U.S. patent application number 14/873296 was filed with the patent office on 2016-02-11 for conditionally immortalized long-term stem cells and methods of making and using such cells.
The applicant listed for this patent is National Jewish Health, THE REGENTS OF THE UNIVERSITY OF COLORADO. Invention is credited to John C. CAMBIER, Sara Ann JOHNSON, Yosef REFAELI, Brian Curtis Turner.
Application Number | 20160040129 14/873296 |
Document ID | / |
Family ID | 37963171 |
Filed Date | 2016-02-11 |
United States Patent
Application |
20160040129 |
Kind Code |
A1 |
CAMBIER; John C. ; et
al. |
February 11, 2016 |
CONDITIONALLY IMMORTALIZED LONG-TERM STEM CELLS AND METHODS OF
MAKING AND USING SUCH CELLS
Abstract
Disclosed are methods for conditionally immortalizing stem
cells, including adult and embryonic stem cells, the cells produced
by such methods, therapeutic and laboratory or research methods of
using such cells, and methods to identify compounds related to cell
differentiation and development or to treat diseases, using such
cells. A mouse model of acute myeloid leukemia (AML) and cells and
methods related to such mouse model are also described.
Inventors: |
CAMBIER; John C.; (Denver,
CO) ; REFAELI; Yosef; (Denver, CO) ; JOHNSON;
Sara Ann; (Denver, CO) ; Turner; Brian Curtis;
(Denver, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National Jewish Health
THE REGENTS OF THE UNIVERSITY OF COLORADO |
Denver
Boulder |
CO
CO |
US
US |
|
|
Family ID: |
37963171 |
Appl. No.: |
14/873296 |
Filed: |
October 2, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14509870 |
Oct 8, 2014 |
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14873296 |
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11583970 |
Oct 18, 2006 |
8883507 |
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14509870 |
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60728131 |
Oct 18, 2005 |
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60765993 |
Feb 6, 2006 |
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Current U.S.
Class: |
435/366 ;
435/375; 530/350 |
Current CPC
Class: |
A61P 37/06 20180101;
C12N 2501/2303 20130101; C12N 2501/145 20130101; A61P 41/00
20180101; C12N 2510/04 20130101; C12N 5/0635 20130101; C12N
2501/105 20130101; C12N 5/0636 20130101; C12N 2501/2306 20130101;
C12N 2501/606 20130101; A61P 7/06 20180101; A61P 35/00 20180101;
C12N 2501/113 20130101; C12N 2740/16322 20130101; A61P 35/02
20180101; C12N 2501/48 20130101; C12N 2501/125 20130101; A61P 37/04
20180101; C07K 2319/50 20130101; A61P 37/00 20180101; C07K 14/4738
20130101; A61P 17/00 20180101; C12N 5/0647 20130101; C07K 14/005
20130101; C12N 5/0646 20130101; C07K 14/4747 20130101; C07K 14/82
20130101; C07K 2319/10 20130101; C12N 15/62 20130101 |
International
Class: |
C12N 5/0789 20060101
C12N005/0789; C07K 14/82 20060101 C07K014/82; C07K 14/005 20060101
C07K014/005; C12N 5/0783 20060101 C12N005/0783; C12N 5/0781
20060101 C12N005/0781 |
Claims
1.-55. (canceled)
56. A fusion protein, comprising: a. a protein transduction domain
of a Tat protein; and b. a MYC protein that is attached to the
protein transduction domain of the Tat protein, wherein the fusion
protein has MYC activity.
57. The fusion protein of claim 56, wherein the Tat protein is
HIV-1 Tat.
58. The fusion protein of claim 56, wherein the MYC protein is
capable of promoting in vitro cell proliferation, cell survival, or
both.
59. The fusion protein of claim 56, wherein the MYC protein
comprises the amino acid sequence of SEQ ID NO: 2.
60. A composition comprising: a. the fusion protein of claim 56;
and b. a fusion protein comprising a Bcl-2 family member that
inhibits apoptosis, and the protein transduction domain of a Tat
protein.
61. The composition of claim 59, wherein the Bcl-2 family member is
Bcl-2.
62. The composition of claim 61, wherein Bcl-2 comprises the amino
acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8.
63. A method for promoting one or more of cell proliferation or
cell survival of one or more cells cultured in vitro comprising:
providing to the one or more cells the fusion protein of claim 56;
and culturing the one or more cells in a media.
64. The method of claim 63, wherein the culturing promotes in vitro
cell proliferation, cell survival, or both; and wherein one or more
of the survival or proliferation of the one or more cells is
increased as compared with corresponding cells not exposed to the
fusion protein or to the composition.
65. The method of claim 63, wherein the one or more cells are
immune cells.
66. The method of claim 65, wherein the one or more immune cells
are activated immune cells.
67. The method of claim 66, wherein the one or more immune cells
are T cells.
68. The method of claim 66, wherein the one or more immune cells
are B cells.
69. The method of claim 66, wherein the one or more immune cells
are NK cells.
70. The method of claim 63, wherein the one or more cells are stem
cells.
71. The method of claim 70, wherein the one or more stem cells are
hematopoietic stem cells.
72. The method of claim 70, wherein the one or more stem cells are
human stem cells.
73. The method of claim 70, wherein the one or more stem cells are
genetically modified.
74. A method for promoting one or more of cell proliferation or
cell survival of one or more cells cultured in vitro comprising:
providing to the one or more cells the composition of claim 60; and
culturing the one or more cells in a media.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/509,870, filed Oct. 8, 2014, which is a continuation of U.S.
application Ser. No. 11/583,970, filed Oct. 18, 2006, now U.S. Pat.
No. 8,883,507, which claims the benefit of U.S. Provisional Patent
Application No. 60/728,131, filed Oct. 18, 2005, and U.S.
Provisional Application No. 60/765,993, filed Feb. 6, 2006. The
entire disclosure of each of U.S. Provisional Patent Application
No. 60/728,131 and U.S. Provisional Patent Application No.
60/765,993, U.S. application Ser. No. 14/509,870, and U.S.
application Ser. No. 11/583,970 is incorporated herein by
reference.
REFERENCE TO SEQUENCE LISTING
[0002] This application contains a Sequence Listing submitted on a
compact disc, in duplicate. Each of the two compact discs, which
are identical to each other pursuant to 37 CFR .sctn.1.52(e)(4),
contains the following file: "2879-117.ST25.txt", having a size in
bytes of 117 KB, recorded on 18 Oct. 2006. The information
contained on the compact disc is hereby incorporated by reference
in its entirety pursuant to 37 CFR .sctn.1.77(b)(4).
FIELD OF THE INVENTION
[0003] The present invention generally relates to conditionally
immortalized long term stem cells, to methods of producing such
cells, and to methods of using such cells, including therapeutic
methods and drug discovery methods.
BACKGROUND OF THE INVENTION
[0004] The ability to manipulate the bone marrow output of various
blood cells has become an important tool in the management of
several diseases. Some of the best new therapies for hematological
malignancies are based on the development of compounds that push
leukemic cells to differentiate into lineages to which they are
committed prior to the transforming event. One such example is the
case of acute promyelocytic leukemia. Upon treatment of patients
with Arsenic Trioxide, the malignant cells are pushed along the
myelomonocytic pathway leading to remission of those tumors.
Another example lies in promotion of successful engraftment of
transplanted bone marrow stem cells (long term reconstituting
hematopoietic stem cells, or lt-HSC) in irradiated individuals. The
appearance of differentiated blood cells can be accelerated by the
systemic administration of cytokines that are known to specifically
induce red blood cell development (erythropoietin, or Epo), or
myeloid cell development (granulocyte-macrophage colony-stimulating
factor, or GM-CSF). Finally, harvesting of lt-HSC from donors has
been greatly simplified by the process of "mobilization" wherein
these cells are induced to move from the bone marrow sites where
they normally reside into peripheral blood by systemic
administration of a cytokine called G-CSF. Stem cells can then by
harvested from peripheral blood obviating the painful and elaborate
collection of bone marrow biopsies. All of these processes rely on
the ability to program and control the biological behavior of
lt-HSC.
[0005] Accordingly, bone marrow (stem cell) transplantation is an
invaluable therapeutic tool for hematologic and immune
reconstitution of individuals who have undergone radiation and/or
chemotherapy (e.g. cancer patients, or have been exposed to
high-level radiation), and is also a critical modality for
treatment of immune deficiency and hematological malignancies. In
addition, bone marrow transplantation would be a highly useful
therapy to combat the negative effects of aging on the immune
system, as well as on other cells and tissues. It is estimated that
stem cell transplantation could benefit more than 35,000 children
and adults per year.
[0006] The operative principle behind bone marrow transplantation
is replacement of radiation sensitive lt-HSC that give rise to all
blood cell types. Recent studies indicate that bone marrow
transplantation may have value in the treatment of heart disease.
Although the basis of this affect is unknown, it, and other
findings, raise the possibility that hematopoietic stem cells
(lt-HSC) may be reprogrammed to give rise to other tissues. If this
is true, lt-HSC may have much broader utility and provide an
alternative to controversial embryonic stem cell therapy.
[0007] The major obstacles confronting clinical application of bone
marrow transplantation lie first in identification of an
appropriately histocompatible marrow donor. This is usually
accomplished using registries that have enrolled more than 6
million potential donors. The selected donor must undergo a
grueling ordeal of induced mobilization stem cell into the blood
followed by 4-5 days of leukapheresis to isolate rare lt-HSC.
Transplantation of these cells must be followed by careful
monitoring and treatment of the recipient to minimize graft versus
host reactions caused by passenger lymphocytes.
[0008] Elucidation of the molecular basis of the impairment in
hematopoietic lineage development has been complicated historically
by the low frequency of relevant cell populations, which prevents
biochemical analysis of signaling and downstream responses. In
fact, this has been a major limiting factor in all studies of
hematopoiesis. In addition, the limited availability of long-term
hematopoietic stem cells (LT-HSCs) has also been a major obstacle
in the treatment of many types of cancer as well as several kinds
of immune deficiencies in humans. To the best of the present
inventors' knowledge, there are currently no available cell lines
that arose spontaneously that resemble lt-HSCs and can
differentiate into normal lineages in vitro, or that can
reconstitute lethally irradiated mice or sublethally irradiated
humans, nor have any methods been described to deliberately
generate such cell lines. Moreover, there are currently no viable
technologies to continuously expand lt-HSCs, such that these cells
need to be obtained from a donor every time they are needed.
[0009] There is also a dire need for additional modalities to treat
hematological malignancies and immune deficiency, and novel
cytokines to increase the output of transplanted lt-HSC. In
addition, an appropriate platform for target identification and
drug discovery does not currently exist. The missing elements are
cell lines that represent different developmental stages in
hematopoietic lineages. Optimally, such cells should retain the
ability to undergo further differentiation in a specific lineage.
Such cell lines are essential for identification of gene products,
and thus new drugable targets, involved in regulation of cell
development, proliferation and survival. In addition, such cell
lines are essential for the screening of small molecule and shRNA
libraries for loss-of function studies, as well as cDNA libraries
for gain of function studies, in search of novel drugs.
[0010] Barriers to current drug discovery in this area include: (a)
isolation of a sufficient number of cells from a particular
developmental stage; (b) propagation of the cells in vitro for a
sufficient length of time; and (c) ability to use conditional
oncogenes to screen for drugs that could affect leukemic cells and
not normal HSCs or progenitors.
[0011] Therefore, there is a great need in the art for a method to
generate lt-HSC cell lines that can be expanded extensively,
frozen, and used again whenever they are required, in the absence
of subsequent harvests from the donor.
SUMMARY OF THE INVENTION
[0012] One embodiment of the present invention relates to a method
to produce conditionally immortalized adult stem cells. The method
includes the steps of: (a) obtaining an expanded population of
adult stem cells; (b) transfecting the stem cells with a nucleic
acid molecule comprising a protooncogene or biologically active
fragment or homologue thereof that promotes cell survival and
proliferation, wherein the protooncogene is inducible; (c)
transfecting the stem cells with a nucleic acid molecule encoding a
protein that inhibits apoptosis of the cell; and (d) expanding the
transfected cells in the presence of a combination of stem cell
growth factors under conditions whereby the protooncogene is
active, to produce conditionally immortalized adult stem cells. In
one aspect of this embodiment, the nucleic acid molecule of (b)
and/or (c) is contained in an integrating vector. In one aspect,
the nucleic acid molecule of (b) and/or (c) is transfected into the
cells using a virus or viral vector selected from: retroviral
vectors, lentivirus vectors, parvovirus, vaccinia virus,
coronavirus, calicivirus, papilloma virus, flavivirus,
orthomixovirus, togavirus, picornavirus, adenoviral vectors,
modified and attenuated herpesviruses. In one aspect, the nucleic
acid molecule of (b) and/or (c) is transfected into the cells using
direct electroporation. In one aspect, the nucleic acid molecule or
(b) and/or (c) is contained in a vector comprising a nucleic acid
sequence encoding a drug-sensitivity protein. In one aspect, the
nucleic acid molecule or (b) and/or (c) is contained in a vector
comprising nucleic acid sequences encoding recognition substrate
sequences for a recombinase flanking the nucleic acid molecule of
(b) or (c).
[0013] In one aspect, this embodiment includes the additional steps
of: (e) removing the conditions of (d) whereby the protooncogene is
active; and (f) culturing the cells of (e) in media comprising
growth factors that induce differentiation of the cells. This
method can further include: (g) adding to the cells of (f), the
conditions of (d) whereby the protooncogene is active, to produce
conditionally immortalized cells in an intermediate stage of cell
differentiation.
[0014] Another embodiment of the present invention relates to a
method to produce conditionally immortalized adult stem cells,
comprising: (a) obtaining an expanded population of adult stem
cells; (b) culturing the stem cells in the presence of: (1) a
combination of stem cell growth factors; (2) a first Tat-fusion
protein, wherein Tat is fused to a protein encoded by a
protooncogene or biologically active fragment or homologue thereof
that promotes cell survival and proliferation; and (3) a second
Tat-fusion protein, wherein Tat is fused to a protein that inhibits
apoptosis in the stem cells.
[0015] Yet another embodiment of the present invention relates to
method to produce conditionally immortalized embryonic stem cells,
comprising: (a) obtaining an expanded population of embryonic stem
cells; (b) transfecting the stem cells with a nucleic acid molecule
comprising a protooncogene or biologically active fragment or
homologue thereof that promotes cell survival and proliferation,
wherein the protooncogene is inducible; (c) transfecting the stem
cells with a nucleic acid molecule encoding a protein that inhibits
apoptosis of the cell; and (d) expanding the transfected cells in
the presence of a combination of stem cell growth factors under
conditions whereby the protooncogene is active, to produce
conditionally immortalized embryonic stem cells.
[0016] Another embodiment of the present invention relates to
method to produce conditionally immortalized stem cells,
comprising: (a) obtaining an expanded population of stem cells; (b)
culturing the stem cells in the presence of: (1) a combination of
stem cell growth factors; (2) a protein encoded by a protooncogene
or biologically active fragment or homologue thereof that promotes
cell survival and proliferation; and; (3) a protein that inhibits
apoptosis in the stem cells. The protein of (2) and (3) are
delivered into the stem cells using any suitable delivery system,
including, but not limited to, Tat fusion, aptamers technology, or
CHARIOT.TM. technology.
[0017] Yet another embodiment of the present invention relates to a
method to produce conditionally immortalized stem cells,
comprising: (a) obtaining an expanded population of stem cells; (b)
delivering into the cells a protein encoded by a protooncogene or
biologically active fragment or homologue thereof that promotes
cell survival and proliferation, or a nucleic acid molecule
encoding the same, wherein the protooncogene is inducible; (c)
inhibiting apoptosis in the stem cells by delivering into the cells
a protein that inhibits apoptosis of the cell, a nucleic acid
molecule encoding the protein that inhibits apoptosis of the cell,
or a nucleic acid molecule or protein that inhibits a proapoptotic
protein in the cells; and (d) expanding the cells in the presence
of a combination of stem cell growth factors under conditions
whereby the protooncogene is active, to produce conditionally
immortalized adult stem cells.
[0018] In any of the embodiments described above, the protooncogene
can be selected from, but is not limited to: MYC-ER and ICN-1-ER.
In any of the embodiments described above, the protein that
inhibits apoptosis can be selected from, but is not limited to a
member of the Bcl-2 family that inhibits apoptosis, such as Bcl-2,
Bcl-X, Bcl-w, BclXL, Mcl-1, Dad-1, or hTERT. When the protooncogene
is MYC-ER or ICN-1-ER, the conditions under which the protooncogene
is active can include the presence of tamoxifen or an agonist
thereof. In one aspect the cells are transfected with or are
delivered (as a protein) MYC-ER and Bcl-2; MYC-ER and hTERT;
ICN-1-ER and Bcl-2; ICN-1-ER and hTERT; or MYC-ER and ICN-1-ER.
[0019] In any of the embodiments described above, the step of
expanding can be conducted in a medium including, but not limited
to, (1) interleukin-6 (IL-6), IL-3 and stem cell factor (SCF); (2)
a serum-free medium comprising stem cell factor (SCF),
thrombopoietin (TPO), insulin-like Growth Factor 2 (IGF-2) and
fibroblast Growth Factor 1 (FGF-1).
[0020] In any of the embodiments described above, the adult stem
cells can include, but are not limited to: hematopoietic stem
cells, intestinal stem cells, osteoblastic stem cells, mesenchymal
stem cells, neural stem cells, epithelial stem cells, cardiac
myocyte progenitor stem cells, skin stem cells, skeletal muscle
stem cells, and liver stem cells. In one aspect, the mesenchymal
stem cells are selected from lung mesenchymal stem cells and bone
marrow stromal cells. In one aspect, the epithelial stem cells are
selected from the group consisting of lung epithelial stem cells,
breast epithelial stem cells, vascular epithelial stem cells and
intestinal epithelial stem cells. In one aspect, the skin stem
cells are selected from the group consisting of epidermal stem
cells and follicular stem cells (hair follicle stem cells). In one
aspect, the neural cells are selected from neuronal dopaminergic
stem cells and motor-neuronal stem cells. In one aspect, the stem
cells are from fresh or cryopreserved cord blood. In one aspect,
the stem cells are hematopoietic progenitor cells obtained from the
peripheral blood of normal or granulocyte colony-stimulating factor
(G-CSF) treated patients.
[0021] In any of the embodiments described above, the method can
further include genetically modifying the stem cells to correct a
genetic defect in the cells, genetically modifying the stem cells
to silence the expression of a gene, and/or genetically modifying
the stem cells to overexpress a gene.
[0022] In any of the embodiments described above, the method can
further include storing the cells. In one aspect, the method
further includes retrieving the cells from storage and culturing
the cells.
[0023] Another embodiment of the present invention relates to cells
produced by any method described above or elsewhere herein.
[0024] Yet another embodiment of the present invention relates to a
method to provide adult stem cells, or cells differentiated
therefrom, to an individual comprising: (a) providing a source of
conditionally immortalized adult stem cells produced by any method
described above or elsewhere herein; (b) removing the conditions
under which the stem cells of (a) are conditionally immortalized;
and (c) administering the stem cells or cells differentiated
therefrom to the individual. In one aspect, the cells were
previously obtained from the individual in (c). In one aspect, the
cells were obtained from a previously frozen stock of said cells.
In one aspect, the cells are freshly obtained from the individual
and conditionally immortalized by any method described above or
elsewhere herein. In one aspect, the individual has cancer. In
another aspect, the individual has leukemia. In another aspect, the
individual has an immune deficiency disorder. In another aspect,
the individual has an anemia disorder. In another aspect, the
individual is undergoing reconstructive surgery. In another aspect,
the individual is undergoing elective cosmetic surgery. In another
aspect, the individual is undergoing transplantation surgery. In
one aspect, the individual is in need of stem cells, or cells
differentiated therefrom, selected from: hematopoietic stem cells,
intestinal stem cells, osteoblastic stem cells, mesenchymal stem
cells, neural stem cells, epithelial stem cells, cardiac myocyte
progenitor stem cells, skin stem cells, skeletal muscle stem cells,
and liver stem cells. In another aspect, the individual is in need
of improved immune cell function. In another aspect, the individual
has a genetic defect that is corrected by the stem cell.
[0025] Yet another embodiment of the present invention relates to a
method to identify compounds that regulate lineage commitment
and/or cell differentiation and development, comprising: (a)
contacting adult stem cells produced by any method described above
or elsewhere herein; and (b) detecting at least one genotypic or
phenotypic characteristic in the stem cells of (a), as compared to
the stem cells in the absence of the compound, wherein detection of
a difference in the characteristic in the presence of the compound
indicates that the compound affects the characteristic in the stem
cell.
[0026] Another embodiment of the present invention relates to a
method to study lineage commitment and/or cell differentiation and
development, comprising evaluating adult stem cells produced by any
method described above or elsewhere herein, or cells differentiated
therefrom, to detect at least one genotypic or phenotypic
characteristic of the cells.
[0027] Yet another embodiment of the present invention relates to
the use of the cells produced by any method described above or
elsewhere herein in a medicament for treating a condition or
disease in which transplantation of stem cells is beneficial.
[0028] Another embodiment of the present invention relates to a
mouse model of acute myeloid leukemia (AML), comprising a mouse
produced by a method comprising: (a) lethally irradiating a mouse;
(b) transferring conditionally immortalized long-term stem cells
produced by any method described above or elsewhere herein and
whole bone marrow cells from a Rag.sup.-/- mouse into the mouse;
and (c) injecting periodic doses of tamoxifen or an agonist thereof
into the mouse until the mouse develops clinical signs of AML. In
one aspect, the cells are transfected with or are delivered (as a
protein) MYC-ER and Bcl-2.
[0029] Another embodiment of the invention relates to tumor cells
obtained from the mouse model of AML described above.
[0030] Yet another embodiment of the invention relates to the use
of the mouse model of AML for preclinical testing of drug
candidates specific for human proteins; to identify, develop,
and/or test a compound for use in the diagnosis of, study of, or
treatment of AML; or to identify, develop, and/or test a target for
use in the diagnosis of, study of, or treatment of AML.
BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION
[0031] FIG. 1 is a graph showing mortality curves following bone
marrow transplantation of transduced cells and activation of MYC
function with 4OHT, in vivo.
[0032] FIG. 2 is a scatter plot showing scatter characteristics and
GFP expression levels of HSCs derived from young and aged mice,
following in vitro transduction. The dot plots represent the flow
cytometric data for the forward (FSC) and side (SSC) scatter
characteristics of the HSCs after three days in culture with IL-3,
IL-6 and SCF. These two criteria correlate with cell size (FSC) and
granularity (SSC).
[0033] FIGS. 3A and 3B are scatter plots showing the phenotypic
comparison of cell lines derived from irradiated recipients
reconstituted using BCL-2, MYC-ER and EGFP-transduced hematopoietic
stem cells from aged (>60% ID.sup.- repertoire) and young
3-83.mu..delta. transgenic mice. Shown is the phenotype of
representative clones 3 (young) and 3 (aged) months after
initiation of culture.
[0034] FIG. 4 is a scatter plot showing the spontaneous
differentiation of the aged LT-HSC line (ABM46) in vitro following
withdrawal of tamoxifen (stem cell and B lineage marker expression
are analyzed by flow cytometry).
[0035] FIGS. 5A and 5B are scatter plots showing the analysis of
hematopoietic cell compartments derived from LT-HSC lines 6 weeks
after adoptive transfer into irradiated young recipients. Data from
three mice are presented in this figure, one mouse received the
aged HSC line ABM42, and two mice received aged HSC line ABM46.
[0036] FIG. 6 is a scatter plot showing that the development of the
B-cell compartment is compromised in mice reconstituted with ABM42
and ABM46 cell lines. Data from three mice are presented in this
figure, one mouse received the aged HSC line ABM42, and two mice
received aged HSC line ABM46.
[0037] FIG. 7 is a scatter plot showing T-cell development in mice
that were reconstituted with ABM42 and ABM46 cell lines. Data from
three mice are presented in this figure, one mouse received the
aged HSC line ABM42, and two mice received aged HSC line ABM46.
[0038] FIG. 8 is a scatter plot and graph showing the phenotypic
comparison of cell lines derived from HSCs obtained from young
C57/BL6 mice that were retrovirally transduced with BCL-2 and
MYC-ER and maintained in continuous in vitro culture for >90
days. The panels represent the results of the flow cytometric
analysis for expression of the viral expression markers (GFP and
Thy1.1), as well as four markers required to define long-term HSCs
in mice, Sca-1, c-kit, CD34 and Flk-2. The four cell lines
contained subpopulations that retained the phenotypes of lt-HSCs
(Sca-1+, c-kit+, CD34-, flk-2-).
[0039] FIG. 9 is a scatter plot and graph showing a phenotypic
comparison of cell lines derived from HSCs obtained from young
C57/BL6 mice that were retrovirally transduced with different
combinations of oncogenes and maintained in continuous in vitro
culture for >90 days (pMIG-MYC and pMIT-Bcl-2 (top panels),
pMIG-MYC.ER and pMIG-hTERT (middle panels), or pMIG-ICN.1.ER and
pMIT-Bcl-2 (bottom panels)).
[0040] FIG. 10 is a scatter plot and graph showing a phenotypic
comparison of cell lines derived from HSCs obtained from young
C57/BL6 mice that were retrovirally transduced with different
combinations of oncogenes and maintained in continuous in vitro
culture for >90 days (pMIG-ICN.1.ER and pMIT-Bcl-2 (top panels),
pMIG-ICN.1 and pMIT-Bcl-2 (second row panels), or pMIG-ICN.1 and
pMIG-Bcl-2 (third row panels), or pMIG-hTERT and pMIT-Bcl-2 (bottom
panels)).
[0041] FIG. 11 is a scatter plot and graph showing a phenotypic
comparison of cell lines derived from HSCs obtained from young
C57/BL6 mice that were retrovirally transduced with different
combinations of oncogenes and maintained in continuous in vitro
culture for >90 days (pMIG-MYC and pMIG-ICN.1 (top panels),
pMIG-MYC.ER and pMIG-ICN.1 (middle panels), or pMIG-ICN.1.ER and
pMIG-MYC (bottom panels).
[0042] FIG. 12 is a scatter plot showing the in vivo reconstitution
of T cell and B cell compartments from cell lines derived from HSCs
obtained from young C57/BL6 mice that were retrovirally transduced
with different combinations of oncogenes and maintained in
continuous in vitro culture for >90 days.
[0043] FIG. 13 is a schematic drawing showing the use of
recognition substrate sequences (RSS's) for recombinases in order
to ensure the excision of recombinant DNA from conditionally
immortalized long-term stem cells of the invention prior to
transplantation.
[0044] FIG. 14 is a graph showing the detection of cells of the NK
and erythroid lineage differentiated from conditionally
immortalized long-term stem cells of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0045] The present invention provides a solution to the problem of
being able to generate, maintain and manipulate stable cell lines
derived from long-term stem cells, and particularly, long-term
hematopoietic stem cells (lt-HSCs), that can give rise to all cell
lineages that would normally arise from such cells when placed
under the appropriate conditions. The present invention generally
relates to methods to produce conditionally immortalized, long-term
stem cells, to the stem cells produced by such methods, and to
methods of using such stem cells. More specifically, using
long-term hematopoietic stem cells as an exemplary stem cell
population, the present inventors have established a powerful
method to produce stem cells that are conditionally immortalized
(e.g., reversibly immortalized or immortalized under specified
conditions which is reversible when such conditions are removed),
such stem cells being capable differentiating into normal cell
lineages in vitro and in vivo, and being capable of reconstituting
subjects in need of such cells. Indeed, the present invention can
eliminate the need for a bone morrow donor, since the invention
provides for the ability to harvest stem cells from a patient prior
to a procedure (e.g. chemotherapy, radiation, etc.) to expand such
cells, and return them to the patient. Moreover, such stem cells
can be expanded extensively, stored (e.g., frozen), and then
retrieved and expanded again, manipulated, and/or used repeatedly
as required or desired. Such stem cells can be manipulated, for
example, to correct a genetic defect or provide a benefit to a
subject (therapeutic or preventative), or differentiated into a
desired cell type. Finally, such cells can be used in a variety of
assays for the identification of new targets involved in regulation
of cell development, proliferation and survival, and the
identification and development of drugs useful in ameliorating or
treating diseases and conditions that would benefit from the
regulation of cell development, proliferation and/or survival.
[0046] The present inventors have developed novel technology that
allows the conditional immortalization of long-term stem cells,
exemplified herein by long-term hematopoietic stem cells (lt-HSCs).
The resulting cell lines can be expanded (propagated) indefinitely
and exponentially in vitro and/or cryopreserved (stored), and have
the ability to rescue lethally irradiated mice and to reconstitute
all blood cell lineages in such animals. Furthermore, the inventors
have been able to generate differentiated blood cells in vitro by
extinguishing the function of the transforming oncogene. Such cells
and the methods of producing them as described herein will allow
the generation of transplantable human stem cells that carry no
recombinant DNA, and thus pose no long term risk to the recipient.
These conditionally immortalized lt-HSC's of the invention can be
stabilized in their mature phenotypes and cell lines established in
which the mature phenotype is preserved after reactivation of the
oncogene. For example, the inventors have been able to develop
CD4.sup.+ .alpha..beta..sup.+ T cells, as well as dendritic cell
lines.
[0047] Applied in the clinical setting, this technology has the
following advantages over bone marrow transplantation:
[0048] 1. Very few lt-HSC are needed to establish clones;
[0049] 2. Clones represent a renewable resource that can be stored
indefinitely and accessed quickly;
[0050] 3. The cost of this therapy should be much less than
conventional bone marrow transplantation;
[0051] 4. Use of lt-HSC clones should mitigate the threat of
graft-versus-host disease, and associated costs.
[0052] 5. The technology can, at least in some cases, mitigate the
need for a bone marrow donor.
[0053] In addition, the present invention provides for the use of
the conditionally transformed long-term stem cells, such as the
lt-HSC cells, to generate cells representing differentiated
lineages (e.g., differentiated hematopoietic lineages, including
intermediate stages of development of hematopoietic lineages). For
example, in addition to countless therapeutic and preventative
applications, these cell lines will allow the identification of
novel compounds that can induce differentiation of malignant cells,
arrest their growth, or induce apoptosis. These cells will also
permit screening for novel cytokines and growth factors that direct
the differentiation of stem cells in a particular pathway. Such
cell lines simply do not exist and will be essential for drug
discovery.
[0054] More specifically, in an effort to overcome the limitations
in the art with regard to the provision and use of long term
populations of adult-derived stem cells (although the invention is
not limited to adult-derived stem cells, as discussed below), the
present inventors have developed novel methods of producing of
conditionally transformed cell lines representing early
hematopoietic stem cell progenitors. In a specific, non-limiting
example of the technology described and exemplified herein, the
strategy involved the transfection (e.g., by retroviral
transduction) of bone marrow stem cells from 5-fluorouracil
(5-FU)-treated 3-83.mu..delta. mice. The inventors utilized the
pMSCV bisistronic retroviral vector with inserts encoding Bcl-2 and
green fluorescent protein (GFP) (as a reporter gene), and MYC-ER
and GFP (again as a reporter gene). MYC was selected because of its
ability to substitute for cytokine-derived survival and
proliferative signals in lymphocytes. By restricting the target
cell, the inventors hypothesized that stem cell tumors would form.
Importantly, MYC-ER function is tamoxifen dependent in this
setting, allowing for the termination of MYC function and
transformation by withdrawing tamoxifen from the animal or
cultures. In cells transduced with MYC-ER, the fusion protein is
produced, but is retained in the cytoplasm until exposed to
tamoxifen. Bcl-2 was selected because of its ability to inhibit
apoptosis of cells that would normally occur as a result of
exposure to the MYC signals and more particularly, when MYC is
"inactivated" or removed by withdrawal of the tamoxifen from the
cells. This novel combination of gene types (i.e., the invention is
not limited to these specific genes, as discussed in more detail
below) is partly responsible for the successful production of
conditionally immortalized stem cells according to the present
invention, and can readily be extended to other similar
combinations of genes, as discussed in detail below.
[0055] Recipients of the transduced stem cells described above
produced tumors (in the presence of 4OHT), and tumor cells from the
bone marrow, spleen and lymph node were harvested and placed in
culture with tamoxifen and a stem cell growth factor cocktail. The
present inventors have discovered that, in the absence of an
appropriate combination of stem cell growth factors, the stem cells
produced by the present method will stop growing and die within a
short period of time. Therefore, the use of a stem cell growth
factor "cocktail" (i.e., combination of appropriate or suitable
growth factors for stem cells) after transfection of the cells with
the combination of genes discussed above is a second important
aspect of the method of the present invention. This cocktail, while
having the general characteristic of promoting and maintaining the
growth of the stem cells, is not limited to a particular
combination of growth factors, and parameters for selection of such
factors are discussed in detail below.
[0056] The stem cells generated by the method of the present
invention could be expanded in culture and were homogeneously
positive for e.g. Sca1, positive for Endoglin and ckit, and
negative for CD34, Flt3, B220, CD19 and mIgM, which are indicative
of the phenotype of lt-HSC, which is well-characterized in the art.
These cells could be frozen (cryopreserved, or stored), and then
easily recovered and cultured after freezing. Importantly, the
recovered cells were homogenous in phenotype and exhibited the
phenotype of lt-HSC (e.g., again, uniformly GFP bright cells were
positive for Sca1, Endoglin and c-kit, and negative for CD34, Flt3,
B220, CD19 and mIgM). This phenotype corresponds perfectly with the
published characteristics of long term repopulating pluripotent
stem cells (Reya et al., 2003, Nature 423:409-14) that provide all
long-term reconstitution in mice.
[0057] The inventors have further developed this method so that it
can be performed completely in vitro (i.e., the initial procedure
was conducted partly in vivo as described above). The inventors
have also demonstrated that other combinations of genes having
similar characteristics as those described above also result in the
conditional immortalization of lt-HSCs. Furthermore, the cell lines
can be differentiated in vitro into hematopoietic lineages by
removing the tamoxifen and providing the appropriate growth
factors, and will differentiate in vivo into all hematopoietic
lineages in recipient animals in which tamoxifen is withheld. In
addition, the cells can be differentiated into intermediate levels
of development that have a stable phenotype and retain their
ability to further differentiate along their committed pathway upon
application or removal of the appropriate signal (described
herein). Such cells are invaluable for various therapeutic
applications. All of these experiments are described in detail
below and in the Examples.
[0058] The methods and cell lines of the present invention provide
a unique opportunity not only to study in detail the molecular,
biochemical and cellular events that are associated with the
commitment of adult stem cells toward various cell lineages and to
study the differentiation and development of stem cells into
various cell lineages, but also provide unique therapeutic and drug
discovery tools.
[0059] For example, the stem cell lines of the present invention
provide a unique source of expandable stem cells for use in a
variety of transplantation, therapeutic and preventative
strategies, including the treatment of cancer, and particularly,
cancer that is treated by radiation. In current therapy for
leukemia, for example, limited access to bone marrow donors and
finite supplies of stem cells from such donors severely limit the
options for reconstitution of a patient after radiation therapy.
The present invention solves this problem by providing a means to
generate a continuously expandable and renewable supply of
autologous stem cells or histocompatible stem cells that can be
stored and recovered as needed. Such technology could ultimately
ablate the need for bone marrow donors altogether. In addition, a
variety of immune deficiency disorders and anemia disorders (e.g.,
aplastic anemia or hemolytic anemia) will also benefit greatly from
this technology, since the present invention provides the ability
to repopulate hematopoietic cells of an individual as needed by the
individual. Furthermore, the aging process is associated with
several important changes in the hematopoietic compartment,
including the increasing inability to mount a productive immune
response, among others. Hematopoietic stem cells from aged mice
have been shown to contain a higher level of mRNAs for DNA-repair
problems. This may ultimately affect their ability to self-renew,
undergo differentiation, undergo proliferation, and survive in
response to bone marrow cytokines. Therefore, an aging individual
can also benefit from the present invention in that a continuous
supply of healthy hematopoietic cells can be provided to correct or
ameliorate such deficiencies.
[0060] The technology of the present invention is not limited to
bone marrow stem cells, but can be applied to virtually any type of
stem cell, and can be extended beyond adult-derived cells to
embryonic stem cells.
[0061] In one example, another application of the present invention
relates to the generation of continuously expandable and renewable
hair follicle stem cells. The development of conditionally
immortalized stem cells from this lineage can be use in the context
of reconstructive surgery for burn victims, for any individual that
undergoes chemotherapy and/or radiation therapy resulting in the
irreversible loss of hair growth, as well as patients following any
surgical procedure affecting the skull. Furthermore, such cells
could be used for elective procedures that involve the induction of
hair growth in individuals affected by hereditary pattern baldness.
Similarly, application of the present invention to stem cells of
the skin will be invaluable for use in wound healing and treatment
of burn victims, as well as plastic reconstructive surgery for
trauma and other patients, as well as elective surgeries,
including, but not limited to, cosmetic surgery. Such cells can be
additionally genetically manipulated to correct inborn or acquired
genetic defects in young and aged individuals. One of skill in the
art will understand based on this disclosure that benefits can be
derived from the use of the present invention on various other stem
cell populations, including, but not limited to, stem cells derived
from lung, breast, and intestinal epithelium and stem cells derived
from neural and cardiac tissue, to name just a few. Other stem cell
types are referenced elsewhere herein.
[0062] In addition, the present invention provides the unique
opportunity for an individual to have access to expandable supplies
of autologous stem cells and cells differentiated therefrom as
needed throughout the life of the individual. For example, as the
body ages, it is known that immune function and immune memory
deteriorates. However, using the technology provided by the present
invention, it will be possible to repopulate an individual with
new, autologous stem cells that are capable of differentiation into
all of the cells of the hematopoietic lineage, thus providing the
aged individual with a "young" immune system. In addition, stem
cells generated by the present method can be stored and used as
part of therapeutic protocols during the lifetime of the
individual, should they be needed (e.g., in the event the
individual develops a cancer or immune deficiency disease or has
another need for newly generated, autologous cells of virtually any
type).
[0063] The present invention also provides unique opportunities for
gene therapy. Specifically, genetic defects can now be corrected or
beneficial gene modifications can be introduced into somatic cells
by manipulating autologous stem cells obtained from an individual
that have been conditionally immortalized and expanded using the
method of the present invention. The stem cells can then be
reintroduced into the individual from which they were obtained.
[0064] The stem cells produced by the method of the invention can
also be used in a variety of drug discovery assays. Since one can
now produce virtually unlimited supplies of homogeneous stem cells
that can readily be stored, recovered, expanded and manipulated,
such stem cells can be used as stem cells or differentiated into
various cell lineages and used in assays to test various compounds
for effects on cell differentiation, gene expression, and cell
processes. The cells can be manipulated prior to contact with the
compounds, such as by genetic manipulation. Stem cells from
individuals with genetic defects can be evaluated in such assays in
order to identify therapeutic compounds (e.g., cancer therapeutics)
and evaluate gene replacement therapies. Indeed, the technology of
the present invention provides an opportunity to target the cells
of a specific individual to identify drug candidates and
therapeutic candidates and strategies that are "tailored" to the
cells of an individual. An example of such an assay is described in
detail below.
[0065] With regard to research and discovery in the area of lineage
commitment and cell differentiation and development, prior to the
present invention, such studies were severely hampered by the lack
of access to and the inability to generate sufficient numbers of
the desired cell population to perform desired experiments. For
example, in order to identify or screen for intermediates in the
differentiation of a particular progenitor cell line, a sufficient
number of cells must be obtained to provide meaningful and
reproducible results. The progenitor cell line should also retain
the ability to further differentiate in the lineage to which it has
already committed, hence making these novel tools that do not
currently exists, nor are there other descriptions of technology
needed to generate those cells. Using technologies available at the
time of the invention, this was not possible. The present invention
solves the problem by providing expandable and essentially
unlimited supplies of homogeneous stem cells that can be used in a
variety of experiments. This technology will greatly enhance
research capabilities in the area of cell differentiation and
discovery.
[0066] As discussed above, the method for conditionally
immortalizing lt-HSCs of the present invention can be adapted for
additional stem cells derived from other tissues. For example, by
adapting the gene delivery and growth factors, if needed, the
present invention can be applied to a variety of different stem
cells as described below. Such cells can also be expanded in vitro,
and proceed to differentiate upon inactivation of the oncogenes, as
described herein for hematopoietic stem cells. These cells can then
be used for therapeutic applications that include tissue repair and
tissue regeneration/engineering. Accordingly, the MYC-ER and Bcl-2
combination of genes, or any of the other combinations described
herein, can be transfected by any method described herein or deemed
suitable by one of skill in the art given this disclosure
(including by a variety of viral-mediated methods), into cells
including, but not limited to, mesenchymal stem cells (including,
but not limited to, lung mesenchymal stem cells, bone marrow
stromal cells), neural stem cells including, but not limited to,
neuronal dopaminergic stem cells and motor-neuronal stem cells),
epithelial stem cells (including, but not limited to, lung
epithelial stem cells, breast epithelial stem cells, and intestinal
epithelial stem cells), cardiac myocyte progenitor stem cells, skin
stem cells (including, but not limited to, epidermal stem cells and
follicular stem cells), skeletal muscle stem cells, endothelial
stem cells (e.g., lung endothelial stem cells), and liver stem
cells, to generate conditionally immortalized cell lines that can
be expanded in vitro and proceed to differentiate upon inactivation
of the oncogenes. In addition to the therapeutic potential of such
cell lines, these lines can be further modified in vitro (or ex
vivo) in order to correct inborn genetic defects, and used for
studying the molecular basis of early lineage commitment and
differentiation. While these cells may be a novel source of
potentially relevant therapeutic targets, these cell lines will
also be useful for the screening of small molecules that either
prevent or induce differentiation, and for the identification of
novel compounds and molecular targets for various therapies,
including, but not limited to, cancer therapy and immune deficiency
therapy.
GENERAL DEFINITIONS
[0067] In accordance with the present invention, reference to an
isolated nucleic acid molecule herein is a nucleic acid molecule
that has been removed from its natural milieu (i.e., that has been
subject to human manipulation), its natural milieu being the genome
or chromosome in which the nucleic acid molecule is found in
nature. As such, "isolated" does not necessarily reflect the extent
to which the nucleic acid molecule has been purified, but indicates
that the molecule does not include an entire genome or an entire
chromosome in which the nucleic acid molecule is found in nature.
An isolated nucleic acid molecule can include a gene. An isolated
nucleic acid molecule that includes a gene is not a fragment of a
chromosome that includes such gene, but rather includes the coding
region and regulatory regions associated with the gene, but no
additional genes that are naturally found on the same chromosome.
An isolated nucleic acid molecule can also include a specified
nucleic acid sequence flanked by (i.e., at the 5' and/or the 3' end
of the sequence) additional nucleic acids that do not normally
flank the specified nucleic acid sequence in nature (i.e.,
heterologous sequences). Isolated nucleic acid molecule can include
DNA, RNA (e.g., mRNA), or derivatives of either DNA or RNA (e.g.,
cDNA, siRNA, shRNA). Although the phrase "nucleic acid molecule"
primarily refers to the physical nucleic acid molecule and the
phrase "nucleic acid sequence" primarily refers to the sequence of
nucleotides on the nucleic acid molecule, the two phrases can be
used interchangeably, especially with respect to a nucleic acid
molecule, or a nucleic acid sequence, being capable of encoding a
protein or domain of a protein.
[0068] Preferably, an isolated nucleic acid molecule of the present
invention is produced using recombinant DNA technology (e.g.,
polymerase chain reaction (PCR) amplification, cloning) or chemical
synthesis. Isolated nucleic acid molecules include natural nucleic
acid molecules and homologues thereof, including, but not limited
to, natural allelic variants and modified nucleic acid molecules in
which nucleotides have been inserted, deleted, substituted, and/or
inverted in such a manner that such modifications provide the
desired effect (e.g., provision of an inducible protooncogene, as
described herein).
[0069] A nucleic acid molecule homologue can be produced using a
number of methods known to those skilled in the art (see, for
example, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Labs Press (1989)). For example, nucleic acid
molecules can be modified using a variety of techniques including,
but not limited to, classic mutagenesis techniques and recombinant
DNA techniques, such as site-directed mutagenesis, chemical
treatment of a nucleic acid molecule to induce mutations,
restriction enzyme cleavage of a nucleic acid fragment, ligation of
nucleic acid fragments, PCR amplification and/or mutagenesis of
selected regions of a nucleic acid sequence, synthesis of
oligonucleotide mixtures and ligation of mixture groups to "build"
a mixture of nucleic acid molecules and combinations thereof.
Nucleic acid molecule homologues can be selected from a mixture of
modified nucleic acids by screening for the function of the protein
encoded by the nucleic acid and/or by hybridization with a
wild-type gene.
[0070] The minimum size of a nucleic acid molecule or
polynucleotide of the present invention is a size sufficient to
encode a protein useful in the present invention, such as a protein
encoded by a protooncogene or functional portion thereof (i.e., a
portion that has the biological activity of the full-length protein
and that is sufficient for use in the method of the invention), or
an anti-apoptotic protein or a functional portion thereof (i.e., a
portion that has the biological activity of the full-length protein
and that is sufficient for use in the method of the invention).
Other nucleic acid molecules that may be useful in the present
invention can include nucleic acid molecules of a minimum size
sufficient to form a probe or oligonucleotide primer that is
capable of forming a stable hybrid with the complementary sequence
of a nucleic acid molecule encoding the natural protein (e.g.,
under moderate, high or very high stringency conditions), which is
typically at least 5 nucleotides in length, and preferably ranges
from about 5 to about 50 or about 500 nucleotides or greater,
including any length in between, in whole number increments (i.e.,
5, 6, 7, 8, 9, 10, . . . 33, 34, . . . 256, 257, . . . 500). There
is no limit, other than a practical limit, on the maximal size of a
nucleic acid molecule of the present invention, in that the nucleic
acid molecule can include a sequence or sequences sufficient to be
useful in any of the embodiments of the invention described
herein.
[0071] As used herein, stringent hybridization conditions refer to
standard hybridization conditions under which nucleic acid
molecules are used to identify similar nucleic acid molecules. Such
standard conditions are disclosed, for example, in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs
Press, 1989. Sambrook et al., ibid., is incorporated by reference
herein in its entirety (see specifically, pages 9.31-9.62). In
addition, formulae to calculate the appropriate hybridization and
wash conditions to achieve hybridization permitting varying degrees
of mismatch of nucleotides are disclosed, for example, in Meinkoth
et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid.,
is incorporated by reference herein in its entirety.
[0072] More particularly, moderate stringency hybridization and
washing conditions, as referred to herein, refer to conditions
which permit isolation of nucleic acid molecules having at least
about 70% nucleic acid sequence identity with the nucleic acid
molecule being used to probe in the hybridization reaction (i.e.,
conditions permitting about 30% or less mismatch of nucleotides).
High stringency hybridization and washing conditions, as referred
to herein, refer to conditions which permit isolation of nucleic
acid molecules having at least about 80% nucleic acid sequence
identity with the nucleic acid molecule being used to probe in the
hybridization reaction (i.e., conditions permitting about 20% or
less mismatch of nucleotides). Very high stringency hybridization
and washing conditions, as referred to herein, refer to conditions
which permit isolation of nucleic acid molecules having at least
about 90% nucleic acid sequence identity with the nucleic acid
molecule being used to probe in the hybridization reaction (i.e.,
conditions permitting about 10% or less mismatch of nucleotides).
As discussed above, one of skill in the art can use the formulae in
Meinkoth et al., ibid. to calculate the appropriate hybridization
and wash conditions to achieve these particular levels of
nucleotide mismatch. Such conditions will vary, depending on
whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated
melting temperatures for DNA:DNA hybrids are 10.degree. C. less
than for DNA:RNA hybrids. In particular embodiments, stringent
hybridization conditions for DNA:DNA hybrids include hybridization
at an ionic strength of 6.times.SSC (0.9 M Na.sup.+) at a
temperature of between about 20.degree. C. and about 35.degree. C.
(lower stringency), more preferably, between about 28.degree. C.
and about 40.degree. C. (more stringent), and even more preferably,
between about 35.degree. C. and about 45.degree. C. (even more
stringent), with appropriate wash conditions. In particular
embodiments, stringent hybridization conditions for DNA:RNA hybrids
include hybridization at an ionic strength of 6.times.SSC (0.9 M
Na.sup.+) at a temperature of between about 30.degree. C. and about
45.degree. C., more preferably, between about 38.degree. C. and
about 50.degree. C., and even more preferably, between about
45.degree. C. and about 55.degree. C., with similarly stringent
wash conditions. These values are based on calculations of a
melting temperature for molecules larger than about 100
nucleotides, 0% formamide and a G+C content of about 40%.
Alternatively, T.sub.m can be calculated empirically as set forth
in Sambrook et al., supra, pages 9.31 to 9.62. In general, the wash
conditions should be as stringent as possible, and should be
appropriate for the chosen hybridization conditions. For example,
hybridization conditions can include a combination of salt and
temperature conditions that are approximately 20-25.degree. C.
below the calculated T.sub.m of a particular hybrid, and wash
conditions typically include a combination of salt and temperature
conditions that are approximately 12-20.degree. C. below the
calculated T.sub.m of the particular hybrid. One example of
hybridization conditions suitable for use with DNA:DNA hybrids
includes a 2-24 hour hybridization in 6.times.SSC (50% formamide)
at about 42.degree. C., followed by washing steps that include one
or more washes at room temperature in about 2.times.SSC, followed
by additional washes at higher temperatures and lower ionic
strength (e.g., at least one wash as about 37.degree. C. in about
0.1.times.-0.5.times.SSC, followed by at least one wash at about
68.degree. C. in about 0.1.times.-0.5.times.SSC).
[0073] In one embodiment of the present invention, any amino acid
sequence described herein, including truncated forms (fragments or
portions) and homologues of such sequences, can be produced with
from at least one, and up to about 20, additional heterologous
amino acids flanking each of the C- and/or N-terminal end of the
given amino acid sequence. The resulting protein or polypeptide can
be referred to as "consisting essentially of" a given amino acid
sequence. According to the present invention, the heterologous
amino acids are a sequence of amino acids that are not naturally
found (i.e., not found in nature, in vivo) flanking the given amino
acid sequence or which would not be encoded by the nucleotides that
flank the naturally occurring nucleic acid sequence encoding the
given amino acid sequence as it occurs in the gene, if such
nucleotides in the naturally occurring sequence were translated
using standard codon usage for the organism from which the given
amino acid sequence is derived. Similarly, the phrase "consisting
essentially of", when used with reference to a nucleic acid
sequence herein, refers to a nucleic acid sequence encoding a given
amino acid sequence that can be flanked by from at least one, and
up to as many as about 60, additional heterologous nucleotides at
each of the 5' and/or the 3' end of the nucleic acid sequence
encoding the given amino acid sequence. The heterologous
nucleotides are not naturally found (i.e., not found in nature, in
vivo) flanking the nucleic acid sequence encoding the given amino
acid sequence as it occurs in the natural gene.
[0074] According to the present invention, a recombinant vector
(also referred to generally as a recombinant nucleic acid molecule,
particularly when it contains a nucleic acid sequence of interest
according to the invention) is an engineered (i.e., artificially
produced) nucleic acid molecule that is used as a tool for
manipulating a nucleic acid sequence of choice and for introducing
such a nucleic acid sequence into a host cell. The recombinant
vector is therefore suitable for use in cloning, sequencing, and/or
otherwise manipulating the nucleic acid sequence of choice, such as
by expressing and/or delivering the nucleic acid sequence of choice
into a host cell. Such a vector typically contains heterologous
nucleic acid sequences, i.e., nucleic acid sequences that are not
naturally or usually found adjacent to a nucleic acid sequence to
be cloned or delivered, although the vector can also contain
regulatory nucleic acid sequences (e.g., promoters, untranslated
regions) which are naturally found adjacent to nucleic acid
molecules of the present invention, or which are useful for
expression of the nucleic acid molecules of the present invention
(discussed in detail below). A vector can be either RNA or DNA,
either prokaryotic or eukaryotic, and typically is a plasmid or a
viral vector. The vector can be maintained as an extrachromosomal
element (e.g., a plasmid) or it can be integrated into the
chromosome of a host cell. The entire vector can remain in place
within a host cell, or under certain conditions, the plasmid DNA
can be deleted, leaving behind the nucleic acid molecule of the
present invention. Under other conditions, the vector is designed
to be excised (removed) from the genome of the host cell at a
selected time (described in more detail below). The integrated
nucleic acid molecule can be under chromosomal promoter control,
under native or plasmid promoter control, or under a combination of
several promoter controls. Single or multiple copies of the nucleic
acid molecule can be integrated into the chromosome. A recombinant
vector of the present invention can contain at least one selectable
marker.
[0075] According to the present invention, the phrase "operatively
linked" refers to linking a nucleic acid molecule to an expression
control sequence (e.g., a transcription control sequence and/or a
translation control sequence) in a manner such that the molecule
can be expressed when transfected (i.e., transformed, transduced,
transfected, conjugated or conduced) into a host cell.
Transcription control sequences are sequences that control the
initiation, elongation, or termination of transcription.
Particularly important transcription control sequences are those
that control transcription initiation, such as promoter, enhancer,
operator and repressor sequences. Suitable transcription control
sequences include any transcription control sequence that can
function in a host cell or organism into which the recombinant
nucleic acid molecule is to be introduced.
[0076] According to the present invention, the term "transfection"
is used to refer to any method by which an exogenous nucleic acid
molecule (i.e., a recombinant nucleic acid molecule) can be
inserted into a cell. The term "transduction" is a specific type of
transfection in which genetic material is transferred from one
source to another, such as by a virus (e.g., a retrovirus) or a
transducing bacteriophage. The term "transformation" can be used
interchangeably with the term "transfection" when such term is used
to refer to the introduction of nucleic acid molecules into
microbial cells, such as bacteria and yeast. In microbial systems,
the term "transformation" is used to describe an inherited change
due to the acquisition of exogenous nucleic acids by the
microorganism and is essentially synonymous with the term
"transfection." However, in animal cells, transformation has
acquired a second meaning that can refer to changes in the growth
properties of cells in culture after they become cancerous, for
example. Therefore, to avoid confusion, the term "transfection" is
preferably used herein with regard to the introduction of exogenous
nucleic acids into animal cells. Therefore, the term "transfection"
will be used herein to generally encompass transfection or
transduction of animal cells, and transformation or transduction of
microbial cells, to the extent that the terms pertain to the
introduction of exogenous nucleic acids into a cell. Transfection
techniques include, but are not limited to, transformation,
transduction, particle bombardment, diffusion, active transport,
bath sonication, electroporation, microinjection, lipofection,
adsorption, infection and protoplast fusion.
[0077] As used herein, reference to an isolated protein or
polypeptide in the present invention includes full-length proteins,
fusion proteins, chimeric proteins, or any fragment (truncated
form, portion) or homologue of such a protein. More specifically,
an isolated protein according to the present invention, is a
protein (including a polypeptide or peptide) that has been removed
from its natural milieu (i.e., that has been subject to human
manipulation), and can include, but is not limited to, purified
proteins, partially purified proteins, recombinantly produced
proteins, membrane bound proteins, proteins complexed with lipids,
soluble proteins, synthetically produced proteins, and isolated
proteins associated with other proteins. As such, "isolated" does
not reflect the extent to which the protein has been purified.
Preferably, an isolated protein of the present invention is
produced recombinantly. In addition, and again by way of example
with respect to the naming of a particular protein (Bcl-2), a
"human Bcl-2 protein" or a protein "derived from" a human Bcl-2
protein refers to a Bcl-2 protein (including a homologue or portion
of a naturally occurring Bcl-2 protein) from a human (Homo sapiens)
or to a Bcl-2 protein that has been otherwise produced from the
knowledge of the structure (e.g., sequence) and perhaps the
function of a naturally occurring Bcl-2 protein from Homo sapiens.
In other words, a human Bcl-2 protein includes any Bcl-2 protein
that has substantially similar structure and function of a
naturally occurring Bcl-2 protein from Homo sapiens or that is a
biologically active (i.e., has biological activity) homologue of a
naturally occurring Bcl-2 protein from Homo sapiens as described in
detail herein. As such, a human Bcl-2 protein can include purified,
partially purified, recombinant, mutated/modified and synthetic
proteins. According to the present invention, the terms
"modification" and "mutation" can be used interchangeably,
particularly with regard to the modifications/mutations to the
amino acid sequence of a protein (or nucleic acid sequences)
described herein.
[0078] As used herein, the term "homologue" is used to refer to a
protein or peptide which differs from a naturally occurring protein
or peptide (i.e., the "prototype" or "wild-type" protein) by
modifications, including minor modifications, to the naturally
occurring protein or peptide, but which maintains the basic protein
and side chain structure of the naturally occurring form. Such
changes include, but are not limited to: changes in one or a few
(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid side chains;
changes one or a few amino acids, including deletions (e.g., a
protein or truncated form of the protein or peptide), insertions
and/or substitutions; changes in stereochemistry of one or a few
atoms; and/or minor derivatizations, including but not limited to:
methylation, glycosylation, phosphorylation, acetylation,
myristoylation, prenylation, palmitation, amidation and/or addition
of glycosylphosphatidyl inositol. A homologue can have either
enhanced, decreased, or substantially similar properties as
compared to the naturally occurring protein or peptide. A homologue
can include an agonist of a protein or an antagonist of a
protein.
[0079] Homologues can be the result of natural allelic variation or
natural mutation. A naturally occurring allelic variant of a
nucleic acid encoding a protein is a gene that occurs at
essentially the same locus (or loci) in the genome as the gene
which encodes such protein, but which, due to natural variations
caused by, for example, mutation or recombination, has a similar
but not identical sequence. Allelic variants typically encode
proteins having similar activity to that of the protein encoded by
the gene to which they are being compared. One class of allelic
variants can encode the same protein but have different nucleic
acid sequences due to the degeneracy of the genetic code. Allelic
variants can also comprise alterations in the 5' or 3' untranslated
regions of the gene (e.g., in regulatory control regions). Allelic
variants are well known to those skilled in the art.
[0080] Homologues can be produced using techniques known in the art
for the production of proteins including, but not limited to,
direct modifications to the isolated, naturally occurring protein,
direct protein synthesis, or modifications to the nucleic acid
sequence encoding the protein using, for example, classic or
recombinant DNA techniques to effect random or targeted
mutagenesis.
[0081] In one embodiment, a homologue of a given protein comprises,
consists essentially of, or consists of, an amino acid sequence
that is at least about 45%, or at least about 50%, or at least
about 55%, or at least about 60%, or at least about 65%, or at
least about 70%, or at least about 75%, or at least about 80%, or
at least about 85%, or at least about 90%, or at least about 95%
identical, or at least about 95% identical, or at least about 96%
identical, or at least about 97% identical, or at least about 98%
identical, or at least about 99% identical (or any percent identity
between 45% and 99%, in whole integer increments), to the amino
acid sequence of the reference protein. In one embodiment, the
homologue comprises, consists essentially of, or consists of, an
amino acid sequence that is less than 100% identical, less than
about 99% identical, less than about 98% identical, less than about
97% identical, less than about 96% identical, less than about 95%
identical, and so on, in increments of 1%, to less than about 70%
identical to the naturally occurring amino acid sequence of the
reference protein.
[0082] As used herein, unless otherwise specified, reference to a
percent (%) identity refers to an evaluation of homology which is
performed using: (1) a BLAST 2.0 Basic BLAST homology search using
blastp for amino acid searches and blastn for nucleic acid searches
with standard default parameters, wherein the query sequence is
filtered for low complexity regions by default (described in
Altschul, S. F., Madden, T. L., Schaaffer, A. A., Zhang, J., Zhang,
Z., Miller, W. & Lipman, D. J. (1997) "Gapped BLAST and
PSI-BLAST: a new generation of protein database search programs."
Nucleic Acids Res. 25:3389-3402, incorporated herein by reference
in its entirety); (2) a BLAST 2 alignment (using the parameters
described below); (3) and/or PSI-BLAST with the standard default
parameters (Position-Specific Iterated BLAST. It is noted that due
to some differences in the standard parameters between BLAST 2.0
Basic BLAST and BLAST 2, two specific sequences might be recognized
as having significant homology using the BLAST 2 program, whereas a
search performed in BLAST 2.0 Basic BLAST using one of the
sequences as the query sequence may not identify the second
sequence in the top matches. In addition, PSI-BLAST provides an
automated, easy-to-use version of a "profile" search, which is a
sensitive way to look for sequence homologues. The program first
performs a gapped BLAST database search. The PSI-BLAST program uses
the information from any significant alignments returned to
construct a position-specific score matrix, which replaces the
query sequence for the next round of database searching. Therefore,
it is to be understood that percent identity can be determined by
using any one of these programs.
[0083] Two specific sequences can be aligned to one another using
BLAST 2 sequence as described in Tatusova and Madden, (1999),
"Blast 2 sequences--a new tool for comparing protein and nucleotide
sequences", FEMS Microbiol Lett. 174:247-250, incorporated herein
by reference in its entirety. BLAST 2 sequence alignment is
performed in blastp or blastn using the BLAST 2.0 algorithm to
perform a Gapped BLAST search (BLAST 2.0) between the two sequences
allowing for the introduction of gaps (deletions and insertions) in
the resulting alignment. For purposes of clarity herein, a BLAST 2
sequence alignment is performed using the standard default
parameters as follows.
[0084] For blastn, using 0 BLOSUM62 matrix:
[0085] Reward for match=1
[0086] Penalty for mismatch=-2
[0087] Open gap (5) and extension gap (2) penalties
[0088] gap x_dropoff (50) expect (10) word size (11) filter
(on)
[0089] For blastp, using 0 BLOSUM62 matrix:
[0090] Open gap (11) and extension gap (1) penalties
[0091] gap x_dropoff (50) expect (10) word size (3) filter
(on).
[0092] According to the present invention, an isolated protein,
including a biologically active homologue or fragment thereof, has
at least one characteristic of biological activity of activity the
wild-type, or natural occurring protein. In general, the biological
activity or biological action of a protein refers to any
function(s) exhibited or performed by the protein that is ascribed
to the naturally occurring form of the protein as measured or
observed in vivo (i.e., in the natural physiological environment of
the protein) or in vitro (i.e., under laboratory conditions).
Modifications, activities or interactions which result in a
decrease in protein expression or a decrease in the activity of the
protein, can be referred to as inactivation (complete or partial),
down-regulation, reduced action, or decreased action or activity of
a protein. Similarly, modifications, activities or interactions
that result in an increase in protein expression or an increase in
the activity of the protein, can be referred to as amplification,
overproduction, activation, enhancement, up-regulation or increased
action of a protein.
Method of Conditional Immortalization of the Invention
[0093] One embodiment of the present invention relates to a method
to produce conditionally immortalized, adult stem cells, and
preferably long-term stem cells. The method generally includes the
following steps: (a) obtaining an expanded population of adult stem
cells; (b) transfecting (transducing) the stem cells with a vector
comprising a protooncogene that promotes cell survival and
proliferation, wherein the protooncogene is regulatable (inducible,
controllable), (c) transfecting (transducing) the stem cells with a
vector encoding a protein that inhibits apoptosis of the cell; and
(d) expanding the transfected cells in the presence of a
combination of stem cell growth factors under conditions whereby
the protooncogene is active. In one embodiment, the vector is an
integrating vector. Cells produced by this method can be cultured,
expanded, stored, recovered, used in therapeutic methods, used in
research and discovery methods, genetically manipulated, induced to
differentiate by removing the conditions whereby the protooncogene
is active, and/or used in any other method described herein or
apparent to one of skill in the art given this disclosure. Steps
(b) and (c) can be performed in any order.
[0094] According to the present invention, the phrase
"conditionally immortalized" refers to cells that are immortalized
(e.g., capable of indefinite growth without differentiation in a
cytokine dependent fashion, while maintaining their ability and
potential to differentiate into a number of different lineages
under the appropriate conditions) in a reversible manner, such that
the cells are immortalized under a specific set of conditions, and
when the conditions are removed or changed (or other conditions
added), the cells are no longer immortalized and may differentiate
into other cell types. The phrase "conditionally immortalized" can
be used interchangeably with the phrase "reversibly immortalized".
For example, referring to the method of the present invention, the
presence of the regulatable protooncogene that promotes cell
survival and proliferation causes the cells to retain an
immortalized phenotype when the stem cell is placed under
conditions that allow the protooncogene to be activated (e.g.,
tamoxifen or an agonist thereof in the case of MYC-ER). In other
words, the cells grow and expand indefinitely in culture, and are
maintained in an undifferentiated state under these specific
conditions. When these conditions are removed (e.g., the tamoxifen
is removed with respect to MYC-ER), the stem cells are no longer
immortalized and can differentiate into various cell lineages given
the appropriate environment (e.g., the appropriate combination of
growth factors).
[0095] Reference to "stem cells", as used herein, refers to the
term as it is generally understood in the art. For example, stem
cells, regardless of their source, are cells that are capable of
dividing and renewing themselves for long periods, are
unspecialized (undifferentiated), and can give rise to
(differentiate into) specialized cell types (i.e., they are
progenitor or precursor cells for a variety of different,
specialized cell types). "Long-term", when used in connection with
stem cells, refers to the ability of stem cells to renew themselves
by dividing into the same non-specialized cell type over long
periods (e.g., many months, such as at least 3 months, to years)
depending on the specific type of stem cell. As discussed herein,
phenotypic characteristics of various long-term stem cells from
different animal species, such as long-term hematopoietic stem
cells (lt-HSC) are known in the art. For example, murine lt-HSC can
be identified by the presence of the following cell surface marker
phenotype: c-kit+, Sca-1+, CD34-, flk2- (see Examples). Adult stem
cells include stem cells that can be obtained from any
non-embryonic tissue or source, and typically generate the cell
types of the tissue in which they reside. The term "adult stem
cell" may be used interchangeably with the term "somatic stem
cell". Embryonic stem cells are stem cells obtained from any
embryonic tissue or source.
[0096] In one embodiment of the invention, the stem cells used in
the present invention can include any adult stem cells obtained
from any source. In another embodiment of the invention, stem cells
can include embryonic stem cells. Stem cells useful in the present
invention include, but are not limited to, hematopoietic stem
cells, mesenchymal stem cells (including, but not limited to, lung
mesenchymal stem cells, bone marrow stromal cells), neural stem
cells, epithelial stem cells (including, but not limited to, lung
epithelial stem cells, breast epithelial stem cells, vascular
epithelial stem cells, and intestinal epithelial stem cells),
intestinal stem cells, cardiac myocyte progenitor stem cells, skin
stem cells (including, but not limited to, epidermal stem cells and
follicular stem cells (hair follicle stem cells)), skeletal muscle
stem cells, osteoblastic precursor stem cells, and liver stem
cells.
[0097] Hematopoietic stem cells give rise to all of the types of
blood cells, including but not limited to, red blood cells
(erythrocytes), B lymphocytes, T lymphocytes, natural killer cells,
neutrophils, basophils, eosinophils, monocytes, macrophages, and
platelets.
[0098] Mesenchymal stem cells (including bone marrow stromal cells)
give rise to a variety of cell types, including, but not limited to
bone cells (osteocytes), cartilage cells (chondrocytes), fat cells
(adipocytes), lung cells, and other kinds of connective tissue
cells such as those in tendons.
[0099] Neural stem cells in the brain give rise to its three major
cell types: nerve cells (neurons) and two categories of
non-neuronal cells, astrocytes and oligodendrocytes.
[0100] Epithelial stem cells in the lining of various tissues give
rise to several cell types that form the epithelium in tissues.
[0101] Skin stem cells occur in the basal layer of the epidermis
and at the base of hair follicles. The epidermal stem cells give
rise to keratinocytes, which migrate to the surface of the skin and
form a protective layer, and the follicular stem cells can give
rise to both the hair follicle and to the epidermis. Other sources
of adult stem cells will be known to those of skill in the art.
[0102] Embryonic stem cells can give rise to all tissues and cells
of the body.
[0103] Methods for obtaining such stem cells and providing initial
culture conditions, such as a liquid culture or semi-solid culture
medium, are known in the art. The cells are initially expanded in
vivo or in vitro, by contacting the source of the stem cells with a
suitable reagent that expands or enriches such cells in the tissue
source or in culture. For example, in the case of hematopoietic
stem cells, the donor individual can be treated with an agent that
enriches for hematopoietic stem cells and encourages such cells to
proliferate without differentiation, such as 5-fluorouracil. Other
suitable agents for expansion of a desired stem cell type will be
known to those of skill in the art. Alternatively, and preferably,
adult stem cells are isolated from a tissue source and then
expanded or enriched in vitro by exposure to a suitable agent. For
example, with regard to hematopoietic stem cells, a method for
producing an expanded culture of adult hematopoietic progenitors is
described in Van Parijs et al., (1999; Immunity, 11, 763-70). Cells
are obtained from an individual by any suitable method for
obtaining a cell sample from an animal, including, but not limited,
to, collection of bone marrow collection of a bodily fluid (e.g.,
blood), collection of umbilical cord blood, tissue punch, and
tissue dissection, including particularly, but not limited to, any
biopsies of skin, intestine, cornea, spinal cord, brain tissue,
scalp, stomach, breast, lung (e.g., including lavage and
bronchioschopy), fine needle aspirates of the bone marrow, amniotic
fluid, placenta and yolk sac.
[0104] In one embodiment, cells useful in the invention can also be
obtained from fresh, or cryopreserved (stored) cord blood,
hematopoietic progenitor populations that can be derived from the
directed differentiation of embryonic stem (ES) cells in vitro,
hematopoietic stem cells (HSCs) obtained from the peripheral blood
of normal or granulocyte colony-stimulating factor (G-CSF)-treated
patients who have been induced to mobilize their lt-HSCs to the
peripheral circulation.
[0105] Once an expanded population of stem cells is obtained (made
available, provided, or produced), the cells are transfected,
either sequentially (in any order) or simultaneously, with: (1) a
vector comprising a protooncogene that promotes cell survival and
proliferation, wherein the protooncogene is regulatable (inducible,
controllable), and (2) a vector encoding a protein that inhibits
apoptosis of the cell. Preferably, the vector is an integrating
vector, defined herein as any vector that has the ability to
integrate into the genome of a cell (e.g., a retroviral vector).
Various vectors and methods of transfection are described in detail
below. The protooncogene is regulatable (inducible or
controllable), so that the protooncogene can be activated and
deactivated (i.e., turned on or turned off) as desired to either
maintain the stem cell in an immortalized state or to allow it to
differentiate into a desired cell type. Protooncongenes can be
selected, or designed, to be regulated by any suitable method,
including in response to any condition, such as the presence or
absence of a compound or agent, temperature, or any other suitable
condition. By way of example, the protooncogenes MYC-ER (the
estrogen receptor (ER)-regulated MYC) and ICN-1-ER (the
ER-regulated intracellular portion of Notch-1) described herein are
both inducible in the presence of tamoxifen. It is noted that such
genes can also be engineered to be responsive to other dimerizing
drugs, such as FK1012, altered forms of Rapamycin, or could be
expressed from vectors that contain a tetracycline responsive
element. The latter scenario regulates expression of the protein,
not the function of a polypeptide present in the cell. Other
similar modifications of this platform technology will be apparent
to those of skill in the art.
[0106] The protooncogene useful in the method of the present
invention is any protooncogene that promotes cell survival and
proliferation. Preferred protooncogenes to use in the method of the
invention include, but are not limited to MYC, ICN-1, hTERT,
(reverse transcriptase component of the human telomerase), NMYC,
S-MYC, L-MYC, Akt (myrystylated). In addition, other suitable genes
to use or methods of the invention or ways to modify genes to
achieve the desired result include, but are not limited to use of
downstream signaling effectors such as pyruvate dehydrogenase
kinase 1 (PDK-1); mammalian target of Rapamycin (mTOR); loss of
phosphatase and tensin homologue (PTEN) by shRNA; Bcl-3, Cyclin D1,
Cyclin D3, Bcl-10, Bcl-6, BCR-ABL (breakpoint cluster region fusion
with ABL) and its various mutant forms, constitutively active forms
of Stat5 and Stat3, AML1-ETO (fusion of acute myelogenous leukemia
1 and runt-related transcription factor 1), MLL-ENL (mixed lineage
leukemia and eleven nineteen leukemia), Hox genes, activated forms
of the interleukin-3 (IL-3) receptor .beta. chain, and other
cytokine receptor chains (epidermal growth factor receptor (EGFR),
c-kit, platelet-derived growth factor receptor (PDGFR), etc.), as
well as wnt (all mammalian forms), .beta.-catenin, sonic hedgehog
(shh-1 and all mammalian forms), bmi-1 and c-jun (all mammalian
forms). Also, the present invention includes inducing the loss (or
inhibition) of cyclin kinase inhibitors by shRNA, including, but
not limited to, p16, p19, p21 and p27. In one embodiment, the
present invention includes the use of regulatable homologues of any
or such protooncogenes (e.g., MYC-ER or ICN-1-ER) or other genes.
The Examples describe the use of both MYC-ER or ICN-1-ER to
successfully produce conditionally immortalized lt-HSC using the
method of present invention.
[0107] The nucleic acid sequence encoding human MYC is represented
herein as SEQ ID NO:1, which encodes an amino acid sequence
represented herein as SEQ ID NO:2. The nucleic acid sequence
encoding hTERT is represented herein as SEQ ID NO:3, which encodes
an amino acid sequence represented herein as SEQ ID NO:4. The
nucleic acid sequence encoding human ICN-1 is represented herein as
SEQ ID NO:11, which encodes an amino acid sequence represented
herein as SEQ ID NO:12. ICN-1 a portion of Notch-1, and
specifically, amino acids 1757-2555 from Notch-1 (see Aster et al.,
Mol Cell Biol. 2000 October; 20(20):7505-15, incorporated herein by
reference in its entirety). The nucleotide and amino acid sequence
for MYC-ER are known in the art and the MYC-ER protein is described
in Soloman et al., Oncogene. 1995 Nov. 2; 11(9):1893-7,
incorporated herein by reference in its entirety. ICN-1-ER was
created by the present inventors and the nucleic acid sequence
encoding this protein is represented herein as SEQ ID NO:13, which
encodes an amino acid sequence represented by SEQ ID NO:14.
[0108] Similarly, a preferred anti-apoptosis gene is Bcl-2,
although other genes that encode proteins that inhibit apoptosis
and particularly, maintain cell survival when the protooncogene is
inactivated in the stem cell, are included in the present
invention. The nucleic acid sequence encoding Bcl-2 alpha is
represented herein as SEQ ID NO:5, which encodes an amino acid
sequence of SEQ ID NO:6. Bcl-2 beta is represented herein as SEQ ID
NO:7, which encodes an amino acid sequence of SEQ ID NO:8. An
"anti-apoptosis" gene is defined herein as any gene that encodes a
protein that can inhibit (reduce, prevent, decrease) a process
associated with apoptosis in a cell or promote (enhance, increase,
stimulate, allow) cell survival, even in the presence of conditions
that could induce apoptosis. Proteins associated with apoptosis,
and the genes encoding such proteins, are well-known in the art.
Such other genes include, but are not limited to, any genes in the
Bcl-2 family that will likely be important in the setting of
conditional transformation of adult stem cells (i.e., not just
hematopoietic stem cells). These genes include, but are not limited
to, other pro-survival members of the Bcl-2 family, such as Bcl-X,
Bcl-w, BclXL, Mcl-1, Dad-1, or hTERT (reverse transcriptase
component of the human telomerase, which has been shown to inhibit
proliferation). Such genes are ectopically overexpressed in the
presence of the regulated oncogene, as described with Bcl-2 in the
working examples herein. In addition, this aspect of the present
invention includes using shRNA mediated gene knockdown (or
disruption or inhibition by any other method) for BH3-only members
of the bcl-2 family that are proapoptotic (e.g., Bim, PUMA, NOXA,
Bax, Bak, BclXS, Bad, Bar, and others), as well as disruption of
Caspases 3, 9, 10, MLL-1 (and all mammalian forms), Enl-1
(Endospermless-1) and all mammalian forms, Apaf-1 and other
elements that form part of the apoptosome.
[0109] The nucleic acid sequence for each of these genes described
above or the coding region thereof is known in the art and is
publicly available, including for humans. Similarly, the amino acid
sequence for proteins encoded by these genes is known in the art
and is publicly available.
[0110] The present inventors have produced several different
long-term, conditionally immortalized stem cells using the method
of the present invention and using different combinations of
protooncogenes and anti-apoptotic genes, including the following
combinations: MYC-ER and Bcl-2; MYC-ER and hTERT (reverse
transcriptase component of the human telomerase); ICN-1-ER and
Bcl-2; ICN-1-ER and hTERT; and MYC-ER and ICN-1-ER.
[0111] It is noted that with regard to either of the protooncogene
or the gene encoding an anti-apoptosis protein used in the present
method, it is not required that the entire gene be used in the
constructs described herein, since any portion of the gene or a
nucleic acid sequence (e.g., cDNA) that encodes the desired
functional protein product, a functional portion thereof, or a
functional homologue thereof is encompassed by the invention.
Accordingly, reference generally herein to the genes or transgenes
used to transfect stem cells is to be understood to be exemplary
and to include the use of any nucleic acid molecules encoding the
entire gene, the entire coding region of the gene, or portions of
the genes or homologues thereof, as long as such nucleic acid
sequences encode functional proteins suitable for use in the
present invention.
[0112] In one embodiment of the present invention, the present
method additional includes the use of shRNAs or siRNAs that are
directed against RNAs encoding proapoptotic proteins, such as the
pro-apoptotic members of the Bcl-2 family, namely those of the
BH3-only type (Bim, Bax, Bak, Puma, Noxa, etc.). The disruption of
a pro-apoptotic gene in the context of a regulated oncogene is
expected to result in a more efficient immortalization of certain
stem cell populations. RNA interference (RNAi) is a process whereby
double stranded RNA, and in mammalian systems, short interfering
RNA (siRNA) or short hairpin RNA (shRNA), is used to inhibit or
silence expression of complementary genes. In the target cell,
siRNA are unwound and associate with an RNA induced silencing
complex (RISC), which is then guided to the mRNA sequences that are
complementary to the siRNA, whereby the RISC cleaves the mRNA.
shRNA is transfected into a target cell in a vector where it is
transcribed, and then processed by DICER enzymes to form siRNA-like
molecules that activate RISC, which, as with siRNA, is then guided
to the mRNA sequences that are complementary to the shRNA, whereby
the RISC cleaves the mRNA.
[0113] The stem cells can be transfected with the vectors
comprising the protooncogene and encoding the anti-apoptosis
protein using any suitable method of transfecting cells, and
particularly mammalian cells, including by using combinations of
techniques. The present inventors have discovered that it is the
particular coordination between the genes (or constructs) that are
expressed that have resulted in the generation of conditionally
immortalized, long term stem cells as described herein. The
Examples have demonstrated the use of retroviral vectors, but other
methods include, but are not limited to, the use of other viruses
and viral vectors derived therefrom, including, but not limited to,
lentivirus vectors, parvovirus, vaccinia virus, coronavirus,
calicivirus, papilloma virus, flavivirus, orthomixovirus,
togavirus, picornavirus, adenoviral vectors, modified and
attenuated herpesviruses. Any such virus can further be modified
with specific surface expressed molecules that target these to HSCs
or other stem cells, such as membrane bound SCF, or other stem-cell
specific growth factor ligands. Other methods of transfection of
mammalian cells include, but are not limited to, direct
electroporation of mammalian expression vectors, such as by using
NUCLEOFECTOR.TM. technology (AMAXA Biosystems). This technology is
a highly efficient non-viral gene transfer method for most primary
cells and for hard-to-transfect cell lines, which is an improvement
on the long-known method of electroporation, based on the use of
cell-type specific combinations of electrical current and solutions
to transfer polyanionic macromolecules directly into the nucleus.
Additionally, suitable methods of transfection can include any
bacterial, yeast or other artificial methods of gene delivery that
are known in the art.
[0114] The step of expanding the transfected stem cells or
culturing the stem cells and exogenous fusion proteins (e.g., the
Tat-fusion proteins described in the variations of this method
described below) in the presence of suitable growth factors can
include the use of any suitable culture conditions, including those
specifically described herein. The combination of suitable stem
cell growth factors can include any stem cell factors that allow
transfected (e.g., transduced) cells of the invention to grow,
survive and proliferate in culture. While specific combinations are
described herein, and while this is an important step of the
present method, this step can be simply described as providing any
combination of growth factors that are suitable for the growth,
proliferation and survival of stem cells, and include any
combinations that are known in the art. Accordingly, the invention
is not limited to a particular combination. One preferred
combination of growth factors includes: interleukin-6 (IL-6), IL-3
and stem cell factor (SCF). Another preferred combination of growth
factors includes stem cell factor (SCF), thrombopoietin (TPO),
insulin-like Growth Factor 2 (IGF-2) and fibroblast Growth Factor 1
(FGF-1), in serum-free media. This latter combination was recently
described in Zhang and Lodich (2005; Murine hematopoietic stem
cells change their surface phenotype during ex vivo expansion,
Blood 105, 4314-20). The stem cells transfected with nucleic acid
molecules encoding the combinations proteins described herein
(e.g., MYC-ER and Bcl-2 as described in the examples) are expected
to also become conditionally immortalized in this cocktail of
growth factors, as with the cocktail described in the Examples
above (using IL-3, IL-6 and SCF). Other growth factors for use in
the invention include, but are not limited to, angiopoietin-like
proteins (e.g., Agptl2, Angptl3, Angptl5, Angptl7, etc.),
proliferin-2 (PLF2), glycogen synthase kinase-3 inhibitors,
inducers of the wnt and Notch signaling pathways, FH3L and related
cytokines, fibroblast growth factor 2 (FGF2) and related cytokines,
wnt-1 and other activators of the Wnt pathway, Sonic hedghog
(shh-1) and other activators of that pathway. Other suitable
combinations of growth factors will be applicable to the method of
the present invention and will be apparent to those of skill in the
art. Indeed, the cell lines generated using the method of the
present invention can readily be used to screen for additional
cytokines and growth factors that could be used for expanding
long-term stem cells, or any of their derived progenitors, in vitro
under neutral or directed conditions.
[0115] According to the present invention, a medium suitable for
culture of animal cells can include any available medium which has
been developed for culture of animal cells and particularly,
mammalian cells, or which can be prepared in the laboratory with
the appropriate components necessary for animal cell growth, such
as assimilable carbon, nitrogen and micronutrients. Such a medium
comprises a base medium, which is any base medium suitable for
animal cell growth, including, but not limited to, Iscove's
Modified Dulbecco's Medium (IMDM), Dulbecco's modified Eagles
medium (DMEM), alpha MEM (Gibco), RPMI 1640, or any other suitable
commercially available media. To the base medium, assimilable
sources of carbon, nitrogen and micro-nutrients are added
including, but not limited to, a serum source, growth factors,
amino acids, antibiotics, vitamins, reducing agents, and/or sugar
sources. It is noted that completed mediums comprising a base
medium and many of the additional components necessary for animal
cell growth are commercially available, and some media are
available for particular types of cell culture. In addition, many
serum-free media are available and may be particularly suited for
the culture of stem cells according to the invention.
Cells and Compositions
[0116] Another embodiment of the present invention relates to a
cell, cell line, or population of cells produced according to the
method of the present invention as described herein. Also included
in the invention are compositions comprising such cells, cell lines
or populations of cells. For therapeutic methods, such compositions
can include a pharmaceutically acceptable carrier, which includes
pharmaceutically acceptable excipients and/or delivery vehicles,
for delivering the cells, cell lines, or cell populations to a
patient. As used herein, a pharmaceutically acceptable carrier
refers to any substance suitable for delivering a therapeutic
composition useful in the method of the present invention to a
suitable in vivo site.
Adaptation of the Method of the Invention to Produce Cell Lineages
at Intermediate Stages of Development
[0117] Another embodiment of the present invention relates to
adaptations of the novel methods described herein to generate cell
lines that capture intermediate stages of development for the
hematopoietic lineages. According to the present invention, an
"intermediate" stage of development or differentiation refers to a
pluripotent stage of cell development or differentiation that is
downstream of the stage of development or differentiation of the
stem cell from which the "intermediate" cell was derived, but is
upstream of the final, or terminal, point of differentiation of a
cell. For example, a pre-B cell is an intermediate stage of a
hematopoietic stem cell, which can still differentiate into a
mature B cell. Intermediate stages of development or
differentiation will be understood by those of skill in the
art.
[0118] More particularly, for many therapeutic and discovery or
research applications, as well as for storage of cells lines, it is
desirable that the cell lines have a stable phenotype and retain
their ability to further differentiate along their committed
pathway once the active oncogene with which the cell has been
transfected is turned off. Accordingly, the present invention
encompasses additional steps of producing cells that have not fully
differentiated (are not terminally differentiated), but rather, are
at an intermediate stage of differentiation. In one non-limiting
example of this embodiment, long-term stem cells produced using the
method described above are randomly differentiated in vitro
following withdrawal of the conditions that maintain the activity
of the protooncogene or other gene that promotes cell survival and
proliferation (e.g., 4-OHT in the case of the tamoxifen-dependent
protooncogenes), or by applying the appropriate conditions that
turn off (inactivate) the protooncogene/oncogene. This step can be
performed while maintaining the culture in neutral cytokine growth
conditions (e.g., IL-3, IL-6 and SCF), or by replacing those
cytokines which could specifically direct differentiation towards a
certain lineage (e.g., IL-7 and Notch ligands for lymphoid
lineages, GM-CSF and IL-4 for dendritic cells, G-CSF for
myelomonocytic cells, etc.) with cytokines that are neutral for
differentiation (do not direct or drive differentiation of the
cells). Once the cultures begin to display differentiation markers
consistent with a specific lineage, the culture media is again
supplemented with the conditions that activate the protooncogene
(e.g., 4-OHT) or exposed to the conditions that otherwise
reactivating the protooncogene, in order to stabilize the phenotype
and generate cell lines having a stable, intermediate
differentiation phenotype.
[0119] By way of exemplification of this method, the inventors have
generated CD4+, .alpha..beta.+ T cells in vitro from ABM42 cells
(lt-HSC produced by the method of the invention; see Examples) by
withdrawal of 4-OHT from the media, and re-addition of 4OHT after
differentiation. The inventors have also generated dendritic cell
lines by incubating ABM46 cells (see Examples) in GM-CSF, IL-4 and
FLT3L and then placing the cultures back in the presence of 4-OHT
after differentiation.
[0120] Another approach for creating such cell lines involves
introducing the ctlt-HSC cells into mice to allow for
differentiation, and arresting, or stabilizing the phenotypes in
vivo after injections of 4-OHT. This method is described in detail
in Example 8. Briefly, and by way of example, lt-HSC generated by
the present method are injected into immuno-compromised animals
(e.g., immuno-compromised mice). The oncogene in the lt-HSCs is
reactivated using injections of the activating agent (e.g., 4-OHT),
cells are later collected, and then the cells can be cultured in
vitro to differentiate the cells, and then stored or used as
desired. This approach, and the other described above, can be used
for both murine and human ctlt-HSC cell lines, such as by using
either NOD/SCID mice as the recipients, or neonatal Rag-1.sup.-/-
mice, which will be given intrahepatic injections.
Application of the Method of the Invention to Embryonic Stem
Cells
[0121] Another embodiment of the invention relates to the
application of the method of conditionally immortalizing stem cells
to embryonic stem (ES) cells. Such methods will be useful for
generating cell lines that are more readily derived from ES cells,
such as cells of the neuronal linage, including neuronal stem
cells.
[0122] In this embodiment, the method of the present invention,
comprising the transduction of cells (in this case, ES cells) with
a protooncogene and a gene that inhibits apoptosis (e.g., MYC-ER
and Bcl-2) can be applied to ES cells to further control the
directed differentiation of these cells. In this embodiment, such
cells can be used to generate transgenic mice, for example, and in
addition, any ES cell and relevant progenitor cell population
derived therefrom can be subjected to the activation of the
protooncogene by exposure to the activating agent, hence allowing
for the generation of novel conditionally transformed stem cell
lines (different tissue types), or mature cell lines for the tissue
type of interest. In addition, the directed differentiation of
transduced ES cells in vitro can also be used to capture
intermediate states of differentiation by as described above. The
use of ES cells or ES-derived cells in this manner provides a novel
platform for drug discovery and target identification in the
setting of different diseases.
[0123] For example, neuronal stem cells can be employed in this
embodiment of the invention, as well as the directed
differentiation of ES cells into the neuronal pathway using the
method of the invention. The isolation and transduction of neuronal
stem cells from the hippocampus has been previously described for
mice. The culture conditions for neurospheres would enable the
proliferation of those cells, rendering them susceptible to
viral-mediated transduction of the genes of the invention (e.g.,
MYC-ER and Bcl-2), in order to generate conditionally transformed
neuronal stem cell lines. Their differentiation in vitro as well as
in vivo following implantation can be monitored by virtue of the
virally encoded reporter genes as well as previously defined
markers of neuronal differentiation. In addition, the
administration of the activating agent (e.g., 4-OHT) to the mice
following transplantation of the conditionally transformed neuronal
stem cell lines may lead to the development of a neurological
malignancy (neuroblastoma, glioblastoma, etc.). Those tumors would
provide a novel model for preclinical studies and target
identification.
[0124] The directed differentiation of ES cells that had been
transduced with, for example, MYC-ER and Bcl-2, can be carried out
in the presence of a previously defined growth medium, as well as
cytokines. The addition of the activating agent (e.g., 4-OHT) at
any time during the culture will enable the stabilization of the
cells at an intermediate phenotype, and leads to the generation of
cell lines that still retain the capacity to undergo further
differentiation. For instance, the generation of dopaminergic
neurons from ES cells is normally done by the addition of Retinoic
acid and FGF8. This type of neuron would be ideal for repairing
brain lesions observed in Alzheimer's patients. However, the
transplantation of fully differentiated neuronal cells may preclude
their successful implantation and engraftment. A conditionally
transformed cell line that was committed to the dopaminergic
neuronal pathway, but still retained its ability to further
differentiate after transplant, as envisioned herein, is expected
to greatly increase the chances of implantation and successful
engraftment. A similar scenario can be proposed for the generation
of motor neurons from ES cells, by adding Retinoic acid and a sonic
hedgehog agonist to the cultures. Those neuronal cells could help
repair spinal cord injuries. Once again, fully differentiated cells
would not be used in this embodiment, but rather, the committed
progenitor cells that retain the capacity to differentiate
(produced by the method of the invention) would be employed.
Variations or Modifications of the Method of Conditional
Immortalization for the Removal of the Transgene
[0125] In one embodiment of the invention, in order to avoid taking
the risk of introducing stem cells that harbor transgenes such as
those described herein (e.g., MYC-ER) into humans and/or mice, the
recombinant constructs are designed so that these DNA fragments
will be excised. This embodiment can be achieved using any suitable
method of first establishing the long-term stem cells according to
the method of the invention, and then exposing the cells (or a
patient) to conditions under which the recombinant DNA will be
removed, excised or completely silenced.
[0126] For example, in one aspect of the invention, a bacterial
recombinase approach is used. In this aspect of the invention,
preferably, two different recombinases are used in order to allow
control over which one of the two genes is excised at any one point
in time. Two examples of such recombinases are the Cre and Flp
recombinases, which are well-known in the art. Briefly, the
recognition substrate sequences (RSS's) for one of the recombinases
is introduced into the retroviral constructs such that they flank
the open reading frame of the oncogene, as well as the reporter
gene (e.g., GFP or Thy1.1). In this case, the cells are incubated
in media containing a Tat-Cre fusion protein (i.e., HIV or other
retroviral Tat protein fused to Cre). This recombinant protein has
been previously described and shown to be able to passively enter
cells, and mediate loxP site-dependent recombination of genomic
DNA. Other methods of gene (nucleic acid molecule) excision are
known to those of skill in the art and could readily be applied to
the present invention. Examples 5 and 13 exemplify this embodiment
of the invention.
[0127] In another embodiment of the invention, to provide another
method of avoiding the risk of introducing stem cells that harbor
transgenes such as those described herein into humans and/or other
animals (e.g., mice), instead of transfecting the stem cells with
the combination of the recombinant constructs for the protooncogene
or the anti-apoptosis protein, the invention is performed by making
use of Tat-fusion proteins as a method to allow the proteins access
to the inside of the cell without having to introduce transgenes
into the cell. For example, recombinant constructs that encode
tat-protooncogene or tat-anti-apoptosis genes (e.g., Tat-MYC-ER or
Tat-Bcl-2) may be used to conditionally immortalize stem cells. In
this embodiment of the invention, the target stem cells will be
cultured under suitable culture conditions, in media that contains
purified recombinant Tat-fusion proteins encoded by the specific
gene combination selected (e.g., MYC-ER and Bcl-2). In this
embodiment of the invention, the protooncogene product or similar
gene product can be inducible, as in the embodiments above.
Alternatively, or in addition, the action of this protein can be
regulated simply by providing or removing the protein from the
culture. While the cell lines that are generated with this approach
will be continuously dependent upon the addition of the exogenous
Tat-fusion proteins, they will not have a specific exogenous
nucleotide sequence introduced into them. The absence of foreign
oncogene sequences is expected to improve the clinical deployment
of the method of the present invention. Human immunodeficiency
virus-1 (HIV-1) Tat, is one exemplary Tat protein, although other
retroviral Tat proteins are known in the art. As a non-limiting
example, the nucleic acid sequence encoding HIV-1 Tat is
represented herein as SEQ ID NO:9, which encodes an amino acid
sequence represented herein by SEQ ID NO:10.
[0128] In another embodiment, to provide another method of avoiding
the risk of introducing stem cells that harbor transgenes such as
those described herein into humans and/or other animals (e.g.,
mice), instead of transfecting the stem cells with the combination
of the recombinant constructs for the protooncogene or the
anti-apoptosis protein, the invention is performed by introducing
proteins (e.g., MYC and Bcl-2) into a cell using aptamer
technology. Aptamers are short strands of synthetic nucleic acids
(usually RNA but also DNA) selected from randomized combinatorial
nucleic acid libraries by virtue of their ability to bind to a
predetermined specific target molecule with high affinity and
specificity. Aptamers assume a defined three-dimensional structure
and are capable of discriminating between compounds with very small
differences in structure. Accordingly aptamers can be conjugated
with the proteins used in the invention or with non-integrating
cDNA encoding the proteins, for example, and used to deliver the
proteins or DNA to the cells. In addition, aptamers can readily be
used to deliver siRNA to cells, for example, when one disrupts
proapoptotic proteins according to the present invention. Aptamer
technology is discussed, for example, in Davidson, 2006, Nature
Biotechnol. 24(8):951-952; and McNamara et al., 2006, Nature
Biotechnol. 24(8):1005-1015). Again, the absence of foreign
oncogene sequences is expected to improve the clinical deployment
of the method of the present invention.
[0129] In another embodiment, to provide another method of avoiding
the risk of introducing stem cells that harbor transgenes such as
those described herein into humans and/or other animals (e.g.,
mice), instead of transfecting the stem cells with the combination
of the recombinant constructs for the protooncogene or the
anti-apoptosis protein, the invention is performed by introducing
the protooncogene and/or anti-apoptosis protein into a cell using
CHARIOT.TM. technology (Krackeler Scientific, Inc., Albany, N.Y.).
With this technology, a non-covalent bond is formed between a
CHARIOT.TM. peptide and the protein of interest. This protects the
protein from degradation and preserves its natural characteristics
during the transfection process. Upon delivery to a cell, the
complex dissociates and CHARIOT.TM. is transported to the nucleus,
while the delivered protein is biologically active and free to
proceed to its cellular target. Efficient delivery can occur in the
presence or absence of serum, and is independent of the endosomal
pathway, which can modify macromolecules during internalization.
This delivery system also bypasses the transcription-translation
process. Accordingly, the proteins useful in the present invention
can be delivered to a cell and released to conditionally
immortalize the cell, without the need for the introduction of a
protooncogene or oncogenes to the cell. As above, the absence of
foreign oncogene sequences is expected to improve the clinical
deployment of the method of the present invention.
[0130] As yet another alternative (or additional) means to control
for the possibility of an insertion of a protooncogene into the
host cell genome by the various viral approaches described herein,
and thereby avoid a transforming event, a drug sensitivity (drug
susceptibility) cassette can be introduced into the viral
constructs to be used such that it will be expressed in every
transduced cell and its differentiated progeny. A drug sensitivity
cassette or a drug susceptibility cassette is a nucleic acid
sequence encoding a protein that renders a cell susceptible or
sensitive to the presence of a particular drug, so that upon
exposure to the drug, the cell activity is inhibited and
preferably, undergoes apoptosis. Those patients in which the levels
of a particular blood cell population increases without apparent
cause (e.g., infection, trauma, stress, etc.), can be given a
course of the drug to which sensitivity has been introduced in
order to ablate those cells and mitigate any possible additional
complications involving cells in which the genetic insertions may
have inadvertently caused an oncogenic mutation. Accordingly, as a
non-limiting example, one could introduce into a construct used in
the method of the invention a cassette that encodes the cDNA for
HPRT in order to render the transduced cells susceptible to
6-thioguanine. Another non-limiting example is the introduction of
the thymidine kinase cDNA from a Herpes-simplex virus family member
(HSV-TK), in order to render the transduced cells susceptible to
relevant inhibitors such as Ganciclovir, Acyclovir, and any
relevant derivatives. In addition, any other such drug sensitivity
cassettes and their relevant agonists would work in this
context.
[0131] Other methods of introducing nucleic acids or proteins
according to the present invention into a cell will be apparent to
those of skill in the art. Those that minimize or eliminate the
risk of introducing recombinant DNA into a host cell genome are
preferred by the invention, many such examples being described
above.
Methods of Use for Conditionally Immortalized Cells of the
Invention
[0132] Another embodiment of the present invention includes any of
the stem cell populations, including mixed and clonal populations,
that are produced by the method of the invention, as well as the
use of the stem cells of the invention in any of the methods
described herein, including differentiation into a desired cell
type, and any method of transplantation, cell replacement, disease
therapy, genetic engineering, drug discovery, and investigation of
cell development and differentiation as described herein.
[0133] Since one can now produce virtually unlimited supplies of
homogeneous stem cells that can readily be stored, recovered,
expanded and manipulated, such stem cells can be used as stem cells
or differentiated into various cell lineages and used in assays to
test various compounds for effects on cell differentiation, gene
expression, and cell processes. Therefore, one embodiment of the
invention relates to a method to identify compounds that effect
cell differentiation, gene expression, and/or cell processes. The
method generally includes the steps of contacting stem cells
produced by the method of the present invention with a compound to
be tested, and measuring a particular result, and particularly a
desired result, such as gene expression, a biological activity,
cell differentiation, cell growth, cell proliferation, etc. (see
below), as compared to in the absence of the compound, to determine
whether or not the test compound had the desired effect on the stem
cell. This method can be used to test for virtually any aspect of
cell differentiation, cell activity or gene expression. In one
aspect, the stem cells are manipulated prior to contact with the
compounds, such as by genetic manipulation. Stem cells from
individuals with genetic defects can be evaluated in such assays in
order to identify therapeutic compounds (e.g., cancer therapeutics)
and to evaluate gene replacement therapies, for example. Indeed,
the technology of the present invention provides an opportunity to
target the cells of a specific individual to identify drug
candidates and therapeutic candidates and strategies that are
"tailored" to the cells of an individual. Furthermore, as discussed
above, such assays can also be used to identify other growth
factors or culture conditions that are suitable for maintaining the
stem cells of the invention in culture. An example of such an assay
is described in detail below in Example 7, although the invention
is not limited to this assay.
[0134] Another embodiment of the invention relates to a method to
study cell lineage commitment and/or differentiation and
development of cells from a stem cell, which generally comprises
culturing the conditionally immortalized stem cells of the present
invention and evaluating such cells for genetic and biological
markers related to cell development and differentiation under
various conditions and in the presence and absence of compounds or
agents that may affect cell lineage commitment or differentiation.
As discussed above, prior to the present invention, such studies
were severely hampered by the lack of access to and the inability
to generate sufficient numbers of the desired cell population to
perform desired experiments. For example, in order to identify or
screen for intermediates in the differentiation of a particular
progenitor cell line, a sufficient number of cells must be obtained
to provide meaningful and reproducible results. Using technologies
available at the time of the invention, this was not possible.
However, the present invention solves the problem by providing
expandable and essentially unlimited supplies of homogeneous stem
cells that can be used in a variety of experiments. This technology
will greatly enhance research capabilities in the area of cell
differentiation and discovery. In one aspect, conditionally
immortalized stem cells of the invention are expanded, and then a
subset are cultured in the absence of the conditions that maintain
the cells in the conditionally immortalized state (e.g., in the
absence of tamoxifen, according to the exemplary method illustrated
herein). The cells can be evaluated for changes in gene expression,
cell surface markers, secretion of biomolecules, or any other
genotypic or phenotypic marker, to study the process of cell
differentiation and lineage commitment. Growth factors or other
factors can be added to the cultures, for example to drive
differentiation down a particular cell lineage pathway, and the
changes in the cells can be evaluated in the presence or absence of
such factors. Furthermore, the cells can be used to evaluate
culture conditions, in vivo conditions, factors, and agents that
influence (regulate) cell differentiation and development.
[0135] Various methods of detection of changes in genotypic or
phenotypic characteristics of cells in any of the assays of the
invention are known in the art. Examples of methods that can be
used to measure or detect gene sequence or expression include, but
are not limited to, polymerase chain reaction (PCR), reverse
transcriptase-PCR (RT-PCR), in situ PCR, quantitative PCR (q-PCR),
in situ hybridization, Southern blot, Northern blot, sequence
analysis, microarray analysis, detection of a reporter gene, or
other DNA/RNA hybridization platforms. Methods to measure protein
levels, include, but are not limited to: Western blot, immunoblot,
enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA),
immunoprecipitation, surface plasmon resonance, chemiluminescence,
fluorescent polarization, phosphorescence, immunohistochemical
analysis, matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectrometry, microcytometry,
microarray, microscopy, fluorescence activated cell sorting (FACS),
flow cytometry, and assays based on a property of the protein
including but not limited to DNA binding, ligand binding,
interaction with other protein partners, cell signal transduction,
enzyme activity, and secretion of soluble factors or proteins.
[0136] In drug screening assays, the term "test compound",
"putative inhibitory compound" or "putative regulatory compound"
refers to compounds having an unknown or previously unappreciated
regulatory activity in a particular process. As such, the term
"identify" with regard to methods to identify compounds is intended
to include all compounds, the usefulness of which as a compound for
a particular purpose (e.g., regulation of cell differentiation) is
determined by a method of the present invention, preferably in the
presence and absence of such a compound. Compounds to be screened
in the methods of the invention include known organic compounds
such as antibodies, products of peptide libraries, and products of
chemical combinatorial libraries. Compounds may also be identified
using rational drug design. Such methods are known to those of
skill in the art and can involve the use of three-dimensional
imaging software programs. For example, various methods of drug
design, useful to design or select mimetics or other therapeutic
compounds useful in the present invention are disclosed in Maulik
et al., 1997, Molecular Biotechnology: Therapeutic Applications and
Strategies, Wiley-Liss, Inc., which is incorporated herein by
reference in its entirety.
[0137] In any of the above-described assays, the conditions under
which a cell, cell lysate, nucleic acid molecule or protein of the
present invention is exposed to or contacted with a putative
regulatory compound, such as by mixing, are any suitable culture or
assay conditions, which can include the use of an effective medium
in which the cell can be cultured (e.g., as described above) or in
which the cell lysate can be evaluated in the presence and absence
of a putative regulatory compound. Cells of the present invention
can be cultured in a variety of containers including, but not
limited to, tissue culture flasks, test tubes, microtiter dishes,
and petri plates. Culturing is carried out at a temperature, pH and
carbon dioxide content appropriate for the cell. Such culturing
conditions are also within the skill in the art, and particularly
suitable conditions for culturing conditionally immortalized stem
cells of the present invention are described in detail elsewhere
herein. Cells are contacted with a putative regulatory compound
under conditions which take into account the number of cells per
container contacted, the concentration of putative regulatory
compound(s) administered to a cell, the incubation time of the
putative regulatory compound with the cell, and the concentration
of compound administered to a cell. Determination of effective
protocols can be accomplished by those skilled in the art based on
variables such as the size of the container, the volume of liquid
in the container, conditions known to be suitable for the culture
of the particular cell type used in the assay, and the chemical
composition of the putative regulatory compound (i.e., size, charge
etc.) being tested.
[0138] In one embodiment of the invention, the cells and methods of
the invention are useful for methods directed at evaluating
pluripotency of ctlt-HSCs derived from human cord blood, CD34+
cells, or adult CD34+ cells isolated from peripheral blood. Such a
method is described in Example 11.
[0139] Yet another embodiment of the invention relates to the use
of ctlt-HSC cell lines as a platform to generate novel models of
Acute Myeloid Leukemia (AML). More particularly, the present
inventors have generated a mouse model of acute myeloid leukemia
using the ctlt-HSCs of the invention. These are leukemias composed
of cells that resemble HSCs, based on their surface marker
expression. In order to generate ctlt-HSCs to promote leukemia in
mice, 10.sup.3-10.sup.5 ctlt-HSCs are transferred along with
10.sup.5 Rag-1.sup.-/- whole bone marrow cells into lethally
irradiated recipient mice. The mice are given weekly doses of 4-OHT
in order to maintain oncogene activity, and monitored for clinical
signs associated with leukemia, as known in the art. Tumors have
been recovered from these animals and they can be propagated in
culture in the absence of 4-OHT. Those cells retain their HSC-like
phenotype, indicating that they are no longer exquisitely dependent
upon MYC hyperactivity in order for proliferation, survival and
arrested differentiation. The leukemic cell lines can also confer
the disease upon secondary transplantation to irradiated recipient
mice. These tools provide a novel platform for studying the biology
and exporting new therapeutic avenues for AML and related diseases.
Furthermore, the introduction of ctlt-HSC cell lines into mice that
are treated with 4-OHT will provide a good built-in positive
control for therapy: the withdrawal of 4-OHT. The secondary cell
lines that arose after the establishment of tumors in vivo can also
be used to understand the relevant therapeutic targets for drug
resistant forms of AML.
[0140] Other embodiments of the present invention relate to the use
of the stem cells generated by the method of the present invention,
as well as cells differentiated from those stem cells, in a variety
of therapeutic and health-related methods. These methods generally
include the steps of obtaining a population, culture or line of
conditionally immortalized stem cells produced by the method of the
present invention, removing the conditions under which such cells
are conditionally immortalized, and then using the cells in a
therapeutic protocol. For example, the cells can be administered
directly to an individual in need of the cells or the cells can be
differentiated into a desired cell type in vitro and then
administered to an individual. In addition, prior to or just after
the removal of the conditions under which the cells are
immortalized, the cells can be genetically modified in vitro to
express or silence a gene or genes, as a novel method of gene
therapy under a controlled environment. The cells can then be
administered to an individual as stem cells or first differentiated
in vitro to a desired cell lineage.
[0141] To obtain the stem cells, in one embodiment, stem cells are
obtained from the individual to be treated, and are then
conditionally immortalized according to the method of the
invention. These cells can be expanded extensively, stored (e.g.,
frozen or cryopreserved), and then retrieved and expanded again,
manipulated, and/or used repeatedly as required. In another
embodiment, one obtains the stem cells by accessing a previously
stored source of conditionally immortalized stem cells from the
individual to be treated. In yet another embodiment, the stem cells
are obtained from a panel of human stem cell lines that were
previously generated and which cover a significant percentage of
the population according to the current criteria used to identify
"matching" donors. In one embodiment, the cells are obtained from
fresh, or cryopreserved cord blood, hematopoietic progenitor
populations that can be derived from the directed differentiation
of ES cells in vitro, HSCs obtained from the peripheral blood of
normal, or G-CSF treated patients who have been induced to mobilize
their lt-HSCs to the peripheral circulation. Other sources of stem
cells will be apparent to those of skill in the art. The cells are
cultured according to the methods described previously herein and
the conditions controlling immortalization can be removed at the
appropriate time. In addition, prior to administration of the cells
to an individual, the cells can be manipulated to excise the genes
or constructs that are responsible for the conditional
immortalization (i.e., the protooncogene and/or the anti-apoptosis
encoding gene), or if the cells are maintained through the use of
soluble fusion proteins in the culture medium, as described above
for the Tat-fusions, these soluble proteins can be removed from the
culture gradually or immediately.
[0142] Therefore, the present invention includes the delivery of
stem cells produced by the method of the invention (including
compositions comprising such stem cells), or cells differentiated
from these cells, to an individual (which can include any animal).
Since the stem cells used in these methods are produced in vitro,
even if stem cells were initially isolated from the patient, the
entire administration process of the cells is essentially an ex
vivo administration protocol. Ex vivo administration refers to
performing part of the regulatory step outside of the patient, such
producing the conditionally immortalized stem cells that were
removed from an individual (which can include producing genetically
modified stem cells in addition to essentially normal stem cells),
and returning the cells, or cells differentiated from these cells,
to the patient. The stem cells produced according to the present
invention or cells differentiated therefrom can be returned to an
individual, or administered to an individual, by any suitable mode
of administration. Such administration can be systemic, mucosal
and/or proximal to the location of a target site. The preferred
routes of administration will be apparent to those of skill in the
art, depending on the type of condition to be prevented or treated
or the reason for administration. Preferred methods of
administration include, but are not limited to, intravenous
administration, intraperitoneal administration, intramuscular
administration, intranodal administration, intracoronary
administration, intraarterial administration (e.g., into a carotid
artery), subcutaneous administration, transdermal delivery,
intratracheal administration, subcutaneous administration,
intraarticular administration, intraventricular administration,
intraspinal, pulmonary administration, impregnation of a catheter,
and direct injection into a tissue (e.g., such as cannulation of
the liver, for example).
[0143] The cells can be administered with carriers or
pharmaceutically acceptable excipients. Carriers are typically
compounds that increase the half-life of a therapeutic composition
in the treated individual. Suitable carriers include, but are not
limited to, polymeric controlled release formulations,
biodegradable implants, liposomes, oils, esters, and glycols. As
used herein, a pharmaceutically acceptable excipient refers to any
substance suitable for delivering cells produced by the method of
the present invention to a suitable in vivo site. Preferred
pharmaceutically acceptable excipients are capable of maintaining a
cells in a form that, upon arrival of the cells at a target tissue
or site in the body, the cells are capable of functioning in a
manner that is beneficial to the individual.
[0144] According to the present invention, an effective
administration protocol comprises suitable dose parameters and
modes of administration that result in delivery of a useful number
of functional cells to a patient in order to provide a transient or
long-term benefit to the patient. Effective dose parameters can be
determined using methods standard in the art for a particular
condition or disease. Such methods include, for example,
determination of survival rates, side effects (i.e., toxicity) and
progression or regression of disease.
[0145] A suitable single dose of stem cells or cells differentiated
therefrom according to the present invention is a dose that is
capable of providing a beneficial number of cells to a patient,
when administered one or more times over a suitable time period.
For example, a preferred single dose of stem cells according to the
present invention is from about 0.5.times.10.sup.4 to about
5.5.times.10.sup.8, or from about 0.5.times.10.sup.5 to about
5.5.times.10.sup.7, or from about 0.5.times.10.sup.6 to about
5.5.times.10.sup.10 stem cells per individual per administration,
with doses from about 1.times.10.sup.8 to about 5.5.times.10.sup.10
being even more preferred. Any dose in between 0.5.times.10.sup.4
and about 5.5.times.10.sup.10 is encompassed by the invention, in
increments of 10.sup.2 cells. Higher or lower doses will be known
to those of skill in the art depending on the type of stem cell or
differentiated cell to be administered, and also depending on the
route of administration. It will be obvious to one of skill in the
art that the number of doses administered to an animal is dependent
upon the extent of the condition or disease and the response of an
individual patient to the treatment. Thus, it is within the scope
of the present invention that a suitable number of doses includes
any number required to treat a given disease.
[0146] As used herein, the phrase "protected from a disease" refers
to reducing the symptoms of the disease; reducing the occurrence of
the disease, and/or reducing the severity of the disease.
Protecting an animal (an individual, a subject) can refer to the
ability of cells produced according to the present invention, when
administered to an animal, to prevent a disease from occurring
and/or to cure or to alleviate disease symptoms, signs or causes.
As such, to protect an animal from a disease includes both
preventing disease occurrence (prophylactic treatment) and treating
an animal that has a disease or that is experiencing initial
symptoms of a disease (therapeutic treatment). The term, "disease"
refers to any deviation from the normal health of a mammal and
includes a state when disease symptoms are present, as well as
conditions in which a deviation (e.g., infection, gene mutation,
genetic defect, etc.) has occurred, but symptoms are not yet
manifested.
[0147] As discussed above, the stem cells of the present invention
can be administered to an individual to treat or prevent a variety
of conditions. For example, the stem cell lines of the present
invention provide a unique source of expandable stem cells for use
in a variety of transplantation and therapeutic strategies,
including the treatment of cancer, and particularly, cancer that is
treated by radiation. In addition, a variety of immune deficiency
disorders and anemia disorders (e.g., aplastic anemia or hemolytic
anemia) will also benefit greatly from this technology, since the
present invention provides the ability to repopulate hematopoietic
cells of an individual as needed by the individual. Another
application of the present invention relates to the generation of
continuously expandable and renewable hair follicle stem cells, for
use, for example in the context of reconstructive surgery for burn
victims, for any individual that undergoes chemotherapy and/or
radiation therapy resulting in the irreversible loss of hair
growth, as well as patients following any surgical procedure
affecting the skull or in elective procedures that involve the
induction of hair growth in individuals affected by hereditary
pattern baldness. Similarly, application of the present invention
to stem cells of the skin will be invaluable for use in wound
healing and treatment of burn victims, as well as plastic
reconstructive surgery for trauma and other patients, as well as
elective surgeries, including, but not limited to, cosmetic
surgery. Such cells can be additionally genetically manipulated to
correct inborn or acquired genetic defects in young and aged
individuals. One of skill in the art will understand based on this
disclosure that benefits can be derived from the use of the present
invention on various other stem cell populations, including, but
not limited to, stem cells derived from lung, breast, and
intestinal epithelium and stem cells derived from neural and
cardiac tissue, to name just a few.
[0148] In addition, as discussed above, the present invention
provides the unique opportunity for an individual to have access to
expandable supplies of autologous stem cells and cells
differentiated therefrom as needed throughout the life of the
individual. Such stem cells generated by the present method can be
stored and used as part of therapeutic protocols during the
lifetime of the individual, should they be needed (e.g., in the
event the individual develops a cancer or immune deficiency
disease).
[0149] Genetic defects can now be corrected or beneficial gene
modifications can be introduced into somatic cells by manipulating
autologous stem cells obtained from an individual that have been
conditionally immortalized and expanded using the method of the
present invention. The stem cells can then be reintroduced into the
individual from whom they were obtained.
[0150] Additional applications of the present invention include the
use of stem cell lines to repair lung injury that occurs as a
result of COPD, IPF, emphysema, asthma and smoking. In addition,
such cells could be used to treat blood vessel damage in the heart,
and help in autoimmune diseases after lethal irradiation (e.g.,
SLE, diabetes, RA).
[0151] In the method of the present invention, cells produced
according to the method of the invention and compositions
comprising the cells can be administered to any animal, including
any member of the Vertebrate class, Mammalia, including, without
limitation, primates, rodents, livestock and domestic pets. A
preferred mammal to treat is a human.
[0152] Various aspects of the present invention are described in
more detail in the following Examples and the attached figures.
However, the present invention is not limited to these examples and
illustrations of the invention.
EXAMPLES
Example 1
[0153] The following example describes the development of a method
to reversibly immortalize long-term hematopoietic stem cells
(lt-HSCs).
[0154] Elucidation of the molecular basis of the impairment in
hematopoietic lineage development has been complicated historically
by the low frequency of relevant cell populations, which prevents
biochemical analysis of signaling and downstream responses. In
fact, this has been a major limiting factor in all studies of
hematopoiesis. In addition, the limited availability of LT-HSCs has
also been a major obstacle in the treatment of many types of cancer
as well as several kinds of immune deficiencies in humans.
[0155] In an effort to overcome this limitation, the present
inventors developed a method to produce conditionally transformed
cell lines representing early hematopoietic stem cell progenitors.
The initial strategy involved retroviral transduction of bone
marrow stem cells from 5FU treated young and immunologically aged
3-83 mice. The inventors utilized the pMSCV bisistronic retroviral
vector with inserts encoding Bcl-2 and GFP, and MYC-ER and GFP [Van
Parijs, L., Y. Refaeli, A. K. Abbas, and D. Baltimore. (1999)
Autoimmunity as a consequence of retrovirus-mediated expression of
C-FLIP in lymphocytes. Immunity, 11, 763-70]. These genes were
selected because the present inventors knew that MYC has the
ability to replace cytokine derived survival and proliferative
signals in lymphocytes. By restricting the target cell, the
inventors hypothesized that stem cell tumors might form.
Importantly, MYC-ER function is tamoxifen dependent in this
setting, allowing the termination of MYC function and
transformation by withdrawing tamoxifen from the animal or
cultures. In cells transduced with MYC-ER, the fusion protein is
produced, but is retained in the cytoplasm until exposed to
tamoxifen.
[0156] More specifically, stem cell populations from 5FU treated
mice were transduced with both retroviruses (encoding MYC-ER and
Bcl-2) and transferred into lethally irradiated recipient mice
(1200 rads). Ten days later, weekly intraperitoneal injections of 1
mg/mouse of 4-hydroxytamoxifen (40HT) emulsified in oil were
initiated to activate MYC function (FIG. 1). Within four weeks,
recipients of young (but not old) transduced stem cells developed
tumors. The tumors were harvested from bone marrow, spleen and
lymph nodes and cultured in vitro with tamoxifen, but without added
cytokines. These cells grew for about 10 days, but then growth
stopped and the cells eventually died. the inventors suspected that
the cells were differentiating and considered that this might have
been due to requirements for cytokines for growth of the cells.
Referring to FIG. 1, the curves represent the kinetics of mortality
after transplantation and activation of MYC function in vivo. The
mice uniformly succumbed to leukemias. While the overexpression of
MYC can replace the cytokine-dependent proliferation and survival
function, it does not seem to be involved in the cytokine-derived
differentiation signals.
[0157] When ill, the mice were euthanized. Bone marrow, spleen and
lymph node cells were harvested and placed in culture with
tamoxifen and a stem cell growth factor cocktail (IL-6, IL-3 and
stem cell factor (SCF)). In parallel, cells were analyzed by flow
cytometry (FIG. 2). Referring to FIG. 2, the dot plots represent
the flow cytometric data for the forward (FSC) and side (SSC)
scatter characteristics of the HSCs after three days in culture
with IL-3, IL-6 and SCF. These two criteria correlate with cell
size (FSC) and granularity (SSC). The two populations have similar
profiles. The histograms represent the levels of GFP expressed in
each cell population. This reflects the efficiency of retroviral
transduction in vitro with retroviruses that encode cDNAs for
MYC-ER and Bcl-2.
[0158] In all cases, ex vivo GFP.sup.+ cells were >90%
Sca-1.sup.+ and Lineage marker negative. After a few days in
culture, cells began to grow and approximately 400 lines were
frozen for later study. After propagation, these cells retained
expression of EGFP and were homogeneously positive for SCA1 and
negative for CD34, F1k2 and lineage markers (FIG. 3). The only
difference in marker expression between young mouse-derived and
aged mouse-derived markers was increased expression of c-kit in
young. Without being bound by theory, the present inventors believe
that this may have resulted from longer culture (3 months vs. 3
weeks) of aged lines in c-kit ligand before markers were analyzed.
Finally, the inventors discovered that these lines can be recovered
easily after freezing and retained their original phenotype.
Importantly, these cell lines are homogenous in phenotype and
exhibit the phenotype of lt-HSC that provide all long term
reconstitution in mice (Reya, T., Duncan, A. W., Ailles, L., Domen,
J., Scherer, D. C., Willert, K., Hintz, L., Nusse, R., and
Weissman, I. L. (2003). A role for Wnt signaling in self-renewal of
hematopoietic stem cells. Nature 423, 409-14).
[0159] Recently, the inventors thawed 10 bone marrow derived lines
produced as described above, and were able to recover 9 out of 10
of these lines easily by culture in the cytokine cocktail and 4OHT.
The inventors phenotyped these tumors, and the results were
extremely promising. Specifically, each line contained two distinct
cell populations based on forward and 90.degree. light scatter. The
nine lines differed only in the proportionality of these
populations. The larger of these populations in cell size were
uniformly GFP bright and positive for Sca1, Endoglin and ckit but
negative for Flt3, B220, CD19 and mIgM. CD34.sup.- also appeared to
be negative, although this required confirmation (FIGS. 3A and 3B).
This phenotype corresponds perfectly with the published
characteristics of long term repopulating pluripotent stem cells
(Reya et al., supra). The inventors observed the same initial
phenotype on the cell lines that they recently obtained from
leukemias that developed from transduced HSCs obtained from young
donor mice (FIGS. 3A and 3B).
[0160] To test the ability of these cells to differentiate,
representative lines were cultured with and without tamoxifen and
in the presence of IL-3, IL-6 and SCF to terminate MYC-ER function
for 7 days before analyzing phenotypic markers. As shown in FIG. 4,
a significant proportion of cells acquired B lineage markers
including B220 (.about.12%), CD19 (.about.10%) and mIgM
(.about.10%). In addition, the inventors have been able to generate
the following lineages in vitro by withdrawal of 4OHT from the
cultures: CD4+ab T-cells, myeloid cells (Mac-1+), ter-119+
erythroid progenitor cells, NK1.1 expressing cells, neutrophils
(Gr-1+ cells). Further experiments will assess the ability of these
cells to give rise to other lineages, as well as the effect of
altering the cytokine regimen on differentiation. Although the
comparison has not been performed, the present inventors expect
differentiation from young animals, as compared to aged animals to
be much more efficient in B cell production. To the best of the
present inventors' knowledge, this is the first example of a
conditionally immortal hematopoietic stem cell line that can be
induced to differentiate in vitro.
Example 2
[0161] The following example describes the results of adoptive
transfer of LT-HSC lines into lethally irradiated recipients.
[0162] If the HSC lines described in Example 1 are to be
appropriate subjects for analysis of the basis of defective B cell
lymphopoiesis in aged animals, they should recapitulate the defect
in vivo. The inventors have begun to address this question by
adoptive transfer of LT-HSC lines into lethally irradiated
recipients. In initial experiments, lines from aged animals
(>60% ID.sup.-) were transferred along with RAG2.sup.-/- bone
marrow, and recipients were not treated with tamoxifen in order to
silence MYC-ER. Six weeks later recipient bone marrow and spleen
cells were harvested and the recovery and phenotype of GFP.sup.+
cells (GFP marks cells derived from HSC lines) was analyzed (FIGS.
5A and 5B).
[0163] In the data from three mice presented in FIGS. 5A and 5B,
one mouse received the aged HSC line ABM42, and two mice received
aged HSC line ABM46. Depending upon the line transferred, 30 to 70%
of cells in the lymphoid scatter gate were GFP.sup.+. As shown in
FIG. 5, both lines tested (ABM46 and ABM42) gave rise to B
(CD19.sup.+) and T (TCR.sup.+, CD4.sup.+, CD8.sup.+) cells,
macrophages (CD11b.sup.+) and granulocytes (GR1.sup.+). There was
some recipient to recipient variation in the proportionality of
these lineages. However, importantly, while both lines tested gave
rise to mature CD4 and CD8 single positive T cells (FIG. 7), B cell
development did not proceed beyond the progenitor stage (FIG. 6).
While B220.sup.+, CD19.sup.+ cells developed, they did not progress
to the mIg.sup.+ stage. This is precisely the outcome predicted by
results of experiments involving autoreconstitution and adoptive
reconstitution using BM HSC from immunologically aged mice
(Johnson, S. A., S. J. Rozzo, and J. C. Cambier, Aging-dependent
exclusion of antigen-inexperienced cells from the peripheral B cell
repertoire. J Immunol, 2002. 168(10): p. 5014-23). In other words,
the same developmental arrest is observed when whole bone marrow
from immunologically aged mice is used for transplantation.
[0164] The inventors have found that this system can be taken a
step further, successfully re-establishing LT-HSC lines from bone
marrow of adoptive recipients of the original HSC lines (data not
shown). This was accomplished simply by culturing bone marrow cells
in stem cell cytokines plus tamoxifen to reactivate MYC. These
cells are now growing and exhibit the original phenotype.
Example 3
[0165] The following example describes a method for reversibly
immortalizing HSCs using a method conducted entirely in vitro.
[0166] In addition to the method for generating conditionally
immortalized long term HSC cell lines described previously herein,
the inventors have been able to carry out this procedure completely
in vitro. The method described above relies upon introducing the
transduced HSC's into mice, and inducing their transformation in
vivo. The advantage of carrying this procedure out in vitro is that
every aspect of the process is carried out in a controlled
environment.
[0167] The method first includes the treatment of donor mice with
5-fluorouracil (5-FU) in order to enrich for HSCs and induce these
cells to proliferate. 5FU enriched hematopoietic stem cells from
the tibia and femurs of mice were collected and then plated in 24
well tissue culture plates in DMEM media containing 15% heat
inactivated fetal calf serum and IL-3, IL-6 and SCF, at a density
of 1.8-2.0.times.10.sup.6 cells per well. The cells were subjected
to three rounds of spin infection in order to retrovirally
transduce the cells with retroviral vectors encoding MYC-ER and
Bcl-2. Briefly, the cells were transfected with pMIG-MYC.ER or
pMIT-Bcl2. The virus containing supernatants were collected and
supplemented with 4 .mu.g/ml of polybrene and 10 mM HEPES, and
passed through a 0.45 .mu.m filter. The two different viral
supernatants were mixed at a 1:1 ratio and added to the wells. The
cells were then centrifuged at 2000 rpm for one hour. The viral
supernatants were replaced at the end of each spin infection. 24
hours after the last round infection, the levels of transduction
were determined by flow cytometric analysis in order to determine
the transduction efficiency. The transduced cells were then
incubated in DMEM medium containing IL-3, IL-6, SCF and 10 nM 4OHT.
The medium was replaced every 3 days and special emphasis was
placed on ensuring a fresh supply of cytokines and 4OHT. Cells are
passed slowly, and as needed.
[0168] Using this in vitro approach, the inventors have been able
to generate conditionally immortalized cell lines with the
following combinations of genes: MYC-ER and Bcl-2; MYC-ER and hTERT
(reverse transcriptase component of the human telomerase); ICN-1-ER
(ER-regulated active element of the intracellular portion of
Notch-1) and Bcl-2; ICN-1-ER and hTERT; and MYC-ER and ICN-1-ER.
The data presented in FIGS. 8-11 show the initial characterization
of most of these cell lines. They yielded lines composed of c-kit+,
Sca-1+, CD34-, flk2- cells, which is a phenotype that is consistent
with the one presented by normal long-term hematopoietic stem
cells. The data presented in FIGS. 8-11 is derived from the flow
cytometric analysis of retrovirally encoded reporter genes (GFP and
thy1.1), as well as four markers for stem cells: c-kit, sca-1, CD34
and flk-2. The cell lines shown in FIGS. 8-11 had been in culture
for 5 weeks prior to phenotyping. These cells have been expanded
and divided in continuous culture for over 35 days to date.
[0169] Referring to FIG. 8, this figure shows the phenotypic
comparison of cell lines derived from HSCs obtained from young
C57/BL6 mice that were retrovirally transduced with BCL-2 and
MYC-ER and maintained in continuous in vitro culture for >90
days. Shown is the phenotype of representative clones 3 (young)
months after 90 days of continuous of culture.
[0170] Referring to FIG. 9, this figure shows the phenotypic
comparison of cell lines derived from HSCs obtained from young
C57/BL6 mice that were retrovirally transduced with different
combinations of oncogenes and maintained in continuous in vitro
culture for >90 days. 5FU enriched HSCs were retroviral
transduced with pMIG-MYC and pMIT-Bcl-2 (top panels), pMIG-MYC.ER
and pMIG-hTERT (middle panels), or pMIG-ICN.1.ER and pMIT-Bcl-2.
The cells were maintained in DMEM supplemented with 15% fetal calf
serum, and a cocktail of IL-6, IL-3 and SCF. Shown is the phenotype
of representative clones 3 (young) months after 90 days of
continuous of culture. The panels represent the results of the flow
cytometric analysis for expression of the viral expression markers
(GFP and Thy1.1), as well as four markers required to define
long-term HSCs in mice, Sca-1, c-kit, CD34 and Flk-2. The four cell
lines contained subpopulations that retained the phenotypes of
lt-HSCs (Sca-1+, c-kit+, CD34-, flk-2-).
[0171] Referring to FIG. 10, this figure shows the phenotypic
comparison of cell lines derived from HSCs obtained from young
C57/BL6 mice that were retrovirally transduced with different
combinations of oncogenes and maintained in continuous in vitro
culture for >90 days. 5FU enriched HSCs were retroviral
transduced with pMIG-ICN.1.ER and pMIT-Bcl-2 (top panels),
pMIG-ICN.1 and pMIT-Bcl-2 (second row panels), or pMIG-ICN.1 and
pMIG-Bcl-2 (third row panels), or pMIG-hTERT and pMIT-Bcl-2 (bottom
panels). The cells were maintained in DMEM supplemented with 15%
fetal calf serum, and a cocktail of IL-6, IL-3 and SCF. Shown is
the phenotype of representative clones 3 (young) months after 90
days of continuous of culture. The panels represent the results of
the flow cytometric analysis for expression of the viral expression
markers (GFP and Thy1.1), as well as four markers required to
define long-term HSCs in mice, Sca-1, c-kit, CD34 and Flk-2. The
four cell lines contained subpopulations that retained the
phenotypes of lt-HSCs (Sca-1+, c-kit+, CD34-, flk-2-).
[0172] Referring to FIG. 11, this figure shows the phenotypic
comparison of cell lines derived from HSCs obtained from young
C57/BL6 mice that were retrovirally transduced with different
combinations of oncogenes and maintained in continuous in vitro
culture for >90 days. 5FU enriched HSCs were retroviral
transduced with pMIG-MYC and pMIG-ICN.1 (top panels), pMIG-MYC.ER
and pMIG-ICN.1 (middle panels), or pMIG-ICN.1.ER and pMIG-MYC. The
cells were maintained in DMEM supplemented with 15% fetal calf
serum, and a cocktail of IL-6, IL-3 and SCF. Shown is the phenotype
of representative clones 3 (young) months after 90 days of
continuous of culture. The panels represent the results of the flow
cytometric analysis for expression of the viral expression markers
(GFP and Thy1.1), as well as four markers required to define
long-term HSCs in mice, Sca-1, c-kit, CD34 and Flk-2. The four cell
lines contained subpopulations that retained the phenotypes of
lt-HSCs (Sca-1+, c-kit+, CD34-, flk-2-).
[0173] These cell lines have also been used to reconstitute
cellular compartments in vivo. Referring to FIG. 12, this figures
shows the results of in vivo reconstitution of T cell and B cell
compartments from cell lines derived from HSCs obtained from young
C57/BL6 mice that were retrovirally transduced with different
combinations of oncogenes and maintained in continuous in vitro
culture for >90 days. Briefly, 5FU enriched HSCs were retroviral
transduced with pMIG-ICN.1-ER and pMIG-hTERT (top panels),
pMIG-MYC.ER and pMIG-hTERT (middle panels), or pMIG-MYC-ER and
pMIT-Bcl-2 (lower panels). The cell lines were maintained in DMEM
supplemented with 15% fetal calf serum, and a cocktail of IL-6,
IL-3 and SCF. Lethally irradiated young C57/BL6 mice were
reconstituted using bone marrow stem cells from Rag2-/- mice and
LT-HSC lines generated in vitro. Six weeks later, bone marrow was
harvested and stained with a panel of specific lineage markers. The
development of mature CD4 and B220 positive/GFP positive cells can
readily be observed. Data from four representative mice are
presented in this figure. In each group, approximately 30% of the
mice retain GFP marker.
Example 4
[0174] The following example describes an extension of the method
for reversibly immortalizing human cord blood and bone marrow
derived HSCs in vitro.
[0175] One additional application of this technology is the ability
to expand human long-term hematopoietic stem cells in vitro through
their conditional immortalization. The inventors have therefore
adapted the in vitro method described in the previous examples for
human cells with a few changes. First, the retroviruses are
packaged preferably with amphotrophic envelopes in order to enable
efficient transduction of human cells. In addition, the source of
the cells is human cord blood obtained anonymously from the a cord
blood bank, following all rules and regulations set forth by the
Institutional Review Boards of the inventors' institutions. The
resulting cells will express reporter genes that may ultimately be
useful for isolating a pure population by high speed cell sorting.
The inventors have noticed that many mature cells resulting from
the murine lt-HSC cell lines lose expression of the surface
markers, potentially due to the methylation of the retroviral
genome upon lineage determination and differentiation. The
inventors expect to see similar behavior in the human cells, in
which case the lt-HSCs and their prevalence in transplant
recipients can be monitored by the presence of reporter genes in
such cells, in combination with cell surface markers for that
population of cells.
Example 5
[0176] The following example describes an approach to the
sequential excision of the DNA fragments encoding MYC-ER and Bcl-2
from conditionally immortalized HSC cells.
[0177] In order to avoid taking the risk of introducing HSCs that
harbor transgenes encoding MYC.ER and Bcl-2 into humans and/or
mice, these two DNA fragments will be excised using a bacterial
recombinase approach. Two different recombinases will be used in
order to allow control over which one of the two genes is excised
at any one point in time. Two examples of such recombinases are the
Cre and Flp recombinases. Briefly, the recognition substrate
sequences (RSS's) for one of the recombinases is introduced into
the retroviral constructs such that they flank the open reading
frame of the oncogene, as well as the reporter gene (GFP or
Thy1.1). In this case, the cells are incubated in media containing
a Tat-Cre fusion protein. This recombinant protein has been
previously described and shown to be able to passively enter cells,
and mediate loxP site-dependent recombination of genomic DNA.
[0178] This approach will allow the achievement of a number of
things in order to enable the generation of many HSCs for
differentiation in vitro and in vivo. First, the cells can
gradually be weaned from the high levels of proliferative and
survival signals they had become accustomed to during the
conditional transformation process. Second, the cells can be
re-adapted to depend on normal cytokines for their homeostatic
functions and differentiation. Third, the sequential loss of
reporter expression will allow the definition of the status and
degree of deletion of each one of the genes in question.
Accordingly, cells that express both reporter genes (GFP and
Thy1.1) harbor both sequences (MYC and Bcl-2, respectively), cells
that express Thy1.1 but no GFP have successfully deleted the MYC
encoding sequences, but still contain Bcl-2 genes, and lastly,
cells that do not express either GFP or Thy1.1 have deleted both of
those alleles. FIG. 12 represents this approach in a diagram.
[0179] In addition, this approach is tested in mice by obtaining
5FU enriched BM-HSCs from a strain of mice in which the expression
of a human MYC transgene can be induced by the withdrawal of
tetracycline and the presence of a bacterial protein called tTA
(tetracycline transactivator protein). The human MYC cDNA was
cloned downstream of a tetracycline regulatory transcription
element (TRE). The TRE-MYC mice are treated with 5FU and used to
harvest BM-HSCs. Those cells are transduced in vitro with
retroviruses expressing Bcl-2 and tTA (pMIT-Bcl2 and pMIG-tTA). The
cells are cultured in the continuous presence of Doxycycline in
order to maintain the MYC transgene silent. Once the cells are
analyzed by flow cytometry, they can be used for transplantation
back into mice that will not be maintained on a doxycycline
containing diet (this is a more stable form of tetracycline is
normally used in vivo).
[0180] Once the lt-HSC cell lines are generated, the effect of
culturing them in the presence of doxycycline in vitro will be
examined in parallel with MYC.ER harboring cell lines that will be
cultured in the absence of 4OHT. The protein levels of MYC are
monitored by western blots and intracellular staining approaches
throughout.
Example 6
[0181] The following example describes the generation of many
hematopoietic lineages in vitro, following the withdrawal of 4OHT
from the liquid tissue culture media.
[0182] The traditional methods used to determine the potency of an
HSC involve the use of semi-solid media (methycellulose) with
defined cytokines in order to potentiate the differentiation of
HSCs into specific lineages. The inventors were interested in
determining the pluripotency of this cell population created using
the method of the present invention in vitro. In order to examine
this issue, the ABM42 and ABM46 cell lines described herein were
maintained in media containing IL-3, IL-6 and SCF, but without
4OHT. In addition to the lineages that the inventors were able to
detect in the reconstituted mice (i.e., lymphoid, myeloid and
granulocytic), GFP+ cells could also be detected that expressed
NK1.1 or ter-119 (FIG. 13). The NK1.1 cells could either be
NK-cell, or NK-T cells. The ter-119 expressing cells are of the
erythroid lineage. These findings indicate that these cell lines
are capable of giving rise to all of the elements of a normal
hematopoietic system and that the cells will be useful for
generation of large quantities of specific elements to be used for
passive therapies. In addition, they will be of great use and
importance to study the early events in hematopoiesis and to
identify novel therapy for therapeutic intervention in genetic
disorders, or complications that arise the normal course of
chemotherapy, or even infectious disease.
Example 7
[0183] The following example describes a method for high throughput
screens of small molecules or biological agents that induce or
inhibit differentiation in conditionally transformed long term
HSCs.
[0184] The following is a general method for screening small
molecules or biological agents that induce or inhibit HSC
differentiation. Previously, these types of large screens were
prohibited by the fact that large numbers of stem cells were
unobtainable. With the present inventors current ability to
conditionally immortalize long term HSCs, it is now feasible to
propose such technologies.
[0185] By way of example, one such method is a myeloid
differentiation read-out that has been adapted from Schneider, et
al. (Schneider, T., and Issekutz, A. C. (1996). Quantitation of
eosinophil and neutrophil infiltration into rat lung by basic
assays for eosinophil peroxidase and myeloperoxidase. Application
in a Brown Norway rat model of allergic pulmonary inflammation. J
Immunol Methods 198, 1-14). Briefly, conditionally transformed long
term HSCs are plated in 96 well, flat bottom plates at various
concentrations of cell numbers (usually
2.times.10.sup.4-5.times.10.sup.4 cells/well). The screens are
carried out either in complete media (DMEM+15% heat inactivated
fetal calf serum, 1.times. penicillin/streptomycin,
1.times.1-glutamine and 1.times. non-essential amino acids,
supplemented with IL-3, IL-6 and SCF) with added 4OHT in order to
maintain the cells in an undifferentiated state, or in the absence
of added 4OHT in order to induce differentiation. These conditions
have been shown to give rise to Mac-1+ cells, consistent with a
myeloid differentiation pattern. Additional cytokines can be added
to direct differentiation in specific paths, although this system
can also be used to screen for specific functions of a panel of
cytokines. In this instance, the complete media will be added
without supplementation with IL-3, IL-6 and SCF, but instead with
the given cytokines to be tested or used to direct differentiation
(e.g., CSF-1, G-CSF, GM-CSF, EPO, TEPO, etc.).
[0186] Small molecules, biological agents or positive control
substances (e.g., Arsenic 0.sub.3) are titrated across the 96 well
plate and incubated with the ltHSCs for time frames ranging from 24
to 72 hours, or longer, if needed and as determined based on the
agents or molecules to be tested. After incubation, the cells are
washed with PBS and resuspended in PBS for overnight storage at
-80.degree. C. to lyse the cells. The cells are then thawed at room
temperature and the plates are centrifuged for 10 min at 3,000 rpm.
The supernatant is then transferred to a new 96 well plate and
mixed with tetramethybenzidine (TMB) for 40 min. The reaction is
stopped with 4N H.sub.2SO.sub.4 and the O.D. is read at 450 nm.
This type of high-throughput assay can be used to test small
molecules or biological agents for the ability to induce or block
the differentiation of conditionally transformed long term HSCs
into a wide variety of cell types. Results of these screens can
then further be tested for the ability to induce or inhibit HSC
differentiation in vivo. Variations on this assay format will be
apparent to those of skill in the art and are encompassed by the
present invention.
Example 8
[0187] The following example describes the use of the method of the
invention to generate cell lines of an intermediate hematopoietic
lineage.
[0188] The following protocol can be used to induce the development
of cell lines representing intermediate stages of hematopoietic
lineage development following transplantation of conditionally
immortalized lt-HSC cell lines into lethally irradiated mice.
First, 10.sup.3-10.sup.5 conditionally transformed lt-HSC cell
lines generated according to the method of the invention are
transferred into cohorts of lethally irradiated recipient mice. The
transplants will also include 10.sup.5 Rag-1.sup.-/- cells as
carriers in order to ensure the initial survival of the irradiated
mice. The mice are treated with weekly injections of 1 mg
tamoxifen, intraperitoneally, in order to immortalize partially
differentiated cells derived from the conditionally transformed
lt-HSC cell lines. Injections begin either 3 days-1 week after the
initial transplant, or 8 weeks after the transplant, once the mice
have been fully reconstituted by the conditionally transformed
lt-HSC cell lines. Cells are collected from the spleen and bone
marrow cells from mice three days after treatment with tamoxifen,
or when they show clinical signs associated with leukemias. The
cells are cultured in either the standard bone marrow culture
conditions with 4-OHT (DMEM, 15% fetal calf serum, pen/strep,
L-glut, non essential amino acids, IL-3, IL-6 and SCF), or in the
presence of other cytokines and medium used for different
hematopoietic cell types. Cell lines are frozen and/or expanded,
and cell lines are also single-cell cloned by limiting dilution and
defined by PCR amplification of proviral integrations, frozen, and
then characterized for surface marker expression by flow cytometry.
These types of approaches are used for both murine and human
ctlt-HSC cell lines, using either NOD/SCID mice as the recipients,
or neonatal Rag-1-/- mice, which will be given intrahepatic
injections.
Example 9
[0189] The following example describes the use of the method of the
invention and the adoption of protocols used to generate mature
CD4+.alpha..beta. T-cells in vitro to develop cell lines
representing intermediate stages of T-cell development.
[0190] In this experiment, conditionally immortalized lt-HSC cell
lines generated according to the method of the invention are plated
in the presence of the normal cytokine cocktail, supplemented with
IL-7 and without tamoxifen. Parallel cultures are established on a
layer of OP-9 stromal cells that express Jagged, a Notch-1 ligand.
Cells are stained for T-cell lineage markers every 48 hours after
the cultures are initiated to monitor for signs of T-cell
development. The wells that show signs of T-lineage commitment and
development are switched to media containing tamoxifen in order to
stabilize the phenotype and establish cell lines. The resulting
cell lines are expanded, cloned and characterized as described in
Example 8. The T-cell lines are specifically stained for individual
TCR-V.beta. alleles in order to determine their T-cell receptor
repertoire usage. Some mature T-cell lines, or cell lines
representing progenitor populations, are transplanted into
Rag-1.sup.-/- mice in order to evaluate their ability to conform to
normal tolerance and homeostatic mechanisms in vivo, as well as
their ability to further differentiate in vivo, when appropriate.
Finally, their ability to respond to antigenic stimulation is
evaluated in vitro and in vivo.
Example 10
[0191] The following example describes the use of the method of the
invention and the adoption of protocols used for the directed
differentiation of HSCs into myeloid cell lineages to develop
intermediate developmental cell lines and myeloid leukemia
models.
[0192] In this experiment, conditionally immortalized lt-HSC cell
lines generated according to the methods of the present invention
are plated in the presence of the normal cytokine cocktail,
supplemented with G-CSF and without tamoxifen. Cells are stained
for myeloid lineage markers every 48 hours after the cultures are
initiated to monitor for signs of myeloid development. The wells
that show signs of myeloid lineage commitment and development are
switched to media containing tamoxifen in order to stabilize the
phenotype and establish cell lines. The resulting cell lines are
expanded, cloned and characterized as described in Example 8. Some
of the resulting cell lines are transplanted back into mice in
order to monitor their ability to repopulate Op/Op mice (mutant
mice that naturally lack macrophages). Those cell lines are also
transplanted into wild type mice that will be maintained on
tamoxifen throughout, in order to determine if these cell lines
will also give rise to myeloid leukemias similar to human AML, CML
and APL. These novel tumors provide novel models for preclinical
therapeutics.
Example 11
[0193] The following example describes the generation of Human
adult ctlt-HSC cell lines and examination of their pluripotential
in vivo using NOD/SCID or RAG.sup.-/- xenotransplant models.
[0194] In this experiment, CD34+ cells (from mobilized blood or
cord blood) are transduced in vitro with retroviral vectors
encoding MYC-ER, Bcl-2 and GFP (for later detection of transplanted
cells), packaged using amphotrophic envelopes (according to the
methods of the present invention). lt-HSC are selected by
propagation in vitro in the presence of 4OHT and growth factors, as
described above using murine HSCs. Pluripotency of the selected
cells is evaluated by transplantation of lt-HSC lines into
sublethally irradiated NOD/SCID or NOD/SCID/.beta.-2M.sup.-/- or
Rag-1.sup.-/- or Rag-2.sup.-/- mice, followed 6-12 weeks later by
analysis of all blood cell lineages by immunofluorescence flow
cytometry. More particularly, following the generation of ctlt-HSC
cell lines using the method of the present invention, one can use
two different and complimentary approaches to examine their
pluripotency. In a first approach, varying amounts of clonal
ctlt-HSC cell lines are introduced into sublethally irradiated
NOD/SCID mice or NOD/SCID/.beta.-2M.sup.-/- mice. In this instance,
10.sup.3-10.sup.5 cells derived from a human ctlt-HSC cell lines
are transferred intravenously after the mice are subjected to a
sublethal irradiation regimen (0.3 Gy). The mice are analyzed for
reconstitution at 6-12 weeks after transplantation. Second,
10.sup.3-10.sup.5 cells derived from a human ctlt-HSC cell lines
are introduced into the liver of neonatal Rag-1.sup.-/- or Rag-2
mice by direct injection. Those xenotransplants will also be
analyzed for appropriate reconstitution 6-12 weeks after
transplantation.
Example 12
[0195] The following example describes the use of conditional
approaches to abrogate expression of MYC and Bcl-2 from the
ctlt-HSCs after transplantation.
[0196] In this experiment, viruses (viral vectors) used to
transform stem cells are re-engineered to contain two loxP sites
flanking the MYC-ER, Bcl-2 and GFP open reading frames (ORFS). When
the cells are transplanted, a regulated form of Cre or CRE-TAT
fusion protein will be used to delete the oncogene-encoding
sequences, thus eliminating risk of insert-driven malignancy in
recipients. This approach is first developed in mice, then applied
to human lt-HSCs.
[0197] In a second approach, lt-HSCs from TRE-MYC mice are used to
generate the cell lines with retroviruses that encode Bcl-2 or
rtTA. These are transplanted into mice. Mice are fed Doxycycline to
abrogate the expression of MYC. One can use lt-HSCs obtained from
TRE-MYCxTRE-Bcl-2 bigenic mice that can be transduced with a
pMIG-rtTA retrovirus to eliminate MYC and Bcl-2 expression.
Example 13
[0198] The following example describes the use of HIV-1 Tat protein
fusions with MYC and/or Bcl-2 to attain conditional transformation
without genetic modification of the lt-HSCs.
[0199] MYC-Tat and Bcl-2-Tat fusion proteins are generated and
purified using established protocols. The fusion proteins are
tested by treatment of cells in which one can easily assay the
effects of overexpressed Bcl-2 (e.g., activated T cells, B-cell
lymphoma cell lines that are rendered resistant to BCMA-Fc, etc.)
or MYC (e.g., anergic B-cells, naive T-cells, activated T-cells).
Combinations of MYC-Tat and Bcl-2-Tat proteins are used to allow
propagation of lt-HSCs prior to transplantation. This approach is
readily developed and tested in the mouse system, then applied to
human.
[0200] The entire disclosure of each of U.S. Provisional patent
Application No. 60/728,131 and U.S. provisional Patent Application
60/765,993 is incorporated herein by reference.
[0201] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention, as set forth in the following claims.
Sequence CWU 1
1
1412377DNAHomo sapiens 1acccccgagc tgtgctgctc gcggccgcca ccgccgggcc
ccggccgtcc ctggctcccc 60tcctgcctcg agaagggcag ggcttctcag aggcttggcg
ggaaaaagaa cggagggagg 120gatcgcgctg agtataaaag ccggttttcg
gggctttatc taactcgctg tagtaattcc 180agcgagaggc agagggagcg
agcgggcggc cggctagggt ggaagagccg ggcgagcaga 240gctgcgctgc
gggcgtcctg ggaagggaga tccggagcga atagggggct tcgcctctgg
300cccagccctc ccgctgatcc cccagccagc ggtccgcaac ccttgccgca
tccacgaaac 360tttgcccata gcagcgggcg ggcactttgc actggaactt
acaacacccg agcaaggacg 420cgactctccc gacgcgggga ggctattctg
cccatttggg gacacttccc cgccgctgcc 480aggacccgct tctctgaaag
gctctccttg cagctgctta gacgctggat ttttttcggg 540tagtggaaaa
ccagcagcct cccgcgacga tgcccctcaa cgttagcttc accaacagga
600actatgacct cgactacgac tcggtgcagc cgtatttcta ctgcgacgag
gaggagaact 660tctaccagca gcagcagcag agcgagctgc agcccccggc
gcccagcgag gatatctgga 720agaaattcga gctgctgccc accccgcccc
tgtcccctag ccgccgctcc gggctctgct 780cgccctccta cgttgcggtc
acacccttct cccttcgggg agacaacgac ggcggtggcg 840ggagcttctc
cacggccgac cagctggaga tggtgaccga gctgctggga ggagacatgg
900tgaaccagag tttcatctgc gacccggacg acgagacctt catcaaaaac
atcatcatcc 960aggactgtat gtggagcggc ttctcggccg ccgccaagct
cgtctcagag aagctggcct 1020cctaccaggc tgcgcgcaaa gacagcggca
gcccgaaccc cgcccgcggc cacagcgtct 1080gctccacctc cagcttgtac
ctgcaggatc tgagcgccgc cgcctcagag tgcatcgacc 1140cctcggtggt
cttcccctac cctctcaacg acagcagctc gcccaagtcc tgcgcctcgc
1200aagactccag cgccttctct ccgtcctcgg attctctgct ctcctcgacg
gagtcctccc 1260cgcagggcag ccccgagccc ctggtgctcc atgaggagac
accgcccacc accagcagcg 1320actctgagga ggaacaagaa gatgaggaag
aaatcgatgt tgtttctgtg gaaaagaggc 1380aggctcctgg caaaaggtca
gagtctggat caccttctgc tggaggccac agcaaacctc 1440ctcacagccc
actggtcctc aagaggtgcc acgtctccac acatcagcac aactacgcag
1500cgcctccctc cactcggaag gactatcctg ctgccaagag ggtcaagttg
gacagtgtca 1560gagtcctgag acagatcagc aacaaccgaa aatgcaccag
ccccaggtcc tcggacaccg 1620aggagaatgt caagaggcga acacacaacg
tcttggagcg ccagaggagg aacgagctaa 1680aacggagctt ttttgccctg
cgtgaccaga tcccggagtt ggaaaacaat gaaaaggccc 1740ccaaggtagt
tatccttaaa aaagccacag catacatcct gtccgtccaa gcagaggagc
1800aaaagctcat ttctgaagag gacttgttgc ggaaacgacg agaacagttg
aaacacaaac 1860ttgaacagct acggaactct tgtgcgtaag gaaaagtaag
gaaaacgatt ccttctaaca 1920gaaatgtcct gagcaatcac ctatgaactt
gtttcaaatg catgatcaaa tgcaacctca 1980caaccttggc tgagtcttga
gactgaaaga tttagccata atgtaaactg cctcaaattg 2040gactttgggc
ataaaagaac ttttttatgc ttaccatctt ttttttttct ttaacagatt
2100tgtatttaag aattgttttt aaaaaatttt aagatttaca caatgtttct
ctgtaaatat 2160tgccattaaa tgtaaataac tttaataaaa cgtttatagc
agttacacag aatttcaatc 2220ctagtatata gtacctagta ttataggtac
tataaaccct aatttttttt atttaagtac 2280attttgcttt ttaaagttga
tttttttcta ttgtttttag aaaaaataaa ataactggca 2340aatatatcat
tgagccaaaa aaaaaaaaaa aaaaaaa 23772454PRTHomo sapiens 2Met Asp Phe
Phe Arg Val Val Glu Asn Gln Gln Pro Pro Ala Thr Met 1 5 10 15 Pro
Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr Asp 20 25
30 Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr Gln
35 40 45 Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu
Asp Ile 50 55 60 Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu
Ser Pro Ser Arg 65 70 75 80 Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val
Ala Val Thr Pro Phe Ser 85 90 95 Leu Arg Gly Asp Asn Asp Gly Gly
Gly Gly Ser Phe Ser Thr Ala Asp 100 105 110 Gln Leu Glu Met Val Thr
Glu Leu Leu Gly Gly Asp Met Val Asn Gln 115 120 125 Ser Phe Ile Cys
Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile Ile 130 135 140 Ile Gln
Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu Val 145 150 155
160 Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly Ser
165 170 175 Pro Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser
Leu Tyr 180 185 190 Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile
Asp Pro Ser Val 195 200 205 Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser
Ser Pro Lys Ser Cys Ala 210 215 220 Ser Gln Asp Ser Ser Ala Phe Ser
Pro Ser Ser Asp Ser Leu Leu Ser 225 230 235 240 Ser Thr Glu Ser Ser
Pro Gln Gly Ser Pro Glu Pro Leu Val Leu His 245 250 255 Glu Glu Thr
Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln Glu 260 265 270 Asp
Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala Pro 275 280
285 Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser Lys
290 295 300 Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser
Thr His 305 310 315 320 Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg
Lys Asp Tyr Pro Ala 325 330 335 Ala Lys Arg Val Lys Leu Asp Ser Val
Arg Val Leu Arg Gln Ile Ser 340 345 350 Asn Asn Arg Lys Cys Thr Ser
Pro Arg Ser Ser Asp Thr Glu Glu Asn 355 360 365 Val Lys Arg Arg Thr
His Asn Val Leu Glu Arg Gln Arg Arg Asn Glu 370 375 380 Leu Lys Arg
Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu Glu 385 390 395 400
Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr Ala 405
410 415 Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu
Glu 420 425 430 Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys
Leu Glu Gln 435 440 445 Leu Arg Asn Ser Cys Ala 450 351552DNAHomo
sapiensmodified_base(31450)..(31450)a, c, g or t 3acttgagccc
aagagttcaa ggctacggtg agccatgatt gcaacaccac acgccagcct 60tggtgacaga
atgagaccct gtctcaaaaa aaaaaaaaaa aattgaaata atataaagca
120tcttctctgg ccacagtgga acaaaaccag aaatcaacaa caagaggaat
tttgaaaact 180atacaaacac atgaaaatta aacaatatac ttctgaatga
ccagtgagtc aatgaagaaa 240ttaaaaagga aattgaaaaa tttatttaag
caaatgataa cggaaacata acctctcaaa 300acccacggta tacagcaaaa
gcagtgctaa gaaggaagtt tatagctata agcagctaca 360tcaaaaaagt
agaaaagcca ggcgcagtgg ctcatgcctg taatcccagc actttgggag
420gccaaggcgg gcagatcgcc tgaggtcagg agttcgagac cagcctgacc
aacacagaga 480aaccttgtcg ctactaaaaa tacaaaatta gctgggcatg
gtggcacatg cctgtaatcc 540cagctactcg ggaggctgag gcaggataac
cgcttgaacc caggaggtgg aggttgcggt 600gagccgggat tgcgccattg
gactccagcc tgggtaacaa gagtgaaacc ctgtctcaag 660aaaaaaaaaa
aagtagaaaa acttaaaaat acaacctaat gatgcacctt aaagaactag
720aaaagcaaga gcaaactaaa cctaaaattg gtaaaagaaa agaaataata
aagatcagag 780cagaaataaa tgaaactgaa agataacaat acaaaagatc
aacaaaatta aaagttggtt 840ttttgaaaag ataaacaaaa ttgacaaacc
tttgcccaga ctaagaaaaa aggaaagaag 900acctaaataa ataaagtcag
agatgaaaaa agagacatta caactgatac cacagaaatt 960caaaggatca
ctagaggcta ctatgagcaa ctgtacacta ataaattgaa aaacctagaa
1020aaaatagata aattcctaga tgcatacaac ctaccaagat tgaaccatga
agaaatccaa 1080agcccaaaca gaccaataac aataatggga ttaaagccat
aataaaaagt ctcctagcaa 1140agagaagccc aggacccaat ggcttccctg
ctggatttta ccaatcattt aaagaagaat 1200gaattccaat cctactcaaa
ctattctgaa aaatagagga aagaatactt ccaaactcat 1260tctacatggc
cagtattacc ctgattccaa aaccagacaa aaacacatca aaaacaaaca
1320aacaaaaaaa cagaaagaaa gaaaactaca ggccaatatc cctgatgaat
actgatacaa 1380aaatcctcaa caaaacacta gcaaaccaaa ttaaacaaca
ccttcgaaag atcattcatt 1440gtgatcaagt gggatttatt ccagggatgg
aaggatggtt caacatatgc aaatcaatca 1500atgtgataca tcatcccaac
aaaatgaagt acaaaaacta tatgattatt tcactttatg 1560cagaaaaagc
atttgataaa attctgcacc cttcatgata aaaaccctca aaaaaccagg
1620tatacaagaa acatacaggc caggcacagt ggctcacacc tgcgatccca
gcactctggg 1680aggccaaggt gggatgattg cttgggccca ggagtttgag
actagcctgg gcaacaaaat 1740gagacctggt ctacaaaaaa cttttttaaa
aaattagcca ggcatgatgg catatgcctg 1800tagtcccagc tagtctggag
gctgaggtgg gagaatcact taagcctagg aggtcgaggc 1860tgcagtgagc
catgaacatg tcactgtact ccagcctaga caacagaaca agaccccact
1920gaataagaag aaggagaagg agaagggaga agggagggag aagggaggag
gaggagaagg 1980aggaggtgga ggagaagtgg aaggggaagg ggaagggaaa
gaggaagaag aagaaacata 2040tttcaacata ataaaagccc tatatgacag
accgaggtag tattatgagg aaaaactgaa 2100agcctttcct ctaagatctg
gaaaatgaca agggcccact ttcaccactg tgattcaaca 2160tagtactaga
agtcctagct agagcaatca gataagagaa agaaataaaa ggcatccaaa
2220ctggaaagga agaagtcaaa ttatcctgtt tgcagatgat atgatcttat
atctggaaaa 2280gacttaagac accactaaaa aactattaga gctgaaattt
ggtacagcag gatacaaaat 2340caatgtacaa aaatcagtag tatttctata
ttccaacagc aaacaatctg aaaaagaaac 2400caaaaaagca gctacaaata
aaattaaaca gctaggaatt aaccaaagaa gtgaaagatc 2460tctacaatga
aaactataaa atgttgataa aagaaattga agagggcaca aaaaaagaaa
2520agatattcca tgttcataga ttggaagaat aaatactgtt aaaatgtcca
tactacccaa 2580agcaatttac aaattcaatg caatccctat taaaatacta
atgacgttct tcacagaaat 2640agaagaaaca attctaagat ttgtacagaa
ccacaaaaga cccagaatag ccaaagctat 2700cctgaccaaa aagaacaaaa
ctggaagcat cacattacct gacttcaaat tatactacaa 2760agctatagta
acccaaacta catggtactg gcataaaaac agatgagaca tggaccagag
2820gaacagaata gagaatccag aaacaaatcc atgcatctac agtgaactca
tttttgacaa 2880aggtgccaag aacatacttt ggggaaaaga taatctcttc
aataaatggt gctggaggaa 2940ctggatatcc atatgcaaaa taacaatact
agaactctgt ctctcaccat atacaaaagc 3000aaatcaaaat ggatgaaagg
cttaaatcta aaacctcaaa ctttgcaact actaaaagaa 3060aacaccggag
aaactctcca ggacattgga gtgggcaaag acttcttgag taattccctg
3120caggcacagg caaccaaagc aaaaacagac aaatgggatc atatcaagtt
aaaaagcttc 3180tgcccagcaa aggaaacaat caacaaagag aagagacaac
ccacagaatg ggagaatata 3240tttgcaaact attcatctaa caaggaatta
ataaccagta tatataagga gctcaaacta 3300ctctataaga aaaacaccta
ataagctgat tttcaaaaat aagcaaaaga tctgggtaga 3360catttctcaa
aataagtcat acaaatggca aacaggcatc tgaaaatgtg ctcaacacca
3420ctgatcatca gagaaatgca aatcaaaact actatgagag atcatctcat
cccagttaaa 3480atggctttta ttcaaaagac aggcaataac aaatgccagt
gaggatgtgg ataaaaggaa 3540acccttggac actgttggtg ggaatggaaa
ttgctaccac tatggagaac agtttgaaag 3600ttcctcaaaa aactaaaaat
aaagctacca tacagcaatc ccattgctag gtatatactc 3660caaaaaaggg
aatcagtgta tcaacaagct atctccactc ccacatttac tgcagcactg
3720ttcatagcag ccaaggtttg gaagcaacct cagtgtccat caacagacga
atggaaaaag 3780aaaatgtggt gcacatacac aatggagtac tacgcagcca
taaaaaagaa tgagatcctg 3840tcagttgcaa cagcatgggg ggcactggtc
agtatgttaa gtgaaataag ccaggcacag 3900aaagacaaac ttttcatgtt
ctcccttact tgtgggagca aaaattaaaa caattgacat 3960agaaatagag
gagaatggtg gttctagagg ggtgggggac agggtgacta gagtcaacaa
4020taatttattg tatgttttaa aataactaaa agagtataat tgggttgttt
gtaacacaaa 4080gaaaggataa atgcttgaag gtgacagata ccccatttac
cctgatgtga ttattacaca 4140ttgtatgcct gtatcaaaat atctcatgta
tgctatagat ataaacccta ctatattaaa 4200aattaaaatt ttaatggcca
ggcacggtgg ctcatgtccg taatcccagc actttgggag 4260gccgaggcgg
gtggatcacc tgaggtcagg agtttgaaac cagtctggcc accatgatga
4320aaccctgtct ctactaaaga tacaaaaatt agccaggcgt ggtggcacat
acctgtagtc 4380ccaactactc aggaggctga gacaggagaa ttgcttgaac
ctgggaggcg gaggttgcag 4440tgagccgaga tcatgccact gcactgcagc
ctgggtgaca gagcaagact ccatctcaaa 4500acaaaaacaa aaaaaagaag
attaaaattg taatttttat gtaccgtata aatatatact 4560ctactatatt
agaagttaaa aattaaaaca attataaaag gtaattaacc acttaatcta
4620aaataagaac aatgtatgtg gggtttctag cttctgaaga agtaaaagtt
atggccacga 4680tggcagaaat gtgaggaggg aacagtggaa gttactgttg
ttagacgctc atactctctg 4740taagtgactt aattttaacc aaagacaggc
tgggagaagt taaagaggca ttctataagc 4800cctaaaacaa ctgctaataa
tggtgaaagg taatctctat taattaccaa taattacaga 4860tatctctaaa
atcgagctgc agaattggca cgtctgatca caccgtcctc tcattcacgg
4920tgcttttttt cttgtgtgct tggagatttt cgattgtgtg ttcgtgtttg
gttaaactta 4980atctgtatga atcctgaaac gaaaaatggt ggtgatttcc
tccagaagaa ttagagtacc 5040tggcaggaag caggtggctc tgtggacctg
agccacttca atcttcaagg gtctctggcc 5100aagacccagg tgcaaggcag
aggcctgatg acccgaggac aggaaagctc ggatgggaag 5160gggcgatgag
aagcctgcct cgttggtgag cagcgcatga agtgccctta tttacgcttt
5220gcaaagattg ctctggatac catctggaaa aggcggccag cgggaatgca
aggagtcaga 5280agcctcctgc tcaaacccag gccagcagct atggcgccca
cccgggcgtg tgccagaggg 5340agaggagtca aggcacctcg aagtatggct
taaatctttt tttcacctga agcagtgacc 5400aaggtgtatt ctgagggaag
cttgagttag gtgccttctt taaaacagaa agtcatggaa 5460gcacccttct
caagggaaaa ccagacgccc gctctgcggt catttacctc tttcctctct
5520ccctctcttg ccctcgcggt ttctgatcgg gacagagtga cccccgtgga
gcttctccga 5580gcccgtgctg aggaccctct tgcaaagggc tccacagacc
cccgccctgg agagaggagt 5640ctgagcctgg cttaataaca aactgggatg
tggctggggg cggacagcga cggcgggatt 5700caaagactta attccatgag
taaattcaac ctttccacat ccgaatggat ttggatttta 5760tcttaatatt
ttcttaaatt tcatcaaata acattcagga ctgcagaaat ccaaaggcgt
5820aaaacaggaa ctgagctatg tttgccaagg tccaaggact taataaccat
gttcagaggg 5880atttttcgcc ctaagtactt tttattggtt ttcataaggt
ggcttagggt gcaagggaaa 5940gtacacgagg agaggcctgg gcggcagggc
tatgagcacg gcagggccac cggggagaga 6000gtccccggcc tgggaggctg
acagcaggac cactgaccgt cctccctggg agctgccaca 6060ttgggcaacg
cgaaggcggc cacgctgcgt gtgactcagg accccatacc ggcttcctgg
6120gcccacccac actaacccag gaagtcacgg agctctgaac ccgtggaaac
gaacatgacc 6180cttgcctgcc tgcttccctg ggtgggtcaa gggtaatgaa
gtggtgtgca ggaaatggcc 6240atgtaaatta cacgactctg ctgatgggga
ccgttccttc catcattatt catcttcacc 6300cccaaggact gaatgattcc
agcaacttct tcgggtgtga caagccatga caaaactcag 6360tacaaacacc
actcttttac taggcccaca gagcacggsc cacacccctg atatattaag
6420agtccaggag agatgaggct gctttcagcc accaggctgg ggtgacaaca
gcggctgaac 6480agtctgttcc tctagactag tagaccctgg caggcactcc
cccagattct agggcctggt 6540tgctgcttcc cgagggcgcc atctgccctg
gagactcagc ctggggtgcc acactgaggc 6600cagccctgtc tccacaccct
ccgcctccag gcctcagctt ctccagcagc ttcctaaacc 6660ctgggtgggc
cgtgttccag cgctactgtc tcacctgtcc cactgtgtct tgtctcagcg
6720acgtagctcg cacggttcct cctcacatgg ggtgtctgtc tccttcccca
acactcacat 6780gcgttgaagg gaggagattc tgcgcctccc agactggctc
ctctgagcct gaacctggct 6840cgtggccccc gatgcaggtt cctggcgtcc
ggctgcacgc tgacctccat ttccaggcgc 6900tccccgtctc ctgtcatctg
ccggggcctg ccggtgtgtt cttctgtttc tgtgctcctt 6960tccacgtcca
gctgcgtgtg tctctgcccg ctagggtctc ggggttttta taggcatagg
7020acgggggcgt ggtgggccag ggcgctcttg ggaaatgcaa catttgggtg
tgaaagtagg 7080agtgcctgtc ctcacctagg tccacgggca caggcctggg
gatggagccc ccgccaggga 7140cccgcccttc tctgcccagc actttcctgc
ccccctccct ctggaacaca gagtggcagt 7200ttccacaagc actaagcatc
ctcttcccaa aagacccagc attggcaccc ctggacattt 7260gccccacagc
cctgggaatt cacgtgacta cgcacatcat gtacacactc ccgtccacga
7320ccgacccccg ctgttttatt ttaatagcta caaagcaggg aaatccctgc
taaaatgtcc 7380tttaacaaac tggttaaaca aacgggtcca tccgcacggt
ggacagttcc tcacagtgaa 7440gaggaacatg ccgtttataa agcctgcagg
catctcaagg gaattacgct gagtcaaaac 7500tgccacctcc atgggatacg
tacgcaacat gctcaaaaag aaagaatttc accccatggc 7560aggggagtgg
ttaggggggt taaggacggt gggggcggca gctgggggct actgcacgca
7620ccttttacta aagccagttt cctggttctg atggtattgg ctcagttatg
ggagactaac 7680cataggggag tggggatggg ggaacccgga ggctgtgcca
tctttgccat gcccgagtgt 7740cctgggcagg ataatgctct agagatgccc
acgtcctgat tcccccaaac ctgtggacag 7800aacccgcccg gccccagggc
ctttgcaggt gtgatctccg tgaggaccct gaggtctggg 7860atccttcggg
actacctgca ggcccgaaaa gtaatccagg ggttctggga agaggcgggc
7920aggagggtca gaggggggca gcctcaggac gatggaggca gtcagtctga
ggctgaaaag 7980ggagggaggg cctcgagccc aggcctgcaa gcgcctccag
aagctggaaa aagcggggaa 8040gggaccctcc acggagcctg cagcaggaag
gcacggctgg cccttagccc accagggccc 8100atcgtggacc tccggcctcc
gtgccatagg agggcactcg cgctgccctt ctagcatgaa 8160gtgtgtgggg
atttgcagaa gcaacaggaa acccatgcac tgtgaatcta ggattatttc
8220aaaacaaagg tttacagaaa catccaagga cagggctgaa gtgcctccgg
gcaagggcag 8280ggcaggcacg agtgatttta tttagctatt ttattttatt
tacttacttt ctgagacaga 8340gttatgctct tgttgcccag gctggagtgc
agcggcatga tcttggctca ctgcaacctc 8400cgtctcctgg gttcaagcaa
ttctcgtgcc tcagcctccc aagtagctgg gatttcaggc 8460gtgcaccacc
acacccggct aattttgtat ttttagtaga gatgggcttt caccatgttg
8520gtcaagctga tctcaaaatc ctgacctcag gtgatccgcc cacctcagcc
tcccaaagtg 8580ctgggattac aggcatgagc cactgcacct ggcctattta
accattttaa aacttccctg 8640ggctcaagtc acacccactg gtaaggagtt
catggagttc aatttcccct ttactcagga 8700gttaccctcc tttgatattt
tctgtaattc ttcgtagact ggggatacac cgtctcttga 8760catattcaca
gtttctgtga ccacctgtta tcccatggga cccactgcag gggcagctgg
8820gaggctgcag gcttcaggtc ccagtggggt tgccatctgc cagtagaaac
ctgatgtaga 8880atcagggcgc aagtgtggac actgtcctga atctcaatgt
ctcagtgtgt gctgaaacat 8940gtagaaatta aagtccatcc ctcctactct
actgggattg agccccttcc ctatcccccc 9000ccaggggcag aggagttcct
ctcactcctg tggaggaagg aatgatactt tgttattttt 9060cactgctggt
actgaatcca ctgtttcatt tgttggtttg tttgttttgt tttgagaggc
9120ggtttcactc ttgttgctca ggctggaggg agtgcaatgg cgcgatcttg
gcttactgca 9180gcctctgcct cccaggttca agtgattctc ctgcttccgc
ctcccatttg gctgggatta 9240caggcacccg ccaccatgcc cagctaattt
tttgtatttt tagtagagac gggggtgggt 9300ggggttcacc atgttggcca
ggctggtctc gaacttctga cctcagatga tccacctgcc 9360tctgcctcct
aaagtgctgg gattacaggt gtgagccacc atgcccagct cagaatttac
9420tctgtttaga aacatctggg tctgaggtag gaagctcacc ccactcaagt
gttgtggtgt 9480tttaagccaa tgatagaatt tttttattgt tgttagaaca
ctcttgatgt tttacactgt 9540gatgactaag acatcatcag cttttcaaag
acacactaac tgcacccata atactggggt
9600gtcttctggg tatcagcaat cttcattgaa tgccgggagg cgtttcctcg
ccatgcacat 9660ggtgttaatt actccagcat aatcttctgc ttccatttct
tctcttccct cttttaaaat 9720tgtgttttct atgttggctt ctctgcagag
aaccagtgta agctacaact taacttttgt 9780tggaacaaat tttccaaacc
gcccctttgc cctagtggca gagacaattc acaaacacag 9840ccctttaaaa
aggcttaggg atcactaagg ggatttctag aagagcgacc tgtaatccta
9900agtatttaca agacgaggct aacctccagc gagcgtgaca gcccagggag
ggtgcgaggc 9960ctgttcaaat gctagctcca taaataaagc aatttcctcc
ggcagtttct gaaagtagga 10020aaggttacat ttaaggttgc gtttgttagc
atttcagtgt ttgccgacct cagctacagc 10080atccctgcaa ggcctcggga
gacccagaag tttctcgccc ccttagatcc aaacttgagc 10140aacccggagt
ctggattcct gggaagtcct cagctgtcct gcggttgtgc cggggcccca
10200ggtctggagg ggaccagtgg ccgtgtggct tctactgctg ggctggaagt
cgggcctcct 10260agctctgcag tccgaggctt ggagccaggt gcctggaccc
cgaggctgcc ctccaccctg 10320tgcgggcggg atgtgaccag atgttggcct
catctgccag acagagtgcc ggggcccagg 10380gtcaaggccg ttgtggctgg
tgtgaggcgc ccggtgcgcg gccagcagga gcgcctggct 10440ccatttccca
ccctttctcg acgggaccgc cccggtgggt gattaacaga tttggggtgg
10500tttgctcatg gtggggaccc ctcgccgcct gagaacctgc aaagagaaat
gacgggcctg 10560tgtcaaggag cccaagtcgc ggggaagtgt tgcagggagg
cactccggga ggtcccgcgt 10620gcccgtccag ggagcaatgc gtcctcgggt
tcgtccccag ccgcgtctac gcgcctccgt 10680cctccccttc acgtccggca
ttcgtggtgc ccggagcccg acgccccgcg tccggacctg 10740gaggcagccc
tgggtctccg gatcaggcca gcggccaaag ggtcgccgca cgcacctgtt
10800cccagggcct ccacatcatg gcccctccct cgggttaccc cacagcctag
gccgattcga 10860cctctctccg ctggggccct cgctggcgtc cctgcaccct
gggagcgcga gcggcgcgcg 10920ggcggggaag cgcggcccag acccccgggt
ccgcccggag cagctgcgct gtcggggcca 10980ggccgggctc ccagtggatt
cgcgggcaca gacgcccagg accgcgctcc ccacgtggcg 11040gagggactgg
ggacccgggc acccgtcctg ccccttcacc ttccagctcc gcctcctccg
11100cgcggacccc gccccgtccc gacccctccc gggtccccgg cccagccccc
tccgggccct 11160cccagcccct ccccttcctt tccgcggccc cgccctctcc
tcgcggcgcg agtttcaggc 11220agcgctgcgt cctgctgcgc acgtgggaag
ccctggcccc ggccaccccc gcgatgccgc 11280gcgctccccg ctgccgagcc
gtgcgctccc tgctgcgcag ccactaccgc gaggtgctgc 11340cgctggccac
gttcgtgcgg cgcctggggc cccagggctg gcggctggtg cagcgcgggg
11400acccggcggc tttccgcgcg ctggtggccc agtgcctggt gtgcgtgccc
tgggacgcac 11460ggccgccccc cgccgccccc tccttccgcc aggtgggcct
ccccggggtc ggcgtccggc 11520tggggttgag ggcggccggg gggaaccagc
gacatgcgga gagcagcgca ggcgactcag 11580ggcgcttccc ccgcaggtgt
cctgcctgaa ggagctggtg gcccgagtgc tgcagaggct 11640gtgcgagcgc
ggcgcgaaga acgtgctggc cttcggcttc gcgctgctgg acggggcccg
11700cgggggcccc cccgaggcct tcaccaccag cgtgcgcagc tacctgccca
acacggtgac 11760cgacgcactg cgggggagcg gggcgtgggg gctgctgctg
cgccgcgtgg gcgacgacgt 11820gctggttcac ctgctggcac gctgcgcgct
ctttgtgctg gtggctccca gctgcgccta 11880ccaggtgtgc gggccgccgc
tgtaccagct cggcgctgcc actcaggccc ggcccccgcc 11940acacgctagt
ggaccccgaa ggcgtctggg atgcgaacgg gcctggaacc atagcgtcag
12000ggaggccggg gtccccctgg gcctgccagc cccgggtgcg aggaggcgcg
ggggcagtgc 12060cagccgaagt ctgccgttgc ccaagaggcc caggcgtggc
gctgcccctg agccggagcg 12120gacgcccgtt gggcaggggt cctgggccca
cccgggcagg acgcgtggac cgagtgaccg 12180tggtttctgt gtggtgtcac
ctgccagacc cgccgaagaa gccacctctt tggagggtgc 12240gctctctggc
acgcgccact cccacccatc cgtgggccgc cagcaccacg caggcccccc
12300atccacatcg cggccaccac gtccctggga cacgccttgt cccccggtgt
acgccgagac 12360caagcacttc ctctactcct caggcgacaa ggagcagctg
cggccctcct tcctactcag 12420ctctctgagg cccagcctga ctggcgctcg
gaggctcgtg gagaccatct ttctgggttc 12480caggccctgg atgccaggga
ctccccgcag gttgccccgc ctgccccagc gctactggca 12540aatgcggccc
ctgtttctgg agctgcttgg gaaccacgcg cagtgcccct acggggtgct
12600cctcaagacg cactgcccgc tgcgagctgc ggtcacccca gcagccggtg
tctgtgcccg 12660ggagaagccc cagggctctg tggcggcccc cgaggaggag
gacacagacc cccgtcgcct 12720ggtgcagctg ctccgccagc acagcagccc
ctggcaggtg tacggcttcg tgcgggcctg 12780cctgcgccgg ctggtgcccc
caggcctctg gggctccagg cacaacgaac gccgcttcct 12840caggaacacc
aagaagttca tctccctggg gaagcatgcc aagctctcgc tgcaggagct
12900gacgtggaag atgagcgtgc gggactgcgc ttggctgcgc aggagcccag
gtgaggaggt 12960ggtggccgtc gagggcccag gccccagagc tgaatgcagt
aggggctcag aaaagggggc 13020aggcagagcc ctggtcctcc tgtctccatc
gtcacgtggg cacacgtggc ttttcgctca 13080ggacgtcgag tggacacggt
gatctctgcc tctgctctcc ctcctgtcca gtttgcataa 13140acttacgagg
ttcaccttca cgttttgatg gacacgcggt ttccaggcgc cgaggccaga
13200gcagtgaaca gaggaggctg ggcgcggcag tggagccggg ttgccggcaa
tggggagaag 13260tgtctggaag cacagacgct ctggcgaggg tgcctgcagg
ttacctataa tcctcttcgc 13320aatttcaagg gtgggaatga gaggtgggga
cgagaacccc ctcttcctgg gggtgggagg 13380taagggtttt gcaggtgcac
gtggtcagcc aatatgcagg tttgtgttta agatttaatt 13440gtgtgttgac
ggccaggtgc ggtggctcac gccggtaatc ccagcacttt gggaagctga
13500ggcaggtgga tcacctgagg tcaggagttt gagaccagcc tgaccaacat
ggtgaaaccc 13560tatctgtact aaaaatacaa aaattagctg ggcatggtgg
tgtgtgcctg taatcccagc 13620tacttgggag gctgaggcag gagaatcact
tgaacccagg aggcggaggc tgcagtgagc 13680tgagattgtg ccattgtact
ccagcctggg cgacaagagt gaaactctgt ctttaaaaaa 13740aaaaagtgtt
cgttgattgt gccaggacag ggtagaggga gggagataag actgttctcc
13800agcacagatc ctggtcccat ctttaggtat gaagagggcc acatgggagc
agaggacagc 13860agatggctcc acctgctgag gaagggacag tgtttgtggg
tgttcagggg atggtgctgc 13920tgggccctgc cgtgtcccca ccctgttttt
ctggatttga tgttgaggaa cctccgctcc 13980agcccccttt tggctcccag
tgctcccagg ccctaccgtg gcagctagaa gaagtcccga 14040tttcaccccc
tccccacaaa ctcccaagac atgtaagact tccggccatg cagacaagga
14100gggtgacctt cttggggctc ttttttttct ttttttcttt ttatggtggc
aaaagtcata 14160taacatgaga ttggcactcc taacaccgtt ttctgtgtac
agtgcagaat tgctaactcg 14220gcggtgttta cagcaggttg cttgaaatgc
tgcgtcttgc gtgactggaa gtccctaccc 14280atcgaacggc agctgcctca
cacctgctgc ggctcaggtg gaccacgccg agtcagataa 14340gcgtcatgca
acccagtttt gctttttgtg ctccagcttc cttcgttgag gagagtttga
14400gttctctgat caggactctg cctgtcattg ctgttctctg acttcagatg
aggtcacaat 14460ctgcccctgg cttatgcagg gagtgaggcg tggtccccgg
gtgtccctgt cacgtgcagg 14520gtgagtgagg cgttgccccc aggtgtccct
gtcacgtgta gggtgagtga ggcgcggccc 14580ccgggtgtcc ctgtcccgtg
cagcgtgatt gaggtgtggc ccccgggtgt ccctgtcacg 14640tgtagggtga
gtgaggcgcc atccccgggt gtccctgtca cgtgtagggt gagtgaggcg
14700tggtccccgg gtgtccctgt cccgtgcagg gtgagtgagg cactgtcccc
gggtgtccct 14760gtcacgtgca gggtgagtga ggcgcggtcc ccgggtgtcc
ctctcaggtg tagggtgagt 14820gaggcgcggc cccagggtgt ccctgtcacg
tgtagggtga gtgaggcacc gtccctgggt 14880gtccctccca ggtatagggt
gagtgaggca ctgtccccgg gtgtccctgt cacgtgcagg 14940gtgagtgagg
cgcggccccc gggtgtccct ctcaggtgca gggtgagtga ggcgctgtcc
15000ctgggtgtcc ctgtctcgtg tagggtgagt gaggctctgt ccccaggtgt
ccttggcgtt 15060tgctcacttg agcttgctcc tgaatgtttg ctctttctat
agccacagct gcgccggttg 15120cccattgcct gggtagatgg tgcaggcgca
gtgctggtcc ccaagcctat cttttctgat 15180gctcggctct tcttggtcac
ctctccgttc cattttgcta cggggacacg ggactgcagg 15240ctctcgcctc
ccgcgtgcca ggcactgcag ccacagcttc aggtccgctt gcctctgttg
15300ggcctggctt gctcaccacg tgcccgccac atgcatgctg ccaatactcc
tctcccagct 15360tgtctcatgc cgaggctgga ctctgggctg cctgtgtctg
ctgccacgtg ttgctggaga 15420catcccagaa agggttctct gtgccctgaa
ggaaagcaag tcaccccagc cccctcactt 15480gtcctgtttt ctcccaagct
gcccctctgc ttggccccct tgggtgggtg gcaacgcttg 15540tcaccttatt
ctgggcacct gccgctcatt gcttaggctg ggctctgcct ccagtcgccc
15600cctcacatgg attgacgtcc agccacaggt tggagtgtct ctgtctgtct
cctgctctga 15660gacccacgtg gagggccggt gtctccgcca gccttcgtca
gacttccctc ttgggtctta 15720gttttgaatt tcactgattt acctctgacg
tttctatctc tccattgtat gctttttctt 15780ggtttattct ttcattcctt
ttctagcttc ttagtttagt catgcctttc cctctaagtg 15840ctgccttacc
tgcaccctgt gttttgatgt gaagtaatct caacatcagc cactttcaag
15900tgttcttaaa atacttcaaa gtgttaatac ttcttttaag tattcttatt
ctgtgatttt 15960tttctttgtg cacgctgtgt tttgacgtga aatcattttg
atatcagtga cttttaagta 16020ttctttagct tattctgtga tttctttgag
cagtgagtta tttgaacact gtttatgttc 16080aagatatgta gagtatcaag
atacgtagag tattttaagt tatcatttta ttattgattt 16140ctaactcagt
tgtgtagtgg tctgtataat accaattatt tgaagtttgc ggagccttgc
16200tttgtgatct agtgtgtgca tggtttccag aactgtccat tgtaaatttg
acatcctgtc 16260aatagtgggc atgcatgttc actatatcca gcttattaag
gtccagtgca aagcttctgt 16320ctccttctag atgcatgaaa ttccaagaag
gaggccatag tccctcacct gggggatggg 16380tctgttcatt tcttctcgtt
tggtagcatt tatgtgaggc attgttaggt gcatgcacgt 16440ggtagaattt
ttatcttcct gatgagtgaa tcttttggag acttctatgt ctctagtaat
16500ctagtaattc tttttttaaa ttgctcttag tactgccaca ctgggcttct
tttgattagt 16560attttcctgc tgtgtctgtt ttctgccttt aatttatata
tatatatata tttttttttt 16620ttttgagaca gagtcttggt ctgtcgccca
gggtgagtgc agtggtgtga tcacaggtca 16680gtgtaacttt taccttctgg
cctgagccgt cctctcacct cagcctcctg agtagctgga 16740actgcagaca
cgcaccgcta cacctggcta atttttaaat tttttctgga gacagggtct
16800tgctgtgttg cccaggctgg tctcaaactc ttggactcaa gggatccatc
tacctcggct 16860tcccaaagtg ctgaattaca ggcatgagcc accatgtctg
gcctaatttt caacactttt 16920atattcttat agtgtgggta tgtcctgtta
acagcatgta ggtgaatttc caatccagtc 16980tgacagtcgt tgtttaactg
gataacctga tttattttca tttttttgtc actagagacc 17040cgcctggtgc
actctgattc tccacttgcc tgttgcatgt cctcgttccc ttgtttctca
17100ccacctcttg ggttgccatg tgcgtttcct gccgagtgtg tgttgatcct
ctcgttgcct 17160cctggtcact gggcatttgc ttttatttct ctttgcttag
tgttaccccc tgatcttttt 17220attgtcgttg tttgcttttg tttattgaga
cagtctcact ctgtcaccca ggctggagtg 17280taatggcaca atctcggctc
actgcaacct ctgcctcctc ggttcaagca gttctcattc 17340ctcaacctca
tgagtagctg ggattacagg cgcccaccac cacgcctggc taatttttgt
17400atttttagta gagataggct ttcaccatgt tggccaggct ggtctcaaac
tcctgacctc 17460aagtgatctg cccgccttgg cctcccacag tgctgggatt
acaggtgcaa gccaccgtgc 17520ccggcatacc ttgatctttt aaaatgaagt
ctgaaacatt gctacccttg tcctgagcaa 17580taagaccctt agtgtatttt
agctctggcc accccccagc ctgtgtgctg ttttccctgc 17640tgacttagtt
ctatctcagg catcttgaca cccccacaag ctaagcatta ttaatattgt
17700tttccgtgtt gagtgtttct gtagctttgc ccccgccctg cttttcctcc
tttgttcccc 17760gtctgtcttc tgtctcaggc ccgccgtctg gggtcccctt
ccttgtcctt tgcgtggttc 17820ttctgtcttg ttattgctgg taaaccccag
ctttacctgt gctggcctcc atggcatcta 17880gcgacgtccg gggacctctg
cttatgatgc acagatgaag atgtggagac tcacgaggag 17940ggcggtcatc
ttggcccgtg agtgtctgga gcaccacgtg gccagcgttc cttagccagt
18000gagtgacagc aacgtccgct cggcctgggt tcagcctgga aaaccccagg
catgtcgggg 18060tctggtggct ccgcggtgtc gagtttgaaa tcgcgcaaac
ctgcggtgtg gcgccagctc 18120tgacggtgct gcctggcggg ggagtgtctg
cttcctccct tctgcttggg aaccaggaca 18180aaggatgagg ctccgagccg
ttgtcgccca acaggagcat gacgtgagcc atgtggataa 18240ttttaaaatt
tctaggctgg gcgcggtggc tcacgcctgt aatcccagca ctttgggagg
18300ccaaggcggg tggatcacga ggtcaggagg tcgagaccat cctggccaac
atgatgaaac 18360cccatctgta ctaaaaacac aaaaattagc tgggcgtggt
ggcgggtgcc tgtaatccca 18420gctactcggg aggctgaggc aggagaattg
cttgaacctg ggagttggaa gttgcagtga 18480gccgacattg caccactgca
ctccagcctg gcaacacagc gagactctgt ctcaaaaaaa 18540aaaaaaaaaa
aaaaaaaaaa aattctagta gccacattaa aaaagtaaaa aagaaaaggt
18600gaaattaatg taataataga ttttactgaa gcccagcatg tccacacctc
atcattttag 18660ggtgttattg gtgggagcat cactcacagg acatttgaca
ttttttgagc tttgtctgcg 18720ggatcccgtg tgtaggtccc gtgcgtggcc
atctcggcct ggacctgctg ggcttcccat 18780ggccatggct gttgtaccag
atggtgcagg tccgggatga ggtcgccagg ccctcagtga 18840gctggatgtg
cagtgtccgg atggtgcacg tctgggatga ggtcgccagg ccctgctgtg
18900agctggatgt gtggtgtctg gatggtgcag gtcaggggtg aggtctccag
gccctcggtg 18960agctggaggt atggagtccg gatgatgcag gtccggggtg
aggtcgccag gccctgctgt 19020gagctggatg tgtggtgtct ggatggtgca
ggtcaggggt gaggtctcca ggccctcggt 19080aagctggagg tatggagtcc
ggatgatgca ggtccggggt gaggtcgcca ggccctgctg 19140tgagctggat
gtgtggtgtc tggatggtgc aggtctgggg tgaggtcacc aggccctgcg
19200gtgagctggg tgtgcggtgt ctggatggtg caggtctgga gtgaggtcgc
cagacggtgc 19260cagaccatgc ggtgagctgg atatgcggtg tccggatggt
gcaggtctgg ggtgaggttg 19320ccaggccctg ctgtgagttg gatgtggggt
gtccggatgc tgcaggtccg gtgtgaggtc 19380accaggccct gctgtgagct
ggatgtgtgg tgtctggatg gtgcaggtct ggggtgaagg 19440tcgccaggcc
cctgcttgtg agctggatgt gtggtgtctg gatggtgcag gtctggagtg
19500aggtcgccag gccctcggtg agctggatgt gcagtgtcca gatggtgcag
gtccggggtg 19560aggtcgccag accctgcggt gagctggatg tgcggtgtct
ggatggtgca ggtctggagt 19620gaggtcgcca ggccctcggt gagctggatg
tatggagtcc ggatggtgcc ggtccggggt 19680gaggtcgcca gaccctgctg
tgagctggat gtgcggtgtc tggatggtac aggtctggag 19740tgaggtcgcc
agaccctgct gtgagctgga tatgcggtgt ccggatggtg caggtcaggg
19800gtgaggtctc caggccctcg gtgagctgga ggtatggagt ccggatgatg
caggtccggg 19860gtgaggtcgc caggccctgc tgtgaactgg atgtgcggcg
tctggatggt gcaggtctgg 19920ggtgtggtcg ccaggccctc ggtgagctgg
aggtatggag tccggatgat gcaggtccgg 19980ggtgaggtcg ccaggccctg
ctgtgagctg gatgtgcggc gtctggatgg tgcaggtctg 20040gggtgtggtc
gccaggccct cggtgagctg gaggtatgga gtccggatga tgcaggtccg
20100gggtgaggtt gccaggccct gctgtgagct ggatgtgctg tatccggatg
gtgcagtccg 20160gggtgaggtc gccaggccct gctgtgagct ggatgtgctg
tatccggatg gtgcaggtct 20220ggggtgaggt caccaggccc tgcggtgagc
tggttgtgcg gtgtccggtt gctgcaggtc 20280cggggtgagt tcgccaggcc
ctcggtgagc tggatgtgcg gtgtccccgt gtccggatgg 20340tgcaggtcca
gggtgaggtc gctaggccct tggtgggctg gatgtgccgt gtccggatgg
20400tgcaggtctg gggtgaggtc gccaggcctt tggtgagctg gatgtgcggt
gtctgcatgg 20460tgcaggtctg gggtgaggtc gccaggccct tggtgggctg
gatgtgtggt gtccggatgg 20520tgcaggtccg gcgtgaggtc gccaggccct
gctgtgagct ggatgtgcgg tgtctggatg 20580gtgcaggtcc ggggtgaggt
agccaaggcc ttcggtgagc tggatgtggg gtgtccggat 20640ggtgcaggtc
cggggtgagg tcgccaggcc ctgcggttag ctggatatgc ggtgtccgga
20700tggtgcaggt ccggggtgag gtcaccaggc cctgcggtta gctggatgtg
cggtgtctgg 20760atggtgcagg tccggggtga ggtcgccagg ccctgctgtg
agctggatgt gctgtatccg 20820gatggtgcag gtccggggtg aggtcgccag
gccctgcagt gagctggatg tgctgtatcc 20880ggatggtgca ggtctggcgt
gaggtcgcca ggccctgcgg ttagctggat atgcggtgtc 20940ggatggtgca
ggtccggggt gaggtcacca ggccctgcgg ttagctggat gtgcggtgtc
21000cggatggtgc aggtctgggg tgaggtcgcc aggccctgct gtgagctgga
tgtgctgtat 21060ccggatggtg caggtccggg gtgaggtcgc caggccctgc
ggtgagctgg atgtgctgta 21120tccggatggt gcaggtctgg cgtgaggtcg
ccaggccctg cggtgagctg gatgtgcagt 21180gtacggatgg tgcaggtccg
gggtgaggtc gccaggccct gcggtgggct gtatgtgtgt 21240tgtctggatg
gtgcaggtcc ggggtgagtt cgccaggccc tgcggtgagc tggatgtgtg
21300gtgtctggat gctgcaggtc cggggtgagt tcgccaggcc ctcggtgagc
tggatatgcg 21360gtgtccccgt gtccgaatgg tgcaggtcca gggtgaggtc
gccaggccct tggtgggctg 21420gatgtgccgt gtccggatgg tgcaggtctg
gggtgaggtc gccaggccct tggtgagctg 21480gatgtgcggt gtccggatgg
tgcaggtccg gggtgaggtc accaggccct cggtgatctg 21540gatgtggcat
gtccttctcg tttaaggggt tggctgtgtt ccggccgcag agcaccgtct
21600gcgtgaggag atcctggcca agttcctgca ctggctgatg agtgtgtacg
tcgtcgagct 21660gctcaggtct ttcttttatg tcacggagac cacgtttcaa
aagaacaggc tctttttcta 21720ccggaagagt gtctggagca agttgcaaag
cattggaatc aggtactgta tccccacgcc 21780aggcctctgc ttctcgaagt
cctggaacac cagcccggcc tcagcatgcg cctgtctcca 21840cttgcctgtg
cttccctggc tgtgcagctc tgggctggga gccaggggcc ccgtcacagg
21900cctggtccaa gtggattctg tgcaaggctc tgactgcctg gagctcacgt
tctcttactt 21960gtaaaatcag gagtttgtgc caagtggtct ctagggtttg
taaagcagaa gggatttaaa 22020ttagatggaa acactaccac tagcctcctt
gcctttccct gggatgtggg tctgattctc 22080tctctctttt ttttttcttt
tttgagatgg agtctcactc tgttgcccag gctggagtgc 22140agtggcataa
tcttggctca ctgcaacctc cacctcctgg gtttaagcga ttcaccagcc
22200tcagcctcct aagtagctgg gattacaggc acctgccacc acgcctggct
aatttttgta 22260cttttaggag agacggggtt tcaccatgtt ggccaggctg
gtctcgaact catgacctca 22320ggtgatccac ccaccttggc ctcccaaagt
gctgggttta caggctaagc caccgtgccc 22380agcccccgat tctcttttaa
ttcatgctgt tctgtatgaa tcttcaatct attggattta 22440ggtcatgaga
ggataaaatc ccacccactt ggcgactcac tgcagggagc acctgtgcag
22500ggagcacctg gggataggag agttccacca tgagctaact tctaggtggc
tgcatttgaa 22560tggctgtgag attttgtctg caatgttcgg ctgatgagag
tgtgagattg tgacagattc 22620aagctggatt tgcatcagtg agggacggga
gcgctggtct gggagatgcc agcctggctg 22680agcccaggcc atggtattag
cttctccgtg tcccgcccag gctgactgtg gagggcttta 22740gtcagaagat
cagggcttcc ccagctcccc tgcacactcg agtccctggg gggccttgtg
22800acaccccatg ccccaaatca ggatgtctgc agagggagct ggcagcagac
ctcgtcagag 22860gtaacacagc ctctgggctg gggaccccga cgtggtgctg
gggccatttc cttgcatctg 22920ggggagggtc agggctttcc ctgtgggaac
aagttaatac acaatgcacc ttacttagac 22980tttacacgta tttaatggtg
tgcgacccaa catggtcatt tgaccagtat tttggaaaga 23040atttaattgg
ggtgaccgga aggagcagac agacgtggtg gtccccaaga tgctccttgt
23100cactactggg actgttgttc tgcctggggg gccttggagg cccctcctcc
ctggacaggg 23160taccgtgcct tttctactct gctgggcctg cggcctgcgg
tcagggcacc agctccggag 23220cacccgcggc cccagtgtcc acggagtgcc
aggctgtcag ccacagatgc ccaggtccag 23280gtgtggccgc tccagccccc
gtgcccccat gggtggtttt gggggaaaag gccaagggca 23340gaggtgtcag
gagactggtg ggctcatgag agctgattct gctccttggc tgagctgccc
23400tgagcagcct ctcccgccct ctccatctga agggatgtgg ctctttctac
ctgggggtcc 23460tgcctggggc cagccttggg ctaccccagt ggctgtacca
gagggacagg catcctgtgt 23520ggaggggcat gggttcacgt ggccccagat
gcagcctggg accaggctcc ctggtgctga 23580tggtgggaca gtcaccctgg
gggttgaccg ccggactggg cgtccccagg gttgactata 23640ggaccaggtg
tccaggtgcc ctgcaagtag aggggctctc agaggcgtct ggctggcatg
23700ggtggacgtg gccccgggca tggccttcag cgtgtgctgc cgtgggtgcc
ctgagccctc 23760actgagtcgg tgggggcttg tggcttcccg tgagcttccc
cctagtctgt tgtctggctg 23820agcaagcctc ctgaggggct ctctattgca
gacagcactt gaagagggtg cagctgcggg 23880agctgtcgga agcagaggtc
aggcagcatc gggaagccag gcccgccctg ctgacgtcca 23940gactccgctt
catccccaag cctgacgggc tgcggccgat tgtgaacatg gactacgtcg
24000tgggagccag aacgttccgc agagaaaaga gggtggctgt gctttggttt
aacttccttt 24060ttaaacagaa gtgcgtttga gccccacatt tggtatcagc
ttagatgaag ggcccggagg 24120aggggccacg ggacacagcc agggccatgg
cacggcgcca acccatttgt gcgcacagtg 24180aggtggccga ggtgccggtg
cctccagaaa agcagcgtgg gggtgtaggg ggagctcctg 24240gggcagggac
aggctctgag gaccacaaga agcagccggg ccagggcctg gatgcagcac
24300ggcccgaggt cctggatccg tgtcctgctg tggtgcgcag cctccgtgcg
cttccgctta 24360cggggcccgg ggaccaggcc acgactgcca ggagcccacc
gggctctgag gatcctggac 24420cttgccccac ggctcctgca ccccacccct
gtggctgcgg tggctgcggt gaccccgtca 24480tctgaggaga gtgtggggtg
aggtggacag aggtgtggca tgaggatccc gtgtgcaaca 24540cacatgcggc
caggaacccg tttcaaacag ggtctgagga agctgggagg ggttctaggt
24600cccgggtctg ggtggctggg gacactgggg aggggctgct tctcccctgg
gtccctatgg
24660tggggtgggc acttggccgg atccactttc ctgactgtct cccatgctgt
ccccgccagg 24720ccgagcgtct cacctcgagg gtgaaggcac tgttcagcgt
gctcaactac gagcgggcgc 24780ggcgccccgg cctcctgggc gcctctgtgc
tgggcctgga cgatatccac agggcctggc 24840gcaccttcgt gctgcgtgtg
cgggcccagg acccgccgcc tgagctgtac tttgtcaagg 24900tgggtgccgg
ggacccccgt gagcagccct gctggacctt gggagtggct gcctgattgg
24960cacctcatgt tgggtggagg aggtactcct gggtgggccg cagggagtgc
aggtgaccct 25020gtcactgttg aggacacacc tggcacctag ggtggaggcc
ttcagccttt cctgcagcac 25080atggggccga ctgtgcaccc tgactgcccg
ggctcctatt cccaaggagg gtcccactgg 25140attccagttt ccgtcagaga
aggaaccgca acggctcagc caccaggccc cggtgccttg 25200caccccagtc
ctgagccagg ggtctcctgt cctgaggctc agagagggga cacagcccgc
25260cctgcccttg gggtctggag tggtgggggt cagagagaga gtgggggaca
ccgccaggcc 25320aggccctgag ggcagaggtg atgtctgagt ttctgcgtgg
ccactgtcag tctcctcgcc 25380tccactcaca caggtggatg tgacgggcgc
gtacgacacc atcccccagg acaggctcac 25440ggaggtcatc gccagcatca
tcaaacccca gaacacgtac tgcgtgcgtc ggtatgccgt 25500ggtccagaag
gccgcccatg ggcacgtccg caaggccttc aagagccacg taaggttcac
25560gtgtgatagt cgtgtccagg atgtgtgtct ctgggatatg aatgtgtcta
gaatgcagtc 25620gtgtctgtga tgcgtttctg tggtggaggt acttccatga
tttacacatc tgtgatatgc 25680gtgtgtggca cgtgtgtgtc gtggtgcatg
tatctgtggc gtgcatattt gtggtgtgtg 25740tgtgtgtggc acgtgtgtgt
ccatggtgtg tgtgcctgtg gtgtgcatgt gtgtgtgtct 25800gtgacacgtg
catgttcatg ctgtgtgctg catgtctgtg atgtgcctat ttgtggtgtg
25860tgtgtgcatg tgtccgtgac atatgcgtgt ctatggcatg ggtgtgtgtg
gccccttggc 25920cttactcctt cctcctccag gcatggtccg caccattgtc
ctcacgctct cgggtgctgg 25980tttggggagc tccacattca gggtcctcac
ttctagcatg ggtgcccctg tcctgtcaca 26040gggctgggcc ttggagactg
taagccaggt ttgagaggag agtagggatg ctggtggtac 26100cttcctggac
ccctggcacc cccaggaccc cagtctggcc tatgccggct ccatgagata
26160taggaaggct gattcaggcc tcgctccccg ggacacactc ctcccagagc
ggccgggggc 26220cttggggctc ggcaggggtg aaaggggccc tgggcttggg
ttcccaccca gtggtcatga 26280gcacgctgga ggggtaagcc ctcaaagtcg
tgccaggccg gggtgcagag gtgaagaagt 26340atccctggag cttcggtctg
gggagaggca catgtggaaa cccacaagga cctctttctc 26400tgacttcttg
agcttgtggg attggttttc atgtgtggga taggtgggga tctgtgggat
26460tggtttttat gagtggggta acacagagtt caaggcgagc tttcttcctg
tagtgggtct 26520gcaggtgctc caacagcttt attgaggaga ccatatcttc
ctttgaacta tggtcgggtt 26580tatagtaagt caggggtgtg gaggcctccc
ctgggctccc tgttctgttt cttccactct 26640ggggtcgtgt ggtgcctgct
gtggtgtgtg gccggtgggc agggcttcca ggcctccttg 26700tgttcattgg
cctggatgtg gccctggcta cgctccgtcc ttggaattcc cctgcgagtt
26760ggaggctttc tttctttctt tttttctttc tttttttttt tttttgataa
cagagtctcg 26820ctcttttttg cccaggctgg agtggtttgg cgtgatcttg
gctcactgca acctgtgctt 26880cctgagttca agcaattctc ttgcctcagc
ctcccaagta gctggaatta taggcgccca 26940ccaccatgct gactaatttt
tgtaatttta gtagagacga ggtttctcca tgttggccag 27000gctggtctcg
aactcctgac ctcaggtgat cctcccacct cggcctccca aagtgctggg
27060atgacaggtg tgaaccgccg cgcccggccg agactcgctt cctgcagctt
ccgtgagatc 27120tgcagcgata gctgcctgca gccttggtgc tgacaacctc
cgttttcctt ctccaggtct 27180cgctaggggt ctttccattt catgactctc
ttcacagaag agtttcacgt gtgctgattt 27240cccggctgtt tcctgcgtaa
ttggtgtctg ctgtttatcg atggcctcct tccatttcct 27300ttaggctttg
tttattgttg tttttccggc tccttgaagg aaaagtttcg attatggatg
27360tttgaacttt cttttctaaa caagcatctg aagttgccgt tttccctcta
aagcagggat 27420cccgaggccc ctggctgtgg agtggcaccg gtctggggcc
tgttaggaac ccggcgcaca 27480gcgggaggct aggtggggtg tggggagcca
gcgttcccgc ctgagccccg cccctctcag 27540atcagcagtg gcatgcggtg
ctcagaggcg cacacaccct actgagaact gtgcgtgaga 27600ggggtctaga
ttctgtgctc cttatgggaa tctaatgcct gatgatctga ggtggaaccg
27660tttgctccca aaaccatccc cttccccact gctgtcctgt ggaaaaatcg
tcttccacga 27720aaccagtccc tggtaccaca atggttgggg accctgtgct
aaagacctgc ttcagcagcc 27780tctcgtcagt gttgatatat tggcttttct
gtgttgagtc cagaataatt acggatttct 27840gtgatgcttt ccgccgacct
cagacccatg ggctatttgt gggcgtgttg cctgctcctg 27900ggttgggaag
ggtgcaggcc ccatgtacct tcctgttact gccttccagg ttggttctca
27960gggttgaatc gtactcgatg tggttttagc ccacggccct gccgccagct
cctgggggct 28020ggggaacatg ctgaagcaca gagtcaccgt gcgcgtcttt
tgatgcctca caagctcgag 28080gcctcctgtg tccgtgttag tgtgtgtcac
gtgcctgctc acatcctgtc ttggggacgc 28140aggggcttag caggtcccgt
agtaaatgac aagcgtcctg ggggagtctg cagaatagga 28200ggtgggggtg
ccggtctctc tcccgcgtct tcagactctt ctcctgcctg tgctgtggct
28260gcacctgcat ccctgcaatc cctccagcac tgggctggag aggcccggga
gctcgagtgc 28320cacttgtgcc acgtgactgt ggatggcagt cggtcacggg
ggtctgatgt gtggtgactg 28380tggatggcgg ttggtcacag gggtctgatg
tgtggtgact gtggatggcg gtcgtggggt 28440ctgatgtggt gactgtggat
ggcggtcgtg gggtctgatg tgtggtgact gtggatggcg 28500gtcgtggggt
ctgatgtggt gactgtggat ggcggtcgtg gggtctgatg tggtgactgt
28560ggatggcggt cgtggggtct gatgtggtga ctgtggatgg cagtcgtggg
gtctgatgtg 28620tggtgactgt ggatggcggt cgtggggtct gatgtggtga
ctgtggatgg cagtcgtggg 28680gtctgatgtg tggtgactgt ggatggcggt
cgtggggtct gatgtgtggt gactgtggat 28740ggcggtcgtg gggtctgatg
tgtggtgact gtggatggcg gtcgtggggt ctgatgtgtg 28800gtgactgtgg
atggcggtcg tggggtctga tgtggtgact gtggatggcg gtcgtggggt
28860ctgatgtgtg gtgactgtgg atggtgatcg gtcacagggg tctgatgtgt
ggtgactgtg 28920gatggcggtc gtggggtctg atgtgtggtg actgtggatg
gtgatcggtc acaggggtct 28980gatgtgtggt gactgtggat ggcggtcgtg
gggtctgatg tgtggtgact gtggatggcg 29040gttggtcccg ggggtctgat
gtgtggtgac tgtggatggc gatcggtcac aggggtctga 29100tgtgtggtga
ctgtggatgg cggtcgtggg gtctgatgtg tggtgactgt ggatggcggt
29160cgtggggtct gatgtgtggt gactgtggat ggcggtcgtg gggtctgatg
tggtgactgt 29220ggatggcggt cgtggggtct gatgtggtga ctgtggatgg
cggtcgtggg gtctgatgtg 29280tggtgactgt ggatggcggt tggtcccggg
ggtctgatgt gtggtgactg tggatggcgg 29340tcgtggggtc tgatgtggtg
actgtggatg gcagtcgtgg ggtctgatgt gtggtgactg 29400tggatggcgg
tcgtggggtc tgatgtgtgg tgactgtgga tggcggtcgt ggggtctgat
29460gtgtggtgac tgtggatggc ggtcgtgggg tctgatgtgt ggtgactgtg
gatggcggtc 29520gtggggtctg atgtggtgac tgtggatggc ggtcgtgggg
tctgatgtgt ggtgactgtg 29580gatggtgatc ggtcacaggg gtctgatgtg
tggtgactgt ggatggcggt cgtggggtct 29640gatgtgtggt gactgtggat
ggcggtcgtg gggtctgatg tggtgactgt ggatggcggt 29700cgtggggtct
gatgtgtggt gactgtggat ggcggtcgta gggtctgatg tgtggtgact
29760gtggatggca gtcggtcaca ggggtctgat gtgtggtgac tgtggatggc
ggtcgtgggg 29820tctgatgtgt ggtgactgtg gatggcggtc gtggggtctg
atgtgtggtg actgtggatg 29880gcggtcgtgg ggtctgatgt gtggtgactg
tggatggcgg tcgtggggtc tgatgtggtg 29940actgtggatg gtgatcggtc
acaggggtct gatgtgtggt agctgcaggt ggagtcccag 30000gtgtgtctgt
agctactttg cgtcctcggc cccccggccc ccgtttccca aacagaagct
30060tcccaggcgc tctctgggct tcatcccgcc atcgggcttg gccgcaggtc
cacacgtcct 30120gatcggaaga aacaagtgcc cagctctggc cggggcaggc
cacatttgtg gctcatgccc 30180tctcctctgc cggcaggtct ctaccttgac
agacctccag ccgtacatgc gacagttcgt 30240ggctcacctg caggagacca
gcccgctgag ggatgccgtc gtcatcgagc aggtctgggc 30300actgccctgc
agggttgggc acggactccc agcagtgggt cctcccctgg gcaatcactg
30360ggctcatgac cggacagact gttggccctg gggggcagtg gggggaatga
gctgtgatgg 30420gggcatgatg agctgtgtgc cttggcgaaa tctgagctgg
gccatgccag gctgcgacag 30480ctgctgcatt caggcacctg ctcacgtttg
actgcgcggc ctctctccag ttccgcagtg 30540cctttgttca tgatttgcta
aatgtcttct ctgccagttt tgatcttgag gccaaaggaa 30600aggtgtcccc
ctcctttagg agggcaggcc atgtttgagc cgtgtcctgc ccagctggcc
30660cctcagtgct gggtctgagg ccaaaggaaa cgtgtccccc ttcttaggag
gacgggccgt 30720gtttgagcca cgccccgctg agcgggcctc tcagtgctgg
gtctgtccac gtggccctgt 30780ggccctttgc agatgtggtc tgtccacgtg
gccctgtggc tctttgcaga tgcctgttag 30840cacttgctcg gctctagggg
acagtcgtgt ccaccgcatg aggctcagag acctctgggc 30900gaatttcctt
ggctcccagg gtgggggtgg aggtggcctg ggctgctggg acccagaccc
30960tgtgcccggc agctgggcag caactcctgg atcacatatg ccatccgggc
cacggtgggc 31020tgtgtgggtg tgagcccagc tggacccaca ggtggcccag
aggagacgtt ctgtgtcaca 31080cactctgcct aagcccatgt gtgtctgcag
agactcggcc cggccagccc acgatggccc 31140tgcattccag cccagccccg
cacttcatca caaacactga ccccaaaagg gacggagggt 31200cttggccacg
tggtcctgcc tgtctcagca cccaccggct cactcccatg tgtctcccgt
31260ctgctttcgc agagctcctc cctgaatgag gccagcagtg gcctcttcga
cgtcttccta 31320cgcttcatgt gccaccacgc cgtgcgcatc aggggcaagt
gagtcaggtg gccaggtgcc 31380attgccctgc gggtggctgg gcgggctggc
agggcttctg ctcacctctc tcctgcccct 31440tccccactgn ccttctgccc
ggggccacca gagtctcctt ttctggcccc cgccccctcc 31500ggctcctggg
ctgcaggctc ccgaggcccc ggaaacatgg ctcggcttgc ggcagccgga
31560gcggagcagg tgccacacga ggcctggaaa tggcaagcgg ggtgtggagt
tgctcctgcg 31620tggaggacga ggggcggggg gtgtgtctgg gtcaggtgtg
cgccgagcgt ttgagcctgc 31680agcttgtcag ctccaagtta ctactgacgc
tggacacccg gctctcacac gcttgtatct 31740ctctctcccg atacaaaagg
attttatccg attctcattc ctgtccctgt cgtgtgaccc 31800ccgcgagggc
gcgggctctt ctctctgtga ctagatttcc catctggaaa gtgcggggtt
31860gaccgtgtag tttgctcctc tcggggggcc tgtggtggcc atggggcagg
cggcctggga 31920gagctgccgt cacacagcca ctgggtgagc cacactcacg
gtggtagagc cacagtgcct 31980ggtgccacat cacgtcctct ggattttaag
taaaaccaca cacctcccgg caggcatctg 32040cctgcgaccc tgtgtgtgcc
tggggagagt ggtagcacgg aggaaattcg tgcacactca 32100aggtcatcag
caaggtcatc cgcagtcagg tggaacgtgg aggcctctct ctgggatcgt
32160ctccagcgga taaaggactg tgcacagctt cggaagcttt tatttaaaaa
tataactatt 32220aattattgca ttataagtaa tcactaatgg tatcagcaat
tataatattt attaaagtat 32280aattagaaat attaagtagt acacacgttc
tggaaaaaca caaattgcac atggcagcag 32340agtgaatttt ggccgaggga
cacgtgtgca catgtgtgta agcggccccc aggcccacag 32400aattcgctga
caaagtcacc tccccagaga agccaccacg ggcctccttc gtggtcgtga
32460attttattaa gatggatcaa gtcacgtacc gtccacgtgt ggcagggctt
tggggaatgt 32520gaggtgatga ctgcgtcctc atgccctgac agacaggagg
tgactgtgtc tgtcctgtcc 32580ctaggacacg gacaggcccg aagctctagt
ccccatcgtg gtccagtttg gcctctgaat 32640aaaaacgtct tcaaaacctg
ttgccccaaa aactaagaac agagagagtt tcccatccca 32700tgtgctcaca
ggggcgtatc tgcttgcgtt gactcgctgg gctggccgga ctcctagagt
32760tggtgcgtgt gcttctgtgc aaaaagtgca gtcctcttgc ccatcactgt
gatatctgca 32820ccagcaagga aagcctcttt tcttttcttt cttttttttt
ttttgagacg gaacgtcact 32880gttgtctgcc tgggcttgag tgcagtggcg
cgatctcaac tcactgcaac ctccgcctcc 32940cgggttccag catttctcct
gcctcagcct cccgagcagc tgagattaca ggcacccacc 33000ccctgcgcct
ggctaatttt tgtattttta gtagagaggg gtttttgcca tgttggccag
33060gctggtctcg aactcctgac ctcaggtgat ccacccacct cggcctccca
aagtgctggg 33120attacaggtg tgagccatca cgcccagccg gaaagcctct
ttttaaggtg accacctata 33180gcgcttcccg aaaataacag gtcttgtttt
tgcagtaggc tgcaagcgtc tcttagcaac 33240aggagtggcg tcctgtgggc
tctggggatg gctgagggtc gcgtggcagc catgccttct 33300gtgtgcacct
ttaggttcca cggggctatt ctgctctcac tgtttgtctg aaaacgcacc
33360cttggcatcc ttgtttggag agtttctgct tctcgttggt catgctgaaa
ctaggggcaa 33420ggttgtatcc gttggcgcgc agcggctaca tgtagggtca
tgagtctttc accgtggaca 33480aattccttga aaaaaaaaaa aggagtccgg
ttaagcattc attccgggtc aagtgtctgg 33540ttctgtgaat aaactctaag
atttaagaaa ccttaatgaa agaaaacctt gatgattcag 33600agcaaggatg
tggtcacacc tgtggctgga tctgtttcag ccgccccagt gcatggtgag
33660agtggggagc agggattgtt tgttcagagg tctcatctgg tatgtttctg
aggtgtttgc 33720cggctgaatg gtagacgtgt cgtttgtgtg tatgaggttc
tgtgtctgtg tgtggctcgg 33780tttgagtgta cgcatgtcca gcacatgccc
tgcccgtctc tcacctgtgt cttcccgccc 33840caggtcctac gtccagtgcc
aggggatccc gcagggctcc atcctctcca cgctgctctg 33900cagcctgtgc
tacggcgaca tggagaacaa gctgtttgcg gggattcggc gggacgggtg
33960aggcctcctc ttccccaggg gggcttgggt gggggttgat ttgcttttga
tgcattcagt 34020gttaatattc ctggtgctct ggagaccatg actgctctgt
cttgaggaac cagacaaggt 34080tgcagcccct tcttggtatg aagccgcacg
ggaggggttg cacagcctga ggactgcggg 34140ctccacgcag gctctgtcca
gcggccatgt ccagaggcct cagggctcag caggcgggag 34200ggccgctgcc
ctgcatgatg agcatgtgaa ttcaacaccg aggaagcaca ccagcttctg
34260tcacgtcacc caggttccgt tagggtcctt ggggagatgg ggctggtgca
gcctgaggcc 34320ccacatctcc cagcaggccc tcgacaggtg gcctggactg
ggcgcctctt cagcccattg 34380cccatcccac ttgcatgggg tctacaccca
aggacgcaca cacctaaata tcgtgccaac 34440ctaatgtggt tcaactcagc
tggcttttat tgacagcagt tacttttttt tttttaatac 34500tttaagttct
agggtacatg tgcacgacgt gcaggttagt tacatatgta tacatgtgcc
34560atgttggtgt gctgcaccca ttaactcatc atttacatta ggtatatctc
ctaatgctat 34620ccctccccac tccccccatc ccatgacagg ccctggtgtg
tgatgttccc caccctgtgt 34680ccaagtgttc tcattgttca gttcccacct
gtgagtgaga acatgtggtg tttggttttc 34740tttccttgca atagtttgct
cagagtgatg gtttccagct tcgtccatgt ccctacaaag 34800gacatgaact
catccttttt tatgactgca tagtattccg tggtgtatat gtgccacatt
34860ttcttaatcc agtctatcat cgatggacat ttgggttggt tgcaagtctt
tgctactgtg 34920aatagtgccg caataaacat acgtgtgcat gtgtctttat
agcagcatga tttataatcc 34980tttgggtata tacccagtaa tgggatggct
gggtcaaatg gtatttctag ttctagatcc 35040ttgaggaatc accacactgt
cttccacaat ggttgaacta gtttacactc ccaccaacag 35100tgtaaaagtg
ttctggtgct ggagaggatg tggacagcag ttattttttt atgaaaatag
35160tatcactgaa caagcagaca gttagtgaag gatgcgtcag gaagcctgca
ggccacacag 35220ccatttctct cgaagactcc gggtttttcc tgtgcatctt
ttgaaactct agctccaatt 35280atagcatgta cagtggatca aggttcttct
tcattaaggt tcaagttcta gattgaaata 35340agtttatgta acagaaacaa
aaatttcttg tacacacaac ttgctctggg atttggagga 35400aagtgtcctc
gagctggcgg cacactggtc agccctctgg gacaggatac ctctggccca
35460tggtcatggg gcgctgggct tgggcctgag ggtcacacag tgcaccatgc
ccagcttcct 35520gtggatagga tctgggtctc ggatcatgct gaggaccaca
gctgccatgc tggtaaaggg 35580caccacgtgg ctcagagggg gcgaggttcc
cagccccagc tttcttaccg tcttcagtta 35640tttttcccta agagtctgag
aagtggggcc gcgcctgatg gccttcgttc gtcttcagct 35700ggcacagaat
tgcacaagct gatggtaaac actgagtact tataatgaat gaggaattgc
35760tgtagcagtt aactgtagag agctcgtctg ttggaaagaa atttaagttt
ttcatttaac 35820cgctttggag aatgttactt tatttatggc tgtgtaaatt
gtttgacatt cagtccctcg 35880tagacagata ctacgtaaaa agtgtaaagt
taaccttgct gtgtattttc ccttatttta 35940ggctgctcct gcgtttggtg
gatgatttct tgttggtgac acctcacctc acccacgcga 36000aaaccttcct
caggtgaggc ccgtgccgtg tgtctgtggg gacctccaca gcctgtgggc
36060tttgcagttg agccccccgt gtcctgcccc tggcaccgca gcgttgtctc
tgccaagtcc 36120tctctctctg ccggtgctgg atccgcaaga gcagaggcgc
ttggccgtgc acccaggcct 36180gggggcgcag gggcaccttc gggagggagt
gggtaccgtg caggccctgg tcctgcagag 36240acgcacccag gttacacacg
tggtgagtgc aggcggtgac ctggctcctg ctgctctttg 36300gaaagtcaag
agtggcggct cctggggccc cagtgagacc cccaggagct gtgcacaggg
36360cctgcagggc cgaggcggca gcctcctccc cagggtgcac ctgagcctgc
ggagagcagg 36420agctgctgag tgagctggcc cacagcgttc gctgcggtca
cgttcctgcg tggggttgtt 36480tgggatcggt gggagaattt ggatttgctg
agtgctgctg tcttgaacca cggagatggc 36540taggagtggg tttcagagtt
gatttttgtg aatcaaacta aaatcaggca caggggacct 36600ggcctcagca
caggggattg tccaatgtgg tccccctcaa gggcgcccca cagagccggt
36660gggcttgttt taaagtgcga tttgacgagg gacgagaaac cttgaaagct
gtaaagggaa 36720ccctcagaaa atgtggccgc caggggtggt ttcaggtgct
ttgctgggct gtgtttgtga 36780aaacccattt ggacccgccc tccaagtcca
ccctccaggt ccaccctcca gggccgccct 36840gggctggggg tatgcctggc
gttccttgtg ccgcagcccg gagcacagca ggctgtgcac 36900atttaaatcc
actaagattc actcgggggg agcccaggtc ccaagcaact gagggctcag
36960gagtcctgag gctgctgagg ggacagagca gacggggaac gctgcttctg
tgtggcaagt 37020tcctgagggt gctggccagg gaggtggctc agagtgtatg
ttggggtccc accgggggca 37080gaactctgtc tctgatgagt cggcagccat
gtaacaggaa ggggtggcca cagggagctg 37140ggaatgcacc aggggagctg
cgcagctggc cgaggtccca gggccaggcc acaggaaggg 37200cagggggacg
cccggggcca cagcagaggc cgcaggaagg gaaggggatg cccaggccag
37260agcagaggct accgggcaca ggggggctcc ctgagctggg tgagcgaggc
tcatgactcg 37320gcgagggaac ctccttgacg tgaagctgac gactggtgtt
gcccagctca cagcccagcc 37380aggtcccgcg cctgagcagg aactcagaac
cctccccttt gtctaaagca cagcagatgc 37440cttcagggca tctaggagaa
aacaggcaaa gtcgttgaga aacgtcttaa aagaaggtgg 37500gatggtggca
atttcttgtc cagattttag tctgccccgg accacagatg agtctataac
37560gggattgtgg tgttgccatg gggacacatg agatggacca tcacagaggc
cactggggct 37620gcacctccca tctgagtcct ggctgtcccg ggtccaggcc
aggttcttgc atgctcacct 37680acctgtcctg cccgggagac agggaaagca
ccccgaagtc tggagcaggg ctgggtccag 37740gctcctcaga gctcctgcca
ggcccagcac cctgctccaa atcaccactt ctctggggtt 37800ttccaaagca
tttaacaagg gtgtcaggtt acctcctggg tgacggcccc gcatcctggg
37860gctgacattg cccctctgcc ttaggaccct ggtccgaggt gtccctgagt
atggctgcgt 37920ggtgaacttg cggaagacag tggtgaactt ccctgtagaa
gacgaggccc tgggtggcac 37980ggcttttgtt cagatgccgg cccacggcct
attcccctgg tgcggcctgc tgctggatac 38040ccggaccctg gaggtgcaga
gcgactactc caggtgagcg cacctggccg gaagtggagc 38100ctgtgcccgg
ctggggcagg tgctgctgca gggccgttgc gtccacctct gcttccgtgt
38160ggggcaggcg actgccaatc ccaaagggtc agaggccaca gggtgcccct
cgtcccatct 38220ggggctgagc agaaatgcat ctttctgtgg gagtgagggt
gctcacaacg ggagcagttt 38280tctgtgctat tttggtaaaa ggaaatggtg
caccagacct gggtgcactg aggtgtcttc 38340agaaagcagt ctggatccga
acccaagacg cccgggccct gctgggcgtg agtctctcaa 38400acccgaacac
aggggccctg ctgggcatga gtccctctga acccgagacc ctggggccct
38460gctgggcgtg agtctctccg aacccagaga cttcagggcc cttttgggcg
tgagtctctc 38520cgctgtgagc cccacactcc aaggctcatc cacagtctac
aggatgccat gagttcatga 38580tcacgtgtga cccatcaggg gacagggcca
tggtgtgggg ggggtctcta caaaattctg 38640gggtcttgtt tccccagagc
ccgagagctc aaggccccgt ctcaggctca gacacaaatg 38700aattgaagat
ggacacagat gcagaaatct gtgctgtttc ttttatgaat aaaaagtatc
38760aacattccag gcagggcaag gtggctcaca cctataatcc cagcactttg
ggaggccgag 38820gtgggtggat cacttgaggc caggagtttg aggccaacct
aaccaacata gtgaaattcc 38880atttctactt aaaaaataca aaaattagcc
tggcctggtg gcacacgcct gtagtccccg 38940ctatgcggga ggctgaggca
ggagaatcat ttgaacccag gaggcagagg ttgcagtgag 39000ccgagatcac
accactgcac tccagcctgg gcaacagagt gagacttcat cttaaaaaaa
39060aaaaaaaaag tatcagcatt ccaaaaccat agtggacagg tgttttttta
ttctgtcctt 39120cgataatatt tactggtgct gtgctagagg ccggaactgg
gggtgccttc ctctgaaagg 39180cacaccttca tgggaagaga aataagtggt
gaatggttgt taaaccagag gtttaaactg 39240gggtcctgtc gttctgagtt
aacagtccag atctggactt tgcctctttc cagaatgctc 39300cctggggttt
gcttcatggg ggagcagcag gtgtggacac cctcgtgatg ggggagcagc
39360aggtgcagac gccctcatga tgggggagtg gcaggtgcag acacccttgt
gcatggtgcc 39420cagcatgtcc ctgttgcagc tccctcccca caaggatgcc
ggtctcctgt gctccccaca 39480gtccctgctt ccctctcaca gccttacctg
gtcctggcct ccactggctt tgtctgcatg 39540atttccacat ttcctgggct
cccagcacct cttcgcctct cccaggcacc tctgcagtgc 39600tggccatacc
agtcagctgt gaactgtcca ctgcttattt tgctccccat gaaatgtatt
39660ttttaggaca ggcacccctg gttccagcct ctggcacagc atcagtgaat
gttattgaag
39720gacaaaggac agacaaacaa atcaggaaaa tgggttctct ctaaacacat
tgcaaagcca 39780cagaggctag tgcaggatgg gtgggcatca ggtcatcaga
tgtgggtcca atgccagaat 39840attctgtgct cccaaaggcc acttggtcag
agtgtgtgct tgcagaggtg gctctaaaag 39900ctcagcagtg gaggcagtgg
ttcgccatac tcagggtgaa ctcacatcct ctgtgtctga 39960agtatacagc
agaggcttga agggcatctg ggagaagaaa acaggcaaaa tgattaagaa
40020aagtgaaaaa ggaaaagtgg taagatggga attttcttgt ccagatttta
gtctcccaaa 40080ccacagctca gatggtagaa tgtggtcaga actgatggac
agaacaatag aacaaaacgg 40140aagccctatc tctcagaaac gtgtgttaat
gtggtatgtg gcacagctga tggaaaagag 40200agtgtgtgtg taattttttt
ttctgagaaa actgactgga agcaaataag ttgtgtcttt 40260acagcatata
ccagagcaga ttctaggtag aagaggagac acatgcaaac aacaccagca
40320acagaaataa aacaaaagac tcaaagggaa gggaggtgaa cgttccctgg
tttggtgttg 40380gggaaggaca cacagggagg cggatgaaac cagtgaggca
acgggcattg ctttcactgc 40440agagaaactc agcttgcctg agccacagtg
aaaatggcca ttccctggag cgtttgtgca 40500cgtgatttat ttaaggcgcc
ctgtgaggtc ctgcacattc atcctctcac tttgttctcc 40560taaccacctg
agaggtagag gaggaaaggc tccaggggag cagccgccct tggtcaccca
40620gctggcaaag ggcatgcatg attgcagcct ggcctcctgc tccggggccc
ttgctctgcc 40680cgaggacccc acacaagtca gacccatagg ctcagggtga
gccggagccc aaggtcgtgt 40740tggggatggc tgtgaaagaa gaaatggacg
tctgatgcac acttgggaag gtcctaccag 40800cagcgtcaaa gaaatgcatg
tgaaactgac agcgagaccc atccctcaaa gaaacgcacg 40860tgaaactgat
ggcgagacct gtccccatcc ctcatgctgg ctccttttct gggcttgcca
40920agagccagca tcaggttgag gcaagctgga aagacttttc tggaaagcag
cttgtttgca 40980tggaagtcct cacaatgtcc tgtgtcttcc cagtaattcc
acttctgaag tgaccagaca 41040ttatcacggg tcttatttac catttccagt
gttccaggca gggggacttg ccacagcaag 41100tcacgaacct gcccaaatac
agggctaagg agatattatg catcacaaaa cttgctctgc 41160cattaaacat
ttttcaaaga atttttgaag aatgtttaat ggcacaaaac gtttatttca
41220atgtagcagt gttcaaagct ggatgtaaaa gaacacaccc caggagcctg
ccgtgaatgt 41280catgtgtgtt catctttgga catggacata catgggcagt
gagtggtggt gaggccctgg 41340aggacatcgg tgggatgcct ccatcctgcc
cctctggaga caccatgtgt gccacgtgca 41400ctcactggag ccctgtttag
ctggtgccac ctggctcttc catccctgag attcaaacac 41460agtgagattc
cccacgccca actcagtgtt ctcccacaaa aaacctgagt cacacctgtg
41520ttcactcgag ggacgcccgg gagccagggc tccacagttt attatgtgtt
tttggctgag 41580ttatgtgcag atctcatcag ggcagatgat gagtgcacaa
acacggccgt gcgaggtttg 41640gatacactca acatcactag ccaggtcctg
gtggagtttg gtcatgcaga gtctggatgg 41700catgtagcat ttggagtcca
tggagtgagc acccagcccc ctcgggctgc agcgcatgcc 41760ccaggcagga
caaggaagcg ggaggaaggc aggaggctct ttggagcaag ctttgcagga
41820gggggctggg tgtggggcag gcacctgtgt ctgacattcc cccctgtgtc
tcagctatgc 41880ccggacctcc atcagagcca gtctcacctt caaccgcggc
ttcaaggctg ggaggaacat 41940gcgtcgcaaa ctctttgggg tcttgcggct
gaagtgtcac agcctgtttc tggatttgca 42000ggtgagcagg ctgatggtca
gcacagagtt cagagttcag gaggtgtgtg cgcaagtatg 42060tgtgtgtgtg
tgtgcgcgcg tgcctgcaag gctgatggtg actggctgca cgtaagagtg
42120cacatgtacg catatacacg tgagcacata catgtgtgca tgtgtgtaca
tgaaggcatg 42180gcagtgtgtg cacaggtgtg caagggcaca agtgtgtgca
catgcgaatg cacacctgac 42240atgcatgtgt gttcgtgcac agtcgtgtgg
gcattcacgt gaggtgcatg cgtgtgggtg 42300tgcagtgtga gtagcatgtg
tgcacataac atgtattgag gggtcctcgt gttcaccccg 42360ctaggtcctc
agcaccagtg ccactcctta caggatgaga cggggtccca ggccttggtg
42420ggctgaggct ctgaagctgc agccctgagg gcattgtccc atctgggcat
ccgcgtccac 42480tccctctcct gtgggcttct gtgtccactc cccctctcct
gtgggcattt acatccactc 42540cactccctct ctcctgtggg catccgcgtc
cactccccct ctctgtgggc atctgcgtcc 42600acctcccctc tctgtgggca
tttgcgtcca ctccctctcc tggttccttc ctgtcttggc 42660cgagcctcgg
gggcaggcag atgacacaga gtcttgactc gcccagggtg gttcgcagct
42720gccgggtgag ggccaggccg gatttcactg ggaagaggga tagtttcttg
tcaaaatgtt 42780cctctttctt gttccatctg aatggatgat aaagcaaaaa
gtaaaaactt aaaatcccag 42840agaggtttct accgtttctc actctttctt
ggcgactcta ggtgaacagc ctccagacgg 42900tgtgcaccaa catctacaag
atcctcctgc tgcaggcgta caggtgagcc gccaccaagg 42960ggtgcaggcc
cagcctccag ggaccctccg cgctctgctc acctctgacc cggggcttca
43020ccttggaact cctgggtttt aggggcaagg aatgtcttac gttttcagtg
gtgctgctgc 43080ctgtgcacag ttctgttcgc gtggctctgt gcaaagcacc
tgttctccat ctctgggtag 43140tggtaggagc cggtgtggcc ccaggtgtcc
ccactgtgcc tgtgcactgg ccgtgggacg 43200tcatggaggc catcccaggg
cagcaggggc atggggtaaa gagatgttta tggggagtct 43260tagcagagga
ggctgggaag gtgtctgaac agtagatggg agatcagatg cccggaggat
43320ttggggtctc agcaaagagg gccgaggtgg gtgcaggtga gggtcgctgg
ccccaccccc 43380gggaaggtgc agcagagctg tggctcccca cacagcccgg
ccagcacctg tgctctgggc 43440atggctgtgc tcctggaacg ttccctgtcc
tggctggtca gggggtgccc ctgccaagaa 43500tcgacaactt tatcacagag
ggaagggcca atctgtggag gccacagggc cagcttctgc 43560ctggagtcag
ggcaggtggt ggcacaagcc tcggggctgt accaaagggc agtcgggcac
43620cacaggcccg ggcctccacc tcaacaggcc tcccgagcca ctgggagctg
aatgccagga 43680ggccgaagcc ctcgccccat gagggctgag aaggagtgtg
agcatttgtg ttacccaggg 43740ccgaggctgc gcgaattacc gtgcacactt
gatgtgaaat gaggtcgtcg tctatcgtgg 43800aaacccagca agggctcacg
ggagagtttt ccattacaag gtcgtaccat gaaaatggtt 43860tttaacccga
gtgcttgcgc cttcatgctc tggcagggag ggcagagcca cagctgcatg
43920ttaccgcctt tgcaccagct ccagaggctt gggaccaggc tgtctcagtt
ccagggtgcg 43980tccggctcag accgccctcc tctctgcctt ctctctctgc
ctcaaatctt ccctcgtttg 44040catctccctg acgcgtgcct gggccctcgt
gcaagctgct tgactccttt ccggaaaccc 44100ttggggtgtg ctggatacag
gtgccactga ggactggagg tgtctgacac tgtggttgac 44160cccagggtcc
agctggcgtg cttggggcct ccttgggcca tgatgaggtc agaggagttt
44220tcccaggtga aaactcctgg gaaactccca gggccatgtg acctgccacc
tgctcctccc 44280atattcagct cagtcttgtc ctcatttccc caccagggtc
tctagctccg aggagctccc 44340gtagagggcc tgggctcagg gcagggcggc
tgagtttccc cacccatgtg gggacccttg 44400ggtagtcgct tgattgggta
gccctgagga ggccgagatg cgatgggcca cgggccgttt 44460ccaaacacag
agtcaggcac gtggaaggcc caggaatccc cttccctcga ggcaggagtg
44520ggagaacgga gagctgggcc ccgatttcac ggcagccagg ctgcagtggg
cgaggctgtg 44580gtggtccacg tggcgctggg ggcggggtct gattcaaatc
cgctggggct cggccttcct 44640ggcccgtgct ggccgcgcct ccacacgggc
ttggggtgga cgccccgacc tctagcaggt 44700ggctatttct ccctttggaa
gagagcccct cacccatgct aggtgtttcc ctcctgggtc 44760aggagcgtgg
ccgtgtggca accccgggac cttaggctta tttatttgtt taaaaacatt
44820ctgggcctgg cttccgttgt tgctaaatgg ggaaaagaca tcccacctca
gcagagttac 44880tgagaggctg aaaccggggt gctggcttga ctggtgtgat
ctcaggtcat tccagaagtg 44940gctcaggaag tcagtgagac caggtacatg
gggggctcag gcagtgggtg agatgaggta 45000cacggggggc tcaggcagtg
ggtgaggcca ggtacatggg gggctcaggc actgggtgag 45060atgaggtaca
cggggggctc aggcagaggg tcagaccagg tacacggggg ctctgatcac
45120acgcacatat gagcacatgt gcacatgtgc tgtttcatgg tagccaggtc
tgtgcacacc 45180tgccccaaag tcccaggaag ctgagaggcc aaagatggag
gctgacaggg ctggcgcggt 45240ggctcacacc tgtagtccca gcactttggg
aggccgaggc gagaggatcc cttgagccca 45300ggagtttaag accagcctga
gcaacatagt agaaccccat ctctatgaaa aataaaaaca 45360aaaattagct
gaacatggtg gtgtgcgcct gtagttccaa tacttgggag gctgaagtgg
45420gaggatcact tgagcccagg aggtggaagc tgcagtgagc tgagattgca
ccactgtact 45480gcagcctggg tgacagagtg agagcccatc tcaacaacaa
caaagaagac tgacaaatgc 45540agtttcttgg aaagaaacat ttagtaggaa
cttaacctac acacagaagc caagtcggtg 45600tctcggtgtc agtgagatga
gatgatgggt cctcacacca tcaccccaga cccagggttt 45660atgcaccaca
ggggcgggtg gctcagaagg gatgcgcagg acgttgatat acgatgacat
45720caaggttgtc tgacgaaggg caggattcat gataagtacc tgctggtaca
caaggaacaa 45780tggataaact ggaaacctta gaggccttcc cggaacaggg
gctaatcaga agccagcatg 45840gggggctggc atccaggatg gagctgcttc
agcctccaca tgcgtgttca tacagatggt 45900gcacagaaac gcagtgtacc
tgtgcacaca cagacacgca gctactcgca cacacaagca 45960cacacacaga
catgcatgca tgcatccgtg tgtgtgcacc tgtgcccatg aggaaaccca
46020tgcatgtgca ttcatgcacg cacacaggca ccggtgggcc catgcccaca
cccacgagca 46080ccgtctgatt aggaggcctt tcctctgacg ctgtccgcca
tcctctcagg tttcacgcat 46140gtgtgctgca gctcccattt catcagcaag
tttggaagaa ccccacattt ttcctgcgcg 46200tcatctctga cacggcctcc
ctctgctact ccatcctgaa agccaagaac gcaggtatgt 46260gcaggtgcct
ggcctcagtg gcagcagtgc ctgcctgctg gtgttagtgt gtcaggagac
46320tgagtgaatc tgggcttagg aagttcttac cccttttcgc atcaggaagt
ggtttaaccc 46380aaccactgtc aggctcgtct gcccgccctc tcgtggggtg
agcagagcac ctgatggaag 46440ggacaggagc tgtctgggag ctgccatcct
tcccaccttg ctctgcctgg ggaagcgctg 46500gggggcctgg tctctcctgt
ttgccccatg gtgggatttg gggggcctgg cctctcctgt 46560ttgccctgtg
gtgggattgg gctgtctccc gtccatggca cttagggccc ttgtgcaaac
46620ccaggccaag ggcttaggag gaggccaggc ccaggctacc ccacccctct
caggagcaga 46680ggccgcgtat caccacgaca gagccccgcg ccgtcctctg
cttcccagtc accgtcctct 46740gcccctggac actttgtcca gcatcaggga
ggtttctgat ccgtctgaaa ttcaagccat 46800gtcgaacctg cggtcctgag
cttaacagct tctactttct gttctttctg tgttgtggaa 46860atttcacctg
gagaagccga agaaaacatt tctgtcgtga ctcctgcggt gcttgggtcg
46920ggacagccag agatggagcc accccgcaga ccgtcgggtg tgggcagctt
tccggtgtct 46980cctgggaggg gagctgggct gggcctgtga ctcctcagcc
tctgttttcc cccagggatg 47040tcgctggggg ccaagggcgc cgccggccct
ctgccctccg aggccgtgca gtggctgtgc 47100caccaagcat tcctgctcaa
gctgactcga caccgtgtca cctacgtgcc actcctgggg 47160tcactcagga
caggcaagtg tgggtggagg ccagtgcggg ccccacctgc ccaggggtca
47220tccttgaacg ccctgtgtgg ggcgagcagc ctcagatgct gctgaagtgc
agacgccccc 47280gggcctgacc ctgggggcct ggagccacgc tggcagccct
atgtgattaa acgctggtgt 47340ccccaggcca cggagcctgg cagggtcccc
aacttcttga acccctgctt cccatctcag 47400gggcgatggc tccccacgct
tgggagcctt ctgacccctg acctgtgtcc tctcacagcc 47460tcttccctgg
ctgctgccct gagctcctgg ggtcctgagc aagttctctc cccgccccgc
47520cgctccagcg tcactgggct gcctgtctgc tcgccccggt ggaggggtgt
ctgtcccttc 47580actgaggttc ccaccagcca gggccacgag gtgcaggccc
tgcctgcccg gccacccaca 47640cgtcctagga gggttggagg atgccacctc
tggcctcttc tggaacggag tctgattttg 47700gccccgcagc ccagacgcag
ctgagtcgga agctcccggg gacgacgctg actgccctgg 47760aggccgcagc
caacccggca ctgccctcag acttcaagac catcctggac tgatggccac
47820ccgcccacag ccaggccgag agcagacacc agcagccctg tcacgccggg
ctctacgtcc 47880cagggaggga ggggcggccc acacccaggc ccgcaccgct
gggagtctga ggcctgagtg 47940agtgtttggc cgaggcctgc atgtccggct
gaaggctgag tgtccggctg aggcctgagc 48000gagtgtccag ccaagggctg
agtgtccagc acacctgccg tcttcacttc cccacaggct 48060ggcgctcggc
tccaccccag ggccagcttt tcctcaccag gagcccggct tccactcccc
48120acataggaat agtccatccc cagattcgcc attgttcacc cctcgccctg
ccctcctttg 48180ccttccaccc ccaccatcca ggtggagacc ctgagaagga
ccctgggagc tctgggaatt 48240tggagtgacc aaaggtgtgc cctgtacaca
ggcgaggacc ctgcacctgg atgggggtcc 48300ctgtgggtca aattgggggg
aggtgctgtg ggagtaaaat actgaatata tgagtttttc 48360agttttgaaa
aaaatctcat gtttgaatcc taatgtgcac tgcatagaca ccactgtatg
48420caattacaga agcctgtgag tgaacggggt ggtggtcagt gcgggcccat
ggcctggctg 48480tgcatttacg gaagtctatg agtgaatggg gttgtggtca
gtgcgggccc atggcctggc 48540tgggcctggg aggtttctga tgctgtgagg
caggagggga aggagggtag gggatagaca 48600gtgggagccc ccaccctgga
agacataaca gtaagtccag gcccgaaggg cagcagggat 48660gctgggggcc
cagcttgggc ggcggggatg atggagggcc tggccagggt ggcagggatg
48720atgggggccc cagctggggt ggcaggggtg atgggggggg ctggtctggg
tggcggggaa 48780gatggggaag cctggctggg ccccctcctc ccctgcctcc
cacctgcagc cgtggatccg 48840gatgtgcttc cctggtgcac atcctctggg
ccatcagctt tcatggaggt ggggggcagg 48900ggcatgacac catcctgtat
aaaatccagg attcctcctc ctgaacgccc caactcaggt 48960tgaaagtcac
attccgcctc tggccattct cttaagagta gaccaggatt ctgatctctg
49020aagggtgggt agggtggggc agtggagggt gtggacacag gaggcttcag
ggtggggctg 49080gtgatgctct ctcatcctct tatcatctcc cagtctcatc
tctcatcctc ttatcatctc 49140ccagtctcat ctgtcttcct cttatctccc
agtctcatct gtcatcctct taccatctcc 49200cagtctcatc tcttatcctc
ttatctccta gtctcatcca gacttacctc ccagggcggg 49260tgccaggctc
gcagtggagc tggacatacg tccttcctca ggcagaagga actggaagga
49320ttgcagagaa caggaggggc ggctcagagg gacgcagtct tggggtgaag
aaacagcccc 49380tcctcagaag ttggcttggg ccacacgaaa ccgagggccc
tgcgtgagtg gctccagagc 49440cttccagcag gtccctggtg gggccttatg
gtatggccgg gtcctactga gtgcaccttg 49500gacagggctt ctggtttgag
tgcagcccgg acgtgcctgg tgtcggggtg ggggcttatg 49560gccactggat
atggcgtcat ttattgctgc tgcttcagag aatgtctgag tgaccgagcc
49620taatgtgtat ggtgggccca agtccacaga ctgtgtcgta aatgcactct
ggtgcctgga 49680gcccccgtat aggagctgtg aggaaggagg ggctcttggc
agccggcctg ggggcgcctt 49740tgccctgcaa actggaaggg agcggccccg
ggcgccgtgg gcggacgacc tcaagtgaga 49800ggttggacag aacagggcgg
ggacttccca ggagcagagg ccgctgctca ggcacacctg 49860ggtttgaatc
acagaccaac aggtcaggcc attgttcagc tatccatctt ctacaaagct
49920ccagattcct gtttctccgg gtgttttttg ttgaaatttt actcaggatt
acttatattt 49980tttgctaaag tattagaccc ttaaaaaagg tatttgcttt
gatatggctt aactcactaa 50040gcacctactt tatttgtctg tttttattta
ttattattat tattattaga gatggtgtct 50100actctgtcac ccaggttgtt
agtgcagtgg cacagtcatg gctcgctgta gccgcaaacc 50160cccaggctca
agtgatcctc cggcctcagc ttcccagagt gctgggatta caggtgtgag
50220ccactgccct tgcctggcac ttttaaaaac cactatgtaa ggtcaggtcc
agtggcttcc 50280acacctgtca tcccagtagt ttgggaagcc gaggcagaag
gattgtctga ggccaggagt 50340ttgagaccag catgggtaac atagggagac
cccatctcta caaaaaatgc aaaaagttat 50400ccgggcgtgg ggtccagcat
ctgtagtccc agctgctcgg gaggctgagt gggaggatcg 50460cttgagcccg
ggaggtcatg gctgcagtga gctgtgattg taccatcgca ctccagcctg
50520ggcaacagag tgagaccctg tctcaaaaaa aaaaaaaaaa aaagaaggag
aaggagaaga 50580gaagaagaag gaagaaggaa agagaagaag aaggaagaag
gaagaaagaa ggagaaggag 50640gcctgctagg tgctaggtag actgtcaaat
ctcagagcaa aatgaaaata acaaagtttt 50700aaagggaaag aaaaacccca
gctctttgga cttccttagg cctgaacttc atctcaagca 50760gcttccttcc
acagacaagc gtgtatggag cgagtgagtt caaagcagaa agggaggaga
50820agcaggcaag ggtggaggct gtgggtgaca ccagccagga cccctgaaag
ggagtggttg 50880ttttcctgcc tcagccccac gctcctgccg gtcctgcacc
tgctgtaacc gtcgatgttg 50940gtgccaggtg cccacctggg aaggatgctg
tgcagggggc ttgccaaact ttggtgggtt 51000tcagaagccc caggcacttg
tggcaggcac aattacagcc cctccccaaa gatgcccacg 51060tccttctcct
ggaacctgtg aatgtgtcac ccgcaaggca gaggctggtg aaggctgcag
51120gtggaatcac ggctgccagt cagccgatct taaggtcatc ctggattatc
tggtgggcct 51180gatatggcca caagggtccc tagaagtgag agagggaggc
aggggagagt cagagagggg 51240acgtgagaag gaccactggc cactgctggc
tttgagatgg aggagggggt ccccagccaa 51300ggaatggggg cagccgctcc
atgctggaaa agcaagcaat cctccccggt cctgagggca 51360cacggccctg
cccacgcctc gatttcaggc cagtgggacc tgtttcagct ttccggcctc
51420cagagctgta agatgatgcg tttgtgttca gccactaagc tgcagtgatt
cgtcacagca 51480gcaaatggaa tagcagtaca gggaaatgaa tacagggaca
gttctcagag tgactctcag 51540cccacccctg gg 5155241132PRTHomo sapiens
4Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1
5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu
Gly 20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala
Ala Phe Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro
Trp Asp Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln
Val Ser Cys Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg
Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe
Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110 Glu Ala Phe
Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125 Asp
Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135
140 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val
145 150 155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro
Pro Leu Tyr 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro
Pro His Ala Ser Gly 180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg
Ala Trp Asn His Ser Val Arg 195 200 205 Glu Ala Gly Val Pro Leu Gly
Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly Gly Ser Ala Ser
Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala
Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255
Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260
265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly
Ala 275 280 285 Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg
Gln His His 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg
Pro Trp Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr
Lys His Phe Leu Tyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg
Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly
Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365 Arg Pro
Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380
Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385
390 395 400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro
Leu Arg 405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg
Glu Lys Pro Gln 420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp
Thr Asp Pro Arg Arg Leu 435 440 445 Val Gln Leu Leu Arg Gln His Ser
Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460 Val Arg Ala Cys Leu Arg
Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn
Glu Arg Arg Phe Leu Arg
Asn Thr Lys Lys Phe Ile Ser 485 490 495 Leu Gly Lys His Ala Lys Leu
Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510 Ser Val Arg Asp Cys
Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525 Val Pro Ala
Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540 Leu
His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe 545 550
555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe
Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile
Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu
Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg Pro Ala Leu Leu
Thr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro Asp Gly Leu Arg
Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr
Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 Arg Val
Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670
Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675
680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro
Pro 690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr
Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr Glu Val Ile Ala
Ser Ile Ile Lys Pro Gln 725 730 735 Asn Thr Tyr Cys Val Arg Arg Tyr
Ala Val Val Gln Lys Ala Ala His 740 745 750 Gly His Val Arg Lys Ala
Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765 Leu Gln Pro Tyr
Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780 Pro Leu
Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795
800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His
805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly
Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu
Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe Ala Gly Ile Arg
Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp Asp Phe Leu Leu
Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Arg
Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890 895 Val Val Asn
Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu 900 905 910 Ala
Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe 915 920
925 Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser
930 935 940 Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu
Thr Phe 945 950 955 960 Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg
Arg Lys Leu Phe Gly 965 970 975 Val Leu Arg Leu Lys Cys His Ser Leu
Phe Leu Asp Leu Gln Val Asn 980 985 990 Ser Leu Gln Thr Val Cys Thr
Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995 1000 1005 Ala Tyr Arg Phe
His Ala Cys Val Leu Gln Leu Pro Phe His Gln 1010 1015 1020 Gln Val
Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp 1025 1030 1035
Thr Ala Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly 1040
1045 1050 Met Ser Leu Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser
Glu 1055 1060 1065 Ala Val Gln Trp Leu Cys His Gln Ala Phe Leu Leu
Lys Leu Thr 1070 1075 1080 Arg His Arg Val Thr Tyr Val Pro Leu Leu
Gly Ser Leu Arg Thr 1085 1090 1095 Ala Gln Thr Gln Leu Ser Arg Lys
Leu Pro Gly Thr Thr Leu Thr 1100 1105 1110 Ala Leu Glu Ala Ala Ala
Asn Pro Ala Leu Pro Ser Asp Phe Lys 1115 1120 1125 Thr Ile Leu Asp
1130 5 6492DNAHomo sapiens 5tttctgtgaa gcagaagtct gggaatcgat
ctggaaatcc tcctaatttt tactccctct 60ccccgcgact cctgattcat tgggaagttt
caaatcagct ataactggag agtgctgaag 120attgatggga tcgttgcctt
atgcatttgt tttggtttta caaaaaggaa acttgacaga 180ggatcatgct
gtacttaaaa aatacaacat cacagaggaa gtagactgat attaacaata
240cttactaata ataacgtgcc tcatgaaata aagatccgaa aggaattgga
ataaaaattt 300cctgcatctc atgccaaggg ggaaacacca gaatcaagtg
ttccgcgtga ttgaagacac 360cccctcgtcc aagaatgcaa agcacatcca
ataaaatagc tggattataa ctcctcttct 420ttctctgggg gccgtggggt
gggagctggg gcgagaggtg ccgttggccc ccgttgcttt 480tcctctggga
aggatggcgc acgctgggag aacagggtac gataaccggg agatagtgat
540gaagtacatc cattataagc tgtcgcagag gggctacgag tgggatgcgg
gagatgtggg 600cgccgcgccc ccgggggccg cccccgcacc gggcatcttc
tcctcccagc ccgggcacac 660gccccatcca gccgcatccc gggacccggt
cgccaggacc tcgccgctgc agaccccggc 720tgcccccggc gccgccgcgg
ggcctgcgct cagcccggtg ccacctgtgg tccacctgac 780cctccgccag
gccggcgacg acttctcccg ccgctaccgc cgcgacttcg ccgagatgtc
840cagccagctg cacctgacgc ccttcaccgc gcggggacgc tttgccacgg
tggtggagga 900gctcttcagg gacggggtga actgggggag gattgtggcc
ttctttgagt tcggtggggt 960catgtgtgtg gagagcgtca accgggagat
gtcgcccctg gtggacaaca tcgccctgtg 1020gatgactgag tacctgaacc
ggcacctgca cacctggatc caggataacg gaggctggga 1080tgcctttgtg
gaactgtacg gccccagcat gcggcctctg tttgatttct cctggctgtc
1140tctgaagact ctgctcagtt tggccctggt gggagcttgc atcaccctgg
gtgcctatct 1200gggccacaag tgaagtcaac atgcctgccc caaacaaata
tgcaaaaggt tcactaaagc 1260agtagaaata atatgcattg tcagtgatgt
accatgaaac aaagctgcag gctgtttaag 1320aaaaaataac acacatataa
acatcacaca cacagacaga cacacacaca cacaacaatt 1380aacagtcttc
aggcaaaacg tcgaatcagc tatttactgc caaagggaaa tatcatttat
1440tttttacatt attaagaaaa aaagatttat ttatttaaga cagtcccatc
aaaactcctg 1500tctttggaaa tccgaccact aattgccaag caccgcttcg
tgtggctcca cctggatgtt 1560ctgtgcctgt aaacatagat tcgctttcca
tgttgttggc cggatcacca tctgaagagc 1620agacggatgg aaaaaggacc
tgatcattgg ggaagctggc tttctggctg ctggaggctg 1680gggagaaggt
gttcattcac ttgcatttct ttgccctggg ggctgtgata ttaacagagg
1740gagggttcct gtggggggaa gtccatgcct ccctggcctg aagaagagac
tctttgcata 1800tgactcacat gatgcatacc tggtgggagg aaaagagttg
ggaacttcag atggacctag 1860tacccactga gatttccacg ccgaaggaca
gcgatgggaa aaatgccctt aaatcatagg 1920aaagtatttt tttaagctac
caattgtgcc gagaaaagca ttttagcaat ttatacaata 1980tcatccagta
ccttaagccc tgattgtgta tattcatata ttttggatac gcacccccca
2040actcccaata ctggctctgt ctgagtaaga aacagaatcc tctggaactt
gaggaagtga 2100acatttcggt gacttccgca tcaggaaggc tagagttacc
cagagcatca ggccgccaca 2160agtgcctgct tttaggagac cgaagtccgc
agaacctgcc tgtgtcccag cttggaggcc 2220tggtcctgga actgagccgg
ggccctcact ggcctcctcc agggatgatc aacagggcag 2280tgtggtctcc
gaatgtctgg aagctgatgg agctcagaat tccactgtca agaaagagca
2340gtagaggggt gtggctgggc ctgtcaccct ggggccctcc aggtaggccc
gttttcacgt 2400ggagcatggg agccacgacc cttcttaaga catgtatcac
tgtagaggga aggaacagag 2460gccctgggcc cttcctatca gaaggacatg
gtgaaggctg ggaacgtgag gagaggcaat 2520ggccacggcc cattttggct
gtagcacatg gcacgttggc tgtgtggcct tggcccacct 2580gtgagtttaa
agcaaggctt taaatgactt tggagagggt cacaaatcct aaaagaagca
2640ttgaagtgag gtgtcatgga ttaattgacc cctgtctatg gaattacatg
taaaacatta 2700tcttgtcact gtagtttggt tttatttgaa aacctgacaa
aaaaaaagtt ccaggtgtgg 2760aatatggggg ttatctgtac atcctggggc
attaaaaaaa aaatcaatgg tggggaacta 2820taaagaagta acaaaagaag
tgacatcttc agcaaataaa ctaggaaatt tttttttctt 2880ccagtttaga
atcagccttg aaacattgat ggaataactc tgtggcatta ttgcattata
2940taccatttat ctgtattaac tttggaatgt actctgttca atgtttaatg
ctgtggttga 3000tatttcgaaa gctgctttaa aaaaatacat gcatctcagc
gtttttttgt ttttaattgt 3060atttagttat ggcctataca ctatttgtga
gcaaaggtga tcgttttctg tttgagattt 3120ttatctcttg attcttcaaa
agcattctga gaaggtgaga taagccctga gtctcagcta 3180cctaagaaaa
acctggatgt cactggccac tgaggagctt tgtttcaacc aagtcatgtg
3240catttccacg tcaacagaat tgtttattgt gacagttata tctgttgtcc
ctttgacctt 3300gtttcttgaa ggtttcctcg tccctgggca attccgcatt
taattcatgg tattcaggat 3360tacatgcatg tttggttaaa cccatgagat
tcattcagtt aaaaatccag atggcaaatg 3420accagcagat tcaaatctat
ggtggtttga cctttagaga gttgctttac gtggcctgtt 3480tcaacacaga
cccacccaga gccctcctgc cctccttccg cgggggcttt ctcatggctg
3540tccttcaggg tcttcctgaa atgcagtggt gcttacgctc caccaagaaa
gcaggaaacc 3600tgtggtatga agccagacct ccccggcggg cctcagggaa
cagaatgatc agacctttga 3660atgattctaa tttttaagca aaatattatt
ttatgaaagg tttacattgt caaagtgatg 3720aatatggaat atccaatcct
gtgctgctat cctgccaaaa tcattttaat ggagtcagtt 3780tgcagtatgc
tccacgtggt aagatcctcc aagctgcttt agaagtaaca atgaagaacg
3840tggacgtttt taatataaag cctgttttgt cttttgttgt tgttcaaacg
ggattcacag 3900agtatttgaa aaatgtatat atattaagag gtcacggggg
ctaattgctg gctggctgcc 3960ttttgctgtg gggttttgtt acctggtttt
aataacagta aatgtgccca gcctcttggc 4020cccagaactg tacagtattg
tggctgcact tgctctaaga gtagttgatg ttgcattttc 4080cttattgtta
aaaacatgtt agaagcaatg aatgtatata aaagcctcaa ctagtcattt
4140ttttctcctc ttcttttttt tcattatatc taattatttt gcagttgggc
aacagagaac 4200catccctatt ttgtattgaa gagggattca catctgcatc
ttaactgctc tttatgaatg 4260aaaaaacagt cctctgtatg tactcctctt
tacactggcc agggtcagag ttaaatagag 4320tatatgcact ttccaaattg
gggacaaggg ctctaaaaaa agccccaaaa ggagaagaac 4380atctgagaac
ctcctcggcc ctcccagtcc ctcgctgcac aaatactccg caagagaggc
4440cagaatgaca gctgacaggg tctatggcca tcgggtcgtc tccgaagatt
tggcaggggc 4500agaaaactct ggcaggctta agatttggaa taaagtcaca
gaattaagga agcacctcaa 4560tttagttcaa acaagacgcc aacattctct
ccacagctca cttacctctc tgtgttcaga 4620tgtggccttc catttatatg
tgatctttgt tttattagta aatgcttatc atctaaagat 4680gtagctctgg
cccagtggga aaaattagga agtgattata aatcgagagg agttataata
4740atcaagatta aatgtaaata atcagggcaa tcccaacaca tgtctagctt
tcacctccag 4800gatctattga gtgaacagaa ttgcaaatag tctctatttg
taattgaact tatcctaaaa 4860caaatagttt ataaatgtga acttaaactc
taattaattc caactgtact tttaaggcag 4920tggctgtttt tagactttct
tatcacttat agttagtaat gtacacctac tctatcagag 4980aaaaacagga
aaggctcgaa atacaagcca ttctaaggaa attagggagt cagttgaaat
5040tctattctga tcttattctg tggtgtcttt tgcagcccag acaaatgtgg
ttacacactt 5100tttaagaaat acaattctac attgtcaagc ttatgaaggt
tccaatcaga tctttattgt 5160tattcaattt ggatctttca gggatttttt
ttttaaatta ttatgggaca aaggacattt 5220gttggagggg tgggagggag
gaagaatttt taaatgtaaa acattcccaa gtttggatca 5280gggagttgga
agttttcaga ataaccagaa ctaagggtat gaaggacctg tattggggtc
5340gatgtgatgc ctctgcgaag aaccttgtgt gacaaatgag aaacattttg
aagtttgtgg 5400tacgaccttt agattccaga gacatcagca tggctcaaag
tgcagctccg tttggcagtg 5460caatggtata aatttcaagc tggatatgtc
taatgggtat ttaaacaata aatgtgcagt 5520tttaactaac aggatattta
atgacaacct tctggttggt agggacatct gtttctaaat 5580gtttattatg
tacaatacag aaaaaaattt tataaaatta agcaatgtga aactgaattg
5640gagagtgata atacaagtcc tttagtctta cccagtgaat cattctgttc
catgtctttg 5700gacaaccatg accttggaca atcatgaaat atgcatctca
ctggatgcaa agaaaatcag 5760atggagcatg aatggtactg taccggttca
tctggactgc cccagaaaaa taacttcaag 5820caaacatcct atcaacaaca
aggttgttct gcataccaag ctgagcacag aagatgggaa 5880cactggtgga
ggatggaaag gctcgctcaa tcaagaaaat tctgagacta ttaataaata
5940agactgtagt gtagatactg agtaaatcca tgcacctaaa ccttttggaa
aatctgccgt 6000gggccctcca gatagctcat ttcattaagt ttttccctcc
aaggtagaat ttgcaagagt 6060gacagtggat tgcatttctt ttggggaagc
tttcttttgg tggttttgtt tattatacct 6120tcttaagttt tcaaccaagg
tttgcttttg ttttgagtta ctggggttat ttttgtttta 6180aataaaaata
agtgtacaat aagtgttttt gtattgaaag cttttgttat caagattttc
6240atacttttac cttccatggc tctttttaag attgatactt ttaagaggtg
gctgatattc 6300tgcaacactg tacacataaa aaatacggta aggatacttt
acatggttaa ggtaaagtaa 6360gtctccagtt ggccaccatt agctataatg
gcactttgtt tgtgttgttg gaaaaagtca 6420cattgccatt aaactttcct
tgtctgtcta gttaatattg tgaagaaaaa taaagtacag 6480tgtgagatac tg
64926239PRTHomo sapiens 6Met Ala His Ala Gly Arg Thr Gly Tyr Asp
Asn Arg Glu Ile Val Met 1 5 10 15 Lys Tyr Ile His Tyr Lys Leu Ser
Gln Arg Gly Tyr Glu Trp Asp Ala 20 25 30 Gly Asp Val Gly Ala Ala
Pro Pro Gly Ala Ala Pro Ala Pro Gly Ile 35 40 45 Phe Ser Ser Gln
Pro Gly His Thr Pro His Pro Ala Ala Ser Arg Asp 50 55 60 Pro Val
Ala Arg Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala 65 70 75 80
Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val His Leu Thr 85
90 95 Leu Arg Gln Ala Gly Asp Asp Phe Ser Arg Arg Tyr Arg Arg Asp
Phe 100 105 110 Ala Glu Met Ser Ser Gln Leu His Leu Thr Pro Phe Thr
Ala Arg Gly 115 120 125 Arg Phe Ala Thr Val Val Glu Glu Leu Phe Arg
Asp Gly Val Asn Trp 130 135 140 Gly Arg Ile Val Ala Phe Phe Glu Phe
Gly Gly Val Met Cys Val Glu 145 150 155 160 Ser Val Asn Arg Glu Met
Ser Pro Leu Val Asp Asn Ile Ala Leu Trp 165 170 175 Met Thr Glu Tyr
Leu Asn Arg His Leu His Thr Trp Ile Gln Asp Asn 180 185 190 Gly Gly
Trp Asp Ala Phe Val Glu Leu Tyr Gly Pro Ser Met Arg Pro 195 200 205
Leu Phe Asp Phe Ser Trp Leu Ser Leu Lys Thr Leu Leu Ser Leu Ala 210
215 220 Leu Val Gly Ala Cys Ile Thr Leu Gly Ala Tyr Leu Gly His Lys
225 230 235 71207DNAHomo sapiens 7tttctgtgaa gcagaagtct gggaatcgat
ctggaaatcc tcctaatttt tactccctct 60ccccgcgact cctgattcat tgggaagttt
caaatcagct ataactggag agtgctgaag 120attgatggga tcgttgcctt
atgcatttgt tttggtttta caaaaaggaa acttgacaga 180ggatcatgct
gtacttaaaa aatacaacat cacagaggaa gtagactgat attaacaata
240cttactaata ataacgtgcc tcatgaaata aagatccgaa aggaattgga
ataaaaattt 300cctgcatctc atgccaaggg ggaaacacca gaatcaagtg
ttccgcgtga ttgaagacac 360cccctcgtcc aagaatgcaa agcacatcca
ataaaatagc tggattataa ctcctcttct 420ttctctgggg gccgtggggt
gggagctggg gcgagaggtg ccgttggccc ccgttgcttt 480tcctctggga
aggatggcgc acgctgggag aacagggtac gataaccggg agatagtgat
540gaagtacatc cattataagc tgtcgcagag gggctacgag tgggatgcgg
gagatgtggg 600cgccgcgccc ccgggggccg cccccgcacc gggcatcttc
tcctcccagc ccgggcacac 660gccccatcca gccgcatccc gggacccggt
cgccaggacc tcgccgctgc agaccccggc 720tgcccccggc gccgccgcgg
ggcctgcgct cagcccggtg ccacctgtgg tccacctgac 780cctccgccag
gccggcgacg acttctcccg ccgctaccgc cgcgacttcg ccgagatgtc
840cagccagctg cacctgacgc ccttcaccgc gcggggacgc tttgccacgg
tggtggagga 900gctcttcagg gacggggtga actgggggag gattgtggcc
ttctttgagt tcggtggggt 960catgtgtgtg gagagcgtca accgggagat
gtcgcccctg gtggacaaca tcgccctgtg 1020gatgactgag tacctgaacc
ggcacctgca cacctggatc caggataacg gaggctgggt 1080aggtgcactt
ggtgatgtga gtctgggctg aggccacagg tccgagatgc gggggttgga
1140gtgcgggtgg gctcctgggg caatgggagg ctgtggagcc ggcgaaataa
aatcagagtt 1200gttgcta 12078205PRTHomo sapiens 8Met Ala His Ala Gly
Arg Thr Gly Tyr Asp Asn Arg Glu Ile Val Met 1 5 10 15 Lys Tyr Ile
His Tyr Lys Leu Ser Gln Arg Gly Tyr Glu Trp Asp Ala 20 25 30 Gly
Asp Val Gly Ala Ala Pro Pro Gly Ala Ala Pro Ala Pro Gly Ile 35 40
45 Phe Ser Ser Gln Pro Gly His Thr Pro His Pro Ala Ala Ser Arg Asp
50 55 60 Pro Val Ala Arg Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro
Gly Ala 65 70 75 80 Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val
Val His Leu Thr 85 90 95 Leu Arg Gln Ala Gly Asp Asp Phe Ser Arg
Arg Tyr Arg Arg Asp Phe 100 105 110 Ala Glu Met Ser Ser Gln Leu His
Leu Thr Pro Phe Thr Ala Arg Gly 115 120 125 Arg Phe Ala Thr Val Val
Glu Glu Leu Phe Arg Asp Gly Val Asn Trp 130 135 140 Gly Arg Ile Val
Ala Phe Phe Glu Phe Gly Gly Val Met Cys Val Glu 145 150 155 160 Ser
Val Asn Arg Glu Met Ser Pro Leu Val Asp Asn Ile Ala Leu Trp 165 170
175 Met Thr Glu Tyr Leu Asn Arg His Leu His Thr Trp Ile Gln Asp Asn
180 185 190 Gly Gly Trp Val Gly Ala Leu Gly Asp Val Ser Leu Gly 195
200 205 9
2594DNAHuman immunodeficiency virus 9atggagccag tagatcctag
actagagccc tggaagcatc caggaagtca gcctaaaact 60gcttgtacca attgctattg
taaaaagtgt tgctttcatt gccaagtttg tttcataaca 120aaagccttag
gcatctccta tggcaggaag aagcggagac agcgacgaag agctcatcag
180aacagtcaga ctcatcaagc ttctctatca aagcagtaag tagtacatgt
aatgcaacct 240ataccaatag tagcaatagt agcattagta gtagcaataa
taatagcaat agttgtgtgg 300tccatagtaa tcatagaata taggaaaata
ttaagacaaa gaaaaataga caggttaatt 360gatagactaa tagaaagagc
agaagacagt ggcaatgaga gtgaaggaga aatatcagca 420cttgtggaga
tgggggtgga gatggggcac catgctcctt gggatgttga tgatctgtag
480tgctacagaa aaattgtggg tcacagtcta ttatggggta cctgtgtgga
aggaagcaac 540caccactcta ttttgtgcat cagatgctaa agcatatgat
acagaggtac ataatgtttg 600ggccacacat gcctgtgtac ccacagaccc
caacccacaa gaagtagtat tggtaaatgt 660gacagaaaat tttaacatgt
ggaaaaatga catggtagaa cagatgcatg aggatataat 720cagtttatgg
gatcaaagcc taaagccatg tgtaaaatta accccactct gtgttagttt
780aaagtgcact gatttgaaga atgatactaa taccaatagt agtagcggga
gaatgataat 840ggagaaagga gagataaaaa actgctcttt caatatcagc
acaagcataa gaggtaaggt 900gcagaaagaa tatgcatttt tttataaact
tgatataata ccaatagata atgatactac 960cagctataag ttgacaagtt
gtaacacctc agtcattaca caggcctgtc caaaggtatc 1020ctttgagcca
attcccatac attattgtgc cccggctggt tttgcgattc taaaatgtaa
1080taataagacg ttcaatggaa caggaccatg tacaaatgtc agcacagtac
aatgtacaca 1140tggaattagg ccagtagtat caactcaact gctgttaaat
ggcagtctag cagaagaaga 1200ggtagtaatt agatctgtca atttcacgga
caatgctaaa accataatag tacagctgaa 1260cacatctgta gaaattaatt
gtacaagacc caacaacaat acaagaaaaa gaatccgtat 1320ccagagagga
ccagggagag catttgttac aataggaaaa ataggaaata tgagacaagc
1380acattgtaac attagtagag caaaatggaa taacacttta aaacagatag
ctagcaaatt 1440aagagaacaa tttggaaata ataaaacaat aatctttaag
caatcctcag gaggggaccc 1500agaaattgta acgcacagtt ttaattgtgg
aggggaattt ttctactgta attcaacaca 1560actgtttaat agtacttggt
ttaatagtac ttggagtact gaagggtcaa ataacactga 1620aggaagtgac
acaatcaccc tcccatgcag aataaaacaa attataaaca tgtggcagaa
1680agtaggaaaa gcaatgtatg cccctcccat cagtggacaa attagatgtt
catcaaatat 1740tacagggctg ctattaacaa gagatggtgg taatagcaac
aatgagtccg agatcttcag 1800acctggagga ggagatatga gggacaattg
gagaagtgaa ttatataaat ataaagtagt 1860aaaaattgaa ccattaggag
tagcacccac caaggcaaag agaagagtgg tgcagagaga 1920aaaaagagca
gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
1980tatgggcgca gcctcaatga cgctgacggt acaggccaga caattattgt
ctggtatagt 2040gcagcagcag aacaatttgc tgagggctat tgaggcgcaa
cagcatctgt tgcaactcac 2100agtctggggc atcaagcagc tccaggcaag
aatcctggct gtggaaagat acctaaagga 2160tcaacagctc ctggggattt
ggggttgctc tggaaaactc atttgcacca ctgctgtgcc 2220ttggaatgct
agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
2280ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
ttgaagaatc 2340gcaaaaccag caagaaaaga atgaacaaga attattggaa
ttagataaat gggcaagttt 2400gtggaattgg tttaacataa caaattggct
gtggtatata aaattattca taatgatagt 2460aggaggcttg gtaggtttaa
gaatagtttt tgctgtactt tctatagtga atagagttag 2520gcagggatat
tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag
2580gcccgaagga atag 25941086PRTHuman immunodeficiency virus 10Met
Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser 1 5 10
15 Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe
20 25 30 His Cys Gln Val Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser
Tyr Gly 35 40 45 Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala His Gln
Asn Ser Gln Thr 50 55 60 His Gln Ala Ser Leu Ser Lys Gln Pro Thr
Ser Gln Pro Arg Gly Asp 65 70 75 80 Pro Thr Gly Pro Lys Glu 85
112397DNAHomo sapiens 11ctgtcccgca agcgccggcg gcagcatggc cagctctggt
tccctgaggg cttcaaagtg 60tctgaggcca gcaagaagaa gcggcgggag cccctcggcg
aggactccgt gggcctcaag 120cccctgaaga acgcttcaga cggtgccctc
atggacgaca accagaatga gtggggggac 180gaggacctgg agaccaagaa
gttccggttc gaggagcccg tggttctgcc tgacctggac 240gaccagacag
accaccggca gtggactcag cagcacctgg atgccgctga cctgcgcatg
300tctgccatgg cccccacacc gccccagggt gaggttgacg ccgactgcat
ggacgtcaat 360gtccgcgggc ctgatggctt caccccgctc atgatcgcct
cctgcagcgg gggcggcctg 420gagacgggca acagcgagga agaggaggac
gcgccggccg tcatctccga cttcatctac 480cagggcgcca gcctgcacaa
ccagacagac cgcacgggcg agaccgcctt gcacctggcc 540gcccgctact
cacgctctga tgccgccaag cgcctgctgg aggccagcgc agatgccaac
600atccaggaca acatgggccg caccccgctg catgcggctg tgtctgccga
cgcacaaggt 660gtcttccaga tcctgatccg gaaccgagcc acagacctgg
atgcccgcat gcatgatggc 720acgacgccac tgatcctggc tgcccgcctg
gccgtggagg gcatgctgga ggacctcatc 780aactcacacg ccgacgtcaa
cgccgtagat gacctgggca agtccgccct gcactgggcc 840gccgccgtga
acaatgtgga tgccgcagtt gtgctcctga agaacggggc taacaaagat
900atgcagaaca acagggagga gacacccctg tttctggccg cccgggaggg
cagctacgag 960accgccaagg tgctgctgga ccactttgcc aaccgggaca
tcacggatca tatggaccgc 1020ctgccgcgcg acatcgcaca ggagcgcatg
catcacgaca tcgtgaggct gctggacgag 1080tacaacctgg tgcgcagccc
gcagctgcac ggagccccgc tggggggcac gcccaccctg 1140tcgcccccgc
tctgctcgcc caacggctac ctgggcagcc tcaagcccgg cgtgcagggc
1200aagaaggtcc gcaagcccag cagcaaaggc ctggcctgtg gaagcaagga
ggccaaggac 1260ctcaaggcac ggaggaagaa gtcccaggac ggcaagggct
gcctgctgga cagctccggc 1320atgctctcgc ccgtggactc cctggagtca
ccccatggct acctgtcaga cgtggcctcg 1380ccgccactgc tgccctcccc
gttccagcag tctccgtccg tgcccctcaa ccacctgcct 1440gggatgcccg
acacccacct gggcatcggg cacctgaacg tggcggccaa gcccgagatg
1500gcggcgctgg gtgggggcgg ccggctggcc tttgagactg gcccacctcg
tctctcccac 1560ctgcctgtgg cctctggcac cagcaccgtc ctgggctcca
gcagcggagg ggccctgaat 1620ttcactgtgg gcgggtccac cagtttgaat
ggtcaatgcg agtggctgtc ccggctgcag 1680agcggcatgg tgccgaacca
atacaaccct ctgcggggga gtgtggcacc aggccccctg 1740agcacacagg
ccccctccct gcagcatggc atggtaggcc cgctgcacag tagccttgct
1800gccagcgccc tgtcccagat gatgagctac cagggcctgc ccagcacccg
gctggccacc 1860cagcctcacc tggtgcagac ccagcaggtg cagccacaaa
acttacagat gcagcagcag 1920aacctgcagc cagcaaacat ccagcagcag
caaagcctgc agccgccacc accaccacca 1980cagccgcacc ttggcgtgag
ctcagcagcc agcggccacc tgggccggag cttcctgagt 2040ggagagccga
gccaggcaga cgtgcagcca ctgggcccca gcagcctggc ggtgcacact
2100attctgcccc aggagagccc cgccctgccc acgtcgctgc catcctcgct
ggtcccaccc 2160gtgaccgcag cccagttcct gacgcccccc tcgcagcaca
gctactcctc gcctgtggac 2220aacaccccca gccaccagct acaggtgcct
gagcacccct tcctcacccc gtcccctgag 2280tcccctgacc agtggtccag
ctcgtccccg cattccaacg tctccgactg gtccgagggc 2340gtctccagcc
ctcccaccag catgcagtcc cagatcgccc gcattccgga ggccttc
239712799PRTHomo sapiens 12Leu Ser Arg Lys Arg Arg Arg Gln His Gly
Gln Leu Trp Phe Pro Glu 1 5 10 15 Gly Phe Lys Val Ser Glu Ala Ser
Lys Lys Lys Arg Arg Glu Pro Leu 20 25 30 Gly Glu Asp Ser Val Gly
Leu Lys Pro Leu Lys Asn Ala Ser Asp Gly 35 40 45 Ala Leu Met Asp
Asp Asn Gln Asn Glu Trp Gly Asp Glu Asp Leu Glu 50 55 60 Thr Lys
Lys Phe Arg Phe Glu Glu Pro Val Val Leu Pro Asp Leu Asp 65 70 75 80
Asp Gln Thr Asp His Arg Gln Trp Thr Gln Gln His Leu Asp Ala Ala 85
90 95 Asp Leu Arg Met Ser Ala Met Ala Pro Thr Pro Pro Gln Gly Glu
Val 100 105 110 Asp Ala Asp Cys Met Asp Val Asn Val Arg Gly Pro Asp
Gly Phe Thr 115 120 125 Pro Leu Met Ile Ala Ser Cys Ser Gly Gly Gly
Leu Glu Thr Gly Asn 130 135 140 Ser Glu Glu Glu Glu Asp Ala Pro Ala
Val Ile Ser Asp Phe Ile Tyr 145 150 155 160 Gln Gly Ala Ser Leu His
Asn Gln Thr Asp Arg Thr Gly Glu Thr Ala 165 170 175 Leu His Leu Ala
Ala Arg Tyr Ser Arg Ser Asp Ala Ala Lys Arg Leu 180 185 190 Leu Glu
Ala Ser Ala Asp Ala Asn Ile Gln Asp Asn Met Gly Arg Thr 195 200 205
Pro Leu His Ala Ala Val Ser Ala Asp Ala Gln Gly Val Phe Gln Ile 210
215 220 Leu Ile Arg Asn Arg Ala Thr Asp Leu Asp Ala Arg Met His Asp
Gly 225 230 235 240 Thr Thr Pro Leu Ile Leu Ala Ala Arg Leu Ala Val
Glu Gly Met Leu 245 250 255 Glu Asp Leu Ile Asn Ser His Ala Asp Val
Asn Ala Val Asp Asp Leu 260 265 270 Gly Lys Ser Ala Leu His Trp Ala
Ala Ala Val Asn Asn Val Asp Ala 275 280 285 Ala Val Val Leu Leu Lys
Asn Gly Ala Asn Lys Asp Met Gln Asn Asn 290 295 300 Arg Glu Glu Thr
Pro Leu Phe Leu Ala Ala Arg Glu Gly Ser Tyr Glu 305 310 315 320 Thr
Ala Lys Val Leu Leu Asp His Phe Ala Asn Arg Asp Ile Thr Asp 325 330
335 His Met Asp Arg Leu Pro Arg Asp Ile Ala Gln Glu Arg Met His His
340 345 350 Asp Ile Val Arg Leu Leu Asp Glu Tyr Asn Leu Val Arg Ser
Pro Gln 355 360 365 Leu His Gly Ala Pro Leu Gly Gly Thr Pro Thr Leu
Ser Pro Pro Leu 370 375 380 Cys Ser Pro Asn Gly Tyr Leu Gly Ser Leu
Lys Pro Gly Val Gln Gly 385 390 395 400 Lys Lys Val Arg Lys Pro Ser
Ser Lys Gly Leu Ala Cys Gly Ser Lys 405 410 415 Glu Ala Lys Asp Leu
Lys Ala Arg Arg Lys Lys Ser Gln Asp Gly Lys 420 425 430 Gly Cys Leu
Leu Asp Ser Ser Gly Met Leu Ser Pro Val Asp Ser Leu 435 440 445 Glu
Ser Pro His Gly Tyr Leu Ser Asp Val Ala Ser Pro Pro Leu Leu 450 455
460 Pro Ser Pro Phe Gln Gln Ser Pro Ser Val Pro Leu Asn His Leu Pro
465 470 475 480 Gly Met Pro Asp Thr His Leu Gly Ile Gly His Leu Asn
Val Ala Ala 485 490 495 Lys Pro Glu Met Ala Ala Leu Gly Gly Gly Gly
Arg Leu Ala Phe Glu 500 505 510 Thr Gly Pro Pro Arg Leu Ser His Leu
Pro Val Ala Ser Gly Thr Ser 515 520 525 Thr Val Leu Gly Ser Ser Ser
Gly Gly Ala Leu Asn Phe Thr Val Gly 530 535 540 Gly Ser Thr Ser Leu
Asn Gly Gln Cys Glu Trp Leu Ser Arg Leu Gln 545 550 555 560 Ser Gly
Met Val Pro Asn Gln Tyr Asn Pro Leu Arg Gly Ser Val Ala 565 570 575
Pro Gly Pro Leu Ser Thr Gln Ala Pro Ser Leu Gln His Gly Met Val 580
585 590 Gly Pro Leu His Ser Ser Leu Ala Ala Ser Ala Leu Ser Gln Met
Met 595 600 605 Ser Tyr Gln Gly Leu Pro Ser Thr Arg Leu Ala Thr Gln
Pro His Leu 610 615 620 Val Gln Thr Gln Gln Val Gln Pro Gln Asn Leu
Gln Met Gln Gln Gln 625 630 635 640 Asn Leu Gln Pro Ala Asn Ile Gln
Gln Gln Gln Ser Leu Gln Pro Pro 645 650 655 Pro Pro Pro Pro Gln Pro
His Leu Gly Val Ser Ser Ala Ala Ser Gly 660 665 670 His Leu Gly Arg
Ser Phe Leu Ser Gly Glu Pro Ser Gln Ala Asp Val 675 680 685 Gln Pro
Leu Gly Pro Ser Ser Leu Ala Val His Thr Ile Leu Pro Gln 690 695 700
Glu Ser Pro Ala Leu Pro Thr Ser Leu Pro Ser Ser Leu Val Pro Pro 705
710 715 720 Val Thr Ala Ala Gln Phe Leu Thr Pro Pro Ser Gln His Ser
Tyr Ser 725 730 735 Ser Pro Val Asp Asn Thr Pro Ser His Gln Leu Gln
Val Pro Glu His 740 745 750 Pro Phe Leu Thr Pro Ser Pro Glu Ser Pro
Asp Gln Trp Ser Ser Ser 755 760 765 Ser Pro His Ser Asn Val Ser Asp
Trp Ser Glu Gly Val Ser Ser Pro 770 775 780 Pro Thr Ser Met Gln Ser
Gln Ile Ala Arg Ile Pro Glu Ala Phe 785 790 795 13814DNAHomo
sapiens 13ccttcacctg cctctgccgc ccaggctaca cgggccacca ctgcgagacc
aacatcaacg 60agtgctccag ccagccctgc cgcctacggg gcacctgcca ggacccggac
aacgcctacc 120tctgcttctg cctgaagggg accacaggac ccaactgcga
gatcaacctg gatgactgtg 180ccagcagccc ctgcgactcg ggcacctgtc
tggacaagat cgatggctac gagtgtgcct 240gtgagccggg ctacacaggg
agcatgtgta acagcaacat cgatgagtgt gcgggcaacc 300cctgccacaa
cgggggcacc tgcgaggacg gcatcaatgg cttcacctgc cgctgccccg
360agggctacca cgaccccacc tgcctgtctg aggtcaatga gtgcaacagc
aacccctgcg 420tccacggggc ctgccgggac agcctcaacg ggtacaagtg
cgactgtgac cctgggtgga 480gtgggaccaa ctgtgacatc aacaacaacg
agtgtgaatc caacccttgt gtcaacggcg 540gcacctgcaa agacatgacc
agtggcatcg tgtgcacctg ccgggagggc ttcagcggtc 600ccaactgcca
gaccaacatc aacgagtgtg cgtccaaccc atgtctgaac aagggcacgt
660gtattgacga cgttgccggg tacaagtgca actgcctgct gccctacaca
ggtgccacgt 720gtgaggtggt gctggccccg tgtgccccca gcccctgcag
aaacggcggg gagtgcaggc 780aatccgagga ctatgagagt tgtcactatg tcct
81414799PRTHomo sapiens 14Leu Ser Arg Lys Arg Arg Arg Gln His Gly
Gln Leu Trp Phe Pro Glu 1 5 10 15 Gly Phe Lys Val Ser Glu Ala Ser
Lys Lys Lys Arg Arg Glu Pro Leu 20 25 30 Gly Glu Asp Ser Val Gly
Leu Lys Pro Leu Lys Asn Ala Ser Asp Gly 35 40 45 Ala Leu Met Asp
Asp Asn Gln Asn Glu Trp Gly Asp Glu Asp Leu Glu 50 55 60 Thr Lys
Lys Phe Arg Phe Glu Glu Pro Val Val Leu Pro Asp Leu Asp 65 70 75 80
Asp Gln Thr Asp His Arg Gln Trp Thr Gln Gln His Leu Asp Ala Ala 85
90 95 Asp Leu Arg Met Ser Ala Met Ala Pro Thr Pro Pro Gln Gly Glu
Val 100 105 110 Asp Ala Asp Cys Met Asp Val Asn Val Arg Gly Pro Asp
Gly Phe Thr 115 120 125 Pro Leu Met Ile Ala Ser Cys Ser Gly Gly Gly
Leu Glu Thr Gly Asn 130 135 140 Ser Glu Glu Glu Glu Asp Ala Pro Ala
Val Ile Ser Asp Phe Ile Tyr 145 150 155 160 Gln Gly Ala Ser Leu His
Asn Gln Thr Asp Arg Thr Gly Glu Thr Ala 165 170 175 Leu His Leu Ala
Ala Arg Tyr Ser Arg Ser Asp Ala Ala Lys Arg Leu 180 185 190 Leu Glu
Ala Ser Ala Asp Ala Asn Ile Gln Asp Asn Met Gly Arg Thr 195 200 205
Pro Leu His Ala Ala Val Ser Ala Asp Ala Gln Gly Val Phe Gln Ile 210
215 220 Leu Ile Arg Asn Arg Ala Thr Asp Leu Asp Ala Arg Met His Asp
Gly 225 230 235 240 Thr Thr Pro Leu Ile Leu Ala Ala Arg Leu Ala Val
Glu Gly Met Leu 245 250 255 Glu Asp Leu Ile Asn Ser His Ala Asp Val
Asn Ala Val Asp Asp Leu 260 265 270 Gly Lys Ser Ala Leu His Trp Ala
Ala Ala Val Asn Asn Val Asp Ala 275 280 285 Ala Val Val Leu Leu Lys
Asn Gly Ala Asn Lys Asp Met Gln Asn Asn 290 295 300 Arg Glu Glu Thr
Pro Leu Phe Leu Ala Ala Arg Glu Gly Ser Tyr Glu 305 310 315 320 Thr
Ala Lys Val Leu Leu Asp His Phe Ala Asn Arg Asp Ile Thr Asp 325 330
335 His Met Asp Arg Leu Pro Arg Asp Ile Ala Gln Glu Arg Met His His
340 345 350 Asp Ile Val Arg Leu Leu Asp Glu Tyr Asn Leu Val Arg Ser
Pro Gln 355 360 365 Leu His Gly Ala Pro Leu Gly Gly Thr Pro Thr Leu
Ser Pro Pro Leu 370 375 380 Cys Ser Pro Asn Gly Tyr Leu Gly Ser Leu
Lys Pro Gly Val Gln Gly 385 390 395 400 Lys Lys Val Arg Lys Pro Ser
Ser Lys Gly Leu Ala Cys Gly Ser Lys 405 410 415 Glu Ala Lys Asp Leu
Lys Ala Arg Arg Lys Lys Ser Gln Asp Gly Lys 420 425 430 Gly Cys Leu
Leu Asp Ser Ser Gly Met Leu Ser Pro Val Asp Ser Leu 435 440 445 Glu
Ser Pro His Gly Tyr Leu Ser Asp Val Ala Ser Pro Pro Leu Leu 450 455
460 Pro Ser Pro Phe Gln Gln Ser Pro Ser Val Pro Leu Asn His Leu Pro
465 470 475 480 Gly Met Pro Asp Thr His Leu Gly Ile Gly His Leu Asn
Val Ala Ala 485 490 495 Lys Pro Glu Met Ala Ala Leu Gly
Gly Gly Gly Arg Leu Ala Phe Glu 500 505 510 Thr Gly Pro Pro Arg Leu
Ser His Leu Pro Val Ala Ser Gly Thr Ser 515 520 525 Thr Val Leu Gly
Ser Ser Ser Gly Gly Ala Leu Asn Phe Thr Val Gly 530 535 540 Gly Ser
Thr Ser Leu Asn Gly Gln Cys Glu Trp Leu Ser Arg Leu Gln 545 550 555
560 Ser Gly Met Val Pro Asn Gln Tyr Asn Pro Leu Arg Gly Ser Val Ala
565 570 575 Pro Gly Pro Leu Ser Thr Gln Ala Pro Ser Leu Gln His Gly
Met Val 580 585 590 Gly Pro Leu His Ser Ser Leu Ala Ala Ser Ala Leu
Ser Gln Met Met 595 600 605 Ser Tyr Gln Gly Leu Pro Ser Thr Arg Leu
Ala Thr Gln Pro His Leu 610 615 620 Val Gln Thr Gln Gln Val Gln Pro
Gln Asn Leu Gln Met Gln Gln Gln 625 630 635 640 Asn Leu Gln Pro Ala
Asn Ile Gln Gln Gln Gln Ser Leu Gln Pro Pro 645 650 655 Pro Pro Pro
Pro Gln Pro His Leu Gly Val Ser Ser Ala Ala Ser Gly 660 665 670 His
Leu Gly Arg Ser Phe Leu Ser Gly Glu Pro Ser Gln Ala Asp Val 675 680
685 Gln Pro Leu Gly Pro Ser Ser Leu Ala Val His Thr Ile Leu Pro Gln
690 695 700 Glu Ser Pro Ala Leu Pro Thr Ser Leu Pro Ser Ser Leu Val
Pro Pro 705 710 715 720 Val Thr Ala Ala Gln Phe Leu Thr Pro Pro Ser
Gln His Ser Tyr Ser 725 730 735 Ser Pro Val Asp Asn Thr Pro Ser His
Gln Leu Gln Val Pro Glu His 740 745 750 Pro Phe Leu Thr Pro Ser Pro
Glu Ser Pro Asp Gln Trp Ser Ser Ser 755 760 765 Ser Pro His Ser Asn
Val Ser Asp Trp Ser Glu Gly Val Ser Ser Pro 770 775 780 Pro Thr Ser
Met Gln Ser Gln Ile Ala Arg Ile Pro Glu Ala Phe 785 790 795
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