U.S. patent application number 16/104047 was filed with the patent office on 2019-02-28 for compositions and method for manipulating pim-1 activity in circulatory system cells.
The applicant listed for this patent is SAN DIEGO STATE UNIVERSITY (SDSU) FOUNDATION. Invention is credited to John A. MURASKI, Mark A SUSSMAN.
Application Number | 20190060417 16/104047 |
Document ID | / |
Family ID | 40639186 |
Filed Date | 2019-02-28 |
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United States Patent
Application |
20190060417 |
Kind Code |
A1 |
SUSSMAN; Mark A ; et
al. |
February 28, 2019 |
COMPOSITIONS AND METHOD FOR MANIPULATING PIM-1 ACTIVITY IN
CIRCULATORY SYSTEM CELLS
Abstract
The invention provides compositions (e.g., pharmaceutical
compositions) comprising nucleic acids encoding the
serine/threonine kinase PIM-1, and methods for making and using
them; including methods for inducing cellular proliferation, and
protecting cardiac cells from hypoxia and cellular apoptosis. The
invention provides compositions (e.g., pharmaceutical compositions)
comprising nucleic acids encoding PIM-1, and methods for enhancing
the regenerative potential of stem cells in the heart.
Inventors: |
SUSSMAN; Mark A; (San Diego,
CA) ; MURASKI; John A.; (San Clemente, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SAN DIEGO STATE UNIVERSITY (SDSU) FOUNDATION |
SAN DIEGO |
CA |
US |
|
|
Family ID: |
40639186 |
Appl. No.: |
16/104047 |
Filed: |
August 16, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15078854 |
Mar 23, 2016 |
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16104047 |
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14082760 |
Nov 18, 2013 |
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15078854 |
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12742871 |
Jul 21, 2010 |
8617534 |
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PCT/US2008/083693 |
Nov 14, 2008 |
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14082760 |
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61091698 |
Aug 25, 2008 |
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60988753 |
Nov 16, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/45 20130101;
A61P 9/00 20180101; A61P 31/12 20180101; C12Y 207/12 20130101; A61K
2035/124 20130101; C12N 9/12 20130101; C12N 15/86 20130101; A61P
9/04 20180101; A61P 9/10 20180101; A61P 43/00 20180101; A61P 31/04
20180101; A61K 35/28 20130101; C12Y 207/11 20130101; A61K 35/28
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 38/45 20060101
A61K038/45; C12N 9/12 20060101 C12N009/12; C12N 15/86 20060101
C12N015/86; A61K 35/28 20060101 A61K035/28 |
Goverment Interests
FEDERAL FUNDING
[0002] This invention was produced in part using funds from the
Federal government under one or more of the following grants
5R01HL067245, 1R01HL091102, 1P01HL085577, and 1P01AG023071, all
from the National Institutes of Health. Accordingly, the Federal
government has certain rights in this invention.
Claims
1-20. (canceled)
21: A composition for enhancing the regenerative potential of
progenitor cardiac cells in a heart, the composition comprising: a
cell population comprising, or a plurality of, isolated human
cardiac progenitor cells comprising a recombinant expression vector
encoding Pim-1 to overexpress Pim-1.
22: The composition of claim 21, wherein the human cardiac
progenitor cells comprise a DNA encoding human Pim-1 under control
of a heterologous non-Pim-1 promoter.
23: The composition of claim 22, wherein the promoter is a
constitutive promoter.
24: The composition of claim 22, wherein the promoter is a
non-human promoter.
25: The composition of claim 21, wherein the cell population is
enriched in human cardiac progenitor cells.
26: The composition of claim 21, wherein the composition is in a
container in vitro.
27: The composition of claim 21, wherein the composition is in a
device suitable for injecting said composition into a cardiac
tissue.
28: The composition of claim 21, wherein the composition is
formulated for administration to heart muscle in vivo.
29. The composition of claim 22, wherein the DNA encoding human
Pim-1 under control of a heterologous non-Pim-1 promoter is
contained in a plasmid.
30: The composition of claim 22, wherein the DNA encoding human
Pim-1 under control of a heterologous non-Pim-1 promoter is
contained in a recombinant virus or an artificial chromosome.
31: The composition of claim 22, wherein the DNA encoding human
Pim-1 under control of a heterologous non-Pim-1 promoter is
contained in a recombinant adeno-associated viral vector; an
adenovirus vector, a retroviral vector; or a lentiviral vector.
32: A composition comprising: an isolated and genetically altered
or engineered progenitor cardiac cell that overexpresses Pim-1,
wherein the altered or engineered progenitor cardiac cell is
prepared by a process comprising: (a) obtaining a cardiac tissue
from a living mammalian subject; (b) isolating from the cardiac
tissue a cardiac progenitor cell capable of differentiating into a
cardiomyocyte; and (c) genetically altering or engineering an
isolated cardiac progenitor cell to contain a recombinant
expression vector encoding Pim-1 to overexpress Pim-1.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. ("USSN") 14/082,760, filed Nov. 18, 2013,
which is a continuation of U.S. patent application Ser. No.
("USSN") 12/742,871, filed Jul. 21, 2010, now U.S. Pat. No.
8,617,534, issued Dec. 31, 2014, which is a continuation of
International Patent Application Serial No. PCT/US2008/083693,
filed Nov. 14, 2008, which claims the benefit of priority under 35
10 U.S.C. .sctn. 119(e) of U.S. Provisional Patent Application Ser.
No. ("USSN") 61/091,698, filed Aug. 25, 2008; and U.S. Ser. No.
60/988,753, filed Nov. 16, 2007. Each of the aforementioned
applications are expressly incorporated herein by reference in
their entirety and for all purposes.
TECHNICAL FIELD
[0003] This invention generally relates to cell and molecular
biology, treatment or prevention of cardiac disease or injury, and
regenerative medicine. Disclosed are compositions (e.g.,
pharmaceutical compositions) comprising nucleic acids encoding the
serine/threonine kinase PIM-1 (and related PIM enzymes), and
medical uses and methods relating to alteration of PIM availability
or availability in cardiac or vascular system cells or tissues;
including inducing or enhancing differentiation, implantation,
survival, and function of stem cells, progenitor cells, or adult
cells in a cardiac or vascular tissue or environment. Also
disclosed are compositions comprising nucleic acids encoding PIM,
and methods for enhancing the regenerative potential of stem cells
and progenitor cells in a vascular or cardiac environment.
BACKGROUND
[0004] Intracellular molecular signaling networks communicate via
kinases that phosphorylate target substrates to regulate critical
aspects of growth and survival. PIM-1, a proto-oncogenic
serine/threonine kinase, was originally discovered as the proviral
integration site for Moloney murine Leukemia virus. PIM-1 is
up-regulated in prostate cancer. The gene is highly expressed in
the liver and spleen during fetal hematopoiesis and primarily in
B-lymphoid and myeloid cell lines.
[0005] PIM-1 exists in two isoforms with molecular weights of 34
and 44 kDa. The 34 kDa isoform is cytosolic and nuclear localized,
while the 44 kDa isoform was recently found to be membrane bound.
PIM-1 may be a relatively promiscuous kinase based upon minimal
target substrate recognition sequence requirements and capacity for
autophosphorylation. Two additional family members, PIM-2 and
PIM-3, may exhibit functional redundancy with PIM-1.
[0006] Induction of PIM-1 expression is mediated by cytokines and
growth factors including LIF, GM-CSF, EGF, and most interleukins,
consistent with a role for PIM-1 in proliferation and survival of
hematopoeitic cells. PIM-1 mediates proliferative actions through
phosphorylation of multiple target substrates, resulting in cell
cycle transition, as well as protective effects via phosphorylation
of multiple targets. Induction of PIM-1 expression has been linked
to AKT (a serine/threonine kinase) in hematopoeitic cells.
SUMMARY
[0007] The invention provides compositions, such as pharmaceutical
compositions, comprising nucleic acids encoding a serine/threonine
kinase PIM, and methods for making and using them; including
methods for inducing cardiac or vascular cellular proliferation,
and protecting cardiac or vascular cells from hypoxia and cellular
apoptosis. In one aspect, the compositions and methods of the
invention are used to express PIM-1 (e.g., by upregulating PIM
kinase expression and/or activity) to protect cardiomyocytes from
hypertrophy and inhibit myocardial apoptosis induced by infarction,
reducing infarct size. In another embodiment, the compositions and
methods of the invention are used to express PIM to induce cardiac
or vascular cellular dedifferentiation and re-expression of stem
cell markers; and in one aspect, to overexpress PIM to enhance the
regenerative potential of stem cells, including stem cell ability
to engraft in the heart after a myocardial infarction
(post-MI).
[0008] One aspect of the disclosure relates to a method, comprising
identifying a patient in need of enhanced PIM activity in a
vascular system tissue, and enhancing levels of PIM in vascular
system tissue of the patient to alter a functional characteristic
of cells in that tissue. In one embodiment, the patient has
experienced cardiac injury and the enhanced PIM levels facilitate
cardiac regeneration to repair that injury. The enhancing step may
advantageously comprise enhancing endogenous production of PIM in
the vascular system tissue. Alternatively, it may comprise
administering to the patient an exogenous PIM. The exogenous PIM
may comprise PIM-1, for example, or another material sharing that
same function, and may comprise a PIM enzyme in association with a
cellular delivery moiety, such as a translocation domain that is
attached to the PIM enzyme. In yet another embodiment, the
enhancing step comprises administering cells to the patient that
produce enhanced levels of PIM. As examples, the administered cells
may be stem cells or vascular system progenitor cells.
Advantageously, the administered cells comprise a PIM-encoding
polynucleotide operatively linked to a non-PIM promoter.
[0009] In one embodiment, the enhancing step comprises
administering cells to vascular tissue of the patient, and
expressing enhanced levels of PIM from the administered cells.
[0010] A different embodiment comprises PIM-delivering or enhancing
material for treatment of vascular system disease or injury. This
material can be, for example, a PIM enzyme linked to a cellular
delivery agent, or a cell for introduction into a human or animal,
wherein the cell has been altered to permit enhanced production of
PIM. In some cases, the cell is a progenitor cell or a stem cell,
and the alteration comprises a PIM-encoding polynucleotide under
control of a non-PIM promoter. Advantageously, the promoter may be
a cardiac-specific promoter, an inducible promoter, an endogenous
promoter, an exogenous promoter, or a constituitive promoter.
Alternatively, the PIM-enhancer may be an inducer of endogenous PIM
expression.
[0011] Yet another embodiment is use of a PIM-delivering or
enhancing material in the preparation of a medicament for treating
vascular system disease or injury.
[0012] Still another embodiment is a composition, comprising a
vascular system cell or a cell that is differentiatable into a
vascular system cell, where the cell comprises a PIM-encoding
polynucleotide sequence operably linked to a non-PIM promoter. The
cell may be, for example, a stem cell or cardiac progenitor cell.
Various types of stem cells that are contemplated include
mesenchymal stem cells, cardiac stem cells, adipose-derived stem
cells, embryonic stem cells, and hematopoietic stem cells.
Advantageously, the promoter is an inducible promoter or a
cardiac-specific promoter.
[0013] Yet another embodiment is a method for treating cardiac
disease or injury, comprising enhancing levels of PIM within
diseased or injured cardiac tissue. The cardiac disease or injury
may include ischemic injury, hypoxic injury, myocardial infarction,
traumatic cardiac injury, cardiac hypertrophy, overpressure injury,
congestive heart failure, apoptosis-inducing injury or disease,
bacterial infection, viral infection, and conditions that create an
enhanced risk of any of the foregoing.
[0014] Another embodiment provides pharmaceutical composition
formulated for administration to heart muscle comprising:
[0015] (i) (a) a PIM-1 encoding nucleic acid;
[0016] (b) a PIM-1 encoding nucleic acid inserted in an expression
construct or expression vehicle, or a naked PIM-1 encoding nucleic
acid operably linked to a promoter;
[0017] (c) the pharmaceutical composition of (b), wherein the
expression construct or expression vehicle comprises or consists of
a vector, a plasmid, a recombinant virus or an artificial
chromosome;
[0018] (d) the pharmaceutical composition of (c), wherein the
expression construct or expression vehicle comprises or consists of
a recombinant adeno-associated viral vector; an adenovirus vector,
a retroviral vector; or a lentiviral vector;
[0019] (e) the pharmaceutical composition of (d), wherein the
expression construct or expression vehicle comprises or consists of
an immunodeficiency virus derived vector;
[0020] (f) the pharmaceutical composition of (e), wherein the
immunodeficiency virus derived vector comprises or consists of a
human immunodeficiency virus (HIV) derived vector; or
[0021] (g) the pharmaceutical composition of (f), wherein the human
immunodeficiency virus (HIV) derived vector comprises or consists
of a human immunodeficiency virus-1 (HIV-1) derived vector;
[0022] (h) the pharmaceutical composition of any of (a) to (g),
wherein the PIM-1 encoding nucleic acid is operably linked to a
promoter;
[0023] (i) the pharmaceutical composition of (h), wherein the
promoter is a constitutive or an inducible promoter; or
[0024] (j) the pharmaceutical composition of (i), wherein the
promoter is constitutively or inducibly active in a heart cell (a
myocyte); and,
[0025] (ii) a pharmaceutically acceptable excipient.
[0026] wherein the pharmaceutical composition formulated for
administration to heart muscle.
[0027] Also contemplated are liposomes comprising a pharmaceutical
compound of the invention; and/or nanoparticles comprising a
pharmaceutical compound of the invention.
[0028] Still other embodiments include uses of a pharmaceutical
compound of the invention, a liposome of the invention, or a
nanoparticle of the invention, for the manufacture of a medicament
for:
[0029] (a) the amelioration, treatment or prevention of cellular
apoptosis and/or damage in a cardiac or vascular cell, tissue or
organ subsequent to cellular, tissue and/or organ hypoxia,
hypoxaemia or anoxia, or subsequent to pressure-overload induced
hypertrophy or heart failure, by increasing PIM-1 kinase activity
in the cardiac or vascular cell, tissue or organ;
[0030] (b) the use of (a), wherein the hypoxia, hypoxaemia or
anoxia is caused by an infarction, trauma, surgery, reimplantation,
transplantation or a toxin;
[0031] (c) inducing cellular dedifferentiation and/or re-expression
of a stem cell marker in a cardiac or vascular cell, tissue or
organ;
[0032] (d) enhancing the retention of engrafted or transplanted
cells, tissues or organs by overexpressing or expressing PIM-1 in
the cells, tissues or organs;
[0033] (e) increasing the expression of bcl-2, bcl-XL and/or
phosphorylation of Bad protein in a cardiac or vascular cell,
tissue or organ;
[0034] (f) the amelioration, treatment or prevention of ischemia
reperfusion injury in a cardiac or vascular cell, tissue or
organ;
[0035] (g) the use of any of (a) to (f), wherein the cardiac or
vascular cell, tissue or organ is or is contained in: a heart cell
(a myocyte), a heart tissue or a heart or other organ;
[0036] (h) overexpressing or expressing PIM-1 in a stem cell or a
pluripotent cell to enhance the regenerative potential and/or
induce proliferation of the stem cell or pluripotent cell;
[0037] (i) overexpressing or expressing PIM-1 in a heart cell (a
myocyte) or heart tissue to increase Bcl-XL expression in the heart
cell (myocyte) or heart tissue to induce cardioprotective
anti-apoptotic signaling and/or to increase myocardial survival
signaling;
[0038] (j) the use of any of (a) to (i), wherein the cell is a stem
cell, an adult stem cell, a hematopoietic stem cell, an
adipose-derived stem cell, a mesenchymal stem cell, a c-kit.sup.+
stem cell, a human stem cell, an autologous or allogeneic stem
cell, an embryonic cell, a tissue-specific resident stem cell, an
allogeneic or autologous cell, a progenitor cell, a placental
and/or cord blood cell, a Sca-1+ cell, or a CD34+ cell; or
[0039] (k) the use of any of (a) to (j), wherein the use is for the
amelioration, treatment or prevention of cellular apoptosis and/or
damage in a cardiac or vascular cell, tissue or organ subsequent to
cellular, tissue and/or organ hypoxia, hypoxaemia or anoxia, or
subsequent to pressure-overload induced hypertrophy or heart
failure; or because of a hypertrophic myocardium, an aged
myocardium, a failing myocardium, an ischemic myocardium, a
remodeled myocardium, a myocardium damaged by inflammation,
infection, chronic stress, disease, diabetes or alcoholism; and/or
oxidative damage.
[0040] Also included are methods for inducing, upregulating or
inserting a PIM-1 nucleic acid or a PIM-1 kinase activity in a
cardiac or vascular cell, tissue or organ, comprising:
[0041] (a) (i) providing a PIM-1 encoding nucleic acid; and
inserting the PIM-1 encoding nucleic acid into the cardiac or
vascular cell, tissue or organ; (ii) providing a cell expressing
and/or secreting a PIM-1 kinase; (iii) administering PIM-1 kinase
or a PIM-1 expressing nucleic acid to the cardiac or vascular cell,
tissue or organ; or, (iv) providing a compound that induces or
upregulates PIM-1 nucleic acid or a PIM-1 kinase activity in a
cardiac or vascular cell, tissue or organ;
[0042] (b) the method of (a), wherein the PIM-1 encoding nucleic
acid comprises or consists of a PIM-1 encoding message (a PIM-1
encoding mRNA), or a PIM-1 gene;
[0043] (c) the method of (a) or (b), wherein the PIM-1 encoding
nucleic acid comprises or consists of a human PIM-1 encoding
nucleic acid, or a human PIM-1 encoding message (mRNA), or a human
PIM-1 gene, or a human PIM-1 gene locus;
[0044] (d) the method of any of (a) to (c), wherein the cell is a
human cell, a stem cell, an adult stem cell, a hematopoietic stem
cell, an adipose-derived stem cell, a mesenchymal stem cell, a
c-kit.sup.+ stem cell, a human stem cell, an autologous or
allogeneic stem cell, an embryonic cell, a tissue-specific resident
stem cell, an allogeneic or autologous cell, a progenitor cell, a
placental and/or cord blood cell, a Sca-1+ cell, or a CD34+
cell;
[0045] (e) the method of any of (a) to (d), wherein the PIM-1
encoding nucleic acid is inserted into a cardiac or vascular cell,
tissue or organ ex vivo or in vivo;
[0046] (f) the method of any of (a) to (e), wherein a PIM-1
encoding nucleic acid is inserted in an expression construct or
expression vehicle;
[0047] (g) the method of any of (f), wherein the expression
construct or expression vehicle comprises or consists of a vector,
a plasmid, a recombinant virus or an artificial chromosome;
[0048] (h) the method of any of (g), wherein the expression
construct or expression vehicle comprises or consists of a
recombinant adeno-associated viral vector; an adenovirus vector, a
retroviral vector; or a lentiviral vector;
[0049] (i) the method of any of (h), wherein the expression
construct or expression vehicle comprises or consists of an
immunodeficiency virus derived vector;
[0050] (j) the method of any of (i), wherein the immunodeficiency
virus derived vector comprises or consists of a human
immunodeficiency virus (HIV) derived vector;
[0051] (k) the method of any of (j), wherein the human
immunodeficiency virus (HIV) derived vector comprises or consists
of a human immunodeficiency virus-1 (HIV-1) derived vector;
[0052] (l) the method of any of (a) to (k), wherein the PIM-1
encoding nucleic acid is inserted into a cell that does not express
wild type (normal) levels of PIM-1 protein;
[0053] (m) the method of (l), wherein the PIM-1 encoding nucleic
acid is inserted into a cell that does not express wild type
(normal) levels of PIM-1 protein-encoding message (mRNA);
[0054] (n) the method of (m), wherein the PIM-1 encoding nucleic
acid is inserted into a cell that does not comprise a wild type
(normal) PIM-1 gene or genomic PIM-1 encoding nucleic acid;
[0055] (o) the method of any of (a) to (n), wherein the PIM-1
encoding nucleic acid is inserted into a cardiac or vascular cell,
tissue or organ ex vivo and the cardiac or vascular cell, tissue or
organ is implanted into an individual in need thereof;
[0056] (p) the method of any of (a) to (o), wherein the PIM-1
encoding nucleic acid is inserted into a heart cell, cardiac or
vascular tissue or cardiac or vascular organ or a myocyte cell ex
vivo and the cell is implanted into a cardiac or vascular cell,
tissue or organ or a myocardium (a heart) in need thereof;
[0057] (q) the method of any of (a) to (n), wherein the PIM-1
encoding nucleic acid is in vivo inserted into a cardiac or
vascular cell, tissue or organ in an individual in need
thereof;
[0058] (r) the method of (q), wherein the PIM-1 encoding nucleic
acid is inserted into a cardiac or vascular cell, tissue or organ
or a heart cell or a myocyte cell or a heart in vivo;
[0059] (s) the method of (r), wherein the individual has congestive
heart failure, or has had a myocardial infarction, or heart muscle
damage;
[0060] (t) the method of any of (a) to (s), wherein the cardiac or
vascular cell, tissue or organ is or is contained in: a heart cell
(a myocyte), a heart tissue or a heart or other organ;
[0061] (u) the method of (a), wherein the compound that induces or
upregulates PIM-1 nucleic acid or a PIM-1 kinase activity in a
cardiac or vascular cell, tissue or organ comprises: an
interleukin, a cytokine and/or a paracrine factor involved in
survival and/or proliferative signaling; an up-regulator of AKT
serine/threonine kinase; insulin-like growth factor-1 (IGF-1);
insulin; leukemia inhibitory factor (LIF); granulocyte-macrophage
colony-stimulating factor (GM-CSF); or epidermal growth factor
(EGF);
[0062] (v) the method of any of (a) to (u), wherein the wherein
PIM-1 activity in the cardiac or vascular cell, tissue or organ is
increased by administering an exogenous PIM-1 kinase to the
population of cells;
[0063] (w) the method of (v), wherein PIM-1 activity is increased
by contacting a population of cells with a transfected cell that
expresses an exogenous PIM-1 gene;
[0064] (x) the method of (v), wherein the population of cells
comprises stem cells; or
[0065] (y) the method of any of (a) to (y), wherein the PIM-1
kinase activity is increased and/or upregulated in the cardiac or
vascular cell, tissue or organ by administering a pharmaceutical
compound of the invention, a liposome of the invention, or a
nanoparticle of the invention, or any combination thereof.
[0066] Still other aspects include methods for treating, preventing
or ameliorating a disease or condition comprising administering to
an individual in need thereof a pharmaceutical compound of the
invention, a liposome of the invention, or a nanoparticle of the
invention, or any combination thereof, wherein the treatment,
prevention and/or amelioration of the disease or condition
comprises:
[0067] (a) the amelioration, treatment or prevention of cellular
apoptosis and/or damage in a cardiac or vascular cell, tissue or
organ subsequent to cellular, tissue and/or organ hypoxia,
hypoxaemia or anoxia, or subsequent to pressure-overload induced
hypertrophy or heart failure; or because of a hypertrophic
myocardium, an aged myocardium, a failing myocardium, an ischemic
myocardium, a remodeled myocardium, a myocardium damaged by
inflammation, infection, chronic stress, disease, diabetes or
alcoholism; and/or oxidative damage, by increasing or upregulating
PIM-1 kinase activity in the cardiac or vascular cell, tissue or
organ;
[0068] (b) the method of (a), wherein the cellular apoptosis and/or
damage, or the hypoxia, hypoxaemia or anoxia, is caused by an
infarction, trauma, surgery, reimplantation, transplantation or a
toxin, or by inflammation, infection, chronic stress, diabetes or
alcoholism; and/or oxidative damage;
[0069] (c) inducing cellular dedifferentiation and/or re-expression
of a stem cell marker in a cardiac or vascular cell, tissue or
organ;
[0070] (d) enhancing the retention of engrafted or transplanted
cells, tissues or organs by overexpressing or expressing PIM-1 in
the cells, tissues or organs;
[0071] (e) increasing the expression of bcl-2, bcl-XL and/or
phosphorylation of Bad protein in a cardiac or vascular cell,
tissue or organ;
[0072] (f) the amelioration, treatment or prevention of ischemia
reperfusion injury in a cardiac or vascular cell, tissue or
organ;
[0073] (g) the method of any of (a) to (f), wherein the cardiac or
vascular cell, tissue or organ is or is contained in: a heart cell
(a myocyte), a heart tissue or a heart or other organ;
[0074] (h) overexpressing or expressing PIM-1 in a stem cell or a
pluripotent cell to enhance the regenerative potential and/or
induce proliferation of the stem cell or pluripotent cell; or
[0075] (i) overexpressing or expressing PIM-1 in a heart cell (a
myocyte) or heart tissue to increase Bcl-XL expression in the heart
cell (myocyte) or heart tissue to induce cardioprotective
anti-apoptotic signaling and/or to increase myocardial survival
signaling.
[0076] (j) the method of any of (a) to (i), wherein the cell is a
stem cell, an adult stem cell, a hematopoietic stem cell, an
adipose-derived stem cell, a mesenchymal stem cell, a c-kit.sup.+
stem cell, a human stem cell, an autologous or allogeneic stem
cell, an embryonic cell, a tissue-specific resident stem cell, an
allogeneic or autologous cell, a progenitor cell, a placental
and/or cord blood cell, a Sca-1+ cell, or a CD34+ cell.
[0077] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
[0078] All publications, patents, patent applications, GenBank
sequences and ATCC deposits, cited herein are hereby expressly
incorporated by reference for all purposes.
DESCRIPTION OF DRAWINGS
[0079] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0080] FIG. 1 illustrates immunoblots demonstrating that
cardioprotective stimuli induces Pim-1 expression, as described in
detail in Example 2, below.
[0081] FIG. 2 illustrates confocal micrographs showing that
cardiomyopathic stimuli induce Pim-1 expression in surviving
myocardium: a widefield view is shown in the micrographs of the
upper row, with selected regions is shown in higher magnification
to reveal cellular detail is shown in the micrographs of the lower
row, as described in detail in Example 2, below.
[0082] FIG. 3 graphically illustrates data showing that Pim-1
preserves hemodynamic function in ischemia-reperfusion injury, as
described in detail in Example 2, below.
[0083] FIG. 4 illustrates immunoblots demonstrating Pim-1
expression is highest in postnatal hearts and decreases with age,
as described in detail in Example 2, below.
[0084] FIG. 5 illustrates immunoblots demonstrating Pim-1
expression in cardiomyocytes from recombinant adenoviral vectors,
as described in detail in Example 2, below.
[0085] FIGS. 6 and 7 show how Pim-1 inhibits apoptosis in
cardiomyocytes: FIG. 6 graphically summarizes that non-infected
cells (NI) or GFP-expressing cells show comparable TUNEL labeling
following doxorubicin treatment, whereas Pimwt expressing cells
show significant reductions of TUNEL positive nuclei (p<0.05);
and FIG. 7 illustrates a micrograph demonstrating that cells
expressing the DN construct show enhanced TUNEL labeling; while
FIG. 6 shows quantitative results, the FIG. 7 panels illustrate
representative fields of infected cardiomyocytes showing GFP
fluorescence (green) overlay with actin filaments revealed by
phalloidin (red) in GFP only, GFP-Pim-wt and GFP-Pim-DN samples, as
described in detail in Example 2, below.
[0086] FIGS. 8A and 8B illustrate that nuclear accumulation of Akt
induces expression of Pim-1 kinase in the myocardium: Immunoblot
(FIG. 8A) and confocal microscopy (FIG. 8B) of sections from 6
month old normal (NTG) and transgenic mice expressing
cardiac-specific nuclear-targeted Akt; a separated grayscale images
in scans correspond to pim-1, actin, and nuclei that correspond to
the overlay colors of green, red, and blue respectively, as
described in detail in Example 2, below.
[0087] FIGS. 9A and 9B illustrate that nuclear accumulation of Akt
induces Pim-1 expression: FIG. 9A illustrates a confocal microscopy
of cultured cardiomyocytes infected with adenoviruses expressing
nuclear-targeted .beta.-galactosidase (B-gal), Akt wild-type (Akt
wt), or nuclear targeted Akt (Akt-nuc) detected with myc-tag
antibody (Tag); FIG. 9B illustrates an immunoblot blot showing
increased Pim-1 expression in cardiomyocyte cells infected with
adenovirus encoding nuclear-targeted Akt (Akt-nuc), as described in
detail in Example 2, below.
[0088] FIG. 10 illustrates an immunoblot blot showing expression of
dominant negative Pim-1 prompts Akt accumulation in cardiomyocytes;
immunoblot shows infection of neonatal rat cardiomyocytes with
adenoviruses expressing Pim-1 in either wild type (wt) or
dominant-negative (DN) forms, as described in detail in Example 2,
below.
[0089] FIG. 11 illustrates data characterizing founder lines and
protein expression in Pim-1 transgenic mice: FIG. 11 left panel
illustrates a PCR of genomic DNA samples from Pim-1 transgenic
mice, and FIG. 11 right panel illustrates an immunoblot of cardiac
lysates, as described in detail in Example 2, below.
[0090] FIGS. 12A-D graphically illustrate data showing that
inactivation of Pim-1 in the myocardium increases apoptosis and
fibrosis: FIG. 12A and FIG. 12B graphically illustrate
echocardiographic measurement of posterior (FIG. 12A) and anterior
(FIG. 12B) wall dimension (PWD and AWD respectively) in NTG and
Pim-DN animals at two week intervals; FIG. 12C graphically
illustrates heart weight to body weight ratios in NTG and Pim-DN
animals at 10 and 22 weeks of age; FIG. 12D graphically illustrates
histogram data representing counts of TUNEL positive myocytes per
mm.sup.2 in 17-22 week old NTG and Pim-DN transgenics, as described
in detail in Example 3, below.
[0091] FIG. 13 shows individual cell surface area measurements from
uninfected control, EGFP, and Pim-wt infected neonatal rat
cardiomyocyte cultures treated and untreated with endothelin-1, as
described in detail in Example 3, below.
[0092] FIGS. 14A-F graphically illustrate data showing Pim-wt
transgenic animals are resistant to pressure overload induced
hypertrophy: FIGS. 14A-F illustrate line graphs representing weekly
echo-cardiographic assessment of NTG and Pim-wt sham and TAC banded
hearts for anterior wall dimension (AWD FIG. 14D, FIG. 14a),
posterior wall dimension (PWD FIG. 14D, FIG. 14B), end diastolic
dimension (EDD, FIG. 14C), end-systolic dimension (ESD, FIG. 14D),
percent fractional shortening (FS, FIG. 14E), and ejection fraction
(EF, FIG. 14F), as described in detail in Example 3, below.
[0093] FIGS. 15A-C graphically illustrate data showing that Pim-1
enhances cardiac function: FIG. 15A, FIG. 15B and FIG. 15C show in
vivo hemodynamic assessment of NTG and Pim-wt hearts 4 and 10 weeks
after sham or TAC operation, as described in detail in Example 3,
below.
[0094] FIGS. 16A-G graphically illustrate data demonstrating that
Pim-1 protects against infarction injury: FIG. 16A graphically
illustrates a histogram representing infarct size 7 days post-MI as
a percent of left-ventricular free wall in Pim-KO hearts; FIG. 16B
graphically illustrates data showing the number of TUNEL positive
myocytes per mm.sup.2 7 days post-MI in Pim-KO hearts; FIG. 16D
graphically illustrates in vivo hemodynamic measurements of NTG and
Pim-KO mice 5 days following MI; FIG. 16E graphically illustrates
immunoblot quantitation of survival protein levels 7 days
post-infarction in Pim-KO and NTG control hearts; FIG. 16F
graphically illustrates infarct size measurements 10 days
post-infarction; FIG. 16G graphically illustrates the number of
TUNEL-labeled CM/m2 in LV 10 days after MI, as described in detail
in Example 3, below.
[0095] FIGS. 17A-C illustrate data showing increased proliferative
rate of Pim-1 engineered CSCs: FIG. 17A illustrates a cell growth
assessment using trypan blue assay of control, CGW, and CGW-Pim-wt
transduced CPCs; FIG. 17B illustrates an MTT assay on control, CGW,
CGW-Pim-wt transduced CPCs; FIG. 17C illustrates the proliferation
rate of Pim-1 expressing CPC's treated with or without 10 uM of
Quercetagentin, a specific Pim-1 activity inhibitor, as described
in detail in Example 4, below.
[0096] FIGS. 18A-F graphically illustrate data showing that
intra-myocardial injection of Pim-1 expressing CPCs improves
cardiac function: FIGS. 18A-C graphically illustrate
electrocardiographic assessment of AWD (FIG. 18A), EF (FIG. 18B),
and FS (FIG. 18C), in sham (.box-solid.), PBS injected
(.circle-solid.), CGW (.tangle-solidup.), and CGW-Pim-WT
(.diamond-solid.) cardiac progenitor cells; FIGS. 18D-F graphically
illustrate in vivo hemodynamic measurements of left ventricular
developed pressure (LVDP) (FIG. 18D), left ventricular end
diastolic pressure (LVEDP) (FIG. 18E), and dP/dT maximum and
minimum (FIG. 18F) were used to assess cardiac function 12 weeks
post-intramyocardial injection of PBS, eGFP, and Pim-1 expressing
CPCs, as described in detail in Example 4, below.
[0097] FIG. 19 graphically illustrates data showing that CGW-Pim-wt
CPCs form myocytes and vasculature in infarcted heart tissue
reducing infarction area; and shows a quantitation of infarction
area 12 weeks post CPC injection, as described in detail in Example
4, below.
[0098] FIGS. 20A and 20B graphically illustrate that long term
cardiac functional recovery is afforded by CGW-Pim-wt expressing
CPCs 32 weeks after intra-myocardial injection: FIGS. 20A and 20B
illustrate electrocardiographic assessment of FS (FIG. 20A), and EF
(FIG. 20B) in sham (.box-solid.), PBS injected (.circle-solid.),
CGW (.tangle-solidup.), and CGW-Pim-WT (.diamond-solid.) cardiac
progenitor cells 32 weeks post CPC transplantation, as described in
detail in Example 4, below.
[0099] FIG. 21 illustrates an exemplary lentiviral constructs of
the invention, as described in detail in Example 4, below.
[0100] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0101] The present disclosure includes the discovery of new roles
for PIM-1, its isoforms, and other PIM enzymes having equivalent or
overlapping targets and substrates. Specifically, these enzymes
have a role in cardiac and other circulatory system protection,
survival, repair, regeneration, and recovery, and in the
implantation, differentiation, function, and survival of stem
cells, progenitor cells, or differentiated cells introduced into
circulatory system tissues. These discoveries form the basis for
new cardiac therapies, including repair of damaged heart tissue and
implantation, expansion, and survival of implanted stem cells or
progenitor cells that differentiate into functional heart tissue.
Prior to this invention, enhancement of PIM activity was not known
to have any prophylactic or therapeutic utility in heart tissue,
heart cells, or in other circulatory system cells or tissues.
[0102] We show that circulatory system disease or injury can be
attenuated, halted, prevented, or reversed, and that damaged
circulatory system tissue can be replaced, repaired, or
regenerated, by enhancement of PIM activity in that tissue. Ways in
which PIM activity can be enhanced are described in more detail
below, but include upregulation of endogenous PIM production,
direct introduction of materials having PIM activity into tissues
or cells, introduction of polynucleotide encoding a PIM material
into existing cells of a human or animal, removing cells from a
subject and altering those cells to express enhanced levels of PIM,
then reintroducing the cells into the subject, introducing
exogenous cells into the subject that have been engineered to
produce enhanced levels of PIM, including stem cells or progenitor
cells that include a PIM-encoding polynucleotide under the control
of a non-PIM promoter, including an inducible promoter, a
constituitive promoter, or a cardiac-specific promoter.
[0103] The term "PIM" is used herein to refer to a serine or
threonine kinase, including the various PIM enzymes, e.g., PIM-1,
PIM-2, and PIM-3, further including any isoforms thereof. For
example, the serine/threonine kinase PIM-1 is known to exist in two
isoforms, and references to PIM and PIM-1 herein are intended to
encompass both isoforms, unless otherwise specified. In addition,
although certain cells, constructs, polynucleotides, techniques,
uses, and methods are described in connection with one particular
PIM, such as PIM-1, such descriptions are exemplary, and should be
taken as also including the other PIM enzymes having similar
activity.
[0104] The term "PIM activity" and "PIM kinase activity" refer to
the enzymatic or physiological activity of the PIM enzymes, e.g.,
the activity of a PIM-1, and encompasses use of other materials
having similar activity. The discoveries set forth herein relate to
altering characteristics of living cells by enhancing a particular
kinase activity in the cells. Of course, as is well known, enzyme
variants exist or can be readily constructed, having conservative
amino acid substitutions, cross-linking, cross-species domain
substitutions, truncations, and the like, while preserving a
physiologically-effective level of enzymatic activity (in this
case, kinase activity for the PIM-1 target). The present
discoveries are not focused only on a particular kinase, but
include the discovery of an entirely new role for PIM kinase
activity in vascular system cells and tissues. Thus, the results
discussed herein flow from alteration of PIM kinase activity,
regardless of the exact modality by which that is achieved.
[0105] The term "vascular system" is used herein to refer to the
blood vessels and the heart, and all the tissues and cells of which
they are comprised, including cardiac smooth muscle,
cardiomyocytes, cardiomyoblasts, vascular wall, endothelium,
vascular smooth muscle, vascular connective tissue, and other known
cells and tissues of the vascular system.
[0106] The term "stem cell" is used broadly to include totipotent,
pluripotent, and multipotent cells that can differentiate into
vascular system cells, including cardiac cells. "Progenitor cells"
overlaps somewhat with multipotent stem cells, and includes cells
that are at least partially differentiated but that are multipotent
or unipotent, in that they have the ability to differentiate into
at least one type of vascular system cells.
[0107] The terms "treat" and "treatment" are used broadly, to
include both prophylactic and therapeutic treatments. Similarly,
when referring to disease or injury of circulatory system tissues,
those terms are used broadly to include fully developed disease or
injury, as well as incipient or threatened disease or injury. Thus,
a patient at risk of or beginning to develop a particular
condition, is considered to have that condition "treated" when
methods as disclosed herein are used to reduce the risk of
development or progression of that condition, as well as when an
already-developed condition is reversed, inhibited, cured, or
ameliorated, and when the rate of development of a condition is
halted or slowed.
[0108] Those being treated are referred to variously as patients,
individuals, subjects, humans, or animals. Treatments identified as
useful for one category are also useful for other categories, and
selection of a particular term (other than "human") is not intended
to be limiting, but rather just a use of an alternative
expression.
[0109] The disclosure includes compositions, such as pharmaceutical
compositions, comprising nucleic acids encoding a PIM
serine/threonine kinase, such as PIM-1, and methods for making and
using them; including methods for inducing cardiac or vascular
cellular proliferation, and protecting cardiac or vascular cells
from hypoxia and cellular apoptosis. In one aspect, the
compositions and methods of the invention are used to express PIM-1
to protect cardiomyocytes from hypertrophy and inhibit myocardial
apoptosis induced by infarction, reducing infarct size. In another
embodiment, the compositions and methods of the invention are used
to express PIM-1 to induce cardiac or vascular cellular
dedifferentiation and re-expression of stem cell markers; and in
one aspect, to overexpress PIM-1 to enhance the regenerative
potential of stem cells, including stem cell ability to engraft in
the heart after a myocardial infarction (post-MI). In another
embodiment, the compositions and methods of the invention are used
to express PIM-1 to increase Bcl-XL expression to induce
cardioprotective anti-apoptotic signaling, thus increasing
myocardial survival signaling.
[0110] Also disclosed are compositions, such as pharmaceutical
compositions, comprising nucleic acids encoding the
serine/threonine kinase PIM-1 and methods for preventing or
inhibiting cell or tissue damage, e.g., cardiomyocyte cell death or
inhibiting an ischemic or reperfusion related injury; including
preventing or inhibiting the irreversible cellular and tissue
damage and cell death caused by ischemia, e.g., ischemia subsequent
to reperfusion (which can exacerbates ischemic damage by activating
inflammatory response and oxidative stress).
[0111] The disclosure further provides compositions, such as
pharmaceutical compositions, comprising nucleic acids encoding a
serine/threonine kinase PIM and methods for regulating cardiac or
vascular cellular proliferation and survival.
[0112] Using human and murine myocardial samples, we have
demonstrated that both human and murine myocardial cells show
elevated PIM-1 expression in failing hearts; where the elevated
PIM-1 has predominantly a nuclear localization. We have also shown
that acute cardiomyopathic challenge also induces PIM-1 expression
with nuclear and perinuclear distribution in mouse myocardium.
[0113] Expression of PIM-1 in postnatal mouse myocardium decreases
with aging, and cardioprotective stimuli associated with AKT
activation and nuclear-targeted AKT in particular increase PIM-1
expression. We disclose that cardiomyocyte apoptosis is inhibited
by PIM-1 via increased expression of bcl-2, bcl-XL, and
phosphorylation of Bad.sup.S112. Ischemia reperfusion injury is
enhanced in PIM-1 knockout mice. Since loss of PIM-1 expression or
activity leads to increased AKT expression without associated
cardioprotective effects, PIM-1 represents a critical and novel
facet of survival signaling downstream of AKT in the
myocardium.
Treatments and Medical Uses
[0114] Detailed strategies for enhancing PIM activity within
circulatory tissues are provided below. Regardless of the method by
which PIM activity is increased, we have discovered that
enhancement of PIM activity has multiple beneficial effects in
cardiac and other circulatory system tissues.
[0115] Initially, the care provider may wish to perform a patient
selection step. This may include, for example, assessing whether a
patient is in need of one or more of the various treatments, or
identifying a patient in need of such treatment. Two significant
categories of need warrant some discussion.
[0116] First, there are individuals with readily-diagnosable
existing conditions, including known disease or injury to cardiac
or other circulatory tissue that is treatable with the
compositions, methods, or techniques contemplated herein. In those
cases, diagnosis or identification of the disease or injury would
constitute diagnosis, selection, or identification of an individual
in need of the specified treatment.
[0117] Second, there are individuals in need of treatment that is
more prophylactic, for example, treatment that takes advantage of
the powerful cardioprotective properties exerted by PIM. In some
cases, identification can take place by recognizing an inchoate
disease or injury that would otherwise progress, for example,
injury or other factors that have or will initiate apoptosis, or
conditions or factors that enhance the risk of developing a
particular condition. Identification and treatment of those
individuals may be desirable to prevent development of a disease or
injury or to slow its development.
[0118] In between these two alternatives are individuals with
existing disease or injury, which disease or injury is likely to
progress. Identification and treatment of those individuals is also
contemplated.
[0119] One significant condition lending itself to treatment
through enhancement of PIM activity in cardiac tissue is myocardial
infarction or other ischemic injury. Prophylactic treatment is
desirable, when high risk of ischemic injury can be identified.
However, in many cases, the patient will be treated after the
injury has occurred. Treatment should be commenced as soon as is
practicable after the injury.
[0120] Similarly, PIM-activity enhancement can be used to treat a
number of other conditions and to create desired physiological
effects, by treating a subject to enhance PIM activity in vascular,
cardiac, or other circulatory system cells or tissues. These
include prevention, reduction, or reversal of cardiac hypertrophy,
including but not limited to maladaptive hypertrophic remodeling;
promoting cardiac cell survival and inhibiting apoptosis of those
cells; enhancing cardiac contractility; improving cardiac ejection
fraction; enhancing vascular growth and repair; and promoting
differentiation of stem cells and progenitor cells toward cardiac
or vascular tissue.
[0121] In another aspect, the methods contemplated herein include,
but are not limited to, inhibition of cardiac apoptosis; inhibition
of cardiac fibrosis; inhibition of cardiac remodeling; inhibition
of cardiac hypertrophy; preservation or reduced loss of ejection
fraction in damaged hearts; enhanced preservation of contractile
function; decrease in cardiac necrosis; reduction in lesion size
following ischemic injury; and increasing cardiac cellularity and
decreasing myocyte volume. All of these methods can be practiced
prophylactically (to prevent or reduce a particular condition that
would otherwise be likely to occur) and therapeutically (to treat a
condition that is already in existence, including treatment to slow
progression of a condition).
[0122] From another perspective, conditions that may lead to
treatment by enhancement of PIM activity include (but are not
limited to) congenital heart conditions; ischemic injury of any
kind to heart tissue; damage from infarction; cardiac reperfusion
injury; traumatic cardiac injury; congestive heart failure injury;
and injury relating to cardiac infection with a pathogen, including
viral, bacterial, and parasitic pathogens.
In addition to identifying an individual having a condition for
which PIM treatment is desirable, methods of treating the
individual can include a step of increasing the level of PIM
activity in a target tissue, such as vascular tissue or cardiac
tissue. This step can be practiced in the various ways disclosed
herein. By way of example, and not limitation, those include
administering factors or drugs to the patient, systemically or
locally, that upregulate endogenous PIM expression; administering
PIM protein, preferably in combination with a delivery modality,
such as a linked transduction domain, a liposome, an antibody, or
the like; administering PIM-encoding polynucleotide to the patient,
including naked DNA administration, administration of the
polynucleotide in a viral vector, liposome, or other delivery
modality; electroporation of cells of a subject to deliver DNA; and
administering an autologous cell to the subject (e.g., into the
heart) that has been altered to enhance PIM expression, including
cardiomyocytes, cardiac progenitor cells, cardiac stem cells,
mesenchymal stem cells, hematopoietic stem cells, adipose-derived
stem cells, and the like.
[0123] When administering cells to a human patient, for example, to
treat a cardiac condition, the number of cells can be any amount
effective to enhance cardiac function or structure or treat a
target condition. Exemplary, non-limiting amounts include 10.sup.5
to 10.sup.10 cells, more typically 10.sup.6 to 10.sup.9 cells.
[0124] Exemplary, non-limiting amounts of PIM protein administered
to an adult human heart can be, for example, from about 10.sup.-4 g
to about 10.sup.-10 g, calculated as the pure PIM protein.
Exemplary, non-limiting amounts of DNA include about 0.05 to 500
ug/kg, or 0.5 to 50 ug/kg body weight, and in the case of viral
particles, formulated at a titer of about at least 10.sup.10,
10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, 10.sup.15, 10.sup.16 ,
or 10.sup.17 physical particles per milliliter. In one aspect, the
PIM-1 encoding nucleic acid is administered in about 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 or more
microliter (.mu.l) injections. Doses and dosage regimens can be
determined by conventional range-finding techniques known to those
of ordinary skill in the art. For example, in alternative
embodiments, about 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9,
10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, 10.sup.15,
10.sup.16 or 10.sup.17 viral (e.g., lentiviral) particles are
delivered to the individual (e.g., a human patient) in one or
multiple doses.
[0125] In other embodiments, an intracardiac single administration
(e.g., a single dose) comprises from about 0.1 .mu.l to 1.0 .mu.l,
10 .mu.l or to about 100 .mu.l of a pharmaceutical composition of
the invention. Alternatively, dosage ranges from about 0.5 ng or
1.0 ng to about 10 .mu.g, 100 .mu.g to 1000 .mu.g of PIM-1
expressing nucleic acid is administered (either the amount in an
expression construct, or as in one embodiment, naked DNA is
injected).
PIM Sequences
[0126] Some embodiments include nucleic acid constructs comprising
a PIM-encoding sequence, e.g., a PIM-1 expressing message or a
PIM-1 gene. In one aspect, PIM-expressing nucleic acids used to
practice this invention include PIM-1 genomic sequences, or
fragments thereof, including coding or non-coding sequences, e.g.,
including introns, 5' or 3' non-coding sequences, and the like.
Also encompassed are PIM-encoding mRNA sequences.
[0127] In one aspect, the PIM-1 expressing, or PIM-1 inducing or
upregulating, composition is a nucleic acid, including a vector,
recombinant virus, and the like; and a recombinant PIM-1 is
expressed in a cell in vitro, ex vivo and/or in vivo.
[0128] In one aspect, a PIM-1 expressing nucleic acid encodes a
human PIM-1, such as Genbank accession no. AAA36447 (see also,
e.g., Domen (1987) Oncogene Res. 1 (1):103-112), SEQ ID NO:1.
[0129] In another aspect, a PIM-1 expressing nucleic acid encodes a
human PIM-1 kinase 44 kDa isoform, see e.g., Genbank accession no.
AAY87461 (see also, e.g., Xie (2006) Oncogene 25 (1), 70-78), SEQ
ID NO:2.
[0130] In a further aspect, a PIM-1 expressing nucleic acid
comprises a human PIM-1 kinase message (mRNA), see e.g., Genbank
accession no. NM_002648 (see also, e.g., Zhang (2007) Mol. Cancer
Res. 5 (9), 909-922), SEQ ID NO:3.
[0131] Also disclosed are human DNA sequences of PIM-2 (SEQ ID
NO:4) and PIM-3 (SEQ ID NO:5).
[0132] In alternative embodiments, nucleic acids of this invention
are operatively linked to a transcriptional regulatory sequence,
e.g., a promoter and/or an enhancer, e.g., cardiac-specific,
promoters to drive (e.g., regulate) expression of Pim-1. Promoters
and enhancers used to practice this invention can be of any type
and/or origin, an in one embodiment promoters specific to the
species receiving the Pim-1 construct are used; e.g., humans can
receive human promoters, mice receive murine promoters, etc. In
other embodiments, promoters from heterologous species can be used;
e.g., mammals or vertebrates receiving promoters that originate
from other mammals or vertebrates, or viral or synthetic promoters
active in the appropriate specie and/or cell type also can be used.
These promoters can comprise, for example, a .alpha.-myosin heavy
chain promoter; a cardiac troponin-T promoter; a MLC-2v promoter;
and any other promoter that drives expression in cardiac tissue but
does not drive significant expression in other tissues. In one
embodiment, promoters and enhancers active in primordial cells or
stem cells, e.g., myocardial stem cells, can be operatively linked
to drive expression of Pim-1.
Nucleic Acid Delivery--Gene Therapy Vehicles
[0133] In one aspect, this disclosure provides constructs or
expression vehicles, e.g., expression cassettes, vectors, viruses
(e.g., lentiviral expression vectors, e.g., see SEQ ID NO:4), and
the like, comprising a PIM- encoding sequence, e.g., a PIM-1
encoding message or a PIM-1.alpha. gene, for use as ex vivo or in
vitro gene therapy vehicles, or for expression of PIM-1 in heart
tissue, a cardiac or vascular cell, tissue or organ to practice the
methods of this invention, and for research, drug discovery or
transplantation.
[0134] In one aspect, an expression vehicle used to practice the
invention can comprise a promoter operably linked to a nucleic acid
encoding a PIM protein (or functional subsequence thereof). For
example, the invention provides expression cassettes comprising
nucleic acid encoding a PIM-1 protein operably linked to a
transcriptional regulatory element, e.g., a promoter.
[0135] In one aspect, an expression vehicle used to practice the
invention is designed to deliver a PIM-1 encoding sequence, e.g., a
PIM-1 gene or any functional portion thereof to a cardiac tissue or
cell of an individual. Expression vehicles, e.g., vectors, used to
practice the invention can be non-viral or viral vectors or
combinations thereof. The invention can use any viral vector or
viral delivery system known in the art, e.g., adenoviral vectors,
adeno-associated viral (AAV) vectors, herpes viral vectors (e.g.,
herpes simplex virus (HSV)-based vectors), retroviral vectors,
lentiviral vectors and baculoviral vectors.
[0136] In one aspect of the invention, an expression vehicle, e.g.,
a vector or a virus, is capable of accommodating a full-length
PIM-1 gene or a message, e.g., a cDNA. In one aspect, the invention
provides a retroviral, e.g., a lentiviral, vector capable of
delivering the nucleotide sequence encoding full-length human PIM-1
in vitro, ex vivo and/or in vivo. An exemplary lentiviral
expression vector backbone (no "payload" included, e.g., no PIM-1
sequence included) that can be used to practice this invention is
set forth in SEQ ID NO:4.
[0137] In one embodiment, a lentiviral vector used to practice this
invention is a "minimal" lentiviral production system lacking one
or more viral accessory (or auxiliary) gene. Exemplary lentiviral
vectors for use in the invention can have enhanced safety profiles
in that they are replication defective and self-inactivating (SIN)
lentiviral vectors. Lentiviral vectors and production systems that
can be used to practice this invention include e.g., those
described in U.S. Pat. Nos. (USPNs) 6,277,633; 6,312,682;
6,312,683; 6,521,457; 6,669,936; 6,924,123; 7,056,699; and
7,198,784; any combination of these are exemplary vectors that can
be employed in the practice of the invention. In an alternative
embodiment, non-integrating lentiviral vectors can be employed in
the practice of the invention. For example, non-integrating
lentiviral vectors and production systems that can be employed in
the practice of the invention include those described in U.S. Pat.
No. 6,808,923.
[0138] The expression vehicle can be designed from any vehicle
known in the art, e.g., a recombinant adeno-associated viral vector
as described, e.g., in U.S. Pat. App. Pub. No. 20020194630,
Manning, et al.; or a lentiviral gene therapy vector, e.g., as
described by e.g., Dull, et al. (1998) J. Virol. 72:8463-8471; or a
viral vector particle, e.g., a modified retrovirus having a
modified proviral RNA genome, as described, e.g., in U.S. Pat. App.
Pub. No. 20030003582; or an adeno-associated viral vector as
described e.g., in U.S. Pat. No. 6,943,153, describing recombinant
adeno-associated viral vectors for use in the eye; or a retroviral
or a lentiviral vector as described in U.S. Pat. Nos. 7,198,950;
7,160,727; 7,122,181 (describing using a retrovirus to inhibit
intraocular neovascularization in an individual having an
age-related macular degeneration); or 6,555,107.
[0139] Any viral vector can be used to practice this invention, and
the concept of using viral vectors for gene therapy is well known;
see e.g., Verma and Somia (1997) Nature 389:239-242; and Coffin et
al ("Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: J
M Coffin, S M Hughes, H E Varmus pp 758-763) having a detailed list
of retroviruses. Any lentiviruses belonging to the retrovirus
family can be used for infecting both dividing and non-dividing
cells with a PIM-1-encoding nucleic acid, see e.g., Lewis et al
(1992) EMBO J. 3053-3058.
[0140] Viruses from lentivirus groups from "primate" and/or
"non-primate" can be used; e.g., any primate lentivirus can be
used, including the human immunodeficiency virus (HIV), the
causative agent of human acquired immunodeficiency syndrome (AIDS),
and the simian immunodeficiency virus (SIV); or a non-primate
lentiviral group member, e.g., including "slow viruses" such as a
visna/maedi virus (VMV), as well as the related caprine
arthritis-encephalitis virus (CAEV), equine infectious anemia virus
(EIAV) and/or a feline immunodeficiency virus (FIV) or a bovine
immunodeficiency virus (BIV).
[0141] In alternative embodiments, lentiviral vectors used to
practice this invention are pseudotyped lentiviral vectors. In one
aspect, pseudotyping used to practice this invention incorporates
in at least a part of, or substituting a part of, or replacing all
of, an env gene of a viral genome with a heterologous env gene, for
example an env gene from another virus. In alternative embodiments,
the lentiviral vector of the invention is pseudotyped with VSV-G.
In an alternative embodiment, the lentiviral vector of the
invention is pseudotyped with Rabies-G.
[0142] Lentiviral vectors used to practice this invention may be
codon optimized for enhanced safety purposes. Different cells
differ in their usage of particular codons. This codon bias
corresponds to a bias in the relative abundance of particular tRNAs
in the cell type. By altering the codons in the sequence so that
they are tailored to match with the relative abundance of
corresponding tRNAs, it is possible to increase expression. By the
same token, it is possible to decrease expression by deliberately
choosing codons for which the corresponding tRNAs are known to be
rare in the particular cell type. Thus, an additional degree of
translational control is available. Many viruses, including HIV and
other lentiviruses, use a large number of rare codons and by
changing these to correspond to commonly used mammalian codons,
increased expression of the packaging components in mammalian
producer cells can be achieved. Codon usage tables are known in the
art for mammalian cells, as well as for a variety of other
organisms. Codon optimization has a number of other advantages. By
virtue of alterations in their sequences, the nucleotide sequences
encoding the packaging components of the viral particles required
for assembly of viral particles in the producer cells/packaging
cells have RNA instability sequences (INS) eliminated from them. At
the same time, the amino acid sequence coding sequence for the
packaging components is retained so that the viral components
encoded by the sequences remain the same, or at least sufficiently
similar that the function of the packaging components is not
compromised. Codon optimization also overcomes the Rev/RRE
requirement for export, rendering optimized sequences Rev
independent. Codon optimization also reduces homologous
recombination between different constructs within the vector system
(for example between the regions of overlap in the gag-pol and env
open reading frames). The overall effect of codon optimization is
therefore a notable increase in viral titer and improved safety.
The strategy for codon optimized gag-pol sequences can be used in
relation to any retrovirus.
[0143] Vectors, recombinant viruses, and other expression systems
used to practice this invention can comprise any nucleic acid which
can infect, transfect, transiently or permanently transduce a cell.
In one aspect, a vector used to practice this invention can be a
naked nucleic acid, or a nucleic acid complexed with protein or
lipid. In one aspect, a vector used to practice this invention
comprises viral or bacterial nucleic acids and/or proteins, and/or
membranes (e.g., a cell membrane, a viral lipid envelope, etc.). In
one aspect, expression systems used to practice this invention
comprise replicons (e.g., RNA replicons, bacteriophages) to which
fragments of DNA may be attached and become replicated. In one
aspect, expression systems used to practice this invention include,
but are not limited to RNA, autonomous self-replicating circular or
linear DNA or RNA (e.g., plasmids, viruses, and the like, see,
e.g., U.S. Pat. No. 5,217,879), and include both the expression and
non-expression plasmids.
[0144] In one aspect, a recombinant microorganism or cell culture
used to practice this invention can comprise "expression vector"
including both (or either) extra-chromosomal circular and/or linear
nucleic acid (DNA or RNA) that has been incorporated into the host
chromosome(s). In one aspect, where a vector is being maintained by
a host cell, the vector may either be stably replicated by the
cells during mitosis as an autonomous structure, or is incorporated
within the host's genome.
[0145] In one aspect, an expression system used to practice this
invention can comprise any plasmid, which are commercially
available, publicly available on an unrestricted basis, or can be
constructed from available plasmids in accord with published
procedures. Plasmids that can be used to practice this invention
are well known in the art.
[0146] In alternative aspects, a vector used to make or practice
the invention can be chosen from any number of suitable vectors
known to those skilled in the art, including cosmids, YACs (Yeast
Artificial Chromosomes), megaYACS, BACs (Bacterial Artificial
Chromosomes), PACs (P1 Artificial Chromosome), MACs (Mammalian
Artificial Chromosomes), a whole chromosome, or a small whole
genome. The vector also can be in the form of a plasmid, a viral
particle, or a phage. Other vectors include chromosomal,
non-chromosomal and synthetic DNA sequences, derivatives of SV40;
bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors
derived from combinations of plasmids and phage DNA, viral DNA such
as vaccinia, adenovirus, fowl pox virus, and pseudorabies. A
variety of cloning and expression vectors for use with prokaryotic
and eukaryotic hosts are described by, e.g., Sambrook. Bacterial
vectors which can be used include commercially available plasmids
comprising genetic elements of known cloning vectors.
Pharmaceutical Compositions
[0147] The invention provides compositions, including
pharmaceutical compositions, and methods for expressing PIM; e.g.,
for expressing PIM-1 or another functionally-equivalent kinase to
protect cardiomyocytes from hypertrophy and to inhibit myocardial
apoptosis induced by infarction, and to reduce infarct size.
(Functional equivalence is considered to exist based on ability to
act on the same substrate and produce the same product, and does
not require identical kinetics.) In another embodiment, the
pharmaceutical compositions of the invention are used to express
PIM-1 to induce cardiac or vascular cellular dedifferentiation and
re-expression of stem cell markers; and in one aspect, to
overexpress PIM-1 to enhance the regenerative potential of stem
cells, including stem cell ability to engraft in the heart after a
myocardial infarction (post-MI).
[0148] In one aspect, the PIM-1 expressing, or PIM-1 inducing or
upregulating, composition is a nucleic acid, including a vector,
recombinant virus, and the like; and a recombinant PIM-1 is
expressed in a cell in vitro, ex vivo and/or in vivo.
[0149] In alternative embodiments, in practicing use of the
pharmaceutical compositions and methods of this invention,
compounds that induce or upregulate PIM nucleic acid or a PIM
kinase activity in the heart or a cardiac or vascular cell, tissue
or organ are administered. For example, compounds that can be
administered in practicing use of the pharmaceutical compositions
and methods of this invention can comprise: an interleukin, a
cytokine and/or a paracrine factor involved in survival and/or
proliferative signaling; an up-regulator of AKT serine/threonine
kinase; insulin-like growth factor-1 (IGF-1); insulin; leukemia
inhibitory factor (LIF); granulocyte-macrophage colony-stimulating
factor (GM-CSF); or epidermal growth factor (EGF). Okadaic acid and
SV40 small T antigen inhibit or block negative regulation of PIM-1
by protein phosphatase 2A, and can thus be used to increase PIM-1
levels. See Maj, et al., Oncogene 26(35):5145-53 (2007).
[0150] In alternative embodiments, the PIM-expressing, or
PIM-inducing or upregulating, compositions of the invention are
formulated with a pharmaceutically acceptable carrier. In
alternative embodiments, the pharmaceutical compositions of the
invention can be administered parenterally, topically, orally or by
local administration, such as by aerosol or transdermally. The
pharmaceutical compositions can be formulated in any way and can be
administered in a variety of unit dosage forms depending upon the
condition or disease and the degree of illness, the general medical
condition of each patient, the resulting preferred method of
administration and the like. Details on techniques for formulation
and administration are well described in the scientific and patent
literature, see, e.g., the latest edition of Remington's
Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.
("Remington's").
[0151] Therapeutic agents of the invention can be administered
alone or as a component of a pharmaceutical formulation
(composition). The compounds may be formulated for administration
in any convenient way for use in human or veterinary medicine.
Wetting agents, emulsifiers and lubricants, such as sodium lauryl
sulfate and magnesium stearate, as well as coloring agents, release
agents, coating agents, sweetening, flavoring and perfuming agents,
preservatives and antioxidants can also be present in the
compositions.
[0152] Formulations of the PIM-expressing, or inducing or
upregulating, compositions of the invention include those suitable
for systemic administration, direct local vascular or cardiac
administration, or alternatively oral/nasal, topical, parenteral,
rectal, and/or intravaginal administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the host
being treated, the particular mode of administration. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will generally be that amount of the
compound which produces a therapeutic effect.
[0153] Pharmaceutical formulations of this invention may comprise
one or more diluents, emulsifiers, preservatives, buffers,
excipients, etc. and may be provided in such forms as liquids,
powders, emulsions, lyophilized powders, sprays, creams, lotions,
controlled release formulations, tablets, pills, gels, on patches,
in implants, etc.
[0154] Pharmaceutical formulations for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in appropriate and suitable dosages. Such carriers enable
the pharmaceuticals to be formulated in unit dosage forms as
tablets, pills, powder, dragees, capsules, liquids, lozenges, gels,
syrups, slurries, suspensions, etc., suitable for ingestion by the
patient. Pharmaceutical preparations for oral use can be formulated
as a solid excipient, optionally grinding a resulting mixture, and
processing the mixture of granules, after adding suitable
additional compounds, if desired, to obtain tablets or dragee
cores. Suitable solid excipients are carbohydrate or protein
fillers include, e.g., sugars, including lactose, sucrose,
mannitol, or sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose;
and gums including arabic and tragacanth; and proteins, e.g.,
gelatin and collagen. Disintegrating or solubilizing agents may be
added, such as the cross-linked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate.
[0155] Dragee cores are provided with suitable coatings such as
concentrated sugar solutions, which may also contain gum arabic,
talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or dragee coatings for product identification or to
characterize the quantity of active compound (i.e., dosage).
Pharmaceutical preparations of the invention can also be used
orally using, e.g., push-fit capsules made of gelatin, as well as
soft, sealed capsules made of gelatin and a coating such as
glycerol or sorbitol. Push-fit capsules can contain active agents
mixed with a filler or binders such as lactose or starches,
lubricants such as talc or magnesium stearate, and, optionally,
stabilizers. In soft capsules, the active agents can be dissolved
or suspended in suitable liquids, such as fatty oils, liquid
paraffin, or liquid polyethylene glycol with or without
stabilizers.
[0156] Aqueous suspensions can contain an active agent (e.g., a
chimeric polypeptide or peptidomimetic of the invention) in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients include a suspending agent, such as
sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing
or wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethylene oxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
(e.g., polyoxyethylene sorbitol mono-oleate), or a condensation
product of ethylene oxide with a partial ester derived from fatty
acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan
mono-oleate). The aqueous suspension can also contain one or more
preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or
more coloring agents, one or more flavoring agents and one or more
sweetening agents, such as sucrose, aspartame or saccharin.
Formulations can be adjusted for osmolarity.
[0157] Oil-based pharmaceuticals can be used to deliver PIM-1
expressing, or PIM-1 inducing or upregulating, compositions of the
invention. Oil-based suspensions can be formulated by suspending an
active agent in a vegetable oil, such as arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin; or a mixture of these. See e.g., U.S. Pat. No. 5,716,928
describing using essential oils or essential oil components for
increasing bioavailability and reducing inter- and intra-individual
variability of orally administered hydrophobic pharmaceutical
compounds (see also U.S. Pat. No. 5,858,401). The oil suspensions
can contain a thickening agent, such as beeswax, hard paraffin or
cetyl alcohol. Sweetening agents can be added to provide a
palatable oral preparation, such as glycerol, sorbitol or sucrose.
These formulations can be preserved by the addition of an
antioxidant such as ascorbic acid. As an example of an injectable
oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.
The pharmaceutical formulations of the invention can also be in the
form of oil-in-water emulsions. The oily phase can be a vegetable
oil or a mineral oil, described above, or a mixture of these.
Suitable emulsifying agents include naturally-occurring gums, such
as gum acacia and gum tragacanth, naturally occurring phosphatides,
such as soybean lecithin, esters or partial esters derived from
fatty acids and hexitol anhydrides, such as sorbitan mono-oleate,
and condensation products of these partial esters with ethylene
oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion
can also contain sweetening agents and flavoring agents, as in the
formulation of syrups and elixirs. Such formulations can also
contain a demulcent, a preservative, or a coloring agent.
[0158] In practicing this invention, the pharmaceutical compounds
can also be administered by in intranasal, intraocular and
intravaginal routes including suppositories, insufflation, powders
and aerosol formulations (for examples of steroid inhalants, see
Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa (1995) Ann.
Allergy Asthma Immunol. 75:107-111). Suppositories formulations can
be prepared by mixing the drug with a suitable non-irritating
excipient which is solid at ordinary temperatures but liquid at
body temperatures and will therefore melt in the body to release
the drug. Such materials are cocoa butter and polyethylene
glycols.
[0159] In practicing this invention, the pharmaceutical compounds
can be delivered by transdermally, by a topical route, formulated
as applicator sticks, solutions, suspensions, emulsions, gels,
creams, ointments, pastes, jellies, paints, powders, and
aerosols.
[0160] In practicing this invention, the pharmaceutical compounds
can also be delivered as microspheres for slow release in the body.
For example, microspheres can be administered via intradermal
injection of drug which slowly release subcutaneously; see Rao
(1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and
injectable gel formulations, see, e.g., Gao (1995) Pharm. Res.
12:857-863 (1995); or, as microspheres for oral administration,
see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.
[0161] In practicing this invention, the pharmaceutical compounds
can be parenterally administered, such as by intravenous (IV)
administration or administration into a body cavity or lumen of the
heart. Use of catheters that temporarily block flow of blood from
the heart while incubating the stem cells or a viral construct in
heart tissue can be used, as well as recirculation systems of
well-known type that isolate the circulation in all or a part of
the heart to increase the dwell time of an introduced agent (e.g.,
stem cell, construct, naked DNA, PIM protein, viral or other
vector) in the heart. These formulations can comprise a solution of
active agent dissolved in a pharmaceutically acceptable carrier.
Acceptable vehicles and solvents that can be employed are water and
Ringer's solution, an isotonic sodium chloride. In addition,
sterile fixed oils can be employed as a solvent or suspending
medium. For this purpose any bland fixed oil can be employed
including synthetic mono- or diglycerides. In addition, fatty acids
such as oleic acid can likewise be used in the preparation of
injectables. These solutions are sterile and generally free of
undesirable matter. These formulations may be sterilized by
conventional, well known sterilization techniques. The formulations
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents, e.g.,
sodium acetate, sodium chloride, potassium chloride, calcium
chloride, sodium lactate and the like. The concentration of active
agent in these formulations can vary widely, and will be selected
primarily based on fluid volumes, viscosities, body weight, and the
like, in accordance with the particular mode of administration
selected and the patient's needs. For IV administration, the
formulation can be a sterile injectable preparation, such as a
sterile injectable aqueous or oleaginous suspension. This
suspension can be formulated using those suitable dispersing or
wetting agents and suspending agents. The sterile injectable
preparation can also be a suspension in a nontoxic
parenterally-acceptable diluent or solvent, such as a solution of
1,3-butanediol. The administration can be by bolus or continuous
infusion (e.g., substantially uninterrupted introduction into a
blood vessel for a specified period of time).
[0162] The pharmaceutical compounds and formulations of the
invention can be lyophilized. The invention provides a stable
lyophilized formulation comprising a composition of the invention,
which can be made by lyophilizing a solution comprising a
pharmaceutical of the invention and a bulking agent, e.g.,
mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A
process for preparing a stable lyophilized formulation can include
lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL
sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a
pH greater than 5.5 but less than 6.5. See, e.g., U.S. patent app.
no. 20040028670.
[0163] The compositions and formulations of the invention can be
delivered by the use of liposomes (see also discussion, below). By
using liposomes, particularly where the liposome surface carries
ligands specific for target cells, or are otherwise preferentially
directed to a specific organ, one can focus the delivery of the
active agent into target cells of the heart or other part of the
circulatory system in vivo. See, e.g., U.S. Pat. Nos. 6,063,400;
6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn
(1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp.
Pharm. 46:1576-1587.
[0164] The formulations of the invention can be administered for
prophylactic and/or therapeutic treatments. In therapeutic
applications, compositions are administered to a subject already
suffering from a condition, infection or disease in an amount
sufficient to cure, alleviate or partially arrest the clinical
manifestations of the condition, infection or disease and its
complications (a "therapeutically effective amount"). For example,
in alternative embodiments, pharmaceutical compositions of the
invention are administered in an amount sufficient to treat,
prevent and/or ameliorate the deleterious effects on the heart of a
myocardial infarction (post-MI); to protect cardiomyocytes from
hypertrophy and to inhibit myocardial apoptosis induced by
infarction, and to reduce infarct size. In another embodiment, the
pharmaceutical compositions of the invention are used to express
PIM-1 to induce cellular dedifferentiation and re-expression of
stem cell markers; and in one aspect, to overexpress PIM-1 to
enhance the regenerative potential of stem cells, including stem
cell ability to engraft in the heart post-MI.
[0165] The amount of pharmaceutical composition adequate to
accomplish this is defined as a "therapeutically effective dose."
The dosage schedule and amounts effective for this use, i.e., the
"dosing regimen," will depend upon a variety of factors, including
the stage of the disease or condition, the severity of the disease
or condition, the general state of the patient's health, the
patient's physical status, age and the like. In calculating the
dosage regimen for a patient, the mode of administration also is
taken into consideration.
[0166] The dosage regimen also takes into consideration
pharmacokinetics parameters well known in the art, i.e., the active
agents' rate of absorption, bioavailability, metabolism, clearance,
and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid
Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie
51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995)
J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613;
Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; the latest
Remington's, supra). The state of the art allows the clinician to
determine the dosage regimen for each individual patient, active
agent and disease or condition treated. Guidelines provided for
similar compositions used as pharmaceuticals can be used as
guidance to determine the dosage regiment, i.e., dose schedule and
dosage levels, administered practicing the methods of the invention
are correct and appropriate.
[0167] Single or multiple administrations of formulations can be
given depending on the dosage and frequency as required and
tolerated by the patient. The formulations should provide a
sufficient quantity of active agent to effectively treat, prevent
or ameliorate a conditions, diseases or symptoms as described
herein. Methods for preparing parenterally or non-parenterally
administrable formulations are known or apparent to those skilled
in the art and are described in more detail in such publications as
Remington's.
[0168] The methods of the invention can further comprise
co-administration with other drugs or pharmaceuticals, e.g.,
compositions for treating heart attacks, congestive heart failure
and related symptoms or conditions. For example, the methods and/or
compositions and formulations of the invention can be co-formulated
with and/or co-administered with antibiotics (e.g., antibacterial
or bacteriostatic peptides or proteins), particularly those
effective against gram negative bacteria, fluids, cytokines,
immunoregulatory agents, anti-inflammatory agents, complement
activating agents, such as peptides or proteins comprising
collagen-like domains or fibrinogen-like domains (e.g., a ficolin),
carbohydrate-binding domains, and the like and combinations
thereof.
Nanoparticles and Liposomes
[0169] The invention also provides nanoparticles and liposomal
membranes comprising the PIM-1-expressing compounds of this
invention which target specific molecules, including biologic
molecules, such as polypeptide, including cardiac or vascular or
stem cell surface polypeptides, including heart cell (e.g.,
myocyte) cell surface polypeptides. In alternative embodiments, the
invention provides nanoparticles and liposomal membranes targeting
diseased and/or injured heart cells, or stem cells, such as any
pluripotent cell.
[0170] In alternative embodiments, the invention provides
nanoparticles and liposomal membranes comprising (in addition to
comprising compounds of this invention) molecules, e.g., peptides
or antibodies, that selectively target diseased and/or injured
heart cells, or stem cells. In alternative embodiments, the
invention provides nanoparticles and liposomal membranes using
interleukin receptors and/or other receptors to target receptors on
cells, e.g., diseased and/or injured heart cells, or stem cells.
See, e.g., U.S. patent application publication no. 20060239968.
[0171] Thus, in one aspect, the compositions of the invention are
specifically targeted to stem cells or heart cells, such as
myocytes.
[0172] The invention also provides nanocells to allow the
sequential delivery of two different therapeutic agents with
different modes of action or different pharmacokinetics, at least
one of which comprises a composition of this invention. A nanocell
is formed by encapsulating a nanocore with a first agent inside a
lipid vesicle containing a second agent; see, e.g., Sengupta, et
al., U.S. Pat. Pub. No. 20050266067. The agent in the outer lipid
compartment is released first and may exert its effect before the
agent in the nanocore is released. The nanocell delivery system may
be formulated in any pharmaceutical composition for delivery to
patients suffering from any disease or condition as described
herein, e.g., such as congestive heart failure or heart attack
(myocardial infarction). For example, in treating myocardial
infarction, an antibody and/or angiogenic agent can be contained in
the outer lipid vesicle of the nanocell, and a composition of this
invention is loaded into the nanocore. This arrangement allows the
antibody and/or angiogenic agent to be released first and delivered
to the injured tissue.
[0173] The invention also provides multilayered liposomes
comprising compounds of this invention, e.g., for transdermal
absorption, e.g., as described in Park, et al., U.S. Pat. Pub. No.
20070082042. The multilayered liposomes can be prepared using a
mixture of oil-phase components comprising squalane, sterols,
ceramides, neutral lipids or oils, fatty acids and lecithins, to
about 200 to 5000 nm in particle size, to entrap a composition of
this invention.
[0174] A multilayered liposome of the invention may further include
an antiseptic, an antioxidant, a stabilizer, a thickener, and the
like to improve stability. Synthetic and natural antiseptics can be
used, e.g., in an amount of 0.01% to 20%. Antioxidants can be used,
e.g., BHT, erysorbate, tocopherol, astaxanthin, vegetable
flavonoid, and derivatives thereof, or a plant-derived
antioxidizing substance. A stabilizer can be used to stabilize
liposome structure, e.g., polyols and sugars. Exemplary polyols
include butylene glycol, polyethylene glycol, propylene glycol,
dipropylene glycol and ethyl carbitol; examples of sugars are
trehalose, sucrose, mannitol, sorbitol and chitosan, or a
monosaccharide or an oligosaccharide, or a high molecular weight
starch. A thickener can be used for improving the dispersion
stability of constructed liposomes in water, e.g., a natural
thickener or an acrylamide, or a synthetic polymeric thickener.
Exemplary thickeners include natural polymers, such as acacia gum,
xanthan gum, gellan gum, locust bean gum and starch, cellulose
derivatives, such as hydroxy ethylcellulose, hydroxypropyl
cellulose and carboxymethyl cellulose, synthetic polymers, such as
polyacrylic acid, poly-acrylamide or polyvinylpyrollidone and
polyvinylalcohol, and copolymers thereof or cross-linked
materials.
[0175] Liposomes can be made using any method, e.g., as described
in Park, et al., U.S. Pat. Pub. No. 20070042031, including method
of producing a liposome by encapsulating a therapeutic product
comprising providing an aqueous solution in a first reservoir;
providing an organic lipid solution in a second reservoir, wherein
one of the aqueous solution and the organic lipid solution includes
a therapeutic product; mixing the aqueous solution with said
organic lipid solution in a first mixing region to produce a
liposome solution, wherein the organic lipid solution mixes with
said aqueous solution so as to substantially instantaneously
produce a liposome encapsulating the therapeutic product; and
immediately thereafter mixing the liposome solution with a buffer
solution to produce a diluted liposome solution.
[0176] The invention also provides nanoparticles comprising
compounds of this invention to deliver a composition of the
invention as a drug-containing nanoparticles (e.g., a secondary
nanoparticle), as described, e.g., in U.S. Pat. Pub. No.
20070077286. In one embodiment, the invention provides
nanoparticles comprising a fat-soluble drug of this invention or a
fat-solubilized water-soluble drug to act with a bivalent or
trivalent metal salt.
Gene Therapy Delivery Methods
[0177] The PIM-1 expressing nucleic acid compositions of the
invention can be delivered for ex vivo or in vivo gene therapy to
deliver a PIM-1 encoding nucleic acid. In one aspect, PIM-1
expressing nucleic acid compositions of the invention, including
non-reproducing viral constructs expressing high levels of the
human PIM-1 protein, are delivered ex vivo or for in vivo gene
therapy.
[0178] The PIM-1 expressing nucleic acid compositions of the
invention can be delivered to and expressed in a variety of cardiac
or vascular cells to induce cellular proliferation, and/or to
protect cardiac or vascular cells from hypoxia and cellular
apoptosis. PIM-1 so expressed (by practicing the composition and
methods of this invention) can protect cardiomyocytes from
hypertrophy and inhibit cell death induced by myocardial infarction
(e.g. heart attack), reducing the amount of muscle affected. In
addition, PIM-1 overexpression (by practicing the composition and
methods of this invention) in cardiac or vascular cells, e.g., in
heart cells, results in cellular reversion; the cardiac or vascular
cells become stem cell like; complete with re-expression of stem
cell markers (such as cardiac stem cell markers).
[0179] In one aspect, overexpression of PIM-1 (by practicing the
compositions and methods of this invention) enhances the
regenerative potential of stem cells and their ability to repair a
damaged or injured organ (e.g., an injured heart after a heart
attack). In one aspect, the invention provides compositions and
methods for overexpressing PIM-1 using a controlled system using
cultured stem cells prior to reintroduction in the adult human to
enhance their ability to repair the organ following injury.
[0180] The invention provides use of PIM-1 for a clinical therapy
for repair of a number of tissues damaged by low oxygen or other
means through use of a conditional control element that allows
control of PIM-1 expression. For example, PIM-1 expressing nucleic
acid delivery vehicles, e.g., expression constructs, such as
vectors or recombinant viruses, can be injected directly into the
organ (e.g., a heart) to protect it from immediate injury.
Expression of the protein can be then activated through an oral
prescription drug (formulations for which are discussed above).
[0181] In one embodiment vectors used to practice this invention,
e.g., to generate a PIM-expressing cell, are bicistronic. In one
embodiment, a MND (or, myeloproliferative sarcoma virus
LTR-negative control region deleted) promoter is used to drive
Pim-1 expression. In one embodiment, a reporter is also used, e.g.,
an enhanced green florescent protein (eGFP) reporter, which can be
driven off a viral internal ribosomal entry site (vIRES). In
alternative embodiments, all constructs are third generation
self-inactivating (SIN) lentiviral vectors and incorporate several
elements to ensure long-term expression of the transgene. For
example, a MND promoter allows for high expression of the
transgene, while the LTR allows for long-term expression after
repeated passage. In alternative embodiments, the vectors also
include (IFN)-.beta.-scaffold attachment region (SAR) element; SAR
elements have been shown to be important in keeping the vector
transcriptionally active by inhibiting methylation and protecting
the transgene from being silenced.
[0182] In alternative embodiments, as a secondary course of
therapy, PIM-1 expressing nucleic acid delivery vehicles, e.g.,
expression constructs, such as vectors or recombinant viruses, can
be used to enhance proliferation during culture of adult stem cells
extracted from the patient's damaged heart or other organ. In
alternative embodiments, blood, fat, or marrow-derived stem cells
can also be used. PIM-1 expression can be activated through
addition of the drug to culture media. After a number of days in
culture, the expression of PIM-1 can be then turned off through
removal of the drug; and, in one aspect, the increased number of
cells produced in culture are reintroduced into the damaged area
contributing to an enhanced repair process.
[0183] The invention can incorporate use of any non-viral delivery
or non-viral vector systems are known in the art, e.g., including
lipid mediated transfection, liposomes, immunoliposomes,
lipofectin, cationic facial amphiphiles (CFAs) and combinations
thereof.
[0184] In one aspect, expression vehicles, e.g., vectors or
recombinant viruses, used to practice the invention are injected
directly into the heart muscle. In one aspect, the PIM-1 encoding
nucleic acid is administered to the individual by direct injection.
Thus, in one embodiment, the invention provides sterile injectable
formulations comprising expression vehicles, e.g., vectors or
recombinant viruses, used to practice the invention.
[0185] In alternative embodiments, it may be appropriate to
administer multiple applications and employ multiple routes, e.g.,
directly into the heart muscle and intravenously, to ensure
sufficient exposure of target cells (e.g., myocytes or stem cells)
to the expression construct. Multiple applications of the
expression construct may also be required to achieve the desired
effect.
[0186] One particular embodiment of the invention is the ex vivo
modification of stem cells of any origin or any pluripotent cell to
enhance PIM-1 expression, followed by administration of the stem
cells to a human or other mammalian host, or to any vertebrate. The
stem cells may be directly or locally administered, for example,
into cardiac tissue in the same manner as in existing stem cell
therapy for cardiac injury or insufficiency. Alternatively,
systemic administration is also contemplated. The stem cells may be
autologous stem cells or heterologous stem cells. They may be
derived from embryonic sources or from infant or adult organisms.
The enhancement of PIM-1 expression may for example be the result
of upregulation of the expression of existing chromosomal
PIM-1-encoding sequence in the stem cells, or may be the result of
insertion of an exogenous polynucleotide operably encoding PIM-1.
As discussed in other contexts herein, a PIM-1-encoding insert in
such stem cells may advantageously be under inducible expression
control. In addition, the use of a "suicide sequence" of known
type
[0187] In alternative embodiments, one or more "suicide sequences"
are also administered, either separately or in conjunction with a
nucleic acid construct of this invention, e.g., incorporated within
the same nucleic acid construct (such as a vector, recombinant
virus, and the like. See, e.g., Marktel S, et al., Immunologic
potential of donor lymphocytes expressing a suicide gene for early
immune reconstitution after hematopoietic T-cell-depleted stem cell
transplantation. Blood 101:1290-1298(2003). Suicide sequences used
to practice this invention can be of known type, e.g., sequences to
induce apoptosis or otherwise cause cell death, e.g., in one
aspect, to induce apoptosis or otherwise cause cell death upon
administration of an exogenous trigger compound or exposure to
another type of trigger, including but not limited to light or
other electromagnetic radiation exposure.
[0188] In one aspect, a PIM-1 encoding nucleic acid-comprising
expression construct or vehicle of the invention is formulated at
an effective amount of ranging from about 0.05 to 500 ug/kg, or 0.5
to 50 ug/kg body weight, and can be administered in a single dose
or in divided doses. However, it should be understood that the
amount of a PIM-1 encoding nucleic acid of the invention, or other
the active ingredient (e.g., a PIM-1 inducing or upregulating
agent) actually administered ought to be determined in light of
various relevant factors including the condition to be treated, the
age and weight of the individual patient, and the severity of the
patient's symptom; and, therefore, the above dose should not be
intended to limit the scope of the invention in any way.
[0189] In one aspect, a PIM-1 encoding nucleic acid-comprising
expression construct or vehicle of the invention is formulated at a
titer of about at least 10.sub.10, 10.sup.11, 10.sup.12, 10.sup.13,
10.sup.14, 10.sup.15, 10.sup.16, or 10.sup.17 physical particles
per milliliter. In one aspect, the PIM-1 encoding nucleic acid is
administered in about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,
120, 130, 140 or 150 or more microliter (.mu.l) injections. Doses
and dosage regimens can be determined by conventional range-finding
techniques known to those of ordinary skill in the art. For
example, in alternative embodiments, about 10.sup.6, 10.sup.7,
10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13,
10.sup.14, 10.sup.15, 10.sup.16 or 10.sup.17 viral (e.g.,
lentiviral) particles are delivered to the individual (e.g., a
human patient) in one or multiple doses.
[0190] In other embodiments, an intracardiac single administration
(e.g., a single dose) comprises from about 0.1 .mu.l to 1.0 .mu.l,
10 .mu.l or to about 100 .mu.l of a pharmaceutical composition of
the invention. Alternatively, dosage ranges from about 0.5 ng or
1.0 ng to about 10 .mu.g, 100 .mu.g to 1000 .mu.g of PIM-1
expressing nucleic acid is administered (either the amount in an
expression construct, or as in one embodiment, naked DNA is
injected). Any necessary variations in dosages and routes of
administration can be determined by the ordinarily skilled artisan
using routine techniques known in the art.
[0191] In one embodiment, a PIM-1 expressing nucleic acid is
delivered in vivo directly to a heart using a viral stock in the
form of an injectable preparation containing pharmaceutically
acceptable carrier such as saline. The final titer of the vector in
the injectable preparation can be in the range of between about
10.sup.8 to 10.sup.14, or between about 10.sup.10 to 10.sup.12,
viral particles; these ranges can be effective for gene
transfer.
[0192] In one aspect, PIM-1 expressing nucleic acids (e.g., vector,
transgene) constructs are delivered to the myocardium by direct
intracoronary injection, e.g., using a standard percutaneous
catheter based methods under fluoroscopic guidance. Alternatively,
PIM-1 expressing nucleic acids (e.g., vector, transgene) constructs
are delivered to organs and tissues, e.g., the heart, directly into
both coronary and/or peripheral arteries, e.g., using a
lipid-mediated gene transfer.
[0193] In these aspects, including direct intracoronary injection,
or directly into both coronary and/or peripheral arteries, can be
at an amount sufficient for the PIM-1 expressing nucleic acids
(e.g., vector, transgene) to be expressed to a degree which allows
for sufficiently effective; e.g., the amount of the PIM-1
expressing nucleic acid (e.g., vector, transgene) injected can be
in the range of between about 10.sup.8 to 10.sup.14, or between
about 10.sup.10 to 10.sup.12, viral particles. The injection can be
made deeply (such as 1 cm within the arterial lumen) into the lumen
of the coronary arteries, and can be made in both coronary
arteries, as the growth of collateral blood vessels is highly
variable within individual patients. By injecting the material
directly into the lumen of the coronary artery by coronary
catheters, it is possible to target the PIM-1 expressing nucleic
acid (e.g., vector, transgene) rather effectively, and to minimize
loss of the recombinant vectors to the proximal aorta during
injection. Any variety of coronary catheter, or Stack perfusion
catheters, and the like can be used. See, e.g., U.S. Patent App.
Pub. No. 20040132190.
[0194] In one aspect, the invention combines a therapeutic PIM-1
nucleic acid with a genetic "sensor" that recognizes and responds
to the oxygen deprivation that follows the reduced blood flow, or
ischemia, from coronary artery disease and heart attack. As soon as
the oxygen declines, the sensor turns on the therapeutic gene,
thereby protecting the heart. In addition to its potential for
patients with heart disease, the aspect of this invention is useful
for any condition in which circulatory system tissues are
susceptible to loss of blood supply, including stroke, shock,
trauma and sepsis.
Direct PIM delivery
[0195] In addition to cellular and nucleic acid approaches, PIM
proteins can also be delivered directly to the affected cardiac or
other circulatory tissues. Because PIM acts intracellularly, it is
preferred to utilize a delivery strategy to facilitate
intracellular delivery of PIM.
[0196] One technique that can be used is to provide the PIM in a
vehicle that in taken up by or that fuses with a target cell. Thus,
for example, PIM can be encapsulated within a liposome or other
vesicle, as described in more detail above in connection with
polynucleotide delivery to cells.
[0197] Alternatively, the PIM may be linked to a transduction
domain, such as TAT protein. In some embodiments, PIM enzyme can be
operably linked to a transduction moiety, such as a synthetic or
non-synthetic peptide transduction domain (PTD), Cell penetrating
peptide (CPP), a cationic polymer, an antibody, a cholesterol or
cholesterol derivative, a Vitamin E compound, a tocol, a
tocotrienol, a tocopherol, glucose, receptor ligand or the like, to
further facilitate the uptake of the PIM by cells.
[0198] A number of protein transduction domains/peptides are known
in the art and facilitate uptake of heterologous molecules linked
to the transduction domains (e.g., cargo molecules). Such peptide
transduction domains (PTD's) facilitate uptake through a process
referred to as macropinocytosis. Macropinocytosis is a nonselective
form of endocytosis that all cells perform.
[0199] Exemplary peptide transduction domains (PTD's) are derived
from the Drosophila homeoprotein antennapedia transcription protein
(AntHD) (Joliot et al., New Biol. 3:1121-34, 1991; Joliot et al.,
Proc. Natl. Acad. Sci. USA, 88:1864-8, 1991; Le Roux et al., Proc.
Natl. Acad. Sci. USA, 90:9120-4, 1993), the herpes simplex virus
structural protein VP22 (Elliott and O'Hare, Cell 88:223-33, 1997),
the HIV-1 transcriptional activator TAT protein (Green and
Loewenstein, Cell 55:1179-1188, 1988; Frankel and Pabo, Cell
55:1189-1193, 1988), and more recently the cationic N-terminal
domain of prion proteins. Preferably, the peptide transduction
domain increases uptake of the biomolecule to which it is fused in
a receptor independent fashion, is capable of transducing a wide
range of cell types, and exhibits minimal or no toxicity (Nagahara
et al., Nat. Med. 4:1449-52, 1998). Peptide transduction domains
have been shown to facilitate uptake of DNA (Abu-Amer, supra),
antisense oligonucleotides (Astriab-Fisher et al., Pharm. Res,
19:744-54, 2002), small molecules (Polyakov et al., Bioconjug.
Chem. 11:762- 71, 2000) and even inorganic 40 nanometer iron
particles (Dodd et al., J. Immunol. Methods 256:89-105, 2001;
Wunderbaldinger et al., Bioconjug. Chem. 13:264-8, 2002; Lewin et
al., Nat. Biotechnol. 18:410-4, 2000; Josephson et al., Bioconjug.,
Chem. 10:186-91, 1999).
[0200] Fusion proteins with such trans-cellular delivery proteins
can be readily constructed using known molecular biology
techniques.
[0201] In addition, any of the polynucleotides encoding PIM
molecules can be linked to the foregoing domains to facilitate
transduction of those polynucleotides into target cells, in vivo or
in vitro.
Kits and Libraries
[0202] The invention provides kits comprising compositions of this
invention and methods of the invention, including PIM-expressing,
or PIM-inducing or upregulating compositions and/or nucleic acids
of the invention, including vectors, recombinant viruses and the
like, transfecting agents, transducing agents, cardiac or vascular
cells and/or cell lines, instructions (regarding the methods of the
invention), or any combination thereof. As such, kits, cells,
vectors and the like are provided herein.
[0203] The invention will be further described with reference to
the following examples; however, it is to be understood that the
invention is not limited to such examples.
EXAMPLE 1
Demonstrating the Therapeutic Efficacy of Upregulating PIM-1
[0204] This example demonstrates that the compositions of the
invention comprising nucleic acids encoding the serine/threonine
kinase PIM-1, and the methods of this invention, are effective for
inducing cellular proliferation, and protecting cells from hypoxia
and cellular apoptosis; and to express PIM-1 kinase to protect
cardiomyocytes from hypertrophy and/or inhibit myocardial apoptosis
induced by infarction, reducing infarct size; and to express PIM-1
to induce cellular dedifferentiation and re-expression of stem cell
markers; and to overexpress PIM-1 to enhance the regenerative
potential of stem cells, including stem cell ability to engraft in
the heart after a myocardial infarction (post-MI). These data
demonstrate that in using compositions and methods described
herein, PIM-1 functions as a defense against apoptotic stimuli
induced during ischemia/reperfusion injury resulting from
myocardial infarction, pressure-overload induced hypertrophy, and
heart failure.
Results
PIM-1 is Expressed in the Human Myocardium and Upregulated in
Failure.
[0205] Immunohistochemistry of normal and failing human heart
samples indicates that PIM-1 expression is distributed throughout
the cytoplasm in normal adult human myocardium. In contrast, in
failing human heart samples, PIM-1 becomes mostly nuclear.
Immunoblotting of human heart lysates demonstrates that PIM-1
expression increases 2.65-fold in the failing human myocardium when
compared to normal controls. A similar pattern is seen in
tropomodulin overexpressing transgenic (TOT) mice, a DCM model.
Though PIM-1 is expressed at low levels in the 6 month old wildtype
(NTG) mouse, expression in the TOT mouse is increased 5.9-fold and
was mostly nuclear.
PIM-1 Expressed in the Mouse Myocardium is Developmentally
Regulated.
[0206] Immunoblot analysis of myocardial lysates from mice at
various time points after birth demonstrates decreasing PIM-1
expression with age. Neonatal heart samples exhibit 6.3-fold more
PIM-1 than 30 week old mice. Postnatal expression levels decline,
but remain significantly elevated, until 8 weeks of age when they
became comparable to 30 week old hearts. Confocal microscopy of
mouse hearts at various developmental time points show PIM-1
expression is predominantly nuclear in neonates, becomes
increasingly cytosolic in early adulthood, and is virtually absent
in the 30 week old adult. This is corroborated by immunoblotting of
subcellular fractionated myocardium for PIM-1. PIM-1 expression is
10.5-fold and 5.2-fold more nuclear and 5.0 and 4.6-fold less
cytosolic in neonatal hearts and 8 week old hearts respectively
when compared to 30 week old mouse myocardium.
PIM-1 Exhibits Cardioprotective Effects In Vivo.
[0207] Using an art-accepted animal model, these data demonstrate
that expression of PIM-1 in vivo has a cardioprotective effect.
PIM-1 localization and expression were examined in hearts from
3-month old normal mice processed four days after sham or
cardio-myopathic injury resulting from infarction (MI) or pressure
overload (TAC). Four days following TAC banding to induce
pressure-overload hypertrophy, a marked peri-nuclear increase in
PIM-1 immunoreactivity is observed in cardiomyocytes surrounding
major vessels. Similarly, peri-nuclear PIM-1 immunoreactivity is
increased in border zone cardiomyocytes, but is unaffected in
healthy regions of remote myocardium. PIM-1 positive border zone
cardiomyocytes are negative for "terminal deoxynucleotidyl
transferase-mediated dUTP-biotin nick end labeling" (TUNEL)
labeling and exhibit increased Bcl-XL expression indicative of
cardioprotective anti-apoptotic signaling, demonstrating a role for
PIM-1 in myocardial survival signaling, also demonstrating the
compositions and methods of the invention can be effective in
myocardial survival signaling by expressing and/or upregulating
PIM-1 kinase expression and/or activity.
[0208] A protective role for PIM-1 was confirmed using hearts of
mice deficient for PIM-1 by genetic deletion subjected to ex vivo
ischemia/reperfusion injury together with age and sex matched
controls. Hearts of PIM-1 knockout mice exhibited statistically
significant decreases in functional recovery following 45 minutes
of reperfusion, as measured by left-ventricular developed pressure.
TUNEL staining of paraffin embedded sections from hearts subjected
to ex vivo ischemia reveals a 2.4-fold increase in the number of
TUNEL positive cardiomyocytes in the PIM-1 knockout mice versus
wildtype controls.
[0209] PIM-1 induces anti-apoptotic protein expression and protects
cardiomyocytes in vitro. GFP-tagged cDNAs for wild-type 34 kDa
PIM-1 (PIM-wt) or a kinase dead (K67M) mutant (PIM-DN) as
previously described.sup.10 were used to generate recombinant
adenoviruses used for infection of neonatal rat cardiomyocyte
cultures. Immunoblotting of lysates from cultures expressing
GFP-PIM-wt or GFP-PIM-DN accumulate 64kDa GFP-PIM-1 fusion proteins
recognized by either GFP or PIM-1 antibodies. Cardiomyocytes
overexpressing GFP-PIM-wt exhibit a statistically significant
decrease in TUNEL labeling compared to EGFP infected controls. In
comparison, GFP-PIM-DN overexpression induced a 30.8% increase in
apoptotic cardiomyocytes (*p<0.05). Cultured cardiomyocytes were
protected from apoptotic challenge with doxorubicin or deoxyglucose
by GFP-PIM-wt overexpression (**p<0.01 for both groups), whereas
GFP-PIM-DN overexpression exacerbated apoptotic effects
(**p<0.01, and *p<0.05 respectively). Consistent with these
results, GFP-PIM-DN induced a 3.6-fold increase in caspase3
cleavage and an 80% increase in cleaved poly (ADP-ribose)
polymerase (PARP). In comparison, GFP-PIM-wt produced significant
increases in bcl-XL and bcl-2 expression (2.2-fold and 25.4-fold,
respectively) when compared to control (*p<0.01). GFP-PIM-wt
also increased phosphorylation of Bad at the serine 112 residue
(S112) by 16.7-fold versus uninfected control while levels of total
Bad remained unchanged (*p<0.01).
PIM-1 is Induced by Cardioprotective Stimuli.
[0210] These data demonstrate that the compositions and methods of
the invention can be used to increase the expression of PIM-1 to
provide a cardioprotective effect, e.g., after a myocardial
infarction. Treatment of neonatal rat cardiomyocyte cultures with
cardioprotective agents including Leukemia Inhibitory Factor (LIF),
Insulin-like Growth Factor (IGF-1), dexamethasone, and PMA, for 2
hours prior to assay induced PIM-1 immunoreactivity compared to
control cells as visualized by confocal microscopy. PIM-1
immunoreactivity was not induced by phenylephrine, endothelin-1,
forskolin, or estradiol (FIG. 4A). LIF, IGF, PMA, and dexamethasone
induced PIM-1 expression by 2.8, 2.7, 2.3, and 2.0-fold
respectively (*p<0.05, **p<0.01). The adenylate cyclase
activator, forskolin, reduced PIM-1 expression by 45% versus
control cultures (**p<0.01).
IGF-1 Induction of PIM-1 Expression is AKT-Dependent.
[0211] PIM-1 expression in response to IGF-1 treatment is
significantly reduced in the presence of the PI3 kinase inhibitor
wortmannin or inactivated AKT (AKT-DN) by 4.0-fold and 9.1-fold
respectively (**p<0.001). A role for nuclear accumulation of AKT
resulting following IGF stimulation.sup.11 was confirmed, as
overexpression of nuclear-targeted AKT.sup.12 increased PIM-1
expression in cultured cardiomyocytes by 2.1-fold compared to
uninfected controls (*p<0.05). In contrast, overexpression of
wildtype AKT (AKT-wt) decreased expression of PIM-1 1.4-fold versus
uninfected control (*p<0.05). Confocal micrographs of cultured
cardiomyocytes demonstrate that expression of nuclear-targeted AKT
induces increased nuclear localization of PIM-1 (FIG. 5C).
Consistent with in vitro results, immunohistochemistry of hearts
from six-month-old cardiac-specific nuclear-targeted AKT
transgenics exhibit increased PIM-1 immunoreactivity and nuclear
localization compared to controls and a representative immunoblot
corroborates increased PIM-1 expression.
PIM-1 and AKT Exhibit Feedback Relationships.
[0212] AKT expression and phosphorylation (S.sup.473) increase in
response to overexpression of GFP-PIM-DN in cultured cardiomyocytes
(FIG. 6A). Comparable findings of increased phospho-AKT.sup.473
were observed with confocal microscopy of immunolabeled myocardial
sections from 2-month old PIM-KO mice. Increased
phospho-AKT.sup.473 and total AKT in immunoblots of whole heart
lysates from PIM-KO mice correlated with the immunostaining,
demonstrating that regulation of Akt expression and activity
depends, in part, upon PIM-1 levels.
Discussion
[0213] Using an art-accepted animal model, these data demonstrate
that the compositions and methods of the invention can be used to
increase the expression of PIM-1 to provide a cardioprotective
effect (a myocardium protective effect), e.g., after a myocardial
infarction. Molecular regulation of cardioprotection endures as a
highly significant research avenue for therapeutic interventional
strategies in the treatment of myocardial injury and heart failure.
With this invention's discovery of a central role for PIM-1 in
cardioprotection, as demonstrated by the data presented herein, a
new facet of signaling has been uncovered with profound
implications for regulation of cardiomyocyte survival and
AKT-mediated effects. Taken together, data presented here provide
the first evidence of PIM-1 expression and protective effects in
the myocardium and demonstrate a reciprocal feedback mechanism
between PIM-1 and AKT. The codependent interrelationship between
AKT and PIM-1 previously documented in the hematopoeitic system'
indicates both molecules work in concert.
[0214] PIM-1 Functions as a Defense Against Apoptotic Stimuli
Induced During Ischemia/Reperfusion Injury
[0215] PIM-1 has not previously been studied in the myocardial
context studies. In non-cardiac cells is has been demonstrated that
PIM-1 as a critical regulator of proliferation and cell survival
signaling, e.g., as reviewed in ref.sup.1 (see below). Using the
compositions and methods of this invention, PIM-1 functions as a
defense against apoptotic stimuli induced during
ischemia/reperfusion injury resulting from myocardial infarction,
pressure-overload induced hypertrophy, and heart failure. Although
PIM-1 level is developmentally down-regulated, expression reappears
in cardiomyocytes following cardiac injury by pressure-overload
induced by TAC banding and myocardial infarction. PIM-1 is one of
several protoncogenes participating in the "immediate early
response" gene profile expressed following cardiac injury.sup.13
including c-fos, c-myc .sup.14, Raf and Ras. PIM-1 has been shown
to cooperate with c-myc in activation of c-Myb dependent cellular
proliferation in other tissues.sup.15-17 suggesting synergistic
effects between oncogenes may help preserve the myocardium in
reaction to injury.
[0216] PIM-1 Potentiates Intracellular Anti-Apoptotic Signaling
[0217] In addition to its proliferative effects, these data also
demonstrate that PIM-1 potentiates intracellular anti-apoptotic
signaling. Consistent with findings in non-cardiac
cells,.sup.17,18,19 adenoviral overexpression of PIM-1 protects
neonatal rat cardiomyocytes from doxorubicin and deoxyglucose
induced apoptosis through induction of bcl-2 and Bcl-XL expression
as well as phosphorylation of Bad (FIG. 3). It is also possible
that PIM-1 serves as the downstream effector of AKT induced p53
inhibition, since induction of mdm2 expression, phosphorylation,
and p53 degradation is mediated by PIM-1.sup.17 and AKT protects
against doxorubicin induced apoptosis through a p53-dependent
mechanism.sup.20. Furthermore, inactivation of PIM-1 induced
apoptotic signaling in the cardiomyocyte context, exacerbating
doxorubicin induced apoptosis. PIM-1 inactivation may also increase
apoptotic activity through increasing generation of reactive-oxygen
species and mitochondrial pore permeability, as has been found in
other cellular contexts.sup.18.
[0218] Several well known cardioprotective factors including LIF,
the PKC activator PMA, the glucocorticoid dexamethasone, and IGF-1
significantly increased PIM-1 expression (FIG. 4), consistent with
published reports showing induction of PIM-1 by PMA treatment of T
cells.sup.21 as well as gp130 receptor ligands including IL-6 and
LIF.sup.(22, reviewed in 1). These ligands and their cognate
receptor are increased in the failing heart.sup.23 24 25 26 as well
as during development and hypertrophy of the myocardium..sup.27
With regard to other inductive stimuli, PMA-mediated PKC activation
is cardioprotective,.sup.28-30 correlative findings show PIM-1 is
rapidly induced by PMA treatment in T cells..sup.21 IGF-1 mediates
myocardial survival signaling.sup.40-42 and stem cell
proliferation.sup.43 although we were unable to find prior
published demonstration of PIM-1 induction by IGF. Results indicate
that IGF-1 induced PIM-1 expression is AKT dependent and that
nuclear-targeted AKT expression induces significant increases in
PIM-1 expression in vitro and in vivo. Conversely, inactivation or
ablation of PIM-1 expression induced AKT expression and activation,
but increased AKT activation is unable to enhance recovery or
reduce apoptosis following ischemia/reperfusion injury in PIM-1
null animals.
[0219] These results indicate that PIM-1 is an important mediator
of cardioprotection downstream from comparatively well documented
AKT signaling in the myocardium responsible for cardioprotection.
Following growth factor or cytokine receptor activation, AKT is
phosphorylated resulting in a conformational change which releases
AKT from the membrane allowing it to transit through the cytosol
and eventually to the nucleus where it affects transcription of
target genes.sup.31 and exerts cardioprotective activity..sup.12
Recent research demonstrates similar substrate specificity shared
by PIM-1 and AKT.sup.3, and that the widely employed PI3K inhibitor
LY294002.TM. binds to and inhibits PIM-1 activity.sup.32.
Therefore, previous studies involving the use of LY294002.TM.
require reinterpretation in the context of AKT-dependent PIM-1
signaling in the myocardium.
[0220] Cardioprotective effects together with heightened expression
in both postnatal/juvenile myocardium and pathologically challenged
hearts implicate PIM-1 in promotion of phenotypic characteristics
typically associated with a youthful myocardium. Indeed, the
cytokine expression profile of neonatal myocardium share marked
similarities with that exhibited by AKT-nuc transgenic hearts. It
appears that many of the beneficial effects previously ascribed to
Akt-nuc.sup.11,12,33 (see references below) may depend, at least in
part, upon induction of PIM-1 expression.
Methods
[0221] Neonatal Rat Cardiomyocyte Cultures infections and
treatments. Neonatal rat cardiomyocyte cultures were prepared as
described previously.sup.12. Cells to be subjected to treatments
were placed in media with 2% serum overnight and then treated with
the appropriate agent and harvested or fixed after the
pre-described timepoint. Cardiomyocytes were infected with
adenovirus for two hours, washed in PBS and then re-fed M199 with
2% FBS and 50 .mu.g/ml pen/strep, and 100 .mu.M glutamine.
[0222] Nuclear and cytosolic extraction. Hearts were washed in PBS,
transferred to 2 ml of 0.57M STEAKM (0.57 M sucrose, 25 mM KCL, 5%
MgCl.sub.2, 1 mM DTT, 0.5 mM PMSF, 500 .mu.l protease inhibitor and
50 .mu.l phosphatase inhibitor), homogenized in ice using polytron
and centrifuged for 10 minutes at 1000.times.g.
[0223] Pellets were resuspended in 1.5 mL of 0.57M STEAKM.TM.,
homogenized in tight fitting pestle and centrifuged at 1000.times.g
for 10 minutes at 4.degree. C. Supernatant was collected for
cytosolic fraction.
[0224] Pellets were re-suspended in 750 .mu.l of 0.57M STEAKM.TM.
with 0.5% TRITON-X.TM. and centrifuged at 1000.times.g for 10
minutes at 4.degree. C. Supernatant was collected for membrane
fraction.
[0225] Pellets were re-suspended in 300 .mu.l of 2.3M STEAKM.TM.
(2.3M sucrose, 25 mM KCL, 5% MgCl.sub.2, 1 mM DTT, 0.5 mM PMSF, 500
.mu.l of protease inhibitor and 50 .mu.l of phosphatase inhibitor).
2 volumes of 0.57M STEAKM.TM. was added and the pellets were gently
mixed.
[0226] A layer of 2.7M STEAKM.TM. (2.7M sucrose, 25 mM KCL, 5%
MgCl.sub.2, 1 mM DTT, 0.5 mM PMSF, 500 .mu.l of protease inhibitor
and 50 .mu.l of phosphatase inhibitor) was made at the bottom of an
ultracentrifuge tube followed by a 2.4M STEAKM.TM. (2.4M sucrose,
25 mM KCL, 5% MgCl.sub.2, 1 mM DTT, 0.5 mM PMSF, 500 .mu.l of
protease inhibitor and 50 .mu.l of phosphatase inhibitor). The
homogenate layer was added. The 3 layers were centrifuged at
112,000.times.g for 1 hour at 4.degree. C. White interface between
2.7 and 2.4M STEAKM.TM. was collected for nuclear fraction. 5
volumes of 0.57 STEAKM.TM. was added and the pellets were
centrifuged at 2000.times.g for 20 minutes at 4.degree. C. The
pellet was re-suspended in sample buffer containing phosphatase
inhibitors and protease inhibitors.
[0227] Immunoblotting. Infected cardiomyocytes were harvested 24
hours after infection in SDS denaturing sample buffer, sonicated
and boiled for 10 minutes, and quantitated using the Bradford
assay. Mouse whole heart lysates were generated from flash frozen
hearts pulverized in a mortar and pestle then resuspended in SDS
denaturing sample buffer, sonicated, boiled for minutes and
quantitated using Bradford assay. Approximately 50 .mu.g of each
sample was loaded on a 4-15% gradient Bis-Acrylamide Tris Glycine
gel and transferred to PVDF. Blots were blocked in 3% BSA for
lhour, and probed with primary antibodies (PIM-1 (Cell Signaling
Technology), c-jun (Cell Signaling Technology), HISTONE3.TM. (Cell
Signaling Technology), GFP (Molecular Probes), bcl-2 (Santa Cruz),
bcl-XL (Cell Signaling Technology), PHOSPHO-BADS112.TM. (Biosource)
AKT (Cell Signaling Technology), GAPDH (Research Diagnostics Inc.),
PHOSPHO-AKTS473TM (Cell Signaling Technology), total PARP
(Biosource), cleaved PARP (Biosource), and cleaved caspase3 (Cell
Signaling Technologies)) diluted in blocking solution overnight at
4.degree. C. Blots were washed in TBS-0.5% Tween three times and
probed with fluorescent or alkaline phosphatase conjugated
secondary antibodies diluted 1:5000 in blocking solution for 1 hour
at room temperature followed by three washes in TBS-0.5% Tween.
Blots were scanned using a TYPHOON 9410 IMAGER.TM. (GE Healthcare)
and quantitated using IMAGEQUANT 5.2.TM. software (GE Healthcare).
All quantitation is based on standardization to loading
controls.
[0228] Adenoviral constructs. AKT-nuc and AKT wildtype adenoviruses
were prepared as described previously, see reference.sup.12. PIM-wt
and PIM-DN adenoviruses were prepared by subcloning of the
NheI/SmaI fragments from pEGFP-N1 PIM-1 and pEGFP-N1PIM-DN.TM.
plasmids described previously.sup.10, into the pDC315io.TM.
(Microbix) adenoviral shuttle vector. .sup.32Sequence verified
shuttle vectors were cotransfected with the genomic
pBHGlox.DELTA.E1,3Cre into 293ig.TM. cells (Microbix) to generate
the adenovirus. Purified plaques were isolated and expanded for use
in experiments.
[0229] Immunohistochemistry. Hearts were fixed and embedded and
cardiomyocytes fixed and permeabilized as described
previously.sup.12. Staining of cultured neonatal rat cardiomyocytes
was performed with antibodies described above diluted 1:25 in PBS
containing 10% horse serum overnight at 4.degree. C. Slides were
washed in PBS and probed with fluorescent conjugated secondary
antibodies (1:100) for one hour at room temperature, and Texas Red
phalloidin (1:50 Molecular Probes) to identify actin filaments.
Slides were washed three times in PBS, and stained for 30 minutes
with TOPRO-3.TM. (1:5000 Molecular Probes) to identify nuclei,
washed once and cover-slipped using VECTRASHIELD.TM. (Vectra Labs).
Paraffin embedded samples were cut at 4 .mu.m and deparaffinized
through a standard series of Xylene and graded Ethanol steps to
water. Antigen retrieval was performed in 10 mM Citrate pH6.0.
PIM-1 and phospho-AKT signals were amplified using the TYRAMIDE
AMPLIFICATION KIT.TM. (Perkin Elmer) with primary concentrations of
1:500 for both antibodies, and secondaries 1:3000. Slides were
washed following the amplification process, and stained with
TOPRO-3 (Molecular Probes) to identify nuclei, washed and
cover-slipped with VECTRASHIELD.TM. (Vectra Labs). Confocal imaging
of stained slides was performed on a Leica LCS confocal microscope.
For comparison purposes, all slides were treated identically and
scanned using the same settings in each experiment.
[0230] Doxorubicin and deoxyglucose induction of apoptosis.
Cardiomyocytes were infected with GFP, PIM-wt, and PIM-DN viruses
as described earlier. Twenty-four hours after infection, cells were
treated with 1 .mu.M Doxorubicin or 1 mM deoxyglucose for 16 hours
then labeled for TUNEL using the IN SITU CELL DEATH DETECTION
KIT.TM., TMR red (Roche Applied Science) per manufacturer
instructions. Number of infected TUNEL positive cells was counted
for each treatment on a Nikon DIAPHOT 420.TM. fluorescent
scope.
[0231] Myocardial infarction and trans-aortic constriction. Mice
were placed under anesthesia, and the chest wall surgically opened.
To induce an acute ischemic event, the left anterior descending
artery (LAD) was located and ligated using 8-0 nylon suture. To
induce pressure overload, the aorta was banded with 8-0 prolene
using a 27 gauge needle as a guide. Sham animals were treated
identically except the LAD or aorta were not ligated. Animal hearts
were harvested as described above and embedded into paraffin.
[0232] Ex Vivo Ischemia/Reperfusion. Ex vivo ischemia/reperfusion
was performed as described previously.sup.34. Sections from four
hearts from each experimental group were cut and analyzed for cell
death using TUNEL labeling (IN SITU CELL DEATH DETECTION KIT.TM.,
TMR red (Roche Applied Science)) according to the kit
directions.
[0233] Statistical Analysis. Statistical analysis was performed
using student T test, and analysis of variance (ANOVA) as
appropriate. P values less than 0.05 were considered
significant.
[0234] References for Example 1:
Wang, Z. et al. PIM-1: a serine/threonine kinase with a role in
cell survival, proliferation, differentiation and tumorigenesis. J
Vet Sci 2, 167-79 (2001). 2. Xie, Y. et al. The 44 kDa PIM-1 kinase
directly interacts with tyrosine kinase Etk/BMX and protects human
prostate cancer cells from apoptosis induced by chemotherapeutic
drugs. Oncogene (2005). 3. Bullock, A. N., Debreczeni, J., Amos,
A., Knapp, S. & Turk, B. E. Structure and substrate specificity
of the PIM-1 kinase. J. Biol. Chem., M510711200 (2005). 4. Palaty,
C. K. et al. Identification of the autophosphorylation sites of the
Xenopus laevis PIM-1 proto-oncogene-encoded protein kinase. J Biol
Chem 272, 10514-21 (1997). 5. Bachmann, M. & Moroy, T. The
serine/threonine kinase PIM-1. Int J Biochem Cell Biol 37, 726-30
(2005). 6. Aho, T. L. et al. PIM-1 kinase promotes inactivation of
the pro-apoptotic Bad protein by phosphorylating it on the Ser112
gatekeeper site. FEBS Lett 571, 43-9 (2004). 7. Hammerman, P. S.,
Fox, C. J., Birnbaum, M. J. & Thompson, C. B. PIM and Akt
oncogenes are independent regulators of hematopoietic cell growth
and survival. Blood 105, 4477-83 (2005). 8. Krumenacker, J. S.,
Narang, V. S., Buckley, D. J. & Buckley, A. R. Prolactin
signaling to PIM-1 expression: a role for phosphatidylinositol
3-kinase. J Neuroimmunol 113, 249-59 (2001). 9. Krishnan, N., Pan,
H., Buckley, D. J. & Buckley, A. Prolactin-regulated PIM-1
transcription: identification of critical promoter elements and Akt
signaling. Endocrine 20, 123-30 (2003). 10. Bhattacharya, N. et al.
PIM-1 associates with protein complexes necessary for mitosis.
Chromosoma 111, 80-95 (2002). 11. Camper-Kirby, D. et al.
Myocardial Akt activation and gender: increased nuclear activity in
females versus males. Circ Res 88, 1020-7 (2001). 12. Shiraishi, I.
et al. Nuclear targeting of Akt enhances kinase activity and
survival of cardiomyocytes. Circ Res 94, 884-91 (2004). 13. Sugden,
P. H. & Clerk, A. Cellular mechanisms of cardiac hypertrophy. J
Mol Med 76, 725-46 (1998). 14. Izumo, S., Nadal-Ginard, B. &
Mandavi, V. Protooncogene Induction and Reprogramming of Cardiac
Gene Expression Produced by Pressure Overload. PNAS 85, 339-343
(1988). 15. Katakami, N. et al. Role of PIM-1 in smooth muscle cell
proliferation. J Biol Chem 279, 54742-9 (2004). 16. Hoefnagel, J.
J. et al. Distinct types of primary cutaneous large B-cell lymphoma
identified by gene expression profiling. Blood (2004). 17. Ionov,
Y. et al. PIM-1 protein kinase is nuclear in Burkitt's lymphoma:
nuclear localization is necessary for its biologic effects.
Anticancer Res 23, 167-78 (2003). 18. Lilly, M., Sandholm, J.,
Cooper, J. J., Koskinen, P. J. & Kraft, A. The PIM-1 serine
kinase prolongs survival and inhibits apoptosis-related
mitochondrial dysfunction in part through a bcl-2-dependent
pathway. Oncogene 18, 4022-31 (1999). 19. Macdonald, A. et al. PIM
kinases phosphorylate multiple sites on Bad and promote 14-3-3
binding and dissociation from Bcl-XL. BMC Cell Biol 7, 1 (2006).
20. Fujiwara, Y. et al. Inhibition of the PI3 kinase/Akt pathway
enhances doxorubicin-induced apoptotic cell death in tumor cells in
a p53-dependent manner. Biochem Biophys Res Commun 340, 560-6
(2006). 21. Wingett, D., Long, A., Kelleher, D. & Magnuson, N.
S. PIM-1 proto-oncogene expression in anti-CD3-mediated T cell
activation is associated with protein kinase C activation and is
independent of Raf-1. J Immunol 156, 549-57 (1996). 22. Rahman, Z.,
Yoshikawa, H., Nakajima, Y. & Tasaka, K. Down-regulation of
PIM-1 and Bcl-2 is accompanied with apoptosis of
interleukin-6-depleted mouse B-cell hybridoma 7TD1 cells. Immunol
Lett 75, 199-208 (2001). 23. Eiken, H. G. et al. Myocardial gene
expression of leukaemia inhibitory factor, interleukin-6 and
glycoprotein 130 in end-stage human heart failure. Eur J Clin
Invest 31, 389-97 (2001). 24. Hirota, H. et al. Circulating
interleukin-6 family cytokines and their receptors in patients with
congestive heart failure. Heart Vessels 19, 237-41 (2004). 25.
Jougasaki, M. et al. Leukemia inhibitory factor is augmented in the
heart in experimental heart failure. Eur J Heart Fail 5, 137-45
(2003). 26. Sheng, Z. et al. Cardiotrophin 1 (CT-1) inhibition of
cardiac myocyte apoptosis via a mitogen-activated protein
kinase-dependent pathway. Divergence from downstream CT-1 signals
for myocardial cell hypertrophy. J Biol Chem 272, 5783-91 (1997).
27. Wollert, K. C. & Chien, K. R. Cardiotrophin-1 and the role
of gp130-dependent signaling pathways in cardiac growth and
development. J Mol Med 75, 492-501 (1997). 28. Sato, T., O'Rourke,
B. & Marban, E. Modulation of mitochondrial ATP-dependent K+
channels by protein kinase C. Circ Res 83, 110-4 (1998). 29. Sato,
T., Saito, T., Saegusa, N. & Nakaya, H. Mitochondrial
Ca2+-activated K+channels in cardiac myocytes: a mechanism of the
cardioprotective effect and modulation by protein kinase A.
Circulation 111, 198-203 (2005). 30. Philipp, S. et al.
Postconditioning protects rabbit hearts through a protein kinase
C-adenosine A2b receptor cascade. Cardiovasc Res 70, 308-14 (2006).
31. Pekarsky, Y. et al. Tcl1 enhances Akt kinase activity and
mediates its nuclear translocation. Proc Natl Acad Sci USA 97,
3028-33 (2000). 32. Jacobs, M. D. et al. PIM-1 Ligand-bound
Structures Reveal the Mechanism of Serine/Threonine Kinase
Inhibition by LY294002. J. Biol. Chem. 280, 13728-13734 (2005). 33.
Rota, M. et al. Nuclear targeting of Akt enhances ventricular
function and myocyte contractility. Circ Res 97, 1332-41 (2005).
34. Kato, T. et al. Atrial natriuretic peptide promotes
cardiomyocyte survival by cGMP-dependent nuclear accumulation of
zyxin and Akt. J Clin Invest 115, 2716-2730 (2005).
EXAMPLE 2
Demonstrating the Therapeutic Efficacy of Upregulating PIM-1
[0235] This example also demonstrates that the compositions of the
invention comprising nucleic acids encoding the serine/threonine
kinase PIM-1, and the methods of this invention, are effective for
inducing cellular proliferation, and protecting cells from hypoxia
and cellular apoptosis; and to express PIM-1 kinase to protect
cardiomyocytes from hypertrophy and/or inhibit myocardial apoptosis
induced by infarction, reducing infarct size.
[0236] Until relatively recently, dogma held that cardiomyocytes
rarely underwent programmed cell death, were impervious to the
effects of aging, and incapable of regeneration. The last decade of
cardiovascular research has produced major paradigm shifts in the
perceptions of cardiomyocyte biology. The emerging picture of the
myocardium is quite unlike previous notions of a tenaciously
steadfast contracting cell that persists throughout the lifespan of
the organism. Instead, cardiomyocytes like many other cell types in
the body possess a finite lifespan characterized by ongoing
processes of birth, survival, death, and (more controversially) 10
regeneration. Consequently, this new perspective has reinvigorated
research into the molecular mechanisms that regulate survival and
the cardiomyocyte life cycle..sup.1
[0237] Cellular proliferation and survival are regulated, in part,
by the action of signaling cascades that lead to activation of
kinases such as protein kinase C (PKC), Akt/PKB, and PIM-1.
Voluminous research in the context of the cardiovascular system has
established both PKC and Akt/PKB as fundamental pillars upon which
cardiomyocyte function is maintained. In contrast, the
cardiovascular role of PIM-1 and influences of this kinase upon
cardiomyocyte structure and/or function are virtually nonexistent.
Despite this dearth of cardiac-related knowledge, published studies
of hematopoeitic and oncogenic cells suggest that the effects of
PIM-1-mediated signaling are as significant and far reaching as
those ascribed to PKC or Akt/PKB. Similarities between these
kinases are readily apparent: 1) phosphorylation of
serine/threonine residues in target substrates, 2) regulation of
cell survival and/or proliferation, and 3) intriguing propensity
for nuclear accumulation..sup.2-4 Between PIM-1 and Akt/PKB there
are additional connections such as similar target substrate
specificities, coordinate regulation of PIM-1 expression by Akt
activation, and blunting of activation by treatment with the PI3-K
inhibitor LY294002..sup.5-7 Collectively, this evidence implicates
PIM-1 for an important role in myocardial signaling, and supportive
findings documenting PIM-1 expression and function in
cardiomyocytes are presented herein.
[0238] PIM-1 was originally identified as a proto-oncogene and
subsequently found to be a highly conserved serine / threonine
kinase. Unlike other serine / threonine kinases (e.g. Akt, MAPK,
PKA or PKC), PIM-1 phosphotransferase activity is not regulated by
upstream kinases--it is active in nascent translated form. Thus,
PIM-1 activity is regulated by concerted control of gene
transcription, mRNA translation, and protein degradation. The
target phosphorylation consensus sequence for PIM-1 is found in
proteins mediating transcription, cell growth, proliferation, and
survival. While PIM-1 overexpression alone is not highly oncogenic,
it does predispose cells to transformation upon exposure to
mutagens..sup.8,9 In general, PIM-1 up-regulation enhances cell
survival whereas loss of PIM-1 increases apoptotic cell death. The
protective effect of PIM-1 is dependent upon kinase activity as
borne out by experiments using a dominant negative kinase dead
mutant construct..sup.10,11 Occasional exceptions wherein PIM-1
activity increases cell death seem to result from differences in
the cellular backgrounds where PIM-1 was studied. Increased PIM-1
expression also associated with cellular
differentiation.sup.4,12,13 as well as proliferation..sup.14,15
Studies with myocardium demonstrate changes in PIM-1 expression
during postnatal development and aging that form the basis of
studies for this invention.
[0239] PIM-1 expression is stimulated by a variety of hormones,
cytokines and mitogens, many of which are associated with
cardioprotective signaling..sup.16,17 These multiple inductive
stimuli lead to an accepted survival kinase in the myocardium:
Akt/PKB. However, the connection of Akt-mediated effects to PIM-1
mediated signaling has been overlooked. In fact, expression of
PIM-1 is increased by Akt activation.sup.18 and studies using
LY294002 to block PI3-K activity were also inadvertently inhibiting
PIM-1 kinase activity as well..sup.5 Despite apparent parallels
between PIM-1 and Akt, these kinases exhibit distinct effects in
regulation of cell growth and survival..sup.6 PIM-1 shares homology
with two related family members that have largely overlapping
functions named PIM-2 and PIM-3. Parallels of signal transduction
between PIM family members and Akt are a primary focus of ongoing
research in non-myocyte cells.
[0240] Independent aspects of PIM-1 mediated signaling are waiting
to be teased apart from overlaps with Akt using knockout mouse
lines in conjunction with overexpression approaches, thereby
providing new insight regarding regulation of myocardial survival
and proliferation. Such studies with hematopoeitic cells have
revealed that PIM and Akt are critical components of overlapping
but independent signaling pathways responsible for enhancement of
growth and survival..sup.6,7 Mouse lines engineered with deletion
of PIM-1 or triple knockouts deficient for all PIM kinases are
viable without severe phenotypic effects..sup.19 However, we have
found cardiac-specific consequences following ischemia reperfusion
damage in the PIM-1 knockout mouse line . This data validates the
role of PIM-1 in response to and protection from cardiomyopathic
challenge.
[0241] Fundamentals of PIM-1 signal transduction are predominantly
based in studies of hematopoeitic and oncogenic cells where the
kinase was first identified. PIM-1 is a downstream effector of many
cytokines that operate through "Signal Transducers and Activators
of Transcription" known by the STAT acronym. Both STAT3 or STATS
bind directly to the PIM-1 promoter and induce expression..sup.20
PIM-1 expression is inhibited by negative feedback loop regulatory
control of the Jak/STAT pathway through interaction with
Suppressors Of Cytokine Signaling (also known as SOCS)..sup.17
PIM-1 protein stability is also decreased through action of
serine/threonine phosphatase PP2a..sup.21 Pivotal roles of STAT,
SOCS, and PP2a signaling in the myocardium.sup.22-24 implicate
PIM-1 as an attractive candidate effector molecule to mediate
biological effects in cardiomyocytes.
[0242] The list of target molecules for PIM-1 kinase continues to
accumulate new members every year, many of which regulate cell
cycle progression and apoptosis. Regulation of cell cycle
proliferation by PIM-1 in vascular smooth muscle cells confirms a
role of PIM-1 in the cardiovascular system..sup.14 In the context
of this proposal, the capacity of PIM-1 to inactivate pro-apoptotic
Bad protein via phosphorylation and enhance Bcl-2
activity.sup.7,25,26 is reminiscent of prior investigations of
cardiomyocyte survival signaling..sup.27,28 The capacity of PIM-1
to inhibit apoptotic cell death by preserving mitochondrial
integrity is a fundamental hypothesis in this proposal studied in
Specific Aim 4. Removal/recycling of mitochondria and other
intracellular organelles by autophagy is regulated in part by
Akt-dependent signaling (reviewed in reference 29, below).
[0243] Recent advances support a central role for PIM kinases in
proliferative and survival signaling. Cytokine-responsive gp130
signaling cascades lie directly upstream of PIM kinase
activation,.sup.30,31 yet extensive studies in the cardiovascular
system have yet to explore the contribution of PIM to reported
protective effects. Furthermore, intermingling of PIM and
Akt-mediated effects are established.sup.5-7 along with the pivotal
role of Akt in the cardiovascular system, (reviewed in references
32 and 33, below) yet the contribution of PIM kinases in myocardial
signaling remains virtually unknown. Specific Aim 3 is designed to
tease apart the relationship of Akt and PIM in the myocardium.
Results point to PIM as a pivotal regulator of proliferation and
survival in the myocardium.
C. PIM-1 is Expressed in Cardiomyocytes Exposed to Cardioprotective
Stimuli
[0244] PIM-1 is expressed in cardiomyocytes exposed to
cardioprotective stimuli. Unlike Akt, PIM-1 is constitutively
active and regulated by protein production/degradation rates.
Constitutive low level production of PIM-1 is detectable in
cardiomyocytes under basal conditions both in cultured cells as
well as normal myocardium. We cultured neonatal rat cardiomyocytes,
which we treated with IGF-1, PMA, dexamethasone, LIF,
phenyl-ephedrine, endothelin-1, estradiol, and forskolin and then
assayed for PIM-1 protein levels. The first four factors
significantly increased PIM-1 expression, whereas little or no
increase was seen with the others over a 2 hour period.
[0245] FIG. 1 illustrates immunoblots demonstrating that
cardioprotective stimuli induces Pim-1 expression. Cultured
neonatal rat cardiomyocytes were treated with various factors to
increase Pim-1 protein level as indicated above each lane including
IGF-1, PMA, dexamethasone, and forskolin. Induction is evident in
response to IGF-1, PMA and dexamethasone, whereas forskolin has no
discernable effect. Similarly, other stimuli including
phenylephrine, endothelin-1, or estradiol did not markedly increase
Pim-1 protein expression in this time frame of exposure, which was
two hours.
[0246] PIM-1 is induced in cardiomyocytes in response to
cardiomyopathic injury. Low level PIM-1 expression is markedly
increased following cardiomyopathic challenge in hearts of mice
subjected to either infarction by coronary occlusion or pressure
overload resulting from transverse aortic constriction (TAC) at
four days after procedures. In comparison, PIM-1 is concentrated
within the nuclei of selected cardiomyocytes in chronic heart
failure from a genetically engineered mouse model (tropomodulin
overexpressing transgenic.sup.34). In all cases, PIM-1 protein
level is elevated relative to sham-operated control mice. The
elevation of PIM-1 under these circumstances is presumably mediated
by paracrine cytokine signaling within the challenged
myocardium.
[0247] FIG. 2 illustrates confocal micrographs showing that
cardiomyopathic stimuli induce Pim-1 expression in surviving
myocardium. Confocal micrographs show induction of Pim-1 protein
expression relative to sham-operated controls (Sham) in response to
acute myocardial injury induced four days after infarction in the
border zone (MI) or near vasculature after pressure overload after
four days (TAC). Both acute injury models show accumulation of
Pim-1 in perinuclear areas, whereas a genetically engineered
transgenic model of chronic dilated cardiomyopathy shows Pim-1
accumulation within select nuclei (TOT). Sections were labeled with
antibody to Pim-1 (green), phalloidin to decorate actin filaments
(red), and TOPRO dye for nuclei (blue). Widefield view (upper row)
with selected regions shown in higher magnification to reveal
cellular detail (lower row).
[0248] Loss of PIM-1 signaling impairs functional recovery
following ischemia-reperfusion injury. Genetically engineered mouse
lines lacking PIM-1 or a triple knockout lacking PIM-1, 2, and 3
created as described.sup.19 have been established in our colony.
Hearts from these mice were subjected to ex vivo treatment.sup.35
leading to ischemia-reperfusion damage. Functional recovery of the
PIM-knockout lines was significantly impaired relative to age,
strain, and gender-matched control hearts. Hemodynamic recovery of
the triple knockout line was comparable to that of the single
isoform PIM-1 knockout line, indicating that the PIM-1 isoform is
the critical member of the PIM family to mediate protective
signaling in response to ischemia-reperfusion challenge.
[0249] FIG. 3 graphically illustrates data showing that Pim-1
preserves hemodynamic function in ischemia-reperfusion injury.
Hearts harvested from mouse normal (FVB, green or upper line) as
well as genetically engineered lines lacking Pim-1 (PIM-1 ko, pink
or lower line) or Pim-1, 2, and 3 (PIM 1,2,3 ko; blue or middle
line) were subjected to ischemia reperfusion challenge and
hemodynamic recovery of function was assessed as previously
described..sup.32 Pim knockout mouse lines show significant
impairment (p<0.01 for time points beyond one hour of
reperfusion).
[0250] PIM-1 expression is developmentally regulated in postnatal
growth. Elevation of PIM-1 in postnatal development is consistent
with our observation that PIM-1 promotes growth and proliferation
of cardiomyocytes in the postnatal heart. In addition, we have also
observed PIM-1 expression in cardiac progenitor cells of adult
hearts coincident with c-kit and Sca-1 stem cell markers.
Correlation of PIM-1 expression in stem cell populations would be
consistent with observations from hematopoeitic cell biology
demonstrating PIM-1 plays a role in proliferation and
survival..sup.6,19
[0251] FIG. 4 illustrates immunoblots demonstrating Pim-1
expression is highest in postnatal hearts and decreases with age.
Immunoblot showing Pim-1 protein expression at the indicated weeks
after birth: data including samples from less than one week, and 2,
3, 4, 5, 8, 12, 19 and 39 weeks of age. Actin is shown as a loading
control to verify comparable protein sample concentration between
lanes.
[0252] PIM-1 is expressed by recombinant adenoviral vectors.
Overexpression of PIM-1 or dominant-negative PIM-1 lacking kinase
activity has been accomplished by creation of adenoviral vectors.
We have engineered these constructs with GFP fluorescent tags to
track their expression without the need for anti-PIM-1 antibodies,
allowing to directly visualize exogenous protein expression. These
constructs have been valuable for understanding the effects of
PIM-1 accumulation in cardiomyocytes.
[0253] FIG. 5 illustrates immunoblots demonstrating Pim-1
expression in cardiomyocytes from recombinant adenoviral vectors.
Cardiomyocytes were infected (or not infected, noting the
"non-infected" control) with GFP-tagged constructs of Pim-1 in
wild-type (GFP-Pimwt) or kinase-dead (GFP-PimDN) forms (and GFP
only). Apparent mobility of GFP-fusion constructs (.about.65 kDa)
differs from native Pim-1 (approximately 33 kDa). Note induction of
native Pim-1 resulting from overexpression of GFP-PimDN, presumably
as a compensatory mechanism. GAPDH shown to demonstrate comparable
loadings between lysates.
[0254] PIM-1 overexpression protects against apoptotic challenge
with doxorubicin. Overexpression of PIM-1 in cultured
cardiomyocytes inhibits apoptosis resulting from exposure to
doxorubicin as measured by TUNEL labeling. Neonatal rat
cardiomyocyte cultures were infected with recombinant adenoviruses
expressing GFP, PIM-1 wild-type (PIMwt), or PIM-1 dominant negative
(DN) overnight prior to apoptotic stimulation with doxorubicin.
With reference to FIG. 1, non-infected cells (NI) or GFP-expressing
cells show comparable TUNEL labeling following doxorubicin
treatment, whereas PIMwt expressing cells show significant
reductions of TUNEL positive nuclei (p<0.05). Cells expressing
the DN construct show enhanced TUNEL labeling. In contrast, the
mutant kinase-dead PIM-1 construct accumulates to lower protein
levels than PIM-1 wild-type, yet significantly increases apoptosis
compared to GFP-expressing control cells.
[0255] FIGS. 6 and 7 show how Pim-1 inhibits apoptosis in
cardiomyocytes. Neonatal rat cardiomyocyte cultures were infected
with recombinant adenoviruses expressing GFP, Pim-1 wild-type
(Pimwt), or Pim-1 dominant negative (DN) overnight prior to
apoptotic stimulation with doxorubicin. As graphically summarized
in FIG. 6, non-infected cells (NI) or GFP-expressing cells show
comparable TUNEL labeling following doxorubicin treatment, whereas
Pimwt expressing cells show significant reductions of TUNEL
positive nuclei (p<0.05). FIG. 7 illustrates a micrograph
demonstrating that cells expressing the DN construct show enhanced
TUNEL labeling; while FIG. 6 shows quantitative results, the FIG. 7
panels illustrate representative fields of infected cardiomyocytes
showing GFP fluorescence (green) overlay with actin filaments
revealed by phalloidin (red) in GFP only, GFP-Pim-wt and GFP-Pim-DN
samples.
[0256] PIM-1 overexpression promotes anti-apoptotic signaling via
cascades involving Bcl-2 family members as well as Mdm2.
Overexpression of PIM-1 increases accumulation of Bcl-2 and Bcl-XL
family members, both of whom antagonize intrinsic apoptotic
signaling by preserving mitochondrial integrity, as illustrated in
FIG. 8A. Additional signaling to promote survival induced by PIM-1
includes accumulation of Mdm2 and phosphorylation of Bad. Mdm2
antagonizes p53-dependent cell death.sup.36 and Bad phosphorylation
inhibits the pro-apoptotic action of this protein..sup.26 Thus,
PIM-1 impacts upon cell survival by promoting the anti-apoptotic
action of Bcl-2 family members as well as enhancing Mdm2.
[0257] PIM-1 expression is induced by nuclear accumulation of
activated Akt. Induction of PIM-1 promoter activity by Akt kinase
indicates that PIM-1 expression lies downstream of Akt
activation..sup.18 This observation has now been validated in both
transgenic mouse hearts expressing nuclear-targeted Akt (as
illustrated in FIG. 8B) as well as cultured cardiomyocytes infected
with an adenoviral vector expressing nuclear-targeted Akt.
[0258] In summary, FIGS. 8A and 8B illustrate that nuclear
accumulation of Akt induces expression of Pim-1 kinase in the
myocardium: Immunoblot (FIG. 8A) and confocal microscopy (FIG. 8B)
of sections from 6 month old normal (NTG) and transgenic mice
expressing cardiac-specific nuclear-targeted Akt..sup.38 Separated
grayscale images in scans correspond to pim-1, actin, and nuclei
that correspond to the overlay colors of green, red, and blue
respectively.
[0259] Nuclear accumulation of Akt promotes increased PIM-1
expression detectable by both immunofluorescence as well as
immunoblot analyses. The implications of this result are profound
for survival signaling in the myocardium, since inhibition of Akt
activation would also lead to reduction in PIM-1 levels.
Furthermore, pharmacologic treatment with LY294002 that has
traditionally been used for inhibition of Akt also inhibits PIM-1
kinase activity..sup.5 These findings provide strong circumstantial
evidence that the protective effects previously ascribed to Akt
activation may be due, in part, to actions of PIM-1 kinase. Since
the role for PIM-1 in myocardial signaling has been overlooked to
date, important aspects of cardiac Akt biology related to cell
survival and growth need reassessment.
[0260] FIG. 9 illustrates that nuclear accumulation of Akt induces
Pim-1 expression. FIG. 9(A) illustrates a confocal microscopy of
cultured cardiomyocytes infected with adenoviruses expressing
nuclear-targeted .beta.-galactosidase (B-gal), Akt wild-type (Akt
wt), or nuclear targeted Akt (Akt-nuc) detected with myc-tag
antibody (Tag). Adenovirally encoded proteins are green in overlay.
Nuclear-targeted Akt promotes accumulation of Pim-1 in the nucleus
(shown blue in overlay). Phalloidin shows actin filaments (red in
overlay). FIG. 9(B) illustrates an immunoblot blot showing
increased Pim-1 expression in cardiomyocyte cells infected with
adenovirus encoding nuclear-targeted Akt (Akt-nuc). GAPDH is shown
to show comparable loading between samples.
[0261] Loss of PIM-1 activity results in compensatory elevation of
Akt. Previously documented overlaps between PIM-1 and Akt in terms
of functional effects and crosstalk warrant further investigation
to determine the role of PIM-1 kinase in myocardial biology.
Experiments using adenoviruses expressing PIM-1 in either wild-type
or dominant negative forms demonstrate that loss of PIM-1 signaling
leads to elevation of Akt protein expression and activity.
Similarly, immunoblot evaluation of the PIM-1 knockout line shows
increased levels of phospho-Akt.sup.473 as well as total Akt
protein (data not shown). The basis for this induction of Akt
expression may lie with compensatory signaling to counterbalance
loss of downstream PIM-1 activity. Since dominant negative PIM-1 is
capable of enhancing cell death, as illustrated in FIG. 10, our
data are consistent with the conclusion that PIM-1 mediates certain
facet(s) of survival signaling in the Akt cascade.
[0262] In summary, FIG. 10 illustrates an immunoblot blot showing
expression of dominant negative Pim-1 prompts Akt accumulation in
cardiomyocytes. Immunoblot showing infection of neonatal rat
cardiomyocytes with adenoviruses expressing Pim-1 in either wild
type (wt) or dominant-negative (DN) forms. Levels of
phospho-Akt.sup.473 as well as total Akt protein levels are
elevated in lysates prepared from the cells expressing DN Pim-1.
Two separate sets of experimental results are shown with controls
of uninfected cells (NI), GFP-expressing cells (GFP), and GAPDH
loading controls to standardize for variation in protein loading
between samples.
[0263] FIG. 11 illustrates data characterizing founder lines and
protein expression in Pim-1 transgenic mice. PCR of genomic DNA
samples (FIG. 11 left) and immunoblot of cardiac lysates (FIG. 11
right). Confirmation of vertical transmission of the
cardiac-specific Pim-1 wild-type (wt) as well as dominant-negative
(DN) transgene is confirmed in samples from the F1 generation. An
immunoblot of cardiac lysates from the Pim-1 wt transgenic line
shows accumulation of Pim-1 and GFP with coincident mobility at
approximately 60 kDa (resulting from Pim-1 with a GFP-tag)
indicative of substantial transgene expression in these 6 week old
mouse hearts. GAPDH shown to indicate comparable loading of samples
between lanes.
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EXAMPLE 3
Pim-1 Kinase Antagonizes Aspects of Myocardial Hypertrophy and
Compensation to Pathological Pressure Overload
[0265] The following data demonstrate that Pim-1 kinase exerts
potent cardioprotective effects in the myocardium downstream of
AKT, and that PIM-1 plays a role in cardiac hypertrophy.
Cardiac-specific expression of Pim-1 (Pim-wt) or the
dominant-negative mutant of Pim-1 (Pim-DN) in transgenic mice
together with adenoviral-mediated overexpression of these Pim-1
constructs was used to delineate the role of Pim-1 in hypertrophy.
Transgenic overexpression of Pim-1 protects mice from pressure
overload induced hypertrophy relative to wild-type controls as
evidenced by improved hemodynamic function, decreased apoptosis,
increases in anti-hypertrophic proteins, smaller myocyte size, and
inhibition of hypertrophic signaling after challenge. Similarly,
Pim-1 overexpression in neonatal rat cardiomyocyte cultures
inhibits hypertrophy induced by endothelin-1. On the cellular
level, hearts of Pim-wt mice show enhanced incorporation of BrdU
into myocytes as well as a hypercellular phenotype compared to
wild-type controls after hypertrophic challenge. In comparison,
transgenic overexpression of Pim-DN leads to dilated cardiomyopathy
characterized by increased apoptosis, fibrosis, and severely
depressed cardiac function. Furthermore, overexpression of Pim-DN
leads to reduced contractility as evidenced by reduced Ca.sup.2+
transient amplitude and decreased percent cell shortening in
isolated myocytes. These data support a pivotal role for Pim-1 in
modulation of hypertrophy by impacting responses on molecular,
cellular, and organ levels.
[0266] Cardiac-Specific Pim-1 Transgenesis
[0267] The wildtype form of human Pim-1 (Pim-wt) and a kinase dead
mutant that functions as a dominant negative protein (Pim-DN) (16)
were fused to GFP under control of the cardiac specific a-myosin
heavy chain promoter. PCR of mouse lines created with these
constructs show incorporation of the transgenes into the genome.
Immunoblot of whole heart lysates from transgenic samples revealed
a 64-kDa GFP-Pim-1 fusion protein that is recognized by both Pim-1
and GFP antibodies. Bona fide inhibitory function of the Pim-DN
construct was validated using the ability of Pim-1 to activate
GATA-1 transcription (Magnuson, unpublished data). Pim-wt
phosphorylates the transcription factor GATA-1 and induces GATA-1
luciferase reporter expression in C2C12 myoblasts, with increasing
titration of Pim-DN inhibiting GATA-1 activity. Based on previous
studies that showed Pim-1 phosphorylates p21 (29), in-vitro kinase
assays confirmed activity of our Pim-wt construct using whole heart
lysates that were prepared from GFP-Pim-1-wt, GFP-Pim-1-DN
transgenic mice and non-transgenic (NTG) mice. GFP-Pim-1 proteins
(wt or KD) were immunoprecipitated from whole heart lysates and
incubated in the presence of [.gamma.-32P] ATP with GST-p21 as
substrates. Samples were resolved on SDS-PAGE, and .sup.32P-labeled
proteins were detected by autoradiography. Pim-wt overexpression
phosphorylates p21 while this activity was abolished in the Pim-DN
construct.
[0268] Pim-1 Inactivation Increases Cardiomyocyte Apoptosis and
Fibrosis
[0269] Hearts from mice created with genetic deletion of Pim-1
(Pim-1 KO) exhibit increased apoptosis in myocytes relative to NTG
(non-transgenic) controls but show no evidence of overt
cardiomyopathic remodeling (12). In comparison, Pim-DN
overexpressing mice suffer from cardiomyopathy characterized by
progressive wall thinning beginning at 3-4 months of age. FIG. 12
compares the characteristics of the wild-type NTG mice with the
mice lacking active PIM-1. In FIG. 12a, echocardiographic
measurement of posterior wall dimension over time shows the PIM-DN
mice have a progressive thinning. This is also seen in the anterior
wall dimension in FIG. 12b. As shown in FIG. 12c, the heart:body
weight ratio at 10 and 22 weeks after birth is also significantly
increased in the PIM-DN mice. Since Pim-DN overexpression induces
cardiomyocyte apoptosis in vitro (12), assessment of apoptotic
myocytes in the myocardium of Pim-DN animals was performed by TUNEL
staining. Pim-DN animals exhibit a two-fold increase in apoptotic
cardiomyocytes per mm.sup.2 relative to age-matched controls
(1.2/mm.sup.2 and 2.4/mm.sup.2 respectively, FIG. 12d**p<0.01)
resulting in increased fibrosis and collagen deposits in the left
ventricle. In addition, the amount of necrosis was quantified
(**p<01) and found to be significantly increased at basal levels
in Pim-DN animals.
[0270] In summary, FIG. 12 graphically illustrates data showing
that inactivation of Pim-1 in the myocardium increases apoptosis
and fibrosis. FIG. 12a and FIG. 12b graphically illustrate
echocardiographic measurement of posterior (12a) and anterior (12b)
wall dimension (PWD and AWD respectively) in NTG (n=5) and Pim-DN
(n=7) animals at two week intervals (*p<0.05, **p<0.01). FIG.
12c graphically illustrates heart weight to body weight ratios in
NTG and Pim-DN animals at 10 and 22 weeks of age (n=6,
**p<0.01). FIG. 12d graphically illustrates histogram data
representing counts of TUNEL positive myocytes per mm.sup.2 in
17-22 week old NTG and Pim-DN transgenics (n=3, **p<0.01).
[0271] Pim-DN Hearts Exhibit Depressed Cardiac Function
[0272] Hearts of Pim-DN mice show progressive dilation from 17
weeks of age (*p<0.05) with attendant depression of fractional
shortening and ejection fraction (36.6% and 74.2% respectively) by
27 weeks of age (*p<0.05, **p<0.01) by echocardiographic
analyses. Morphometric analysis performed on both NTG and Pim-DN
hearts additionally confirmed that Pim-DN hearts were significantly
dilated. In vivo hemodynamic assessments verified impaired
hemodynamics with diminished .+-.dP/dt, increased left-ventricular
end diastolic pressure (LVEDP), and decreased left-ventricular
developed pressure (LVDP). Mechanistically, Pim-DN myocytes
displayed reduced Ca.sup.2+ transient amplitude coupled with
decreased percent cell shortening in respect to NTG myocytes.
Additionally, the time constant (.tau.) of the Ca.sup.2+ transient
decay was larger in Pim-DN myocytes. These results indicate that
depressed contractile function of Pim-DN myocytes is mediated, at
least in part, by a decline in Ca.sup.2+ release from the
sarcoplasmic reticulum together with a slower reuptake. Thus
inactivation of Pim-1 by Pim-DN in the myocardium has a negative
effect on cardiac function.
[0273] Overexpression of Pim-1 Inhibits Hypertrophy In Vitro
[0274] Induction of Pim-1 in the damaged myocardium is thought to
be a protective survival response (12) occurring in cardiomyocytes
such as those in the infarct border zone where Pim-1 colocalizes to
cells expressing atrial natriuretic peptide. ANP is both
anti-hypertrophic and cardioprotective (24), so the coincidence of
these proteins prompted assessment of the role that Pim-1
accumulation plays in mitigation of hypertrophic signaling.
[0275] The impact of Pim-wt overexpression upon cardiomyocyte
hypertrophy was initially examined using neonatal rat
cardiomyocytes (NRCMs) infected with adenoviruses encoding
EGFP-Pim-wt or EGFP protein followed by stimulation with
endothelin-1 (ET-1) for 24 hours. FIG. 13 shows individual cell
surface area measurements from uninfected control, EGFP, and Pim-wt
infected neonatal rat cardiomyocyte cultures treated and untreated
with endothelin-1. As illustrated in FIG. 13, Pim-wt overexpression
inhibits ET-1 induced hypertrophy (*p<0.05, **p<0.01) as
assessed by cell surface area measurements relative to the increase
in cell size seen in control and EGFP infected myocytes treated
with ET-1. Molecular profiling of the hypertrophic signature of
untreated cultures shows that Pim-wt expression decreases mRNA
levels for atrial natriuretic peptide (ANP) by 60.6% and B-type
natriuretic peptide (BNP) by 39.8% while increasing a-skeletal
actin levels 89% compared to EGFP infected controls. However, upon
treatment with ET-1, Pim-wt cultures exhibit a 2.5-fold increase in
ANP levels and 10.2-fold decrease in .beta.-myosin heavy chain
levels versus ET-1 treated EGFP controls. Unfortunately, cultured
cardiomyocytes overexpressing Pim-DN protein show diminished
viability after the necessary time course to infect and treat with
ET-1 as evidenced by high levels of TUNEL positive cells. As
cardiomyocytes overexpressing Pim-DN protein begin to round up and
detach from the plate, their morphology is drastically changed,
thereby preventing an accurate assessment of cell size (12).
However, the effect of Pim-DN on hypertrophy can be seen using the
specific Pim-1 activity inhibitor quercetagetin. Pim-1 expressing
NRCM cultures were treated for 1 hour with or without 10 nM
quercetagetin prior to 48 hour incubation with ET-1 and cell size
assessment. Cells treated with quercetagetin and ET-1 had
significantly larger surface compared to ET-1 stimulated cells
where Pim-1 activity was not blunted by inhibitor. Collectively
these results support an anti-hypertrophic role when Pim-1 is
overexpressed, albeit at levels well above normal physiological
induction in this cell culture system.
[0276] In summary, FIG. 13 graphically illustrates data showing
that Pim-1 overexpression protects cardiomyocytes from endothelin-1
induced hypertrophy. Individual cell surface area measurements from
uninfected control, EGFP, and Pim-wt infected neonatal rat
cardiomyocyte cultures treated and untreated with endothelin-1
(ET-1) (n=4, *p<0.05, **p<0.01).
[0277] Pim-1 Overexpression Inhibits Remodeling Induced by Pressure
Overload Hypertrophy
[0278] Consequences of Pim-1 overexpression upon hypertrophy in
vivo was assessed with Pim-wt mice subjected to trans-aortic
constriction (TAC) to induce pressure overload relative to age and
gender matched NTG controls. With reference to FIGS. 14a-f, line
graphs were generated representing weekly echocardiographic
assessment of NTG and Pim-wt sham and TAC banded hearts for
anterior wall dimension (AWD 14d, 14a), posterior wall dimension
(PWD 14d, 14b), end diastolic dimension (EDD, c), end-systolic
dimension (ESD, 14d), percent fractional shortening (FS, 14e), and
ejection fraction (EF, 14f) (NTG sham n=6, Pim-wt sham n=6, NTG TAC
n=9, Pim-wt TAC n=9; *p<0.05, **p<0.01) (FIG. 14a-14b,
*p<0.05, **p<0.01).
[0279] Results show that TAC of control NTG hearts prompts
remodeling at two weeks after challenge evidenced by anterior and
posterior wall thickening. In comparison, Pim-wt animals do not
show significant increases in wall thickness for up to 14 weeks
after challenge (FIG. 14a-14b). Similarly, NTG controls show left
ventricular chamber enlargement measured by end diastolic diameter
(EDD) within 8 weeks after banding, and end systolic diameter (ESD)
increases significantly within 4 weeks. Neither EDD nor ESD
parameters show significant changes in Pim-wt transgenics
throughout the same time period (FIGS. 14c-14d, **p<0.01 vs.
sham, $p<0.01 vs. Pim-wt TAC). Furthermore, NTG controls show
marked decreases in both fractional shortening (FS) and ejection
fraction after challenge, while myocardial function is maintained
in Pim-wt hearts (FIGS. 14e-14f; **p<0.01 vs. Pim-wt TAC).
Interestingly, although decreases in cardiac function are seen in
NTG animals, Pim-1 protein is modestly elevated in response to
pressure overload during early hypertrophy and progression to heart
failure. Endogenous levels of Pim-1 expression increase early
during adaptive hypertrophy and decline shortly thereafter, while
during late -phase hypertrophy (9 weeks post-TAC), Pim-1 appears
localized to nuclei within vasculature. These observations support
the conclusion that Pim-1 is induced in response to stress.
[0280] In summary, FIG. 14 graphically illustrates data showing
Pim-wt transgenic animals are resistant to pressure overload
induced hypertrophy. FIG. 14a-f) Line graphs representing weekly
echocardiographic assessment of NTG and Pim-wt sham and TAC banded
hearts for anterior wall dimension (AWDd, a), posterior wall
dimension (PWDd, b), end diastolic dimension (EDD, c), end-systolic
dimension (ESD, d), percent fractional shortening (FS, e), and
ejection fraction (EF, f) (NTG sham n=6, Pim-wt sham n=6, NTG TAC
n=9, Pim-wt TAC n=9; *p<0.05, **p<0.01).
[0281] Pim-wt Hearts are Resistant to TAC Induced Hypertrophy
[0282] NTG mice exhibit significant increases in heart size and
succumb at a significantly faster rate compared to Pim-wt
transgenic mice following TAC challenge. Molecular mRNA markers of
hypertrophy including ANP, BNP, a-skeletal actin (.alpha.SKA),
.beta.-myosin heavy chain (.beta.-MHC) and c-fos are significantly
increased in NTG TAC challenged hearts compared to shams. In
comparison, molecular hypertrophic markers are not significantly
increased in hearts of Pim-wt mice subjected to TAC challenge,
although Pim-wt hearts do express more c-fos mRNA under basal
conditions. Quantitation of apoptotic myocytes by TUNEL labeling in
sections reveals a 3.72-fold increase in NTG TAC-challenged hearts
compared to shams (3.2/mm.sup.2 and 0.86/mm.sup.2 respectively),
whereas Pim-wt animals exhibit no significant increase in TUNEL
positive cells (1.31/mm.sup.2 versus 1.05/mm.sup.2). Consistent
with improved myocardial viability, Pim-wt TAC-challenged hearts
show decreased peri-vascular fibrosis as well as decreased necrosis
relative to NTG TAC-challenged counterparts. Additionally, Pim-wt
TAC banded hearts have significantly increased levels of
anti-apoptotic proteins including Bcl-x1, Bcl-2, and increased
phosphorylation of BAD relative to NTG counterparts. These data
support the idea that protection afforded by Pim-1 overexpression
is due in part to increased survival signaling.
[0283] Pim-wt Hearts Exhibit Increased Contractile Function in
Response to TAC Banding
[0284] Decreased fibrosis is present in Pim-wt hearts after TAC
banding, suggesting Pim-1 overexpression preserves contractile
function. Actions of Pim-1 overexpression upon cardiac
contractility were examined using Pim-wt and NTG controls assessed
by in vivo hemodynamic measurements conducted at 4 weeks and 10
weeks after TAC challenge. FIGS. 15a-c illustrate an in vivo
hemodynamic assessment of NTG and Pim-wt hearts 4 and 10 weeks
(black and gray bars respectively) after sham or TAC operation (14
and 20 weeks of age respectively). FIG. 15a shows .+-.dP/dt
measurements; FIG. 15b shows left ventricular developed pressure
(LVDP), and FIG. 15c illustrates left ventricular end-diastolic
pressure (LVEDP). While contractile function is depressed in NTG
TAC-challenged hearts at both time points, Pim-wt hearts possess
better function after TAC challenge with slight decreases in +dP/dt
and no significant change in -dP/dt compared to sham operated NTG
controls . Comparison of 4-week and 10-week dP/dt assessments show
significant decreases in function for both NTG and Pim-wt TAC
challenged hearts, although performance of Pim-wt TAC-challenged
hearts is relatively improved. Measurements reveal increases in
left ventricular developed pressure and end-diastolic pressure in
NTG hearts 4 and 10 weeks after TAC, but Pim-wt hearts show
relative preservation of LVDP (FIG. 15b, Pim-wt 19.75% increase,
**p<0.01) and no change in LVEDP (FIG. 15c **p<0.01,
$$p<0.01 vs. NTG TAC). Hemodynamic function reflected in
.+-.dP/dt and LVDP is improved in Pim-wt hearts compared to NTG at
4 and 10 week time points (FIGS. 15a-15b, .psi.p<0.05,
.psi.p<0.01).
[0285] In summary, FIG. 15 graphically illustrates data showing
that Pim-1 enhances cardiac function. FIG. 15a-c) In vivo
hemodynamic assessment of NTG and Pim-wt hearts 4 and 10 weeks
(black and gray bars respectively) after sham or TAC operation (14
and 20 weeks of age respectively). .+-.dP/dt measurements (FIG.
15a), left ventricular developed pressure (LVDP, b), left
ventricular end-diastolic pressure (LVEDP, FIG. 15c). For 4-week
animals NTG sham n=4, NTG TAC n=3, Pim-wt sham n=4, Pim-wt TAC n=4.
For 10-week animals NTG sham n=5, NTG TAC n=10, Pim-wt sham n=14,
Pim-wt TAC n=7 (*p<0.05, **p<0.01 vs. sham, ##p<0.01 vs.
4-week TAC, $p<0.05, $$p<0.01 vs. NTG TAC, .psi.p<0.05,
.psi..psi.p<0.01 vs. NTG sham).
[0286] The mechanistic basis for preservation of contractile
function in Pim-wt hearts may rest with the cellular response in
TAC challenged animals. NTG and Pim-wt groups injected with BrdU
for 10 days were used to assess stimulation of DNA synthesis and
potential cellular proliferation after TAC challenge. Pim-wt hearts
possess 67% more BrdU+ myocytes relative to NTG controls after TAC
challenge. The majority of BrdU+ cells in Pim-wt hearts post-TAC
are diploid, supporting the premise that increases in BrdU+ cells
stemmed from new myocyte formation and not enhanced DNA synthesis
in pre-existing cells. Consistent with improved contractility, we
now show that in addition to increased SERCA2a levels (12), Pim-wt
hearts also show increased levels of phosphorylated phospholamban
(PLB) while Pim-DN animals show significant decreases in
phospho-PLB compared to NTG control animals. These results support
the conclusion that in the face of decreased cardiac function,
overexpression of Pim-1 allows the heart to maintain function
through increased contractility through elevation of SERCA2a and
phosphorylated PLB.
[0287] Pim-1 Increases Myocardial Cellularity
[0288] The volume and cellularity of myocytes resulting from
myocardial Pim-1 overexpression was assessed by quantitation of
myocyte volume distribution. Results show Pim-wt hearts possess an
increased percentage of small myocytes relative to NTG controls
that is also reflected in decreased average myocyte size in these
hearts, resulting in a hypercellular phenotype of approximately 33%
more myocytes in Pim-wt compared to NTG. Additionally, isolated
Pim-DN myocytes were 11% larger than NTG myocytes, indicating an
inverse effect wherein impaired Pim-1 activity prompts formation of
larger myocytes in the transgenic heart.
[0289] Inhibition of hypertrophy in vivo and in vitro indicates
Pim-1 contributes to Akt-mediated blunting of hypertrophic
remodeling. Pim-1 is only upregulated in localized regions close to
acute injury or damage and is not increased throughout the
myocardium until initiation of transit to end stage failure. Thus,
Pim-1 likely serves as a survival and protective response to blunt
maladaptive hypertrophic remodeling in early phases of reactive
signaling. In comparison, Pim-1 elevation occurring in late stage
decompensation probably represents a terminal effort to preserve
function, although beneficial effects can be overridden by the
sequelae of end stage failure. The differential expression of
endogenous Pim-1 during transition from adaptive to maladaptive
hypertrophy possibly represents a mechanism by which Pim-1 exerts
cardioprotection.
[0290] Nuclear AKT delayed but did not overcome compensatory
remodeling after TAC challenge (9), but Pim-wt transgenic hearts
exhibit persistent blunting of myocardial hypertrophy (see e.g.,
FIG. 14) without increases in apoptosis, changes in hypertrophic
signaling markers or deterioration of function (see e.g., FIG. 15).
In addition, Pim-wt transgenic hearts perform significantly better
functionally than NTG counterparts at 14 and 20 weeks of age (see
e.g., FIG. 15). A potential basis for this remarkable resiliency to
pressure overload is in part related to Pim-1-mediated induction of
sarco/endoplasmic reticulum Ca.sup.+2ATP-ase 2a (SERCA2a) (12) and
phospholamban (PLB) expression. Overexpression of Pim-DN in the
myocardium increases myocyte apoptosis (see e.g., FIG. 12d)
associated with dilated cardiomyopathy, whereas genetic deletion of
Pim-1 also results in increased apoptosis although dilated
cardiomyopathy is not observed (12). The cardiomyopathic
consequences of Pim-DN overexpression suggest this kinase is
critical to cardiomyocyte viability, as compensatory up-regulation
of Pim-2 occurs in the Pim-1 knockout line could help account for
lack of cardiomyopathic changes (12). These findings are also
consistent with our previous study that shows the dominant negative
form of Pim-1 induces PARP and caspase 3 cleavage, increasing
cardiomyocyte apoptosis in vitro (12). Furthermore for the effect
on hypertrophy, Pim-DN seem to be able to mount a hypertrophic
response as evidenced by increased anterior wall thickness at one
week after TAC challenge (not shown).
[0291] Overexpression of Pim-1 in the pathologically challenged
myocardium results in numerous salutary effects including decreased
apoptosis, increased expression of anti-apoptotic proteins, and
decreased fibrosis and necrosis. Pim-1 also increases the
percentage of small myocytes and an overall increase in the number
of myocytes constituting the myocardium. Consequently, PIM-1
overexpression provides an increased capacity to withstand TAC
challenge by virtue of increased cell numbers of small cells and
decreased cell death.
[0292] Genetic Ablation of Pim-1 increases infarction injury.
Protective effects of Pim-1 were assessed following MI in Pim-KO
animals. Left ventricular free wall infarct size is increased 22.7%
in Pim-KO hearts compared to wild type controls. (FIG. 5a). Pim-KO
mice possess a minor but significant increase in TUNEL positive
myocytes in the left ventricle relative to wild type controls
(Table S2, $p<0.01), and this differential is exacerbated
following MI up to a 4.0 fold increase in TUNEL positive myocytes
relative to wild-type samples (FIG. 5b **p<0.01). Hemodynamic
performance is comparable between Pim-KO and wild type controls
under normal conditions (FIG. S5), but developed ventricular
pressure in Pim-KO mice is depressed and end diastolic pressure is
increased with respect to wild type following infarction (FIG. 5c).
Further, diastolic wall stress is significantly increased in both
left ventricular free wall and septum after infarction in Pim-KO
hearts (FIG. S5, **p<0.01). Pim-KO mice were noted to possess
decreased lymphocyte proliferation and hematopoetic cell
differentiation.sup.13-15 that could possibly decrease inflammatory
responses following MI, but no significant differences were found
in circulating c-kit+cell number after MI or c-kit+/sca-1+ bone
marrow cell number pre or post-MI (FIG. S5). Likewise, inflammatory
cell recruitment following MI as indicated by CD45 staining is
comparable between Pim-KO hearts versus controls (FIG. S5).
[0293] Pim-KO Hearts Exhibit Altered Protective Signaling.
[0294] Pim-1 may be a relatively promiscuous kinase based upon
minimal target substrate recognition sequence requirements' and
capacity for autophosphorylation.sup.17, so molecular mechanisms
responsible for Pim-1-mediated cardioprotection were examined.
[0295] Pim-1-KO heart samples possess increases in
phospho-AKT.sup.T308 (90.72%), phospho-AKT.sup.S473 (2.76-fold),
total AKT (2.10-fold), phospho-STAT3.sup.Y705 (2.61-fold), total
STAT3 (68.6%) and Pim-2 (4.6-fold) relative to wild type samples.
However, no increases were observed for bcl-2, bcl-XL,
phospho-BAD.sup.S112, or Pim-3 expression compared to wild type
controls (FIG. 16d). These survival-signaling molecules were also
examined seven days after MI, when Pim-KO mice exhibit a 2.57-fold
increase in Pim-3 expression, but decreases in bcl-X.sub.L
(2.1-fold), phospho-BAD.sup.S112 (75.9%), phospho-AKT.sup.T308
(92.55%), phospho-AKT.sup.S473 (2.24-fold), total AKT (73.66%),
phospho-STAT3.sup.Y705 (2.72-fold), total STAT3 (2.0-fold), with no
significant changes in bcl-2 or Pim-2 expression compared to
sham-operated controls (FIG. 5e). In comparison, wild type heart
samples show significant increases in bcl-XL (57.0%),
phospho-BAD.sup.S112 (64.58%), phospho-AKT.sup.T308 (98.32%),
phospho-AKT.sup.S473 (2.81-fold), total AKT (2.26-fold),
phospho-STAT3.sup.Y705 (3.43-fold) total STAT3 (2.02-fold), with no
change in Pim-2 or Pim-3 expression compared to sham (FIG. 16e).
Thus, Pim-KO hearts exhibit significant increases in Pim-2 and
Pim-3 compared to NTG hearts post-MI, but profound decreases in the
other survival signaling molecules are observed (FIG. 16e).
[0296] In summary, FIG. 16 graphically illustrates data
demonstrating that Pim-1 protects against infarction injury. FIG.
16a graphically illustrates a histogram representing infarct size 7
days post-MI as a percent of left-ventricular free wall in Pim-KO
hearts (*p<0.05 v NTG MI). FIG. 16b graphically illustrates data
showing the number of TUNEL positive myocytes per mm.sup.2 7 days
post-MI in Pim-KO hearts (**p<0.01). FIG. 16c graphically
illustrates in vivo hemodynamic measurements of NTG and Pim-KO mice
5 days following MI (*p<0.05). Left ventricular developed
pressure (LVDP), left ventricular end-diastolic pressure (LVEDP),
and .+-.change in pressure over change in time. FIG. 16e
graphically illustrates Immunoblot quantitation of survival protein
levels 7 days post-infarction in Pim-KO and NTG control hearts
(*p<0.05 vs. sham, #p<0.01 vs. sham, $p<0.01 vs. NTG MI).
FIG. 16f graphically illustrates Infarct size measurements 10 days
post-infarction (n=3, **p<0.01). FIG. 16g graphically
illustrates the number of TUNEL-labeled CM/m2 in LV 10 days after
MI.
Materials and Methods
[0297] Generation of Transgenic Animals and Animal Use
[0298] Pim-wt and Pim-DN cDNA fragments (16) were subcloned into
the .alpha.-MHC plasmid for transgenesis. Prior publications
describe methods for TAC banding and echo (9), as well as HW:BW
ratio determination and hemodynamics (12). Further details provided
in the online supplement. All animal studies were approved by the
Institutional Animal Use and Care Committee.
[0299] Confocal Microscopy, Immunoblotting and Assays
[0300] GFP-Pim-1 proteins immunoprecipitated from heart lysates
were used in an in vitro kinase assay with GST-p21 as substrate.
For luciferase assays, C2C12 cells transfected with indicated
plasmids and pGATA-Luc reporter construct were analyzed for
GATA-dependent luciferase activity. Methods for immunofluorescence
microscopy were done as described in reference 26, listed below,
immunoblotting were done as described in reference 24, listed
below, quantitative RT-PCR and TUNEL ("terminal deoxynucleotidyl
transferase-mediated dUTP-biotin nick end labeling") staining were
done as described in reference 9, listed below.
[0301] In Vitro Cell Culture and Analyses
[0302] Neonatal rat cardiomyocyte cultures were prepared as
described previously (10). Adult myocyte isolation, volume
calculations, cell shortening and Ca.sup.2+ transient experiments
performed as previously described in references 12, 22, 25, listed
below.
FIGURE LEGENDS
References for Example 3
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[0306] 4. Kumar, D., Lou, H. & Singal, P. K. (2002) Oxidative
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EXAMPLE 4
Pim-1 Engineered Cardiac Stem (Progenitor) Cells
[0333] Pim-1 Increases the Proliferation of Cardiac Progenitor
Cells
[0334] To evaluate the growth rate of CGW-Pim-wt CPCs (cardiac
progenitor cells), the number of viable cells was determined by
trypan blue exclusion over a six day time course (FIG. 17A). At day
six CGW-Pim-wt CPCs continued to expand significantly over that of
CGW (r.05) and non-treated (p<0.001) CPCs. CGW-Pim-wt CPCs also
exhibited an increased metabolic rate over CGW CPCs at one
(p<0.001) and three days (p<0.001), as determined by MTT
assay (FIG. 17B). A specific Pim-1 activity inhibitor,
Quercetagentin, was used to confirm that the growth advantage
acquired by CGW-Pim-wt CPCs was due to overexpression of Pim-1.
When added to the culture media, the Pim-1 inhibitor significantly
decreased the growth rate of CGW-Pim-wt CPCs at day two and three
(p<0.001) compared to untreated CGW-Pim-wt CPCs (FIG. 17C).
[0335] In summary, FIG. 17 illustrates data showing increased
proliferative rate of Pim-1 engineered CSCs: FIG. 17A illustrates a
cell growth assessment using trypan blue assay of control, CGW, and
CGW-Pim-wt transduced CPCs; FIG. 17B illustrates an MTT assay on
control, CGW, CGW-Pim-wt transduced CPCs (mean.+-.SEM, n=3); FIG.
17C illustrates the proliferation rate of Pim-1 expressing CPC's
treated with or without 10 uM of Quercetagentin, a specific Pim-1
activity inhibitor (mean.+-.SEM, n=3) *p<0.05, **p<0.01,
***p<0.001.
[0336] Pim-1 Overexpressing CPCs Improve Cardiac Function
Post-Myocardial Infarction
[0337] Previous studies have shown that Pim-1 transgenic mice
elicit a significant resiliency to pathological challenge. To test
whether Pim-1 modified CPCs would also confer substantial
resistance to infarction damage, twelve week old female FVB mice
were given a myocardial infarction and intramyocardially injected
with CGW or CGW-Pim-wt CPCs surrounding the border zone
(n=15-20/group). Echocardiography measurement at two weeks showed
mice that received CGW-Pim-wt CPCs had a thicker anterior wall
dimension (AWD) compared to that of saline (p<0.001) and CGW CPC
(p<0.01) injected mice (FIG. 18A). In order to determine if
intramyocardial injection of Pim-1 modified CPCs provides long term
increased functional improvement after pathological challenge; mice
were followed for 12 weeks post infarction. At 6 weeks post
infarction mice that received CGW-Pim-wt CPCs maintained EF and FS
(FIG. 18B, C), and were statistically improved over CGW CPC
injected mice. By 7 weeks, CGW CPCs failed to maintain cardiac
function (FS and EF) and were not statistically different than
those animals that received saline injections. At 12 weeks,
echocardiography again indicated that CGW-Pim-wt CPC injected
animals continued to maintain EF and FS, whereby animals that
received control CPCs had a 2-fold and 1.6-fold decrease,
respectively (FIG. 18B, C). Hemodynamic measurements of DP (FIG.
18D), Ped (FIG. 18E), and changes in .+-.dp/dt (FIG. 18F),
confirmed significant enhanced cardiac function in CGW-Pim-wt CPC
injected mice over CGW CPC and saline injected animals.
[0338] In summary, FIG. 18 graphically illustrates data showing
that intra-myocardial injection of Pim-1 expressing CPCs improves
cardiac function. FIG. 18A-C graphically illustrates
electrocardiographic assessment of AWD (FIG. 18A), EF (FIG. 18B),
and FS (FIG. 18C), in sham (.box-solid.), PBS injected
(.circle-solid.), CGW (.tangle-solidup.), and CGW-Pim-WT
(.diamond-solid.) cardiac progenitor cells 12 weeks post CPC
transplantation. Echocardiography measurements represent n.gtoreq.9
animals for each group. FIG. 18 D-F graphically illustrates in vivo
hemodynamic measurements of left ventricular developed pressure
(LVDP) (18D), left ventricular end diastolic pressure (LVEDP)
(18E), and dP/dT maximum and minimum (18F) were used to assess
cardiac function 12 weeks post-intramyocardial injection of PBS,
eGFP, and Pim-1 expressing CPCs (n.gtoreq.5). ANOVA statistical
tests were run for echocardiography and in-vivo hemodynamic
measurements, using Tukey's post-hoc test. Results are represented
as mean.+-.SEM.
[0339] Injection of Pim-1 Modified CPCs Results in a Reduction of
Infarct Size
[0340] Quantitation of tropomyosin over left ventricular free wall
area (LVFW) showed mice injected with Pim-1 modified CPCs had a
significant 2-fold decrease in infarct area (p=0.02) (FIG. 3B).
FIG. 19 graphically illustrates data showing that CGW-Pim-wt CPCs
form myocytes and vasculature in infarcted heart tissue reducing
infarction area; and shows a quantitation of infarction area 12
weeks post CPC injection. Results are represented as mean.+-.SEM,
n=3 animals, *p<0.02.
[0341] Long Term Cardiac Functional Improvement is Only Afforded by
Pim-1 Modified CPCs
[0342] In an effort to extend our previous studies we repeated our
initial experiments and monitored injection of PBS, CGW, and
CGW-Pim-wt CPC injected mice over 32 weeks by echocardiography and
hemodynamic assessment. At 3 days all groups of mice had decreased
FS (FIG. 20A) and EF (FIG. 20B), and were not statistically
different from saline controls. As was previously seen, mice that
received CGW CPCs had an initial early improvement at one week with
onset of cardiac failure at six weeks, becoming statistically
insignificant to saline controls by eight weeks. However, mice that
received Pim-1 modified CPCs had an increase in FS and EF at 1 week
that was maintained through 32 weeks (FIG. 20A-B).
[0343] In summary, FIGS. 20A-B graphically illustrates that long
term cardiac functional recovery is afforded by CGW-Pim-wt
expressing CPCs 32 weeks after intra-myocardial injection:
illustrates electrocardiographic assessment of FS (FIG. 20A), EF
(FIG. 20B), in sham (.box-solid.), PBS injected (.circle-solid.),
CGW (.tangle-solidup.), and CGW-Pim-WT (.diamond-solid.) cardiac
progenitor cells 32 weeks post CPC transplantation.
Echocardiography measurements represent an n.gtoreq.7 animals for
each group.
[0344] Exemplary Bicistronic Vectors of the Invention
[0345] Vectors are bicistronic whereby the MND promoter drives
Pim-1 expression and the reporter, enhanced green florescent
protein (eGFP), is driven off a viral internal ribosomal entry site
(vIRES). All constructs are third generation self-inactivating
(SIN) lentiviral vectors and incorporate several elements to ensure
long-term expression of the transgene. The MND (MND,
myeloproliferative sarcoma virus LTR-negative control region
deleted) promoter allows for high expression of the transgene,
while the LTR allows for long-term expression after repeated
passage. The vectors also include (IFN)-.quadrature.-scaffold
attachment region (SAR) element. The SAR element has been shown to
be important in keeping the vector transcriptionally active by
inhibiting methylation and protecting the transgene from being
silenced.
[0346] In order to investigate the potential myocardial benefits of
long term overexpression of Pim-1 in CPCs, a bicistronic lentiviral
vector was designed to deliver the human Pim-1 gene, CGW-Pim-wt, as
well as a control vector, CGW (FIG. S1A). Expression of the Pim-1
gene is controlled through a myeloproliferative sarcoma virus
LTR-negative control region deleted (MND) promoter, while the eGFP
reporter is driven off of a viral internal ribosomal entry site
(vIRES).
[0347] FIG. 21 illustrates an exemplary lentiviral constructs of
the invention for e.g., gene expression in cardiac progenitor
cells, e.g., gene expression in c-kit+ cardiac progenitor cells
(CPCs). The figures illustrates a self-inactivating (SIN)
lentiviral vectors, termed CGW (GFP control) and CGW-Pim-wt; they
were designed such that the Pim-1 gene is driven off an MND
promoter while the eGFP reporter is driven off an internal
ribosomal entry site.
[0348] The following sequence is an exemplary lentiviral vector
backbone for practicing the invention, e.g., to express PIM-1 in a
cell, including a human cell, e.g., a stem cell or a cardiac or
myocyte cell.
TABLE-US-00001 (SEQ ID NO: 4)
gacggatcgggagatctcccgatcccctatggtcgactctcagtacaatctgctctgatgccgcatagttaagc-
cagtatctgctc
cctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccga-
caattgcatgaa
gaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattat-
tgactagttatt
aatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaat-
ggcccgcctggc
tgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggacttt-
ccattgacgtca
atgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcccccta-
ttgacgtcaatg
acggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctac-
gtattagtcatc
gctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttcc-
aagtctccaccc
cattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccc-
cattgacgcaaa
tgggcggtaggcgtgtacggtgggaggtctatataagcagcgcgttttgcctgtactgggtctctctggttaga-
ccagatctgagc
ctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtag-
tgtgtgcccgtc
tgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcc-
cgaacagggacc
tgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggc-
gaggggcggcga
ctggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaa-
gcgggggagaat
tagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatggg-
caagcagggagc
tagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaa-
ccatcccttcag
acaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagat-
aaaagacaccaa
ggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttc-
agacctggagga
ggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagc-
acccaccaaggc
aaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcag-
caggaagcacta
tgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaat-
ttgctgagggct
attgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgt-
ggaaagatacct
aaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatg-
ctagttggagta
ataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagc-
ttaatacactcc
ttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagttt-
gtggaattggtt
taacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatag-
tttttgctgtac
tttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgagggga-
cccgacaggccc
gaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatccgatccaca-
aatggcagtatt
catccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaa-
cagacatacaaa
ctaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtt-
tggcctgcagag
atccagagttaggcagggacattcaccattatcgtttcagacccacctcccaaccccggtcatatgggaatgaa-
agaccccacctg
taggtttggcaagctaggatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggt-
aagcagttcctg
ccccggctcagggccaagaacagttggaacaggagaatatgggccaaacaggatatctgtggtaagcagttcct-
gccccggctcag
ggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccaggg-
tgccccaaggac
ctgaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctcc-
ccgagctctata
taagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaaga-
tcagttaattaa
gaattcgcccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgttt-
gtctatatgtta
ttttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcc-
taggggtctttc
ccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagac-
aaacaacgtctg
tagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataa-
gatacacctgca
aaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgt-
attcaacaaggg
gctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtg-
tttagtcgaggt
taaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataatatggccac-
aaccatggtgag
caagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagt-
tcagcgtgtccg
gcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtg-
ccctggcccacc
ctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttctt-
caagtccgccat
gcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtga-
agttcgagggcg
acaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctg-
gagtacaactac
aacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaa-
catcgaggacgg
cagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaacc-
actacctgagca
cccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgcc-
gggatcactctc
ggcatggacgagctgtacaagtaaagcggccgcactgttctcatcacatcatatcaaggttatataccatcaat-
attgccacagat
gttacttagccttttaatatttctctaatttagtgtatatgcaatgatagttctctgatttctgagattgagtt-
tctcatgtgtaa
tgattatttagagtttctctttcatctgttcaaatttttgtctagttttattttttactgatttgtaagacttc-
tttttataatct
gcatattacaattctctttactggggtgttgcaaatattttctgtcattctatggcctgacttttcttaatggt-
tttttaatttta
aaaataagtcttaatattcatgcaatctaattaacaatcttttctttgtggttaggactttgagtcataagaaa-
tttttctctaca
ctgaagtcatgatggcatgcttctatattattttctaaaagatttaaagttttgccttctccatttagacttat-
aattcactggaa
tttttttgtgtgtatggtatgacatatgggttcccttttattttttacatataaatatatttccctgtttttct-
aaaaaagaaaaa
gatcatcattttcccattgtaaaatgccatatttttttcataggtcacttacatatatcaatgggtctgtttct-
gagctctactct
attttatcagcctcactgtctatccccacacatctcatgctttgctctaaatcttgatatttagtggaacattc-
tttcccattttg
ttctacaagaatatttttgttattgtctttgggctttctatatacattttgaaatgaggttgacaagtttctag-
agttaactcgag
ggatcaagcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgtt-
gctccttttacg
ctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctcctt-
gtataaatcctg
gttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacg-
caacccccactg
gttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaa-
ctcatcgccgcc
tgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgac-
gtcctttccatg
gctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccag-
cggaccttcctt
cccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctt-
tgggccgcctcc
ccgcatcgataccgtcgagacctagaaaaacatggagcaatcacaagtagcaacacagcagctaccaatgctga-
ttgtgcctggct
agaagcacaagaggaggaggaggtgggttttccagtcacacctcaggtacctttaagaccaatgacttacaagg-
cagctgtagatc
ttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatatccttgat-
ctgtggatctac
cacacacaaggctacttccctgattggcagaactacacaccagggccagggatcagatatccactgacctttgg-
atggtgctacaa
gctagtaccagttgagcaagagaaggtagaagaagccaatgaaggagagaacacccgcttgttacaccctgtga-
gcctgcatggga
tggatgacccggagagagaagtattagagtggaggtttgacagccgcctagcatttcatcacatggcccgagag-
ctgcatccggac
tgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaa-
gcctcaataaag
cttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagaccct-
tttagtcagtgt
ggaaaatctctagcagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgt-
tgtttgcccctc
ccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgc-
attgtctgagta
ggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcat-
gctggggatgcg
gtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcgg-
cgcattaagcgc
ggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttct-
tcccttcctttc
tcgccacgttcgccggctttccccgtcaagctctaaatcggggcatccctttagggttccgatttagtgcttta-
cggcacctcgac
cccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgac-
gttggagtccac
gttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttat-
aagggattttgg
ggatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgt-
gtcagttagggt
gtggaaagtccccaggctccccaggcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtg-
tggaaagtcccc
aggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactc-
cgcccatcccgc
ccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgag-
gccgcctctgcc
tctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttg-
tatatccatttt
cggatctgatcagcacgtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgag-
gaactaaaccat
ggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgacc-
ggctcgggttct
cccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccag-
gaccaggtggtg
ccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtc-
cacgaacttccg
ggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccgg-
ccggcaactgcg
tgcacttcgtggccgaggagcaggactgacacgtgctacgagatttcgattccaccgccgccttctatgaaagg-
ttgggcttcgga
atcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaa-
cttgtttattgc
agcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattcta-
gttgtggtttgt
ccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtca-
tagctgtttcct
gtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgc-
ctaatgagtgag
ctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaat-
gaatcggccaac
gcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgtt-
cggctgcggcga
gcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtg-
agcaaaaggcca
gcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatc-
acaaaaatcgac
gctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtg-
cgctctcctgtt
ccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacg-
ctgtaggtatct
cagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcct-
tatccggtaact
atcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcaga-
gcgaggtatgta
ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgc-
tctgctgaagcc
agttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttg-
tttgcaagcagc
agattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaac-
gaaaactcacgt
taagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaa-
atcaatctaaag
tatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctat-
ttcgttcatcca
tagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatg-
ataccgcgagac
ccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgc-
aactttatccgc
ctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttg-
ttgccattgcta
caggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagtt-
acatgatccccc
atgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatc-
actcatggttat
ggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaacca-
agtcattctgag
aatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaact-
ttaaaagtgctc
atcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacc-
cactcgtgcacc
caactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaa-
aaaagggaataa
gggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgt-
ctcatgagcgga
tacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctga-
cgtc
EXAMPLE 5
Exemplary PIM Sequences Used to Practice This Invention
[0349] The invention provides compositions and methods comprising
use of PIM-expressing nucleic acids and PIM polypeptides.
[0350] For example, in one embodiment the Human PIM-1 protein is
used to practice the compositions and methods of this invention; an
exemplary Human PIM-1 protein that can be used is Genbank accession
no. AAA36447 (see also, e.g., Domen (1987) Oncogene Res. 1
(1):103-112) (SEQ ID NO:5):
TABLE-US-00002 (SEQ ID NO: 5) 1 mllskinsla hlraapcndl hatklapgke
keplesqyqv gpllgsggfg svysgirvsd 61 nlpvaikhve kdrisdwgel
pngtrvpmev vllkkvssgf sgvirlldwf erpdsfvlil 121 erpepvqdlf
dfitergalq eelarsffwq vleavrhchn cgvlhrdikd enilidlnrg 181
elklidfgsg allkdtvytd fdgtrvyspp ewiryhryhg rsaavwslgi llydmvcgdi
241 pfehdeeiir gqvffrqrvs secqhlirwc lalrpsdrpt feeiqnhpwm
qdvllpqeta 301 eihlhslspg psk
[0351] In one embodiment, a Human PIM-1 protein isoform is used to
practice the compositions and methods of this invention; an
exemplary Human PIM-1 protein isoform that can be used is the human
pim-1 kinase 44 kDa isoform, see e.g., Genbank accession no.
AAY87461 (see also, e.g., Xie (2006) Oncogene 25 (1), 70-78) (SEQ
ID NO:6):
TABLE-US-00003 (SEQ ID NO: 6) 1 mphepheplt ppfsalpdpa gapsrrqsrq
rpqlssdsps afrasrshsr natrshshsh 61 sprhslrhsp gsgscgsssg
hrpcadilev gmllskinsl ahlraapcnd lhatklapgk 121 ekeplesqyq
vgpllgsggf gsvysgirvs dnlpvaikhv ekdrisdwge 1pngtrvpme 181
vvllkkvssg fsgvirlldw ferpdsfvli lerxepvqd1 fdfitergal qeelarsffw
241 qvleavrhch ncgvlhrdik denilidlnr gelklidfgs gallkdtvyt
dfdgtrvysp 301 pewiryhryh grsaavwslg illydmvcgd ipfehdeeii
rgqvffrqry ssecqhlirw 361 clalrpsdrp tfeeiqnhpw mqdvllpqet
aeihlhslsp gpsk
[0352] In one embodiment, a Human PIM-1 message (mRNA) is used to
practice the compositions and methods of this invention; an
exemplary Human PIM-1 message that can be used is Genbank accession
no. NM_002648 (see also, e.g., Zhang (2007) Mol. Cancer Res. 5 (9),
909-922) (SEQ ID NO:7):
TABLE-US-00004 (SEQ ID NO: 7) 1 cccgagagga gtcggtggca gcggcggcgg
cgggaccggc agcagcagca gcagcagcag 61 cagcaaccac tagcctcctg
ccccgcggcg ctgccgcacg agccccacga gccgctcacc 121 ccgccgttct
cagcgctgcc cgaccccgct ggcgcgccct cccgccgcca gtcccggcag 181
cgccctcagt tgtcctccga ctcgccctcg gccttccgcg ccagccgcag ccacagccgc
241 aacgccaccc gcagccacag ccacagccac agccccaggc atagccttcg
gcacagcccc 301 ggctccggct cctgcggcag ctcctctggg caccgtccct
gcgccgacat cctggaggtt 361 gggatgctct tgtccaaaat caactcgctt
gcccacctgc gcgccgcgcc ctgcaacgac 421 ctgcacgcca ccaagctggc
gcccggcaag gagaaggagc ccctggagtc gcagtaccag 481 gtgggcccgc
tactgggcag cggcggcttc ggctcggtct actcaggcat ccgcgtctcc 541
gacaacttgc cggtggccat caaacacgtg gagaaggacc ggatttccga ctggggagag
601 ctgcctaatg gcactcgagt gcccatggaa gtggtcctgc tgaagaaggt
gagctcgggt 661 ttctccggcg tcattaggct cctggactgg ttcgagaggc
ccgacagttt cgtcctgatc 721 ctggagaggc ccgagccggt gcaagatctc
ttcgacttca tcacggaaag gggagccctg 781 caagaggagc tggcccgcag
cttcttctgg caggtgctgg aggccgtgcg gcactgccac 841 aactgcgggg
tgctccaccg cgacatcaag gacgaaaaca tccttatcga cctcaatcgc 901
ggcgagctca agctcatcga cttcgggtcg ggggcgctgc tcaaggacac cgtctacacg
961 gacttcgatg ggacccgagt gtatagccct ccagagtgga tccgctacca
tcgctaccat 1021 ggcaggtcgg cggcagtctg gtccctgggg atcctgctgt
atgatatggt gtgtggagat 1081 attcctttcg agcatgacga agagatcatc
aggggccagg ttttcttcag gcagagggtc 1141 tcttcagaat gtcagcatct
cattagatgg tgcttggccc tgagaccatc agataggcca 1201 accttcgaag
aaatccagaa ccatccatgg atgcaagatg ttctcctgcc ccaggaaact 1261
gctgagatcc acctccacag cctgtcgccg gggcccagca aatagcagcc tttctggcag
1321 gtcctcccct ctcttgtcag atgcccgagg gaggggaagc ttctgtctcc
agcttcccga 1381 gtaccagtga cacgtctcgc caagcaggac agtgcttgat
acaggaacaa catttacaac 1441 tcattccaga tcccaggccc ctggaggctg
cctcccaaca gtggggaaga gtgactctcc 1501 aggggtccta ggcctcaact
cctcccatag atactctctt cttctcatag gtgtccagca 1561 ttgctggact
ctgaaatatc ccgggggtgg ggggtggggg tgggtcagaa ccctgccatg 1621
gaactgtttt cttcatcatg agttctgctg aatgccgcga tgggtcaggt aggggggaaa
1681 caggttggga tgggatagga ctagcaccat tttaagtccc tgtcacctct
tccgactctt 1741 tctgagtgcc ttctgtgggg actccggctg tgctgggaga
aatacttgaa cttgcctctt 1801 ttacctgctg cttctccaaa aatctgcctg
ggttttgttc cctatttttc tctcctgtcc 1861 tccctcaccc cctccttcat
atgaaaggtg ccatggaaga ggctacaggg ccaaacgctg 1921 agccacctgc
ccttttttct gcctccttta gtaaaactcc gagtgaactg gtcttccttt 1981
ttggttttta cttaactgtt tcaaagccaa gacctcacac acacaaaaaa tgcacaaaca
2041 atgcaatcaa cagaaaagct gtaaatgtgt gtacagttgg catggtagta
tacaaaaaga 2101 ttgtagtgga tctaattttt aagaaatttt gcctttaagt
tattttacct gtttttgttt 2161 cttgttttga aagatgcgca ttctaacctg
gaggtcaatg ttatgtattt atttatttat 2221 ttatttggtt cccttcctat
tccaagcttc catagctgct gccctagttt tctttcctcc 2281 tttcctcctc
tgacttgggg accttttggg ggagggctgc gacgcttgct ctgtttgtgg 2341
ggtgacggga ctcaggcggg acagtgctgc agctccctgg cttctgtggg gcccctcacc
2401 tacttaccca ggtgggtccc ggctctgtgg gtgatgggga ggggcattgc
tgactgtgta 2461 tataggataa ttatgaaaag cagttctgga tggtgtgcct
tccagatcct ctctggggct 2521 gtgttttgag cagcaggtag cctgctggtt
ttatctgagt gaaatactgt acaggggaat 2581 aaaagagatc ttattttttt
ttttatactt ggcgtttttt gaataaaaac cttttgtctt 2641 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa
[0353] In one embodiment, a Human PIM-1 gene is used to practice
the compositions and methods of this invention; an exemplary Human
PIM-1 gene that can be used is (SEQ ID NO:8):
TABLE-US-00005 LOCUS NC_000023 5826 bp DNA linear CON 03-MAR-2008
DEFINITION Homo sapiens chromosome X, reference assembly, complete
sequence. ACCESSION NC_000023 REGION:
complement(48655403..48661228) VERSION NC_000023.9 GI: 89161218
PROJECT GenomeProject: 168 SOURCE Homo sapiens (human) ORGANISM
Homo sapiens REFERENCE 1 (bases 1 to 5826) AUTHORS International
Human Genome Sequencing Consortium. TITLE Finishing the euchromatic
sequence of the human genome JOURNAL Nature 431 (7011),
931-945(2004) PUBMED 15496913 (SEQ ID NO: 8) 1 cgcgcgcggc
gaatctcaac gctgcgccgt ctgcgggcgc ttccgggcca ccagtttctc 61
tgctttccac cctggcgccc cccagccctg gctccccagc tgcgctgccc cgggcgtcca
121 cgccctgcgg gcttagcggg ttcagtgggc tcaatctgcg cagcgccacc
tccatgttga 181 ccaagcctct acaggggcct cccgcgcccc ccgggacccc
cacgccgccg ccaggtgagt 241 acatcctccc ctactgcaac cagacggggt
gggctggaat gatgggttgc agcgcggggg 301 gagggagtcg tggctgggct
cagcacgccg ccaccctgac ttcctcgcct ccgcctgcgt 361 aggaggcaag
gatcgggaag cgttcgaggc cgagtatcga ctcggccccc tcctgggtaa 421
ggggggcttt ggcaccgtct tcgcaggaca ccgcctcaca gatcgactcc aggtatccgt
481 catgagggtc ttgggagggt caggtgcgtg tggcgggggc gggggtcctg
gccctggaat 541 gctggttgac cgaggagtga gcctgcagag tgtgtagagg
accaggtgtg tgtgtgtgtg 601 tgtccgtgtc cgtgtccgag gagtgagcct
gcagtgtgtg tagagggcca ggtgtgtgtg 661 cgtgcgcgtg tgtgtgtcgg
tctaggaggt tatgggcggg gggggggggc agggggcttc 721 agattccgga
gttccttgac cccggggtcc aggctgtgta tgtgtgggaa agcagggacc 781
tagatgtgag atttgtggga cttttggagg taggtgtcca gtgtggagtc atgcggacca
841 ggaccctggt acagagttgg ggtgtcgtag agctaaatag gaagattgtg
ggcctggggt 901 atcaggaaat ctagaactca ggacttggag tgatgagtcc
tgatgcctga gaacggagag 961 cccagggcta aggaaggtgg gagagataaa
cttggttccg aggacctgga gggcagggga 1021 gacgccctgg tacgcgttct
gtggggtgct gtggttgggg accagaaaga ctagagtgct 1081 ggtagatgga
ggaatactgg aggtaggcag aaggtctaga ctgggagggg tctggggatc 1141
acctgctggc ctccttatca cggccttctt ctccaggtgg ccatcaaagt gattccccgg
1201 aatcgtgtgc tgggctggtc ccccttggtg agtaccttcg gagcccttcc
taacctacct 1261 actccatcac tgatgtattc acctccttgc ttttccaggg
gatgtatgac tccctgggcc 1321 ctgtaacagt gagaatactg ccagtccatt
tatactccct tggggtgaca tacagttctg 1381 attcacccca attcccctag
agccctggat tctcccctcc aacaaacctt taccatcctt 1441 cctccaaaca
ctgctggggg actgcccgca gggcgtgctg gtggggaaca aggggcagag 1501
gtcactggtt gccatggtga tggtggctgc ttctctcttg ccgttataac gctaacggac
1561 atcagggcgg gtctgggcaa gttgtagagt tgggagcgcc ccctggcggg
ctctagggga 1621 aactgcgcct gcgcagtcca tgggacccaa agggagaggg
tgcgcctgcg caatatcggt 1681 atttttgcat ctcggtgaga aaacgtctgc
tgccgtgcaa gtcagcagcc tggccaggag 1741 agggctctac ctcatcccag
aaggttgctg ctcgaagtgt acctgcgcag ggcttgggga 1801 ggcagtgggg
ggcggatttt gtggccccca gcgtttatac tttttttttt ttggagacac 1861
agtctccctc tgttgcccag gctggagtga ggtgacgcga tctcggctca ctgcaacctc
1921 cgtctcctgg gttcaagtga ttctcctgcc tcagcctccc aagtagctgg
gactacagga 1981 gcgcacaacc atgcccggct aatttttgta tttttagtag
agacagggtt tcaccatgtt 2041 ggccaggcgg gttttgaact gctgacctca
ggtgatccgc ctgcctcggc cactcaaagt 2101 gctgggatta caggcatgag
ccaccacgcc cggctgcatt tatgactttt ttttttcctt 2161 gagacggagt
ttcgctctgc tgcctgggct ggagtgcagt ggcgtgatct cagctcactg 2221
cagcctccac ctcctgggtt caagcgattc tcctgcctca ggctcctgag tagctggaat
2281 tacaggcacc cgctgccatg cccggctaag ttttacgttt ttagtagaga
ccgtgtttca 2341 ccatgttggc caggctggtc tcgaacccct gacctagtga
tctgcccgcc ttgggcctcc 2401 caaagtgctg ggattacagg cgtgagccac
cgcgcccagc ctctaatttt gtatttttag 2461 tagagacggg gtttctccat
gttggtcagg ctggtctcga actcccgacc tcaggtgatc 2521 tgcccgtctc
ggcctcccaa agtgctggga ttacaggcgt gagccactgc gcagggccac 2581
atttaggctt tttattggct ggttctaggt gcttggtgat gctgacaaaa cacatgataa
2641 cactaagtcc ttttgtgcta ggtcctttgt aataaatcac tcagctgttt
aacaaattag 2701 gtatattgac cacctactat atgacagaca taattctaga
cactcagcaa agtattacat 2761 aagtattgag agctcatttt gtgctaggtc
cttttttact aattgttttc acctgtttaa 2821 caaatattta ttcagcccta
ctctgttagc agccactgtt ctagtgcttc atatacgtcc 2881 gtgaacaaaa
caaaccatta cacaataagt gtttattgag tgctaactgc ttgtcagagc 2941
ccatgctatt aagtgctgtc atctgtttaa catttattga tcacctgtgt aaggtactat
3001 tctaatctgg gatatgtcag ggaacaaaac aaaacacata atggtggtgc
tgcttctgct 3061 gaaagccttc agttgataac cagatttttc tttgtatttt
tgcttgtttg ttttgagaca 3121 gctggagtgc agtggtgtga tcttcactgc
aacctctgcc ttcttggctc aagcgaccct 3181 cccacctgag cctcccaagt
agctgggact acaggtgcat gccaccaagc ctggctaatt 3241 tttgtgtttg
tgccattttg cccaggctga tcttgaactc ttgggctcaa gcaatccacc 3301
cacatcagcc tcccaaagtg ctgggattgc agggatgagc cactgtgcct ggccgaactt
3361 ctttcgttta ttcaaatgtt tattgatcta cgacatgcga gatttgtgca
ggctctttgc 3421 tggtttcacc ctctcaatcg ctgtgtgagt ttgtgtcttt
agggaaagtg aggcccagga 3481 agggaagtga gttgcttagc gacacactgt
caggaaaagg ggccctgagt tgagcttagg 3541 taaaaagcct cagagctgtt
gccctgacat ctgtcttttt tctctccctg cttcccaccc 3601 cacctgtgcc
cccagtcaga ctcagtcaca tgcccactcg aagtcgcact gctatggaaa 3661
gtgggtgcag gtggtgggca ccctggcgtg atccgcctgc ttgactggtt tgagacacag
3721 gagggcttca tgctggtcct cgagcggcct ttgcccgccc aggatctctt
tgactatatc 3781 acagagaagg gcccactggg tgaaggccca agccgctgct
tctttggcca agtagtggca 3841 gccatccagc actgccattc ccgtggagtt
gtccatcgtg acatcaagga tgagaacatc 3901 ctgatagacc tacgccgtgg
ctgtgccaaa ctcattgatt ttggttctgg tgccctgctt 3961 catgatgaac
cctacactga ctttgatggt aaggcttctc taaatctccc tggagggatt 4021
gtttttactt gatggccttg tgacctttgg cctccagtgg tggggtgtcc tgtaatcctt
4081 gacccatact gcattatata agatgatcga ttgctaatac tggggattct
cagccttgcc 4141 ctctgataaa gtccatcttt taatggtgtg ctaaccttat
tctgggctcc tattctggtg 4201 aggggatcct gttaccatcc tgagtattct
ttctctggta aggggatcct gttacttttc 4261 agtgctttta ttctgttgag
gggactctgt tattttagct gctttttatc tagtgagggg 4321 actctgcttt
tatcttgagt gctcttaatt gtggtgaggc catccttcct ggagagtttg 4381
gggttggaga agggcatcat gagattgagt tggtctaacc cctggcttgt gtgcagggac
4441 aagggtgtac agccccccag agtggatctc tcgacaccag taccatgcac
tcccggccac 4501 tgtctggtca ctgggcatcc tcctctatga catggtgtgt
ggggacattc cctttgagag 4561 ggaccaggag attctggaag ctgagctcca
cttcccagcc catgtctccc caggtgaggc 4621 ctcactgacc ccagcccaga
agactccatc cttctcaggg accagtaccc cctactgact 4681 gctaatcttc
cctctctgct tcttggccta cagactgctg tgccctaatc cgccggtgcc 4741
tggcccccaa accttcttcc cgaccctcac tggaagagat cctgctggac ccctggatgc
4801 aaacaccagc cgaggatgta cccctcaacc cctccaaagg aggccctgcc
cctttggcct 4861 ggtccttgct accctaagcc tggcctggcc tggcctggcc
cccaatggtc agaagagcca 4921 tcccatggcc atgtcacagg gatagatgga
catttgttga cttggtttta caggtcatta 4981 ccagtcatta aagtccagta
ttactaaggt aagggattga ggatcagggg ttagaagaca 5041 taaaccaagt
ctgcccagtt cccttcccaa tcctacaaag gagccttcct cccagaacct 5101
gtggtccctg attctggagg gggaacttct tgcttctcat tttgctaagg aagtttattt
5161 tggtgaagtt gttcccattc tgagccccgg gactcttatt ctgatgatgt
gtcaccccac 5221 attggcacct cctactacca ccacacaaac ttagttcata
tgctcttact tgggcaaggg 5281 tgctttcctt ccaatacccc agtagctttt
attttagtaa agggaccctt tcccctagcc 5341 tagggtccca tattgggtca
agctgcttac ctgcctcagc ccaggattct ttattctggg 5401 ggaggtaatg
ccctgttgtt accccaaggc ttcttttttt tttttttttt tttgggtgag 5461
gggaccctac tctgttatcc caagtgctct tattctggtg agaagaacct tacttccata
5521 atttgggaag gaatggaaga tggacaccac cggacaccac cagacactag
gatgggatgg 5581 atggtttttt gggggatggg ctaggggaaa taaggcttgc
tgtttgttct cctggggcgc 5641 tccctccaac ttttgcagat tcttgcaacc
tcctcctgag ccgggattgt ccaattacta 5701 aaatgtaaat aatcacgtat
tgtggggagg ggagttccaa gtgtgccctc ctctcttctc 5761 ctgcctggat
tatttaaaaa gccatgtgtg gaaacccact atttaataaa agtaatagaa 5821
tcagaa
[0354] In one embodiment, exemplary Human PIM-1 polypeptides and
message that can be used are:
TABLE-US-00006 (SEQ ID NO: 9)
MLLSKFGSLAHLCGPGGVDHLPVKILQPAKADKESFEKAYQVGA (SEQ ID NO: 10)
VLGSGGFGTVYAGSRIADGLPVAVKHVVKERVTEWGSLGGATVPLEVVLLRKVGAAGG
ARGVIRLLDWFERPDGFLLVLERPEPAQDLFDFITERGALDEPLARRFFAQVLAAVRH
CHSCGVVHRDIKDENLLVDLRSGELKLIDFGSGALLKDTVYTDFDGTRVYSPPEWIRY
HRYHGRSATVWSLGVLLYDMVCGDIPFEQDEEILRGRLLFRRRVSPECQQLIRWCLSL RPSERP
SLDQIAAHPWMLGADGGAPESCDLRLCTLDPDDVASTTSSSESL'' (SEQ ID NO: 11) 1
agcggaccga cgcgacacgc cgtgcgcctc cgcggctgcg ctacgaaaac gagtcccgga
61 gcggccccgc gcccgccgca cccggccctc gcccacccga agacaggcgc
ccagctgccc 121 cgccgtctcc ccagctagcg cccggccgcc gccgcctcgc
gggccccggg cggaaggggg 181 cggggtcccg attcgccccg cccccgcgga
gggatacgcg gcgccgcggc ccaaaacccc 241 cgggcgaggc ggccggggcg
ggtgaggcgc tccgcctgct gctcgtctac gcggtccccg 301 cgggccttcc
gggcccactg cgccgcgcgg accgcctcgg gctcggacgg ccggtgtccc 361
cggcgcgccg ctcgcccgga tcggccgcgg cttcggcgcc tggggctcgg ggctccgggg
421 aggccgtcgc ccgcgatgct gctctccaag ttcggctccc tggcgcacct
ctgcgggccc 481 ggcggcgtgg accacctccc ggtgaagatc ctgcagccag
ccaaggcgga caaggagagc 541 ttcgagaagg cgtaccaggt gggcgccgtg
ctgggtagcg gcggcttcgg cacggtctac 601 gcgggtagcc gcatcgccga
cgggctcccg gtggctgtga agcacgtggt gaaggagcgg 661 gtgaccgagt
ggggcagcct gggcggcgcg accgtgcccc tggaggtggt gctgctgcgc 721
aaggtgggcg cggcgggcgg cgcgcgcggc gtcatccgcc tgctggactg gttcgagcgg
781 cccgacggct tcctgctggt gctggagcgg cccgagccgg cgcaggacct
cttcgacttt 841 atcacggagc gcggcgccct ggacgagccg ctggcgcgcc
gcttcttcgc gcaggtgctg 901 gccgccgtgc gccactgcca cagctgcggg
gtcgtgcacc gcgacattaa ggacgaaaat 961 ctgcttgtgg acctgcgctc
cggagagctc aagctcatcg acttcggttc gggtgcgctg 1021 ctcaaggaca
cggtctacac cgacttcgac ggcacccgag tgtacagccc cccggagtgg 1081
atccgctacc accgctacca cgggcgctcg gccaccgtgt ggtcgctggg cgtgcttctc
1141 tacgatatgg tgtgtgggga catccccttc gagcaggacg aggagatcct
ccgaggccgc 1201 ctgctcttcc ggaggagggt ctctccagag tgccagcagc
tgatccggtg gtgcctgtcc 1261 ctgcggccct cagagcggcc gtcgctggat
cagattgcgg cccatccctg gatgctgggg 1321 gctgacgggg gcgccccgga
gagctgtgac ctgcggctgt gcaccctcga ccctgatgac 1381 gtggccagca
ccacgtccag cagcgagagc ttgtgaggag ctgcacctga ctgggagcta 1441
ggggaccacc tgccttggcc agacctggga cgcccccaga ccctgacttt ttcctgcgtg
1501 ggccgtctcc tcctgcggaa gcagtgacct ctgacccctg gtgaccttcg
ctttgagtgc 1561 cttttgaacg ctggtcccgc gggacttggt tttctcaagc
tctgtctgtc caaagacgct 1621 ccggtcgagg tcccgcctgc cctgggtgga
tacttgaacc ccagacgccc ctctgtgctg 1681 ctgtgtccgg aggcggcctt
cccatctgcc tgcccacccg gagctctttc cgccggcgca 1741 gggtcccaag
cccacctccc gccctcagtc ctgcggtgtg cgtctgggca cgtcctgcac 1801
acacaatgca agtcctggcc tccgcgcccg cccgcccacg cgagccgtac ccgccgccaa
1861 ctctgttatt tatggtgtga ccccctggag gtgccctcgg cccaccgggg
ctatttattg 1921 tttaatttat ttgttgaggt tatttcctct gagcagtctg
cctctcccaa gccccagggg 1981 acagtgggga ggcaggggag ggggtggctg
tggtccaggg accccaggcc ctgattcctg 2041 tgcctggcgt ctgtcctggc
cccgcctgtc agaagatgaa catgtatagt ggctaactta 2101 aggggagtgg
gtgaccctga cacttccagg cactgtgccc agggtttggg ttttaaatta 2161
ttgactttgt acagtctgct tgtgggctct gaaagctggg gtggggccag agcctgagcg
2221 tttaatttat tcagtacctg tgtttgtgtg aatgcggtgt gtgcaggcat
cgcagatggg 2281 ggttctttca gttcaaaagt gagatgtctg gagatcatat
ttttttatac aggtatttca 2341 attaaaatgt ttttgtacat aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aa (SEQ ID NO: 12)
Agcttcgaattatgctcttgtccaaaatcaactcgcttgcccacctgcgcgccgcgcc
ctgcaacgacctgcacgccaccaagctggcgcccggcaaggagaaggagcccctggag
tcgcagtaccaggtgggcccgctactgggcagcggcggcttcggctcggtctactcag
gcatccgcgtctccgacaacttgccggtggccatcaaacacgtggagaaggaccggat
ttccgactggggagagctgcctaatggcactcgagtgcccatggaagtggtcctgctg
aagaaggtgagctcgggtttctccggcgtcattaggctcctggactggttcgagaggc
ccgacagtttcgtcctgatcctggagaggcccgagccggtgcaagatctcttcgactt
catcacggaaaggggagccctgcaagaggagctggcccgcagcttcttctggcaggtg
ctggaggccgtgcggcactgccacaactgcggggtgctccaccgcgacatcaaggacg
aaaacatccttatcgacctcaatcgcggcgagctcaagctcatcgacttcgggtcggg
ggcgctgctcaaggacaccgtctacacggacttcgatgggacccgagtgtatagccct
ccagagtggatccgctaccatcgctaccatggcaggtcggcggcagtctggtccctgg
ggatcctgctgtatgatatggtgtgtggagatattcctttcgagcatgacgaagagat
catcaggggccaggttttcttcaggcagagggtctcttcagaatgtcagcatctcatt
agatggtgcttggccctgagaccatcagataggccaaccttcgaagaaatccagaacc
atccatggatgcaagatgttctcctgccccaggaaactgctgagatccacctccacag
cctgtcgccggggcccagcagcctgtcgccggggcccagcaaacaattggtaccgcgg gcccgg
(SEQ ID NO: 13) atgctct tgtccaaaat caactcgctt gcccacctgc gcgccgcgcc
ctgcaacgac 421 ctgcacgcca ccaagctggc gcccggcaag gagaaggagc
ccctggagtc gcagtaccag 481 gtgggcccgc tactgggcag cggcggcttc
ggctcggtct actcaggcat ccgcgtctcc 541 gacaacttgc cggtggccat
caaacacgtg gagaaggacc ggatttccga ctggggagag 601 ctgcctaatg
gcactcgagt gcccatggaa gtggtcctgc tgaagaaggt gagctcgggt 661
ttctccggcg tcattaggct cctggactgg ttcgagaggc ccgacagttt cgtcctgatc
721 ctggagaggc ccgagccggt gcaagatctc ttcgacttca tcacggaaag
gggagccctg 781 caagaggagc tggcccgcag cttcttctgg caggtgctgg
aggccgtgcg gcactgccac 841 aactgcgggg tgctccaccg cgacatcaag
gacgaaaaca tccttatcga cctcaatcgc 901 ggcgagctca agctcatcga
cttcgggtcg ggggcgctgc tcaaggacac cgtctacacg 961 gacttcgatg
ggacccgagt gtatagccct ccagagtgga tccgctacca tcgctaccat 1021
ggcaggtcgg cggcagtctg gtccctgggg atcctgctgt atgatatggt gtgtggagat
1081 attcctttcg agcatgacga agagatcatc aggggccagg ttttcttcag
gcagagggtc 1141 tcttcagaat gtcagcatct cattagatgg tgcttggccc
tgagaccatc agataggcca 1201 accttcgaag aaatccagaa ccatccatgg
atgcaagatg ttctcctgcc ccaggaaact 1261 gctgagatcc acctccacag
cctgtcgccg gggcccagca aatag (SEQ ID NO: 14) 100 a tgctcctgtc
caagatcaac 121 tccctggccc acctgcgcgc cgcgccctgc aacgacctgc
acgccaccaa gctggcgccg 181 ggcaaagaga aggagcccct ggagtcgcag
taccaggtgg gcccgctgtt gggcagcggt 241 ggcttcggct cggtctactc
tggcatccgc gtcgccgaca acttgccggt ggccattaag 301 cacgtggaga
aggaccggat ttccgattgg ggagaactgc ccaatggcac ccgagtgccc 361
atggaagtgg tcctgttgaa gaaggtgagc tcggacttct cgggcgtcat tagacttctg
421 gactggttcg agaggcccga tagtttcgtg ctgatcctgg agaggcccga
accggtgcaa 481 gacctcttcg actttatcac cgaacgagga gccctacagg
aggacctggc ccgaggattc 541 ttctggcagg tgctggaggc cgtgcggcat
tgccacaact gcggggttct ccaccgcgac 601 atcaaggacg agaacatctt
aatcgacctg agccgcggcg aaatcaaact catcgacttc 661 gggtcggggg
cgctgctcaa ggacacagtc tacacggact ttgatgggac ccgagtgtac 721
agtcctccag agtggattcg ctaccatcgc taccacggca ggtcggcagc tgtctggtcc
781 cttgggatcc tgctctatga catggtctgc ggagatattc cgtttgagca
cgatgaagag 841 atcatcaagg gccaagtgtt cttcaggcaa actgtctctt
cagagtgtca gcaccttatt 901 aaatggtgcc tgtccctgag accatcagat
cggccctcct ttgaagaaat ccggaaccat 961 ccatggatgc agggtgacct
cctgccccag gcagcttctg agatccatct gcacagtctg 1021 tcaccggggt
ccagcaagta g
[0355] While the invention is susceptible to various modifications
and alternative forms, specific examples thereof have been shown by
way of example in the drawings and are herein described in detail.
It should be understood, however, that the invention is not to be
limited to the particular forms or methods disclosed, but to the
contrary, the invention is to cover all modifications, equivalents,
and alternatives falling within the spirit and scope of the
appended claims.
Sequence CWU 1
1
141313PRTHomo sapiensDOMAIN(1)..(313)human PIM-1 1Met Leu Leu Ser
Lys Ile Asn Ser Leu Ala His Leu Arg Ala Ala Pro 1 5 10 15 Cys Asn
Asp Leu His Ala Thr Lys Leu Ala Pro Gly Lys Glu Lys Glu 20 25 30
Pro Leu Glu Ser Gln Tyr Gln Val Gly Pro Leu Leu Gly Ser Gly Gly 35
40 45 Phe Gly Ser Val Tyr Ser Gly Ile Arg Val Ser Asp Asn Leu Pro
Val 50 55 60 Ala Ile Lys His Val Glu Lys Asp Arg Ile Ser Asp Trp
Gly Glu Leu 65 70 75 80 Pro Asn Gly Thr Arg Val Pro Met Glu Val Val
Leu Leu Lys Lys Val 85 90 95 Ser Ser Gly Phe Ser Gly Val Ile Arg
Leu Leu Asp Trp Phe Glu Arg 100 105 110 Pro Asp Ser Phe Val Leu Ile
Leu Glu Arg Pro Glu Pro Val Gln Asp 115 120 125 Leu Phe Asp Phe Ile
Thr Glu Arg Gly Ala Leu Gln Glu Glu Leu Ala 130 135 140 Arg Ser Phe
Phe Trp Gln Val Leu Glu Ala Val Arg His Cys His Asn 145 150 155 160
Cys Gly Val Leu His Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp 165
170 175 Leu Asn Arg Gly Glu Leu Lys Leu Ile Asp Phe Gly Ser Gly Ala
Leu 180 185 190 Leu Lys Asp Thr Val Tyr Thr Asp Phe Asp Gly Thr Arg
Val Tyr Ser 195 200 205 Pro Pro Glu Trp Ile Arg Tyr His Arg Tyr His
Gly Arg Ser Ala Ala 210 215 220 Val Trp Ser Leu Gly Ile Leu Leu Tyr
Asp Met Val Cys Gly Asp Ile 225 230 235 240 Pro Phe Glu His Asp Glu
Glu Ile Ile Arg Gly Gln Val Phe Phe Arg 245 250 255 Gln Arg Val Ser
Ser Glu Cys Gln His Leu Ile Arg Trp Cys Leu Ala 260 265 270 Leu Arg
Pro Ser Asp Arg Pro Thr Phe Glu Glu Ile Gln Asn His Pro 275 280 285
Trp Met Gln Asp Val Leu Leu Pro Gln Glu Thr Ala Glu Ile His Leu 290
295 300 His Ser Leu Ser Pro Gly Pro Ser Lys 305 310 2405PRTHomo
sapiensDOMAIN(1)..(405)human pim-1 kinase 44 kDa
isoformSITE(214)..(214)Xaa = any naturally occurring amino acid
2Met Pro His Glu Pro His Glu Pro Leu Thr Pro Pro Phe Ser Ala Leu 1
5 10 15 Pro Asp Pro Ala Gly Ala Pro Ser Arg Arg Gln Ser Arg Gln Arg
Pro 20 25 30 Gln Leu Ser Ser Asp Ser Pro Ser Ala Phe Arg Ala Ser
Arg Ser His 35 40 45 Ser Arg Asn Ala Thr Arg Ser His Ser His Ser
His Ser Pro Arg His 50 55 60 Ser Leu Arg His Ser Pro Gly Ser Gly
Ser Cys Gly Ser Ser Ser Gly 65 70 75 80 His Arg Pro Cys Ala Asp Ile
Leu Glu Val Gly Met Leu Leu Ser Lys 85 90 95 Ile Asn Ser Leu Ala
His Leu Arg Ala Ala Pro Cys Asn Asp Leu His 100 105 110 Ala Thr Lys
Leu Ala Pro Gly Lys Glu Lys Glu Pro Leu Glu Ser Gln 115 120 125 Tyr
Gln Val Gly Pro Leu Leu Gly Ser Gly Gly Phe Gly Ser Val Tyr 130 135
140 Ser Gly Ile Arg Val Ser Asp Asn Leu Pro Val Ala Ile Lys His Val
145 150 155 160 Glu Lys Asp Arg Ile Ser Asp Trp Gly Glu Leu Pro Asn
Gly Thr Arg 165 170 175 Val Pro Met Glu Val Val Leu Leu Lys Lys Val
Ser Ser Gly Phe Ser 180 185 190 Gly Val Ile Arg Leu Leu Asp Trp Phe
Glu Arg Pro Asp Ser Phe Val 195 200 205 Leu Ile Leu Glu Arg Xaa Glu
Pro Val Gln Asp Leu Phe Asp Phe Ile 210 215 220 Thr Glu Arg Gly Ala
Leu Gln Glu Glu Leu Ala Arg Ser Phe Phe Trp 225 230 235 240 Gln Val
Leu Glu Ala Val Arg His Cys His Asn Cys Gly Val Leu His 245 250 255
Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp Leu Asn Arg Gly Glu 260
265 270 Leu Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu Leu Lys Asp Thr
Val 275 280 285 Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr Ser Pro Pro
Glu Trp Ile 290 295 300 Arg Tyr His Arg Tyr His Gly Arg Ser Ala Ala
Val Trp Ser Leu Gly 305 310 315 320 Ile Leu Leu Tyr Asp Met Val Cys
Gly Asp Ile Pro Phe Glu His Asp 325 330 335 Glu Glu Ile Ile Arg Gly
Gln Val Phe Phe Arg Gln Arg Val Ser Ser 340 345 350 Glu Cys Gln His
Leu Ile Arg Trp Cys Leu Ala Leu Arg Pro Ser Asp 355 360 365 Arg Pro
Thr Phe Glu Glu Ile Gln Asn His Pro Trp Met Gln Asp Val 370 375 380
Leu Leu Pro Gln Glu Thr Ala Glu Ile His Leu His Ser Leu Ser Pro 385
390 395 400 Gly Pro Ser Lys Leu 405 32684DNAHomo
sapiensmisc_feature(1)..(2684)human pim-1 kinase message (mRNA)
3cccgagagga gtcggtggca gcggcggcgg cgggaccggc agcagcagca gcagcagcag
60cagcaaccac tagcctcctg ccccgcggcg ctgccgcacg agccccacga gccgctcacc
120ccgccgttct cagcgctgcc cgaccccgct ggcgcgccct cccgccgcca
gtcccggcag 180cgccctcagt tgtcctccga ctcgccctcg gccttccgcg
ccagccgcag ccacagccgc 240aacgccaccc gcagccacag ccacagccac
agccccaggc atagccttcg gcacagcccc 300ggctccggct cctgcggcag
ctcctctggg caccgtccct gcgccgacat cctggaggtt 360gggatgctct
tgtccaaaat caactcgctt gcccacctgc gcgccgcgcc ctgcaacgac
420ctgcacgcca ccaagctggc gcccggcaag gagaaggagc ccctggagtc
gcagtaccag 480gtgggcccgc tactgggcag cggcggcttc ggctcggtct
actcaggcat ccgcgtctcc 540gacaacttgc cggtggccat caaacacgtg
gagaaggacc ggatttccga ctggggagag 600ctgcctaatg gcactcgagt
gcccatggaa gtggtcctgc tgaagaaggt gagctcgggt 660ttctccggcg
tcattaggct cctggactgg ttcgagaggc ccgacagttt cgtcctgatc
720ctggagaggc ccgagccggt gcaagatctc ttcgacttca tcacggaaag
gggagccctg 780caagaggagc tggcccgcag cttcttctgg caggtgctgg
aggccgtgcg gcactgccac 840aactgcgggg tgctccaccg cgacatcaag
gacgaaaaca tccttatcga cctcaatcgc 900ggcgagctca agctcatcga
cttcgggtcg ggggcgctgc tcaaggacac cgtctacacg 960gacttcgatg
ggacccgagt gtatagccct ccagagtgga tccgctacca tcgctaccat
1020ggcaggtcgg cggcagtctg gtccctgggg atcctgctgt atgatatggt
gtgtggagat 1080attcctttcg agcatgacga agagatcatc aggggccagg
ttttcttcag gcagagggtc 1140tcttcagaat gtcagcatct cattagatgg
tgcttggccc tgagaccatc agataggcca 1200accttcgaag aaatccagaa
ccatccatgg atgcaagatg ttctcctgcc ccaggaaact 1260gctgagatcc
acctccacag cctgtcgccg gggcccagca aatagcagcc tttctggcag
1320gtcctcccct ctcttgtcag atgcccgagg gaggggaagc ttctgtctcc
agcttcccga 1380gtaccagtga cacgtctcgc caagcaggac agtgcttgat
acaggaacaa catttacaac 1440tcattccaga tcccaggccc ctggaggctg
cctcccaaca gtggggaaga gtgactctcc 1500aggggtccta ggcctcaact
cctcccatag atactctctt cttctcatag gtgtccagca 1560ttgctggact
ctgaaatatc ccgggggtgg ggggtggggg tgggtcagaa ccctgccatg
1620gaactgtttt cttcatcatg agttctgctg aatgccgcga tgggtcaggt
aggggggaaa 1680caggttggga tgggatagga ctagcaccat tttaagtccc
tgtcacctct tccgactctt 1740tctgagtgcc ttctgtgggg actccggctg
tgctgggaga aatacttgaa cttgcctctt 1800ttacctgctg cttctccaaa
aatctgcctg ggttttgttc cctatttttc tctcctgtcc 1860tccctcaccc
cctccttcat atgaaaggtg ccatggaaga ggctacaggg ccaaacgctg
1920agccacctgc ccttttttct gcctccttta gtaaaactcc gagtgaactg
gtcttccttt 1980ttggttttta cttaactgtt tcaaagccaa gacctcacac
acacaaaaaa tgcacaaaca 2040atgcaatcaa cagaaaagct gtaaatgtgt
gtacagttgg catggtagta tacaaaaaga 2100ttgtagtgga tctaattttt
aagaaatttt gcctttaagt tattttacct gtttttgttt 2160cttgttttga
aagatgcgca ttctaacctg gaggtcaatg ttatgtattt atttatttat
2220ttatttggtt cccttcctat tccaagcttc catagctgct gccctagttt
tctttcctcc 2280tttcctcctc tgacttgggg accttttggg ggagggctgc
gacgcttgct ctgtttgtgg 2340ggtgacggga ctcaggcggg acagtgctgc
agctccctgg cttctgtggg gcccctcacc 2400tacttaccca ggtgggtccc
ggctctgtgg gtgatgggga ggggcattgc tgactgtgta 2460tataggataa
ttatgaaaag cagttctgga tggtgtgcct tccagatcct ctctggggct
2520gtgttttgag cagcaggtag cctgctggtt ttatctgagt gaaatactgt
acaggggaat 2580aaaagagatc ttattttttt ttttatactt ggcgtttttt
gaataaaaac cttttgtctt 2640aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaa 2684410570DNAHomo sapiensmisc_featureHuman DNA
sequence of PIM-2 4gacggatcgg gagatctccc gatcccctat ggtcgactct
cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag
gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg
ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt
aacgccaata gggactttcc 420attgacgtca atgggtggag tatttacggt
aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc
cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag
gtctatataa gcagcgcgtt ttgcctgtac tgggtctctc 840tggttagacc
agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
900cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
gtgtgactct 960ggtaactaga gatccctcag acccttttag tcagtgtgga
aaatctctag cagtggcgcc 1020cgaacaggga cctgaaagcg aaagggaaac
cagaggagct ctctcgacgc aggactcggc 1080ttgctgaagc gcgcacggca
agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt 1140tgactagcgg
aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
1200gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
aaatataaat 1260taaaacatat agtatgggca agcagggagc tagaacgatt
cgcagttaat cctggcctgt 1320tagaaacatc agaaggctgt agacaaatac
tgggacagct acaaccatcc cttcagacag 1380gatcagaaga acttagatca
ttatataata cagtagcaac cctctattgt gtgcatcaaa 1440ggatagagat
aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
1500gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
agatatgagg 1560gacaattgga gaagtgaatt atataaatat aaagtagtaa
aaattgaacc attaggagta 1620gcacccacca aggcaaagag aagagtggtg
cagagagaaa aaagagcagt gggaatagga 1680gctttgttcc ttgggttctt
gggagcagca ggaagcacta tgggcgcagc gtcaatgacg 1740ctgacggtac
aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
1800agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
caagcagctc 1860caggcaagaa tcctggctgt ggaaagatac ctaaaggatc
aacagctcct ggggatttgg 1920ggttgctctg gaaaactcat ttgcaccact
gctgtgcctt ggaatgctag ttggagtaat 1980aaatctctgg aacagatttg
gaatcacacg acctggatgg agtgggacag agaaattaac 2040aattacacaa
gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
2100gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
taacataaca 2160aattggctgt ggtatataaa attattcata atgatagtag
gaggcttggt aggtttaaga 2220atagtttttg ctgtactttc tatagtgaat
agagttaggc agggatattc accattatcg 2280tttcagaccc acctcccaac
cccgagggga cccgacaggc ccgaaggaat agaagaagaa 2340ggtggagaga
gagacagaga cagatccatt cgattagtga acggatccga tccacaaatg
2400gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
tgcaggggaa 2460agaatagtag acataatagc aacagacata caaactaaag
aattacaaaa acaaattaca 2520aaaattcaaa attttcgggt ttattacagg
gacagcagag atccagtttg gcctgcagag 2580atccagagtt aggcagggac
attcaccatt atcgtttcag acccacctcc caaccccggt 2640catatgggaa
tgaaagaccc cacctgtagg tttggcaagc taggatcaag gttaggaaca
2700gagagacagc agaatatggg ccaaacagga tatctgtggt aagcagttcc
tgccccggct 2760cagggccaag aacagttgga acaggagaat atgggccaaa
caggatatct gtggtaagca 2820gttcctgccc cggctcaggg ccaagaacag
atggtcccca gatgcggtcc cgccctcagc 2880agtttctaga gaaccatcag
atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg 2940ccttatttga
actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc
3000gagctctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag
acgccatcca 3060cgctgttttg acctccatag aagatcagtt aattaagaat
tcgcccctct ccctcccccc 3120cccctaacgt tactggccga agccgcttgg
aataaggccg gtgtgcgttt gtctatatgt 3180tattttccac catattgccg
tcttttggca atgtgagggc ccggaaacct ggccctgtct 3240tcttgacgag
cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga
3300atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg
tctgtagcga 3360ccctttgcag gcagcggaac cccccacctg gcgacaggtg
cctctgcggc caaaagccac 3420gtgtataaga tacacctgca aaggcggcac
aaccccagtg ccacgttgtg agttggatag 3480ttgtggaaag agtcaaatgg
ctctcctcaa gcgtattcaa caaggggctg aaggatgccc 3540agaaggtacc
ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg
3600tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg
gttttccttt 3660gaaaaacacg atgataatat ggccacaacc atggtgagca
agggcgagga gctgttcacc 3720ggggtggtgc ccatcctggt cgagctggac
ggcgacgtaa acggccacaa gttcagcgtg 3780tccggcgagg gcgagggcga
tgccacctac ggcaagctga ccctgaagtt catctgcacc 3840accggcaagc
tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
3900tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
cgccatgccc 3960gaaggctacg tccaggagcg caccatcttc ttcaaggacg
acggcaacta caagacccgc 4020gccgaggtga agttcgaggg cgacaccctg
gtgaaccgca tcgagctgaa gggcatcgac 4080ttcaaggagg acggcaacat
cctggggcac aagctggagt acaactacaa cagccacaac 4140gtctatatca
tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
4200aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
ccccatcggc 4260gacggccccg tgctgctgcc cgacaaccac tacctgagca
cccagtccgc cctgagcaaa 4320gaccccaacg agaagcgcga tcacatggtc
ctgctggagt tcgtgaccgc cgccgggatc 4380actctcggca tggacgagct
gtacaagtaa agcggccgca ctgttctcat cacatcatat 4440caaggttata
taccatcaat attgccacag atgttactta gccttttaat atttctctaa
4500tttagtgtat atgcaatgat agttctctga tttctgagat tgagtttctc
atgtgtaatg 4560attatttaga gtttctcttt catctgttca aatttttgtc
tagttttatt ttttactgat 4620ttgtaagact tctttttata atctgcatat
tacaattctc tttactgggg tgttgcaaat 4680attttctgtc attctatggc
ctgacttttc ttaatggttt tttaatttta aaaataagtc 4740ttaatattca
tgcaatctaa ttaacaatct tttctttgtg gttaggactt tgagtcataa
4800gaaatttttc tctacactga agtcatgatg gcatgcttct atattatttt
ctaaaagatt 4860taaagttttg ccttctccat ttagacttat aattcactgg
aatttttttg tgtgtatggt 4920atgacatatg ggttcccttt tattttttac
atataaatat atttccctgt ttttctaaaa 4980aagaaaaaga tcatcatttt
cccattgtaa aatgccatat ttttttcata ggtcacttac 5040atatatcaat
gggtctgttt ctgagctcta ctctatttta tcagcctcac tgtctatccc
5100cacacatctc atgctttgct ctaaatcttg atatttagtg gaacattctt
tcccattttg 5160ttctacaaga atatttttgt tattgtcttt gggctttcta
tatacatttt gaaatgaggt 5220tgacaagttt ctagagttaa ctcgagggat
caagcttatc gataatcaac ctctggatta 5280caaaatttgt gaaagattga
ctggtattct taactatgtt gctcctttta cgctatgtgg 5340atacgctgct
ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
5400ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
ttgtcaggca 5460acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
actggttggg gcattgccac 5520cacctgtcag ctcctttccg ggactttcgc
tttccccctc cctattgcca cggcggaact 5580catcgccgcc tgccttgccc
gctgctggac aggggctcgg ctgttgggca ctgacaattc 5640cgtggtgttg
tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg
5700gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
cggaccttcc 5760ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
cttcgccttc gccctcagac 5820gagtcggatc tccctttggg ccgcctcccc
gcatcgatac cgtcgagacc tagaaaaaca 5880tggagcaatc acaagtagca
acacagcagc taccaatgct gattgtgcct ggctagaagc 5940acaagaggag
gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac
6000ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
tggaagggct 6060aattcactcc caacgaagac aagatatcct tgatctgtgg
atctaccaca cacaaggcta 6120cttccctgat tggcagaact acacaccagg
gccagggatc agatatccac tgacctttgg 6180atggtgctac aagctagtac
cagttgagca agagaaggta gaagaagcca atgaaggaga 6240gaacacccgc
ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt
6300attagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag
agctgcatcc 6360ggactgtact gggtctctct ggttagacca gatctgagcc
tgggagctct ctggctaact 6420agggaaccca ctgcttaagc ctcaataaag
cttgccttga gtgcttcaag tagtgtgtgc 6480ccgtctgttg tgtgactctg
gtaactagag atccctcaga cccttttagt cagtgtggaa 6540aatctctagc
agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
6600agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
gccactccca 6660ctgtcctttc ctaataaaat gaggaaattg catcgcattg
tctgagtagg tgtcattcta 6720ttctgggggg tggggtgggg caggacagca
agggggagga ttgggaagac aatagcaggc 6780atgctgggga tgcggtgggc
tctatggctt ctgaggcgga aagaaccagc tggggctcta 6840gggggtatcc
ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
6900gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
ttcttccctt 6960cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
aaatcggggc atccctttag 7020ggttccgatt tagtgcttta cggcacctcg
accccaaaaa acttgattag ggtgatggtt 7080cacgtagtgg gccatcgccc
tgatagacgg tttttcgccc tttgacgttg gagtccacgt 7140tctttaatag
tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
7200cttttgattt ataagggatt ttggggattt cggcctattg gttaaaaaat
gagctgattt 7260aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
cagttagggt gtggaaagtc 7320cccaggctcc ccaggcaggc agaagtatgc
aaagcatgca tctcaattag tcagcaacca
7380ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
catctcaatt 7440agtcagcaac catagtcccg cccctaactc cgcccatccc
gcccctaact ccgcccagtt 7500ccgcccattc tccgccccat ggctgactaa
ttttttttat ttatgcagag gccgaggccg 7560cctctgcctc tgagctattc
cagaagtagt gaggaggctt ttttggaggc ctaggctttt 7620gcaaaaagct
cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa
7680ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga
actaaaccat 7740ggccaagttg accagtgccg ttccggtgct caccgcgcgc
gacgtcgccg gagcggtcga 7800gttctggacc gaccggctcg ggttctcccg
ggacttcgtg gaggacgact tcgccggtgt 7860ggtccgggac gacgtgaccc
tgttcatcag cgcggtccag gaccaggtgg tgccggacaa 7920caccctggcc
tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt
7980cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg
gcgagcagcc 8040gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac
tgcgtgcact tcgtggccga 8100ggagcaggac tgacacgtgc tacgagattt
cgattccacc gccgccttct atgaaaggtt 8160gggcttcgga atcgttttcc
gggacgccgg ctggatgatc ctccagcgcg gggatctcat 8220gctggagttc
ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
8280caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
gttgtggttt 8340gtccaaactc atcaatgtat cttatcatgt ctgtataccg
tcgacctcta gctagagctt 8400ggcgtaatca tggtcatagc tgtttcctgt
gtgaaattgt tatccgctca caattccaca 8460caacatacga gccggaagca
taaagtgtaa agcctggggt gcctaatgag tgagctaact 8520cacattaatt
gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
8580gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
gctcttccgc 8640ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
cggcgagcgg tatcagctca 8700ctcaaaggcg gtaatacggt tatccacaga
atcaggggat aacgcaggaa agaacatgtg 8760agcaaaaggc cagcaaaagg
ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 8820taggctccgc
ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
8880cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
tgcgctctcc 8940tgttccgacc ctgccgctta ccggatacct gtccgccttt
ctcccttcgg gaagcgtggc 9000gctttctcaa tgctcacgct gtaggtatct
cagttcggtg taggtcgttc gctccaagct 9060gggctgtgtg cacgaacccc
ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 9120tcttgagtcc
aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
9180gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
ggcctaacta 9240cggctacact agaaggacag tatttggtat ctgcgctctg
ctgaagccag ttaccttcgg 9300aaaaagagtt ggtagctctt gatccggcaa
acaaaccacc gctggtagcg gtggtttttt 9360tgtttgcaag cagcagatta
cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 9420ttctacgggg
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
9480attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
ttaaatcaat 9540ctaaagtata tatgagtaaa cttggtctga cagttaccaa
tgcttaatca gtgaggcacc 9600tatctcagcg atctgtctat ttcgttcatc
catagttgcc tgactccccg tcgtgtagat 9660aactacgata cgggagggct
taccatctgg ccccagtgct gcaatgatac cgcgagaccc 9720acgctcaccg
gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
9780aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
gggaagctag 9840agtaagtagt tcgccagtta atagtttgcg caacgttgtt
gccattgcta caggcatcgt 9900ggtgtcacgc tcgtcgtttg gtatggcttc
attcagctcc ggttcccaac gatcaaggcg 9960agttacatga tcccccatgt
tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 10020tgtcagaagt
aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
10080tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
caaccaagtc 10140attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
ccggcgtcaa tacgggataa 10200taccgcgcca catagcagaa ctttaaaagt
gctcatcatt ggaaaacgtt cttcggggcg 10260aaaactctca aggatcttac
cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 10320caactgatct
tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
10380gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
tcatactctt 10440cctttttcaa tattattgaa gcatttatca gggttattgt
ctcatgagcg gatacatatt 10500tgaatgtatt tagaaaaata aacaaatagg
ggttccgcgc acatttcccc gaaaagtgcc 10560acctgacgtc 105705313PRTHomo
sapiensmisc_featureHuman DNA sequence of PIM-3 5Met Leu Leu Ser Lys
Ile Asn Ser Leu Ala His Leu Arg Ala Ala Pro 1 5 10 15 Cys Asn Asp
Leu His Ala Thr Lys Leu Ala Pro Gly Lys Glu Lys Glu 20 25 30 Pro
Leu Glu Ser Gln Tyr Gln Val Gly Pro Leu Leu Gly Ser Gly Gly 35 40
45 Phe Gly Ser Val Tyr Ser Gly Ile Arg Val Ser Asp Asn Leu Pro Val
50 55 60 Ala Ile Lys His Val Glu Lys Asp Arg Ile Ser Asp Trp Gly
Glu Leu 65 70 75 80 Pro Asn Gly Thr Arg Val Pro Met Glu Val Val Leu
Leu Lys Lys Val 85 90 95 Ser Ser Gly Phe Ser Gly Val Ile Arg Leu
Leu Asp Trp Phe Glu Arg 100 105 110 Pro Asp Ser Phe Val Leu Ile Leu
Glu Arg Pro Glu Pro Val Gln Asp 115 120 125 Leu Phe Asp Phe Ile Thr
Glu Arg Gly Ala Leu Gln Glu Glu Leu Ala 130 135 140 Arg Ser Phe Phe
Trp Gln Val Leu Glu Ala Val Arg His Cys His Asn 145 150 155 160 Cys
Gly Val Leu His Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp 165 170
175 Leu Asn Arg Gly Glu Leu Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu
180 185 190 Leu Lys Asp Thr Val Tyr Thr Asp Phe Asp Gly Thr Arg Val
Tyr Ser 195 200 205 Pro Pro Glu Trp Ile Arg Tyr His Arg Tyr His Gly
Arg Ser Ala Ala 210 215 220 Val Trp Ser Leu Gly Ile Leu Leu Tyr Asp
Met Val Cys Gly Asp Ile 225 230 235 240 Pro Phe Glu His Asp Glu Glu
Ile Ile Arg Gly Gln Val Phe Phe Arg 245 250 255 Gln Arg Val Ser Ser
Glu Cys Gln His Leu Ile Arg Trp Cys Leu Ala 260 265 270 Leu Arg Pro
Ser Asp Arg Pro Thr Phe Glu Glu Ile Gln Asn His Pro 275 280 285 Trp
Met Gln Asp Val Leu Leu Pro Gln Glu Thr Ala Glu Ile His Leu 290 295
300 His Ser Leu Ser Pro Gly Pro Ser Lys 305 310 6404PRTHomo
sapiensmisc_featureHuman pim-1 kinase 44 kDa
isoformmisc_feature(214)..(214)Xaa can be any naturally occurring
amino acid 6Met Pro His Glu Pro His Glu Pro Leu Thr Pro Pro Phe Ser
Ala Leu 1 5 10 15 Pro Asp Pro Ala Gly Ala Pro Ser Arg Arg Gln Ser
Arg Gln Arg Pro 20 25 30 Gln Leu Ser Ser Asp Ser Pro Ser Ala Phe
Arg Ala Ser Arg Ser His 35 40 45 Ser Arg Asn Ala Thr Arg Ser His
Ser His Ser His Ser Pro Arg His 50 55 60 Ser Leu Arg His Ser Pro
Gly Ser Gly Ser Cys Gly Ser Ser Ser Gly 65 70 75 80 His Arg Pro Cys
Ala Asp Ile Leu Glu Val Gly Met Leu Leu Ser Lys 85 90 95 Ile Asn
Ser Leu Ala His Leu Arg Ala Ala Pro Cys Asn Asp Leu His 100 105 110
Ala Thr Lys Leu Ala Pro Gly Lys Glu Lys Glu Pro Leu Glu Ser Gln 115
120 125 Tyr Gln Val Gly Pro Leu Leu Gly Ser Gly Gly Phe Gly Ser Val
Tyr 130 135 140 Ser Gly Ile Arg Val Ser Asp Asn Leu Pro Val Ala Ile
Lys His Val 145 150 155 160 Glu Lys Asp Arg Ile Ser Asp Trp Gly Glu
Leu Pro Asn Gly Thr Arg 165 170 175 Val Pro Met Glu Val Val Leu Leu
Lys Lys Val Ser Ser Gly Phe Ser 180 185 190 Gly Val Ile Arg Leu Leu
Asp Trp Phe Glu Arg Pro Asp Ser Phe Val 195 200 205 Leu Ile Leu Glu
Arg Xaa Glu Pro Val Gln Asp Leu Phe Asp Phe Ile 210 215 220 Thr Glu
Arg Gly Ala Leu Gln Glu Glu Leu Ala Arg Ser Phe Phe Trp 225 230 235
240 Gln Val Leu Glu Ala Val Arg His Cys His Asn Cys Gly Val Leu His
245 250 255 Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp Leu Asn Arg
Gly Glu 260 265 270 Leu Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu Leu
Lys Asp Thr Val 275 280 285 Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr
Ser Pro Pro Glu Trp Ile 290 295 300 Arg Tyr His Arg Tyr His Gly Arg
Ser Ala Ala Val Trp Ser Leu Gly 305 310 315 320 Ile Leu Leu Tyr Asp
Met Val Cys Gly Asp Ile Pro Phe Glu His Asp 325 330 335 Glu Glu Ile
Ile Arg Gly Gln Val Phe Phe Arg Gln Arg Val Ser Ser 340 345 350 Glu
Cys Gln His Leu Ile Arg Trp Cys Leu Ala Leu Arg Pro Ser Asp 355 360
365 Arg Pro Thr Phe Glu Glu Ile Gln Asn His Pro Trp Met Gln Asp Val
370 375 380 Leu Leu Pro Gln Glu Thr Ala Glu Ile His Leu His Ser Leu
Ser Pro 385 390 395 400 Gly Pro Ser Lys 72684DNAHomo
sapiensmisc_featureHuman PIM-1 message (mRNA) 7cccgagagga
gtcggtggca gcggcggcgg cgggaccggc agcagcagca gcagcagcag 60cagcaaccac
tagcctcctg ccccgcggcg ctgccgcacg agccccacga gccgctcacc
120ccgccgttct cagcgctgcc cgaccccgct ggcgcgccct cccgccgcca
gtcccggcag 180cgccctcagt tgtcctccga ctcgccctcg gccttccgcg
ccagccgcag ccacagccgc 240aacgccaccc gcagccacag ccacagccac
agccccaggc atagccttcg gcacagcccc 300ggctccggct cctgcggcag
ctcctctggg caccgtccct gcgccgacat cctggaggtt 360gggatgctct
tgtccaaaat caactcgctt gcccacctgc gcgccgcgcc ctgcaacgac
420ctgcacgcca ccaagctggc gcccggcaag gagaaggagc ccctggagtc
gcagtaccag 480gtgggcccgc tactgggcag cggcggcttc ggctcggtct
actcaggcat ccgcgtctcc 540gacaacttgc cggtggccat caaacacgtg
gagaaggacc ggatttccga ctggggagag 600ctgcctaatg gcactcgagt
gcccatggaa gtggtcctgc tgaagaaggt gagctcgggt 660ttctccggcg
tcattaggct cctggactgg ttcgagaggc ccgacagttt cgtcctgatc
720ctggagaggc ccgagccggt gcaagatctc ttcgacttca tcacggaaag
gggagccctg 780caagaggagc tggcccgcag cttcttctgg caggtgctgg
aggccgtgcg gcactgccac 840aactgcgggg tgctccaccg cgacatcaag
gacgaaaaca tccttatcga cctcaatcgc 900ggcgagctca agctcatcga
cttcgggtcg ggggcgctgc tcaaggacac cgtctacacg 960gacttcgatg
ggacccgagt gtatagccct ccagagtgga tccgctacca tcgctaccat
1020ggcaggtcgg cggcagtctg gtccctgggg atcctgctgt atgatatggt
gtgtggagat 1080attcctttcg agcatgacga agagatcatc aggggccagg
ttttcttcag gcagagggtc 1140tcttcagaat gtcagcatct cattagatgg
tgcttggccc tgagaccatc agataggcca 1200accttcgaag aaatccagaa
ccatccatgg atgcaagatg ttctcctgcc ccaggaaact 1260gctgagatcc
acctccacag cctgtcgccg gggcccagca aatagcagcc tttctggcag
1320gtcctcccct ctcttgtcag atgcccgagg gaggggaagc ttctgtctcc
agcttcccga 1380gtaccagtga cacgtctcgc caagcaggac agtgcttgat
acaggaacaa catttacaac 1440tcattccaga tcccaggccc ctggaggctg
cctcccaaca gtggggaaga gtgactctcc 1500aggggtccta ggcctcaact
cctcccatag atactctctt cttctcatag gtgtccagca 1560ttgctggact
ctgaaatatc ccgggggtgg ggggtggggg tgggtcagaa ccctgccatg
1620gaactgtttt cttcatcatg agttctgctg aatgccgcga tgggtcaggt
aggggggaaa 1680caggttggga tgggatagga ctagcaccat tttaagtccc
tgtcacctct tccgactctt 1740tctgagtgcc ttctgtgggg actccggctg
tgctgggaga aatacttgaa cttgcctctt 1800ttacctgctg cttctccaaa
aatctgcctg ggttttgttc cctatttttc tctcctgtcc 1860tccctcaccc
cctccttcat atgaaaggtg ccatggaaga ggctacaggg ccaaacgctg
1920agccacctgc ccttttttct gcctccttta gtaaaactcc gagtgaactg
gtcttccttt 1980ttggttttta cttaactgtt tcaaagccaa gacctcacac
acacaaaaaa tgcacaaaca 2040atgcaatcaa cagaaaagct gtaaatgtgt
gtacagttgg catggtagta tacaaaaaga 2100ttgtagtgga tctaattttt
aagaaatttt gcctttaagt tattttacct gtttttgttt 2160cttgttttga
aagatgcgca ttctaacctg gaggtcaatg ttatgtattt atttatttat
2220ttatttggtt cccttcctat tccaagcttc catagctgct gccctagttt
tctttcctcc 2280tttcctcctc tgacttgggg accttttggg ggagggctgc
gacgcttgct ctgtttgtgg 2340ggtgacggga ctcaggcggg acagtgctgc
agctccctgg cttctgtggg gcccctcacc 2400tacttaccca ggtgggtccc
ggctctgtgg gtgatgggga ggggcattgc tgactgtgta 2460tataggataa
ttatgaaaag cagttctgga tggtgtgcct tccagatcct ctctggggct
2520gtgttttgag cagcaggtag cctgctggtt ttatctgagt gaaatactgt
acaggggaat 2580aaaagagatc ttattttttt ttttatactt ggcgtttttt
gaataaaaac cttttgtctt 2640aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaa 268485826DNAHomo sapiensmisc_featureexemplary Human
PIM-1 gene 8cgcgcgcggc gaatctcaac gctgcgccgt ctgcgggcgc ttccgggcca
ccagtttctc 60tgctttccac cctggcgccc cccagccctg gctccccagc tgcgctgccc
cgggcgtcca 120cgccctgcgg gcttagcggg ttcagtgggc tcaatctgcg
cagcgccacc tccatgttga 180ccaagcctct acaggggcct cccgcgcccc
ccgggacccc cacgccgccg ccaggtgagt 240acatcctccc ctactgcaac
cagacggggt gggctggaat gatgggttgc agcgcggggg 300gagggagtcg
tggctgggct cagcacgccg ccaccctgac ttcctcgcct ccgcctgcgt
360aggaggcaag gatcgggaag cgttcgaggc cgagtatcga ctcggccccc
tcctgggtaa 420ggggggcttt ggcaccgtct tcgcaggaca ccgcctcaca
gatcgactcc aggtatccgt 480catgagggtc ttgggagggt caggtgcgtg
tggcgggggc gggggtcctg gccctggaat 540gctggttgac cgaggagtga
gcctgcagag tgtgtagagg accaggtgtg tgtgtgtgtg 600tgtccgtgtc
cgtgtccgag gagtgagcct gcagtgtgtg tagagggcca ggtgtgtgtg
660cgtgcgcgtg tgtgtgtcgg tctaggaggt tatgggcggg gggggggggc
agggggcttc 720agattccgga gttccttgac cccggggtcc aggctgtgta
tgtgtgggaa agcagggacc 780tagatgtgag atttgtggga cttttggagg
taggtgtcca gtgtggagtc atgcggacca 840ggaccctggt acagagttgg
ggtgtcgtag agctaaatag gaagattgtg ggcctggggt 900atcaggaaat
ctagaactca ggacttggag tgatgagtcc tgatgcctga gaacggagag
960cccagggcta aggaaggtgg gagagataaa cttggttccg aggacctgga
gggcagggga 1020gacgccctgg tacgcgttct gtggggtgct gtggttgggg
accagaaaga ctagagtgct 1080ggtagatgga ggaatactgg aggtaggcag
aaggtctaga ctgggagggg tctggggatc 1140acctgctggc ctccttatca
cggccttctt ctccaggtgg ccatcaaagt gattccccgg 1200aatcgtgtgc
tgggctggtc ccccttggtg agtaccttcg gagcccttcc taacctacct
1260actccatcac tgatgtattc acctccttgc ttttccaggg gatgtatgac
tccctgggcc 1320ctgtaacagt gagaatactg ccagtccatt tatactccct
tggggtgaca tacagttctg 1380attcacccca attcccctag agccctggat
tctcccctcc aacaaacctt taccatcctt 1440cctccaaaca ctgctggggg
actgcccgca gggcgtgctg gtggggaaca aggggcagag 1500gtcactggtt
gccatggtga tggtggctgc ttctctcttg ccgttataac gctaacggac
1560atcagggcgg gtctgggcaa gttgtagagt tgggagcgcc ccctggcggg
ctctagggga 1620aactgcgcct gcgcagtcca tgggacccaa agggagaggg
tgcgcctgcg caatatcggt 1680atttttgcat ctcggtgaga aaacgtctgc
tgccgtgcaa gtcagcagcc tggccaggag 1740agggctctac ctcatcccag
aaggttgctg ctcgaagtgt acctgcgcag ggcttgggga 1800ggcagtgggg
ggcggatttt gtggccccca gcgtttatac tttttttttt ttggagacac
1860agtctccctc tgttgcccag gctggagtga ggtgacgcga tctcggctca
ctgcaacctc 1920cgtctcctgg gttcaagtga ttctcctgcc tcagcctccc
aagtagctgg gactacagga 1980gcgcacaacc atgcccggct aatttttgta
tttttagtag agacagggtt tcaccatgtt 2040ggccaggcgg gttttgaact
gctgacctca ggtgatccgc ctgcctcggc cactcaaagt 2100gctgggatta
caggcatgag ccaccacgcc cggctgcatt tatgactttt ttttttcctt
2160gagacggagt ttcgctctgc tgcctgggct ggagtgcagt ggcgtgatct
cagctcactg 2220cagcctccac ctcctgggtt caagcgattc tcctgcctca
ggctcctgag tagctggaat 2280tacaggcacc cgctgccatg cccggctaag
ttttacgttt ttagtagaga ccgtgtttca 2340ccatgttggc caggctggtc
tcgaacccct gacctagtga tctgcccgcc ttgggcctcc 2400caaagtgctg
ggattacagg cgtgagccac cgcgcccagc ctctaatttt gtatttttag
2460tagagacggg gtttctccat gttggtcagg ctggtctcga actcccgacc
tcaggtgatc 2520tgcccgtctc ggcctcccaa agtgctggga ttacaggcgt
gagccactgc gcagggccac 2580atttaggctt tttattggct ggttctaggt
gcttggtgat gctgacaaaa cacatgataa 2640cactaagtcc ttttgtgcta
ggtcctttgt aataaatcac tcagctgttt aacaaattag 2700gtatattgac
cacctactat atgacagaca taattctaga cactcagcaa agtattacat
2760aagtattgag agctcatttt gtgctaggtc cttttttact aattgttttc
acctgtttaa 2820caaatattta ttcagcccta ctctgttagc agccactgtt
ctagtgcttc atatacgtcc 2880gtgaacaaaa caaaccatta cacaataagt
gtttattgag tgctaactgc ttgtcagagc 2940ccatgctatt aagtgctgtc
atctgtttaa catttattga tcacctgtgt aaggtactat 3000tctaatctgg
gatatgtcag ggaacaaaac aaaacacata atggtggtgc tgcttctgct
3060gaaagccttc agttgataac cagatttttc tttgtatttt tgcttgtttg
ttttgagaca 3120gctggagtgc agtggtgtga tcttcactgc aacctctgcc
ttcttggctc aagcgaccct 3180cccacctgag cctcccaagt agctgggact
acaggtgcat gccaccaagc ctggctaatt 3240tttgtgtttg tgccattttg
cccaggctga tcttgaactc ttgggctcaa gcaatccacc 3300cacatcagcc
tcccaaagtg ctgggattgc agggatgagc cactgtgcct ggccgaactt
3360ctttcgttta ttcaaatgtt tattgatcta cgacatgcga gatttgtgca
ggctctttgc 3420tggtttcacc ctctcaatcg ctgtgtgagt ttgtgtcttt
agggaaagtg aggcccagga 3480agggaagtga gttgcttagc gacacactgt
caggaaaagg ggccctgagt tgagcttagg 3540taaaaagcct cagagctgtt
gccctgacat ctgtcttttt tctctccctg cttcccaccc 3600cacctgtgcc
cccagtcaga ctcagtcaca tgcccactcg aagtcgcact gctatggaaa
3660gtgggtgcag gtggtgggca ccctggcgtg atccgcctgc ttgactggtt
tgagacacag 3720gagggcttca tgctggtcct cgagcggcct ttgcccgccc
aggatctctt tgactatatc 3780acagagaagg gcccactggg tgaaggccca
agccgctgct tctttggcca agtagtggca 3840gccatccagc actgccattc
ccgtggagtt gtccatcgtg acatcaagga tgagaacatc 3900ctgatagacc
tacgccgtgg ctgtgccaaa ctcattgatt ttggttctgg tgccctgctt
3960catgatgaac cctacactga ctttgatggt aaggcttctc taaatctccc
tggagggatt 4020gtttttactt gatggccttg tgacctttgg cctccagtgg
tggggtgtcc tgtaatcctt 4080gacccatact gcattatata agatgatcga
ttgctaatac tggggattct cagccttgcc 4140ctctgataaa gtccatcttt
taatggtgtg ctaaccttat tctgggctcc tattctggtg 4200aggggatcct
gttaccatcc tgagtattct ttctctggta aggggatcct gttacttttc
4260agtgctttta ttctgttgag gggactctgt tattttagct gctttttatc
tagtgagggg 4320actctgcttt tatcttgagt gctcttaatt gtggtgaggc
catccttcct ggagagtttg 4380gggttggaga agggcatcat gagattgagt
tggtctaacc cctggcttgt gtgcagggac 4440aagggtgtac agccccccag
agtggatctc tcgacaccag taccatgcac tcccggccac 4500tgtctggtca
ctgggcatcc tcctctatga catggtgtgt ggggacattc cctttgagag
4560ggaccaggag attctggaag ctgagctcca cttcccagcc catgtctccc
caggtgaggc 4620ctcactgacc ccagcccaga agactccatc cttctcaggg
accagtaccc cctactgact 4680gctaatcttc cctctctgct tcttggccta
cagactgctg tgccctaatc cgccggtgcc 4740tggcccccaa accttcttcc
cgaccctcac tggaagagat cctgctggac ccctggatgc 4800aaacaccagc
cgaggatgta cccctcaacc cctccaaagg aggccctgcc cctttggcct
4860ggtccttgct accctaagcc tggcctggcc tggcctggcc cccaatggtc
agaagagcca 4920tcccatggcc atgtcacagg gatagatgga catttgttga
cttggtttta caggtcatta 4980ccagtcatta aagtccagta ttactaaggt
aagggattga ggatcagggg ttagaagaca 5040taaaccaagt ctgcccagtt
cccttcccaa tcctacaaag gagccttcct cccagaacct 5100gtggtccctg
attctggagg gggaacttct tgcttctcat tttgctaagg aagtttattt
5160tggtgaagtt gttcccattc tgagccccgg gactcttatt ctgatgatgt
gtcaccccac 5220attggcacct cctactacca ccacacaaac ttagttcata
tgctcttact tgggcaaggg 5280tgctttcctt ccaatacccc agtagctttt
attttagtaa agggaccctt tcccctagcc 5340tagggtccca tattgggtca
agctgcttac ctgcctcagc ccaggattct ttattctggg 5400ggaggtaatg
ccctgttgtt accccaaggc ttcttttttt tttttttttt tttgggtgag
5460gggaccctac tctgttatcc caagtgctct tattctggtg agaagaacct
tacttccata 5520atttgggaag gaatggaaga tggacaccac cggacaccac
cagacactag gatgggatgg 5580atggtttttt gggggatggg ctaggggaaa
taaggcttgc tgtttgttct cctggggcgc 5640tccctccaac ttttgcagat
tcttgcaacc tcctcctgag ccgggattgt ccaattacta 5700aaatgtaaat
aatcacgtat tgtggggagg ggagttccaa gtgtgccctc ctctcttctc
5760ctgcctggat tatttaaaaa gccatgtgtg gaaacccact atttaataaa
agtaatagaa 5820tcagaa 5826944PRTHomo sapiens 9Met Leu Leu Ser Lys
Phe Gly Ser Leu Ala His Leu Cys Gly Pro Gly 1 5 10 15 Gly Val Asp
His Leu Pro Val Lys Ile Leu Gln Pro Ala Lys Ala Asp 20 25 30 Lys
Glu Ser Phe Glu Lys Ala Tyr Gln Val Gly Ala 35 40 10282PRTHomo
sapiens 10Val Leu Gly Ser Gly Gly Phe Gly Thr Val Tyr Ala Gly Ser
Arg Ile 1 5 10 15 Ala Asp Gly Leu Pro Val Ala Val Lys His Val Val
Lys Glu Arg Val 20 25 30 Thr Glu Trp Gly Ser Leu Gly Gly Ala Thr
Val Pro Leu Glu Val Val 35 40 45 Leu Leu Arg Lys Val Gly Ala Ala
Gly Gly Ala Arg Gly Val Ile Arg 50 55 60 Leu Leu Asp Trp Phe Glu
Arg Pro Asp Gly Phe Leu Leu Val Leu Glu 65 70 75 80 Arg Pro Glu Pro
Ala Gln Asp Leu Phe Asp Phe Ile Thr Glu Arg Gly 85 90 95 Ala Leu
Asp Glu Pro Leu Ala Arg Arg Phe Phe Ala Gln Val Leu Ala 100 105 110
Ala Val Arg His Cys His Ser Cys Gly Val Val His Arg Asp Ile Lys 115
120 125 Asp Glu Asn Leu Leu Val Asp Leu Arg Ser Gly Glu Leu Lys Leu
Ile 130 135 140 Asp Phe Gly Ser Gly Ala Leu Leu Lys Asp Thr Val Tyr
Thr Asp Phe 145 150 155 160 Asp Gly Thr Arg Val Tyr Ser Pro Pro Glu
Trp Ile Arg Tyr His Arg 165 170 175 Tyr His Gly Arg Ser Ala Thr Val
Trp Ser Leu Gly Val Leu Leu Tyr 180 185 190 Asp Met Val Cys Gly Asp
Ile Pro Phe Glu Gln Asp Glu Glu Ile Leu 195 200 205 Arg Gly Arg Leu
Leu Phe Arg Arg Arg Val Ser Pro Glu Cys Gln Gln 210 215 220 Leu Ile
Arg Trp Cys Leu Ser Leu Arg Pro Ser Glu Arg Pro Ser Leu 225 230 235
240 Asp Gln Ile Ala Ala His Pro Trp Met Leu Gly Ala Asp Gly Gly Ala
245 250 255 Pro Glu Ser Cys Asp Leu Arg Leu Cys Thr Leu Asp Pro Asp
Asp Val 260 265 270 Ala Ser Thr Thr Ser Ser Ser Glu Ser Leu 275 280
112392DNAHomo sapiens 11agcggaccga cgcgacacgc cgtgcgcctc cgcggctgcg
ctacgaaaac gagtcccgga 60gcggccccgc gcccgccgca cccggccctc gcccacccga
agacaggcgc ccagctgccc 120cgccgtctcc ccagctagcg cccggccgcc
gccgcctcgc gggccccggg cggaaggggg 180cggggtcccg attcgccccg
cccccgcgga gggatacgcg gcgccgcggc ccaaaacccc 240cgggcgaggc
ggccggggcg ggtgaggcgc tccgcctgct gctcgtctac gcggtccccg
300cgggccttcc gggcccactg cgccgcgcgg accgcctcgg gctcggacgg
ccggtgtccc 360cggcgcgccg ctcgcccgga tcggccgcgg cttcggcgcc
tggggctcgg ggctccgggg 420aggccgtcgc ccgcgatgct gctctccaag
ttcggctccc tggcgcacct ctgcgggccc 480ggcggcgtgg accacctccc
ggtgaagatc ctgcagccag ccaaggcgga caaggagagc 540ttcgagaagg
cgtaccaggt gggcgccgtg ctgggtagcg gcggcttcgg cacggtctac
600gcgggtagcc gcatcgccga cgggctcccg gtggctgtga agcacgtggt
gaaggagcgg 660gtgaccgagt ggggcagcct gggcggcgcg accgtgcccc
tggaggtggt gctgctgcgc 720aaggtgggcg cggcgggcgg cgcgcgcggc
gtcatccgcc tgctggactg gttcgagcgg 780cccgacggct tcctgctggt
gctggagcgg cccgagccgg cgcaggacct cttcgacttt 840atcacggagc
gcggcgccct ggacgagccg ctggcgcgcc gcttcttcgc gcaggtgctg
900gccgccgtgc gccactgcca cagctgcggg gtcgtgcacc gcgacattaa
ggacgaaaat 960ctgcttgtgg acctgcgctc cggagagctc aagctcatcg
acttcggttc gggtgcgctg 1020ctcaaggaca cggtctacac cgacttcgac
ggcacccgag tgtacagccc cccggagtgg 1080atccgctacc accgctacca
cgggcgctcg gccaccgtgt ggtcgctggg cgtgcttctc 1140tacgatatgg
tgtgtgggga catccccttc gagcaggacg aggagatcct ccgaggccgc
1200ctgctcttcc ggaggagggt ctctccagag tgccagcagc tgatccggtg
gtgcctgtcc 1260ctgcggccct cagagcggcc gtcgctggat cagattgcgg
cccatccctg gatgctgggg 1320gctgacgggg gcgccccgga gagctgtgac
ctgcggctgt gcaccctcga ccctgatgac 1380gtggccagca ccacgtccag
cagcgagagc ttgtgaggag ctgcacctga ctgggagcta 1440ggggaccacc
tgccttggcc agacctggga cgcccccaga ccctgacttt ttcctgcgtg
1500ggccgtctcc tcctgcggaa gcagtgacct ctgacccctg gtgaccttcg
ctttgagtgc 1560cttttgaacg ctggtcccgc gggacttggt tttctcaagc
tctgtctgtc caaagacgct 1620ccggtcgagg tcccgcctgc cctgggtgga
tacttgaacc ccagacgccc ctctgtgctg 1680ctgtgtccgg aggcggcctt
cccatctgcc tgcccacccg gagctctttc cgccggcgca 1740gggtcccaag
cccacctccc gccctcagtc ctgcggtgtg cgtctgggca cgtcctgcac
1800acacaatgca agtcctggcc tccgcgcccg cccgcccacg cgagccgtac
ccgccgccaa 1860ctctgttatt tatggtgtga ccccctggag gtgccctcgg
cccaccgggg ctatttattg 1920tttaatttat ttgttgaggt tatttcctct
gagcagtctg cctctcccaa gccccagggg 1980acagtgggga ggcaggggag
ggggtggctg tggtccaggg accccaggcc ctgattcctg 2040tgcctggcgt
ctgtcctggc cccgcctgtc agaagatgaa catgtatagt ggctaactta
2100aggggagtgg gtgaccctga cacttccagg cactgtgccc agggtttggg
ttttaaatta 2160ttgactttgt acagtctgct tgtgggctct gaaagctggg
gtggggccag agcctgagcg 2220tttaatttat tcagtacctg tgtttgtgtg
aatgcggtgt gtgcaggcat cgcagatggg 2280ggttctttca gttcaaaagt
gagatgtctg gagatcatat ttttttatac aggtatttca 2340attaaaatgt
ttttgtacat aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 239212992DNAHomo
sapiens 12agcttcgaat tatgctcttg tccaaaatca actcgcttgc ccacctgcgc
gccgcgccct 60gcaacgacct gcacgccacc aagctggcgc ccggcaagga gaaggagccc
ctggagtcgc 120agtaccaggt gggcccgcta ctgggcagcg gcggcttcgg
ctcggtctac tcaggcatcc 180gcgtctccga caacttgccg gtggccatca
aacacgtgga gaaggaccgg atttccgact 240ggggagagct gcctaatggc
actcgagtgc ccatggaagt ggtcctgctg aagaaggtga 300gctcgggttt
ctccggcgtc attaggctcc tggactggtt cgagaggccc gacagtttcg
360tcctgatcct ggagaggccc gagccggtgc aagatctctt cgacttcatc
acggaaaggg 420gagccctgca agaggagctg gcccgcagct tcttctggca
ggtgctggag gccgtgcggc 480actgccacaa ctgcggggtg ctccaccgcg
acatcaagga cgaaaacatc cttatcgacc 540tcaatcgcgg cgagctcaag
ctcatcgact tcgggtcggg ggcgctgctc aaggacaccg 600tctacacgga
cttcgatggg acccgagtgt atagccctcc agagtggatc cgctaccatc
660gctaccatgg caggtcggcg gcagtctggt ccctggggat cctgctgtat
gatatggtgt 720gtggagatat tcctttcgag catgacgaag agatcatcag
gggccaggtt ttcttcaggc 780agagggtctc ttcagaatgt cagcatctca
ttagatggtg cttggccctg agaccatcag 840ataggccaac cttcgaagaa
atccagaacc atccatggat gcaagatgtt ctcctgcccc 900aggaaactgc
tgagatccac ctccacagcc tgtcgccggg gcccagcagc ctgtcgccgg
960ggcccagcaa acaattggta ccgcgggccc gg 99213942DNAHomo sapiens
13atgctcttgt ccaaaatcaa ctcgcttgcc cacctgcgcg ccgcgccctg caacgacctg
60cacgccacca agctggcgcc cggcaaggag aaggagcccc tggagtcgca gtaccaggtg
120ggcccgctac tgggcagcgg cggcttcggc tcggtctact caggcatccg
cgtctccgac 180aacttgccgg tggccatcaa acacgtggag aaggaccgga
tttccgactg gggagagctg 240cctaatggca ctcgagtgcc catggaagtg
gtcctgctga agaaggtgag ctcgggtttc 300tccggcgtca ttaggctcct
ggactggttc gagaggcccg acagtttcgt cctgatcctg 360gagaggcccg
agccggtgca agatctcttc gacttcatca cggaaagggg agccctgcaa
420gaggagctgg cccgcagctt cttctggcag gtgctggagg ccgtgcggca
ctgccacaac 480tgcggggtgc tccaccgcga catcaaggac gaaaacatcc
ttatcgacct caatcgcggc 540gagctcaagc tcatcgactt cgggtcgggg
gcgctgctca aggacaccgt ctacacggac 600ttcgatggga cccgagtgta
tagccctcca gagtggatcc gctaccatcg ctaccatggc 660aggtcggcgg
cagtctggtc cctggggatc ctgctgtatg atatggtgtg tggagatatt
720cctttcgagc atgacgaaga gatcatcagg ggccaggttt tcttcaggca
gagggtctct 780tcagaatgtc agcatctcat tagatggtgc ttggccctga
gaccatcaga taggccaacc 840ttcgaagaaa tccagaacca tccatggatg
caagatgttc tcctgcccca ggaaactgct 900gagatccacc tccacagcct
gtcgccgggg cccagcaaat ag 94214942DNAHomo sapiens 14atgctcctgt
ccaagatcaa ctccctggcc cacctgcgcg ccgcgccctg caacgacctg 60cacgccacca
agctggcgcc gggcaaagag aaggagcccc tggagtcgca gtaccaggtg
120ggcccgctgt tgggcagcgg tggcttcggc tcggtctact ctggcatccg
cgtcgccgac 180aacttgccgg tggccattaa gcacgtggag aaggaccgga
tttccgattg gggagaactg 240cccaatggca cccgagtgcc catggaagtg
gtcctgttga agaaggtgag ctcggacttc 300tcgggcgtca ttagacttct
ggactggttc gagaggcccg atagtttcgt gctgatcctg 360gagaggcccg
aaccggtgca agacctcttc gactttatca ccgaacgagg agccctacag
420gaggacctgg cccgaggatt cttctggcag gtgctggagg ccgtgcggca
ttgccacaac 480tgcggggttc tccaccgcga catcaaggac gagaacatct
taatcgacct gagccgcggc 540gaaatcaaac tcatcgactt cgggtcgggg
gcgctgctca aggacacagt ctacacggac 600tttgatggga cccgagtgta
cagtcctcca gagtggattc gctaccatcg ctaccacggc 660aggtcggcag
ctgtctggtc ccttgggatc ctgctctatg acatggtctg cggagatatt
720ccgtttgagc acgatgaaga gatcatcaag ggccaagtgt tcttcaggca
aactgtctct 780tcagagtgtc agcaccttat taaatggtgc ctgtccctga
gaccatcaga tcggccctcc 840tttgaagaaa tccggaacca tccatggatg
cagggtgacc tcctgcccca ggcagcttct 900gagatccatc tgcacagtct
gtcaccgggg tccagcaagt ag 942
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