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16197434 |
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13425633 |
Mar 21, 2012 |
8691762 |
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14192924 |
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PCT/US2011/000860 |
May 16, 2011 |
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13425633 |
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61395775 |
May 17, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/00 20130101;
A61K 38/27 20130101; A61K 38/30 20130101; A61P 5/06 20180101; A61K
49/0008 20130101; G01N 33/53 20130101; A61P 25/00 20180101; G01N
2333/72 20130101; G01N 2333/65 20130101; A61K 38/06 20130101 |
International
Class: |
A61K 49/00 20060101
A61K049/00; G01N 33/53 20060101 G01N033/53; A61K 38/06 20060101
A61K038/06; A61K 38/27 20060101 A61K038/27; A61K 38/30 20060101
A61K038/30; A61K 31/00 20060101 A61K031/00 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
number MH093725 awarded by the National Institute of Mental Health.
The government has certain rights in the invention.
Claims
1. A method for treating a subject with 22q13 deletion syndrome or
SHANK3 deletion or duplication, SHANK3 mutation or reduced
expression of SHANK3, the method comprising administering to the
subject insulin-like growth factor 1 (IGF-1), an active IGF-1
fragment including the tripeptide (1-3)IGF-1 or an analog thereof,
growth hormone, an AMPAkine, or a compound that enhances glutamate
neurotransmission, in an amount and manner effective to treat a
subject with 22q13 deletion syndrome or SHANK3 deletion or
duplication, mutation or reduced expression.
2.-4. (canceled)
5. The method of claim 1, wherein the analog of (1-3)IGF-1 is
selected from the group consisting of (1-3)IGF-1 amide, (1-3)IGF-1
stearate, Gly-Pro-D-glutamate, glycine-proline-threonine
(Gly-Pro-Thr), glycine-glutamic acid-proline (Gly-Glu-Pro),
glutamic acid-glycine-proline (Glu-Gly-Pro), and glutamic
acid-proline-glycine (Glu-Pro-Gly).
6. The method of claim 1, wherein IGF-1, IGF-1-derived peptide or
analog, growth hormone, AMPAkine, compound that enhances glutamate
neurotransmission, or agent is administered locally.
7. The method of claim 1, wherein IGF-1, IGF-1-derived peptide or
analog, growth hormone, AMPAkine, compound that enhances glutamate
neurotransmission, or agent is administered systemically.
8-13. (canceled)
14. The method of claim 1, wherein the subject is human.
15. The method of claim 14, wherein the subject has autism,
Asperger syndrome, autism spectrum disorder, pervasive
developmental disorder, mental retardation, hypotonia, speech
deficits, or a developmental delay and/or defect.
16. The method of claim 1, wherein IGF-1, IGF-1-derived peptide or
analog, growth hormone, AMPAkine, compound that enhances glutamate
neurotransmission, or agent alleviates one or more of hypotonia; a
motor deficit; absent speech; increased tolerance to pain; thin,
flaky toenails; poor thermoregulation; chewing non-food items;
teeth grinding; autistic behaviors; tongue thrusting; hair pulling;
and aversion to clothes.
17. The method of claim 1, wherein the compound that enhances
glutamate neurotransmission inhibits an inhibitory
neurotransmitter.
18. The method of claim 17, wherein the inhibitory neurotransmitter
is GABA.
19. A method for treating a subject with 22q13 deletion syndrome or
SHANK3 deletion or duplication, SHANK3 mutation or reduced
expression of SHANK3, the method comprising administering to the
subject insulin-like growth factor 1 (IGF-1) or an active IGF-1
fragment including the tripeptide (1-3)IGF-1 or an analog thereof,
in an amount and manner effective to treat a subject with 22q13
deletion syndrome or SHANK3 deletion or duplication, mutation or
reduced expression, wherein the subject has autism spectrum
disorder, autism, Asperger syndrome, pervasive developmental
disorder, mental retardation, hypotonia, a speech deficit, or a
developmental delay and/or defect.
20. (canceled)
21. A method for treating a human subject with Phelan-McDermid
Syndrome comprising administering insulin-like growth factor 1
(IGF-1) to the human subject in an amount and manner effective to
ameliorate delayed speech in a human subject with Phelan-McDermid
Syndrome.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of and claims
priority of PCT International Patent Application No.
PCT/US2011/000860, filed May 16, 2011, which designates the United
States of America, and claims the benefit of U.S. Provisional
Patent Application No. 61/395,775, filed May 17, 2010, the contents
of which are herein incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates generally to methods, and
assays for compounds, for treating subjects with 22q13 deletion
syndrome or SHANK3 deletion, duplication, mutation or reduced
expression.
BACKGROUND OF THE INVENTION
[0004] Throughout this application various publications are
referred to in short form. Full citations for these references may
be found at the end of the specification. The disclosures of these
publications are hereby incorporated by reference in their entirety
into the subject application to more fully describe the art to
which the subject invention pertains.
[0005] 22q13 Deletion Syndrome.
[0006] Chromosome 22q13 deletion syndrome, also known as
Phelan-McDermid Syndrome, was first described in case reports in
the early 90s, culminating in a review of the 24 published cases
and 37 additional cases by Phelan et al. (2001). The studies
conclusively demonstrated that individuals identified with 22q13
deletion syndrome had global developmental delay and absent or
severely delayed expressive speech. Furthermore, the overwhelming
majority of cases had hypotonia (97%) with normal or accelerated
growth (95%). The developmental delay is associated with mental
retardation typically in the mild-to-moderate range. Other, less
universal, features included large hands (>75%), dysplastic
toenails (>75%), and decreased perspiration. Behavior
characteristics include mouthing or chewing non-food items
(>75%), decreased perception of pain (>75%), and autism or
autistic-like traits. Approximately 75% of individuals with a 22q13
deletion syndrome diagnosis have either a 22 q terminal deletion
(i.e., a chromosome break in 22q with loss of the segment distal to
the break), or an interstitial deletion (i.e., two breaks within
the same chromosome arm and loss of the intervening segment). The
remaining 25% of individuals diagnosed with 22q13.3 deletion
syndrome had deletions resulting from an unbalanced translocation
or other structural rearrangement, including ring 22.
[0007] Hypotonia, global developmental delay and speech deficits
together represent some of the most consistent findings, each in
>95% of all patients. The hypotonia in newborns with the
syndrome can be associated with weak cry, poor head control, and
feeding difficulties leading to failure to thrive. In terms of
developmental delay, in addition to the mental retardation noted
above, there is also evidence for a delay to major milestones, such
that, for example, the average age for rolling over is
approximately eight months, for crawling approximately 16 months,
and for walking approximately three years. Poor muscle tone, lack
of balance, and decreased upper body strength contribute to the
delay in walking and ultimately, gait is often broad-based and
unsteady. Finally, while infants with the syndrome typically babble
at the appropriate age and children may acquire a limited
vocabulary, by approximately age four years many children have
significant deficits in the ability to speak. With intensive
therapy, the individuals with the syndrome may have some speech and
increase their vocabularies. It is interesting to note that
receptive communication skills are more advanced than expressive
language skills as demonstrated by the ability of affected children
to follow simple commands, demonstrate humor, and express
emotions.
[0008] Role of SHANK3 in 22q13 Deletion Syndrome.
[0009] Three lines of evidence implicated a single gene, SHANK3
(for SH3 and multiple ankyrin repeat domains 3, also referred to as
proline-rich synapse associated protein 2/PROSAP2), in 22q13
deletion syndrome. First, careful analysis of the extent of the
deletion in independent cases indicated a small critical region
encompassing SHANK3. Thus, an analysis of 33 cases with various
forms of monosomy of chromosome 22 (include ring 22, which as noted
above is phenotypical similarly to the deletion syndrome) showed
that the 12 with simple deletions had deletions of variable in size
(from 160 kb to 9 Mb), with a minimal critical region responsible
for the phenotype including SHANK3, ACR, and RABL2B (Luciani et
al., 2003). Similarly, an analysis of 56 patients with the syndrome
again demonstrated a very variable size of the deletion (130 kb to
9 Mb) with deletion of SHANK3 found in all cases explicitly tested,
including the smallest deletion, with the minimal region
encompassing the same three genes (Wilson et al., 2003).
Remarkably, the severity of the behavioral phenotype was not
correlated with the size of the deletion, indicating that
haploinsufficiency of just one or more of these three genes was
primarily responsible for the phenotype. Higher resolution studies
have now identified patients with even smaller deletions, which
exclude ACR and RABL2B from the minimal region, leaving only SHANK3
as the causal gene for the deletion syndrome (Bonaglia et al.,
2011).
[0010] The second line of evidence was the demonstration of a
recurrent breakpoint in SHANK3 in some cases with 22q13 deletion
syndrome. The first report of a translocation with a breakpoint in
SHANK3 associated with 22q13 deletion syndrome already made the
point that disruption of SHANK3 likely underlied the disorder
(Bonaglia et al., 2001). This group went on the identify two
additional cases (Bonaglia et al., 2006), both with a breakpoint
within the same 15-bp repeat unit in the SHANK3 gene (which
overlapped with another SHANK3 breakpoint described by Wong et al.,
1997). The presence of recurrent disruptions in SHANK3 led to the
conclusion that disruption of this one gene is sufficient for the
generation of 22q13 deletion syndrome.
[0011] Role of SHANK3 in Autism Spectrum Disorders (ASD).
[0012] Mutations directly in SHANK3 also resulting in the main
features of 22q13 deletion syndrome represent the final line of
evidence. Thus, while it has become increasingly recognized that
22q13 deletion syndrome can present with ASD and in fact 22q13
deletions are commonly associated with ASD in literature surveys
(Vorstman et al., 2006), three recent studies explored the separate
question as to whether SHANK3 disruption and mutations can be found
in cohorts with apparently idiopathic ASD. In the first such study
(Durand et al., 2007), SHANK3 was analyzed by both FISH and by
direct sequencing in as many as 227 individuals with ASD. Three
variants were identified. First, an individual with a de novo
deletion of SHANK3 was identified; this individual had autism
(narrowly defined), absent language, and moderate mental
retardation. Second, a paternally inherited translocation was
identified that resulted in a deletion of the 22q13 region
(including SHANK3) in a girl with autism and severe language delay,
and a duplication of the same region in her brother with Asperger
syndrome. Finally, Durand et al. (2007) identified two brothers
with autism, severely impaired speech, and severe mental
retardation, which carried a single-base insertion in SHANK3. The
insertion, which was maternal in origin (likely due to germline
mosaicism in the mother), resulted in a frameshift at the
COOH-terminal of the protein that disrupts domains involved in
Homer and cortactin binding and the sterile alpha motif (SAM)
domain involved in assembly of the SHANK3 platform. Overexpression
of the mutant form in cultured hippocampal neurons did not lead to
synaptic localization of the heterologous protein, in contrast to
the wild-type SHANK3 protein.
[0013] In a follow up to Durand et al. (2007), Moessner et al.
(2007), examined both sequence and SHANK3 gene dosage in 400
individuals with ASD. Two deletions were identified, as well as 1
de novo mutation. Furthermore, an additional deletion was
identified in two siblings from an additional collection. The
mutation, found in a girl with autism, results in a Q321R change in
the ankyrin repeat domain at the NH2 terminal of SHANK3.
[0014] In a third study, Gauthier et al. (2009) sequenced SHANK3 in
427 ASD subjects and identified a de novo deletion at an intronic
donor splice site and a missense variant transmitted from an
epileptic father.
[0015] A de novo splice site variant of the SHANK3 gene has also
been reported in a patient with mental retardation and severe
language delay (Hamdan et al., 2011). In addition, Shank3 mutant
mice display autistic-like behaviours (Bozdagi et al. 2010; Bangash
et al., 2011; Peca et al., 2011; Wang et al., 2011).
[0016] Remarkably, SHANK3 mutations can also result in
schizophrenia, including atypical schizophrenia associated with
mental retardation and/or early onset as recently shown by Gauthier
et al. (2010).
[0017] Altogether, these studies strongly support a role for
disruptions of SHANK3 in developmental delay and ASD. Clearly,
haploinsufficiency of SHANK3, caused either by a chromosomal
abnormality or a mutation, can result in a profound phenotype.
Furthermore, even overexpression of SHANK3 can result in
developmental disorders (considering, for example, the case with
Asperger syndrome and three copies of the SHANK3 locus reported in
Durand et al., 2007 or the case with three copies and ADHD reported
in Moessner et al., 2007). Recent, very large scale studies in
clinical samples demonstrate that ca. 0.3% of patients with
intellectual disability referred to for chromosome microarray have
a SHANK3 deletion or duplication (Cooper et al., 2011). With the
advent of clinical sequencing, point mutations in SHANK3 are also
being identified in the clinical setting and evidence from research
studies indicates a similar rate (ca. 0.3%) making SHANK3 deletions
and mutations one of the more common monogenic causes of
developmental delay syndromes, intellectual disability and ASD.
[0018] Function of SHANK Proteins in the Structure of the
Synapse.
[0019] The post-synaptic density (PSD) is an electron-dense
structure underlying the postsynaptic membrane in glutamatergic
synapses in the central nervous system (Okabe, 2007). The PSD is
most commonly found on dendritic spines of pyramidal neurons of the
neocortex and hippocampus and Purkinje cells of the cerebellum, as
well as on dendritic shafts at sites of contact with interneurons
in the neocortex and hippocampus, as well as motoneurons in the
spinal cord. As such the PSD represents a critical organelle for
glutamatergic transmission. It has been shown that the SHANK
proteins (including SHANK3) are a major part of the PSD. Multiple
analytical approaches, including the characterization of antibodies
directed against PSD preparations, two-hybrid screens, gel
electrophoresis and mass spectrometry and other modern proteomic
approaches have placed the SHANK proteins in the PSD (reviewed in
Boeckers, 2006 and Okabe, 2007). Moreover, recent quantitative
methods have estimated that there are about 300 individual SHANK
molecules in a single postsynaptic site, representing something in
the order of 5% of the total protein molecules and total protein
mass in the site (Sugiyama et al., 2005). As it has been postulated
that SHANK proteins may nucleate the protein framework for the PSD,
a recent study examined the ability of the sterile alpha motif
(SAM) of SHANK3 to form polymers by self-association (Baron et al.,
2006). As with other SAM domains (Qiao and Bowie, 2005), the SAM
domain of SHANK3 was able to self-associate, giving rise to large
sheets of parallel fibers. These studies support the hypothesis
that sheets of the SHANK proteins can form the scaffold or platform
onto which the PSD is constructed. Such a role for the SHANK
proteins has led to them being called "master scaffolding proteins"
of the PSD.
[0020] The SHANK Protein Interactome.
[0021] With the SHANK proteins (including SHANK3) forming a
molecular platform onto which the PSD protein complex can be
constructed, other proteins and protein complexes of the PSD can
associate with the SHANK platform. Of the various protein complexes
associated with glutamatergic synapses, there is good evidence that
the NMDA receptor complex (NRC), the metabotropic glutamate
receptor complex (mGC), and the AMPA receptor complex (ARC)
associate with the SHANK platform (see Boeckers, 2006).
[0022] The NRC (Husi et al., 2000), analyzed after isolation by
affinity purification, includes receptors, scaffolding proteins,
signaling proteins, and cytoskeletal proteins. Amongst the
scaffolding proteins identified in the NRC are the SHANK proteins,
and it is thought that NMDA receptors are anchored to the SHANK
platform through the mediation of PSD-95 and SAPAP/GKAP (see
Boeckers, 2006). Thus, NMDA receptors are tethered to the
postsynaptic membrane by interaction with PDZ domains of PSD-95,
while the guanylate kinase domain of PSD-95 interacts with the
SAPAP/GKAP proteins, which in turn bind to the SHANK proteins.
[0023] Similarly, mGC is linked to the SHANK platform, at least in
part via Homer. The mGC (Farr et al., 2004), analyzed after
immunoisolation of mGluR5 and associated molecules, includes SHANK
and Homer proteins, both of which have been previously associated
with metabotropic glutamate receptors using other methods. Homer
proteins bind the cytoplasmic domain of mGlu receptors (Brakeman et
al., 1997) and couple mGlu receptors - - - and hence the mGC - - -
to the SHANK platform (Tu et al., 1999). As SHANK proteins are able
to bind to the IP3 receptor, this interaction also links mGlu
receptors to the IP3 receptor (Sala et al., 2005).
[0024] Finally, the components of the ARC are bound to the SHANK
platform. There is evidence for a direct interaction between the
GluR1 AMPA receptor and SHANK3 (Uchino et al., 2006). Moreover,
there is evidence for an indirect interaction in which
transmembrane AMPA regulatory protein (TARP) subunits, including
stargazin, bind both AMPA receptors and PSD-95 (e.g., Bats et al.,
2007). The interaction of AMPA receptors with PSD-95 in turn allows
for the linking of AMPA receptors with the SHANK platform via
SAPAP/GKAP.
[0025] There are additional important interactions that involve the
SHANK platform, but even focusing on these three protein complexes,
NRC, mGC, and ARC, it is clear that the SHANK proteins are
critically involved in the molecular architecture of glutamatergic
synapses. Moreover, as SHANK proteins also interact with F-actin
(the major cytoskeletal component of spines) through cortactin
(Naisbitt et al., 1999) and additional mechanisms (see Boeckers,
2006), the SHANK platform is also likely involved in the dynamic
remodeling of glutamatergic synapses over short and longer time
frames (e.g., Hering and Sheng, 2003).
[0026] Modulation of SHANK3 Expression and Synapse Formation.
[0027] Overexpression of SHANK1 leads to increased spine size in
neurons in culture (Sala et al., 2001). This effect, which could be
further enhanced with the cotransfection of Homerl, also led to the
recruitment of Homer, PSD-95, and GKAP to the spines, along with
glutamate receptors, the IP3 receptor, and F-actin and bassoon,
with enhancement of synaptic function, as measured
electrophysiologically (Sala et al., 2001). More recent studies
with SHANK3 support these conclusions (Roussignol et al., 2005).
Thus, introduction of an siRNA construct inhibiting SHANK3
expression led to reduced number of spines in hippocampal neurons
in culture. Furthermore, Roussignol et al. (2005) demonstrated that
the introduction of SHANK3 into aspiny cerebellar neurons was
sufficient to induce functional dendritic spines in these cells,
which then express functional NMDA and AMPA receptors. Altogether,
these studies in cultured cells support a critical role for SHANK
proteins in the development and function of the PSD and the
glutamatergic synapse.
[0028] Recently, SHANK1 homozygous knockout mice were described
which showed alterations in PSD thickness and PSD protein make-up,
changes in spine morphology, and decrease glutamatergic synaptic
strength (but no changes in long term potentiation (LTP)) (Hung et
al. 2008). These changes were associated with an increase in
anxiety behavior, deficiencies on rotarod, impaired memory in a
contextual fear task and in retention in a radial maze, but
increased acquisition in the radial maze, confirming a role for
SHANK proteins in glutamatergic transmission and behavior.
[0029] Regulation of SHANK3 Expression by Methylation.
[0030] Proper expression of SHANK3 is an important element of spine
formation and brain development. Methylation of genes is one
important means of regulating expression. Interestingly, in a
genome-wide analysis, SHANK3 was identified as one of several genes
where there was a clear relationship between methylation status at
CpG islands in the gene and expression (Ching et al., 2005). The
authors demonstrated that SHANK3 is expressed in brain tissue,
where the gene is predominantly unmethylated, and not expressed in
lymphocytes, where the CpG island studied in the SHANK3 gene was
nearly completely methylated.
[0031] The study of Ching et al. (2005) was followed by a more
recent study that looked in greater detail at SHANK3 as well as at
the CpG islands in SHANK1 and SHANK2 (Beni et al., 2007). The
authors identified 5 CpG islands in SHANK3 (one of which - - -
identified by Beni et al. (2007) as CpG 4 - - - was the CpG island
studied by Ching et al., 2005) and an equivalent number in SHANK1
and SHANK2. Only SHANK3 demonstrated tissue-specific methylation of
CpG islands, with a relationship between methylation and
tissue-specific expression. These studies demonstrated not only
that methylation at several of the CpG islands of SHANK3 correlated
with SHANK3 expression, but also that modulating the methylation of
SHANK3 in cells in culture altered SHANK3 expression. Thus,
treating primary neuronal cultures with methionine to increase
methylation resulted in decreased expression of SHANK3, while
treating HeLa cells with the demethylating agent 5-AdC resulted in
decreased methylation of SHANK3 and increased expression of this
gene in these cells, which do not normally express SHANK3.
Significantly, the decreased expression of SHANK3 in primary
neurons treated with methionine was associated with decreased
numbers of dendritic spines and with decreased spine width, similar
to what was observed by this same group with siRNA treatment of
such cells (see above and Roussignol et al., 2005).
[0032] It has been shown that a proportion (0.5-1%) of children
diagnosed with autism or autism spectrum disorders have deletions,
duplications or mutations in SHANK3. While individuals with a
diagnosis of 22q13 deletion syndrome are relatively rare, autism
and autism spectrum disorders occur with a frequency of about 1 in
100 children. Considering this, as well as the rates of
intellectual disability syndromes in the population, it can be
estimated that at least 1/6,000-1/16,000 individuals will have
deletions, duplications or mutations in SHANK3 with associated
phenotypes. This translates to .about.20-60,000 individuals in the
USA alone with life-long disability due to alterations in SHANK3
expression. Thus, there is a compelling need for treatments for
subjects with 22q13 deletions or duplications or SHANK3 mutations.
The present invention addresses this need.
SUMMARY OF THE INVENTION
[0033] The present invention provides methods for treating subjects
with 22q13 deletion syndrome or SHANK3 deletion or duplication,
SHANK3 mutation or reduced expression of SHANK3, in need thereof,
the methods comprising administering to the subject insulin-like
growth factor 1 (IGF-1), an active IGF-1 fragment including the
tripeptide (1-3)IGF-1 or an analog thereof, growth hormone, or an
AMPAkine, or another compound that directly or indirectly enhances
glutamate neurotransmission, including by inhibiting inhibitory
(most typically .gamma.-aminobutyric acid (GABA)) transmission, in
an amount and manner effective to treat a subject with 22q13
deletion syndrome or SHANK3 deletion or duplication, mutation or
reduced expression.
[0034] The present invention further provides methods for treating
subjects with 22q13 deletion syndrome or SHANK3 deletion or
duplication, mutation or reduced expression in need thereof, the
methods comprising administering to the subject an agent that
activates the growth hormone receptor, or a downstream signaling
pathway thereof, or the insulin-like growth factor 1 (IGF-1)
receptor, or a downstream signaling pathway thereof, or a
downstream signaling pathway of (1-3)IGF-1, in an amount and manner
effective to treat a subject with 22q13 deletion syndrome or SHANK3
deletion or duplication, mutation or reduced expression.
[0035] The present invention also provides methods for screening
for agents for treating subjects with 22q13 deletion syndrome or
SHANK3 deletion or duplication, mutation or reduced expression, the
methods comprising determining whether or not the agent enhances
long-term potentiation or increases glutamate transmission, wherein
an agent that enhances long-term potentiation or increases
glutamate transmission is a candidate for treating a subject with
22q13 deletion syndrome or SHANK3 deletion or duplication, mutation
or reduced expression, whereas an agent that does not enhance
long-term potentiation or increase glutamate transmission is not a
candidate for treating a subject with 22q13 deletion syndrome or
SHANK3 deletion or duplication, mutation or reduced expression.
[0036] The present invention also provides methods for screening
for agents for treating subjects with 22q13 deletion syndrome or
SHANK3 deletion or duplication, mutation or reduced expression, the
methods comprising determining whether or not the agent activates
the growth hormone receptor, or a downstream signaling pathway
thereof, or the insulin-like growth factor 1 (IGF-1) receptor, or a
downstream signaling pathway thereof, or a downstream signaling
pathway of (1-3)IGF-1, wherein an agent that activates the growth
hormone receptor, or a downstream signaling pathway thereof, or the
insulin-like growth factor 1 (IGF-1) receptor, or a downstream
signaling pathway thereof, or a downstream signaling pathway of
(1-3)IGF-1 is a candidate for treating a subject with 22q13
deletion syndrome or SHANK3 deletion or duplication, mutation or
reduced expression, whereas an agent that does not activate the
growth hormone receptor, or a downstream signaling pathway thereof,
or the insulin-like growth factor 1 (IGF-1) receptor, or a
downstream signaling pathway thereof, or a downstream signaling
pathway of (1-3)IGF-1 is not a candidate for treating a subject
with 22q13 deletion syndrome or SHANK3 deletion or duplication,
mutation or reduced expression.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1A-1B. Long-term potentiation is impaired in Shank3
heterozygotes. Long-term potentiation (LTP) was induced either by
(B) high frequency stimulus (HFS) (4.times.100 Hz, separated by 5
min) or (A) theta-burst stimulus (TBS) (10 bursts of four pulses at
100 Hz separated by 200 ms) in hippocampal slices in mice. In both
conditions, LTP as assessed by field recordings of excitatory
postsynaptic potential (EPSP) was impaired in the heterozygous
animals (Het) compared to wildtype (WT).
[0038] FIG. 2A-2B. Basal synaptic transmission is reduced in
Shank3-deficient mice. Mice with a targeted disruption of one copy
of the Shank3 gene ("Shank3 heterozygotes") and knockouts were
compared to wild type littermate controls. Both the input-output
curve (figure) and the amplitude of miniature excitatory
postsynaptic currents (EPSCs) (not shown) from hippocampal CA1
pyramidal neurons for Shank3 heterozygotes are significantly lower
than those in control mice indicating a reduction in basal
transmission due to a postsynaptic effect. (A) Field excitatory
postsynaptic potential (fEPSP) slope versus stimulus intensity for
wild type (WT), heterozygous (Het), and knockout (KO). Average
slope of input-output function: WT (+/+), 1.38.+-.0.3; Het (+/-),
1.07.+-.0.2; KO (-/-), 0.91.+-.0.2, F2,21=7.30, p<0.01. (B) Both
LTP induction and maintenance is impaired in Shank3 knockouts,
indicating a more severe phenotype in the knockout mice. fEPSP
slope versus time for WT, Het and KO. In the +/+control group,
fEPSP slope recorded in area CA1 significantly increased over
baseline after TBS and was sustained for at least 60 min
(154.7.+-.2.9% of baseline at 60 min, 159.3.+-.2.6% at 40 min
post-TBS). In Shank3 -/- mice, the initial potentiation was
significantly lower and decayed rapidly to baseline by 40 min
(101.9.+-.2.4% at 40 min post-TBS, N=4-7 mice per genotype,
F(2,14)=85.2, p<0.001). Shank3+/-mice also showed reduced
TBS-induced LTP but normal initial potentiation.
[0039] FIG. 3A-3B. Both Shank3 heterozygous and homozygous mice do
not show alteration in long-term depression. Long-term depression
is induced either by (A) low frequency stimulus (LFS, 900 pulses at
1 Hz; 15 min duration) or (B) paired-pulse low frequency stimulus
(PP-LFS, 1 Hz for 20 min; 50 ms interstimulus interval), which is
known to induce mGluR-dependent form of long-term depression. N=3
for each group. N=3 for each group.
[0040] FIG. 4A-4B. Decrease in the AMPA component of fEPSP in
SHANK3 heterozygotes. (A) fEPSP slope versus fiber volley amplitude
for the NMDA component of neurotransmission, carried out in the
presence of CNQX, a blocker of AMPA receptors. (B) fEPSP slope
versus fiber volley amplitude for the AMPA component of
neurotransmission, carried out in the presence of APV, a blocker of
NMDA receptors (N=4 mice per genotype, two to three slices per
mouse; P=0.001).
[0041] FIG. 5A-5C. Effects of (1-3)IGF-1 treatment on long-term
potentiation at Schaffer collateral-CA1 synapses. (1-3)IGF-1 was
administered daily via i.p. injections (0.01 mg/g body weight)
starting at P13-15 and continuing for 2 weeks for
electrophysiological recordings. (A) Hippocampal slices from
wildtype (WT) and Shank3 heterozygous (Het) mice injected with
vehicle (saline, 0.01% bovine serum albumin (BSA)) or (1-3)IGF-1
were subjected to an LTP inducing stimulation, producing
long-lasting potentiation as shown by normalized field EPSP slope
as a function of time. Vehicle-treated heterozygotes showed reduced
LTP, which was reversed by (1-3)IGF-1 (ANOVA, F(2,11)=8.98, p=0.007
at 90 min. The inset shows representative EPSP traces at 90 min
after LTP induction from saline-injected (1) and
(1-3)IGF-1-injected (2) heterozygous mice (scale bar: 0.5 mV, 10
ms). (B) Input-output curves, plotting field EPSP slopes (mV/ms) as
a function of stimulation strength (mA) were significantly
suppressed in slices from Shank3 heterozygous mice, but were not
different from the wild type in heterozygous mice injected with
(1-3)IGF-1. (C) (1-3)IGF-1 peptide (same protocol used in the
treatment of heterozygote mice) treatment reversed the impairment
in LTP in Shank3 KO mice. fEPSP versus time for KO, KO injected
with IGF1 peptide (N=2), and WT mice.
[0042] FIG. 6. IGF1 treatment activates PI3K-Akt pathway in the
hippocampus. PI3K binds to AMPARs and is required to maintain AMPAR
surface expression during long-term potentiation. Figure shows the
increase in the phosphorylation of Akt (pAkt1) after IGF1 treatment
in heterozygote mice, compared to the vehicle injected mice (n=3,
P=0.0366, unpaired t test). The data implicates the PI3K-Akt
pathway in the beneficial effects of (1-3)IGF-1.
[0043] FIG. 7. Effect of intranasal recombinant IGF-1 treatment on
field EPSP in Shank3 heterozygotes (Het). Two-week old mice were
anesthetized with a mixture of ketamine and xylazine. Recombinant
human IGF-1 (rhIGF-1) or saline was administered intranasally at 48
h intervals for a total of 10 doses (15 .mu.l solution containing
60 .mu.g IGF-1 or vehicle per mouse was given over 10-15 min
period). N=2.
[0044] FIG. 8. Ampakine CX1837 restores long-term potentiation in
Shank3 heterozygous mice. Effects of CX1837 treatment on long-term
potentiation at Schaffer collateral-CA1 synapses in Shank3
heterozygous mice. Ampakine CX1837 or HPCD vehicle is administered
daily via i.p. injections (1.5 mg/kg body weight) starting at 2
weeks old and continued for 4 weeks for electrophysiological
recordings.
[0045] FIG. 9. Effects of growth hormone treatment on long-term
potentiation at Schaffer collateral-CA1 synapses in Shank3
heterozygous mice. Growth hormone is administered daily via i.p.
injections (1 mg/kg body weight) starting at P13-15 and continued
for 2 weeks for electrophysiological recordings.
[0046] FIG. 10A-10B. Recombinant human IGF-1 (rhIGF-1) reverses
deficits in long-term potentiation and basal synaptic properties at
Schaffer collateral-CA1 synapses in Shank3 heterozygous mice in a
dose-dependent fashion. rhIGF-1 is administered daily via i.p.
injections (120 or 240 .mu.g/kg body weight) starting at P13-15 and
continued for 2 weeks for electrophysiological recordings. A. LTP
was induced with high-frequency stimulation and normalized field
EPSP slope was plotted as a function of time. Vehicle-treated
heterozygotes showed reduced LTP, which was reversed by the higher,
but not the lower dose of rhIGF-1 (ANOVA, F(2,11)=14.39, p=0.002).
The inset shows representative EPSP traces at 90 min after LTP
induction from saline-injected (1) and rhIGF-1-injected (2)
heterozygous mice (scale bar: 0.5 mV, 10 ms). B. AMPA receptor
responses were assessed in the mice. Slices were incubated in the
presence of APV and mean field EPSP slope as a function of fiber
volley is shown for slices derived from wildtype (WT), Shank3
heterozygous (Het) mice, and Het injected with rhIGF-1. Deficits in
AMPA receptor signaling observed in Shank3 heterozygotes were
reversed with 2-week rhIGF-1 treatment.
[0047] FIG. 11. IGF-1 treatment reverses motor deficits in Shank3
heterozygous mice. Male wildtype (WT) and Shank3 heterozygous (Het)
mice, treated with vehicle or recombinant human IGF-1 were tested
for motor performance and learning by measuring latencies to fall
off a rotating rod. Mice were challenged with three 2-minute trials
(each separated by 15 minutes) where the rotation was gradually
increased from 0 to 45 rpm. Heterozygous mice injected with saline
exhibit reduced latencies to fall compared to wildtype mice. After
IGF-1 treatment, heterozygous mice exhibit significantly longer
latencies in comparison to vehicle-injected mice of the same
genotype.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The present invention provides a method for treating a
subject with 22q13 deletion syndrome or SHANK3 deletion or
duplication, SHANK3 mutation or reduced expression of SHANK3, in
need thereof, the method comprising administering to the subject
insulin-like growth factor 1 (IGF-1), an active IGF-1 fragment
comprising the tripeptide (1-3)IGF-1 or an analog thereof, growth
hormone, or an AMPAkine, or another compound that directly or
indirectly enhances glutamate neurotransmission, including by
inhibiting inhibitory (most typically GABA) transmission, in an
amount and manner effective to treat a subject with 22q13 deletion
syndrome or SHANK3 deletion or duplication, mutation or reduced
expression. Reduced SHANK3 expression can be due, for example, to
abnormal methylation of the gene encoding SHANK3.
[0049] The invention thus provides a method for treating a subject
with 22q13 deletion syndrome or SHANK3 deletion or duplication,
SHANK3 mutation or reduced expression of SHANK3, the method
comprising administering to the subject insulin-like growth factor
1 (IGF-1) or an active IGF-1 fragment including the tripeptide
(1-3)IGF-1 or an analog thereof, in an amount and manner effective
to treat a subject with 22q13 deletion syndrome or SHANK3 deletion
or duplication, mutation or reduced expression, wherein the subject
has autism spectrum disorder, autism, Asperger syndrome, pervasive
developmental disorder, mental retardation, hypotonia, a speech
deficit, or developmental delay and/or defects.
[0050] The invention further provides a method for treating a
subject with autism spectrum disorder, autism or Asperger syndrome
comprising administering to the subject insulin-like growth factor
1 (IGF-1) or an active IGF-1 fragment including the tripeptide
(1-3)IGF-1 or an analog thereof, in an amount and manner effective
to treat a subject with autism spectrum disorder, autism, or
Asperger syndrome.
[0051] The present invention also provides a method for treating a
subject with 22q13 deletion syndrome or SHANK3 deletion or
duplication, mutation or reduced expression in need thereof, the
method comprising administering to the subject an agent that
activates the growth hormone receptor, or a downstream signaling
pathway thereof, or the insulin-like growth factor 1 (IGF-1)
receptor, or a downstream signaling pathway thereof, or a
downstream signaling pathway of (1-3)IGF-1, in an amount and manner
effective to treat a subject with 22q13 deletion syndrome or SHANK3
deletion or duplication, mutation or reduced expression. As
discussed herein, growth hormone stimulates production of IGF-1,
and the main downstream signaling pathways of the IGF-1 receptor
are the phosphoinositide 3-kinase (PI3K),
3-phosphoinositide-dependent protein kinase 1 (PDK), Akt, mammalian
target of rapamycin (mTOR), and extracellular-signal-regulated
kinase (ERK) pathways. Examples of such agents include growth
hormone, insulin-like growth factor 1 (IGF-1), the tripeptide
(1-3)IGF-1 and analogs thereof.
[0052] As used herein, to treat a subject with 22q13 deletion
syndrome or SHANK3 deletion or duplication, SHANK3 mutation or
reduced expression of SHANK3 means to alleviate a sign or symptom
associated with 22q13 deletion syndrome or SHANK3 deletion or
duplication, mutation or reduced expression. The syndrome is
characterized by general hypotonia, motor deficits, absent to
delayed speech, and global developmental delays. Individuals with a
22q13 deletion or SHANK3 mutation can suffer from a range of
symptoms, with mild to very serious physical and behavioral
characteristics. Possible symptoms include, but are not limited to,
absent to severely delayed speech; hypotonia; increased tolerance
to pain; thin, flaky toenails; ptosis; poor thermoregulation;
chewing non-food items; teeth grinding; autistic behaviors; tongue
thrusting; hair pulling; aversion to clothes; as well as other
physical and behavioral symptoms, including autism spectrum
disorders and atypical schizophrenia.
[0053] The present invention also provides a method for screening
for agents for treating a subject with 22q13 deletion syndrome or
SHANK3 deletion or duplication, mutation or reduced expression, the
method comprising determining whether or not the agent enhances
long-term potentiation or increases glutamate transmission, wherein
an agent that enhances long-term potentiation or increases
glutamate transmission is a candidate for treating a subject with
22q13 deletion syndrome or SHANK3 deletion or duplication, mutation
or reduced expression, whereas an agent that does not enhance
long-term potentiation or increase glutamate transmission is not a
candidate for treating a subject with 22q13 deletion syndrome or
SHANK3 deletion or duplication, mutation or reduced expression. The
assay can be carried out, for example, using mice with a disruption
of at least one copy of SHANK3 (e.g., Shank3 heterozygous
mice).
[0054] The present invention also provides a method for screening
for agents for treating a subject with 22q13 deletion syndrome or
SHANK3 deletion or duplication, mutation or reduced expression, the
method comprising determining whether or not the agent activates
the growth hormone receptor, or a downstream signaling pathway
thereof, or the insulin-like growth factor 1 (IGF-1) receptor, or a
downstream signaling pathway thereof, or a downstream signaling
pathway of (1-3)IGF-1, wherein an agent that activates the growth
hormone receptor, or a downstream signaling pathway thereof, or the
insulin-like growth factor 1 (IGF-1) receptor, or a downstream
signaling pathway thereof, or a downstream signaling pathway of
(1-3)IGF-1 is a candidate for treating a subject with 22q13
deletion syndrome or SHANK3 deletion or duplication, mutation or
reduced expression, whereas an agent that does not activate the
growth hormone receptor, or a downstream signaling pathway thereof,
or the insulin-like growth factor 1 (IGF-1) receptor, or a
downstream signaling pathway thereof, or a downstream signaling
pathway of (1-3)IGF-1 is not a candidate for treating a subject
with 22q13 deletion syndrome or SHANK3 deletion or duplication,
mutation or reduced expression. Downstream signaling pathways of
the IGF-1 receptor include the PI3K, PDK, Akt, mTOR and ERK
pathways.
[0055] The assays can be carried out, for example, using a brain
slice preparation, such as a hippocampal slice preparation, such
as, for example, described herein in Experimental Details.
Preferably, the brain slice is from an animal, such as a mouse,
with a disruption of at least one copy of SHANK3 (e.g., a Shank3
heterozygous mouse).
[0056] Growth hormone (GH) is a protein based polypeptide hormone
which stimulates growth and cell reproduction and regeneration in
humans and other animals. Growth hormone is synthesized, stored,
and secreted by the somatotroph cells in the anterior pituitary
gland. Growth hormone is used clinically to treat children's growth
disorders and adult growth hormone deficiency. Growth hormone
stimulates production of IGF-1. Growth hormone can refer either to
the natural hormone produced by the pituitary or biosynthetic
growth hormone for therapy. Somatotropin refers to the growth
hormone produced naturally in animals, whereas the term somatropin
refers to growth hormone produced by recombinant DNA technology. In
preferred embodiments, growth hormone is human growth hormone
having the amino acid sequence (SEQ ID NO:1) (Accession
AAH90045)
TABLE-US-00001 1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnam
lrahrlhqla fdtyqefeea 61 yipkeqkysf lqnpqtslcf sesiptpsnr
eetqqksnle llrisllliq swlepvqflr 121 svfanslvyg asdsnvydll
kdleegiqtl mgrledgspr tgqifkqtys kfdtnshndd 181 allknyglly
cfrkdmdkve tflrivqcrs vegscgf
or recombinant human growth hormone having the amino acid sequence
(SEQ ID NO:2)
TABLE-US-00002 1 fptiplsrlf dnamlrahrl hqlafdtyqe feeayipkeq
kysflqnpqt slcfsesipt 61 psnreetqqk snlellrisl lliqswlepv
qflrsvfans lvygasdsnv ydllkdleeg 121 iqtlmgrled gsprtgqifk
qtyskfdtns hnddallkny gllycfrkdm dkvetflriv 181 qcrsvegscg f.
Recombinant human growth hormone is available, e.g., from Cell
Sciences.RTM., Canton Mass. (Catalog No. CRH200A-C) (SEQ ID
NO:2).
[0057] Insulin-like growth factor 1 (IGF-1), also known as
somatomedin C or mechano growth factor, is a protein that is
encoded by the IGF1 gene in humans. IGF-1 is a hormone similar in
molecular structure to insulin. It plays an important role in
childhood growth and continues to have an anabolic effect in
adults. A synthetic analog of IGF-1, mecasermin, is used for the
treatment of growth failure. IGF-1 is produced primarily by the
liver as an endocrine hormone as well as in target tissues in a
paracrine/autocrine fashion. Production is stimulated by growth
hormone and can be retarded by undernutrition, growth hormone
insensitivity, lack of growth hormone receptors, or failure of the
downstream signaling pathway post growth hormone receptors
including SHP2 and STAT5B. IGF-1 has substantial human safety data
and is approved for use in children. In preferred embodiment, IGF-1
is recombinant human IGF-1 (a 7.6 kDa protein) having the amino
acid sequence (SEQ ID NO:3)
TABLE-US-00003 1 gpetlcgael vdalqfvcgd rgfyfnkptg ygsssrrapq
tgivdeccfr scdlrrlemy 61 caplkpaksa.
[0058] The tripeptide (1-3)IGF-1 has the amino acid sequence
glycine-proline-glutamic acid (Gly-Pro-Glu or GPE). (1-3)IGF-1 can
be obtained from Bachem (Torrance, Calif.) as H-Gly-Pro-Glu-OH
(Catalog No. H-2468). The peptide has the advantage that it can
penetrate the blood-brain barrier. Analogs of (1-3)IGF-1 that can
be used include, but are not limited to, (1-3) IGF-1 amide, (1-3)
IGF-1 stearate, Gly-Pro-Dglutamate, glycine-proline-threonine
(Gly-Pro-Thr), glycine-glutamic acid-proline (Gly-Glu-Pro),
glutamic acid-glycine-proline (Glu-Gly-Pro), and glutamic
acid-proline-glycine (Glu-Pro-Gly).
[0059] AMPAkines are a class of compounds that strongly interact
with glutamergic AMPA
(.alpha.-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid)
receptors. Glutamergic AMPA receptors are non-NMDA-type ionotropic
transmembrane receptors for glutamate that mediates fast synaptic
transmission in the central nervous system (CNS). To date, four
structural classes of AMPAkines have been developed: pyrrolidine
derivatives of racetam drugs such as piracetam and aniracetam;
CX-series of drugs encompassing a range of benzoylpiperidine and
benzoylpyrrilidine structures; benothiazide derivatives such as
cyclothiazide and IDRA-21; and biarylpropylsulfonamides such as
LY-392,098, LY-404,187, LY-451,646, and LY-503,430. AMPAkines bind
to glutamergic AMPA receptors, boosting the activity of glutamate,
a neurotransmitter, and making it easier to encode memory and
learning. Some AMPAkines may increase levels of trophic factors
such as brain-derived neurotrophic factor. Preferred AMPAkines
include CX AMPAkines (Cortex Pharmaceuticals, Inc., Irvine,
Calif.), such as for example CX-516 (Ampalex), CX-546, CX-614,
CX-691 (Farampator), CX-717, CX-701, CX-1739, CX-1763 and CX-1837
(see, e.g., U.S. Pat. Nos. 5,650,409, 5,736,543, 5,985,871,
6,166,008, 6,313,115, and 7,799,913, and U.S. Patent Application
Publications No. 2002/0055508, 2010/0041647, 2010/0173903,
2010/0267728, 2010/02866177, and 2011/0003835, the contents of
which are herein incorporated by reference).
[0060] Compounds that directly or indirectly enhance glutamate
neurotransmission including, for example, by inhibiting inhibitory
(most typically GABA) transmission, include, for example, glycine
transporter 1 (GLYT1) inhibitors, brain-derived neurotrophic factor
(BDNF), and cyclothiazide. Cyclothiazide acts both on AMPA
receptors and GABA(A) receptors. GLYT1 inhibitors are a functional
class of compounds and include compounds that act as GABA(A)
receptor negative allosteric modulators and inhibitors. Specific
GLYT1 inhibitors include, for example, NFPS, Org 24461, and
sarcosine.
[0061] IGF-1 interacts with its receptor (IGF-1R) to initiate
downstream responses such as proliferation and differentiation. The
IGF-1R is a transmembrane receptor that is activated by IGF-1 and
by the related growth factor IGF-2. It belongs to the large class
of tyrosine kinase receptors and mediates the effects of IGF-1.
Tyrosine kinase receptors, including the IGF-1R, mediate their
activity by causing the addition of a phosphate group to particular
tyrosines on certain proteins within a cell. This addition of
phosphate induces what are called "cell signaling" cascades - and
the usual result of activation of the IGF-1R is survival and
proliferation in mitosis-competent cells, and growth (hypertrophy)
in tissues such as skeletal muscle and cardiac muscle. IGF-1R
activates several downstream signaling pathways. The main
downstream signaling pathways of IGF-1R are the PI3K, PDK, Akt,
mTOR and ERK pathways.
[0062] Phosphoinositide 3-kinases (PI 3-kinases or PI3Ks) are a
family of enzymes involved in cellular functions such as cell
growth, proliferation, differentiation, motility, survival and
intracellular trafficking. PI3Ks are downstream of IGF-1R and
interact with the IRS (Insulin receptor substrate) in order to
regulate cell function uptake through a series of phosphorylation
events. The phosphoinositol-3-kinase family is divided into three
different classes: Class I, Class II, and Class III. The
classifications are based on primary structure, regulation, and in
vitro lipid substrate specificity. PI3K has also been implicated in
Long-term potentiation (LTP). PI3Ks are necessary for the survival
of progenitors and mature oligodendrocytes and for the
IGF-1-mediated cell survival, proliferation, and protein
synthesis.
[0063] AKT protein family, which members are also called protein
kinases B (PKB) plays an important role in mammalian cellular
signaling. In humans, there are three genes in the "Akt family":
Akt1, Akt2, and Akt3. These genes code for enzymes that are members
of the serine/threonine-specific protein kinase family. Akt1 is
involved in cellular survival pathways, by inhibiting apoptotic
processes. Akt1 is also able to induce protein synthesis pathways,
and is therefore a key signaling protein in the cellular pathways
that lead to skeletal muscle hypertrophy, and general tissue
growth. Akt2 is an important signaling molecule in the insulin
signaling pathway. It is required to induce glucose transport. Akt
can be phosphorylated by PDK1 and mTORC2. Beside downstream
effectors of PI3K, Akt can be activated in a PI3K-independent
manner. Akt2 is required for the insulin-induced translocation of
glucose transporter 4 (GLUT4) to the plasma membrane. Glycogen
synthase kinase 3 (GSK-3) could be inhibited upon phosphorylation
by Akt, which results in promotion of glycogen synthesis. Akt
inhibitors and dominant-negative Akt expression can block IGF-1
stimulated protein synthesis in oligodendrocyte progenitors.
[0064] 3-phosphoinositide dependent protein kinase-1 (PDK1) is a
protein which in humans is encoded by the PDPK1 gene and is a
master kinase crucial for the activation of AKT/PKB and many other
AGC kinases including PKC, S6K, and SGK. An important role for PDK1
is in the signalling pathways activated by several growth factors
and hormones including insulin signalling. PDK1 functions
downstream of PI3K through PDK1's interaction with membrane
phospholipids including phosphatidylinositols, phosphatidylinositol
(3,4)-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate.
PI3K indirectly regulates PDPK1 by phosphorylating
phosphatidylinositols which in turn generates phosphatidylinositol
(3,4)-bisphosphate and phosphatidylinositol
(3,4,5)-trisphosphate.
[0065] The mammalian target of rapamycin (mTOR), also known as
mechanistic target of rapamycin or FK506 binding protein
12-rapamycin associated protein 1 (FRAP1), is a protein which in
humans is encoded by the FRAP1 gene. mTOR is a serine/threonine
protein kinase that regulates cell growth, cell proliferation, cell
motility, cell survival, protein synthesis, and transcription. mTOR
integrates the input from upstream pathways, including insulin,
growth factors (such as IGF-1 and IGF-2), and mitogens. mTOR also
senses cellular nutrient and energy levels and redox status.
[0066] Extracellular-signal-regulated kinases (ERKs), or classical
MAP kinases, are widely expressed protein kinase intracellular
signalling molecules that are involved in functions including the
regulation of meiosis, mitosis, and postmitotic functions in
differentiated cells. Many different stimuli, including growth
factors (such as IGF-1 and IGF-2), cytokines, virus infection,
ligands for heterotrimeric G protein-coupled receptors,
transforming agents, and carcinogens, activate the ERK pathway.
[0067] The 22q13 deletion syndrome or SHANK3 deletion or
duplication, mutation or reduced expression can be treated by local
or systemic administration of the IGF-1, IGF-1-derived peptide or
analog, growth hormone, AMPAkine, or other compound that directly
or indirectly enhances glutamate neurotransmission, including by
inhibiting inhibitory (most typically GABA) transmission, or other
therapeutic agent. Local treatment may comprise intramuscular or
intratissue injection. Systemic treatment may comprise enteral or
intravenous methods. The IGF-1, IGF-1-derived peptide or analog,
growth hormone, AMPAkine, or other compound that directly or
indirectly enhances glutamate neurotransmission, including by
inhibiting inhibitory (most typically GABA) transmission, or agent
may be administered in a pharmaceutical composition comprising the
IGF-1, IGF-1-derived peptide or analog, growth hormone, AMPAkine,
or other compound that directly or indirectly enhances glutamate
neurotransmission, including by inhibiting inhibitory (most
typically GABA) transmission, or agent in a pharmaceutically
acceptable carrier.
[0068] The pharmaceutically acceptable carrier must be compatible
with the IGF-1, IGF-1-derived peptide or analog, growth hormone,
AMPAkine, or other compound that directly or indirectly enhances
glutamate neurotransmission, including by inhibiting inhibitory
(most typically GABA) transmission, or agent, and not deleterious
to the subject. Examples of acceptable pharmaceutical carriers
include carboxymethylcellulose, crystalline cellulose, glycerin,
gum arabic, lactose, magnesium stearate, methylcellulose, powders,
saline, sodium alginate, sucrose, starch, talc, and water, among
others. Formulations of the pharmaceutical composition may
conveniently be presented in unit dosage and may be prepared by any
method known in the pharmaceutical art. For example, the IGF-1,
IGF-1-derived peptide or analog, growth hormone, AMPAkine, or other
compound that directly or indirectly enhances glutamate
neurotransmission, including by inhibiting inhibitory (most
typically GABA) transmission, or agent may be brought into
association with a carrier or diluent, as a suspension or solution.
Optionally, one or more accessory ingredients, such as buffers,
flavoring agents, surface-active ingredients, and the like, may
also be added. The choice of carriers will depend on the method of
administration. The pharmaceutical composition can be formulated
for administration by any method known in the art, including but
not limited to, intravenously and intracranially.
[0069] The amount of IGF-1, IGF-1-derived peptide or analog, growth
hormone, AMPAkine, or other compound that directly or indirectly
enhances glutamate neurotransmission, including by inhibiting
inhibitory (most typically GABA) transmission, or agent
therapeutically necessary will depend on the severity of the 22q13
deletion syndrome or SHANK3 mutation as well as the manner of
administration of the IGF-1, IGF-1-derived peptide or analog,
growth hormone, AMPAkine, or other compound that directly or
indirectly enhances glutamate neurotransmission, including by
inhibiting inhibitory (most typically GABA) transmission, or agent.
One skilled in the art can easily determine the amount and manner
of administration of IGF-1, IGF-1-derived peptide or analog, growth
hormone, AMPAkine, or other compound that directly or indirectly
enhances glutamate neurotransmission, including by inhibiting
inhibitory (most typically GABA) transmission, or agent
necessary.
[0070] According to the method of the present invention, the IGF-1,
IGF-1-derived peptide or analog, growth hormone, AMPAkine, or other
compound that directly or indirectly enhances glutamate
neurotransmission, including by inhibiting inhibitory (most
typically GABA) transmission, or other therapeutic agent may be
administered to a subject by any known procedure including, but not
limited to, oral administration, parenteral administration,
transdermal administration, intranasal administration, and
administration through an osmotic mini-pump.
[0071] For oral administration, the formulation of the IGF-1,
IGF-1-derived peptide or analog, growth hormone, AMPAkine, or other
compound that directly or indirectly enhances glutamate
neurotransmission, including by inhibiting inhibitory (most
typically GABA) transmission, or other therapeutic agent may be
presented as capsules, tablets, powder, granules, or as a
suspension. The formulation may have conventional additives, such
as lactose, mannitol, corn starch, or potato starch. The
formulation may also be presented with binders, such as crystalline
cellulose, cellulose derivatives, acacia, corn starch, or gelatins.
Additionally, the formulation may be presented with disintegrators,
such as corn starch, potato starch, or sodium
carboxymethylcellulose. The formulation also may be presented with
dibasic calcium phosphate anhydrous or sodium starch glycolate.
Finally, the formulation may be presented with lubricants, such as
talc or magnesium stearate.
[0072] For a parenteral administration, the IGF-1, IGF-1-derived
peptide or analog, growth hormone, AMPAkine, or other compound that
directly or indirectly enhances glutamate neurotransmission,
including by inhibiting inhibitory (most typically GABA)
transmission, or other therapeutic agent may be combined with a
sterile aqueous solution which is preferably isotonic with the
blood of the subject. Such a formulation may be prepared by
dissolving a solid active ingredient in water containing
physiologically-compatible substances, such as sodium chloride,
glycine, and the like, and having a buffered pH compatible with
physiological conditions, so as to produce an aqueous solution,
then rendering said solution sterile. The formulations may be
present in unit or multi-dose containers, such as sealed ampoules
or vials. The formulation may be delivered by any mode of
injection, including, without limitation, epifascial, intrasternal,
intravascular, intravenous, parenchymatous, or subcutaneous.
[0073] For transdermal administration, the IGF-1, IGF-1-derived
peptide or analog, growth hormone, AMPAkine, or other compound that
directly or indirectly enhances glutamate neurotransmission,
including by inhibiting inhibitory (most typically GABA)
transmission, or other therapeutic agent may be combined with skin
penetration enhancers, such as propylene glycol, polyethylene
glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and
the like, which increase the permeability of the skin to the IGF-1,
IGF-1-derived peptide or analog, growth hormone, AMPAkine, or other
compound that directly or indirectly enhances glutamate
neurotransmission, including by inhibiting inhibitory (most
typically GABA) transmission, or other therapeutic agent. The
IGF-1, IGF-1-derived peptide or analog, growth hormone, AMPAkine,
or other compound that directly or indirectly enhances glutamate
neurotransmission, including by inhibiting inhibitory (most
typically GABA) transmission, or other therapeutic agent
compositions also may be further combined with a polymeric
substance, such as ethylcellulose, hydroxypropyl cellulose,
ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to
provide the composition in gel form, which may be dissolved in
solvent such as methylene chloride, evaporated to the desired
viscosity, and then applied to backing material to provide a
patch.
[0074] The IGF-1, IGF-1-derived peptide or analog, growth hormone,
AMPAkine, or other compound that directly or indirectly enhances
glutamate neurotransmission, including by inhibiting inhibitory
(most typically GABA) transmission, or other therapeutic agent may
also be released or delivered from an osmotic mini-pump. The
release rate from an elementary osmotic mini-pump may be modulated
with a microporous, fast-response gel disposed in the release
orifice. An osmotic mini-pump would be useful for controlling the
release of, or targeting delivery of, the IGF-1, IGF-1-derived
peptide or analog, growth hormone, AMPAkine, or other compound that
directly or indirectly enhances glutamate neurotransmission,
including by inhibiting inhibitory (most typically GABA)
transmission, or other therapeutic agent.
[0075] Long-term potentiation (LTP) is a long-lasting enhancement
in signal transmission between two neurons that results from
stimulating them synchronously. It is one of several phenomena
underlying synaptic plasticity, the ability of chemical synapses to
change their strength. LTP shares many features with long-term
memory, making it an attractive candidate for a cellular mechanism
of learning. For example, LTP and long-term memory are triggered
rapidly, each depends upon the synthesis of new proteins, each has
properties of associativity, and each can last for many months. LTP
may account for many types of learning, from the relatively simple
classical conditioning present in all animals, to the more complex,
higher-level cognition observed in humans. At a cellular level, LTP
enhances synaptic transmission. It improves the ability of two
neurons, one presynaptic and the other postsynaptic, to communicate
with one another across a synapse.
[0076] Chemical synapses are functional connections between neurons
throughout the nervous system. In a typical synapse, information is
largely passed from the first (presynaptic) neuron to the second
(postsynaptic) neuron via a process of synaptic transmission.
Through experimental manipulation, a non-tetanic stimulus can be
applied to the presynaptic cell, causing it to release a
neurotransmitter such as glutamate onto the postsynaptic cell
membrane. There, glutamate binds to receptors such as AMPA
receptors (AMPARs) embedded in the postsynaptic membrane. The AMPA
receptor is one of the main excitatory receptors in the brain, and
is responsible for most of its rapid, moment-to-moment excitatory
activity. Glutamate binding to the AMPA receptor triggers the
influx of positively charged sodium ions into the postsynaptic
cell, causing a short-lived depolarization called the excitatory
postsynaptic potential (EPSP). Extracellular-signal-regulated
kinases (ERKs) play a role in late LTP, where gene expression and
protein synthesis is brought about by the persistent activation of
protein kinases activated during early LTP. ERK phosphorylates a
number of cytoplasmic and nuclear molecules that ultimately result
in the protein synthesis and morphological changes observed in late
LTP. ERK-mediated changes in transcription factor activity may
trigger the synthesis of proteins that underlie the maintenance of
L-LTP.
[0077] An excitatory postsynaptic potential (EPSP) is a temporary
depolarization of postsynaptic membrane potential caused by the
flow of positively charged ions into the postsynaptic cell as a
result of opening of ligand-sensitive channels. ESPSs in living
cells are caused chemically. When an active presynaptic cell
releases neurotransmitters into the synapse, some bind to receptors
on the postsynaptic cell. Many of these receptors contain an ion
channel capable of passing positively charged ions either into or
out of the cell. The depolarizing current causes an increase in
membrane potential, the ESPS. The amino acid glutamate is the
neurotransmitter most often associated with EPSPs.
[0078] Glutamate is the most abundant excitatory neurotransmitter
in the vertebrate nervous system. At chemical synapses, glutamate
is stored in vesicles. Nerve impulses trigger release of glutamate
from the pre-synaptic cell. In the opposing post-synaptic cell,
glutamate receptors, such as the NMDA receptor, bind glutamate and
are activated. Because of its role in synaptic plasticity,
glutamate is involved in cognitive functions like learning and
memory in the brain. The form of plasticity known as long-term
potentiation takes place at glutamatergic synapses in the
hippocampus, neocortex, and other parts of the brain. Glutamate
does not work only as a point to point transmitter but also through
spill-over synaptic crosstalk between synapses in which summation
of glutamate released from neighboring synapse creates
extrasynaptic signaling/volume transmission.
[0079] Glutamate transporters are found in neuronal and glial
membranes. They rapidly remove glutamate from the extracellular
space. In brain injury or disease, they can work in reverse and
excess glutamate can accumulate outside cells. This process causes
calcium ions to enter cells via NMDA receptor channels, leading to
neuronal damage and eventual cell death, and is called
excitotoxicity.
[0080] The subject can be any mammal, in particular a human. The
subject can have, for example, one or more of autism, Asperger
syndrome, autism spectrum disorder, pervasive developmental
disorder, mental retardation, hypotonia, speech deficits and
developmental delay and/or defects.
EXPERIMENTAL DETAILS
[0081] Brief Experimental Procedures:
[0082] Hippocampal slices (350 .mu.m) are prepared from 1-3 month
old Shank3 heterozygous, Shank3 knockout, and wildtype littermates,
treated with (1-3) IGF-1 peptide, full-length IGF1, growth hormone,
AMPAkine or saline or other appropriate control. Slices are
perfused with Ringer's solution, bubbled with 95% O.sub.2/5%
CO.sub.2, at 32.degree. C., during extracellular recordings.
Baseline of field excitatory postsynaptic potentials (fEPSPs)
recorded from stratum radiatum in area CA1, evoked by stimulation
of the Schaffer collateral-commissural afferents with bipolar
tungsten electrodes placed into area CA3. Long-term potentiation
(LTP) is induced either by a high-frequency stimulus (four trains
of 100 Hz, 1 s stimulation separated by 5 min), or by theta-burst
stimulation (TBS) (10 bursts of four pulses at 100 Hz separated by
200 ms), with a success rate >90% for control and
genetically-modified animals with all stimulation protocols. To
induce long-term depression (LTD), Schaffer collaterals were
stimulated by a low frequency stimulus (900 pulses at 1 Hz for 15
min) or paired-pulse low frequency stimulus. LTD was induced with a
success rate >90% for control animals. In order to examine Akt
phosphorylation hippocampus and cortex are dissected from the other
hemisphere from Shank3 heterozygous and wildtype littermates, used
to make slices and are immediately snap-frozen on dry ice. Western
blot analysis is performed for total- and phospho-Akt levels.
Results are presented in FIGS. 1-10. Mice were also tested for
motor performance and learning by measuring latencies to fall off a
rotating rod. Mice were challenged with three 2-minute trials (each
separated by 15 minutes) where the rotation was gradually increased
from 0 to 45 rpm, and results are presented in FIG. 11.
[0083] A mouse with hemizygous loss of full-length Shank3 (Bozdagi
et al., 2010) was used to investigate whether IGF-1 could reverse
synaptic deficits in a preclinical model. Tests were first made
with an active peptide of IGF-1 ((1-3)IGF-1), which has been shown
to cross the blood-brain barrier (O'Kusky et al., 2000) and rescue
Rett syndrome symptoms in Mecp2-deficient mice. Intraperitoneal
injection at 10 .mu.g/g/day for 2 weeks restored normal hippocampal
LTP in Shank3 heterozygous mice (FIG. 5a). While LTP was
significantly reduced in vehicle treated heterozygotes,
heterozygous mice treated with IGF-1 showed a complete rescue.
Similarly, LTP at 90 minutes after induction was significantly
(P=0.007) reduced in vehicle-treated heterozygotes, compared to
wildtype littermates, but not when comparing peptide treated
heterozygotes to wildtype animals. In addition, the significantly
(P=0.029) reduced input-output (I/O) function observed in
heterozygotes [obtained by plotting field excitatory postsynaptic
potential (fEPSP) slope versus stimulus intensity], was reversed
after 2 weeks administration of active peptide of IGF-1 (FIG. 5b),
indicating that deficits in synaptic transmission are rescued by
(1-3)IGF-1.
[0084] IGF-1 has been approved for clinical use as a recombinant
full-length protein. Full-length IGF-1 enters the CNS through an
interaction with lipoprotein-related receptor 1 (LRP1), after
activity dependent cleavage of IGF binding protein-3 (IGFBP-3) by
matrix metalloproteinase-9 (MMP9) (Nishijima et al., 2010). To
investigate whether peripherally administered full-length IGF-1
could also reverse synaptic deficits, IGF-1 was administered by
intraperitoneal injection at 240 .mu.g/kg/day, starting at P13-15
and continuing for 2 weeks. This dose was chosen because it
represents the maximum dose according to the current FDA label for
IGF-1. This treatment was also effective in rescuing defective LTP
in heterozygous mice (FIG. 10a). Furthermore, specific deficits in
the AMPA receptor component of I/O function (Bozdagi et al., 2010)
were reversed by this treatment (FIG. 10b). Mean slope of the I/O
function was 0.625.+-.0.08 for wildtype; 0.31.+-.0.045 for Shank3
heterozygous and 0.618.+-.0.075 for IGF-1 injected heterozygous
mice (comparison between heterozygotes and IGF-1-injected
heterozygotes, p=0.004). Importantly, lower dose IGF-1 (120
.mu.g/kg/day for 2 weeks) was not associated with significant
reversal of deficits in LTP (FIG. 10a), showing a dose-response
effect and providing preclinical dosing information.
[0085] Phelan-McDermid syndrome frequently presents with hypotonia
and at least transient motor deficits, and subtle motor deficits
have been observed in Shank3-heterozygous mice (Bozdagi et al.,
2010). To determine whether treatment with full-length IGF-1 may
improve motor performance in Shank3-deficient mice, male
heterozygous mice were treated with either vehicle or IGF-1 (240
.mu.g/kg/day for 2 weeks). Significant enhancement of motor
performance was observed following treatment (FIG. 11).
[0086] To date, pharmacological treatments for ASD and other
neurodevelopmental disorders are primarily ameliorative, focusing
on managing associated symptoms such as anxiety, aggression,
repetitive behaviors, or epilepsy (King et al., 2006).
Pharmacological treatments addressing "core" deficits, such as
cognitive impairments, social deficits, and absent or delayed
speech, do not yet exist. Recently, however, the field has begun to
see the evaluation of therapies targeted to etiology ("personalized
medicine") - - - each arising from the basic analysis of model
systems - - - in neurodevelopmental disorders including Fragile X
syndrome, tuberous sclerosis, and Rett syndrome (Bear et al., 2004;
Ehninger et al., 2009; Tropea et al., 2009).
[0087] It is interesting to note that IGF-1 activates the mTOR/Akt
pathway as this has been implicated in other neurodevelopmental
disorders (Veenstra-Vanderweele et al., 2012). It was therefore
predicted that phospho-Akt/Akt ratios would be increased after
IGF-1 treatment, and this is what was observed in studies in
hippocampal lysates (0.36.+-.0.03 and 0.55.+-.0.04 in Shank3
heterozygous mice injected for 2 weeks with vehicle [n=8] or
full-length IGF-1 protein [n=10], respectively; 1-tailed
p=0.040).
[0088] Loss of one functional copy of the SHANK3 gene, through
either mutation or deletion, is found in about 0.5% of ASD
(Abrahams and Geschwind, 2008) and in about 0.3% of the
developmentally delayed population (Cooper et al., 2011). As such
it represents one of the more frequent causes of these disorders
and a significant health burden. In addition, there is emerging
evidence that the SHANK3 pathway may play a role in other
neurodevelopmental disorders, as evidenced by large-scale proteomic
and gene expression studies (Darnell et al., 2011; Sakai et al.,
2011). Even more broadly, deficits in proteins associated with the
postsynaptic density, which is in no small degree sculpted by
SHANK3 (Roussignol et al., 2005), are associated with
neurodevelopmental disorders (Laumonnier et al., 2007). Mutations
in SHANK3 are also associated with schizophrenia (Gauthier et al.,
2010). This indicates that therapies for SHANK3 deficiency and
synaptic development represent important targets that can have
broad positive impact in neurodevelopmental disorders (Melom and
Littleton, 2011). These results show that IGF-1, approved for use
in children, can lead to functional improvements in a mouse model
of ASD and developmental delay, representing an important
preclinical step.
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Sequence CWU 1
1
31217PRTHomo sapiens 1Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu
Ala Phe Gly Leu Leu 1 5 10 15 Cys Leu Pro Trp Leu Gln Glu Gly Ser
Ala Phe Pro Thr Ile Pro Leu 20 25 30 Ser Arg Leu Phe Asp Asn Ala
Met Leu Arg Ala His Arg Leu His Gln 35 40 45 Leu Ala Phe Asp Thr
Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys 50 55 60 Glu Gln Lys
Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe 65 70 75 80 Ser
Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys 85 90
95 Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp
100 105 110 Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser
Leu Val 115 120 125 Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu
Lys Asp Leu Glu 130 135 140 Glu Gly Ile Gln Thr Leu Met Gly Arg Leu
Glu Asp Gly Ser Pro Arg 145 150 155 160 Thr Gly Gln Ile Phe Lys Gln
Thr Tyr Ser Lys Phe Asp Thr Asn Ser 165 170 175 His Asn Asp Asp Ala
Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe 180 185 190 Arg Lys Asp
Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys 195 200 205 Arg
Ser Val Glu Gly Ser Cys Gly Phe 210 215 2191PRTHomo sapiens 2Phe
Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10
15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu
20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln
Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr
Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu
Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu
Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val
Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp
Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp
Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140
Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145
150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu
Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser
Cys Gly Phe 180 185 190 370PRTHomo sapiens 3Gly Pro Glu Thr Leu Cys
Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 1 5 10 15 Val Cys Gly Asp
Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly 20 25 30 Ser Ser
Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 35 40 45
Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 50
55 60 Lys Pro Ala Lys Ser Ala 65 70
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