U.S. patent application number 17/146336 was filed with the patent office on 2021-05-06 for method and composition for generating basal forebrain cholinergic neurons (bfcns).
The applicant listed for this patent is Icahn School of Medicine at Mount Sinai, New York Stem Cell Foundation, Inc.. Invention is credited to Michelle Ehrlich, Sam Gandy, Ilya Kruglikov, Scott Noggle, Maitane Ortiz-Virumbrales.
Application Number | 20210130773 17/146336 |
Document ID | / |
Family ID | 1000005345569 |
Filed Date | 2021-05-06 |
United States Patent
Application |
20210130773 |
Kind Code |
A1 |
Noggle; Scott ; et
al. |
May 6, 2021 |
METHOD AND COMPOSITION FOR GENERATING BASAL FOREBRAIN CHOLINERGIC
NEURONS (BFCNs)
Abstract
The invention relates to methods and compositions for developing
basal forebrain cholinergic neurons (BFCNs) from stem cells, and in
particular, BFCNs having repaired electrophysiological defects
relating to one or more mutations in PSEN2, and to the use of such
BFCNs in cell-based therapies to treat Alzheimer's disease.
Inventors: |
Noggle; Scott; (New York,
NY) ; Ortiz-Virumbrales; Maitane; (New York, NY)
; Gandy; Sam; (New York, NY) ; Kruglikov;
Ilya; (New York, NY) ; Ehrlich; Michelle; (New
York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
New York Stem Cell Foundation, Inc.
Icahn School of Medicine at Mount Sinai |
New York
New York |
NY
NY |
US
US |
|
|
Family ID: |
1000005345569 |
Appl. No.: |
17/146336 |
Filed: |
January 11, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15990208 |
May 25, 2018 |
10889800 |
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17146336 |
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62586571 |
Nov 15, 2017 |
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62574639 |
Oct 19, 2017 |
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62571741 |
Oct 12, 2017 |
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62511271 |
May 25, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/41 20130101;
C12N 5/0619 20130101; C12N 2506/02 20130101; C12N 2800/80 20130101;
G01N 33/5058 20130101; C12N 2506/45 20130101; C12N 2510/00
20130101; C12N 5/0696 20130101; C12N 15/11 20130101; G01N 2800/2814
20130101; C12N 9/22 20130101; C12N 2310/20 20170501; C12N 2501/15
20130101; C12N 2501/999 20130101; A61K 35/30 20130101 |
International
Class: |
C12N 5/0793 20060101
C12N005/0793; C12N 15/11 20060101 C12N015/11; C12N 9/22 20060101
C12N009/22; A61K 35/30 20060101 A61K035/30; G01N 33/50 20060101
G01N033/50; C12N 5/074 20060101 C12N005/074 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
Nos. AG005138, AG046170 and AG042965 awarded by the National
Institutes of Health. The government has certain rights in this
invention.
Claims
1. A method of generating basal forebrain cholinergic neurons
(BFCNs) comprising: culturing pluripotent stem cells (PSCs) in a
basal media comprising an inhibitor of transforming growth factor
beta (TGF-.beta.) signaling and an activator of sonic hedgehog
(Shh) signaling to induce neuroectodermal differentiation, wherein
the basal media lacks basic fibroblast growth factor (bFGF),
TGF-.beta., lithium chloride (Li--Cl), GABA and pipecolic acid,
thereby generating BFCNs.
2. A method of treating a disease or disorder in a subject
comprising administering a BFCN generated using the method of claim
1 to the subject, thereby treating the disease or disorder.
3. The method of claim 2, wherein the disease or disorder is an
amyloidogenic disease.
4. The method of claim 2, wherein the disease or disorder is
associated with diminished neuronal excitability in BFCNs of the
subject.
5. The method of claim 3, wherein the disease or disorder is
selected from the group consisting of systemic amyloidosis,
Alzheimer's disease, mature onset diabetes, Parkinson's disease,
Huntington's disease, fronto-temporal dementia, and prion-related
transmissible spongiform encephalopathies.
6. The method of claim 2, wherein the BFCN comprises a genome
repaired PSEN2 mutation.
7. The method of claim 6, wherein the PSEN2 mutation is
PSEN2.sup.N141I.
8. A method of restoring neuronal excitability in basal forebrain
cholinergic neurons (BFCNs) in a subject, comprising: a) isolating
a BFCN from the subject, wherein the BFCN has a mutation in
presenilin 2 (PSEN2) resulting in impaired neuronal excitability of
the BFCN; b) generating an induced pluripotent stem cell (iPSC)
using the BFCN of (a); c) repairing the PSEN2 mutation in the iPSC;
d) culturing the iPSC of (c) using the method of claim 1 to
generate a BFCN having the repaired mutation; and e) administering
the iPSC of (d) to the subject, thereby restoring neuronal
excitability in BFCNs in the subject.
9. The method of claim 8, wherein the mutation is
PSEN2.sup.N141I.
10. The method of claim 8, wherein the subject has, or is at risk
of having Alzheimer's disease.
11. A method of identifying a compound for treatment or prevention
of a disease or disorder associated with diminished neuronal
excitability in basal forebrain cholinergic neurons (BFCNs), the
method comprising: a) contacting a BFCN or neuronal embryoid body
(NEB) generated by the method of claim 1 with a candidate compound,
wherein the BFCN comprises a mutation in presenilin 2 (PSEN2)
resulting in impaired neuronal excitability of the BFCN; and b)
detecting neuronal excitability of the BFCN after contact with the
candidate compound.
12. The method of claim 11, wherein the mutation is
PSEN2.sup.N141I.
13. The method of claim 11, wherein the disease or disorder is
Alzheimer's disease.
14. A basal forebrain cholinergic neuron (BFCN) generated using the
method of claim 1.
15. The BFCN of claim 14, wherein the genome has a recombinantly
introduced marker.
16. A kit for generating a basal forebrain cholinergic neuron
(BFCN) comprising a culture media having an inhibitor of
transforming growth factor beta (TGF-.beta.) signaling and an
activator of sonic hedgehog (Shh) signaling, wherein the basal
media lacks basic fibroblast growth factor (bFGF), TGF-.beta.,
lithium chloride (Li--Cl), GABA and pipecolic acid, wherein the
inhibitor comprises SB431542 and LDN193189, and wherein the
activator comprises Smoothened Agonist (SAG) and purmorphamine.
17. The kit of claim 16, wherein the kit further comprises reagents
for generating an induced pluripotent stem cell (iPSC).
18. The kit of claim 16, wherein the kit further comprises a
gene-editing reagent.
19. The kit of claim 16, wherein the kit further comprises a
reagent for detecting a genetic mutation.
20. The kit of claim 19, wherein the mutation is is
PSEN2.sup.N141I.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 15/990,208 filed May 25, 2018, now issued as
U.S. Pat. No. 10,889,800; which claims the benefit under 35 USC
.sctn. 119(e) to U.S. Application Ser. No. 62/586,571 filed Nov.
15, 2017, U.S. Application Ser. No. 62/574,639 filed Oct. 19, 2017,
U.S. Application Ser. No. 62/571,741 filed Oct. 12, 2017 and U.S.
Application Ser. No. 62/511,271 filed May 25, 2017, all now
expired. The disclosure of each of the prior applications is
considered part of and is incorporated by reference in the
disclosure of this application.
INCORPORATION OF SEQUENCE LISTING
[0003] The material in the accompanying sequence listing is hereby
incorporated by reference into this application. The accompanying
sequence listing text file, named NYSC1390-5_ST25.txt, was created
on Jan. 8, 2021 and is 53 kB in size. The file can be accessed
using Microsoft Word on a computer that uses Windows OS.
BACKGROUND OF THE INVENTION
Field of the Invention
[0004] The present invention relates generally to the field of
medicine, and more specifically to methods and compositions for
developing basal forebrain cholinergic neurons (BFCNs) from stem
cells, and in particular, BFCNs comprising repaired
electrophysiological defects relating to one or more mutations in
the presenilin 2 gene (PSEN2), and the use of such BFCNs in
cell-based therapies to treat Alzheimer's disease.
Background Information
[0005] Alzheimer's disease (AD) is a progressive disease resulting
in senile dementia. Broadly speaking the disease falls into two
categories: late onset, which occurs in old age (65+ years) and
early onset, which develops well before the senile period, i.e.,
between 35 and 60 years. In both types of disease, the pathology is
the same but the abnormalities tend to be more severe and
widespread in cases beginning at an earlier age. The disease is
characterized by at least two types of lesions in the brain, senile
plaques and neurofibrillary tangles. Senile plaques are areas of
disorganized neuropil up to 150 .mu.m across with extracellular
amyloid deposits at the center visible by microscopic analysis of
sections of brain tissue. Neurofibrillary tangles are intracellular
deposits of microtubule associated tau protein consisting of two
filaments twisted about each other in pairs.
[0006] The principal constituent of the plaques is a peptide termed
A.beta. or .beta.-amyloid peptide. A.beta. peptide is an internal
fragment of 39-43 amino acids of a precursor protein termed amyloid
precursor protein (APP). Several mutations within the APP protein
have been correlated with the presence of Alzheimer's disease. Such
mutations are thought to cause Alzheimer's disease by increased or
altered processing of APP to A.beta., particularly processing of
APP to increased amounts of the long form of A.beta. (i.e.,
A.beta.1-42 and A.beta.1-43). Mutations in other genes, such as the
presenilin genes, PSEN1 and PSEN2, are thought indirectly to affect
processing of APP to generate increased amounts of long form
A.beta.. These observations suggest that A.beta., and particularly
its long form, is a causative element in Alzheimer's disease.
[0007] There are 5 million people currently affected by Alzheimer's
disease in the US and, according to the Alzheimer's Association,
this number will increase to 16 million by the year 2050.
Unfortunately, we only have direct evidence for genetic causation
that accounts for 3-5% of these patients. This percentage
encompasses the autosomal dominant early onset familial Alzheimer's
disease (EOFAD) variants caused by inherited fully penetrant
autosomal dominant mutations in the APP, or PSEN1, PSEN2 that
constitute the .gamma.-secretase apparatus [87], and changes in
their function increases the production of A.beta.42 oligomers
and/or deposition of amyloid plaques.
[0008] After decades studying murine models of AD that do not fully
recapitulate the pathophysiology of this disease in the human brain
[5, 57, 58], a complementary new concept of AD modeling in vitro
has emerged upon the breakthrough by [81] allowing adult human
tissue reprogramming into iPSC using defined factors, and their
subsequent in vitro differentiation into specific brain cell
types.
[0009] BFCNs are one of the most vulnerable neuronal populations
whose deterioration explains, in part, the cognitive decline in AD
patients. Apart from the evidence for BFCN failure and atrophy,
other studies have revealed that human embryonic stem cell-derived
BFCNs transplanted into AD mouse models can be associated with
improvement in the learning behavior of the implanted mouse [94].
These findings highlight the relevance of iPSC- and ESC-derived
BFCNs as not only early clinical indicators but also as a potential
strategy for subtype-specific cell-based therapy for AD [39]. In
order to move this cell-based therapeutic strategy forward, there
has been an urgent need for a refined differentiation protocol to
generate human ESC- and/or iPSC-derived BFCNs.
SUMMARY OF THE INVENTION
[0010] The present invention provides a highly reproducible
protocol to efficiently derive BFCNs from pluripotent stem cells
(PSCs), including embryonic stem cells (ESCs) and induced
pluripotent stem cells (iPSCs).
[0011] Accordingly, in one embodiment, the invention provides a
method of generating BFCNs. The method includes culturing PSCs in a
basal media comprising an inhibitor of transforming growth factor
beta (TGF-.beta.) signaling and an activator of sonic hedgehog
(Shh) signaling to induce neuroectodermal differentiation. In some
aspects, the basal media is a modified mTeSR1 formulation that
lacks factors that support pluripotency including basic fibroblast
growth factor (bFGF), TGF-.beta., lithium chloride (Li--Cl), GABA
and pipecolic acid. In some aspects, culturing is performed in the
presence of dual SMAD inhibitors, such as SB431542 and LDN193189
along with one or more agonists of smoothened protein, such as
smoothened agonist (SAG) and purmorphamine. After about 9, 10, 11
or 12 days of culturing, CD271+ cells are selected and in a
neuronal basal medium, such as Brainphys.TM. to generate neuronal
embryoid bodies (NEBs). The neuronal basal medium is optionally
supplemented with one or more of B27 supplement, an inhibitor of
rho-associated protein kinase (ROCK), nerve growth factor (NGF) and
brain derived neurotrophic factor (BDNF). After about 7, 8, 9 or 10
days of culturing the CD271+ cells, the formed NEBs are harvested,
dissociated, and plated as monolayer cultures and further cultured
in a neuronal basal medium optionally supplemented with B27
supplement, NGF and BDNF. To ensure differentiation into BFCNs, the
cultured cells are analyzed for positive expression of Tuj1, MAP2,
BF1, Nkx2.1 and p75.
[0012] In another embodiment, the method utilizes iPSCs which may
be treated with a gene editing system to repair one or more
mutations, such as a mutation of presenilin 1 (PSEN1) or presenilin
2 (PSEN2). In one aspect, the mutation is PSEN2.sup.N141I, repair
of which restores neuronal excitability in BFCNs.
[0013] Accordingly, in another embodiment, the invention provides a
method of treating a disease or disorder in a subject. The method
includes administering to a subject a BFCN generated using the
culturing method described herein. In certain aspects, the disease
or disorder is an amyloidogenic disease, such as systemic
amyloidosis, Alzheimer's disease, mature onset diabetes,
Parkinson's disease, Huntington's disease, fronto-temporal
dementia, and prion-related transmissible spongiform
encephalopathies. In embodiments, a BFCN having a mutation that
impairs neuronal excitability, such as PSEN2.sup.N141I, may be
obtained from a subject and used to generate an iPSC, which in turn
may be treated with a gene editing system to repair the mutation.
The gene edited iPSC is then cultured as described herein to
produce BFCNs having restored neuronal excitability which are
administered to the subject to treat the disease or disorder. In
certain aspects, the disease or disorder is AD.
[0014] In a related embodiment, the invention provides a method of
restoring neuronal excitability in BFCNs in a subject. The method
includes: a) isolating a BFCN from the subject, wherein the BFCN
has a mutation in PSEN2 resulting in impaired neuronal excitability
of the BFCN; b) generating an iPSC using the BFCN of (a); c)
repairing the PSEN2 mutation in the iPSC; d) culturing the iPSC of
(c) using the differentiation protocol described herein to generate
a BFCN having the repaired mutation; and e) administering the iPSC
of (d) to the subject, thereby restoring neuronal excitability in
BFCNs in the subject.
[0015] Also provided is a method of identifying a compound for
treatment or prevention of a disease or disorder associated with
diminished neuronal excitability in BFCNs. The method includes: a)
contacting a BFCN or neuronal embryoid body (NEB) generated using
the differentiation protocol described herein with a candidate
compound, wherein the BFCN comprises a mutation in PSEN2 resulting
in impaired neuronal excitability of the BFCN; and b) detecting
neuronal excitability of the BFCN after contact with the candidate
compound. An increase in neuronal excitability of the BFCN after
contact with the candidate compound identifies the compound as a
compound potentially capable of restoring neuronal excitability in
BFCNs.
[0016] The invention further provides a BFCN generated using the
differentiation protocol as described herein. The BFCN may include
a gene edited repair of PSEN2, as well as a detectable marker
recombinantly introduced into the BFCN genome.
[0017] The invention also provides a kit for generating a BFCN. The
kit includes a culture media having an inhibitor of TGF-.beta.
signaling and an activator of Shh signaling. In embodiments, the
culture media is a modified mTeSR1 formulation that lacks factors
that support pluripotency including bFGF, TGF-.beta., lithium
chloride (Li--Cl), GABA and pipecolic acid. In embodiments, the
culture media includes dual SMAD inhibitors, such as SB431542 and
LDN193189, along with one or more agonists of smoothened protein,
such as smoothened agonist (SAG) and purmorphamine. The kit may
also include a neuronal basal medium, such as Brainphys.TM.
optionally supplemented with one or more of B27 supplement, an
inhibitor of ROCK, NGF and BDNF. In embodiments, the kit includes
reagents for detection of CD271+ cells as well as cells which
positively express Tuj1, MAP2, BF1, Nkx2.1 and p75.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A-1E. Overview schematic of basal cholinergic
differentiation protocol. (FIG. 1A) Cells are plated and allowed to
reach 100% confluency (Day 0), before the initiation of dual SMAD
inhibition and the subsequent introduction of ventralizing agents
(Day 2). At day 10 the monolayer is dissociated, sorted for p75+
cells, and kept as NEBs until day 19. Then the culture is
dissociated again into a monolayer (See Methods for more details).
(FIG. 1B) Left panel shows sustained EGFP expression driven by
Nkx2.1 induction in NKx2.1-EGFP hESCs upon SHH plus purmorphamine
or SAG plus purmorphamine treatment, maintained at Day 14, after
removal of treatment at Day 8. Right panel shows Nkx2.1, Lhx8 and
BF1 relative gene expression to GAPDH measured by qPCR, in
NKx2.1-EGFP cell line in the presence of the indicated ventralizing
agents, or unpatterned (UNP) at Day 12. n=3, in technical
triplicates. (FIG. 1C) Confocal microscope images of Nestin, Sox2
and DRAQS immunostaining in fControl and control lines at Day 11,
showing typical neural rosettes (left panel), or Tuj1, Nkx2.1 right
and DRAQS in the right panel. Images representative of 3
independent experiments. (FIG. 1D) Fluorescence microscope images
of immunostained NEB cryosections or dissociated NEBs into a
monolayer with the BFCN markers Nkx2.1/Tuj1/p75/BF1/MAP2/ChAT.
(FIG. 1E) Dissociated NEBs into a monolayer immunostained at Day 50
with MAP2, ChAT and Hoescht. Fluorescence microscope images the
effect of NGF addition to SAG plus purmorphamine treatment alone.
Images are representative of at least 3 independent
experiments.
[0019] FIGS. 2A-2F. Basal cholinergic markers in PSEN2.sup.N141I
neuroprecursors. (FIG. 2A) Table showing the cell lines used. Four
iPS lines reprogrammed from fibroblasts were used; two controls
(949 and 050643, labelled as fControl and Control, respectively)
that do not carry the PSEN2.sup.N141I mutation nor the .English
Pound.4 allele; and two AD patients (948 and 950, labelled as AD1
and AD2, respectively) who carry the mutation and the .English
Pound.4 allele. Three of the four iPS lines were family related
(fControl, AD1, and AD2). (FIG. 2B) Representative Sanger
sequencing chromatograms showing a fragment of exon 5 of PSEN2.
Arrow marks site of the missense point mutation Chr1:227,073,304
A>T. (FIG. 2C) Immunocytochemistry and RT-PCR for early neuronal
and basal forebrain markers. n=3, 3 independent experiments with
technical triplicates. (FIG. 2D) RTPCR fold changes for TUJ1 and
BF1. n=3, 3 independent experiments with technical triplicates.
(FIG. 2E) Representative histograms for P75 staining. n>6. (F)
A.beta.40 and A.beta.42 ELISA quantifications. n=3, 3 independent
experiments with technical triplicates. ***, p<0.001. *,
p<0.05.
[0020] FIGS. 3A-3B. Neuronal and basal cholinergic markers by
immunocytochemistry. (FIG. 3A) Immunostaining for TrkA on DIV 21.
(FIG. 3B) Immunostainings for ChAT and vAChT at different
magnifications at DIV65; and Tuj1 and MAP2. Images are
representative of at least 3 independent experiments.
[0021] FIGS. 4A-4D. CRISPR/Cas9-mediated correction of
PSEN2.sup.N141I iPS lines. (FIG. 4A) Schematic showing guide RNAs
used in the targeting of CRISPR/Cas9, as well as donor ssODNs
utilized to introduce wild-type genotype. Sequence identifiers from
top to bottom: SEQ ID NOs: 26-31. (FIG. 4B) Left 2 panels show GFP
positive HEK293T cells indicating Cas9 system with guide RNA
expression, NT refers to non-transfected; right 2 panels show
sample of GFP positive iPSCs after lipofection with
pCas9-gN141I-GFP vector. (FIG. 4C) Sanger sequencing results from
iPSC lines, showing corrections in the N141I mutation. (FIG. 4D)
A.beta. 42/40 ratio detected by ELISA in 72 h conditioned media
from mutant, control or Cispr-Cas9 corrected BFCNs (DIV 34). n=4, 4
independent experiments with technical triplicates. *, p<0.05;
**, p<0.01 Student T-test.
[0022] FIGS. 5A-5B. BFCNs carrying various PSEN mutations are not
consistently more susceptible to A.beta.42 oligomer toxicity. (FIG.
5A) Sample images of BFCNs from the indicated genotypes treated
with propidium iodide to visualize cell death in response to 72-h
exposure to A.beta.42 oligomers (5 .mu.M). (FIG. 5B) % LDH Release
recorded from media collected after 72-h exposure. n=3, 3
independent experiments with technical triplicates. *, p<0.05;
**, p<0.01 as detected by 2-Way ANOVA Bonferroni post hoc
tests.
[0023] FIGS. 6A-6F. NLRP2 inflammasome mRNA levels are
over-expressed in some PSEN2.sup.N141I cells, but it is not driven
by mutation. RT-PCR expression of (FIG. 6A) NLRP2, (FIG. 6B) NLRP3,
and (FIG. 6C) ASB9 in cholinergic neuroprecursors. (FIG. 6D)
Western blot showing NLRP2, PSEN2 and .beta.-Actin. RT-PCR
expression of NLRP2 in Neuroprecursors (FIG. 6E) and BFCNs (F).
n=3, 3 independent experiments with technical triplicates, for all
panels. ***, p<0.001.
[0024] FIGS. 7A-7B. Electrophysiological and morphological features
of BFCN. (FIG. 7A) Top row--compound sodium and potassium currents
produced by a voltage protocol shown in bottom row. Current trace
produced by a voltage step to -20 mV shown in red. Inset shows
first 25 ms of a current produced by a voltage step to -20 mV
(scale bars 200 pA, 5 ms). (FIG. 7B) Differential interference
contrast image of a patched BFCN recorded in (A). Ninety-four
neurons (22 wild-type control, 21 familial control, 18 AD1, 28 AD2
and 5 iAD1_control). Scale bar is 30 .mu.m.
[0025] FIGS. 8A-8C. Electrophysiological deficits in BFCNs from AD
lines. (FIG. 8A) Co-localization of biocytin-labelled neurons with
cholinergic markers ChAT and VAChT. Arrows indicate positions of
recorded neurons somas, scale bar is 50 .mu.m. (FIG. 8B)
Representative firing patterns of BFCNs produced by a 1 sec
negative and positive square current injection are depicted. A
grand total of 94 individual neurons were studied
electrophysiologically: 22 wild-type control neurons, 21 familial
control neurons, 18 AD1 neurons, 28 AD2 neurons, and 5
iAD1_(CRISPR-corrected) neurons. The experiments on the 94 neurons
required days to weeks. On each experimental day, representatives
from each genotype were included, with at least three samples from
each genotype studied each day. (FIG. 8C) Summary data on maximum
number of action potentials that neurons are capable of sustaining
(left) and height of a single action potential at rheobase (right)
across all conditions. Individual data points are shown as circles,
group means are shown as bars. **, p<0.01 Tukey HSD test.
[0026] FIGS. 9A-9D. Intrinsic electrophysiological properties of
BFCNs. Summary data on all recorded BFCNs from five groups.
Ninety-four neurons (22 wild-type control, 21 familial control, 18
AD1, 28 AD2 and 5 iAD1_control). Histograms show individual values
from each neuron (circle) and group means (bars) for membrane
resistance (FIG. 9A), capacitance (FIG. 9B), resting potential
(FIG. 9C) and rheobase current (FIG. 9D). Statistical significance
was tested with ANOVA and Tukey's post hoc comparisons.
[0027] FIGS. 10A-10B. Quality control of iPSC lines. (FIG. 10A)
Immunofluorescence shows expression of pluripotency markers SSEA4,
Nanog, Tra160 and in 7889(S)B iPSC line. (FIG. 10B) Three germ
layers (endoderm, mesoderm, and ectoderm) from teratomas generated
by 7889(S)B iPSC line.
[0028] FIGS. 11A-11B. Amyloid .beta. levels in mature BFCNs. (FIG.
11A) Levels of A.beta.40 on BFCNs (DIV 34). *, P<0.01 vs. other
lines in study according to One-Way ANOVA Bonferroni Post-hoc test.
(FIG. 11B) Levels of A.beta.42 on BFCNs (DIV 34). n=3, 3
independent experiments with technical triplicates. *, P<0.01
based on Student's T-test.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention is based on the discovery of a robust,
fast, and reproducible differentiation protocol to generate BFCNs
from PSCs using a chemically defined medium.
[0030] The following is a detailed description of the invention
provided to aid those skilled in the art in practicing the present
invention. Those of ordinary skill in the art may make
modifications and variations in the embodiments described herein
without departing from the spirit or scope of the present
invention. Unless otherwise defined, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention belongs.
The terminology used in the description of the invention herein is
for describing particular embodiments only and is not intended to
be limiting of the invention. All publications, patent
applications, patents, figures and other references mentioned
herein are expressly incorporated by reference in their
entirety.
[0031] Although any methods and materials similar or equivalent to
those described herein can also be used in the practice or testing
of the present invention, the preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and described the methods and/or
materials in connection with which the publications are cited.
[0032] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
following references, the entire disclosures of which are
incorporated herein by reference, provide one of skill with a
general definition of many of the terms (unless defined otherwise
herein) used in this invention: Singleton et al., Dictionary of
Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge
Dictionary of Science and Technology (Walker ed., 1988); The
Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer
Verlag (1991); and Hale & Marham, the Harper Collins Dictionary
of Biology (1991). Generally, the procedures of molecular biology
methods described or inherent herein and the like are common
methods used in the art. Such standard techniques can be found in
reference manuals such as for example Sambrook et al. (2000,
Molecular Cloning--A Laboratory Manual, Third Edition, Cold Spring
Harbor Laboratories); and Ausubel et al. (1994, Current Protocols
in Molecular Biology, John Wiley & Sons, New-York).
[0033] The terminology used in the description is for describing
particular embodiments only and is not intended to be limiting of
the invention. Where a range of values is provided, it is
understood that each intervening value, to the tenth of the unit of
the lower limit unless the context clearly dictates otherwise (such
as in the case of a group containing a number of carbon atoms in
which case each carbon atom number falling within the range is
provided), between the upper and lower limit of that range and any
other stated or intervening value in that stated range is
encompassed within the invention. The upper and lower limits of
these smaller ranges may independently be included in the smaller
ranges is also encompassed within the invention, subject to any
specifically excluded limit in the stated range. Where the stated
range includes one or both of the limits, ranges excluding either
both of those included limits are also included in the
invention.
[0034] The following terms are used to describe the present
invention. In instances where a term is not specifically defined
herein, that term is given an art-recognized meaning by those of
ordinary skill applying that term in context to its use in
describing the present invention.
[0035] The articles "a" and "an" as used herein and in the appended
claims are used herein to refer to one or to more than one (i.e.,
to at least one) of the grammatical object of the article unless
the context clearly indicates otherwise. By way of example, "an
element" means one element or more than one element.
[0036] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements).
[0037] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e., "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of."
[0038] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from anyone or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
nonlimiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements).
[0039] It should also be understood that, in certain methods
described herein that include more than one step or act, the order
of the steps or acts of the method is not necessarily limited to
the order in which the steps or acts of the method are recited
unless the context indicates otherwise.
[0040] The term "PSEN2 gene" refers herein to a gene that encodes a
PSEN2 polypeptide. The PSEN2 gene is represented by NCBI Reference
Sequence: NC_000001.11 (SEQ ID NO: 1) as well as known orthologs.
The term "PSEN2 polypeptide" refers herein to a polypeptide that is
represented by NCBI Reference Sequence: NP_000438.2 (SEQ ID NO: 2)
as well as known orthologs.
[0041] The term "amyloidogenic disease" includes any disease
associated with (or caused by) the formation or deposition of
insoluble amyloid fibrils. Exemplary amyloidogenic diseases
include, but are not limited to systemic amyloidosis, Alzheimer's
disease, mature onset diabetes, Parkinson's disease, Huntington's
disease, fronto-temporal dementia, and the prion-related
transmissible spongiform encephalopathies (kuru and
Creutzfeldt-Jacob disease in humans and scrapie and BSE in sheep
and cattle, respectively). Different amyloidogenic diseases are
defined or characterized by the nature of the polypeptide component
of the fibrils deposited. For example, in subjects or patients
having Alzheimer's disease, .beta.-amyloid protein (e.g.,
wild-type, variant, or truncated .beta.-amyloid protein) is the
characterizing polypeptide component of the amyloid deposit.
Accordingly, Alzheimer's disease is an example of a "disease
characterized by deposits of A.beta." or a "disease associated with
deposits of A.beta.", e.g., in the brain of a subject or patient.
The terms .beta.-amyloid protein", .beta.-amyloid peptide",
".beta.-amyloid", "A.beta." and "A.beta. peptide" are used
interchangeably herein.
[0042] The term "patient" or "subject" is used throughout the
specification to describe an animal, preferably a human or a
domesticated animal, to whom treatment, including prophylactic
treatment, with the compositions according to the present
disclosure is provided. For treatment of conditions or disease
states which are specific for a specific animal such as a human
patient, the term patient refers to that specific animal, including
a domesticated animal such as a dog or cat or a farm animal such as
a horse, cow, sheep, etc. In general, in the present disclosure,
the term patient refers to a human patient unless otherwise stated
or implied from the context of the use of the term.
[0043] The terms ".beta.-amyloid protein", ".beta.-amyloid
peptide", ".beta.-amyloid", "A.beta." and "A.beta. peptide" are
used interchangeably herein. A.beta. peptide (e.g., A.beta. 39,
A.beta. 40, A.beta. 41, A.beta. 42 and A.beta. 43) is about 4-kDa
internal fragment of 39-43 amino acids of APP. A.beta. 40, for
example, consists of residues 672-711 of APP and A.beta. 42
consists of residues 672-713 of APP. A.beta. peptides include
peptides resulting from secretase cleavage of APP and synthetic
peptides having the same or essentially the same sequence as the
cleavage products. A.beta. peptides can be derived from a variety
of sources, for example, tissues, cell lines, or body fluids (e.g.,
sera or cerebrospinal fluid). For example, an A .beta. can be
derived from APP-expressing cells such as Chinese hamster ovary
(CHO) cells stably transfected with APP.sub.717V F, as described,
for example, in Walsh et al. (2002), Nature, 416, pp 535-539. An A
.beta. preparation can be derived from tissue sources using methods
previously described (see, e.g., Johnson-Wood et al. (1997), Proc.
Natl. Acad. Sci. USA 94:1550). Alternatively, A.beta. peptides can
be synthesized using methods which are well known to those in the
art. See, for example, Fields et al., Synthetic Peptides: A User's
Guide, ed. Grant, W.H. Freeman & Co., New York, N.Y., 1992, p
77). Hence, peptides can be synthesized using the automated
Merrifield techniques of solid phase synthesis with the
.alpha.-amino group protected by either t-Boc or F-moc chemistry
using side chain protected amino acids on, for example, an Applied
Biosystems Peptide Synthesizer Model 430A or 431. Longer peptide
antigens can be synthesized using well known recombinant DNA
techniques. For example, a polynucleotide encoding the peptide or
fusion peptide can be synthesized or molecularly cloned and
inserted in a suitable expression vector for the transfection and
heterologous expression by a suitable host cell. A .beta. peptide
also refers to related A.beta. sequences that results from
mutations in the A.beta. region of the normal gene.
[0044] As used herein the phrase "substantially pure" refers to a
population of cells wherein at least 95% of the cells have the
recited phenotype. In all embodiments that refer to a
"substantially pure" cell population, alternative embodiments in
which the cell populations have a lower or higher level of purity
are also contemplated. For example, in some embodiments, instead of
a given cell population being "substantially pure" the cell
population may be one in which at least 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of
the cells, or 100% of the cells, have the recited phenotype.
[0045] The terms "co-administration", "co-administered" and
"co-administering" or "combination therapy" refer to both
concurrent administration (administration of two or more agents at
the same time) and time varied administration (administration of
one or more agents at a time different from that of the
administration of an additional agent or agents), as long as the
agents are present in the area to be treated to some extent,
preferably at effective amounts, at the same time.
[0046] The term "therapeutically effective amount" means the amount
required to achieve a therapeutic effect. The therapeutic effect
could be any therapeutic effect ranging from prevention, symptom
amelioration, symptom treatment, to disease termination or cure,
e.g., the treatment of Alzheimer's disease or an associated
condition.
[0047] As used herein, the term "administering" is meant to refer
to a means of providing the composition to the subject in a manner
that results in the composition being inside the subject's body.
Such an administration can be by any route including, without
limitation, subcutaneous, intradermal, intravenous, intra-arterial,
intraperitoneal, and intramuscular.
[0048] The term "effective" is used to describe an amount of a
compound, composition or component which, when used within the
context of its intended use, effects an intended result. The term
effective subsumes all other effective amount or effective
concentration terms, which are otherwise described or used in the
present application.
[0049] As used herein, the term "comprising" is intended to mean
that the compositions and methods include the recited elements, but
do not exclude other elements. "Consisting essentially of", when
used to define compositions and methods, shall mean excluding other
elements of any essential significance to the combination. Thus, a
composition consisting essentially of the elements as defined
herein would not exclude trace contaminants from the isolation and
purification method and pharmaceutically acceptable carriers, such
as phosphate buffered saline, preservatives, and the like.
"Consisting of" shall mean excluding more than trace elements of
other ingredients and substantial method steps for administering
the compositions of this invention. Embodiments defined by each of
these transition terms are within the scope of this invention.
[0050] Ranges provided herein are understood to be shorthand for
all of the values within the range. For example, a range of 1 to 50
is understood to include any number, combination of numbers, or
sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, and 50.
[0051] As used herein, "kits" are understood to contain at least
the non-standard laboratory reagents of the invention and one or
more non-standard laboratory reagents for use in the methods of the
invention.
[0052] The term "obtaining" is understood herein as manufacturing,
purchasing, or otherwise coming into possession of.
[0053] As used herein, the term "treated," "treating" or
"treatment" includes the diminishment or alleviation of at least
one symptom associated or caused by the state, disorder or disease
being treated. A subject that has been treated can exhibit a
partial or total alleviation of symptoms (for example, Alzheimer's
disease or associated condition), or symptoms can remain static
following treatment according to the invention. The term
"treatment" is intended to encompass prophylaxis, therapy and
cure.
[0054] As used herein, the term "control" refers to a sample or
standard used for comparison with an experimental sample. In some
embodiments, the control is a sample obtained from a healthy
patient. In other embodiments, the control is a historical control
or standard reference value or range of values (such as a
previously tested sample, subject, or group of samples or
subjects).
Methods
[0055] BFCNs are believed to be one of the first cell types to be
affected in all forms of AD, and their dysfunction is clinically
correlated with impaired short-term memory formation and retrieval.
As detailed in the Example of this disclosure, the inventors
present an optimized in vitro protocol to generate human BFCNs from
iPSCs, using cell lines from PSEN2 mutation carriers and controls.
Cell lines harboring the PSEN2.sup.N141I mutation displayed an
increase in the A.beta.42/40 in iPSC-derived BFCNs. Neurons derived
from PSEN2.sup.N141I lines generated fewer maximum number of spikes
in response to a square depolarizing current injection. The height
of the first action potential at rheobase current injection was
also significantly decreased in PSEN2.sup.N141I BFCNs. CRISPR/Cas9
correction of the PSEN2 point mutation abolished the
electrophysiological deficit, restoring both the maximal number of
spikes and spike height to the levels recorded in controls.
Increased A.beta.42/40 was also normalized following
CRISPR/Cas-mediated correction of the PSEN2.sup.N141I mutation. The
genome editing data set forth herein confirms the robust
consistency of mutation-related changes in A.beta.42/40 ratio while
also showing a PSEN2-mutation-related alteration in
electrophysiology.
[0056] Accordingly, in one embodiment, the invention provides a
method of generating BFCNs. The method may first include preparing
PSC colonies. PSCs are seeded (plated) at low density and grown in
an adherent culture for about 1-2 days. "Low density" means about
8,000 to about 11,000 cells/cm.sup.2. Cells are preferably seeded
at about 9,500 to about 10,500 cells/cm.sup.2, more preferably at
about 10,000 cells/cm.sup.2. After about 1-2 days (or greater,
i.e., 3, 4, 5, 6, 7, 8, 9, 10 or more), the PSCs form colonies,
which are preferably about 75 .mu.m to about 300 .mu.m in diameter,
more preferably about 100 .mu.m to about 250 .mu.m in diameter.
[0057] The term "PSCs" has its usual meaning in the art, i.e.,
self-replicating cells that have the ability to develop into
endoderm, ectoderm, and mesoderm cells. Preferably, PSCs are hPSCs.
PSCs include ESCs and iPSCs, preferably hESCs and hiPSCs. PSCs can
be seeded on a surface comprising a matrix, such as a gel or
basement membrane matrix. A preferable matrix is the protein
mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma
cells, sold under trade names including MATRIGEL.RTM.,
CULTREX.RTM., and GELTREX.RTM.. Other suitable matrices include,
without limitation, collagen, fibronectin, gelatin, laminin,
poly-lysine, vitronectin, and combinations thereof.
[0058] In some embodiments, media suitable for use in maintaining
pluripotent stem cells is used. In embodiments such a medium is
mTeSR1 medium from Stem Cell Technologies. However, one of skill in
the art will recognize that there are several other types of media
that are equivalent to mTeSR medium in terms of their suitability
for use in maintaining pluripotent stem cells, any of which could
be used. Typically such media will contain one or more pluripotency
factors to facilitate the maintenance of cells in a pluripotent
state. The composition of mTeSR1 medium is known in the art and
described in, for example, Ludwig et al., 2006 (Nat Methods. 2006
August; 3(8):637-46; "Feeder-Independent Culture of Human Embryonic
Stem Cells"), the contents of which are hereby incorporated by
reference.
[0059] The pluripotent stem cells used in the method of the
invention can be any suitable type of pluripotent stem cells. Where
iPSCs are used, such cells may have been "reprogrammed" to the
pluripotent state from a non-pluripotent state using any suitable
means known in the art, including, but not limited to, modified
RNA-based methods, Sendai virus based methods, and the like.
Furthermore, such cells may have been reprogrammed to the
pluripotent state using any suitable cocktail of reprogramming
factors known in the art.
[0060] In one embodiment, after PSCs are prepared and grown to
confluence in, for example mTeSR1 media, the method includes
culturing the PSCs in a basal media comprising an inhibitor of
transforming growth factor beta (TGF-.beta.) signaling and an
activator of sonic hedgehog (Shh) signaling to induce
neuroectodermal differentiation. The basal media utilized is a
modified mTeSR1 medium, which is a variant of mTeSR1 medium
(sometimes referred to herein as "mTeSR1 Custom" medium) that does
not comprise lithium chloride, GABA, pipecolic acid, bFGF or
TGF.beta.1. Inhibitors of TGF.beta. signaling include, for example,
one or more of SB431542, GW788388, LDN193189, LY2109761, LY2157299,
and LY364947. Activators of Shh signaling include agonists of
Smoothened, such as Smoothened Agonist (SAG;
3-chloro-N-[(1r,4r)-4-(methylamino)cyclohexyl]-N-[3-(pyridin-4-yl)b-
enzyl]benzo[b]thiophene-2-carboxamide) and purmorphamine.
[0061] After about 6, 7, 8, 9, 10, 11 or 12 days (or greater, i.e.,
15, 16, 17, 18, 19, 20, 25, 30 or more) of culturing, CD271+ cells
are selected and cultured in a neuronal basal medium, such as
Brainphys.TM. to generate neuronal embryoid bodies (NEBs). The
neuronal basal medium is optionally supplemented with one or more
of B27 supplement, an inhibitor of rho-associated protein kinase
(ROCK), nerve growth factor (NGF) and brain derived neurotrophic
factor (BDNF). Inhibitors of ROCK include, for example, GSK269962,
GSK429286, H-1152, HA-1077, RKI-1447, thiazovivin, Y-27632, or
derivatives thereof.
[0062] To select for CD271+ cells, overconfluent cells are lifted
from the culture surface and purified by FACS and re-plated. This
process allows for the formation of cell aggregates or spheres,
also referred to herein as NEBs. For purposes of the present
invention, the terms "NEB," "aggregate" and "sphere" are used
interchangeably and refer to a multicellular three-dimensional
structure, preferably, but not necessarily, of at least about 100
cells.
[0063] Lifting can be performed mechanically, with a cell scraper
or other suitable implement, or chemically. Chemical lifting can be
achieved using a proteolytic enzyme, for example, collagenase,
trypsin, trypsin-like proteinase, recombinant enzymes, such as that
sold under the trade name TRYPLE.TM., naturally derived enzymes,
such as that sold under the trade name ACCUTASE.TM., and
combinations thereof. Chemical lifting can also be done using a
chelator, such as EDTA, or a compound such as urea. Mechanical
lifting or detachment offers the advantage of minimal cell death,
however it produces aggregates of variable size, thus suitable
spheres need to be selected through a manual picking process. Good
spheres are defined as those having a round-shape, golden/brown
color, with darker core and with a diameter between about 300 .mu.m
and about 800 .mu.m. Detaching the cells using chemical methods,
such as enzymatic digestion predominantly produces spheres that are
appropriate for further culture. Therefore manual picking of
spheres is not required, and the detachment steps can be adapted
for automation and used in high throughput studies. However,
enzymatic digestion increases cell death, resulting in a lower
number of spheres.
[0064] After about 5, 6, 7, 8, 9 or 10 days (or greater, i.e., 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more) of culturing
selected CD271+ cells, the formed NEBs are harvested, dissociated,
and plated as monolayer cultures and further cultured in a neuronal
basal medium, such as Brainphys.TM., optionally supplemented with
B27 supplement, NGF and BDNF. The surface on which the cells are
plated and cultured can comprise an extracellular matrix protein
(e.g., collagen, fibronectin, laminin) and/or a positively charged
poly-amino acid (e.g., poly-arginine, poly-lysine, poly-ornithine).
Preferably the surface comprises laminin and/or poly-ornithine.
[0065] To ensure differentiation into BFCNs, the cultured cells are
analyzed for positive expression of Tuj1, MAP2, BF1, Nkx2.1 and
p75.
[0066] Many of the embodiments of the present invention involve
certain factors to be used in (or excluded from) the compositions
and methods described herein, for example as media supplements.
These include, but are not limited to, bFGF, GABA, pipecolic acid,
lithium chloride, TGF-.beta., NGF and BDNF. Each of these factors
is well known in the art, including the full names of each of these
factors in the cases where acronyms or other abbreviations are
used. Furthermore, all of these factors are available to the public
from multiple sources, including commercial sources. Exemplary
amounts/concentrations for use of each of these factors in the
methods and compositions of the present invention are provided in
the Examples section of this patent disclosure. For all embodiments
where specified amounts are referred to, amounts that are "about"
the specified amount are also intended. Furthermore, one of skill
in the art will recognize that in some situations further
deviations from the specified amounts can be used, and will be able
to determine how much of each factor to use by performing routine
testing, optimization, dose-response studies, and the like, for
example to reduce or increase the specified amounts, as long as the
amounts used still achieve the stated effect, e.g., the stated
differentiation effect. For example, in some embodiments specified
amounts of the specified agents may be reduced to 10%, or 20%, or
30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% of the stated
amounts. Similarly, in some embodiments the specified amounts of
the specified agents may be increased by 10%, by 20%, by 30%, by
40%, by 50%, by 60%, by 70%, by 80%, by 90%, by 100%, by 150%, by
200%, by 300%, by 400%, or by 500% of the stated amounts.
Similarly, where specified factors are referred to, one of skill in
the art will recognize that analogs, variants, or derivatives of
such factors can also be used as long as the analogs, variants, or
derivatives have the same general function/activity as the
specified factors.
[0067] As discussed herein, the inventors have discovered that
mutation of PSEN2 results in impaired neuronal excitability in
BFCNs. As discussed in the Example herein the inventors observed
significant mutation-related, editing-reversible differences in
excitability of BFCNs via repair of PSEN2.sup.N141I mutation which
restored neuronal excitability. Accordingly, the method of the
invention may utilize iPSCs which may be treated with a gene
editing system to repair one or more mutations, such as a mutation
of presenilin 1 (PSEN1) or presenilin 2 (PSEN2). In one embodiment,
the mutation is PSEN2.sup.N141I, repair of which restores neuronal
excitability in BFCNs.
[0068] As used herein the term "gene editing" or "genome editing"
refers to a type of genetic engineering in which DNA is inserted,
replaced, or removed from a target DNA, e.g., the genome of a cell,
using one or more nucleases and/or nickases. The nucleases create
specific double-strand breaks (DSBs) at desired locations in the
genome, and harness the cell's endogenous mechanisms to repair the
induced break by homology-directed repair (HDR) (e.g., homologous
recombination) or by nonhomologous end joining (NHEJ). The nickases
create specific single-strand breaks at desired locations in the
genome. In one non-limiting example, two nickases can be used to
create two single-strand breaks on opposite strands of a target
DNA, thereby generating a blunt or a sticky end. Any suitable
nuclease can be introduced into a cell to induce genome editing of
a target DNA sequence including, but not limited to,
CRISPR-associated protein (Cas) nucleases, zinc finger nucleases
(ZFNs), transcription activator-like effector nucleases (TALENs),
meganucleases, other endo- or exo-nucleases, variants thereof,
fragments thereof, and combinations thereof. In particular
embodiments, nuclease-mediated genome editing of a target DNA
sequence (e.g., a safe harbor gene) by homology-directed repair
(HDR) (e.g., homologous recombination) is used for generating a
genetically modified human neural stem cell in accordance with the
methods described herein.
[0069] The term "DNA nuclease" refers to an enzyme capable of
cleaving the phosphodiester bonds between the nucleotide subunits
of DNA, and may be an endonuclease or an exonuclease. According to
the invention, the DNA nuclease may be an engineered (e.g.,
programmable or targetable) DNA nuclease which can be used to
induce genome editing of a target DNA sequence such as a safe
harbor gene. Any suitable DNA nuclease can be used including, but
not limited to, CRISPR-associated protein (Cas) nucleases, zinc
finger nucleases (ZFNs), transcription activator-like effector
nucleases (TALENs), meganucleases, other endo- or exo-nucleases,
variants thereof, fragments thereof, and combinations thereof.
[0070] In various embodiments of the invention, a gene editing
system utilizes a DNA nuclease to edit a gene and repair a
mutation. In specific embodiments, a mutation is repaired using one
or more of the following gene editing system: CRISPR/Cas system,
Cre/Lox system, TALEN system and homologous recombination.
[0071] The differentiation protocol of the present invention may be
utilized to treat a disease or disorder in a subject. The method
includes administering to a subject a BFCN generated using the
culturing method described herein. In various embodiments, the
disease or disorder is an amyloidogenic disease, such as systemic
amyloidosis, Alzheimer's disease, mature onset diabetes,
Parkinson's disease, Huntington's disease, fronto-temporal
dementia, and prion-related transmissible spongiform
encephalopathies. In embodiments, a BFCN having a mutation that
impairs neuronal excitability, such PSEN2.sup.N141I, may be
obtained from a subject and used to generate an iPSC, which in turn
may be treated with a gene editing system to repair the mutation.
The gene edited iPSC is then cultured as described herein to
produce BFCNs having restored neuronal excitability which are
administered to the subject to treat the disease or disorder.
[0072] In a related manner, neuronal excitability in BFCNs in a
subject may be restored. This method includes: a) isolating a BFCN
from the subject, wherein the BFCN has a genetic mutation resulting
in impaired neuronal excitability of the BFCN; b) generating an
iPSC using the BFCN of (a); c) repairing the mutation in the iPSC;
d) culturing the iPSC of (c) using the differentiation protocol
described herein to generate a BFCN having the repaired mutation;
and e) administering the iPSC of (d) to the subject, thereby
restoring neuronal excitability in BFCNs in the subject. In
embodiments, the mutations is of PSEN1 or PSEN2, such as
PSEN2.sup.N141I.
[0073] The invention also encompasses BFCNs generated using the
differentiation protocol as described herein. The BFCN may include
a gene edited repair of PSEN2, as well as a detectable marker
recombinantly introduced into the BFCN genome. In some embodiments,
BFCNs are differentiated from PSCs, and in such embodiments, the
PSCs can be iPSCs. The iPSCs can be derived from a somatic cell of
a subject. In one aspect, the subject has an amyloidogenic disease
or disorder.
[0074] Alongside its potential for autologous cell transplantation,
iPSC technology is emerging as a tool for developing new drugs and
gaining insight into disease pathogenesis. Han, S. S. W. et al.,
Neuron. 70:626-644 (2011). The methods and cells of the invention
can aid the development of high-throughput in vitro screens for
compounds that promote restoration of neuronal excitability. To
that end, the disclosure provides a method of identifying a
compound that can be used for treatment or prevention of a disease
or disorder associated with diminished neuronal excitability in
BFCNs. The method includes: a) contacting a BFCN or neuronal
embryoid body (NEB) generated using the differentiation protocol
described herein with a candidate compound, wherein the BFCN
comprises a mutation in PSEN2 resulting in impaired neuronal
excitability of the BFCN; and b) detecting neuronal excitability of
the BFCN after contact with the candidate compound. A beneficial
effect on neuronal excitability is evident in partial or complete
restoration of neuronal excitability, thereby being indicative of a
candidate therapeutic agent for treating a disease or disorder
associated with diminished neuronal excitability in BFCNs, such as
an amyloidogenic disease or disorder. Preferably, the method is
conducted in a high-throughput format.
[0075] The invention also provides a model system for a
neurological disease, preferably a disease or disorder associated
with diminished neuronal excitability in BFCNs, such as an
amyloidogenic disease or disorder. In one aspect, the model system
comprises a BFCN differentiated from an iPSC derived from a subject
having a disease or disorder associated with diminished neuronal
excitability in BFCNs, such as an amyloidogenic disease or
disorder. The model system can further comprise a non-human mammal
into which the myelin-producing cell has been transplanted. In one
embodiment, the non-human mammal is a mouse or a rat. Model systems
provided by the invention can be used to study diseases or
disorders, including understanding underlying mechanisms and
defining therapeutic targets.
[0076] In some embodiments the present invention provides tissue
culture media, tissue culture media supplements, and various kits
useful in performing the various methods described herein.
[0077] In one embodiment, the invention provides a kit for
generating a BFCN via the differentiation protocol of the
invention. The kit includes a culture media having an inhibitor of
TGF-.beta. signaling and an activator of Shh signaling. In
embodiments, the culture media is a modified mTeSR1 formulation
that lacks factors that support pluripotency including bFGF,
TGF-.beta., lithium chloride (Li--Cl), GABA and pipecolic acid. In
embodiments, the culture media includes dual SMAD inhibitors, such
as SB431542 and LDN193189, along with one or more agonists of
smoothened protein, such as smoothened agonist (SAG) and
purmorphamine.
[0078] The kit may also include an additional neuronal basal
medium, such as Brainphys.TM. optionally supplemented with one or
more of B27 supplement, an inhibitor of ROCK, NGF and BDNF.
[0079] The kit may also include reagents for detection and
isolation of CD271+ cells via FACS as well as detection of
expression of Tuj1, MAP2, BF1, Nkx2.1 and p75.
[0080] The kit may optionally comprise instructions for use, one or
more containers, one or more antibodies, or any combination
thereof. A label typically accompanies the kit, and includes any
writing or recorded material, which may be electronic or computer
readable form (e.g., disk, optical disc, memory chip, or tape)
providing instructions or other information for use of the kit
contents.
[0081] The following example is provided to further illustrate the
advantages and features of the present invention, but it is not
intended to limit the scope of the invention. While this example is
typical of those that might be used, other procedures,
methodologies, or techniques known to those skilled in the art may
alternatively be used.
Example I
CRISPR/Cas9-Correctable Mutation-Related Molecular and
Physiological Phenotypes in iPSC-Derived Alzheimer's
PSEN2.sup.N141I Mutation
[0082] Basal forebrain cholinergic neurons (BFCNs) are believed to
be one of the first cell types to be affected in all forms of AD,
and their dysfunction is clinically correlated with impaired
short-term memory formation and retrieval. We present an optimized
in vitro protocol to generate human BFCNs from iPSCs, using cell
lines from presenilin 2 (PSEN2) mutation carriers and controls. As
expected, cell lines harboring the PSEN2.sup.N141I mutation
displayed an increase in the A.beta.42/40 in iPSC-derived BFCNs.
Neurons derived from PSEN2.sup.N141I lines generated fewer maximum
number of spikes in response to a square depolarizing current
injection. The height of the first action potential at rheobase
current injection was also significantly decreased in
PSEN2.sup.N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point
mutation abolished the electrophysiological deficit, restoring both
the maximal number of spikes and spike height to the levels
recorded in controls. Increased A.beta.42/40 was also normalized
following CRISPR/Cas-mediated correction of the PSEN2.sup.N141I
mutation. The genome editing data confirms the robust consistency
of mutation-related changes in A.beta.42/40 ratio while also
showing a PSEN2-mutation-related alteration in
electrophysiology.
[0083] The "amyloid hypothesis" is one of the most popular
formulations for the pathogenesis of Alzheimer's disease (AD).
Recent examples of clinicopathological and/or clini-coradiological
dissociation have led to the consideration of alternative models in
order to explain, respectively, why neuropathological AD is not
always associated with dementia [24], and why about one-third of
patients with clinical AD have negative amyloid brain scans [40].
It has been proposed that clinical AD can be caused by one of
several "feed-forward" scenarios linking amyloidosis, tauopathy,
neuroinflammation, and neurodegeneration [22]. Mutations in the
gene encoding presenilin 2 (PSEN2) are associated with autosomal
dominant early onset familial Alzheimer's disease (EOFAD). The
linkage of a locus on human chromosome 1q31-42 linked to EOFAD led
to the identification of the PSEN2.sup.N141I point mutation in the
Volga German kindreds in 1995 [43]. This mutation causes elevation
in the A.beta.42-43/40 ratio, thereby promoting assembly of A.beta.
oligomers and fibrils [83].
[0084] In considering the progression of AD, human basal forebrain
cholinergic neurons (BFCNs) are one of the first cell types whose
dysfunction underlies the early loss of short-term memory recall in
all forms of AD. The "cholinergic hypothesis of AD" was formulated
in the mid-1970s [6, 20, 61], and the discoveries of reduced
acetylcholine release from neurons of the nucleus basalis of
Meynert confirmed the presence of a presynaptic cholinergic deficit
in the basal forebrain of AD patients [1, 71]. Based on those
observations, acetylcholinesterase inhibitors were developed and
continue as the most widely used symptomatic treatments for AD [21,
28, 33, 82]. Eventually, post-mortem brain biochemical and
volumetric studies at different stages of the disease identified
several other regions of the brain that were also affected early in
the course of AD [63]. These studies have traditionally focused on
the hippocampus and cortex, but more recently, attention has begun
shifting back to the basal forebrain and adding other areas, such
as the striatum [27, 62]. The latest analyses suggest that
cholinergic basal forebrain volume measurement may be a better
predictor of the transition from MCI to AD than the previous
standard, hippocampal volume [10].
[0085] We previously reported the generation of iPSC-derived
neurons from banked fibroblasts from subjects harboring
PSEN1.sup.A246E and PSEN1.sup.M146L mutations [77]. In
characterizing the gene expression profiles from these iPSC-derived
neurons, we observed an unexpected association of elevated
expression of the inflammasome gene NLRP2 in undifferentiated PSEN1
mutant iPSCs and their and neuronally differentiated progeny [77].
This led us to examine NLRP2 expression in our PSEN2 mutant lines
and employ CRISPR/Cas9 [15] to investigate if activation of the
inflammasome was tightly linked to the pathogenic mutation in
PSEN2. While we did not find altered expression of NLRP2 in
gene-corrected PSEN2 lines, we observed significant
mutation-related, editing-reversible differences in excitability of
BFCNs.
Materials and Methods
[0086] Generation and Maintenance of iPSC Lines
[0087] 7889(s)B, 050643 (Control), 948 (AD1), 949(fControl), and
950 (AD2) iPSC lines were obtained via the NYSCF Repository
following the guidelines from [60]. The derivation and
characterization of Nkx2.1-GFP ESC line was previously published
[30]. ES and iPS cell lines were expanded and maintained in
serum-free mTeSR1.TM. media (Stem Cell Technologies). Cells were
lifted using StremPro.TM. Accutase (ThermoFisher) and media was
supplemented with 10 .mu.M ROCK inhibitor (Y27632, Stemgent) during
cell passaging.
[0088] For all studies in this paper, cell lines underwent at least
3 independent differentiations from the iPSC stage to the mature
neuron stage. Data were routinely compared across these
independently derived genotype-identical neurons (or in some cases
neuronal precursors), and if comparable results were obtained
across independently genotype-identical derived cells, they were
considered to be qualified representatives of their genotype and so
were passed along for genotype-specific experimentation.
A.beta.42 Oligomer Preparation
[0089] A.beta.42 oligomers were prepared as previously reported
[23, 78]. Briefly, we dissolved 1 mg of A.beta.42 (American Peptide
Company) in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (Sigma). This
preparation was aliquoted and dried using a SpeedVac.TM.
centrifuge. The pellet was then resuspended in DMSO to obtain a 5
mM solution which was sonicated in a water bath for 10 min. From
here aliquots were stored at -20 C and used within 2 weeks by
diluting with 100 .mu.l of PBS and leaving for 12 h at 4.degree. C.
in order for oligomerization to proceed. This final solution was
diluted 1:16 in cell media for studies, allowing cells to be
exposed to 5 .mu.M of A.beta.42 oligomers. Control wells were
diluted with 1:16 PBS. Cells were exposed to oligomers or PBS
without media change for a period of 3 days.
Cell Death Assays
[0090] Cells were assayed in a 96-well plate format. Oligomer or
vehicle solutions were added to media and allowed to incubate for a
period of 3 days. Media was then collected and assayed using a
lactate dehydrogenase toxicity assay (Thermo Fisher Scientific). 50
.mu.l of media and an equal amount of reaction mix buffer were
incubated for a period of 30 min. An additional set of wells per
experiment were treated with 2% Triton.TM. X-100 for a 5-min period
in order to lyse all cells, and media from these wells was also
collected and incubated as described. After incubation absorbance
was recorded at 490 nm and 680 nm, signal and background
absorbance, respectively. Signal values were subtracted from
background, and values were adapted to the total LDH content as
determined by Triton X-100 treated wells. Propidium iodide (Thermo
Fisher Scientific) was added to cell media for a 1 .mu.M final
concentration and allowed to incubate for 5 min. Cells were then
washed twice with media and imaged. Images were captured using
CELIGO' image cytometer and accompanying software (Nexcelom
Bioscience). Each biological variable was assessed in technical
triplicates within each designated "Experiment", and each
designated "Experiment" was performed in at least three complete
"start to finish" iterations.
Differentiation of Basal Forebrain Cholinergic Neurons from iPS and
ES Cells
[0091] Human ES or iPSC were plated as single cells after chemical
dissociation using Accutase.TM. (Sigma-Aldrich) into Cul-trex.TM.
(Trevigen) coated plates, at a density of 4-8.times.10.sup.5 cells
per well in 6-well plates or petri dishes and adapting cell
numbers. Cells were initially maintained in mTeSR1.TM. media (Stem
Cell Technologies) until reaching full confluency. On "day 0" of
differentiation, media was replaced by Custom mTeSR1.TM. media
(Stem Cell Technologies) lacking factors promoting pluripotency
i.e., bFGF, TGF-.beta., Li--Cl, GABA and pipecolic acid. The
addition of dual SMAD inhibitors (SB431542 10 .mu.M plus LDN193189
250 nM, Selleckchem) at day 0 drives cells towards neuroectoderm
specification. At day 2 of differentiation, media was replaced by
Custom mTeSR1 containing dual SMAD inhibitors plus two ventralizing
agents: SAG at 500 nM (R&D) and Purmorphamine at 2 .mu.M
(Stemgent.TM.). Cells were fed every 2 days with this media until
day 9, when media was progressively switched to Brainphys.TM. media
(Stemcell Technologies) supplemented with B27 (Life Technologies)
[3]. Neural progenitors were harvested at day 11 using Accutase,
p75+(CD271) NPCs were purified by FACS and plated at a density of
80,000 cells per well into non-adherent 96 well V-bottom plates in
Brainphys.TM.+B27 supplemented with 10 .mu.M ROCK inhibitor
(Y27632, Stemgent), Nerve Growth Factor, NGF (Alamone labs, 50
ng/mL) and Brain Derived Neurotrophic Factor, BDNF (R&D, 50
ng/mL). Cells were allowed to aggregate and form Neuronal Embryoid
Bodies (NEBs) and were fed every other day until day 19. At day 19
NEBs were dissociated using Accutase (Sigma-Aldrich) and were
plated as monolayer cultures on plates coated with branched
polyethynilimine (0.1%, Sigma-Aldrich) and laminin (10 mg/mL, Life
Technology) in Brainphys.TM. media+B27 supplement with BDNF and
NGF. The media was changed every 2 days until analysis. As an
alternative, 3D NEBs were dissected manually into 3-4 pieces for
expansion and further grown, or were cryopreserved. Initial
versions of the protocol used Neurobasal.TM. as a base media
instead of Brainphys.TM..
Genomic DNA Isolation and Sequencing
[0092] Genomic DNA was isolated from PSEN2 mutant or control iPSC
lines using High Pure.TM. PCR Template Preparation Kit (Roche)
following manufacturer instructions. Genomic samples were treated
with RNAse (QIAGEN) prior to amplification. A fragment from exon 5
of PSEN2 containing PSEN2.sup.N141I mutation was amplified using
the following primers: Forward 5'-CATCAGCCCTTTGCCTTCT-3' (SEQ ID
NO: 3), Reverse: 5'-CTCACCTTGTAGCAGCGGTA-3' (SEQ ID NO: 4),
generating a 173 bp fragment, regardless of the genotype. For
detection of ApoE allelic variants, a fragment of 244 bp was
amplified prior to sequencing using the primers: Forward:
5'-ACAGAATTCGCCCCGGCCTGGTACAC-3' (SEQ ID NO: 5), Reverse:
5'-TAAGCTTGGCACGGCTGTCCAAGGA-3' (SEQ ID NO: 6). Both PCR were
performed with the following settings: 10 min 94 C, 40 cycles (30 s
94 C, 20 s 62 C, 10 s 72 C) 7 min 72 C. PCR products were run in a
2% agarose gel to check the size of the amplified fragment. After
amplification, samples were cleaned using EXOSAP-it.TM. (Thermo
Fisher Scientific) and then sequenced using the following primers:
PSEN2 (Forward: 5'-TCAGCATCTACACGCCATTC-3' (SEQ ID NO: 7), Reverse:
5'-AGCACCACCAAGAAGATGGT-3') (SEQ ID NO: 8), from [53]; ApoE
(Forward: 5'-ATTCGCCCCGGCCTGGTACAC TGCCA-3' (SEQ ID NO: 9),
Reverse: 5'-CTGTCCAAGGAGCTGC AGGCGGCGCAG-3' (SEQ ID NO: 10)), from
[36].
CRISPR/Cas9 Gene Correction
[0093] iAD1 Control and iAD2 Control lines were originated from 948
(AD1) and 950 (AD2) iPSC lines by CRISPR/Cas9-mediated correction
of the PSEN2.sup.N141I/WT heterozygous point mutation to
PSEN2.sup.WT/WT. g1N141I single guide RNA (sgRNA) was cloned into
pSpCas9(BB)-2A-GFP (PX458) vector, generating pSpCas9-g1N141I-GFP
vector to direct gene editing to the sequence in exon 5 of PSEN2
where the Volga mutation is located. Single stranded
oligonucleotides (ssODN) are efficient templates for the
CRISPR/Cas9 correction [13, 66]. ssODN #A-N141I (sequence detail
below) was used as donor sequence for gene correction. We designed
asymmetric ssODN sequences with a long homology arm of 91 bp, and a
short homology arm of 36 bp since asymmetrical ssODNs showed a
higher efficiency of homology-directed repair using CRISPR/Cas9
[68].
TABLE-US-00001 TABLE 1 Sequence SEQ Name Bases Sequence ID NO
g1N141I 25 /5Phos/CACCGCATCATGATC 11 guide RNA F AGCGTCATCG g1N141I
25 /5Phos/AAACCGATGACGCTG 12 guide RNA R ATCATGATGC Donor 127
GAGAGAAGCGTGGCTGGAGGGC 13 ssODN#A AGGGCCAGGGCCTCACCTTGTA N141I
GCAGCGGTACTTGTAGAGCACC ACCAAGAAGATGGTCATAACCA
CGATGACGCTGATCATGATGAG GGTGTTCAGCACGGAGT Underline = Base of the
ssODN that Corrects the Point Mutation
[0094] The donor sequence and pSpCas9-g1N141I-GFP vector were
transduced in the AD1 and AD2 iPSC lines, plated at 50-70%
confluency, using Amaxa Human Stem Cell Nucleofector.TM. kit (Lonza
VPH-5002) and re-plated for recovery. GFP.sup.+ cells were sorted
in a BD FACSAria IIu Cell Sorter.TM. and were seeded at 30-50 cells
per well in 96-well format to detect and pick single clones.
Positive clones were expanded, qDNA was extracted and analyzed for
successful HDR was determined using a custom designed TaqMan.TM.
genotyping assay with a probe specific for the SNP (dbSNP ID:
rs63750215) located in Chr1:227,073,304 A>T. Selected clones
were analyzed by Sanger sequencing to confirm correction of
Chr1:227,073,304 location and discard possible insertions or
deletions in the surrounding areas.
Fluorescence-Activated Cell Sorting (FACS)
[0095] Neural progenitors at day 12 of differentiation were
dissociated with Accutase (Sigma-Aldrich) for 5 min at 37 C and
inactivated in Neurobasal media. Cells were spun at 1000 rpm for 4
min and the pellets were resuspended in FACS buffer (DPBS, 0.5% BSA
Fraction V-Solution, 100 U/mL Penicillin-Streptomycin, 0.5%. EDTA
and 20 mM Glucose) with PE Mouse anti-human CD271 antibody (clone
C40-1457, BD) at 1:100, also known as p75 or NGFR, and incubated
for 20 min at room temperature (RT) in the dark. After the
incubation time, cells were washed with FACS buffer and the pellet
was resuspended in 2 mL of FACS buffer with 10 .mu.M ROCK inhibitor
(Y27632, Stemgent). p75 positive cells were purified in a BD
FACSAria IIu Cell Sorter.TM. and data was analyzed using FlowJo.TM.
software.
Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
[0096] Human iPSC from PSEN2 mutants or control patients were grown
in a monolayer and lysed directly in the cell culture wells with
RLT buffer. Total RNA purification was performed with the
RNeasy.TM. Micro kit (Qiagen), and was carried out according to the
manufacturer's instructions. cDNA was synthesized using
SuperScript.TM. III Reverse Transcriptase (RT) (Invitrogen,
Carlsbad, Calif.). Semi-quantitative real-time PCR was performed on
StepOnePlus.TM. Real-Time PCR System (Applied Biosystems, Foster
City, Calif.) using the primers listed in Table 2 below. We
normalized expression levels to GAPDH. The PCR cycling parameters
were: 50.degree. C. for 2 min, 95.degree. C. for 10 min, followed
by 40 cycles of 95.degree. C. for 15 s and 60.degree. C. for 1 min.
Each biological variable was assessed in technical triplicates
within each designated "Experiment", and each designated
"Experiment" was performed in at least three complete "start to
finish" iterations. Expression levels were normalized to the
control line, and results were expressed as AVG.+-.SEM.
TABLE-US-00002 TABLE 2 Forward SEQ Reverse SEQ Primer ID Primer ID
Gene 5'-3' NO 5'-3' NO BDNF TAACGGCGGCAGAC 14 GAAGTATTGCTTCAG 15
AAAAAGA TTGGCCT BF1 AGAAGAACGGCAAG 16 TGTTGAGGGACAGAT 17 TACGAGA
TGTGGC Nkx2.1 TAACGGCGGCAGAC 18 GAAGTATTGCTTCAG 19 AAAAAGA TTGGCCT
NLRP2, From [77] 20 From [77] 21 ASB9 NLRP3 ACGAATCTCCGACC 22
CCATGGCCACAACAA 23 ACCACT CTGAC Tuj1 GAAGTGTCCCAGGA 24
CTCTTGAGTAGCTGG 25 CATGATAA GATTGAG
A.beta. Assays
[0097] Cells were conditioned for 3 days after day 8 of dual SMAD
inhibition to measure secretion of A.beta. by neural progenitors in
vitro. A.beta. levels were quantified using human/rat 3 amyloid 40
ELISA Kit and 3 amyloid 42 ELISA Kit high sensitive (Wako). Each
biological variable was assessed using technical triplicates within
each designated "Experiment", and each designated "Experiment" was
performed in at least three complete "start to finish"
iterations.
Immunostaining/ICC
[0098] Cells were fixed with PFA 4% directly on the wells of 12, 48
or 96 well plates for 20 min, washed 3 times with DPBS
1.times.(ThermoFisher). For the staining, cells were incubated in
blocking solution (DPBS 1.times. with 0.1% Triton.TM. X-100 plus 5%
Donkey serum) for two hours at room temperature. The corresponding
primary antibodies were diluted at suitable concentration in
blocking solution, and incubated overnight at 4 C. The primary
antibodies used are represented in the table below. Cells were
washed three times with DPBST (DPBS 1.times.+0.1% Triton.TM. X-100)
and suitable secondary antibody was added in blocking solution for
1 h at room temperature. Then cells were washed three times with
DPBST and incubated with DRAQS or Hoescht 33,342 (1 .mu.g/mL,
diluted in DPBS 1.times.) for 10 min at room temperature for
nuclear counterstain. Cells were visualized using an inverted
fluorescence microscope (Olympus.TM. IX71 microscope) or a confocal
microscope (Zeiss.TM. LSMS Pascal microscope) under 10.times.,
20.times. or 63.times. magnification.
Western Blots
[0099] Human iPSC from PSEN2 mutants or control patients were grown
in a monolayer and lysed directly in the cell culture wells with
RIPA buffer (Thermo Scientific) with protease and phosphatase
inhibitors. The protein concentration was measured using the BCA
protein assay kit (Thermo Scientific). After protein estimation, 20
.mu.g of cell lysate were separated by SDS-PAGE electrophoresis on
a 4-12% Bis-Tris gel (Bolt.TM. protein gels) and transferred onto
nitrocellulose membranes by electrophoresis blotting. The membranes
were blocked with blocking buffer 1.times.TBST (tris-buffered
saline +0.1% Tween) plus 5% nonfat dry milk for 1 h in agitation at
room temperature and washed three times with TBST. After washing,
membranes were incubated at 4.degree. C. overnight in agitation,
with the primary antibodies against NLRP2 (1:1000), PSEN2 (1:200)
or 3-actin (1:1000). After rinsing, the membranes were incubated
with horseradish peroxidase (HRP)-conjugated suitable secondary
antibodies for 1 h at room temperature. Finally, protein bands were
visualized with a chemiluminescent reagent according to the
manufacturer's instructions. 3-actin was used as loading
control.
Electrophysiology
[0100] Whole cell patch-clamp recordings were obtained from single
neurons between differentiation days 38 and 55. Cells were seeded
at low density onto plastic coverslips which were placed in a
perfusion based enclosed recording chamber. Neurons were localized
using differential interference contrast optics under an Olympus
BX61WI microscope fitted with a Hamamatsu Orca.TM. R.sup.2 CCD
camera. Recordings were carried out at room temperature using
MultiClamp.TM. 700 B amplifier (Molecular Devices, Sunnyvale,
Calif., USA). Signals were sampled at 10 kHz and filtered at 6 kHz
using a Digidata.TM. 1440 A analog to digital converter (Molecular
Devices). Amplifier control and data acquisition was done using
pClamp.TM. 10.0 software (Molecular Devices).
[0101] During recordings neurons were perfused with oxygenated
Brainphys.TM. media (StemCell Technologies Inc). Medium resistance
recording pipettes (4-6 M.OMEGA.) were filled with an intracellular
solution consisting of (in mM) 130 K-gluconate, 10 KCl, 2 Mg-ATP,
0.2 Na-GTP, 0.6 CaCl2, 2 MgCl2, 0.6 EGTA, and 5 HEPES titrated to
pH 7.1 and osmolarity of 310 mOsm. In some experiments, the
intracellular solution also contained 4 mg/mL biocytin
(Sigma-Aldrich) for post-hoc identification of individual neurons,
which were visualized with streptavidin-conjugated Alexa 488 (Life
Sciences) as described elsewhere [42]. After initial break-in,
access resistance (Rs) was constantly monitored and recordings were
discarded if Rs exceeded 20 M.OMEGA. or changed more than 30%. The
voltage protocol for compound Na+ and K+ currents characterization
was as follows: cells were held at -80 mV potential followed by 500
ms steps from -100 mV to 30 mV with 10 mV increment at a frequency
of 0.1 Hz. Following transition to current-clamp mode, resting
membrane potential was recorded and cells were hyperpolarized by a
negative DC current injection to -70 mV to ensure consistency of
excitability measurements. Action potentials were evoked with
square 1 s current steps from -10 pA to 40 pA with 1 pA steps.
[0102] Electrophysiological recordings were analyzed using
ClampFit.TM. software (Molecular Devices, Sunnyvale, Calif., USA)
and statistical significance of the results was measured using
ANOVA test with Tukey's post-hoc comparison of means. Salts and
other reagents were purchased from Sigma-Aldrich (St. Louis, Mo.,
USA).
Statistical Analysis
[0103] qPCR gene expression experiments and A.beta.42/40 ELISAs
were analyzed for statistical significance using Student t-test.
LDH Release assays were analyzed by 2-Way ANOVA Bonferroni post hoc
tests. ANOVA test with Tukey's post hoc comparisons were used for
analysis of electrophysiology results. The experiments needed to
study each of the 94 neurons recorded for electrophysiology
analyses required days to weeks. On each experimental day,
representatives from each genotype were included, with at least
three samples from each genotype studied on each day. *, p<0.05;
**, p<0.01; ***, p<0.001.
Results
Optimization of Protocol for BFCN Differentiation
[0104] The scheme of BFCN differentiation is described in FIG. 1A.
iPSCs from control subjects or AD patients were plated in
feeder-free conditions and allowed to reach 100% con-fluency prior
to differentiation using mTeSR1 basal media. Both branches of TGF-0
signaling were inhibited (dual SMAD inhibition) to induce
neuroectodermal fate on "day 0" [12]. Differentiations (day 2-10)
were performed using a modified mTeSR1 formulation, lacking factors
that support pluripotency (bFGF, TGF-.beta., Li--Cl, GABA and
pipecolic acid). To specify these cells to basal forebrain
cholinergic neurons, ventralization for medial ganglionic eminences
(MGE) induction is required [19, 85, 91]. As such cells were
treated with the Sonic Hedgehog (Shh) analog (SAG) at 500 nM and
Purmorphamine at 2 .mu.M from days 2 to 8. SAG is a suitable
substitute to activate Shh signaling, as demonstrated during
differentiation of ChAT.sup.+ motor neurons and glutamatergic
interneurons [91], with lower cost than recombinant Shh and some
advantages in neuronal survival properties over Shh itself [7, 35].
We used the Nkx2.1-GFP embryonic stem cell (ESC) reporter line as a
tool to adjust the combination, dosage and timing of ventralizing
agents more beneficial for specification of BFCNs from induced
Nkx2.1 basal forebrain precursors.
[0105] However, given the potential of Nkx2.1 intermediate neural
precursors to generate multiple neuronal subtypes, such as TH+ and
GABA+ hypothalamic neurons, we analyzed the expression of the
downstream cholinergic specification factor Lhx8 over expression of
the GABAergic interneuron specific transcription factor Lhx6 [26]
under different specification conditions (FIG. 1B). These data
agree with those from [50] supporting the existence of a
synergistic effect of SAG and purmorphamine on Nkx2.1 induction
although an effect that is less than the effect of Shh plus
purmorphamine (FIG. 1B). Nkx2.1-driven GFP levels were maintained
after Day 14, even after withdrawal of SAG+purmorphamine at day 8
(FIG. 1B). We observed higher Lhx8 induction upon SAG plus
purmorphamine treatment than SAG alone, or even Shh plus
purmorphamine (FIG. 1B). Interestingly, intermediate Nkx2.1 levels
driven by SAG plus purmorphamine correlate with higher induction of
Lhx8 and BF1 gene expression (FIG. 1B). Our choice of starting SHH
pathway-driven ventralization at day 2 was based on reports
demonstrating other MGE-derived populations being generated by
earlier (e.g., hypothalamic neurons) or later (e.g., GABAergic
interneurons.) ventralization in the context of dual smad
inhibition protocols.
[0106] Following the patterning stage, we gradually switched from
Custom mTESR1.TM. media to Brainphys' media with B-27 supplement to
support neuronal survival and growth [3]. At day 11, we observed
neural rosettes positive for Nestin and Sox2 markers (FIG. 1C);
also, we observed Tuj1+ neurites as early as day 11 (FIG. 1C). To
obtain cholinergic populations of a higher purity, we developed a
P75+ FACS strategy to isolate progenitors specific for cholinergic
neurons due to the fact that BFCNs are the only CNS neuron type to
express robust levels of P75 under non-pathogenic conditions in the
adult brain). Support for this strategy includes a previously
published protocol using FACS to isolate high expressing P75+ cells
from the embryonic murine septum [65]. This population correlated
with best expression of cholinergic-related markers.
[0107] At day 11/12, we lifted the cells using chemical
dissociation (Accutase) and purified day 11-12 p75+ (CD271) neural
progenitors and generated 3D neuronal embryoid bodies (NEBs) by
spinning down neural progenitors in V-bottom 96 well plates. On day
19 NEBs were dissociated and re-plated as a monolayer on plates
coated with branched polyethylenimine (Aldrich catalog number
408727) and laminin. Monolayer cultures were maintained with the
addition of growth factors BDNF, NGF and DAPT until day 26, when
cultures no longer had DAPT added. Immunostaining of both
cryosections of NEB structures and fixed monolayers, resulting from
chemical dissociation of NEBs from several control iPSC and H9 hESC
lines, demonstrated expression of BFCN lineage markers Tuj1, MAP2,
BF1, Nkx2.1 and p75, at final stages of the differentiation
protocol (FIG. 1D). NGF addition to neuronal cultures showed an
advantageous effect on maturation, neurite outgrowth and presence
of ChAT (FIG. 1E).
Generation and QC of PSEN2.sup.N141I iPSC Lines
[0108] PSEN2.sup.N141I mutant iPSC and control lines were generated
from fresh skin biopsies. Established fibroblast lines were grown
from skin punches donated by a kindred of 2 carriers for presenilin
2 Volga familial AD mutation (PSEN2.sup.N141I) and one non-affected
member. Additionally, we included a non-family related control.
Fibroblasts were reprogrammed using modified RNA method to
introduce the Yamanaka factors (Oct4, KLF4, SOX2 and c-Myc), and
the iPSC lines obtained were subject to several quality control
processes to ensure robust cell-renewal and pluripotency, including
alkaline-phosphatase (AP) enzymatic activity, gene expression
analysis and immunostaining for pluripotency markers, as well as
karyotyping for detection of chromosome abnormalities, following
the automated iPSC reprogramming and QC methods developed by [60].
A summary of the genotypes, sex and age of the subjects included in
the study is shown in FIG. 2A. The two PSEN2.sup.N141I iPSC lines
were also heterozygous for APOE .epsilon.4 (.epsilon.3/.epsilon.4),
whereas the control iPSC lines were homozygous
.epsilon.3/.epsilon.3. The characterization of the iPSC lines,
expression of pluripotency markers and quality control results are
shown in FIGS. 10A-10B. Briefly, all iPSC clones selected
demonstrated pluripotency by embryoid body formation and
differentiation into the three germ layers (FIG. 10A), incorporated
herein by reference). Finally, the lines were fingerprinted (Cell
Line Genetics) to ensure they matched the parental fibroblast lines
(data not shown). All the parental fibroblast lines and the iPSC
lines were subject to Sanger sequencing to determine PSEN2 and APOE
genotypes. A 173 bp fragment from the exon 5 of PSEN2, surrounding
the area where the PSEN2.sup.N141I point mutation is located
(Chr1:227,073,304 A>T), was amplified by PCR and sequenced using
the primers published in [53]; similarly a fragment of 244 bp from
APOE locus that contains two SNPs which determine the three allelic
variants was amplified by PCR from genomic DNA, and subsequently
sequenced to distinguish between .epsilon.2/.epsilon.3/.epsilon.4
variants, using the primers from [36]. Sample chromatograms showing
the presence of PSEN2N141I point mutation are shown in FIG. 2B, and
all genotypes are summarized in FIG. 2A.
Characterization of PSEN2.sup.N141I Neural Progenitors
[0109] To study the effect of the PSEN2.sup.N141I mutation in early
stages of the differentiation of cholinergic neurons, we analyzed
the neural progenitors (NPCs) obtained at DIV 11-16 along the BFCN
differentiation protocol. The analysis of this intermediate
immature population allows us to detect possible early alterations
in the generation of BFCNs that would otherwise not be detected in
terminally differentiated cholinergic neurons. Such defects could
potentially play roles in mature neurons and contribute to the
pathophysiology of AD. We analyzed the expression of early neuronal
markers in PSEN2.sup.N141I mutant and control NPCs by gene
expression and immunofluorescence methods. Although, we found a
lower RNA expression of Tuj1 (.beta.III-Tubulin), a general
neuronal marker, in mutant NPCs at day 11 of differentiation, we
did not detected quantifiable differences by immunocytochemistry
circa days 16-21 (FIGS. 2C and 2D). NPC monolayer cultures at day
11 were also immunostained for typical NPC markers: Sox2, and Pax6;
with Pax6 levels dropping as expected along with Nkx2.1 induction
(not shown). We observed comparable expression of Sox2 and Nestin
in PSEN2.sup.N141I cultures at day 11 (FIG. 2C, top panel). At day
21, mutant NPCs expressed comparable levels of Nkx2.1 (MGE marker),
but reduced levels of BF1 (forebrain marker) by qPCR; however, BF1
protein expression did not seem affected by immunostaining at this
differentiation stage (FIG. 2C bottom panel, and D). We did not
observe differences in the surface expression of NGFR (p75/CD271)
in DIV11-12 PSEN2.sup.N141I cells, in terms of percentage of
positive cells or fluorescence mean peak value (FIG. 2E).
[0110] As previously published by [59, 73], the expression of
mutant PSEN2.sup.N141I causes an increase in the A.beta.42/40 ratio
in the brains of transgenic mice; additionally, this enhanced
A.beta.42 production was observed in neural cell lines upon induced
overexpression of mutant PSEN2.sup.N141I protein [83] and in iPSC
derived from PSEN2.sup.N141I mutant patients [93]. Consistently, we
observed a 2-fold increase in the A.beta.42/40 ratio, a 50%
increase in the amount of secreted A.beta.40 and 2.5-fold increase
in A.beta.42 species in the conditioned media from PSEN2N141'
neural progenitors at DIV 11 (***p<0.001) (FIG. 2E). The levels
of secreted A.beta.40 and 42 observed in our study and the levels
found in [93], using a different neuronal differentiation method
applied to FAD1/PS2 iPSC lines derived from fibroblasts from the
Coriell repository are very similar in both absolute number and in
fold-increase.
Characterization of Mature BFCNs from PSEN2.sup.N141I iPSC Lines
and Controls
[0111] With the aim of determining the impact of PSEN2.sup.N141I
mutation on the differentiation, gene expression, function, and
communication of BFCNs, we characterized cells at later time points
for appropriate expression markers; our goal was to explore whether
PSEN2.sup.N141I iPSC were able to complete BFCN maturation process
and if so, if any abnormalities along later stages of BFCN
differentiation may account for the pathophysiology of EOFAD (FIGS.
3A-3B). In addition to p75, which preferentially binds pro-NGF, we
analyzed the expression of TrkA, the primary mature NGF receptor,
was also expressed in PSEN2.sup.N141I BFCNs and control (FIG. 3A).
This suggested that PSEN2.sup.N141I BFCNs are susceptible to
receiving and benefiting from NGF pro-survival and differentiation
signals as expected and further confirms their proper identity. We
observed comparable expression of additional cholinergic neuron
specific markers choline acetyltransferase (ChAT) and vesicular
acetylcholine transporter (vAChT) in PSEN2.sup.N141I BFCNs and
controls (FIG. 3B). Other general neuronal markers such as Tuj1,
and the mature marker microtubule-associated protein 2 (MAP2)
showed no apparent differences by immunofluorescence (FIG. 3B).
CRISPR/Cas9-Mediated Correction of PSEN2.sup.N141I Mutation and
Effect on A.beta. 42/40 Ratio
[0112] To determine if the molecular alterations in the processing
and cleavage of APP and/or the exacerbated activation of NLRP2
inflammasome, as previously observed in PSEN1 mutants [77], can be
attributed to PSEN2.sup.N141I mutation only, we modified the PSEN2
locus in our iPSC lines employing CRISPR/Cas9 technology. We did
this by correcting the PSEN2.sup.N141I point mutation in the two
PSEN2 mutant iPSC lines (AD1, AD2). For this purpose, a specific
guide RNA (g1N141I) was designed using an online tool
(tools.genome-engineering.org) to direct Cas9 to the region of
PSEN2 exon 5 surrounding PSEN2.sup.N141I mutation (23 bp upstream
of Chr1:227,073,304 A>T). g1N141I was cloned into
pSpCas9(BB)-2A-GFP (PX458) vector. Expression was assessed by GFP
fluorescence upon transfection of pSpCas9-g1N141I-GFP in HEK293T
(FIG. 4A).
[0113] In order to correct the mutation, we designed an asymmetric
ssODN HDR (homology directed repair) template, ssODN #A-N141I, with
a long homology arm of 91 bp, and a short homology arm of 36 bp
since asymmetrical donor sequences with a shorter arm oriented to
the area closer to the PAM side demonstrated a superior efficiency
of homology-directed repair using CRISPR/Cas9 system [13]. We then
proceeded to trans-duce pSpCas9-g1N141I-GFP and ssODN#A-N141I into
the iPSC lines using Amaxa nucleofection (FIG. 4A). Forty-eight
hours post-nucleofection cells were dissociated and the GFP.sup.+
population was purified by FACS and replated at low density feeder
free for isolation of single gene-corrected clones (FIG. 4B).
Subsequently, clones were grown and gDNA extracted post expansion.
The screening of positive clones that demonstrated successful HDR
was determined by qPCR using a custom designed TaqMan.TM.
genotyping assay with a probe specific for the SNP (dbSNP ID:
rs63750215) located in Chr1:227,073,304 A>T. We were able to
distinguish by this method between homozygous PSEN2.sup.N141I
heterozygous PSEN2.sup.N141I and PSEN2 single clones derived from
the original iPSC lines, and pre-selected clones were subjected to
Sanger sequencing to confirm Chr1:227,073,304 location and detect
possible insertions, deletions or mismatches introduced by
CRISPR/Cas9 modification in the surrounding area and corroborate
successful HDR (FIG. 4C).
[0114] Successfully corrected clones were expanded and subjected to
the BFCN differentiation protocol in parallel to the other 4 lines
used in the study. We collected media from BFCNs (DIV 34) and
re-tested for amyloid beta production. In support of our previous
finding in NPCs at DIV11-12 (FIG. 2F), we observed that mature
BFCNs also display significant increases in A.beta.42/40 ratio
(FIG. 4D) and overall A.beta. production (FIGS. 11A-11B).
Importantly, these results also showed a normalization of
A.beta.42/40 ratio to control levels in corrected lines (iAD1
Control and iAD2 Control, are corrected clones of AD1 and AD2,
respectively) (FIG. 4D). These results also strengthen previous
findings linking the PSEN2.sup.N141I mutation to abnormal APP
processing and reinforcing that presenilins contains the catalytic
site of .gamma.-secretase [90].
Assessment of Sensitivity to A.beta.42 Oligomer Toxicity in
iPSC-Derived PSEN2.sup.N141I Neurons
[0115] Previous reports have shown that iPSC lines carrying FAD
mutations may display an enhanced susceptibility to noxious
stimuli, such as high concentrations of A.beta.42 oligomers [2]. We
therefore tested whether our BFCNs from PSEN2.sup.N141I mutants
would display enhanced toxicity to A.beta.42 oligomers in the media
(FIGS. 5A-5B). We assessed neurotoxicity by measuring the
percentage of lactate dehydrogenase (LDH) released by dead cells,
thus providing an indirect measurement for toxicity. Using this
methodology by 2-way ANOVA we detected a significant effect in
toxicity driven by 5 .mu.M A.beta.42 oligomer addition to the
culture media, after 72-h exposure (***, p<0.01). Post hoc
Bonferroni analysis revealed significant differences between the
AD2 line and its corrected isogenic control (iAD2 Control).
However, this apparent enhanced sensitivity to A.beta.42 oligomer
toxicity was not observed in the AD1 line and its corresponding
control. These results indicate that differences in susceptibility
to A.beta.42 are not exclusively linked to mutant PSEN2 genotype,
and that likely additional genetic factors different between AD1
and AD2 subjects affect susceptibility to this stress, further
emphasizing the importance of multiple isogenic models.
Assessment of NLRP2 mRNA in iPSC-Derived PSEN2.sup.N141I
Neurons
[0116] We previously reported that NLRP2 mRNA was elevated in PSEN1
mutant iPSC and NPCs, [77] which was also the case for PSEN1 mutant
cortical neurons (unpublished observation). Therefore, we wanted to
analyze the status of the components of the inflammasome in the
context of PSEN2.sup.N141I mutation. When we assayed by qPCR the
mRNA levels of NLRP2 in NPCs at DIV12, we observed an increase over
100-fold in AD1 and AD2 lines, as compared to control lines (FIG.
6A). This correlated with a notable increase in NLRP2 protein, as
observed by SDS-PAGE in whole cell lysates from day 11 PSEN2
mutants (FIG. 6D). Noticeably, however we did not detect bands for
NLRP2 by immu-noblot in the AD2 line lysates. Further, we were
unable to corroborate some other transcriptional events previously
seen in PSEN1 mutant iPS neural precursors, such as the elevated
ASB9 that encodes an E3 ligase that directs mitochondrial creatine
kinase for degradation. Instead, we observed a trend toward
decreased levels in PSEN2 mutation carriers by 20-30%.
Assessment of Excitability of iPSC-Derived PSEN2.sup.N141I
BFCNs
[0117] Using BFCN differentiation protocol, we were able to
generate electrophysiologically active cholinergic neurons in a
dish from two PSEN2.sup.N141I mutant AD patients, wild-type and
familial controls starting from differentiation day 35. We were
initially unable to obtain mature action potential waveforms from
BFCNs grown in neurobasal media at this stage, but switching to
Brainphys' media significantly improved electrophysiological
properties of cultured neurons [3]. These findings are in line with
electrophysiological characterization of other iPSC generated
neurons used to compare both media [3]. The benefits of the
protocol containing Brainphys' media in two additional cell lines
(including the H9 embryonic stem cell line) with comparable
endpoint expression of ChAT and VAChT as well as
electrophysiological responses were repeated. In order to
investigate the electrophysiological properties of BFCN, we
recorded from a total of 94 neurons (22 wild-type control, 21
familial control, 18 AD1, 28 AD2 and 5 iAD1_control) using whole
cell patch-clamp method. In all experimental groups, recorded
neurons displayed voltage-activated currents through sodium and
potassium ion channels, ability to generate action potentials and
displayed classical neuronal morphologies (FIGS. 7A-7B). In subset
of experiments, recorded neurons were labeled with biocytin through
a patch pipette, which allowed for post hoc cell identification and
ICH characterization. We found that all biocytin-labelled cells
were also immuno-positive for ChAT and VAChT (n=12, FIG. 8A).
[0118] Significant differences between the groups in terms of
neuronal membrane resistance and capacitance were not observed,
membrane resting potential and the minimum current required for
generation of a single action potential (FIGS. 9A-9D). However, it
was observed that there were significant mutation-related,
editing-reversible differences in excitability of BFCNs.
[0119] Neurons derived from AD1 and AD2 lines (as compared to WT
and familial controls) were able to generate fewer maximum number
of spikes in response to a square depolarizing current injection
(ANOVA test with Tukey's post hoc comparisons, FIGS. 8B, 8C).
Height of the first action potential at rheobase current injection
was also significantly decreased in AD1 and AD2 BFCNs (FIG. 1C).
Importantly, CRISPR/Cas9 correction of the PSEN2 point mutation in
the AD1 mutant iPSC line abolished the observed
electro-physiological deficit, restoring both the maximal number of
spikes and spike height to the levels recorded in wild-type and
familial controls (ANOVA test with Tukey's post hoc comparisons,
FIGS. 8A-8C).
Discussion
[0120] There are 5 million people currently affected by Alzheimer's
disease in the US and, according to the Alzheimer's Association,
this number will increase to 16 million by the year 2050.
Unfortunately, we only have direct evidence for genetic causation
that accounts for 3-5% of these patients. This percentage
encompasses the EOFAD variants, caused by inherited fully penetrant
autosomal dominant mutations in the amyloid protein precursor
(APP), or PSEN1, PSEN2 that constitute the .gamma.-secretase
apparatus [87], and changes in their function increases the
production of A.beta.42 oligomers and/or deposition of amyloid
plaques.
[0121] After decades studying murine models of AD that do not fully
recapitulate the pathophysiology of this disease in the human brain
[5, 57, 58], a complementary new concept of AD modeling in vitro
has emerged upon the breakthrough by [81] allowing adult human
tissue reprogramming into iPSC using defined factors, and their
subsequent in vitro differentiation into specific brain cell
types.
[0122] BFCNs are one of the most vulnerable neuronal populations
whose deterioration explains, in part, the cognitive decline in AD
patients. Apart from the evidence for BFCN failure and atrophy,
other studies have revealed that human embryonic stem cell-derived
BFCNs transplanted into AD mouse models can be associated with
improvement in the learning behavior of the implanted mouse [94].
These findings highlight the relevance of iPSC- and ESC-derived
BFCNs as not only early clinical indicators but also as a potential
strategy for subtype-specific cell-based therapy for AD [39]. In
order to move this cell-based therapeutic strategy forward, there
has been an urgent need for a refined differentiation protocol to
generate human ESC- and/or iPSC-derived BFCNs.
[0123] Our first goal was to develop an improved protocol for the
generation of BFCNs and intermediate neural progenitors (NPCs),
followed by the use of these methods when differentiating cell
lines from both control subjects and those harboring the
PSEN2.sup.N141I mutation. Using fibroblasts isolated from 3
sisters, 2 carrying the PSEN2 mutation and displaying cognitive
decline, with the third wild-type for the mutation, iPSCs were
developed [60]. In order to approach the dissection of the fidelity
of linkage of various phenotypes to the pathogenic mutation, we
began by optimizing published BFCNs protocols [4, 17, 46, 50, 89]
including the purification of an intermediate CD271.sup.+ (p75)
forebrain progenitor population by Fluorescence Activated Cell
Sorting (FACS) to generate 3D ventralized neural embryoid bodies
(vNEBs), which can be later dissociated to look at neuronal
populations in a monolayer.
[0124] After induction of BFCN differentiation in these cell lines,
we have analyzed: (1) capacity to generate
Tuj1.sup.+/BF1.sup.+/ChAT.sup.+ neurons in vitro; (2) expression of
genes/proteins of interest related to neuronal differentiation or
inflammation; (3) generation of soluble and oligomeric A.beta.40
and 42; (4) electrophysiological (ePhys) properties; and (5)
selective vulnerability of BFCNs to one or more innate or
microenvironmental factors within or in close approximation to
those cells.
[0125] Several studies in AD mouse models highlight
electrophysiological defects associated to late stages of AD
pathology. Synaptic function in the hippocampus was reduced in
APP23 mouse models [70]. Similarly, cholinergic neurons from the
prefrontal cortex of TgCRND8 mice are unable to sustain cholinergic
excitation as compared to control mice [64]. Here we report
deficient electrophysiological properties in PSEN2.sup.N141I
iPSC-derived BFCNs in vitro. Notably, correction of this point
mutation re-established neuronal excitability to the level of the
control iPSC-derived neurons.
[0126] We have optimized an in vitro BFCN differentiation protocol
from human iPSC, focusing on generating a homogeneous population of
electrophysiologically active ChAT+/VAChT+ neurons in a
reproducible and fast way. The innovations introduced to the
protocol granted a homogeneous expression of Nkx2.1, a
transcriptional marker for MGE subregions, as soon as day 8 and
very robust by day 11, compared to day 20 suggested in previously
published protocols [38]; in defined serum-free media conditions
and without forcing overexpression of factors implicated in
cholinergic fate. We were able to record mature action potentials
in neurons from day 38 in culture, accompanying co-expression of
cholinergic specific markers, which is an earlier time point as
compared to other existing protocols using ES or iPSC [4, 17, 46,
50, 89]. Therefore, our protocol has potential application to
high-throughput drug screening in homogeneous cholinergic cultures.
In addition, the 3D structure of NEBs themselves if left
undisssociated organoid form would also allow mechanistic analysis
in a more physiological setting.
[0127] After applying this optimized protocol to PSEN2.sup.N141I
mutant iPSC lines, we found an increase in A.beta.42/40 ratio in
the conditioned media. We did not observe any evident defects in
the neuronal differentiation process and expression of BFCN
markers. Interestingly, we observed a decrease on BDNF gene
expression in PSEN2.sup.N141I NPCs, similar to results described in
a report [18] wherein BDNF changes were observed in homozygous and
heterozygous APP.sup.swe/PSEN1.sup.M146V mice. The two mutant lines
are also carriers of one APOE .epsilon.4 allele. The presence of
this allelic variant, the most common and well characterized risk
factor polymorphism for LOAD [16], may modulate the age of onset
and severity of the phenotype [49]. Therefore, these iPSC lines
combining both the EOFAD PSEN2 Volga mutation (or CRISPR/Cas9
corrected) and APOE E4 allele constitute a tremendously useful tool
to study the pathophysiology of early onset AD in vitro, especially
when apoE-secreting iPSC-derived astrocytes are also present.
[0128] Searching for adjacent mechanisms or events that may be a
cause or a consequence of elevated .beta.-amyloid production,
researchers have found overactivated inflammation and
electrophysiological defects associated with AD mutations. The
concept of these defects being independent from .beta.-amyloid
deposition and their demonstration using CRISPR/Cas9 technology to
correct EOFAD mutations would open the debate to the need of
combined AD treatments not only targeting .beta.-amyloid plaques
(Gandy et al., in press), but also to overcome parallel
inflammatory processes or excitotoxicity/defective neuronal
firing.
[0129] NLRPs are components of the inflammasome, which induces the
secretion of mature pro-inflammatory cytokine IL-13 in response to
pathogens and toxic stimuli [11, 41]. NLRP2 appears dysregulated in
astrocytes [45, 51], and NLRP3 in microglia [34] in the context of
Alzheimer's disease as well as in other neurological diseases like
Parkinson's disease [14, 32]; additionally, NLRP2/3 are altered in
pathologies that show comorbidity with AD: obesity, type-2
diabetes. We previously reported an unexpected association of
elevated expression of the inflammasome gene NLRP2 in iPSC-derived
neurons from banked fibroblasts from subjects harboring
PSEN1.sup.A246E and PSEN1.sup.M146L mutations [77]. This
association reminded us of the association of the inflammatory skin
disease acne inversa (AI) with mutations in presenilin 1,
nicastrin, APH-1 and PEN-2, raising in our minds the question of
whether some .gamma.-secretase component mutations might be
associated not only with proamyloidogenic actions but also with
proinflammatory mechanisms.
[0130] Despite our observations PSEN2.sup.N141I mutant cells had
elevated NLRP2 compared to controls, we were not able to attribute
this upregulation to the familial PSEN2 mutation, as gene
correction did not significantly reduce NLRP2 levels. Our results
suggest that, although inflammasome dysregulation may occur in the
brains of EOFAD patients, there may be factors triggering this
event apart from any effect of PSENs on inflammasome biology that
are reflected in reprogrammed PSEN2 mutant cell lines. Some
potential explanations for this PSEN2-independent NLRP2
upregulation include effects of the apoE4 allele present in both
PSEN2 subjects (not preset in controls) or epigenetic effects on
fibroblasts collected from the EOFAD subjects that are maintained
through the reprogramming process.
[0131] Electrophysiological defects in neurons have been associated
with PSEN1 and PSEN2 mutations. Some of these defects are
attributed to altered function of voltage-gated K+ channels,
potentially through the cleavage of channel components mediated by
the PS/.gamma.-secretase apparatus [44, 72]. Presenilin mutations
also disrupt calcium signaling by increasing the levels of calcium
stored in the endoplasmic reticulum that result in increased
stimulus-induced released into the cytosol, rather than altered
influx of calcium. One of the mechanisms behind neuronal calcium
dysregulation was described in cortical neurons from
PSEN1.sup.M146V mice, mediated by inositol triphosphate (IP3) [79];
and, more directly, the formation of dual function protein-ion
channels by unprocessed PSEN1 and PSEN2 themselves, modulating the
exit of calcium from the endoplasmic reticulum [29, 55, 80, 84].
Given the important role of presenilins on potassium and calcium
flux and neuronal excitability, mutations in PSEN1 and PSEN2 may
lead to reduced neuronal excitability and neurotoxicity. Mice
carrying mutant forms of APP exhibited aberrant action potentials
associated to a decrease in sodium currents with no alteration in
potassium currents, only after plaque burden was considerable [9].
There is evidence that APP overexpression causes hyperexcitability
in mouse cortical neurons [75, 86, 92].
[0132] Mucke and Selkoe [52] have highlighted a toxic effect of A3
resulting in synaptic and network dysfunction. In fibroblasts and
neural cell lines, A3-mediated accumulation of mitochondrial
Ca.sup.2+ was elevated when mutant forms of PS1 were expressed
[31]. Neuronal firing patterns in mouse hippocampal neurons were
altered by exposure to A3 [67, 69]. A3 exposure was also associated
with altered K.sup.+ channel conductance in pyramidal neurons [54].
PSEN1 mutations have been observed to associate with altered
Ca.sup.2+ mitochondrial channels in the cerebellum, apparently
causing reduced spike activity in Purkinje cells in the absence of
amyloid plaque deposition [74]. A.beta.42 may accentuate the
defects present in Ca.sup.2+ homeostasis by modulation of
additional voltage-dependent ion channels [8, 25, 76, 88].
[0133] Apart from mouse data and immortalized neuronal cell lines,
electrophysiological defects in iPSC-derived neurons upon exposure
to A.beta. have been shown: hiPSC-derived cortical pyramidal
neurons and GABAergic interneurons have deficient action potentials
upon exposure to A3 [56], and neurons differentiated from hiPSC
harboring PS1.sup.A426E mutation also showed deficient firing
patterns [47]. However, there are no previously published data on
characterization of electrophysiological properties of PSEN2 mutant
iPSC-derived BFCNs.
[0134] Hyper- or hypoexcitatory effects and differences in firing
frequency vary with the gene mutation and are highly dependent on
the neuronal subtype [37, 48]. All these events may contribute to
the progressive neurodegeneration present in the pathogenesis of
AD, and we specifically document events that may account for the
neuronal defects associated to early stages of EOFAD human
pathogenesis. Here we report defective electrophysiological
properties in iPSC-derived BFCNs that are specifically associated
with the PSEN2N141I familial mutation. Interestingly, although some
of the previous studies attribute this impairment in neuronal
activity to the build-up of plaques in the brain of AD mice, we
found a substantial impairment in the induced action potentials in
the absence of amyloid plaques, solely in the presence of a
discrete excess of A.beta.42 oligomers in the culture media,
consistent with other reports [18]. Correction of this point
mutation re-established the firing patterns to those of the
wildtype iPSC-derived neurons.
[0135] Modulators of potassium channels in neurons have proven
efficacy in memory improvement in AD mouse models [44]. Modulation
of Ca' channels and excitotoxicity may open a new wave of AD drugs.
Understanding the mechanism through which PSEN2 mutations affect
the electrophysiological activity in different subsets of neuronal
populations and unraveling the connection between PSEN2, other
genetic modulatory factors and inflammation will potentially lead
to, not only alternative symptomatic treatments, but also to novel
drugs decreasing the Ca.sup.2+-mediated vulnerability to ROS and
potentially stopping the neuronal loss and progression of the
disease, if administered at early stages.
[0136] It is clear that mutant presenilins alter neuronal
excitability even before the formation of A.beta. plaques [18, 74].
One plausible hypothesis is that APP and presenilins may exert
effects that modulate neuronal excitability through currently
unrecognized mechanisms acting separate from their roles in the
biogenesis of A3. Accumulation of A.beta. could synergize with the
altered electrophysiological mechanisms in a pathway leading to AD.
With the wealth of data supporting neuronal excitotoxicity as a key
mechanism implicated in AD, further studies focusing on clarifying
the possible role(s) of PSENs and/or A.beta. in physiological or
pathological events are warranted.
CONCLUSIONS
[0137] We have optimized an in vitro protocol to generate human
BFCNs from iPSCs from presenilin 2 (PSEN2) mutation carriers and
controls. As expected, PSEN2.sup.N141I was associated with an
increase in the A.beta.42/40 in iPSC-derived BFCNs, and this was
reversed by CRISPR/Cas9-mediated gene editing. Unexpectedly,
iPSC-derived BFCNs or cortical neurons from PSEN2.sup.N141I
carriers showed diminished basal excitability as quantified by a
reduction of both spike frequency and spike amplitude. This
electrophysiological phenotype was also abolished following
CRISPR/Cas9 correction of the PSEN2.sup.N141I mutation. The gene
editing data confirm that there was a robust consistency of
mutation-related changes that characterized all the expected
findings and genotypes from all cells.
[0138] Abbreviations
[0139] AD: Alzheimer's Disease;
[0140] ApoE: Apolipoprotein E;
[0141] APP: Amyloid Protein Precursor;
[0142] AVG: Average;
[0143] A3: Amyloid Beta;
[0144] BDNF: Brain Derived Neurotrophic Factor;
[0145] BF1: Brain Factor 1;
[0146] BFCNs: Basal Forebrain Cholinergic Neurons;
[0147] ChAT: Acetylcholine Transferase;
[0148] DAPT:
(N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl
ester);
[0149] DIV: Days in vitro;
[0150] DNA: Deoxyribonucleic Acid;
[0151] DPBS: Dulbecco's Phosphate-Buffered Saline;
[0152] DPBST: Dulbecco's Phosphate-Buffered Saline+0.1% Triton
X-100;
[0153] EGTA: Ethylene-bis(oxyethylenenitrilo)tetraacetic acid;
[0154] EOFAD: Early Onset Familial Alzheimer's Disease;
[0155] ESC: Embryonic Stem Cells;
[0156] FACS: Fluorescence-Activated Cell Sorting;
[0157] GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase;
[0158] GFP: Green Fluorescent Protein;
[0159] HDR: Homology Directed Repair;
[0160] HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol;
[0161] HRP: Horseradish Peroxidase;
[0162] IPSCs: Induced Pluripotent Stem Cells;
[0163] LDH: Lactate Dehydrogenase;
[0164] MAP2: Microtubule-Associated Protein 2;
[0165] MGE: Medial Ganglionic Eminences;
[0166] NEBs: Neuronal Embryoid Bodies;
[0167] NGF: Nerve Growth Factor;
[0168] NLRP2: NLR Family Pyrin Domain Containing 2;
[0169] NPC: Neural Progenitor Cells;
[0170] PFA: Paraformaldehyde;
[0171] PSEN: Presenilin;
[0172] RNA: Ribonucleic Acid;
[0173] Rock: Rho-Associated, coiled-coil containing protein
kinase;
[0174] RT: Reverse Transcriptase;
[0175] RT-qPCR: Real-time quantitative polymerase chain
reaction;
[0176] SAG: Smoothened Agonist;
[0177] SDS-PAGE: Sodium Dodecyl Sulfate PolyAcrylamide Gel
Electrophoresis;
[0178] SEM: Standard Error of the Mean;
[0179] sgRNA: Single Guide RNA;
[0180] Shh: Sonic HedgeHog;
[0181] SNP: Single Nucleotide Polymorphism;
[0182] ssODN: Single Stranded Oligonucleotides;
[0183] TBST: Tris-Buffered Saline+0.1% Tween;
[0184] VACht: Vesicular Acetylcholine Transporter;
[0185] WT: Wild Type
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[0280] Although the invention has been described with reference to
the examples herein, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 31 <210> SEQ ID NO 1 <211> LENGTH: 33258
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: chromosome 1, GRCh38.p12 Primary Assembly
<400> SEQUENCE: 1 ggggcctggg ccggcgccgg gtccggccgg gcgctcagcc
agctgcgtaa actccgctgg 60 agcgcggcgg cagagcaggt gagcgggcgg
tgccgggggg tgcccaggcc agggccctgt 120 cgcctgcggc gctgagggcc
cggggtgggg ctgcgccctg agggccctgc cctgccctcc 180 gcacgcctct
ggccacggtc ccttccccgg ctgtgggtct gcggcccctg cgtgcgcagc 240
gctcctggcc tctgcggcca gcgcgggggc ggagagagga gagtgcccgg caggcggcgg
300 ctgggccggc ccggaactgg gtcgtggaag gatcgcgggg agcggccctc
aggccttcgg 360 cctcactgcg tccccacttc cctgcgcccg cctgccgccg
agccccggct gggggtgggc 420 gcggcgcgag cggttaaagg gccggtgcat
ttaaaggagc ggtgcacgtg ggtctctgag 480 gcgtgtagca ggcgggggcg
ttttgttctt cttctctctc gccggagacc tccgttgcgc 540 cgagtccatt
cggcctctag caccgggtcc tgggcatgct ttccccggga aggaggcgcg 600
cgggggctct gcccgcacgt gaggggcagg gccgcaggct caagcctaga gccggtttct
660 gttagcagcg gtgtttggct gttttatcag gcatttccag cagtgaggag
acagccagaa 720 gcaagctttt ggagctgaag gaacctgaga cagaagctag
tcccccctct gaattttact 780 gatgaagaaa ctgaggccac agagctaaag
tgacttttcc caaggtcgcc caggtacgat 840 atagcagagc caggcttcga
ccccagtgtc ctggcttcta gatctgctgt ccatccctcc 900 gagcagacct
cacccctgtt tattgcctta ataagtattc cctttgaaag gtatgaacgg 960
tgttgagtga agtaactgca tccctattta caaatggaga acctgagagc attccataga
1020 gacgattgta gactaactta actcagaagc gacagcctgg ggttgccaag
gctgtctacg 1080 aagtaacttg attaggaccg accccagctt ccagtaagga
agcctctgat gcctctgtag 1140 ccaattctgc agacacctga gcctccaagg
ccttcagcca agacctttgg cggtaattgg 1200 agtctcggga taagctgctt
caggtgtgtg agcctcaggt tcttctctcc tgaatgtggt 1260 tgtgggcagc
cggtgactgg cgcaggtgca gaaggggcct ggttcttggc cccacctcag 1320
agctgcgtcc tcacgacgcc cacgttgagc cttgggttcc agggcagaga ctggagtgag
1380 ggcttggggg catgttgctt tgaagtggga tggatgtatc aggtttttgg
ggaaaactct 1440 gtaccctttg gtgttgaagt gcccatgtgc caagtcttga
gtccagcatg ttcacatgtg 1500 gggagtgagt ggcttgttcc tgtctatttg
aaagagcagc aaggaggagg aggagcaagg 1560 gctaggggct gctgctgggg
tgcctggagc tgtggtgcat aatgtcacac ctgtctcccc 1620 tccgtagctg
ctcaccgtcc ccccaagggg ggtttgcctc ttgcctactt tggcctttct 1680
ctgttatcga tgttaataat gacatatatc tcgcttatga gttggtcata ataaaaagct
1740 atcttgtaca gaatattaga atttaagatc ttaagaattt caatgacact
gaaatagttt 1800 attattacct ttttacagaa gaggaaacaa gttcagggag
ttaagcagct agtccagtta 1860 tgtggcttca gtgctttaag caccaggatt
tgaacatagc tggctacact gtctttatct 1920 cttgagtttt tgcgcaggag
gttcctgtat tcaactccta cccgtgtctc tccactactg 1980 ctgggaaagt
tttgtggagt ccccatgagc aacttcctga caaacaaaca aaattttttt 2040
aaagaaacca aagcagtgtg tgtaggtcac atgcagtgtg tctaatgaaa acatctctgg
2100 cgggttttca gctgttgctt tgactttcgg acactgttta gttggggact
gataagacag 2160 caaatatttc tgcaagtatt cccacctgtt ctattcccag
ctgccacagc tgcggaaagg 2220 cgggggtgag gctgagaggc cccgagagga
acattttcca ctgggctcca atcctggaga 2280 tgggatgacc atcatgttaa
tgtctggaga aaagaatgat ttcaggctgg gtgctgtggc 2340 tcatgtctgt
aatcccagca ctttgggagg ccgaggtggg tggatcacct gaggtcggaa 2400
gtttgagacc agcctgacca acatggagaa accccatctt tactaaaaat acaaaattag
2460 ccgggcgtgg tggcacatgc ctgtaatccc agctactcag gaggctgagg
caggagaatc 2520 gcttgaaccc aggaggcgga ggttgcagtg agccgagatc
gggccatggc actccagcct 2580 gggcaacaag agcaaaactc catctcaaaa
aaaaaaaaat ggtttcacat cagtcctcag 2640 gaaagatcag atgtcagtga
gggagatcat ttcttgagag cctcttcact gagtgggaga 2700 atgggctgct
tgttcatctt tgtgaaaatt ctagaacggg aagaacaatt caaagggtgt 2760
ccaccattct gctgtacctt aaccagaaac ttactggact ctttttaaaa taaaagtaat
2820 tcatgtttat tctagaaaat tagggaaaaa aaattttttt tgagatggag
tttcactctt 2880 gttgcccagg ctggagtgcg atggtatgat ctcagctaac
tgcaacctct gcttgccggg 2940 ttctagcgac tctcctgcct cagcctcctg
ggtagctggg attacaggtg cctgccacca 3000 ctcccagcta atttttgtat
ttttattaga ggcggggttt caccatgttg gccaggctgg 3060 tctcaaactc
ctgacctcag gtgatccgcc aaccttggcc tcccaaaatg ctggtattac 3120
aagcataagc cattgcgctg ggctgagata accactctta acatgcattt ccttccacac
3180 tgttcacata tgcatattta tctattaaaa caatagaggg agggaaccgt
aggtgaagtt 3240 tgtgtatcct gttttttctg ttatgccttc agaattttcc
ttttattgag tactcatgga 3300 aaagcagatt tgatggctgt gtagagcatt
tgaattattt atcctaccag tcccacagca 3360 ggacactttc ccaccctccc
ttttgcccca gagaagcagt gccctctgtc ctccccatgc 3420 ccatatgtgg
gcactcccca ccatggagcc aaacctacct gggcaagtag cagagggaga 3480
gcagagtgag ccctgggggc aggagagaga cttgagagtt ttgaggtgac agatgagctg
3540 gtgagtgagt gattagggag catttcttga cacatacctg cccgtggtga
aggcatgtgt 3600 cttgtgagtg tgctcccaga aagcctgtgt agtgtgtggt
gggcctgcct gtgtgaccaa 3660 accctggcca ctgggtacgt gaccctcaca
agtgctgact gggctgagaa gagctccttg 3720 atgggcagtt tggagacttg
agttgtaact gtggcttttg gccatgggac attaactgat 3780 tacttttgcc
cctctaggct tcagttgtcc ttaattataa tacagggagc tgactaggtg 3840
gcgttgatgg cctttacggt ccttgccagc tctgacattg tcctatggat atgtcctttc
3900 atttgataat atgtttacgt ggccatagtg cctggggctg ggccgggaat
ggaaacttga 3960 tctctggggc ctggcctttg aagccagttc atgtgtctgg
tggttcagca gatccgtaac 4020 tttccaagag gcacatccat aggctaccgt
gtcctttctc actgtgtccc tcctccattt 4080 catcttcttt ataactacga
cttattgaac atctactgtg tgctggacac tttacaggtt 4140 atctctaggt
tttacgataa tcttgcaagg tatgcctgtt ctgcttttta cagcagagga 4200
aatgagctgt gtcagattag actgtctgag gcctcttggc cagggagtat gtggttcaaa
4260 tcacataggc aggcgatctg aaccctgtca gtctccaaag cctctgcttt
tgaccgctga 4320 cttgctgctg cttgtttaaa aataaatgtg tttctggagc
ctactccaga ggggcgtgct 4380 aggggctccc tctcccactt ccccacaaac
cacccttttc cctggctgct tcaggaaatg 4440 agagaactct gcctgggccc
caggcacttc tgagtgggac agggctgtta gaggtaagtc 4500 tagagcctgg
cccaaaattc aggaggcccc atcagagggc ccctggggcc tgtggtccgg 4560
gagggtggta gggcagtacc tcacttccct ttgagactca ggccccagct ctggcttagg
4620 ccagggagaa ccatccccaa gtggtatgtg ttactatatg agctgagatg
gatggtcagc 4680 tggaccaaat acatagtcgg gtacccaggg ccagggggag
gaaggtgagc agggaagctg 4740 tgggcaattg tctgggtatc acctgacctt
agcaaactct tccttgtttt aagcgaggac 4800 gtgggacttc tcagacgtca
ggagagtgat gtgagggagc tgtgtgacca tagaaagtga 4860 cgtgttaaaa
accagcgctg ccctctttga aagccaggga gcatcattca tttagcctgc 4920
tgagaagaag aaaccaagtg tccgggattc agacctctct gcggccccaa gtgttcgtgg
4980 taagtgcagt gactcccaac ctgcttttga accctctttt tccattagga
ttttctccgt 5040 ggaggcagat ttccatggga gtttgctgtg gcattttgaa
atctgtttct tacctagttc 5100 cattggcctt aaatgttaag gccaaagcct
ttacatttct ctgtaatgaa aagaaggtcg 5160 aggaaattgg gtcattgggt
ttccataatg attgcaggaa ctgctgacac aagcacggct 5220 ggggagattc
tctaggtcag actcccttgg tttggctaat tcagcagttt gatcccattc 5280
agctgattaa tgggaatgtg cagtggcttc tttggatgtt tgattttgca tcctaatcca
5340 aagcagctat cagcctcagc acttccttgt tggaaggctt tccagaacgt
agtctatgtt 5400 ggacacttcc ttctgcctct ctgcattttc ctgccacttc
tctagagaat ggggtgcagg 5460 gggtgggaga cggggaaagc tggtcgctga
gtggctgatg ggacttgaca tcacccagcc 5520 ccacccccac ctgcccgtga
gtcagcctcc ggggagagtt catcgcgtca ccggcactct 5580 aatgtggaca
gacacctagc agtgttgttt atctgcacac gtttgggtgg tgatttttcc 5640
ctccaaggat ttcagagcac cagcaggctt cagagcagac ttaggtggct tgcaaagcag
5700 gccctcagga attcagaggg tagcagaagt ccatcccaga tgctctgttt
tccttcagga 5760 gctaggtaaa tcagaggggc tgagggacaa atgaaaaaag
ttacagcctt tgagtcccat 5820 ctgctcctcc tggccaatga gaggggatct
gggaggggca gatgtagagg aaaatctgtc 5880 taaatgttga tgctcgttat
tttcctttaa agaattaata gcctaaaata aaccctacag 5940 atacagtctg
tgtttattat ggcgacttag agaaatgcag aaaaatatca agaaaataaa 6000
aaccactctt ggttctacca tgcaaagata atcatcttta atgttttgta atatttccga
6060 tcttttatat acatacattt tataaggaca ttcagatgat caggttcgta
aagttttatg 6120 ttcggttaaa tttaacagcg tgtcattgtt caggttatta
aatgtttgaa ataagatttt 6180 tggtggtcct gtcacagtct ccatgaagta
gcatttcagg atcgaaaggt atgctgtgtt 6240 taaagtgttg attcttactc
ctttcagtta aggccagtgc agtttgtcca ggtagtgact 6300 gagacccagt
ttttccacac tctcctccgc agtgggcatt gttttgggcc tttttcagcc 6360
caagagctct cttctcccca tgccgctctg ctggtctgag atttttccac tcctcctcct
6420 ccctagttgc tctctgacca gactctaggt attcaggaga aagtgttcat
tgtctcactc 6480 tctcatgtgg caatcaagta gtgccaagca gtgagagggt
gaaggtgggt gggtgaggga 6540 cactcacctt gctgagaaag ggccccagcc
tgttcgggtg attataaagc agagacagtg 6600 ccaggaaaag tctgacactg
gctgagaatc acccggggac caaccatccc gaatgcggat 6660 ccctgacact
gggtgaggat ggagcttgga gatctgcatt gttaataagc agcctagcag 6720
agtggtgaag agtccagaca cactacctag gtccaagggt aaccttgagc taattacttt
6780 ttgagcctct gtttcctcat cagtaccatg gggaagaata gtagcacctt
gctccaggat 6840 gtttagtgcc ggctaagggc tcagcaggtg ctggtccatc
tccaccagcc cccagtggcc 6900 tgggccacct ttgagaaaca gtgatcctaa
gggattcagc atttcctaag ttggtgcctc 6960 ccacctgtca cccccacccc
accaggctag gagggttgtg attagagggt gcccttgctg 7020 tgacagctga
gactagctct tccctgatta ttccttaatg acagctctct ccttccctgc 7080
tttcttgaag tcttggtcct cgttgttgtg ggcacagctt caggggaggc cttggaggaa
7140 tttttgaaag tggaatgagg gaagcagcct gctcaaggga acacttgttt
tctggtgagg 7200 aggccgcatg tatgaatgac gtttgtgggt tagaaagcat
gttttgtagt ttttccttgt 7260 ttcttcctga agacatgtca ggtcttgatg
agaccgggcc tgggcacagg gcaggcagtc 7320 agcgagtgtg gatgatgacg
acagtggtca ccaggtcact gtctagacca ggtcactgtc 7380 tagcgcagtg
tcacatggaa agggtatggt cctttaaccc taccctcccc agcacaacta 7440
tcacagatgt cagggaacct ctgctcacag aactgctttc cagggattgt cttttttttc
7500 tttttctttt tctttctttt ttttttgaga cagagtctca ctctgtcgcc
caggctgaag 7560 tgcagtggtg cgatctcagc tcactgcagc ctccgcctcc
tgggttcaag tgattatctt 7620 gctacagcct tctgagtagc tgggattaca
ggtgcctgcc accatgtcca gctaattttt 7680 gtatttttat tagagacggg
gtttccccat gttggctagg ctggtcttga actcctgacc 7740 tcaggtgatc
tgcccacctc agacaggcat gagcaccgca cccagcccca gggagcgtct 7800
tattagtggt tggcaactga atggagacgt gggaattgta aggaactgat tctacttgat
7860 cctgggtccc ctgcttctcc atcttcaccc acccatcagc tccctttctc
ctttaaacag 7920 gcacctttgc tctctgctta tccatttttg ttgtgcattg
ctatttggga gcctaagaaa 7980 cacaacatcc tctgaatgct ccagctgttg
tgggtctgaa gggtgagcct gccctctgtc 8040 attggaggct gcagcctgtg
gctttttagg tacagggact cccagaactg ctcctccagt 8100 catagcagag
ataaatcaca ggagcttaag aggcatggga agaacagagg gaggagatcg 8160
tagcttccct gttcattcac acccaaaaca aaactgtcat actagaaaag gaggtattaa
8220 aagagccacc tgtacagcct cgtatctcat ccagcacact gctgcagatg
gaatattatg 8280 atttagcttg agaaaatgca gcaactcttt gttgtggtgc
ccctctttga gtaagagtga 8340 attccccatt gccagagtgg atagtgaggg
aaaccctggg tccaggcagg agtctgttta 8400 ggatttatct agtgaggctg
agccagagga ggaccttaca gttttttctc ttcaatttct 8460 tttatttatt
tatttatttt tgtagagatg gggttttgcc atgttaccca ggctggtctt 8520
gaactcctgg gctcaagcga tctgtctgcc tcagcctccc aaactgttgg gattacaggc
8580 gtgagccgag ccaccatacc cggcccttct cctgcatttc cacctgataa
tttctctcat 8640 ttccatagat gatgaaggaa ctaaagccaa gaactttcca
aggtcctgca gctctttggg 8700 ggatgtgaag ctgtgctcta tttgtatgga
ttttgctggt tcccagaact tccctgtggc 8760 cctggggcct agtctgaggg
tactctgagt gaagagggag gagggcccac acctcttctg 8820 caaaggctgc
ttttgtaaag ttcacttcag ttcacatctt cctcctggtc agaaagcttc 8880
gggggctctc ctctgctgca ttaagctctt actcctccat caggcaccaa actcctccct
8940 ggcatggccc atcctaccag gtccccacac ttgagccaca tccaattgct
cgatattatc 9000 aggataggtt atgttatgtt cccaactcat atgtttactt
aagtggttac ctctttccag 9060 aatgagcccc ctcctccaaa ctctgcctgg
tgaaatattc ctaacctttg cagcttcaca 9120 tccctcttac ttcttgtgac
ctgaggcatc tactcctgac aactgataga ctgtgtcccc 9180 tcctgtcggg
tgcattgtcc ttgtcactac cctcctggct tttagctggc tttgcttccc 9240
gctgttgtta ctcctgtact tgtctcatct atcctaaaca gaaggtgctg caggctgggg
9300 agtttgttca tgttgaaatc cctgtgatgg aggtgagcag aggcagtctc
tgcctgtgcc 9360 tcttatttgg ggatgaagtt aaagtccctg taggaataat
ccaggccata gccggggttg 9420 ctgtcttcag aaagaagggc agccacaggt
cttgttaagg ggattgaaat tggctgactt 9480 ggtggaagga acctgcctgc
tttgtttaaa aaccacatat agctgagtgt agtggttcac 9540 actctgtaat
accagtgctt tgggaggctg aggcaggagg atcacttgag gccaggagtt 9600
tgagactagc ttgggcaaca acgtgagacc ctcatttcta caaaatattt taaaaattag
9660 cctagtatgg tggcgtgcat ctgcagccct agctactgag gaggctgaag
tgggagaatt 9720 gcttgaaccc aggagttcaa ggctgcagtg agctatgatt
gcaccactgt actccaacgt 9780 aagtgaccag tgagaccctg tctctagaaa
taaaaataaa aaaaatcaca tatattgtgg 9840 ggtgacttac ttggagacga
actttcagca gagcgcacac ctgctatccc tgcccagggt 9900 gtgaagctca
gccctgaggg tctctggaca gcgatcactc agcctctgga cagcgatcac 9960
tcagcctctg gacagacagc gatcactcag cctctggaca gcgatcactc agcctctgga
10020 cagcgatcac tcagcctctg gacagcgata actcagcctc tgtccccgtc
tgagatgttg 10080 gcagggactg tcagatttgc caggcattgt ttgaagttct
tcccagccca gaaacctgca 10140 tgtgtagatt ttggtacact gggtccccca
cttggtacta ctgtgtgaaa ccccacttgg 10200 cactgtttta gggggcaggc
ttccctcctg tccccttggc cttggccttc ccctgggtcc 10260 cgccctcagt
ggcacttccc cacctcacac gtctgctctc atggcttagg tctccacttc 10320
taacctcagg agacctggtc ctcagacacc tcccagacag cttccccatt ttatcccata
10380 gacactcaaa gggtgaaatt catggtcttt cccgagactc tcttctccgg
tcttccctgt 10440 cttagtcccc acctggctgc cattctagac tgtgttttct
ctcctgcgtc aggtcctgcc 10500 cttgacctct tgaccccttc tggactctgc
cctcacttgc atccatcctg ctgctgtcct 10560 cagctctcct cacctgccac
agttgtctct gggggtcact ttccctctct ggtcagtggc 10620 cagactgact
tttataaacc tggttcagat cttgtctgtc aagattgtcc tcagggtgat 10680
gtgtgtctcc ttaacatggt gcctgagacc ctgatcatct ccactgcccg ccccacagtg
10740 tgaggccctc actggaacat tgtgccttct gcccttccct cctcctggga
aaaccagtct 10800 ccatcagata ggctcttctc tagaaaacat tcgtgatctc
tgagatttgg ttccactttt 10860 gtgcttctgc acctaccatc aaacacccgg
attgtatcat ttgtcacatt agatgatttt 10920 tgttttgttt taagacaagg
gtgtttctta ctcatctttt tatccccaga gcccagcatg 10980 atctttggtg
cataatagat gcaccacaga tgtttgctga ttgaatgaat gagcacactg 11040
acagtttgga gctgccctga ctttcgtggc tatgcgtttt gccccctggg atgtgagtca
11100 cctcaggcca gccccaggca aggccgctgc tgcctccatg gtaactctca
aggcctcttg 11160 ttttatggca gtcgtttgat tgacaggcat ctcttggaag
cttttggggc aggacttgtg 11220 tccaagtctc caggtcgcct ccagccaccc
cctgagtcct ccactgcctt tgtctcacag 11280 gaaagtggaa caaggtcctt
gtgctccttt ttccaggtgc ttccagaggc agggctatgc 11340 tcacattcat
ggcctctgac agcgaggaag aagtgtgtga tgagcggacg tccctaatgt 11400
cggctgagag ccccacgccg cgctcctgcc aggagggcag gcagggccca gaggatggag
11460 agaacactgc ccagtgggta ggtcccacca gcagctgggg gccttcaaac
aggtccctgc 11520 ggctactgta ccttacagat gaaaaccaga cattcattcc
ctgatgcggg agggagaagg 11580 gaagtaatga tgaggattgg ccgaaaaggt
gggtggctgg ccatgatgga ccttccatct 11640 gcagggtttc ataggactgc
gcattcacag ccagagatgg acttggcagt gggctgaagg 11700 acgctgtcca
ctctgccacc ttgggtttac ctctctcatg caggtcactg tttccactgt 11760
aataggagag tttgtttgga tgcctgggtg ctaggacagg taacacagaa gcttaggatg
11820 gtagcagggg aagcattttt tggcagatgg ccagacatgg taagtgtgag
aggagtctgc 11880 ctgatacacg attgactttt gagctgggga tatttgggct
tcactgtgat cattcagccc 11940 ccaggggagg agattgtaac gttagaaaga
gtaggatatc gttgggagag ccacttagtt 12000 gtgtcctttc tctcccgatc
agggcagaac atctgaattt gcctgaaccc tgttctctgt 12060 tttgcccatt
atagaattaa aaaatgtctc tgtgtggact gttttcttgc agccagtctt 12120
aatcctgctt gctgaaattt gagctcactt ctccatgttc tccttgagaa cggaaccatc
12180 gtccctaagc cctgagtgaa atcacaccag cttaaggcca ctgctctgcc
actcctcagc 12240 cttttcttgt ttgttatctc cgggaagttt tgtacacttt
ggttgtttca gtttctgttc 12300 atgagtagtc ttctttcttg gctgaacgtc
tagattggga ctctctctgc agagaaccgg 12360 tactgaagca actgtcattt
tcagtttttg tttcatttgg ctttttcttt agctgttcac 12420 ctcattagca
aggcagccca tgaccttgac ttgccacagt tccaaaacac aaattcttac 12480
agatcggttt gtgctagtgt ctggcaggtg tcctgccctc cctcgttacc tcctcatttg
12540 tgcctgccca ccttcccaga gcctgcgtct tctcagatgc ttaacacctg
tttagcctct 12600 ctagttcaga gctacaaatt tacatgcttg attctgtggg
gcagaaagtt caaagtaatt 12660 tcttcctctg caaattccca gtatcttagt
cacacgcaaa gagagtgtcc ctgtgcactg 12720 actcctctag ctagtgattt
gtcagccaaa aatgtttatt tatctcctgg cctgtttcct 12780 cccatatcag
tatggccaca tgaacagaat tgagtgacct cctgagtccc tgtattagga 12840
aggggaaaga tcttttgatt cattaaccat taagttgatt cattaaccat taagtcttgg
12900 gcctgcagac catagcaacc ttccttcctt catttatggt gcttcatcca
gctccaaatc 12960 ttctctactt tgtcctcaca aacttttcat atgccctagt
agctcataga ctgctcctta 13020 tatctggaaa gcaacattca aacttctcat
ttctggttcc aaaaatccgt gcattacatg 13080 gataggctgc cgtgggggac
attctgcggc cctcacgatg tggtttccca cagagaagcc 13140 aggagaacga
ggaggacggt gaggaggacc ctgaccgcta tgtctgtagt ggggttcccg 13200
ggcggccgcc aggcctggag gaagagctga ccctcaaata cggagcgaag cacgtgatca
13260 tgctgtttgt gcctgtcact ctgtgcatga tcgtggtggt agccaccatc
aagtctgtgc 13320 gcttctacac agagaagaat ggacagctgt gagttggggg
gctgggggga gcagggtggg 13380 gtgagggctg agttgccagg gggtgggggg
cgcagcagcc tgtgttggtc actgtacctg 13440 cagctccaca ccagcagcgg
taaagagcag ggatgaagaa ccgcccaggt tcatggcctg 13500 gctcactgcc
tcctggattg tgacctactt gggcatgctt ttaacatccc tatgcctcag 13560
cttccttgtt cgtataatgg gttgataacg cagttactgg gagaattaag tgagttaata
13620 tgagtgaagg gcttagaaga gtgtctactg cacgtgagtg ctcaggcaag
ctggatcctg 13680 ctgcagaaag caagctcttg atcctgggca tggctgtgcc
actgatccct gtgtgactgc 13740 aaacaaatca cttcctctct gagtctctgc
ttccctgaat gtgaaacaag gtggttggac 13800 cagatatttc tcagctcact
tccagccttg tgaggaagac ttataaagcc tttcgtttat 13860 tttagtaaaa
tacatgcaga ggcagcagcg tagaaaaatg agaagcttcc tccacttctt 13920
ccccctcccc tttctgtggt cctcactgct aagcaccttc tgtaaacttt tttttttttt
13980 tttaaagtta gggatttttg tttcatttcg tgtgtgttgg ttttttttgt
tgttgttgtt 14040 tcttttaaag aaaggaataa ggccaggtgt ggtgtctcat
gcctgtaatc ccagcacttt 14100 gggagactga ggtgagagga ttatttgagc
ccaggagttt gagaccagcc tgggaaatgt 14160 ggcgagaccc tgtctgtaca
aaaaatgcaa aaattagcca ggtgtggtgg tacatgcctg 14220 tagtctcagc
tacttgggag actgaggtgg aagaacacct gagcccagaa gtcgaggctg 14280
cagtgagcca tgattgcgcc actgcactgc agcctcagca acagagtgag accctgtctc
14340 aaaatttttt taaaaaatta aaaaagaagt agagtcccat cctcagaaag
cttatagtgt 14400 gtgggggatt cagcgcagaa caggtgaaag catggagaga
atgcagccag cggtttgttt 14460 gcagcagtcc aggctgggaa gagtgaggtt
tgagtgaatt gcttcctgtg tctgcttcct 14520 gagcttatga gctgcaagga
cagcagttgc ttcagcggat gggggtcggg tagtagcagg 14580 tggaggagtg
ctgggctggg tggagctggt ggagaggtgt gggtgggtgg gggaatgaga 14640
actggatggg tgagagaagt gcctagggag cctttaatcc ctgtgggggt ggggaaagca
14700 gcagggaggt catctagccc tcgtcctcac tgctgcactg ggcccagttg
gcaggctgag 14760 agccacaggt ctgtggtcag ggtgccagga aatgagctgg
aggacaggaa ctgctcatgg 14820 ggatggtgcc cgcactccat cagggcagca
tgtgggcagc atgggcatcc caggcacctc 14880 ccctagcagg tccagaatca
ctcaaggtgg ggagcctcga ggagcagtca gggccgggag 14940 catcagccct
ttgccttctc cctcagcatc tacacgccat tcactgagga cacaccctcg 15000
gtgggccagc gcctcctcaa ctccgtgctg aacaccctca tcatgatcag cgtcatcgtg
15060 gttatgacca tcttcttggt ggtgctctac aagtaccgct gctacaaggt
gaggccctgg 15120 ccctgccctc cagccacgct tctctccgtc tgccccacac
catggcggca gggcccgtga 15180 aacagccgcc tttagaaaaa cacaaattag
aggaaaatag acccagattt tttgtactcc 15240 tccccacccc atcctgtctc
ccaccgtgga tgacctaata ctgttgtctt ttatttttat 15300 ttattttctt
tttcttgaaa catggtctca ctccattgcc caggctggag tgcagtggtg 15360
cgatcatgac tcactgcagc ctcaacctcc tgggctcaag ttctcccacc cagcccctca
15420 agtagctagg actacaggtt tgcaccacca tacctggcta attaaaaaat
ttttttttgt 15480 gcaggctaga tctcacagtg ttgcccaggc tggtctcaaa
ctcctggact caagtgatct 15540 cccaccttgg cctcccaaag ttctgggatt
acatgtgtga gccattgcat ccagcctgtt 15600 gtcttttaaa tttacacatt
atcccacttg agttcctcat tgcagtgttc caagcatcat 15660 ttctcatatt
tcaaagttaa ttttgttttg cttctctttc tgaagttcta ttttaggctc 15720
ccctcacccc gatacttccc ctgaagattt atttttagtt ttccttttcc ttttcgggca
15780 aggatgtgca gaggccatgc tgaggtcttg cagccctggg agacttttgg
gttgtagctg 15840 cctatagctg ccgagtagcc ccagggagta gtggaagggc
agatcccatc tggccagaat 15900 catgggcact gcctgtcccc aaagatgcca
taagctttta gacagcggct tcaggctttt 15960 ctcccaggta aggggttgaa
cccctaacga tggaaaggaa attaagctgg gcattaccta 16020 ttttaaaact
gtttacacac aggtgcctca cagcattttt tgttcaggcc gctgccatcc 16080
atggagcagg tagatagaag tgcagagtgc ccaggctaga gggatgggac agggacagtg
16140 cagggaggga gctgagcccc cttccagcgg gggcagcaga ggggaaagcc
atgggagggg 16200 ctgcaggatg tgtcctgagc tgaagcttat caacaagtaa
tgagtaccag ctgggcattg 16260 tggtgcacgc ctgtggtccc aactacttgg
gagactgagg caggaggatc gcctgacccc 16320 aggagttcaa gtctagcctg
ggcaatgtaa gaccctgtct ctaaaaaaat aataataaaa 16380 taagtaacaa
ttacctgtgt aactgtgacg aggcagggtt tgaacattgc cgctgggagg 16440
ttggcagatg gtgggaagca gggtggaggg ctgctggttt ggagcagagg atacagattg
16500 catggggtca agctagaaat tgcgtggcag atgtgaagag ctggccccac
tgcgggcagt 16560 aggtgtctgg tggccagtcc cagaggctgt gaagaggggc
tcagccatct gtctagtagg 16620 gcttccttgg aggttccacg atacaggcag
atggtggtgg cccgggcagc caggtggtgg 16680 ctgggatgaa gagggttggc
aggtcccaga ggcagcccct tccccttttg gctgtgtgtg 16740 cagcagggcc
gtggaggctg cttttagtcc aggtagacca gggccacgct gaggtcccag 16800
tgggctgagc tggtgactga tgagttggtc ctcaggggtg aggctggtgg gaagtgatgt
16860 cactgtcccg ccgatggcca gctaagggac tgggttagga tcagccccct
cttgtccttc 16920 actctcccat ccttggccag gagaagagga acaggtcttt
ctgaggacct gcttgtagac 16980 ctttgggtag gaggggactt cccaggttct
ctgttgaggc cactctatct aaaatagcac 17040 cccagtgagt ctcctatcac
tgtatcctaa cattattttc tccatggccc tcatcattac 17100 ctgctgatat
actgtatgtt tgtctatatg tcatctaaca cccctcacac tggaacacaa 17160
tgcccgtggg cagagacttt gctagccttg gttccagagc ctagaacagt gcctggcaag
17220 taggagacac ccagcattac ctttctaagt gaaccagtag agatgggggg
agaccgcaag 17280 gctatgccgg cagacctgag ggagtcctgt ctgcatgcgc
tgcaggatga cctgagggga 17340 actccttgga cttctgtgcc ctctttatct
gtaaggtggc cacctgatcc cttccagcgt 17400 aggcatgaag tagcctaatg
aagagcattc aggcttgggt atcagtctca ggatcctggg 17460 ggccttagaa
tttgtggcgc ttggggacac cttgtgatcg tgcaatttct gttgtctagt 17520
tcatccatgg ctggttgatc atgtcttcac tgatgctgct gttcctcttc acctatatct
17580 accttgggta agtgacagat aagcagcagg gtccctggga gcccctctcc
atgtggcaca 17640 agtggacatg ggcatgagga cctgggcggg gaaagatgac
catcgagctc cagtcttccc 17700 cagtgccagc cgttttggga acccaggcct
ccgtcgccct ctctcatggc cttgacacag 17760 gggagtggaa gtggggctgc
atggtggacc acatgtttct gtctcgttcc tgatttaaaa 17820 tgaacccttc
atggagaagg ctctctgtga accccagggg gatagaaacc ccccaaaatt 17880
tacattctga tttttaggct aggcctgggt actttctggt ttgtgggaaa aattatctgt
17940 tctatcgccc cttgatttgg gatatcagcc tgacccaggg gcccaaagag
actgggagga 18000 caagagaaaa cactttccca aggacctttc catgtgcaca
gggtcttcca ggtcatgccc 18060 atgcacattt ctgtgatctg ttccaagcat
ccccaccttg ttttagaaaa tgctgcaaat 18120 ggtaaattgt aaggacagtg
aaggtcgggg aaggaaatgt tagtaaagag ggccaggttg 18180 ggactgaatg
gtggtaaact gctaggctgt aatgcctcca ctgagtccca gtcacaggct 18240
ccaccttggt cctgcaggga agtgctcaag acctacaatg tggccatgga ctaccccacc
18300 ctcttgctga ctgtctggaa cttcggggca gtgggcatgg tgtgcatcca
ctggaagggc 18360 cctctggtgc tgcagcaggc ctacctcatc atgatcagtg
cgctcatggc cctagtgttc 18420 atcaagtacc tcccagagtg gtccgcgtgg
gtcatcctgg gcgccatctc tgtgtatggt 18480 aggtgggcag caaggctggt
gggggcagtg ggggcgatgt ccagggccaa atcgtcccca 18540 gtgctgcaca
aggagggcag gtgctgaagg gcttgcatcc ctttctgcag aggcctgggt 18600
gggatccctc ctgagagagt cgcctttgta aaacagaggg gggtccacta tttctggaac
18660 actcctggtg gtctagataa aacgcagtag tcactgagct cctcatttac
tttttttttt 18720 tttgagatgg agtcttgctc tgtcgcccag gctggagtgt
agtggcgcca tcttggctga 18780 ctgcaacctc cgcctcccgg gttcaagtga
ttctcctgcc tcagcctcct gagtagatag 18840 gattataggc atgtgccacc
acgctgggct aatttttgta tttttagtag agatggggtt 18900 tcaccatgtt
ggccaggctg atctcgaact cctgaccttg tgatcggccc gcctcagcct 18960
cccaaagtac tgggattaca ggcatgagcc actacaccca gcctcatttt ccattattac
19020 tgctatgctg attgagcaag tgcactgtta agcactggac acgctgtaag
tgatttgttc 19080 atcaagacag tcctttgggt accatgcata tacataaccc
caaatgttag ctgctatttg 19140 atattagcat gattatcatt gccagtattg
ttacttccat tttaaggtta aagaattgga 19200 ggctcagaga agtgggactc
cccagcctgg ccaccgcgtc tcgggtgcac agctcctcca 19260 tgcttgcagt
tgcctgcgag gccctactct ggctcacacc agggcctgct ctaagttgtg 19320
actggagaat gagaatttgg gatgccagcc cagaggcaag gcatgctctg agagctccac
19380 ccggggctcc tgtgctacag ggcaggctct tcttcagggg gctgcccggg
gatagtttga 19440 caaggatgtc tctgtcttcc tagatctcgt ggctgtgctg
tgtcccaaag ggcctctgag 19500 aatgctggta gaaactgccc aggagagaaa
tgagcccata ttccctgccc tgatatactc 19560 atgtgagtga gccccccgtg
cctctgcctg actcggggtc agcaggcagc ctgtgggggg 19620 acaggggcct
gcttcctggc cgtggctttc agagttgact gggcgatccc aggagggtct 19680
ccactttcag aagccaggga gggcagtatc ttgttattac acagtaagaa gcttagaaag
19740 ttaggacagg aagcaggcat ctgctgggat gtgctgcagt ccctgacttc
atcccgtcca 19800 tcctccagcg gcatgctgcg gtgcaggttg cattcctgtg
atcccgcagc cacccctcag 19860 ctctccaggc tcttgagaag ggactttgga
gagggattct tcagggcagg gggtcgggga 19920 gcaaggagct tctgggcttc
cttgacagca gcgtggctga ttggcattaa tcctaactga 19980 agggaaggca
cacgggatgg cccctggcct cggggtcaat gtgtagagat ttggacttac 20040
acatgcagtc aacaaaggca catcaagtcc ccattttgtg acaggcactg tgctaggcat
20100 tgggggaccc agcaggaaag aagaccacag ggtcccaggc ctcatggagc
tcacggccct 20160 gtgattgtga tgccctcggt ctgttgatgg cggggcttaa
atagcctgaa tttctggagc 20220 tctggcgtct gcaaggtggc ctgggaaaga
gtttatggaa cagctacaga gttctaggta 20280 ccttcatgca gttgaggatt
cgagcccgta gaggagaatc gcctgcagcg tggccccacg 20340 ggaaagcaca
ttccaggcgc attccgagga tgagcggaga ccatgtatgg aaaggtagtg 20400
ccaggactgt catgagtgtc ccagggctcg ggggattcac ccgtgaactg tgaggtcttg
20460 gctctgatag acctggttct tatgctttag gaggggagac aaacagtaac
agaatagaca 20520 aatgcaagag agagtgactc tggacccctc ccacaacggc
ctcctaacaa tggagcatga 20580 gcagatacct gcaggatgga gggtcctgtg
caggctttct gggacgcaga ctggccacct 20640 cccccaggcc ctgcaggcag
ccactgttag caccgcctga gatgtgaacc ttttctcctc 20700 ccccagctgc
catggtgtgg acggttggca tggcgaagct ggacccctcc tctcagggtg 20760
ccctccagct cccctacgac ccggagatgg gtgagtatct tggggagcta acagcctctc
20820 atcactgggg ggcagctccc tacctgcacc cagctctgct cggcctggct
tccctgagag 20880 gcatgagttc aggaggggca gagggaaagg tccgttgaaa
accagccgga cacatgcggc 20940 ttgaagattc agcaagtgtt ggaccctcgg
tcctctgcca gcctctgttg catcgttctg 21000 ctgggcgtgg gtgggtggag
tgggggaagc cctggtgtca ggtgctggtg ctcaggggga 21060 ccccttcttg
gagctttgtt ccctggtaac actctgacca gctgttgttt ctctctcttg 21120
ttgtcccctc ctcacggtga tgacggacat cttctcttcc tggacaccca gaagaagact
21180 cctatgacag ttttggggag ccttcatacc ccgaagtctt tgagcctccc
ttgactggct 21240 acccagggga ggagctggag gaagaggagg aaagtaaggt
gcccatgttc acacggcctg 21300 cttcagccta cggcgggagc ggagacagag
ggtggaggct ccctgcagcc tgggtggagg 21360 agggcatgag gggaggggcc
ccttttccca tcagaggcat ctctgtgaaa gtagaagatg 21420 cctgcagcgc
tggggtcttc tcagcaggcc ccatgtagtt gtccggcatg tattgagtat 21480
gggccacgtg cccgtgctgt gctgggtgag gcccagccct ggtgggaccc acaggctaag
21540 gagacacggg cagtaatcac atagactgag aagccaagga ctatgaaggg
ggccatgggg 21600 ttggggaggg gcggcaggag agcatgccac ggggcttctt
gacctggttg gcaggggtga 21660 gagaaagtca gctgaggaag taactgctga
gctgagctct gaaggttgag tcacagcagt 21720 cactagagga gaggagcaca
gggtggggag catttcctga cagacagact caggaatcag 21780 aggaagccgg
ggcgggatgc agagagcaga agtgtgggag agccttgcaa acaggcctgg 21840
agacatgcga agataggagt tcatcctggc gtcagtacac ggtgcctgcc taacacccaa
21900 tgccagccca ctgctgcgtg ccaggcagca ccctggagca gggagatgct
gcactgtcgt 21960 aacagcccct gccttgagag gtgccttacg ggagcagcct
ggtgacagtg gcttggcata 22020 caggactcca gtgacacggg aggggcaagc
tagggaaaga tcactctgcg gtgggtctgg 22080 aaggaggagc aggtgcgcac
cctccaggca ggcttggggg aggtatttat tccaaggcca 22140 actggtgtgc
tgcagaccag gagttagcac agatcccacg gggcccgcag gactggcctc 22200
cctccagaca ccagccacaa gctctagagg gtctagatgc cacttgtgct tctgaccggc
22260 tgcaaattta gggctcccat gaccccctta ggttcaataa cttgctagaa
tgactcacag 22320 aactcaggaa agcactacac ttaaaattgc agtttgtttt
ttgttgtcgt tttgttttgg 22380 agacagggtc tcgctctgtt gcccaggctg
gagtgcagta gcacgattgt ggctcactgc 22440 aaccttgact tcctgggctc
aagtgatcct cccaccttgg cctcctgagt agctgggatt 22500 acaggcacgt
gctaccacac ctggctcatt tatattttta gtagagacaa ggttttgact 22560
tgttgcccgg gctggtttcg aactcctggg ctcaagtgat ccacctgcct tggccttcca
22620 aagtactggg attataggtc tgagccacag cacccggcca aaattagttt
tattataaga 22680 gatgcaactc aggaccagcc aaatgaagag acagtgaaga
agtaatgctg atggatcaca 22740 cctggtgggg gaaggaggac agctggggcc
aggagcagga gggacacctg cagggctgga 22800 agggcagggg aggtgggcct
ccatggtttg tgtttattgc ataaccattt ttattgtcta 22860 cagtgagcaa
agttatccta taaacaagtg tcagggacca ttgcactaaa gaaaacaaac 22920
gagagcattt tggaagctct aatttcctga tcagtaatgg gtagactaat tcccagttat
22980 atttacctgt tgtaaggtga aaggttcttc agaggacctc tgtcttggtg
ttatatgggc 23040 ttttgaatgt actgaaatta aattccctaa aaatctgtga
ttcagacttc atactaaatt 23100 gtacagcagt gcccagccca aggccttgca
tttctatttg ttgttttctt tactctctaa 23160 gtgcccaaca ctggttttac
ctgagtttca gaactgcccg cttttctctg cccaggttgt 23220 aagtcaccca
gtccacaggt gtcccctgct ttcccactgg ccactgattt ggggaggcag 23280
ctgtccatgt ccccagtcca catcttagct tctagaggcc aggtggggtg ggctgggctg
23340 ggcaagagca gctgggcctt ctgggccaga gtttctcttc tttttccatt
ctgtgcacgc 23400 ctcttcagta cgggttactg tctctcctca cacagggggc
gtgaagcttg gcctcgggga 23460 cttcatcttc tacagtgtgc tggtgggcaa
ggcggctgcc acgggcagcg gggactggaa 23520 taccacgctg gcctgcttcg
tggccatcct cattgtgagt ggctggggat gcgtccagct 23580 gcctcgtggt
gggggccccc agggtcctca ttgtggtggg ggcaggtctc aggatcccta 23640
gggatttttc atttcttctc ttccctctga gggacaagag cagggagcgg ggctggaagg
23700 gtcagcttga gaccaaggct cacaggaggt gtgctcgccc ctaggtgggc
tccagcctgt 23760 ggaggacagt gcaggggagg gtgaggagtg taccggcccc
agcgtggctg agcacacagc 23820 ctccaggccg aggacccagc tgacagcttt
gcgcagtgat gataccctcg aggtggttgt 23880 gatgacatca gatttgcaga
aaagaaaatt gcttaagggc cttgcccatg ggcgcaaagc 23940 tagtgaggac
catgttttcc ccctcctcca tgccattggg acaccacagg gtctgaatct 24000
ggggcactag gggtggcccc gttactgtga accacagcag tgaaatgtgg aggccctgta
24060 gtcagttaac gtgaccagat acacataatg gggagacgtc ctgccgtgac
ttcatctcag 24120 agatttcgct gtcacgttag aggaggagga gcgtctgagc
cgtgcgcttg gcatctgccc 24180 cttagtgaaa accctgggca tggcatgatt
aaggttgatg ctccagtgtc cagaaggttt 24240 tctttttgcc cacaagtata
tcagggatgg gatggtggac ccaggctcct ccaccaccag 24300 actgccttac
ctgagccctg ctggccccaa agatatagaa ggcaccctgg ttccctgtgc 24360
tcacctggac cactgcctgc atcagctggg tcaggggagg atgggcagcc cccacacctg
24420 cttcccaggg gcaggttgcc tggcggctct gattcccttg gtgccagctg
ctgagaacct 24480 tactgccatt tcagttgagc ccacctagct ctcatataaa
tacatgttcc ctgagggcat 24540 cttaccatcc catgtgacca ctccagccag
acaggggagg cagcacggcc tcggggcaca 24600 gcactgctcc aggagtcagg
aggcctgcct tctggttcac tcactaacag gtgaggtgat 24660 ctaatggggg
tgagaacttc tgcccttaac acctcaagag ctgttgcagg accagggaag 24720
ataatggggt gtctagcgcc gttatccgac tggtcctcga acaagctcct gtgcccaggg
24780 actagaccat gactcacagc tcctgtccac accagggatc accacgctca
ccctcccctc 24840 catgtcctgc agggcttgtg tctgaccctc ctgctgcttg
ctgtgttcaa gaaggcgctg 24900 cccgccctcc ccatctccat cacgttcggg
ctcatctttt acttctccac ggacaacctg 24960 gtgcggccgt tcatggacac
cctggcctcc catcagctct acatctgagg gacatggtgt 25020 gccacaggct
gcaagctgca gggaattttc attggatgca gttgtatagt tttacactct 25080
agtgccatat atttttaaga cttttctttc cttaaaaaat aaagtacgtg tttacttggt
25140 gaggaggagg cagaaccagc tctttggtgc cagctgtttc atcaccagac
tttggctccc 25200 gctttgggga gcgcctcgct tcacggacag gaagcacagc
aggtttatcc agatgaactg 25260 agaaggtcag attagggcgg ggagaagagc
atccggcatg agggctgaga tgcgcaaaga 25320 gtgtgctcgg gagtggcccc
tggcacctgg gtgctctggc tggagaggaa aagccagttc 25380 cctacgagga
gtgttcccaa tgctttgtcc atgatgtcct tgttatttta ttgcctttag 25440
aaactgagtc ctgttcttgt tacggcagtc acactgctgg gaagtggctt aatagtaata
25500 tcaataaata gatgagtcct gttagaatct tggagtttgg tccgttgtaa
atgttgaccc 25560 ctctccctgc atcttgggca cccctgggat aacttgtgct
gtgagcccag gatggaggca 25620 gtttgccctg tttgaaggaa cttttaatga
tctcgcctct ctgcacacat ttctttaact 25680 agaaagtttc ctaagcaaag
gagttaggag agcagggtgg cctgacatct gccagccctg 25740 agctgtaagg
ctgtggatgc tgagcaggtc cctggactca gttgtgcacg gtggcacaga 25800
cactgccagg tggttgccaa aacatccagt ggttccttca gcaagtgttc accctctgca
25860 gaagcctgtg agggcctgag ctcagaaacc actctccttt ccttctctgg
ctttggccct 25920 gggcactgtg gtgggagagt ggacagtttg gctttgcctt
ctctgtacat caatcatggg 25980 ttgcaaagag aatctcagaa gtgcctcttc
ctgagcacag tggctcacac ctgtaatccc 26040 aatacttcgg gaggtcgagt
cggaaggatc acttgagccc aggagtttga gaccagccgg 26100 ggcaacatag
tgagactttg tacaaaaaaa aatttaaaaa ttagccaagc atggtggcat 26160
gcatctgtag tctcagctac tctggaggct gaggtgggag gatcacttaa gcccaggagg
26220 ctgaggctgc aatgagccga gatcaagcag gtgttaggta tatcagacag
ctgagaagac 26280 gcaagtgtgc cctggggttc aaactggtac ccctgtctcc
ctgttccagg aataacatga 26340 gtgccgggac aatgcatctt tattatgaga
ggaatgagaa ttgtgtatct tgacatttga 26400 caggagcttg ctttccccca
ggctgtttga ggaagggcag aggaaaatgt ggtgccctaa 26460 gaaggaagga
cagaggaggc cgaacactgg cgggtggaat cccactgatt agtagtgcag 26520
gtcagagacc tgggatgggg ggcattgccg tcatggaagc cacagcgggg agcgggtaaa
26580 gcagacaggg atggtccctg atggtgacaa ctcgcaagag gttaagggga
aagaaaaact 26640 gaaaagctta ttcaatttgg caattatggc agtgtttatc
ttcagaagag cagttttagg 26700 gtggggtttc caaagatggg attggacata
tattttgaat cattaagctt gaggtctttc 26760 aaaggcctgg ccaagggttg
ctgggtggag accacattca gaggtaaagg cagaaattgg 26820 gggcccttaa
gtagacagcg agggaggaag aaatgaaggg gcctggtgat ggttagggtg 26880
aagtgttaag actgagaaaa caaggacatg tgagaagacg agggaagagc attggagaga
26940 acaaagacac tggaggagat gctacttgga ggtccccaga gagcagggag
acaaatgaac 27000 ccagaacaca aatggcaaag aagaaaaatg agagaatttg
taaaagacag cattcgaaca 27060 tgccgaacaa gagcagggta ctggtgttca
aacacctgta tctcccccgt gtaacccgtc 27120 aactaatatc tttccatatt
tgctccagat ttgtctttag aaataaaacc cacgttctga 27180 agtcctgttt
gtatgtggcc ccagtcctgt tgcctccgcc tcctgtcctg aagtcgattt 27240
ctgcccttct catctatggt tagttttgtt ttgtatgttg gcatgttttc ttaactttac
27300 agaaatggta tcatactgta catatttgat aattttttaa aatattgcat
tctggaggca 27360 tgtataaatg tagctccagt tcatttattt tatttatttt
ttgagatgga gttttgctct 27420 tgtcacccag gctagagtgc aatggcgtga
tgttggctca ctgcaacctc tgcctcctgg 27480 gttcaagcaa ttctcctgtc
tcaatttcct gagtagctgg gattacagtt gcccgccacc 27540 atgcctggct
aattttgtat tttagtagag acggggtttc accacgttag ccaggctggt 27600
ctcaaactcc tgactgcagg tgatccacgc accttggcct ccaaaagtgc tgggattaca
27660 ggcgtgagcc accgtgccca gcccagttat tttaactatt gtatagtgtt
ccattgtatg 27720 agttctactg tttatatgct attgatcgac ctgtaggggt
tttgcagtgt ttctgtatta 27780 cagctgtgct gcagtgagca tcccatcaca
ttgtgtggat ttgaggaagt attggaattc 27840 ccccaattga ctggacattc
ccaattaccc tccaagtatg tgtctgttta tccttccatc 27900 cgcaatctga
gagttcccca actctataat acttggtgtc atcagacttt tcatcttgtc 27960
tgattggatg ggtgtcattt cctttaggtt ttataattat cttttcatat gtgtattggc
28020 tgtacaaggt tccttctctg ttcattatta ttaatttttt tagacagagt
ctcgcgctgt 28080 cgcccaggct ggagtgcagc agcgtgatct tggctcactg
caagctccgc ctcccgggtt 28140 catgccattc tcctgcctca gcctcctgag
tagctgggat tacaggtgcc tgccatcacg 28200 cccggctagt ttttttgtat
tttgagtaga gatggggttt caccgtgtta gccaggaggg 28260 tctcgatctc
ctgacctcgt gatccacccg cctcggcctc ccaaagtgct gggattacag 28320
gtgtgagtca ctgcgcccag cccaagtttc cttctctgtt acttgttcat atcctctgcc
28380 catttttcac ttggattttt tgtcttacgg atatttaagc ctcttaaaat
atatattctg 28440 gagagatgct aatctttgat taattatatg cattgcaaat
gtctggtaca ttgtggcttg 28500 cctctcttcc ctgcctttag gagtgttttg
ctggacccaa gtaattttta aatgttaatg 28560 ttattaaatc tatcagtttt
ttgcttgtat ggcttatgcc attgaatctt gttttaagag 28620 atccttccct
accctcaagg ttttctaaat ttttattttc ataataagat ttttagttca 28680
tctgaaatgt atttttatga ttgtatttag tagggaccta attttgtttt tctttgtaac
28740 caggtgtccc agcactgttt actgaacagt ctctcctttc tcgctggtct
gtagaactct 28800 cctgacatat accaagtttc cataagtggg tggatgggtt
cctgagctct ctactgttaa 28860 tagaacttgc tctctcgcag gccaatgcct
caccaggtga ttgaagcaga gaaacttagg 28920 tggtgaaagg agaagatggg
gcctgtcctg agagtttctg ttcctgagat gctagaggca 28980 gagggtatgt
aaatctgaag ttacactgga tctcctaaaa cagtataaag ctacagaagt 29040
ataatagtgt ggaatggtgg tgggagtcag taagggttag gtcactgcag tggtttaaac
29100 aagatgggct agaatccttt cacaggcaca ggcagcttgg agagggtgca
atagtgcatg 29160 gtatcagggg tcagatgcct ctttttcctt tgagatcagt
aagtggcttt cacctcatga 29220 cctaggctgg ctgctgtgtg ctagccgtca
agtcacactc catccagcat gaaaggaggt 29280 tagaaaaggg tgcatttcct
cttcttaaaa acatgtctca aagttgcaca cagcactttt 29340 gcctatattc
aattggccat tagtcccacg gccatacctg tctgagactg agagactggg 29400
aaatgtcttt atttcaagtg gccatatatc cacctaaaca agataaggga tacgtggtta
29460 tggcgtgtct tttggtttac caatgcagat aatgaagtta ccaaaacaat
gagaaaatgg 29520 ggtcgtgagg gatcatgtga atcacaagct gatgtcttca
aagacggtgg aaatgggccc 29580 cgggaggcag cagatgacag cagtggggat
taaggtagac ctccatcctg gggttaaaat 29640 gaggggaagg tgatggagct
ggaccagcag tcagaatggt cagtggttag gagaccctct 29700 gccccccacc
gctgccacca ttggctctct acagaatgcc tgcgagtggc ttagagtgac 29760
caaggatgag gtgcagatcc atgtgcaccc ccctgccccc tctgtggaca attttcatgc
29820 ctgacagcac agtctatgtg gattgcaagc cgatgaaact atgcaaagta
gaagcatgcc 29880 tgcagtttgt gattcggtga tgtgttttat gcttatgtga
gtcgaatggg gcggcagggt 29940 cctgtggtca cccgctgaga aggaagggtc
ctgtaaccac tgcctttctt tcagctactt 30000 gagaaaggtg ttgtgaggga
ccgtggattt tgggacagct ttgaatggtg gtagggagga 30060 agggtccggt
ctgagtgaat ggccagaaag ctgtggggaa gcttttagga cattggccaa 30120
gagctccctg aaggcagcca gggagatact tgtcagtaca tgtgactaat ggccaactga
30180 atataagcag aagtgctgtg ttgctgtgtg caacactgga caccttagga
aggacctcga 30240 gacagtggtt gtggactctg tagagagtaa cagtgacagt
agcaaaccct tacccagtgc 30300 caaccttgtg ctaggctcgc actaaatgag
tttaccttca attctcgtaa caataggagg 30360 taactactat tctaatttcc
attttataga tgaggaaact aaggcacaga gatcactgac 30420 ttgcccaaaa
tcaagcaggg agtagttagt atataagccc acggtatgtg gtttgtagaa 30480
taggtgctct tgactagcag aaataggtcc tccctgcagt gtgtaattga taacaagcat
30540 gggctgccat cttcctgtcg aggccactca aaacacccaa caggctacgc
acggtggctc 30600 acacctgtaa tcccagcact gtgggaggcc gaggtgggcg
ggtcacctga ggtcaggagt 30660 tcgagaccag cctggccgac atggtgaaac
tccgtctcta ctaacagtac aaaaattagc 30720 tgggcgtggt ggcgggcacc
tgtaatccca gctactcagg aggctgagac agaagaatca 30780 cttgaaccag
ggaggcagag gttgcagtga gacaagatca cgccattgca ctccagcctg 30840
tgtgacaaaa gcgaaactgt ctcaaaaaaa aaaaaaaaaa aagtatgatt ttataatccc
30900 agcaccttgg gaggctgagt cgtgagaatc acttgagccc aggagtttaa
gaccaatcta 30960 ggcaacatgg caagacccca tctctgccaa aaataaaaaa
tagtctaatt ttagctattc 31020 atgtgtgtgt gaagtggtgt ctcttcgtgg
ctttgatctg catttcccta atgctgacta 31080 atgacgttgg gcacctgttc
atgtgcttac tggtcagata tctttctttt gttacatttt 31140 attaagtttt
aaaatttaaa gtcaaagatt tccctatgag aatgactttt aaaatgacca 31200
aaaaggggaa gataacatta attcttgaag agaaggcctc tgagaaaaat acagttgtag
31260 caagctgcta ctttgcaaat gacccatgca ttttaatttt cccctaagga
aggccaagga 31320 agagtcttat cacctcaggg caggagatgt agggacttgg
gtcatttaat aagagtggta 31380 ggtttgaaaa ctcaaaccca gaagactcct
tagagtttct cccaggaggt agggaagggg 31440 ccgcatccat ggagagagga
ggatgtgact tagagcagtg gtccccaatc tttagggacc 31500 agggactggt
gtcatggtag acagtttttc cacagatagg ggttgggggg atgatttgga 31560
gctgaaactg ctccacctca ggtcatcagg cattagattc tcatgtggag tgtgccactt
31620 agatccttgg cgtgcacagt tcacaatggg gttcgaggtc ctatgagaat
ccgatgccac 31680 tgatttgaca ggaggcggag ctcaggtggt aatgctcatc
tccaccgctt accacctgct 31740 gtgcagcctg gttcctaata ggctatagac
tggtactggt ccatggcctg ggggttgggg 31800 acccctgatt tagaggaagt
aagggcatgg cttaccgtgg gccctggggt gttctgggaa 31860 tggggaggat
ggagagaaga gaggaggtag ggaagacctc cccttgctcc ccatttggga 31920
tttggggaga aagtcaggtc tcaggctcaa cagtacctga tcctgtacca tcttccaaag
31980 ggaagtcagt ggggttggaa ggtaggcagg ggttatcttc tctgagccac
ggcacaagac 32040 agaagtttcc caccattcct gagggggcag gtggtaggtc
cccaagcaga gagccagcag 32100 tccctctctg aggcctgcaa tggaatgggg
tggggtgtcc actgagccaa gggtctgtca 32160 gtgagagctg gggaggctgg
gctggcttgc aagcacctgt tataaccaaa ccaggaaatc 32220 aggttccgag
tcttgccagc aagggcctac agctgccagc agagatggac agccaggaga 32280
ccccaattgg ccacccagag ccaccctcct ctgcctaccc caccctccag tactccagag
32340 cctactcgga ggggaacaga aacctgagag gctgaacaca cacacatgga
gaaacaaacg 32400 tagtaaaata tttggggaat caggaagaat tatttgtact
attcctgcaa cctttctata 32460 ggcttgaaat tatcaaaata aatttttaaa
aattgtaata acattctcat actaaaacac 32520 tgagtttttt tctttcattt
tttgattttt tctttttgac tccagcatga cttactctaa 32580 caatgggtgg
tctcgatttt gaaatacttt cttctccaag cctttcatga caccctgtct 32640
ctgttggttc tgaaaatgtt ggattttgtc tcagcccttg cttctggaaa cagccaaggt
32700 taagaaaacc ccccatgctt tgtgttctag cagacagctt cctgcaaaga
gccatcttcc 32760 cagagcactt aggcctctta gatgtctccc ttgtttaatt
atgacaagag cacacacaca 32820 gaccctccaa attcccattc ttagtcttct
aaatgattag ctgagctgct tttccccact 32880 gattaatcgg aataaaatgc
tcattaacca aacttccctc ctttccccag gtccctaaac 32940 tttcctgagt
cggcagacat cccctctgga gaagaggttg gccccagagt cgaacatcct 33000
ctgatctacc tgatcctgct gcccttccat tccacttccc cacatctgtt ctttctggtc
33060 gtgtttactc ccctattaaa aaaacaaaac cagaaaacgt gtttgcctag
atcttgagac 33120 tctggaagat cttaacagtc agaggttccc cctatttgca
atgatctcct ttcctgcccc 33180 ttcctatcct tgcaataatc cttttgaata
aagtctctcc ttactaaatc cagttcctaa 33240 aaattaattt ttttagag 33258
<210> SEQ ID NO 2 <211> LENGTH: 448 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
presenilin-2 isoform 1 <400> SEQUENCE: 2 Met Leu Thr Phe Met
Ala Ser Asp Ser Glu Glu Glu Val Cys Asp Glu 1 5 10 15 Arg Thr Ser
Leu Met Ser Ala Glu Ser Pro Thr Pro Arg Ser Cys Gln 20 25 30 Glu
Gly Arg Gln Gly Pro Glu Asp Gly Glu Asn Thr Ala Gln Trp Arg 35 40
45 Ser Gln Glu Asn Glu Glu Asp Gly Glu Glu Asp Pro Asp Arg Tyr Val
50 55 60 Cys Ser Gly Val Pro Gly Arg Pro Pro Gly Leu Glu Glu Glu
Leu Thr 65 70 75 80 Leu Lys Tyr Gly Ala Lys His Val Ile Met Leu Phe
Val Pro Val Thr 85 90 95 Leu Cys Met Ile Val Val Val Ala Thr Ile
Lys Ser Val Arg Phe Tyr 100 105 110 Thr Glu Lys Asn Gly Gln Leu Ile
Tyr Thr Pro Phe Thr Glu Asp Thr 115 120 125 Pro Ser Val Gly Gln Arg
Leu Leu Asn Ser Val Leu Asn Thr Leu Ile 130 135 140 Met Ile Ser Val
Ile Val Val Met Thr Ile Phe Leu Val Val Leu Tyr 145 150 155 160 Lys
Tyr Arg Cys Tyr Lys Phe Ile His Gly Trp Leu Ile Met Ser Ser 165 170
175 Leu Met Leu Leu Phe Leu Phe Thr Tyr Ile Tyr Leu Gly Glu Val Leu
180 185 190 Lys Thr Tyr Asn Val Ala Met Asp Tyr Pro Thr Leu Leu Leu
Thr Val 195 200 205 Trp Asn Phe Gly Ala Val Gly Met Val Cys Ile His
Trp Lys Gly Pro 210 215 220 Leu Val Leu Gln Gln Ala Tyr Leu Ile Met
Ile Ser Ala Leu Met Ala 225 230 235 240 Leu Val Phe Ile Lys Tyr Leu
Pro Glu Trp Ser Ala Trp Val Ile Leu 245 250 255 Gly Ala Ile Ser Val
Tyr Asp Leu Val Ala Val Leu Cys Pro Lys Gly 260 265 270 Pro Leu Arg
Met Leu Val Glu Thr Ala Gln Glu Arg Asn Glu Pro Ile 275 280 285 Phe
Pro Ala Leu Ile Tyr Ser Ser Ala Met Val Trp Thr Val Gly Met 290 295
300 Ala Lys Leu Asp Pro Ser Ser Gln Gly Ala Leu Gln Leu Pro Tyr Asp
305 310 315 320 Pro Glu Met Glu Glu Asp Ser Tyr Asp Ser Phe Gly Glu
Pro Ser Tyr 325 330 335 Pro Glu Val Phe Glu Pro Pro Leu Thr Gly Tyr
Pro Gly Glu Glu Leu 340 345 350 Glu Glu Glu Glu Glu Arg Gly Val Lys
Leu Gly Leu Gly Asp Phe Ile 355 360 365 Phe Tyr Ser Val Leu Val Gly
Lys Ala Ala Ala Thr Gly Ser Gly Asp 370 375 380 Trp Asn Thr Thr Leu
Ala Cys Phe Val Ala Ile Leu Ile Gly Leu Cys 385 390 395 400 Leu Thr
Leu Leu Leu Leu Ala Val Phe Lys Lys Ala Leu Pro Ala Leu 405 410 415
Pro Ile Ser Ile Thr Phe Gly Leu Ile Phe Tyr Phe Ser Thr Asp Asn 420
425 430 Leu Val Arg Pro Phe Met Asp Thr Leu Ala Ser His Gln Leu Tyr
Ile 435 440 445 <210> SEQ ID NO 3 <211> LENGTH: 19
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Forward 5'-3' Primer <400> SEQUENCE: 3
catcagccct ttgccttct 19 <210> SEQ ID NO 4 <211> LENGTH:
20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Reverse 3'-5' Primer <400> SEQUENCE: 4
ctcaccttgt agcagcggta 20 <210> SEQ ID NO 5 <211>
LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Forward 5'-3' Primer <400> SEQUENCE: 5
acagaattcg ccccggcctg gtacac 26 <210> SEQ ID NO 6 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Reverse 3'-5' Primer <400> SEQUENCE: 6
taagcttggc acggctgtcc aagga 25 <210> SEQ ID NO 7 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Forward 5'-3' Primer <400> SEQUENCE: 7
tcagcatcta cacgccattc 20 <210> SEQ ID NO 8 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Reverse 3'-5' Primer <400> SEQUENCE: 8
agcaccacca agaagatggt 20 <210> SEQ ID NO 9 <211>
LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Forwad 5'-3' Primer <400> SEQUENCE: 9
attcgccccg gcctggtaca ctgcca 26 <210> SEQ ID NO 10
<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <223> OTHER INFORMATION: Reverse 3'-5' Primer
<400> SEQUENCE: 10 ctgtccaagg agctgcaggc ggcgcag 27
<210> SEQ ID NO 11 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
g1N141I guide RNA F <400> SEQUENCE: 11 caccgcatca tgatcagcgt
catcg 25 <210> SEQ ID NO 12 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: g1N141I guide RNA R <400> SEQUENCE: 12
aaaccgatga cgctgatcat gatgc 25 <210> SEQ ID NO 13 <211>
LENGTH: 100 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: misc_feature
<223> OTHER INFORMATION: Donor ssODN#A N141I <400>
SEQUENCE: 13 gagagaagcg tggctggagg gcagggccag ggcctcacct tgtagcagcg
gtacttgtag 60 agcaccacca agaagatggt cagggtgttc agcacggagt 100
<210> SEQ ID NO 14 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Forward 5' Primer <400> SEQUENCE: 14 taacggcggc agacaaaaag a
21 <210> SEQ ID NO 15 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Reverse 5'-3' Primer <400> SEQUENCE: 15 gaagtattgc ttcagttggc
ct 22 <210> SEQ ID NO 16 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Forward 5' Primer <400> SEQUENCE: 16 agaagaacgg caagtacgag a
21 <210> SEQ ID NO 17 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Reverse 5'-3' Primer <400> SEQUENCE: 17 tgttgaggga cagattgtgg
c 21 <210> SEQ ID NO 18 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Forward 5' Primer <400> SEQUENCE: 18 taacggcggc agacaaaaag a
21 <210> SEQ ID NO 19 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Reverse 5'-3' Primer <400> SEQUENCE: 19 gaagtattgc ttcagttggc
ct 22 <210> SEQ ID NO 20 <400> SEQUENCE: 20 000
<210> SEQ ID NO 21 <400> SEQUENCE: 21 000 <210>
SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: misc_feature <223> OTHER INFORMATION: Forward 5'
Primer <400> SEQUENCE: 22 acgaatctcc gaccaccact 20
<210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Reverse 5'-3' Primer <400> SEQUENCE: 23 ccatggccac aacaactgac
20 <210> SEQ ID NO 24 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Forward 5' Primer <400> SEQUENCE: 24 gaagtgtccc aggacatgat aa
22 <210> SEQ ID NO 25 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Reverse 5'-3' Primer <400> SEQUENCE: 25 ctcttgagta gctgggattg
ag 22 <210> SEQ ID NO 26 <211> LENGTH: 46 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Forward 5'-3' Primer <400> SEQUENCE: 26 ctccgtgctg atcaccctca
tcatgatcag cgtcatcggt tatgac 46 <210> SEQ ID NO 27
<211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <223> OTHER INFORMATION: Reverse 3'-5' Primer
<400> SEQUENCE: 27 gaggcacgac tagtgggagt agtactagtc
gcagtagcac caatactg 48 <210> SEQ ID NO 28 <211> LENGTH:
20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Forward 5'-3' Primer <400> SEQUENCE: 28
catcatgatc agcgtcatcg 20 <210> SEQ ID NO 29 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: g1N2411 guide RNA F <400> SEQUENCE: 29
caccgcatca tgatcagcgt catcg 25 <210> SEQ ID NO 30 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: g1N2411 guide RNA R <400> SEQUENCE: 30
aaaccgatga cgctgatcat gatgc 25 <210> SEQ ID NO 31 <211>
LENGTH: 118 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: misc_feature
<223> OTHER INFORMATION: Donor ssODN#A N141I <400>
SEQUENCE: 31 gagagaagcg tggctggagg gcagggccag ggcctcacct tgtagcagcg
gtacttgtag 60 agcaccacca agaagatggt cataaccacg atgacgctga
tcatgatgag ggtgttca 118
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 31 <210>
SEQ ID NO 1 <211> LENGTH: 33258 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: misc_feature <223> OTHER INFORMATION: chromosome 1,
GRCh38.p12 Primary Assembly <400> SEQUENCE: 1 ggggcctggg
ccggcgccgg gtccggccgg gcgctcagcc agctgcgtaa actccgctgg 60
agcgcggcgg cagagcaggt gagcgggcgg tgccgggggg tgcccaggcc agggccctgt
120 cgcctgcggc gctgagggcc cggggtgggg ctgcgccctg agggccctgc
cctgccctcc 180 gcacgcctct ggccacggtc ccttccccgg ctgtgggtct
gcggcccctg cgtgcgcagc 240 gctcctggcc tctgcggcca gcgcgggggc
ggagagagga gagtgcccgg caggcggcgg 300 ctgggccggc ccggaactgg
gtcgtggaag gatcgcgggg agcggccctc aggccttcgg 360 cctcactgcg
tccccacttc cctgcgcccg cctgccgccg agccccggct gggggtgggc 420
gcggcgcgag cggttaaagg gccggtgcat ttaaaggagc ggtgcacgtg ggtctctgag
480 gcgtgtagca ggcgggggcg ttttgttctt cttctctctc gccggagacc
tccgttgcgc 540 cgagtccatt cggcctctag caccgggtcc tgggcatgct
ttccccggga aggaggcgcg 600 cgggggctct gcccgcacgt gaggggcagg
gccgcaggct caagcctaga gccggtttct 660 gttagcagcg gtgtttggct
gttttatcag gcatttccag cagtgaggag acagccagaa 720 gcaagctttt
ggagctgaag gaacctgaga cagaagctag tcccccctct gaattttact 780
gatgaagaaa ctgaggccac agagctaaag tgacttttcc caaggtcgcc caggtacgat
840 atagcagagc caggcttcga ccccagtgtc ctggcttcta gatctgctgt
ccatccctcc 900 gagcagacct cacccctgtt tattgcctta ataagtattc
cctttgaaag gtatgaacgg 960 tgttgagtga agtaactgca tccctattta
caaatggaga acctgagagc attccataga 1020 gacgattgta gactaactta
actcagaagc gacagcctgg ggttgccaag gctgtctacg 1080 aagtaacttg
attaggaccg accccagctt ccagtaagga agcctctgat gcctctgtag 1140
ccaattctgc agacacctga gcctccaagg ccttcagcca agacctttgg cggtaattgg
1200 agtctcggga taagctgctt caggtgtgtg agcctcaggt tcttctctcc
tgaatgtggt 1260 tgtgggcagc cggtgactgg cgcaggtgca gaaggggcct
ggttcttggc cccacctcag 1320 agctgcgtcc tcacgacgcc cacgttgagc
cttgggttcc agggcagaga ctggagtgag 1380 ggcttggggg catgttgctt
tgaagtggga tggatgtatc aggtttttgg ggaaaactct 1440 gtaccctttg
gtgttgaagt gcccatgtgc caagtcttga gtccagcatg ttcacatgtg 1500
gggagtgagt ggcttgttcc tgtctatttg aaagagcagc aaggaggagg aggagcaagg
1560 gctaggggct gctgctgggg tgcctggagc tgtggtgcat aatgtcacac
ctgtctcccc 1620 tccgtagctg ctcaccgtcc ccccaagggg ggtttgcctc
ttgcctactt tggcctttct 1680 ctgttatcga tgttaataat gacatatatc
tcgcttatga gttggtcata ataaaaagct 1740 atcttgtaca gaatattaga
atttaagatc ttaagaattt caatgacact gaaatagttt 1800 attattacct
ttttacagaa gaggaaacaa gttcagggag ttaagcagct agtccagtta 1860
tgtggcttca gtgctttaag caccaggatt tgaacatagc tggctacact gtctttatct
1920 cttgagtttt tgcgcaggag gttcctgtat tcaactccta cccgtgtctc
tccactactg 1980 ctgggaaagt tttgtggagt ccccatgagc aacttcctga
caaacaaaca aaattttttt 2040 aaagaaacca aagcagtgtg tgtaggtcac
atgcagtgtg tctaatgaaa acatctctgg 2100 cgggttttca gctgttgctt
tgactttcgg acactgttta gttggggact gataagacag 2160 caaatatttc
tgcaagtatt cccacctgtt ctattcccag ctgccacagc tgcggaaagg 2220
cgggggtgag gctgagaggc cccgagagga acattttcca ctgggctcca atcctggaga
2280 tgggatgacc atcatgttaa tgtctggaga aaagaatgat ttcaggctgg
gtgctgtggc 2340 tcatgtctgt aatcccagca ctttgggagg ccgaggtggg
tggatcacct gaggtcggaa 2400 gtttgagacc agcctgacca acatggagaa
accccatctt tactaaaaat acaaaattag 2460 ccgggcgtgg tggcacatgc
ctgtaatccc agctactcag gaggctgagg caggagaatc 2520 gcttgaaccc
aggaggcgga ggttgcagtg agccgagatc gggccatggc actccagcct 2580
gggcaacaag agcaaaactc catctcaaaa aaaaaaaaat ggtttcacat cagtcctcag
2640 gaaagatcag atgtcagtga gggagatcat ttcttgagag cctcttcact
gagtgggaga 2700 atgggctgct tgttcatctt tgtgaaaatt ctagaacggg
aagaacaatt caaagggtgt 2760 ccaccattct gctgtacctt aaccagaaac
ttactggact ctttttaaaa taaaagtaat 2820 tcatgtttat tctagaaaat
tagggaaaaa aaattttttt tgagatggag tttcactctt 2880 gttgcccagg
ctggagtgcg atggtatgat ctcagctaac tgcaacctct gcttgccggg 2940
ttctagcgac tctcctgcct cagcctcctg ggtagctggg attacaggtg cctgccacca
3000 ctcccagcta atttttgtat ttttattaga ggcggggttt caccatgttg
gccaggctgg 3060 tctcaaactc ctgacctcag gtgatccgcc aaccttggcc
tcccaaaatg ctggtattac 3120 aagcataagc cattgcgctg ggctgagata
accactctta acatgcattt ccttccacac 3180 tgttcacata tgcatattta
tctattaaaa caatagaggg agggaaccgt aggtgaagtt 3240 tgtgtatcct
gttttttctg ttatgccttc agaattttcc ttttattgag tactcatgga 3300
aaagcagatt tgatggctgt gtagagcatt tgaattattt atcctaccag tcccacagca
3360 ggacactttc ccaccctccc ttttgcccca gagaagcagt gccctctgtc
ctccccatgc 3420 ccatatgtgg gcactcccca ccatggagcc aaacctacct
gggcaagtag cagagggaga 3480 gcagagtgag ccctgggggc aggagagaga
cttgagagtt ttgaggtgac agatgagctg 3540 gtgagtgagt gattagggag
catttcttga cacatacctg cccgtggtga aggcatgtgt 3600 cttgtgagtg
tgctcccaga aagcctgtgt agtgtgtggt gggcctgcct gtgtgaccaa 3660
accctggcca ctgggtacgt gaccctcaca agtgctgact gggctgagaa gagctccttg
3720 atgggcagtt tggagacttg agttgtaact gtggcttttg gccatgggac
attaactgat 3780 tacttttgcc cctctaggct tcagttgtcc ttaattataa
tacagggagc tgactaggtg 3840 gcgttgatgg cctttacggt ccttgccagc
tctgacattg tcctatggat atgtcctttc 3900 atttgataat atgtttacgt
ggccatagtg cctggggctg ggccgggaat ggaaacttga 3960 tctctggggc
ctggcctttg aagccagttc atgtgtctgg tggttcagca gatccgtaac 4020
tttccaagag gcacatccat aggctaccgt gtcctttctc actgtgtccc tcctccattt
4080 catcttcttt ataactacga cttattgaac atctactgtg tgctggacac
tttacaggtt 4140 atctctaggt tttacgataa tcttgcaagg tatgcctgtt
ctgcttttta cagcagagga 4200 aatgagctgt gtcagattag actgtctgag
gcctcttggc cagggagtat gtggttcaaa 4260 tcacataggc aggcgatctg
aaccctgtca gtctccaaag cctctgcttt tgaccgctga 4320 cttgctgctg
cttgtttaaa aataaatgtg tttctggagc ctactccaga ggggcgtgct 4380
aggggctccc tctcccactt ccccacaaac cacccttttc cctggctgct tcaggaaatg
4440 agagaactct gcctgggccc caggcacttc tgagtgggac agggctgtta
gaggtaagtc 4500 tagagcctgg cccaaaattc aggaggcccc atcagagggc
ccctggggcc tgtggtccgg 4560 gagggtggta gggcagtacc tcacttccct
ttgagactca ggccccagct ctggcttagg 4620 ccagggagaa ccatccccaa
gtggtatgtg ttactatatg agctgagatg gatggtcagc 4680 tggaccaaat
acatagtcgg gtacccaggg ccagggggag gaaggtgagc agggaagctg 4740
tgggcaattg tctgggtatc acctgacctt agcaaactct tccttgtttt aagcgaggac
4800 gtgggacttc tcagacgtca ggagagtgat gtgagggagc tgtgtgacca
tagaaagtga 4860 cgtgttaaaa accagcgctg ccctctttga aagccaggga
gcatcattca tttagcctgc 4920 tgagaagaag aaaccaagtg tccgggattc
agacctctct gcggccccaa gtgttcgtgg 4980 taagtgcagt gactcccaac
ctgcttttga accctctttt tccattagga ttttctccgt 5040 ggaggcagat
ttccatggga gtttgctgtg gcattttgaa atctgtttct tacctagttc 5100
cattggcctt aaatgttaag gccaaagcct ttacatttct ctgtaatgaa aagaaggtcg
5160 aggaaattgg gtcattgggt ttccataatg attgcaggaa ctgctgacac
aagcacggct 5220 ggggagattc tctaggtcag actcccttgg tttggctaat
tcagcagttt gatcccattc 5280 agctgattaa tgggaatgtg cagtggcttc
tttggatgtt tgattttgca tcctaatcca 5340 aagcagctat cagcctcagc
acttccttgt tggaaggctt tccagaacgt agtctatgtt 5400 ggacacttcc
ttctgcctct ctgcattttc ctgccacttc tctagagaat ggggtgcagg 5460
gggtgggaga cggggaaagc tggtcgctga gtggctgatg ggacttgaca tcacccagcc
5520 ccacccccac ctgcccgtga gtcagcctcc ggggagagtt catcgcgtca
ccggcactct 5580 aatgtggaca gacacctagc agtgttgttt atctgcacac
gtttgggtgg tgatttttcc 5640 ctccaaggat ttcagagcac cagcaggctt
cagagcagac ttaggtggct tgcaaagcag 5700 gccctcagga attcagaggg
tagcagaagt ccatcccaga tgctctgttt tccttcagga 5760 gctaggtaaa
tcagaggggc tgagggacaa atgaaaaaag ttacagcctt tgagtcccat 5820
ctgctcctcc tggccaatga gaggggatct gggaggggca gatgtagagg aaaatctgtc
5880 taaatgttga tgctcgttat tttcctttaa agaattaata gcctaaaata
aaccctacag 5940 atacagtctg tgtttattat ggcgacttag agaaatgcag
aaaaatatca agaaaataaa 6000 aaccactctt ggttctacca tgcaaagata
atcatcttta atgttttgta atatttccga 6060 tcttttatat acatacattt
tataaggaca ttcagatgat caggttcgta aagttttatg 6120 ttcggttaaa
tttaacagcg tgtcattgtt caggttatta aatgtttgaa ataagatttt 6180
tggtggtcct gtcacagtct ccatgaagta gcatttcagg atcgaaaggt atgctgtgtt
6240 taaagtgttg attcttactc ctttcagtta aggccagtgc agtttgtcca
ggtagtgact 6300 gagacccagt ttttccacac tctcctccgc agtgggcatt
gttttgggcc tttttcagcc 6360 caagagctct cttctcccca tgccgctctg
ctggtctgag atttttccac tcctcctcct 6420 ccctagttgc tctctgacca
gactctaggt attcaggaga aagtgttcat tgtctcactc 6480 tctcatgtgg
caatcaagta gtgccaagca gtgagagggt gaaggtgggt gggtgaggga 6540
cactcacctt gctgagaaag ggccccagcc tgttcgggtg attataaagc agagacagtg
6600 ccaggaaaag tctgacactg gctgagaatc acccggggac caaccatccc
gaatgcggat 6660 ccctgacact gggtgaggat ggagcttgga gatctgcatt
gttaataagc agcctagcag 6720 agtggtgaag agtccagaca cactacctag
gtccaagggt aaccttgagc taattacttt 6780 ttgagcctct gtttcctcat
cagtaccatg gggaagaata gtagcacctt gctccaggat 6840 gtttagtgcc
ggctaagggc tcagcaggtg ctggtccatc tccaccagcc cccagtggcc 6900
tgggccacct ttgagaaaca gtgatcctaa gggattcagc atttcctaag ttggtgcctc
6960 ccacctgtca cccccacccc accaggctag gagggttgtg attagagggt
gcccttgctg 7020
tgacagctga gactagctct tccctgatta ttccttaatg acagctctct ccttccctgc
7080 tttcttgaag tcttggtcct cgttgttgtg ggcacagctt caggggaggc
cttggaggaa 7140 tttttgaaag tggaatgagg gaagcagcct gctcaaggga
acacttgttt tctggtgagg 7200 aggccgcatg tatgaatgac gtttgtgggt
tagaaagcat gttttgtagt ttttccttgt 7260 ttcttcctga agacatgtca
ggtcttgatg agaccgggcc tgggcacagg gcaggcagtc 7320 agcgagtgtg
gatgatgacg acagtggtca ccaggtcact gtctagacca ggtcactgtc 7380
tagcgcagtg tcacatggaa agggtatggt cctttaaccc taccctcccc agcacaacta
7440 tcacagatgt cagggaacct ctgctcacag aactgctttc cagggattgt
cttttttttc 7500 tttttctttt tctttctttt ttttttgaga cagagtctca
ctctgtcgcc caggctgaag 7560 tgcagtggtg cgatctcagc tcactgcagc
ctccgcctcc tgggttcaag tgattatctt 7620 gctacagcct tctgagtagc
tgggattaca ggtgcctgcc accatgtcca gctaattttt 7680 gtatttttat
tagagacggg gtttccccat gttggctagg ctggtcttga actcctgacc 7740
tcaggtgatc tgcccacctc agacaggcat gagcaccgca cccagcccca gggagcgtct
7800 tattagtggt tggcaactga atggagacgt gggaattgta aggaactgat
tctacttgat 7860 cctgggtccc ctgcttctcc atcttcaccc acccatcagc
tccctttctc ctttaaacag 7920 gcacctttgc tctctgctta tccatttttg
ttgtgcattg ctatttggga gcctaagaaa 7980 cacaacatcc tctgaatgct
ccagctgttg tgggtctgaa gggtgagcct gccctctgtc 8040 attggaggct
gcagcctgtg gctttttagg tacagggact cccagaactg ctcctccagt 8100
catagcagag ataaatcaca ggagcttaag aggcatggga agaacagagg gaggagatcg
8160 tagcttccct gttcattcac acccaaaaca aaactgtcat actagaaaag
gaggtattaa 8220 aagagccacc tgtacagcct cgtatctcat ccagcacact
gctgcagatg gaatattatg 8280 atttagcttg agaaaatgca gcaactcttt
gttgtggtgc ccctctttga gtaagagtga 8340 attccccatt gccagagtgg
atagtgaggg aaaccctggg tccaggcagg agtctgttta 8400 ggatttatct
agtgaggctg agccagagga ggaccttaca gttttttctc ttcaatttct 8460
tttatttatt tatttatttt tgtagagatg gggttttgcc atgttaccca ggctggtctt
8520 gaactcctgg gctcaagcga tctgtctgcc tcagcctccc aaactgttgg
gattacaggc 8580 gtgagccgag ccaccatacc cggcccttct cctgcatttc
cacctgataa tttctctcat 8640 ttccatagat gatgaaggaa ctaaagccaa
gaactttcca aggtcctgca gctctttggg 8700 ggatgtgaag ctgtgctcta
tttgtatgga ttttgctggt tcccagaact tccctgtggc 8760 cctggggcct
agtctgaggg tactctgagt gaagagggag gagggcccac acctcttctg 8820
caaaggctgc ttttgtaaag ttcacttcag ttcacatctt cctcctggtc agaaagcttc
8880 gggggctctc ctctgctgca ttaagctctt actcctccat caggcaccaa
actcctccct 8940 ggcatggccc atcctaccag gtccccacac ttgagccaca
tccaattgct cgatattatc 9000 aggataggtt atgttatgtt cccaactcat
atgtttactt aagtggttac ctctttccag 9060 aatgagcccc ctcctccaaa
ctctgcctgg tgaaatattc ctaacctttg cagcttcaca 9120 tccctcttac
ttcttgtgac ctgaggcatc tactcctgac aactgataga ctgtgtcccc 9180
tcctgtcggg tgcattgtcc ttgtcactac cctcctggct tttagctggc tttgcttccc
9240 gctgttgtta ctcctgtact tgtctcatct atcctaaaca gaaggtgctg
caggctgggg 9300 agtttgttca tgttgaaatc cctgtgatgg aggtgagcag
aggcagtctc tgcctgtgcc 9360 tcttatttgg ggatgaagtt aaagtccctg
taggaataat ccaggccata gccggggttg 9420 ctgtcttcag aaagaagggc
agccacaggt cttgttaagg ggattgaaat tggctgactt 9480 ggtggaagga
acctgcctgc tttgtttaaa aaccacatat agctgagtgt agtggttcac 9540
actctgtaat accagtgctt tgggaggctg aggcaggagg atcacttgag gccaggagtt
9600 tgagactagc ttgggcaaca acgtgagacc ctcatttcta caaaatattt
taaaaattag 9660 cctagtatgg tggcgtgcat ctgcagccct agctactgag
gaggctgaag tgggagaatt 9720 gcttgaaccc aggagttcaa ggctgcagtg
agctatgatt gcaccactgt actccaacgt 9780 aagtgaccag tgagaccctg
tctctagaaa taaaaataaa aaaaatcaca tatattgtgg 9840 ggtgacttac
ttggagacga actttcagca gagcgcacac ctgctatccc tgcccagggt 9900
gtgaagctca gccctgaggg tctctggaca gcgatcactc agcctctgga cagcgatcac
9960 tcagcctctg gacagacagc gatcactcag cctctggaca gcgatcactc
agcctctgga 10020 cagcgatcac tcagcctctg gacagcgata actcagcctc
tgtccccgtc tgagatgttg 10080 gcagggactg tcagatttgc caggcattgt
ttgaagttct tcccagccca gaaacctgca 10140 tgtgtagatt ttggtacact
gggtccccca cttggtacta ctgtgtgaaa ccccacttgg 10200 cactgtttta
gggggcaggc ttccctcctg tccccttggc cttggccttc ccctgggtcc 10260
cgccctcagt ggcacttccc cacctcacac gtctgctctc atggcttagg tctccacttc
10320 taacctcagg agacctggtc ctcagacacc tcccagacag cttccccatt
ttatcccata 10380 gacactcaaa gggtgaaatt catggtcttt cccgagactc
tcttctccgg tcttccctgt 10440 cttagtcccc acctggctgc cattctagac
tgtgttttct ctcctgcgtc aggtcctgcc 10500 cttgacctct tgaccccttc
tggactctgc cctcacttgc atccatcctg ctgctgtcct 10560 cagctctcct
cacctgccac agttgtctct gggggtcact ttccctctct ggtcagtggc 10620
cagactgact tttataaacc tggttcagat cttgtctgtc aagattgtcc tcagggtgat
10680 gtgtgtctcc ttaacatggt gcctgagacc ctgatcatct ccactgcccg
ccccacagtg 10740 tgaggccctc actggaacat tgtgccttct gcccttccct
cctcctggga aaaccagtct 10800 ccatcagata ggctcttctc tagaaaacat
tcgtgatctc tgagatttgg ttccactttt 10860 gtgcttctgc acctaccatc
aaacacccgg attgtatcat ttgtcacatt agatgatttt 10920 tgttttgttt
taagacaagg gtgtttctta ctcatctttt tatccccaga gcccagcatg 10980
atctttggtg cataatagat gcaccacaga tgtttgctga ttgaatgaat gagcacactg
11040 acagtttgga gctgccctga ctttcgtggc tatgcgtttt gccccctggg
atgtgagtca 11100 cctcaggcca gccccaggca aggccgctgc tgcctccatg
gtaactctca aggcctcttg 11160 ttttatggca gtcgtttgat tgacaggcat
ctcttggaag cttttggggc aggacttgtg 11220 tccaagtctc caggtcgcct
ccagccaccc cctgagtcct ccactgcctt tgtctcacag 11280 gaaagtggaa
caaggtcctt gtgctccttt ttccaggtgc ttccagaggc agggctatgc 11340
tcacattcat ggcctctgac agcgaggaag aagtgtgtga tgagcggacg tccctaatgt
11400 cggctgagag ccccacgccg cgctcctgcc aggagggcag gcagggccca
gaggatggag 11460 agaacactgc ccagtgggta ggtcccacca gcagctgggg
gccttcaaac aggtccctgc 11520 ggctactgta ccttacagat gaaaaccaga
cattcattcc ctgatgcggg agggagaagg 11580 gaagtaatga tgaggattgg
ccgaaaaggt gggtggctgg ccatgatgga ccttccatct 11640 gcagggtttc
ataggactgc gcattcacag ccagagatgg acttggcagt gggctgaagg 11700
acgctgtcca ctctgccacc ttgggtttac ctctctcatg caggtcactg tttccactgt
11760 aataggagag tttgtttgga tgcctgggtg ctaggacagg taacacagaa
gcttaggatg 11820 gtagcagggg aagcattttt tggcagatgg ccagacatgg
taagtgtgag aggagtctgc 11880 ctgatacacg attgactttt gagctgggga
tatttgggct tcactgtgat cattcagccc 11940 ccaggggagg agattgtaac
gttagaaaga gtaggatatc gttgggagag ccacttagtt 12000 gtgtcctttc
tctcccgatc agggcagaac atctgaattt gcctgaaccc tgttctctgt 12060
tttgcccatt atagaattaa aaaatgtctc tgtgtggact gttttcttgc agccagtctt
12120 aatcctgctt gctgaaattt gagctcactt ctccatgttc tccttgagaa
cggaaccatc 12180 gtccctaagc cctgagtgaa atcacaccag cttaaggcca
ctgctctgcc actcctcagc 12240 cttttcttgt ttgttatctc cgggaagttt
tgtacacttt ggttgtttca gtttctgttc 12300 atgagtagtc ttctttcttg
gctgaacgtc tagattggga ctctctctgc agagaaccgg 12360 tactgaagca
actgtcattt tcagtttttg tttcatttgg ctttttcttt agctgttcac 12420
ctcattagca aggcagccca tgaccttgac ttgccacagt tccaaaacac aaattcttac
12480 agatcggttt gtgctagtgt ctggcaggtg tcctgccctc cctcgttacc
tcctcatttg 12540 tgcctgccca ccttcccaga gcctgcgtct tctcagatgc
ttaacacctg tttagcctct 12600 ctagttcaga gctacaaatt tacatgcttg
attctgtggg gcagaaagtt caaagtaatt 12660 tcttcctctg caaattccca
gtatcttagt cacacgcaaa gagagtgtcc ctgtgcactg 12720 actcctctag
ctagtgattt gtcagccaaa aatgtttatt tatctcctgg cctgtttcct 12780
cccatatcag tatggccaca tgaacagaat tgagtgacct cctgagtccc tgtattagga
12840 aggggaaaga tcttttgatt cattaaccat taagttgatt cattaaccat
taagtcttgg 12900 gcctgcagac catagcaacc ttccttcctt catttatggt
gcttcatcca gctccaaatc 12960 ttctctactt tgtcctcaca aacttttcat
atgccctagt agctcataga ctgctcctta 13020 tatctggaaa gcaacattca
aacttctcat ttctggttcc aaaaatccgt gcattacatg 13080 gataggctgc
cgtgggggac attctgcggc cctcacgatg tggtttccca cagagaagcc 13140
aggagaacga ggaggacggt gaggaggacc ctgaccgcta tgtctgtagt ggggttcccg
13200 ggcggccgcc aggcctggag gaagagctga ccctcaaata cggagcgaag
cacgtgatca 13260 tgctgtttgt gcctgtcact ctgtgcatga tcgtggtggt
agccaccatc aagtctgtgc 13320 gcttctacac agagaagaat ggacagctgt
gagttggggg gctgggggga gcagggtggg 13380 gtgagggctg agttgccagg
gggtgggggg cgcagcagcc tgtgttggtc actgtacctg 13440 cagctccaca
ccagcagcgg taaagagcag ggatgaagaa ccgcccaggt tcatggcctg 13500
gctcactgcc tcctggattg tgacctactt gggcatgctt ttaacatccc tatgcctcag
13560 cttccttgtt cgtataatgg gttgataacg cagttactgg gagaattaag
tgagttaata 13620 tgagtgaagg gcttagaaga gtgtctactg cacgtgagtg
ctcaggcaag ctggatcctg 13680 ctgcagaaag caagctcttg atcctgggca
tggctgtgcc actgatccct gtgtgactgc 13740 aaacaaatca cttcctctct
gagtctctgc ttccctgaat gtgaaacaag gtggttggac 13800 cagatatttc
tcagctcact tccagccttg tgaggaagac ttataaagcc tttcgtttat 13860
tttagtaaaa tacatgcaga ggcagcagcg tagaaaaatg agaagcttcc tccacttctt
13920 ccccctcccc tttctgtggt cctcactgct aagcaccttc tgtaaacttt
tttttttttt 13980 tttaaagtta gggatttttg tttcatttcg tgtgtgttgg
ttttttttgt tgttgttgtt 14040 tcttttaaag aaaggaataa ggccaggtgt
ggtgtctcat gcctgtaatc ccagcacttt 14100 gggagactga ggtgagagga
ttatttgagc ccaggagttt gagaccagcc tgggaaatgt 14160 ggcgagaccc
tgtctgtaca aaaaatgcaa aaattagcca ggtgtggtgg tacatgcctg 14220
tagtctcagc tacttgggag actgaggtgg aagaacacct gagcccagaa gtcgaggctg
14280 cagtgagcca tgattgcgcc actgcactgc agcctcagca acagagtgag
accctgtctc 14340 aaaatttttt taaaaaatta aaaaagaagt agagtcccat
cctcagaaag cttatagtgt 14400 gtgggggatt cagcgcagaa caggtgaaag
catggagaga atgcagccag cggtttgttt 14460 gcagcagtcc aggctgggaa
gagtgaggtt tgagtgaatt gcttcctgtg tctgcttcct 14520 gagcttatga
gctgcaagga cagcagttgc ttcagcggat gggggtcggg tagtagcagg 14580
tggaggagtg ctgggctggg tggagctggt ggagaggtgt gggtgggtgg gggaatgaga
14640 actggatggg tgagagaagt gcctagggag cctttaatcc ctgtgggggt
ggggaaagca 14700 gcagggaggt catctagccc tcgtcctcac tgctgcactg
ggcccagttg gcaggctgag 14760 agccacaggt ctgtggtcag ggtgccagga
aatgagctgg aggacaggaa ctgctcatgg 14820 ggatggtgcc cgcactccat
cagggcagca tgtgggcagc atgggcatcc caggcacctc 14880 ccctagcagg
tccagaatca ctcaaggtgg ggagcctcga ggagcagtca gggccgggag 14940
catcagccct ttgccttctc cctcagcatc tacacgccat tcactgagga cacaccctcg
15000 gtgggccagc gcctcctcaa ctccgtgctg aacaccctca tcatgatcag
cgtcatcgtg 15060 gttatgacca tcttcttggt ggtgctctac aagtaccgct
gctacaaggt gaggccctgg 15120 ccctgccctc cagccacgct tctctccgtc
tgccccacac catggcggca gggcccgtga 15180 aacagccgcc tttagaaaaa
cacaaattag aggaaaatag acccagattt tttgtactcc 15240 tccccacccc
atcctgtctc ccaccgtgga tgacctaata ctgttgtctt ttatttttat 15300
ttattttctt tttcttgaaa catggtctca ctccattgcc caggctggag tgcagtggtg
15360 cgatcatgac tcactgcagc ctcaacctcc tgggctcaag ttctcccacc
cagcccctca 15420 agtagctagg actacaggtt tgcaccacca tacctggcta
attaaaaaat ttttttttgt 15480 gcaggctaga tctcacagtg ttgcccaggc
tggtctcaaa ctcctggact caagtgatct 15540 cccaccttgg cctcccaaag
ttctgggatt acatgtgtga gccattgcat ccagcctgtt 15600 gtcttttaaa
tttacacatt atcccacttg agttcctcat tgcagtgttc caagcatcat 15660
ttctcatatt tcaaagttaa ttttgttttg cttctctttc tgaagttcta ttttaggctc
15720 ccctcacccc gatacttccc ctgaagattt atttttagtt ttccttttcc
ttttcgggca 15780 aggatgtgca gaggccatgc tgaggtcttg cagccctggg
agacttttgg gttgtagctg 15840 cctatagctg ccgagtagcc ccagggagta
gtggaagggc agatcccatc tggccagaat 15900 catgggcact gcctgtcccc
aaagatgcca taagctttta gacagcggct tcaggctttt 15960 ctcccaggta
aggggttgaa cccctaacga tggaaaggaa attaagctgg gcattaccta 16020
ttttaaaact gtttacacac aggtgcctca cagcattttt tgttcaggcc gctgccatcc
16080 atggagcagg tagatagaag tgcagagtgc ccaggctaga gggatgggac
agggacagtg 16140 cagggaggga gctgagcccc cttccagcgg gggcagcaga
ggggaaagcc atgggagggg 16200 ctgcaggatg tgtcctgagc tgaagcttat
caacaagtaa tgagtaccag ctgggcattg 16260 tggtgcacgc ctgtggtccc
aactacttgg gagactgagg caggaggatc gcctgacccc 16320 aggagttcaa
gtctagcctg ggcaatgtaa gaccctgtct ctaaaaaaat aataataaaa 16380
taagtaacaa ttacctgtgt aactgtgacg aggcagggtt tgaacattgc cgctgggagg
16440 ttggcagatg gtgggaagca gggtggaggg ctgctggttt ggagcagagg
atacagattg 16500 catggggtca agctagaaat tgcgtggcag atgtgaagag
ctggccccac tgcgggcagt 16560 aggtgtctgg tggccagtcc cagaggctgt
gaagaggggc tcagccatct gtctagtagg 16620 gcttccttgg aggttccacg
atacaggcag atggtggtgg cccgggcagc caggtggtgg 16680 ctgggatgaa
gagggttggc aggtcccaga ggcagcccct tccccttttg gctgtgtgtg 16740
cagcagggcc gtggaggctg cttttagtcc aggtagacca gggccacgct gaggtcccag
16800 tgggctgagc tggtgactga tgagttggtc ctcaggggtg aggctggtgg
gaagtgatgt 16860 cactgtcccg ccgatggcca gctaagggac tgggttagga
tcagccccct cttgtccttc 16920 actctcccat ccttggccag gagaagagga
acaggtcttt ctgaggacct gcttgtagac 16980 ctttgggtag gaggggactt
cccaggttct ctgttgaggc cactctatct aaaatagcac 17040 cccagtgagt
ctcctatcac tgtatcctaa cattattttc tccatggccc tcatcattac 17100
ctgctgatat actgtatgtt tgtctatatg tcatctaaca cccctcacac tggaacacaa
17160 tgcccgtggg cagagacttt gctagccttg gttccagagc ctagaacagt
gcctggcaag 17220 taggagacac ccagcattac ctttctaagt gaaccagtag
agatgggggg agaccgcaag 17280 gctatgccgg cagacctgag ggagtcctgt
ctgcatgcgc tgcaggatga cctgagggga 17340 actccttgga cttctgtgcc
ctctttatct gtaaggtggc cacctgatcc cttccagcgt 17400 aggcatgaag
tagcctaatg aagagcattc aggcttgggt atcagtctca ggatcctggg 17460
ggccttagaa tttgtggcgc ttggggacac cttgtgatcg tgcaatttct gttgtctagt
17520 tcatccatgg ctggttgatc atgtcttcac tgatgctgct gttcctcttc
acctatatct 17580 accttgggta agtgacagat aagcagcagg gtccctggga
gcccctctcc atgtggcaca 17640 agtggacatg ggcatgagga cctgggcggg
gaaagatgac catcgagctc cagtcttccc 17700 cagtgccagc cgttttggga
acccaggcct ccgtcgccct ctctcatggc cttgacacag 17760 gggagtggaa
gtggggctgc atggtggacc acatgtttct gtctcgttcc tgatttaaaa 17820
tgaacccttc atggagaagg ctctctgtga accccagggg gatagaaacc ccccaaaatt
17880 tacattctga tttttaggct aggcctgggt actttctggt ttgtgggaaa
aattatctgt 17940 tctatcgccc cttgatttgg gatatcagcc tgacccaggg
gcccaaagag actgggagga 18000 caagagaaaa cactttccca aggacctttc
catgtgcaca gggtcttcca ggtcatgccc 18060 atgcacattt ctgtgatctg
ttccaagcat ccccaccttg ttttagaaaa tgctgcaaat 18120 ggtaaattgt
aaggacagtg aaggtcgggg aaggaaatgt tagtaaagag ggccaggttg 18180
ggactgaatg gtggtaaact gctaggctgt aatgcctcca ctgagtccca gtcacaggct
18240 ccaccttggt cctgcaggga agtgctcaag acctacaatg tggccatgga
ctaccccacc 18300 ctcttgctga ctgtctggaa cttcggggca gtgggcatgg
tgtgcatcca ctggaagggc 18360 cctctggtgc tgcagcaggc ctacctcatc
atgatcagtg cgctcatggc cctagtgttc 18420 atcaagtacc tcccagagtg
gtccgcgtgg gtcatcctgg gcgccatctc tgtgtatggt 18480 aggtgggcag
caaggctggt gggggcagtg ggggcgatgt ccagggccaa atcgtcccca 18540
gtgctgcaca aggagggcag gtgctgaagg gcttgcatcc ctttctgcag aggcctgggt
18600 gggatccctc ctgagagagt cgcctttgta aaacagaggg gggtccacta
tttctggaac 18660 actcctggtg gtctagataa aacgcagtag tcactgagct
cctcatttac tttttttttt 18720 tttgagatgg agtcttgctc tgtcgcccag
gctggagtgt agtggcgcca tcttggctga 18780 ctgcaacctc cgcctcccgg
gttcaagtga ttctcctgcc tcagcctcct gagtagatag 18840 gattataggc
atgtgccacc acgctgggct aatttttgta tttttagtag agatggggtt 18900
tcaccatgtt ggccaggctg atctcgaact cctgaccttg tgatcggccc gcctcagcct
18960 cccaaagtac tgggattaca ggcatgagcc actacaccca gcctcatttt
ccattattac 19020 tgctatgctg attgagcaag tgcactgtta agcactggac
acgctgtaag tgatttgttc 19080 atcaagacag tcctttgggt accatgcata
tacataaccc caaatgttag ctgctatttg 19140 atattagcat gattatcatt
gccagtattg ttacttccat tttaaggtta aagaattgga 19200 ggctcagaga
agtgggactc cccagcctgg ccaccgcgtc tcgggtgcac agctcctcca 19260
tgcttgcagt tgcctgcgag gccctactct ggctcacacc agggcctgct ctaagttgtg
19320 actggagaat gagaatttgg gatgccagcc cagaggcaag gcatgctctg
agagctccac 19380 ccggggctcc tgtgctacag ggcaggctct tcttcagggg
gctgcccggg gatagtttga 19440 caaggatgtc tctgtcttcc tagatctcgt
ggctgtgctg tgtcccaaag ggcctctgag 19500 aatgctggta gaaactgccc
aggagagaaa tgagcccata ttccctgccc tgatatactc 19560 atgtgagtga
gccccccgtg cctctgcctg actcggggtc agcaggcagc ctgtgggggg 19620
acaggggcct gcttcctggc cgtggctttc agagttgact gggcgatccc aggagggtct
19680 ccactttcag aagccaggga gggcagtatc ttgttattac acagtaagaa
gcttagaaag 19740 ttaggacagg aagcaggcat ctgctgggat gtgctgcagt
ccctgacttc atcccgtcca 19800 tcctccagcg gcatgctgcg gtgcaggttg
cattcctgtg atcccgcagc cacccctcag 19860 ctctccaggc tcttgagaag
ggactttgga gagggattct tcagggcagg gggtcgggga 19920 gcaaggagct
tctgggcttc cttgacagca gcgtggctga ttggcattaa tcctaactga 19980
agggaaggca cacgggatgg cccctggcct cggggtcaat gtgtagagat ttggacttac
20040 acatgcagtc aacaaaggca catcaagtcc ccattttgtg acaggcactg
tgctaggcat 20100 tgggggaccc agcaggaaag aagaccacag ggtcccaggc
ctcatggagc tcacggccct 20160 gtgattgtga tgccctcggt ctgttgatgg
cggggcttaa atagcctgaa tttctggagc 20220 tctggcgtct gcaaggtggc
ctgggaaaga gtttatggaa cagctacaga gttctaggta 20280 ccttcatgca
gttgaggatt cgagcccgta gaggagaatc gcctgcagcg tggccccacg 20340
ggaaagcaca ttccaggcgc attccgagga tgagcggaga ccatgtatgg aaaggtagtg
20400 ccaggactgt catgagtgtc ccagggctcg ggggattcac ccgtgaactg
tgaggtcttg 20460 gctctgatag acctggttct tatgctttag gaggggagac
aaacagtaac agaatagaca 20520 aatgcaagag agagtgactc tggacccctc
ccacaacggc ctcctaacaa tggagcatga 20580 gcagatacct gcaggatgga
gggtcctgtg caggctttct gggacgcaga ctggccacct 20640 cccccaggcc
ctgcaggcag ccactgttag caccgcctga gatgtgaacc ttttctcctc 20700
ccccagctgc catggtgtgg acggttggca tggcgaagct ggacccctcc tctcagggtg
20760 ccctccagct cccctacgac ccggagatgg gtgagtatct tggggagcta
acagcctctc 20820 atcactgggg ggcagctccc tacctgcacc cagctctgct
cggcctggct tccctgagag 20880 gcatgagttc aggaggggca gagggaaagg
tccgttgaaa accagccgga cacatgcggc 20940 ttgaagattc agcaagtgtt
ggaccctcgg tcctctgcca gcctctgttg catcgttctg 21000 ctgggcgtgg
gtgggtggag tgggggaagc cctggtgtca ggtgctggtg ctcaggggga 21060
ccccttcttg gagctttgtt ccctggtaac actctgacca gctgttgttt ctctctcttg
21120 ttgtcccctc ctcacggtga tgacggacat cttctcttcc tggacaccca
gaagaagact 21180 cctatgacag ttttggggag ccttcatacc ccgaagtctt
tgagcctccc ttgactggct 21240 acccagggga ggagctggag gaagaggagg
aaagtaaggt gcccatgttc acacggcctg 21300 cttcagccta cggcgggagc
ggagacagag ggtggaggct ccctgcagcc tgggtggagg 21360 agggcatgag
gggaggggcc ccttttccca tcagaggcat ctctgtgaaa gtagaagatg 21420
cctgcagcgc tggggtcttc tcagcaggcc ccatgtagtt gtccggcatg tattgagtat
21480 gggccacgtg cccgtgctgt gctgggtgag gcccagccct ggtgggaccc
acaggctaag 21540 gagacacggg cagtaatcac atagactgag aagccaagga
ctatgaaggg ggccatgggg 21600 ttggggaggg gcggcaggag agcatgccac
ggggcttctt gacctggttg gcaggggtga 21660 gagaaagtca gctgaggaag
taactgctga gctgagctct gaaggttgag tcacagcagt 21720 cactagagga
gaggagcaca gggtggggag catttcctga cagacagact caggaatcag 21780
aggaagccgg ggcgggatgc agagagcaga agtgtgggag agccttgcaa acaggcctgg
21840 agacatgcga agataggagt tcatcctggc gtcagtacac ggtgcctgcc
taacacccaa 21900 tgccagccca ctgctgcgtg ccaggcagca ccctggagca
gggagatgct gcactgtcgt 21960 aacagcccct gccttgagag gtgccttacg
ggagcagcct ggtgacagtg gcttggcata 22020 caggactcca gtgacacggg
aggggcaagc tagggaaaga tcactctgcg gtgggtctgg 22080
aaggaggagc aggtgcgcac cctccaggca ggcttggggg aggtatttat tccaaggcca
22140 actggtgtgc tgcagaccag gagttagcac agatcccacg gggcccgcag
gactggcctc 22200 cctccagaca ccagccacaa gctctagagg gtctagatgc
cacttgtgct tctgaccggc 22260 tgcaaattta gggctcccat gaccccctta
ggttcaataa cttgctagaa tgactcacag 22320 aactcaggaa agcactacac
ttaaaattgc agtttgtttt ttgttgtcgt tttgttttgg 22380 agacagggtc
tcgctctgtt gcccaggctg gagtgcagta gcacgattgt ggctcactgc 22440
aaccttgact tcctgggctc aagtgatcct cccaccttgg cctcctgagt agctgggatt
22500 acaggcacgt gctaccacac ctggctcatt tatattttta gtagagacaa
ggttttgact 22560 tgttgcccgg gctggtttcg aactcctggg ctcaagtgat
ccacctgcct tggccttcca 22620 aagtactggg attataggtc tgagccacag
cacccggcca aaattagttt tattataaga 22680 gatgcaactc aggaccagcc
aaatgaagag acagtgaaga agtaatgctg atggatcaca 22740 cctggtgggg
gaaggaggac agctggggcc aggagcagga gggacacctg cagggctgga 22800
agggcagggg aggtgggcct ccatggtttg tgtttattgc ataaccattt ttattgtcta
22860 cagtgagcaa agttatccta taaacaagtg tcagggacca ttgcactaaa
gaaaacaaac 22920 gagagcattt tggaagctct aatttcctga tcagtaatgg
gtagactaat tcccagttat 22980 atttacctgt tgtaaggtga aaggttcttc
agaggacctc tgtcttggtg ttatatgggc 23040 ttttgaatgt actgaaatta
aattccctaa aaatctgtga ttcagacttc atactaaatt 23100 gtacagcagt
gcccagccca aggccttgca tttctatttg ttgttttctt tactctctaa 23160
gtgcccaaca ctggttttac ctgagtttca gaactgcccg cttttctctg cccaggttgt
23220 aagtcaccca gtccacaggt gtcccctgct ttcccactgg ccactgattt
ggggaggcag 23280 ctgtccatgt ccccagtcca catcttagct tctagaggcc
aggtggggtg ggctgggctg 23340 ggcaagagca gctgggcctt ctgggccaga
gtttctcttc tttttccatt ctgtgcacgc 23400 ctcttcagta cgggttactg
tctctcctca cacagggggc gtgaagcttg gcctcgggga 23460 cttcatcttc
tacagtgtgc tggtgggcaa ggcggctgcc acgggcagcg gggactggaa 23520
taccacgctg gcctgcttcg tggccatcct cattgtgagt ggctggggat gcgtccagct
23580 gcctcgtggt gggggccccc agggtcctca ttgtggtggg ggcaggtctc
aggatcccta 23640 gggatttttc atttcttctc ttccctctga gggacaagag
cagggagcgg ggctggaagg 23700 gtcagcttga gaccaaggct cacaggaggt
gtgctcgccc ctaggtgggc tccagcctgt 23760 ggaggacagt gcaggggagg
gtgaggagtg taccggcccc agcgtggctg agcacacagc 23820 ctccaggccg
aggacccagc tgacagcttt gcgcagtgat gataccctcg aggtggttgt 23880
gatgacatca gatttgcaga aaagaaaatt gcttaagggc cttgcccatg ggcgcaaagc
23940 tagtgaggac catgttttcc ccctcctcca tgccattggg acaccacagg
gtctgaatct 24000 ggggcactag gggtggcccc gttactgtga accacagcag
tgaaatgtgg aggccctgta 24060 gtcagttaac gtgaccagat acacataatg
gggagacgtc ctgccgtgac ttcatctcag 24120 agatttcgct gtcacgttag
aggaggagga gcgtctgagc cgtgcgcttg gcatctgccc 24180 cttagtgaaa
accctgggca tggcatgatt aaggttgatg ctccagtgtc cagaaggttt 24240
tctttttgcc cacaagtata tcagggatgg gatggtggac ccaggctcct ccaccaccag
24300 actgccttac ctgagccctg ctggccccaa agatatagaa ggcaccctgg
ttccctgtgc 24360 tcacctggac cactgcctgc atcagctggg tcaggggagg
atgggcagcc cccacacctg 24420 cttcccaggg gcaggttgcc tggcggctct
gattcccttg gtgccagctg ctgagaacct 24480 tactgccatt tcagttgagc
ccacctagct ctcatataaa tacatgttcc ctgagggcat 24540 cttaccatcc
catgtgacca ctccagccag acaggggagg cagcacggcc tcggggcaca 24600
gcactgctcc aggagtcagg aggcctgcct tctggttcac tcactaacag gtgaggtgat
24660 ctaatggggg tgagaacttc tgcccttaac acctcaagag ctgttgcagg
accagggaag 24720 ataatggggt gtctagcgcc gttatccgac tggtcctcga
acaagctcct gtgcccaggg 24780 actagaccat gactcacagc tcctgtccac
accagggatc accacgctca ccctcccctc 24840 catgtcctgc agggcttgtg
tctgaccctc ctgctgcttg ctgtgttcaa gaaggcgctg 24900 cccgccctcc
ccatctccat cacgttcggg ctcatctttt acttctccac ggacaacctg 24960
gtgcggccgt tcatggacac cctggcctcc catcagctct acatctgagg gacatggtgt
25020 gccacaggct gcaagctgca gggaattttc attggatgca gttgtatagt
tttacactct 25080 agtgccatat atttttaaga cttttctttc cttaaaaaat
aaagtacgtg tttacttggt 25140 gaggaggagg cagaaccagc tctttggtgc
cagctgtttc atcaccagac tttggctccc 25200 gctttgggga gcgcctcgct
tcacggacag gaagcacagc aggtttatcc agatgaactg 25260 agaaggtcag
attagggcgg ggagaagagc atccggcatg agggctgaga tgcgcaaaga 25320
gtgtgctcgg gagtggcccc tggcacctgg gtgctctggc tggagaggaa aagccagttc
25380 cctacgagga gtgttcccaa tgctttgtcc atgatgtcct tgttatttta
ttgcctttag 25440 aaactgagtc ctgttcttgt tacggcagtc acactgctgg
gaagtggctt aatagtaata 25500 tcaataaata gatgagtcct gttagaatct
tggagtttgg tccgttgtaa atgttgaccc 25560 ctctccctgc atcttgggca
cccctgggat aacttgtgct gtgagcccag gatggaggca 25620 gtttgccctg
tttgaaggaa cttttaatga tctcgcctct ctgcacacat ttctttaact 25680
agaaagtttc ctaagcaaag gagttaggag agcagggtgg cctgacatct gccagccctg
25740 agctgtaagg ctgtggatgc tgagcaggtc cctggactca gttgtgcacg
gtggcacaga 25800 cactgccagg tggttgccaa aacatccagt ggttccttca
gcaagtgttc accctctgca 25860 gaagcctgtg agggcctgag ctcagaaacc
actctccttt ccttctctgg ctttggccct 25920 gggcactgtg gtgggagagt
ggacagtttg gctttgcctt ctctgtacat caatcatggg 25980 ttgcaaagag
aatctcagaa gtgcctcttc ctgagcacag tggctcacac ctgtaatccc 26040
aatacttcgg gaggtcgagt cggaaggatc acttgagccc aggagtttga gaccagccgg
26100 ggcaacatag tgagactttg tacaaaaaaa aatttaaaaa ttagccaagc
atggtggcat 26160 gcatctgtag tctcagctac tctggaggct gaggtgggag
gatcacttaa gcccaggagg 26220 ctgaggctgc aatgagccga gatcaagcag
gtgttaggta tatcagacag ctgagaagac 26280 gcaagtgtgc cctggggttc
aaactggtac ccctgtctcc ctgttccagg aataacatga 26340 gtgccgggac
aatgcatctt tattatgaga ggaatgagaa ttgtgtatct tgacatttga 26400
caggagcttg ctttccccca ggctgtttga ggaagggcag aggaaaatgt ggtgccctaa
26460 gaaggaagga cagaggaggc cgaacactgg cgggtggaat cccactgatt
agtagtgcag 26520 gtcagagacc tgggatgggg ggcattgccg tcatggaagc
cacagcgggg agcgggtaaa 26580 gcagacaggg atggtccctg atggtgacaa
ctcgcaagag gttaagggga aagaaaaact 26640 gaaaagctta ttcaatttgg
caattatggc agtgtttatc ttcagaagag cagttttagg 26700 gtggggtttc
caaagatggg attggacata tattttgaat cattaagctt gaggtctttc 26760
aaaggcctgg ccaagggttg ctgggtggag accacattca gaggtaaagg cagaaattgg
26820 gggcccttaa gtagacagcg agggaggaag aaatgaaggg gcctggtgat
ggttagggtg 26880 aagtgttaag actgagaaaa caaggacatg tgagaagacg
agggaagagc attggagaga 26940 acaaagacac tggaggagat gctacttgga
ggtccccaga gagcagggag acaaatgaac 27000 ccagaacaca aatggcaaag
aagaaaaatg agagaatttg taaaagacag cattcgaaca 27060 tgccgaacaa
gagcagggta ctggtgttca aacacctgta tctcccccgt gtaacccgtc 27120
aactaatatc tttccatatt tgctccagat ttgtctttag aaataaaacc cacgttctga
27180 agtcctgttt gtatgtggcc ccagtcctgt tgcctccgcc tcctgtcctg
aagtcgattt 27240 ctgcccttct catctatggt tagttttgtt ttgtatgttg
gcatgttttc ttaactttac 27300 agaaatggta tcatactgta catatttgat
aattttttaa aatattgcat tctggaggca 27360 tgtataaatg tagctccagt
tcatttattt tatttatttt ttgagatgga gttttgctct 27420 tgtcacccag
gctagagtgc aatggcgtga tgttggctca ctgcaacctc tgcctcctgg 27480
gttcaagcaa ttctcctgtc tcaatttcct gagtagctgg gattacagtt gcccgccacc
27540 atgcctggct aattttgtat tttagtagag acggggtttc accacgttag
ccaggctggt 27600 ctcaaactcc tgactgcagg tgatccacgc accttggcct
ccaaaagtgc tgggattaca 27660 ggcgtgagcc accgtgccca gcccagttat
tttaactatt gtatagtgtt ccattgtatg 27720 agttctactg tttatatgct
attgatcgac ctgtaggggt tttgcagtgt ttctgtatta 27780 cagctgtgct
gcagtgagca tcccatcaca ttgtgtggat ttgaggaagt attggaattc 27840
ccccaattga ctggacattc ccaattaccc tccaagtatg tgtctgttta tccttccatc
27900 cgcaatctga gagttcccca actctataat acttggtgtc atcagacttt
tcatcttgtc 27960 tgattggatg ggtgtcattt cctttaggtt ttataattat
cttttcatat gtgtattggc 28020 tgtacaaggt tccttctctg ttcattatta
ttaatttttt tagacagagt ctcgcgctgt 28080 cgcccaggct ggagtgcagc
agcgtgatct tggctcactg caagctccgc ctcccgggtt 28140 catgccattc
tcctgcctca gcctcctgag tagctgggat tacaggtgcc tgccatcacg 28200
cccggctagt ttttttgtat tttgagtaga gatggggttt caccgtgtta gccaggaggg
28260 tctcgatctc ctgacctcgt gatccacccg cctcggcctc ccaaagtgct
gggattacag 28320 gtgtgagtca ctgcgcccag cccaagtttc cttctctgtt
acttgttcat atcctctgcc 28380 catttttcac ttggattttt tgtcttacgg
atatttaagc ctcttaaaat atatattctg 28440 gagagatgct aatctttgat
taattatatg cattgcaaat gtctggtaca ttgtggcttg 28500 cctctcttcc
ctgcctttag gagtgttttg ctggacccaa gtaattttta aatgttaatg 28560
ttattaaatc tatcagtttt ttgcttgtat ggcttatgcc attgaatctt gttttaagag
28620 atccttccct accctcaagg ttttctaaat ttttattttc ataataagat
ttttagttca 28680 tctgaaatgt atttttatga ttgtatttag tagggaccta
attttgtttt tctttgtaac 28740 caggtgtccc agcactgttt actgaacagt
ctctcctttc tcgctggtct gtagaactct 28800 cctgacatat accaagtttc
cataagtggg tggatgggtt cctgagctct ctactgttaa 28860 tagaacttgc
tctctcgcag gccaatgcct caccaggtga ttgaagcaga gaaacttagg 28920
tggtgaaagg agaagatggg gcctgtcctg agagtttctg ttcctgagat gctagaggca
28980 gagggtatgt aaatctgaag ttacactgga tctcctaaaa cagtataaag
ctacagaagt 29040 ataatagtgt ggaatggtgg tgggagtcag taagggttag
gtcactgcag tggtttaaac 29100 aagatgggct agaatccttt cacaggcaca
ggcagcttgg agagggtgca atagtgcatg 29160 gtatcagggg tcagatgcct
ctttttcctt tgagatcagt aagtggcttt cacctcatga 29220 cctaggctgg
ctgctgtgtg ctagccgtca agtcacactc catccagcat gaaaggaggt 29280
tagaaaaggg tgcatttcct cttcttaaaa acatgtctca aagttgcaca cagcactttt
29340 gcctatattc aattggccat tagtcccacg gccatacctg tctgagactg
agagactggg 29400 aaatgtcttt atttcaagtg gccatatatc cacctaaaca
agataaggga tacgtggtta 29460 tggcgtgtct tttggtttac caatgcagat
aatgaagtta ccaaaacaat gagaaaatgg 29520 ggtcgtgagg gatcatgtga
atcacaagct gatgtcttca aagacggtgg aaatgggccc 29580 cgggaggcag
cagatgacag cagtggggat taaggtagac ctccatcctg gggttaaaat 29640
gaggggaagg tgatggagct ggaccagcag tcagaatggt cagtggttag gagaccctct
29700 gccccccacc gctgccacca ttggctctct acagaatgcc tgcgagtggc
ttagagtgac 29760 caaggatgag gtgcagatcc atgtgcaccc ccctgccccc
tctgtggaca attttcatgc 29820 ctgacagcac agtctatgtg gattgcaagc
cgatgaaact atgcaaagta gaagcatgcc 29880 tgcagtttgt gattcggtga
tgtgttttat gcttatgtga gtcgaatggg gcggcagggt 29940 cctgtggtca
cccgctgaga aggaagggtc ctgtaaccac tgcctttctt tcagctactt 30000
gagaaaggtg ttgtgaggga ccgtggattt tgggacagct ttgaatggtg gtagggagga
30060 agggtccggt ctgagtgaat ggccagaaag ctgtggggaa gcttttagga
cattggccaa 30120 gagctccctg aaggcagcca gggagatact tgtcagtaca
tgtgactaat ggccaactga 30180 atataagcag aagtgctgtg ttgctgtgtg
caacactgga caccttagga aggacctcga 30240 gacagtggtt gtggactctg
tagagagtaa cagtgacagt agcaaaccct tacccagtgc 30300 caaccttgtg
ctaggctcgc actaaatgag tttaccttca attctcgtaa caataggagg 30360
taactactat tctaatttcc attttataga tgaggaaact aaggcacaga gatcactgac
30420 ttgcccaaaa tcaagcaggg agtagttagt atataagccc acggtatgtg
gtttgtagaa 30480 taggtgctct tgactagcag aaataggtcc tccctgcagt
gtgtaattga taacaagcat 30540 gggctgccat cttcctgtcg aggccactca
aaacacccaa caggctacgc acggtggctc 30600 acacctgtaa tcccagcact
gtgggaggcc gaggtgggcg ggtcacctga ggtcaggagt 30660 tcgagaccag
cctggccgac atggtgaaac tccgtctcta ctaacagtac aaaaattagc 30720
tgggcgtggt ggcgggcacc tgtaatccca gctactcagg aggctgagac agaagaatca
30780 cttgaaccag ggaggcagag gttgcagtga gacaagatca cgccattgca
ctccagcctg 30840 tgtgacaaaa gcgaaactgt ctcaaaaaaa aaaaaaaaaa
aagtatgatt ttataatccc 30900 agcaccttgg gaggctgagt cgtgagaatc
acttgagccc aggagtttaa gaccaatcta 30960 ggcaacatgg caagacccca
tctctgccaa aaataaaaaa tagtctaatt ttagctattc 31020 atgtgtgtgt
gaagtggtgt ctcttcgtgg ctttgatctg catttcccta atgctgacta 31080
atgacgttgg gcacctgttc atgtgcttac tggtcagata tctttctttt gttacatttt
31140 attaagtttt aaaatttaaa gtcaaagatt tccctatgag aatgactttt
aaaatgacca 31200 aaaaggggaa gataacatta attcttgaag agaaggcctc
tgagaaaaat acagttgtag 31260 caagctgcta ctttgcaaat gacccatgca
ttttaatttt cccctaagga aggccaagga 31320 agagtcttat cacctcaggg
caggagatgt agggacttgg gtcatttaat aagagtggta 31380 ggtttgaaaa
ctcaaaccca gaagactcct tagagtttct cccaggaggt agggaagggg 31440
ccgcatccat ggagagagga ggatgtgact tagagcagtg gtccccaatc tttagggacc
31500 agggactggt gtcatggtag acagtttttc cacagatagg ggttgggggg
atgatttgga 31560 gctgaaactg ctccacctca ggtcatcagg cattagattc
tcatgtggag tgtgccactt 31620 agatccttgg cgtgcacagt tcacaatggg
gttcgaggtc ctatgagaat ccgatgccac 31680 tgatttgaca ggaggcggag
ctcaggtggt aatgctcatc tccaccgctt accacctgct 31740 gtgcagcctg
gttcctaata ggctatagac tggtactggt ccatggcctg ggggttgggg 31800
acccctgatt tagaggaagt aagggcatgg cttaccgtgg gccctggggt gttctgggaa
31860 tggggaggat ggagagaaga gaggaggtag ggaagacctc cccttgctcc
ccatttggga 31920 tttggggaga aagtcaggtc tcaggctcaa cagtacctga
tcctgtacca tcttccaaag 31980 ggaagtcagt ggggttggaa ggtaggcagg
ggttatcttc tctgagccac ggcacaagac 32040 agaagtttcc caccattcct
gagggggcag gtggtaggtc cccaagcaga gagccagcag 32100 tccctctctg
aggcctgcaa tggaatgggg tggggtgtcc actgagccaa gggtctgtca 32160
gtgagagctg gggaggctgg gctggcttgc aagcacctgt tataaccaaa ccaggaaatc
32220 aggttccgag tcttgccagc aagggcctac agctgccagc agagatggac
agccaggaga 32280 ccccaattgg ccacccagag ccaccctcct ctgcctaccc
caccctccag tactccagag 32340 cctactcgga ggggaacaga aacctgagag
gctgaacaca cacacatgga gaaacaaacg 32400 tagtaaaata tttggggaat
caggaagaat tatttgtact attcctgcaa cctttctata 32460 ggcttgaaat
tatcaaaata aatttttaaa aattgtaata acattctcat actaaaacac 32520
tgagtttttt tctttcattt tttgattttt tctttttgac tccagcatga cttactctaa
32580 caatgggtgg tctcgatttt gaaatacttt cttctccaag cctttcatga
caccctgtct 32640 ctgttggttc tgaaaatgtt ggattttgtc tcagcccttg
cttctggaaa cagccaaggt 32700 taagaaaacc ccccatgctt tgtgttctag
cagacagctt cctgcaaaga gccatcttcc 32760 cagagcactt aggcctctta
gatgtctccc ttgtttaatt atgacaagag cacacacaca 32820 gaccctccaa
attcccattc ttagtcttct aaatgattag ctgagctgct tttccccact 32880
gattaatcgg aataaaatgc tcattaacca aacttccctc ctttccccag gtccctaaac
32940 tttcctgagt cggcagacat cccctctgga gaagaggttg gccccagagt
cgaacatcct 33000 ctgatctacc tgatcctgct gcccttccat tccacttccc
cacatctgtt ctttctggtc 33060 gtgtttactc ccctattaaa aaaacaaaac
cagaaaacgt gtttgcctag atcttgagac 33120 tctggaagat cttaacagtc
agaggttccc cctatttgca atgatctcct ttcctgcccc 33180 ttcctatcct
tgcaataatc cttttgaata aagtctctcc ttactaaatc cagttcctaa 33240
aaattaattt ttttagag 33258 <210> SEQ ID NO 2 <211>
LENGTH: 448 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: misc_feature
<223> OTHER INFORMATION: presenilin-2 isoform 1 <400>
SEQUENCE: 2 Met Leu Thr Phe Met Ala Ser Asp Ser Glu Glu Glu Val Cys
Asp Glu 1 5 10 15 Arg Thr Ser Leu Met Ser Ala Glu Ser Pro Thr Pro
Arg Ser Cys Gln 20 25 30 Glu Gly Arg Gln Gly Pro Glu Asp Gly Glu
Asn Thr Ala Gln Trp Arg 35 40 45 Ser Gln Glu Asn Glu Glu Asp Gly
Glu Glu Asp Pro Asp Arg Tyr Val 50 55 60 Cys Ser Gly Val Pro Gly
Arg Pro Pro Gly Leu Glu Glu Glu Leu Thr 65 70 75 80 Leu Lys Tyr Gly
Ala Lys His Val Ile Met Leu Phe Val Pro Val Thr 85 90 95 Leu Cys
Met Ile Val Val Val Ala Thr Ile Lys Ser Val Arg Phe Tyr 100 105 110
Thr Glu Lys Asn Gly Gln Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr 115
120 125 Pro Ser Val Gly Gln Arg Leu Leu Asn Ser Val Leu Asn Thr Leu
Ile 130 135 140 Met Ile Ser Val Ile Val Val Met Thr Ile Phe Leu Val
Val Leu Tyr 145 150 155 160 Lys Tyr Arg Cys Tyr Lys Phe Ile His Gly
Trp Leu Ile Met Ser Ser 165 170 175 Leu Met Leu Leu Phe Leu Phe Thr
Tyr Ile Tyr Leu Gly Glu Val Leu 180 185 190 Lys Thr Tyr Asn Val Ala
Met Asp Tyr Pro Thr Leu Leu Leu Thr Val 195 200 205 Trp Asn Phe Gly
Ala Val Gly Met Val Cys Ile His Trp Lys Gly Pro 210 215 220 Leu Val
Leu Gln Gln Ala Tyr Leu Ile Met Ile Ser Ala Leu Met Ala 225 230 235
240 Leu Val Phe Ile Lys Tyr Leu Pro Glu Trp Ser Ala Trp Val Ile Leu
245 250 255 Gly Ala Ile Ser Val Tyr Asp Leu Val Ala Val Leu Cys Pro
Lys Gly 260 265 270 Pro Leu Arg Met Leu Val Glu Thr Ala Gln Glu Arg
Asn Glu Pro Ile 275 280 285 Phe Pro Ala Leu Ile Tyr Ser Ser Ala Met
Val Trp Thr Val Gly Met 290 295 300 Ala Lys Leu Asp Pro Ser Ser Gln
Gly Ala Leu Gln Leu Pro Tyr Asp 305 310 315 320 Pro Glu Met Glu Glu
Asp Ser Tyr Asp Ser Phe Gly Glu Pro Ser Tyr 325 330 335 Pro Glu Val
Phe Glu Pro Pro Leu Thr Gly Tyr Pro Gly Glu Glu Leu 340 345 350 Glu
Glu Glu Glu Glu Arg Gly Val Lys Leu Gly Leu Gly Asp Phe Ile 355 360
365 Phe Tyr Ser Val Leu Val Gly Lys Ala Ala Ala Thr Gly Ser Gly Asp
370 375 380 Trp Asn Thr Thr Leu Ala Cys Phe Val Ala Ile Leu Ile Gly
Leu Cys 385 390 395 400 Leu Thr Leu Leu Leu Leu Ala Val Phe Lys Lys
Ala Leu Pro Ala Leu 405 410 415 Pro Ile Ser Ile Thr Phe Gly Leu Ile
Phe Tyr Phe Ser Thr Asp Asn 420 425 430 Leu Val Arg Pro Phe Met Asp
Thr Leu Ala Ser His Gln Leu Tyr Ile 435 440 445 <210> SEQ ID
NO 3 <211> LENGTH: 19 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <223> OTHER INFORMATION: Forward 5'-3' Primer
<400> SEQUENCE: 3 catcagccct ttgccttct 19 <210> SEQ ID
NO 4 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <223> OTHER INFORMATION: Reverse 3'-5' Primer
<400> SEQUENCE: 4 ctcaccttgt agcagcggta 20 <210> SEQ ID
NO 5 <211> LENGTH: 26 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature
<223> OTHER INFORMATION: Forward 5'-3' Primer <400>
SEQUENCE: 5 acagaattcg ccccggcctg gtacac 26 <210> SEQ ID NO 6
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <223> OTHER INFORMATION: Reverse 3'-5' Primer
<400> SEQUENCE: 6 taagcttggc acggctgtcc aagga 25 <210>
SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: misc_feature <223> OTHER INFORMATION: Forward 5'-3'
Primer <400> SEQUENCE: 7 tcagcatcta cacgccattc 20 <210>
SEQ ID NO 8 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: misc_feature <223> OTHER INFORMATION: Reverse 3'-5'
Primer <400> SEQUENCE: 8 agcaccacca agaagatggt 20 <210>
SEQ ID NO 9 <211> LENGTH: 26 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: misc_feature <223> OTHER INFORMATION: Forwad 5'-3'
Primer <400> SEQUENCE: 9 attcgccccg gcctggtaca ctgcca 26
<210> SEQ ID NO 10 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Reverse 3'-5' Primer <400> SEQUENCE: 10 ctgtccaagg agctgcaggc
ggcgcag 27 <210> SEQ ID NO 11 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: g1N141I guide RNA F <400> SEQUENCE: 11
caccgcatca tgatcagcgt catcg 25 <210> SEQ ID NO 12 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: g1N141I guide RNA R <400> SEQUENCE: 12
aaaccgatga cgctgatcat gatgc 25 <210> SEQ ID NO 13 <211>
LENGTH: 100 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: misc_feature
<223> OTHER INFORMATION: Donor ssODN#A N141I <400>
SEQUENCE: 13 gagagaagcg tggctggagg gcagggccag ggcctcacct tgtagcagcg
gtacttgtag 60 agcaccacca agaagatggt cagggtgttc agcacggagt 100
<210> SEQ ID NO 14 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Forward 5' Primer <400> SEQUENCE: 14 taacggcggc agacaaaaag a
21 <210> SEQ ID NO 15 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Reverse 5'-3' Primer <400> SEQUENCE: 15 gaagtattgc ttcagttggc
ct 22 <210> SEQ ID NO 16 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Forward 5' Primer <400> SEQUENCE: 16 agaagaacgg caagtacgag a
21 <210> SEQ ID NO 17 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Reverse 5'-3' Primer <400> SEQUENCE: 17 tgttgaggga cagattgtgg
c 21 <210> SEQ ID NO 18 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Forward 5' Primer <400> SEQUENCE: 18 taacggcggc agacaaaaag a
21 <210> SEQ ID NO 19 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Reverse 5'-3' Primer <400> SEQUENCE: 19 gaagtattgc ttcagttggc
ct 22 <210> SEQ ID NO 20 <400> SEQUENCE: 20 000
<210> SEQ ID NO 21 <400> SEQUENCE: 21 000 <210>
SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: misc_feature <223> OTHER INFORMATION: Forward 5'
Primer <400> SEQUENCE: 22 acgaatctcc gaccaccact 20
<210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Reverse 5'-3' Primer <400> SEQUENCE: 23 ccatggccac aacaactgac
20 <210> SEQ ID NO 24 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Forward 5' Primer <400> SEQUENCE: 24 gaagtgtccc aggacatgat aa
22 <210> SEQ ID NO 25 <211> LENGTH: 22 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
Reverse 5'-3' Primer
<400> SEQUENCE: 25 ctcttgagta gctgggattg ag 22 <210>
SEQ ID NO 26 <211> LENGTH: 46 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: misc_feature <223> OTHER INFORMATION: Forward 5'-3'
Primer <400> SEQUENCE: 26 ctccgtgctg atcaccctca tcatgatcag
cgtcatcggt tatgac 46 <210> SEQ ID NO 27 <211> LENGTH:
48 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Reverse 3'-5' Primer <400> SEQUENCE: 27
gaggcacgac tagtgggagt agtactagtc gcagtagcac caatactg 48 <210>
SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: misc_feature <223> OTHER INFORMATION: Forward 5'-3'
Primer <400> SEQUENCE: 28 catcatgatc agcgtcatcg 20
<210> SEQ ID NO 29 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
g1N2411 guide RNA F <400> SEQUENCE: 29 caccgcatca tgatcagcgt
catcg 25 <210> SEQ ID NO 30 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: g1N2411 guide RNA R <400> SEQUENCE: 30
aaaccgatga cgctgatcat gatgc 25 <210> SEQ ID NO 31 <211>
LENGTH: 118 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: misc_feature
<223> OTHER INFORMATION: Donor ssODN#A N141I <400>
SEQUENCE: 31 gagagaagcg tggctggagg gcagggccag ggcctcacct tgtagcagcg
gtacttgtag 60 agcaccacca agaagatggt cataaccacg atgacgctga
tcatgatgag ggtgttca 118
* * * * *