U.S. patent application number 12/032615 was filed with the patent office on 2008-09-25 for preparative regimen for engraftment, growth and differentiation of non-hematopoeitic cells in vivo after transplantation.
This patent application is currently assigned to Varian Medical Systems Technologies, Inc.. Invention is credited to Alan Alfieri, Chandan Guha, Jayanta Roy-Chowdury, Robert M. Sutherland.
Application Number | 20080233088 12/032615 |
Document ID | / |
Family ID | 39690553 |
Filed Date | 2008-09-25 |
United States Patent
Application |
20080233088 |
Kind Code |
A1 |
Guha; Chandan ; et
al. |
September 25, 2008 |
PREPARATIVE REGIMEN FOR ENGRAFTMENT, GROWTH AND DIFFERENTIATION OF
NON-HEMATOPOEITIC CELLS IN VIVO AFTER TRANSPLANTATION
Abstract
The invention relates to methods of obtaining an expanded
population of mammalian ex vivo cells and/or for treating a
mammalian subject by (a) administering to a subject an effective
amount of an agent that confers a growth disadvantage to at least a
subset of endogenous cells at the site of engraftment; (b)
administering to the subject an effective amount of a mitogenic
stimulus for the ex vivo cells; and (c) administering the ex vivo
cells to the subject, wherein the ex vivo cells engraft at the site
and proliferate to a greater extent than the subset of endogenous
cells, to repopulate at least a portion of the engraftment site
with the ex vivo cells. The repopulated cells can be harvested for
further use or be left at the engraftment site of a subject to be
treated. The invention also provides methods of treating brain
injury in a subject by engrafting ex vivo cells at the site of
injury.
Inventors: |
Guha; Chandan; (Scarsdale,
NY) ; Sutherland; Robert M.; (Menlo Park, CA)
; Alfieri; Alan; (Garden City, NY) ; Roy-Chowdury;
Jayanta; (New Rochelle, NY) |
Correspondence
Address: |
Townsend and Townsend and Crew LLP (Varian)
Two Embarcadero Center, Eighth Floor
San Francisco
CA
94111
US
|
Assignee: |
Varian Medical Systems
Technologies, Inc.
Palo Alto
CA
Montefiore Medical Center
Bronx
NY
|
Family ID: |
39690553 |
Appl. No.: |
12/032615 |
Filed: |
February 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60890444 |
Feb 16, 2007 |
|
|
|
Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
Y02A 50/463 20180101;
A61K 38/1833 20130101; A61K 35/407 20130101; A61K 38/1833 20130101;
A61L 2430/28 20130101; A61K 38/204 20130101; A61L 2400/06 20130101;
A61K 35/407 20130101; A61K 38/00 20130101; A61K 2300/00 20130101;
A61L 27/3804 20130101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Claims
1. A method of treating a mammalian subject with an organ having a
reduced function by grafting mammalian ex vivo cells which
supplement the function and by promoting proliferation of the
engrafted cells at the site of engraftment, the method comprising
the steps of: (a) administering to the subject an effective amount
of an agent that confers a growth disadvantage to at least a subset
of endogenous cells at the site of engraftment; (b) administering
to the subject an effective amount of a mitogenic stimulus for the
ex vivo cells; and (c) administering the ex vivo cells to the
subject, wherein the ex vivo cells engraft at the site and
proliferate to a greater extent than the subset of endogenous
cells, to repopulate at least a portion of the engraftment site
with the ex vivo cells, wherein the repopulated ex vivo cells
supplement or provide the function.
2. The method of claim 1, wherein the organ with the reduced
function is the intestine, liver, lung, kidney, pancreas, eye,
testes, ovary, brain, spinal cord, or skin.
3. The method of claim 1, wherein the organ with the reduced
function is selected from the group consisting of intestine and
brain.
4. The method of claim 1, wherein the organ with the reduced
function was damaged by irradiation, trauma, chemical or drug
exposure, a genetic illness, or an infectious disease.
5. The method of claim 1, wherein the ex vivo cells are selected
from the group consisting of adult somatic cells, adult progenitor
cells, adult stem cells, embryonic progenitor cells, fetal cells,
and embryonic stem cells from the same species as the subject or
from a different mammalian species than the subject.
6. The method of claim 1, wherein the ex vivo cells are
heterologous to the subject.
7. The method of claim 1, wherein the ex vivo cells are autologous
to the subject.
8. The method of claim 1, wherein the ex vivo cells are cultured,
expanded or modified prior to their administration to the
subject.
9. The method of claim 1, wherein the ex vivo cells are harvested
and sorted from a heterogeneous cell population prior to their
administration to the subject.
10. The method of claim 1, wherein the agent that confers a growth
disadvantage to at least a subset of endogenous cells is
radiation.
11. The method of claim 10, wherein the radiation is selected from
the group consisting of x-ray and gamma ray.
12. The method of claim 1, wherein the agent that confers a growth
disadvantage to at least a subset of endogenous cells is selected
from the group consisting of cytotoxic chemicals, ultrasound, heat,
biological agents, misfolded proteins, and proteins or nucleic
acids that suppress cell division.
13. The method of claim 1, wherein the protein that suppresses cell
division is an antibody.
14. The method of claim 1, wherein the mitogenic stimuli comprises
one or more growth factors selected from the group consisting of
HGF, EGF, FGF, VEGF, NGF, 1'-6, TNF-alpha, CTNF, R-spondin 1,
Noggin, and TWEAK.
15-18. (canceled)
19. The method of claim 1, wherein the organ with reduced function
was damaged by a neurodegenerative disease.
20-31. (canceled)
32. A method of obtaining an expanded population of ex vivo cells,
comprising the steps of: (a) administering to a non-human mammalian
subject an effective amount of an agent that confers a growth
disadvantage to at least a subset of endogenous cells at a site of
engraftment; (b) administering to the non-human mammalian subject
an effective amount of a mitogenic stimuli for the ex vivo cells;
and (c) administering the ex vivo cells t the subject, wherein the
ex vivo cells engraft at the site and proliferate to a greater
extent than the subset of endogenous cells, thereby repopulating at
least a portion of the engraftment site with the ex vivo cells, and
(d) harvesting the repopulated cells from the engraftment site;
thereby obtaining the expanded ex vivo cell population.
33. The method of claim 32, wherein the ex vivo cells are
human.
34. The method of claim 32, wherein the ex vivo cells are selected
from the group consisting of adult somatic cells, adult progenitor
cells, adult stem cells, embryonic progenitor cells, fetal cells,
xenogenic cells, and embryonic stem cells.
35. The method of claim 32, wherein the ex vivo cells heterologous
to the non-human subject.
36. The method of claim 32, wherein the agent that confers a growth
disadvantage to at least a subset of endogenous cells of the organ
is radiation.
37. The method of claim 32, wherein the radiation is selected from
the group consisting of x-ray and gamma ray.
38. The method of claim 32, wherein the agent that confers a growth
disadvantage to at least a subset of endogenous cells of the
damaged organ is selected from the group consisting of cytotoxic
chemicals, ultrasound, heat, biological agents, degenerative
proteins, and proteins that suppress cell division.
39. The method of claim 32, wherein the mitogenic composition
comprises a growth factor selected from the group consisting of
HGF, EGF, FGF, VEGF, NGF, 11-6, TNF-alpha, CTNF, R-spondin 1,
Noggin, and TWEAK.
40. The method of claim 32, wherein the ex vivo cell is derived
from brain, intestinal, or liver tissue.
41. A method of treating a subject having an organ with reduced
function, comprising engrafting cells obtained by the method of
claim 32.
42. A method of treating a subject having an organ with reduced
function, comprising engrafting ex vivo cells obtained by the
method of claim 33.
43. The method of claim 42, wherein the organ is intestine, liver,
lung, kidney, pancreas, eye, testes, brain, ovary, or skin.
44. A method of treating a mammalian subject having a damaged
central nervous system with a reduced function by engrafting
mammalian ex vivo cells at the site of injury, the method
comprising the steps of: (a) administering to the subject an
effective amount of an agent that increases the engraftment of ex
vivo cells at the site of injury; (b) optionally administering to
the subject an effective amount of a mitogenic stimuli for the ex
vivo cells; and (c) administering the ex vivo cells to the subject,
wherein the ex vivo cells engraft at the site of injury and
repopulate at least a portion of the site with the ex vivo cells,
wherein the repopulated ex vivo cells supplement the function,
thereby treating the subject.
45. The method of claim 44, wherein the ex vivo cells are selected
from the group consisting of adult somatic cells, adult neuron
progenitor cells, adult stem cells, embryonic progenitor cells,
fetal cells, xenogenic cells, and embryonic stem cells which are
capable of populating the site of injury with neurons.
46. A method of obtaining an expanded population of ex vivo cells,
comprising the steps of: (a) administering to an irradiated organ
ex vivo or in culture an effective amount of a mitogenic stimuli
for the ex vivo cells; wherein the irradiation confers a growth
disadvantage to at least a subset of endogenous cells at a site
where the ex vivo cells engraft; and (c) administering the ex vivo
cells to the irradiated organ, wherein the ex vivo cells engraft at
the site and proliferate to a greater extent than the subset of
endogenous cells, thereby repopulating at least a portion of the
engraftment site with the ex vivo cells, and (d) harvesting the
repopulated cells from the engraftment site; thereby obtaining the
expanded ex vivo cell population.
47. The method of claim 46, wherein the ex vivo cells are
human.
48. The method of claim 46, wherein the ex vivo cells are selected
from the group consisting of adult somatic cells, adult progenitor
cells, adult stem cells, embryonic progenitor cells, fetal cells,
xenogenic cells, and embryonic stem cells.
49. The method of claim 46, wherein the ex vivo cell is
heterologous to the non-human subject.
50. The method of claim 46, wherein the agent that confers a growth
disadvantage to at least a subset of endogenous cells of the organ
is radiation.
51. The method of claim 46, wherein the radiation is selected from
the group consisting of x-ray and gamma ray.
52. The method of claim 46, wherein the agent that confers a growth
disadvantage to at least a subset of endogenous cells of the
damaged organ is selected from the group consisting of cytotoxic
chemicals, ultrasound, heat, biological agents, degenerative
proteins, and proteins that suppress cell division.
53. The method of claim 46, wherein the mitogenic composition
comprises a growth factor selected from the group consisting of
HGF, EGF, FGF, VEGF, NGF, 11-6, TNF-alpha, CTNF, R-spondin 1,
Noggin, and TWEAK.
54. The method of claim 46, wherein the ex vivo cell is derived
from brain, intestinal, or liver tissue.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority benefit of U.S. Provisional
Patent Application Ser. No. 60/890,444 which was filed on Feb. 16,
2007, which is incorporated herein by reference in its entirety for
all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] NOT APPLICABLE
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ON A COMPACT DISK
[0003] NOT APPLICABLE
FIELD OF THE INVENTION
[0004] This invention relates to methods of cell transplantation
therapy in which the subject is administered an effective amount of
an agent that confers a growth disadvantage to at least a subset of
endogenous cells at the site of engraftment.
BACKGROUND OF THE INVENTION
[0005] Orthotopic whole organ transplantation is expensive and
invasive. Moreover, there is an acute shortage of donor organs.
Accordingly, cell transplantation has been considered as a
potential alternative to whole organ transplantation. Cell
transplantation has been around for two decades but has not been
clinically very useful because even when primary or stem cells can
engraft in organs they often cannot selectively proliferate or
repopulate the intended organ. Accordingly, cell transplantation
without a preparative regimen of is ineffective because of poor
engraftment of the transplanted cells in the host organ.
Administration of growth factors alone cannot offer selective
proliferative/growth advantage to the transplanted cells over the
residual host cells and have not been used clinically.
[0006] There is a great clinical need for cell transplantation
treatments which can restore an organ to health and provide for its
biological functions. For instance, with respect to liver disease,
more than 40,000 patients die of terminal liver diseases every year
in the United States alone, and it is estimated that approximately
20 million suffer from liver diseases (Hagmann, M., Science,
287:1185, 1187 (2000)). For those with inherited metabolic liver
diseases or terminal liver failure, orthotopic liver
transplantation (OLT) is the only treatment option, but most die
without OLT because of a critical shortage of donor livers. Out of
1.3 million patients who may benefit from OLT, only about 4000
patients receive it each year (Hagmann, M., Science, 287:1185, 1187
(2000)). In theory, many patients with primary or metastatic
cancers in the liver could also be cured, or have their survival
and/or the quality of life improved, by total hepatectomy with OLT.
In practice, however, cancer patients are rarely considered for OLT
because of the long waiting lists for donor liver.
[0007] In a clinical trial of hepatocyte transplantation (HT), 7.5
billion normal allogeneic hepatocytes (representing .about.5% of
the hepatocyte mass) were transplanted in a 10-year old girl with
Crigler-Najjar syndrome type 1 (Fox, I. et al., N Engl J Med,
338:1422-1426 (1998)). Although the study demonstrated the
long-term safety of HT, only partial correction of the metabolic
disorder was achieved, because of the lack of proliferation of the
engrafted donor hepatocytes. Similarly, a clinical trial of ex vivo
hepatic gene therapy in patients with familial hypercholesterolemia
failed to demonstrate convincing therapeutic effect (Raper, S. et
al., Ann Surg, 223:116-126 (1996); Grossman, M. et al., Nat Med,
1:1148-1154 (1995)) because only a fraction of the transplanted
cells engrafted and those that engrafted failed to proliferate in
the host liver.
[0008] While it has been established that HT can be employed safely
in humans, its applicability remains limited by: [0009] (i) a
critical shortage of donor hepatocytes, [0010] (ii) the number of
hepatocytes that can be transplanted safely, without causing portal
hypertension, [0011] (iii) the inability of the transplanted
hepatocytes to proliferate in the host liver, and [0012] (iv) lack
of a noninvasive method to evaluate the repopulation of
transplanted hepatocytes in the liver.
[0013] New strategies to provide a selective growth advantage to
the engrafted cells over the host endogenous cells of the target
organ are needed. These strategies can allow the diseased host
cells to be replaced progressively with normal ones.
BRIEF SUMMARY OF THE INVENTION
[0014] In a first aspect, the invention provides a method of
treating a mammalian subject with an organ or tissue having a
reduced function by engrafting mammalian ex vivo cells which
supplement the function of the organ or tissue and by promoting
proliferation of the engrafted cells at the site of engraftment by
(a) administering to the subject an effective amount of an agent
that confers a growth disadvantage to at least a subset of
endogenous cells at the site of engraftment, (b) administering to
the subject an effective amount of a mitogenic stimulus or stimuli
for the ex vivo cells, and (c) administering the ex vivo cells to
the subject, wherein the ex vivo cells engraft at the site and
proliferate to a greater extent than the subset of endogenous
cells, thereby repopulating at least a portion of the engraftment
site with the ex vivo cells, so that the repopulated ex vivo cells
supplement the function. In some embodiments, the organ with the
reduced function is intestine, brain, liver, lung, kidney,
pancreas, eye, testes, ovary, or skin. In other embodiments, the
organ or tissue with the reduced function was damaged by
irradiation, trauma, chemical or drug exposure, a genetic illness,
or an infectious disease. In additional embodiments, the ex vivo
cells can be adult somatic cells, adult progenitor cells, adult
stem cells, embryonic progenitor cells, fetal cells, and embryonic
stem cells from the same species as the subject or from a different
mammalian species than the subject. The ex vivo cells can be
autologous or heterologous with respect to the subject. In yet
other embodiments, the ex vivo cells are cultured prior to their
administration to the subject. In still other embodiments, the ex
vivo cells are harvested and sorted from a heterogeneous cell
population prior to their administration to the subject. In some
embodiments, the engrafted cells are of the same cell type as the
cells of the organ or tissue at the site of engraftment.
[0015] In other embodiments, the agent that confers a growth
disadvantage to at least a subset of endogenous cells is radiation
(e.g. x-ray and gamma ray), a cytotoxic chemical, ultrasound, heat,
biological agent, degenerative proteins, and proteins or nucleic
acids that suppress cell division. The protein may be an antibody
directed to a cell surface receptor that regulates cell division or
apoptosis.
[0016] In other embodiments, the mitogenic stimulus may comprise
one or more growth factors for the cell type to be engrafted. In
some embodiments, the growth factors are one, two, three or more
growth factors selected from the group consisting of HGF, EGF, FGF,
VEGF, NGF, Il-6, TNF-alpha, CTNF, R-spondin 1, Noggin, and TWEAK.
In other embodiments, the administered ex vivo cells comprise,
contain, or include a heterologous gene that increases their
intrinsic proliferation capacity or survival. In some embodiments,
the mitogenic stimulus can comprise a biological agent, antibody or
peptide factor that increases the proliferation capacity of the
donor cells or comprise a procedure that deliberately injures the
engraftment site to enable entry and integration of the
transplanted cells into the host parenchyma and/or to provide a
compensatory growth signal for the ex vivo cells. In some further
embodiments, the procedure is a surgical resection, portal vein
branch ligation, portal vein embolism by chemotherapeutic agents or
toxins, radiofrequency ablation, radiosurgical ablation, or high
frequency ultrasonic ablation of the a portion of the donor
organ.
[0017] The subject to be treated is a mammal, preferably, a human.
The ex vivo cells can be heterologous to the engraftment site. In
some other embodiments, the engraftment site is the organ or tissue
having the reduced function or elsewhere. In some embodiments, the
ex vivo cells express a heterologous nucleic acid for gene therapy
of the reduced function. In further embodiments of any of the
above, immunosuppressive therapy is also administered to the host
following administration of the ex vivo cells.
[0018] There is, in some further embodiments, a proviso that the
organ or tissue is not bone marrow or a proviso that the damage is
not due to cancer or a cancer. There is, in some further
embodiments, a proviso that the damage to be treated is one which
was not caused by exposure to the agent to be used to confer the
growth disadvantage. There is, in some further embodiments, a
proviso that the damaged organ or tissue is not the pancreas or
islet cell. In some embodiments, there is a proviso that the
mammalian growth factor is not HGF or a nucleic acid encoding HGF
(e.g., an adenoviral vector comprising a nucleic acid encoding
HGF).
[0019] In some embodiments, the ex vivo cell is derived from a
precursor cell which is capable of differentiating into a cell type
of the engraftment site or of the organ or tissue with the reduced
function. In some embodiments, the ex vivo cell can be an
epithelial cell, a hepatocyte, a nerve cell, a muscle cell, a
kidney cell, a pancreatic islet .beta.-cell or a precursor thereto.
In some embodiments, the ex vivo cells are genetically modified to
modulate or eliminate the expression of an endogenous gene or to
express a gene encoding a protein not endogenous expressed in the
cell. In some embodiments, the cells have been genetically modified
to enhance their proliferation rate or survival without
immortalization. In some further embodiments, the cells are
genetically modified to become immortalized.
[0020] Where the ex vivo cells are sensitive to the growth
disadvantaging properties of the agent that confers a growth
disadvantage to at least a subset of the endogenous cells of the
organ, step (a) is performed before step (c). Otherwise steps (a),
(b) and (c) may be performed concurrently or in any order. With a
preferred order having step (a) being performed first and then step
(b) followed by step (c), or steps (b) and (c) being performed
concurrently, or step (c) being performed before step (b). In
instances, where the ex vivo cells are antigenically different from
the host, immunosuppressive therapy may be further
administered.
[0021] In some embodiments, the ex vivo cell expresses a protein
not expressed by the host tissue at its engraftment site and the
presence of the protein is used to selectively detect the ex vivo
cells as opposed to the host cells at the engraftment site. In a
further embodiment, the protein is an enzyme whose reaction
products are detected using magnetic resonance spectroscopic
imaging. In some further embodiments still, the protein is creatine
kinase and a reaction product (e.g., phosphocreatine) containing
.sup.31P is detected using magnetic resonance spectroscopic imaging
in vivo.
[0022] In additional embodiments, the invention also provides a
method of treating a subject with a damaged organ or tissue/organ
or tissue having a reduced function by grafting ex vivo cells onto
the organ or tissue and promoting proliferation of the engrafted
cells by (a) administering to the subject an effective amount of an
agent that confers a growth disadvantage to at least a subset of
endogenous cells of the damaged organ; (b) administering to the
subject an effective amount of a mitogenic stimulus for the ex vivo
cells; and (c) administering the ex vivo cells to the organ,
wherein the ex vivo cells engraft and proliferate and the
endogenous cells having a growth disadvantage fail to proliferate
or proliferate at a reduced rate or lesser rate than the engrafted
ex vivo cells, thereby repopulating the damaged organ or tissue
with ex vivo cells and treating the subject with the damaged organ
or tissue. Where the ex vivo cells are sensitive to the growth
disadvantaging properties of the agent that confers a growth
disadvantage to at least a subset of the endogenous cells of the
organ, step (a) is performed before step (c). Otherwise steps (a),
(b) and (c) may be performed concurrently or in any order. With a
preferred order having step (a) being performed first and then step
(b) followed by step (c), or steps (b) and (c) being performed
concurrently, or step (c) being performed before step (b). The
damage can be due to trauma, surgery, or disease. The disease may
be inherited/genetic or caused by an environmental agent (e.g.,
dietary deficiency, drug treatment, radiation, chemical exposure,
or infectious agent). In some embodiments, the damaged organ is the
liver, intestine, pancreas, heart, kidney, or a part of the central
nervous system (e.g., brain, spinal cord). In other embodiments,
the damage to be treated is not cancer and/or a result of a cancer.
In yet other embodiments, the subject does not have cancer. The
subject can be a mammal (e.g., human, a non-human primate (e.g., a
chimpanzee, baboon), a rodent (e.g., a mouse, a rat) a pig, a
rabbit). In instances, where the ex vivo cells are antigenically
different from the host, immunosuppressive therapy may be further
administered. In some embodiments, the methods is practiced in
utero to treat a congenital disease. Accordingly, in some
embodiments the organ or tissue to be engrafted in utero is a fetal
organ or tissue or the organ whose function is to be supplemented
in utero is a fetal organ or tissue.
[0023] In some embodiments, the ex vivo cells are adult somatic
cells, adult progenitor cells, adult stem cells, embryonic
progenitor cells, fetal cells, xenogenic cells, or embryonic stem
cells. The ex vivo cells can be fetal hepatocytes, amniotic
epithelial cells, "oval" cells and "small" hepatocytes. In still
other embodiments, the ex vivo cells are heterologous to the
subject. In still other embodiments, the ex vivo cells are
autologous to the subject. In additional embodiments, the ex vivo
cells are the same cell type as an endogenous cell type of the
organ or tissue or heterologous to the organ or tissue. In other
embodiments, the ex vivo cells are not an endogenous cell type of
the organ or tissue. The ex vivo cells can be cultured prior to
administration. In some embodiments, the ex vivo cells express a
heterologous nucleic acid for gene therapy. In some embodiments,
the grafted cells repopulate the damaged organ or tissue with
healthy cells of the same type and/or function as those whose
growth was targeted to be disadvantaged, or disadvantaged, by
administration of the agent that confers a growth disadvantage. In
some embodiments, the ex vivo cells are bone marrow progenitor
cells, embryonic stem cells, neural progenitor cells, endothelial
progenitor cells, progenitor cells obtained from peripheral blood,
cord blood cells, amniotic epithelial cells, fetal cells,
organ-specific stem cells, such as, oval cells, primary
hepatocytes, primary parenchymal cells, human umbilical
microvascular endothelial cells, blood outgrowth endothelial cells,
glia, or human brain microvascular endothelial cells.
[0024] The agent that confers a growth disadvantage to at least a
subset of endogenous cells of the damaged organ can be radiation
(e.g., X-rays, gamma rays), cytotoxic chemicals (e.g., a compound
which is hepatotoxic or toxic to liver parenchymal cells or more
preferably selectively toxic for such; ultrasound; heat (e.g.,
focused ultrasound induced heating); biological agents;
degenerative proteins; or proteins that suppress cell division. In
some embodiments, the agent is partial liver irradiation (e.g.,
using 3-D conformal RT). For instance, hepatic irradiation can be
delivered to portions of the liver while other portions are
shielded (e.g., the right anterior lobes of the liver can be
irradiated after shielding the left anterior and right posterior
and caudate lobes using lead shields). The radiation in some
embodiments is Stereotactic Radiosurgery (SRS), Intensity-Modulated
Radiation Therapy (IMRT), dynamic adaptive radiation therapy, or
image-guided radiation therapy (IGRT). Accordingly, in some
embodiments, use of spatially confined, focally ablative regimen of
single-fraction or hypofractionated irradiation for stem cell
engraftment and growth of human primary parenchymal cells is
contemplated.
[0025] The mitogenic composition can be a mammalian growth factor
(e.g., hepatocyte growth factor (HGF), TGF-beta (transforming
growth factor-beta), neurotrophins (NGF, BDNF, and NT3),
Granulocyte-colony stimulating factor (G-CSF),
Granulocyte-macrophage colony stimulating factor (GM-CSF),
Platelet-derived growth factor (PDGF), Erythropoietin (EPO),
Thrombopoietin (TPO), Myostatin (GDF-8), Growth Differentiation
factor-9 (GDF9), basic fibroblast growth factor (bFGF or FGF2),
epidermal growth factor (EGF), fibroblast growth factor (FGF),
vascular endothelial growth factor (VEGF), nerve growth factor
(NGF), interleukin-6 (Il-6), tumor necrosis factor-alpha
(TNF-alpha), circulating tumour necrosis factor (CTNF), R-spondin
1, Noggin, c-met activating antibody, and tumor necrosis
factor-like weak inducer of apoptosis (TWEAK, growth receptor
ligand) which can be selective for the type of transplated cell.
The mitogen can be administered in the form of gene therapy or as a
protein preparation. The protein may be recombinant. In some
embodiments, the proliferative stimuli or mitogenic composition can
be either a combination of growth factors such as HGF+
tri-iodo-thyronine (T3), or HGF+EGF, or a combination of growth
factor and compensatory regenerative stimuli.
[0026] In some embodiments, the organ damage is a degenerative
disease (e.g, a neurodegenerative disease). In other embodiments,
the organ damage is a disease selected from the group consisting of
liver cancer, hepatitis A, hepatitis B, hepatitis C, hepatic
fibrosis, cirrhosis, Wilson's disease, alpha1-antitrypsin disease,
Niemann-Pick disease, Tyrosinemia Type I, Protophophyria, Ornithine
transcarbamylase deficiency, Parkinson's disease, Alzheimer's
disease, and Crohn's disease; hepatitis, inflammation of the liver,
caused mainly by various viruses but also by some poisons and/or
chemicals (e.g., carbon tetrachloride), autoimmunity or hereditary
conditions. hemochromatosis; primary sclerosing cholangitis;
primary biliary cirrhosis, autoimmune disease of small bile ducts;
Budd-Chiari syndrome, obstruction of the hepatic vein; and
Gilbert's syndrome, a genetic disorder of bilirubin metabolism;
biliary atresia, alpha-1 antitrypsin deficiency, alagille syndrome,
and progressive familial intrahepatic cholestasis. In some
embodiments, the organ with the reduced function is the kidney,
liver, brain, heart, eye, testes, ovary, skin, lung, pancreas,
spleen, or kidney. In some embodiments, the ex vivo cell is derived
from a precursor cell which is capable of differentiating into a
cell type of the engraftment site or organ with the reduced
function. In some embodiments, the ex vivo cell can be an
epithelial cell, a hepatocytes, a nerve cell, a muscle cell, a
pancreatic islet .beta.-cell or a precursor thereto.
[0027] Accordingly, the invention provides a method of treating a
subject having organ damage due to radiation exposure, infection,
or toxic exposure, the method comprising the steps of administering
to the subject an effective amount of a mitogenic composition; and
administering ex vivo cells to the subject, wherein the ex vivo
cells engraft and proliferate, thereby repopulating the damaged
organ. In some embodiments, the grafted cells can serve to
repopulate the damaged organ with healthy cells of the same type
and/or function as those damaged by the radiation exposure,
infection or toxic exposure. In some embodiments, the damaged organ
is the liver, intestine, pancreas, heart, kidney, lungs, brain,
spleen eye, testes, ovary, or spinal cord, or a part of the central
nervous system (e.g., brain, spinal cord). In other embodiments,
the damage to be treated is not cancer and/or a result of a cancer.
In yet other embodiments, the subject does not have cancer. The
subject can be a human, a non-human animal, a mammal (e.g., human,
a non-human primate (e.g., a chimpanzee, baboon), a rodent (e.g., a
mouse, a rat) a pig, a rabbit).
[0028] In some embodiments, the ex vivo cells are also adult
somatic cells, adult progenitor cells, adult stem cells, embryonic
progenitor cells, or embryonic stem cells. In still other
embodiments, the ex vivo cells are heterologous to the subject. In
still other embodiments, the ex vivo cells are autologous to the
subject. In additional embodiments, the ex vivo cells are the same
cell type as an endogenous cell type of the organ or heterologous
to the organ. In other embodiments, the ex vivo cells are not an
endogenous cell type of the organ. The ex vivo cells can be
cultured prior to administration. In some embodiments, the ex vivo
cells express a heterologous nucleic acid for gene therapy. The
mitogenic composition in this aspect can be a mammalian growth
factor (e.g., HGF, EGF, FGF, VEGF, NGF, Il-6, TNF-alpha, CTNF,
R-spondin 1, Noggin, and TWEAK).
[0029] In some embodiments, the invention provides methods of
treating an organ damaged by exposure to toxic chemicals. The toxic
effects and target organs for a variety of chemicals are disclosed
in Registry of Toxic Effects of Chemical Substances (RTECS), RTECS
(NIOSH 1980 or later editions, including 1995). RTECS is a database
of toxicity information compiled from the published scientific
literature. Prior to 2001, RTECS was maintained by US National
Institute for Occupational Safety and Health (NIOSH). Now it is
maintained by Elsevier MDL. See, also, Olson et al. Ed., Poisoning
and Drug Overdose, 5th Edition, published by McGraw Hill/Lange for
a listing of toxic agents and their effects and target organs.
[0030] There is in some further embodiments, a proviso that the
organ is not bone marrow or a proviso that the damage is not due to
cancer or cancer.
[0031] In one embodiment, endothelial (progenitor, microvascular
and/or differentiated) cell transplantation is used to rescue
radiation-induced gastro-intestinal injury, especially after
exposure to irradiation to the intestines or whole body.
[0032] In another embodiment, the invention provides a method of
grafting ex vivo cells onto a liver or other organ and promoting
proliferation of the engrafted cells in a subject by (a)
administering to the subject an effective amount of an agent that
confers growth disadvantage to at least a subset of endogenous
cells of the liver or other organ; (b) administering to the subject
an effective amount of a mitogenic composition; and (c)
administering the ex vivo cells to the subject, wherein the ex vivo
cells engraft and proliferate and the endogenous cells having a
growth disadvantage proliferate to a lesser extent, thereby
repopulating the liver or other organ with the ex vivo cells. Where
the ex vivo cells are sensitive the growth disadvantaging
properties of the agent that confers a growth disadvantage to at
least a subset of the endogenous cells of the organ, step (a) is
performed before step (c). Otherwise steps (a), (b) and (c) may be
performed concurrently or in any order. With a preferred order
having step (a) being performed first and then step (b) followed by
step (c), or steps (b) and (c) being performed concurrently, or
step (c) being performed before step (b). In yet other embodiments,
there is the further step, after steps (a) to (c), of transplanting
the ex vivo cells into a human subject. In embodiments, the ex vivo
cells are adult somatic cells, adult progenitor cells, adult stem
cells, embryonic progenitor cells, or embryonic stem cells which
can differentiate into a parenchymal cell of the host organ or (in
the case of a heterotopic engraftment) of another organ whose
functional activity is deficient in the host. In still other
embodiments, the ex vivo cells are heterologous to the subject. In
some embodiments, the ex vivo cells are human. In still other
embodiments, the ex vivo cells are autologous, allogeneic, or
xenogenic to the subject. In additional embodiments, the ex vivo
cells are the same cell type as an endogenous cell type of the
organ or heterologous to the organ. In other embodiments, the ex
vivo cells are not an endogenous cell type of the organ and are
cultured prior to administration. In some embodiments, the ex vivo
cells express a heterologous nucleic acid for gene therapy. In
other embodiments, the ex vivo cells have been genetically modified
to eliminate a gene or to increase or decrease the expression of a
protein. In still another embodiment, the ex vivo cell is an
immortalized cell. In other embodiments of any of the above, the ex
vivo cell is optionally further modified to contain genes whose
expression is under the control of a promoter sensitive to a
specific agent (drug, compound, heat, radiation) wherein the genes
so controlled express a protein functioning as a maker or
modulating cell function, cell growth, cell replication, apoptosis,
or survival. The heterologous nucleic acid may express a protein
needed by the subject. The organ may be damaged or healthy. In some
embodiments of any of the above, the ex vivo cells are liver cells
and the target organ is the liver. The subject can be a human or a
mammal (e.g., a non-human primate (e.g., a chimpanzee, baboon,
macaque), a rodent (e.g., a mouse, a rat) a pig, a rabbit). In
instances, where the ex vivo cells are antigenically different from
the host, immunosuppressive therapy may be further administered. In
further embodiments of the above, the invention provides a
preparative regimen of hepatic irradiation and hepatic mitotic
signals, such as, hepatocyte growth factors to grow adult primary
human hepatocytes or human stem cells in a non-human liver.
Development of chimeric immunodeficient or tolerized
immunocompetent (non-human mammals (e.g., mice, SCID mice, nude
mice) with human liver cells or human stem cells is contemplated
wherein such mice can be used as a source of human origin cells
(e.g., stem and parenchymal cells) for ex vivo transplants in
humans in need thereof.
[0033] The agent that confers a growth disadvantage to at least a
subset of endogenous cells of the engraftment site or damaged organ
can be ionizing radiation (e.g., X-rays, gamma rays, ARC therapy,
TOMO therapy, particle therapy, electron therapy, proton therapy,
carbon ion therapy). cytotoxic chemicals, ultrasound, heat,
biological agents, degenerative proteins, or proteins that suppress
cell division. The mitogenic composition can be a mammalian growth
factor (e.g., HGF, EGF, FGF, VEGF, NGF, Il-6, TNF-alpha, CTNF,
R-spondin 1, Noggin, and TWEAK). In some embodiments, the agent is
partial liver irradiation (e.g., using 3-D conformal RT). For
instance, hepatic irradiation can be delivered to portions of the
liver while other portions are shielded (e.g., the right anterior
lobes of the liver can be irradiated after shielding the left
anterior and right posterior and caudate lobes using lead shields).
The radiation in other embodiments is SRS, Intensity-Modulated
Radiation Therapy (IMRT), dynamic adaptive radiation therapy, or
image-guided radiation therapy (IGRT).
[0034] With respect to the liver, preparative hepatic irradiation
can be used to facilitate stem cell/liver cell transplantation for
i) the treatment of inherited liver diseases, ii) liver failure,
iii) ex vivo hepatic gene therapy, iv) rescuing patients with liver
cancer, following chemotherapy or radiation therapy, and v)
expanding human hepatocytes in animal liver for generating animal
models for human-specific hepatic infections and human hepatic
responses to hepatoxic agents, metabolism of drugs, or the response
of the human liver to therapeutic treatments in various disease
states (e.g., infection (e.g., HCV, HBV), toxicity).
[0035] Accordingly, in some embodiments, the invention provides a
method of obtaining an expanded population of ex vivo cells by (a)
administering to a non-human mammalian subject an effective amount
of an agent that confers a growth disadvantage to at least a subset
of endogenous cells at a site of engraftment; (b) administering to
the non-human mammalian subject an effective amount of a mitogenic
stimuli for the ex vivo cells; and (c) administering the ex vivo
cells to the subject, wherein the ex vivo cells engraft at the site
and proliferate to a greater extent than the subset of endogenous
cells, thereby repopulating at least a portion of the engraftment
site with the ex vivo cells, and (d) harvesting the repopulated
cells from the engraftment site; thereby obtaining the expanded ex
vivo cell population. In some of these embodiments, the ex vivo
cells are human. In other further embodiments, the ex vivo cells
are selected from the group consisting of adult somatic cells,
adult progenitor cells, adult stem cells, embryonic progenitor
cells, fetal cells, xenogenic cells, and embryonic stem cells. In
some embodiments, the ex vivo cells heterologous to the non-human
subject. In yet other embodiments, the agent that confers a growth
disadvantage to at least a subset of endogenous cells of the organ
is radiation (e.g., x-ray and gamma ray). In still another further
embodiment, the agent that confers a growth disadvantage to at
least a subset of endogenous cells of the engraftment site is
selected from the group consisting of cytotoxic chemicals,
ultrasound, heat, biological agents, degenerative proteins, and
proteins that suppress cell division. In additional further
embodiments, the mitogenic stimulus comprises one or more (e.g., 1,
2, 3, or 4) growth factors selected from the group consisting of
HGF, EGF, FGF, VEGF, NGF, Il-6, TNF-alpha, CTNF, R-spondin 1,
Noggin, and TWEAK. In still additional further embodiments, the ex
vivo cell is derived from brain, intestinal, or liver tissue.
[0036] In a further related aspect, the invention provides a method
of treating a subject having a damaged organ or tissue or an organ
or tissue with reduced function by engrafting cells obtained by the
above methods. In some embodiments, the organ is intestine, liver,
lung, kidney, pancreas, eye, testes, brain, ovary, or skin. In
another aspect accordingly, the invention provides a chimeric
non-human animal and methods of making such an animal as described
above which has greater than 50, 60, 70 percent or near total
replacement (e.g., 80, 85, 90, 95, 98 percent) of its host organ by
engrafted ex vivo cells (e.g, a host liver repopulated with 50, 60,
70 percent, or near total replacement by, human primary hepatocytes
or human organ specific stem cells).
[0037] In another aspect, the invention provides a method of
treating a mammalian subject having a damaged central nervous
system (e.g., brain, spinal cord) with a reduced function by
engrafting mammalian ex vivo cells at the site of injury, by (a)
administering to the subject an effective amount of an agent that
increases the engraftment of ex vivo cells at the site of injury;
(b) optionally administering to the subject an effective amount of
a mitogenic stimuli for the ex vivo cells; and (c) administering
the ex vivo cells to the subject, wherein the ex vivo cells engraft
at the site of injury and repopulate at least a portion of the site
with the ex vivo cells, wherein the repopulated ex vivo cells
supplement the function, thereby treating the subject. In this
aspect, the ex vivo cells can be selected from the group consisting
of adult somatic cells, adult neuron progenitor cells, adult stem
cells, embryonic progenitor cells, fetal cells, xenogenic cells,
and embryonic stem cells which are capable of populating the site
of injury with neurons.
[0038] In another aspect, the invention provides methods for the
isolation and/or purification of HPCs from donor livers by
selecting cells therefrom for the presence of the EpCAM marker.
Accordingly, in some embodiments, above aspects and embodiments of
the invention employ HPCs which have been selected for or sorted
according to the presence of the EpCAM marker.
ABBREVIATIONS
[0039] ALT, Alanine transaminase
[0040] AST, Aspartate transaminase
[0041] ATP, Adenosine tri-phosphate
[0042] BrdU, bromodeoxyuridine
[0043] CRT, conformal radiation therapy
[0044] DCF, 2',7'-dichlorofluorescin diacetate
[0045] DPPIV, Dipeptidyl peptidase IV
[0046] F344, Fischer 344 rats
[0047] GGT, .gamma.-glutamyl transpeptidase
[0048] HGF, Hepatocyte Growth factor
[0049] HIR, Hepatic irradiation
[0050] HT, Hepatocyte transplantation
[0051] IR, ionizing radiation
[0052] LDH, Lactose dehydrogenase
[0053] OLT, Orthotopic Liver Transplantation
[0054] PET, Positron emission tomography
[0055] PH, Partial hepatectomy
[0056] PVBL, Portal vein branch ligation
[0057] RILD, radiation-induced liver disease
[0058] RT, Radiation therapy
[0059] SRS, Stereotactic radiosurgery
[0060] T3, tri-iodo-thyronine
[0061] UGT1A1, bilirubin-UDP-glucuronosyltransferase
[0062] VOD, veno-occlusive disease
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] FIG. 1. HT ameliorates histomorphological changes of RILD.
A, hepatocellular loss around central veins (C) in rats receiving
PH+HIR at 2 weeks. B, centrizonal steatosis in PH+HIR rats at 1
week. C, amelioration of perivenous hepatocellular loss and
steatosis after 1 week of HT. D and E, Extensive proliferation of
oval cells and bile duct proliferation extending from portal tract
(P) in PH+HIR rats at 17 weeks. F, amelioration of bile duct
proliferation and fibrosis in transplanted rat at 17 weeks.
[0064] FIG. 2. HT Improves survival in rats that received PH+HIR.
Kaplan-Meier analysis of survival shows improved survival (P=0.02,
log-rank test) of PH+HIR-treated rats after HT versus PH+HIR
alone.
[0065] FIG. 3. DPPIV staining of livers. A (.times.4) and B
(.times.320), PH+HT. C (.times.2) and D (.times.320), PH+HIR+HT.
Note the near-total replacement of the irradiated host liver by
DPPIV+ve transplanted cells (red stain) 12 weeks PH+HIR+HT.
[0066] FIG. 4. Complete long-term normalization of serum bilirubin
levels by extensive repopulation of Gunn rat (GR) liver by normal
hepatocytes, transplanted after PH+HIR. Results shown are 5 months
after HT in GR treated with PH+HIR. A. UGT1A1 immunoblot analysis
of GR liver homogenates. Note the presence of UGT1A1 band in GR
treated with PH+HIR. B-D. UGT1A1 immunohistochemical staining of
liver sections--B, Wistar-RHA; C, GR; D, GR after PH+HIR+HT. E.
Serum bilirubin levels after HT in Gunn rats receiving PH (n=4,
.quadrature.), HIR (n=4, ), or HIR+PH (n=9, .DELTA.).
[0067] FIG. 5. Extensive liver repopulation by DPPIV+ve
hepatocytes) in livers of DPPIV-ve F344, congeneic rats, that were
subjected to HIR+PVBL. Selective ligation of the portal venous
branch results in their atrophy and induction of regeneration in
the residual lobes. The right branch of the portal vein was
ligated, followed by irradiation of the anterior liver lobes after
blocking the right posterior and the caudate lobes in DPPIV-ve F344
rats. Note selective repopulation of the anterior lobes (median and
right anterior) by the transplanted cells in these animals (B-E,
Anterior lobe; F, Caudate lobe). A, blanching and apoptosis of
right median lobe 48 hours after PVBL. B. Anterior lobes at 3
weeks. C. Anterior lobes at 3 months. D. Anterior lobes 5 months.
E. DPPIV histochemistry of median lobe 5 months after PVBL+HR+HT
(2.times.). Note the near total replacement of the host hepatocytes
with transplanted DPPIV+ve cells. B to E. Selective lobar
repopulation of transplanted hepatocytes after right middle vein
branch ligation (PVBL)+partial liver irradiation (anterior lobes)
HIR. Note minimal repopulation in the posterior lobes that were
shielded from HIR. F. Posterior Lobe 5 months.
[0068] FIG. 6. Preparative regimen of HIR (50 Gy)+Tri-iodothyronine
(T3) (400 .mu.g/day subQ, every 10 days after HT). A, Extensive
repopulation in Gunn rats. B, Normalization of serum bilirubin in
Gunn rats after HIR+T3+HT. Note that HT after HIR or T3 alone fails
to normalize the serum bilirubin. C, T3 induces DNA synthesis in
liver cells, which is not suppressed by inderal. D-E, Extensive
liver repopulation after T3+HIR. F-G, Moderate liver repopulation
after T3+Inderal+HIR. This regimen could be clinically useful, as
it allows hepatocyte repopulation without the cardiac side effects
of tachycardia produced by T3.
[0069] FIG. 7. HT following HIR and systemic administration of
recombinant adenovirus expressing human Hepatocyte Growth (HGF).
Beta-Gal+ve Rosa hepatocytes were transplanted in congeneic C57Bl/6
mice that received HIR (50 Gy)+Adeno-HGF injection. Histochemical
staining of frozen liver sections for beta-galactosidase 2 (a), 4
(b), 8 (c) and 20 (d) weeks after HT.
[0070] FIGS. 8A and 8B. Focal HIR promotes selective lobar
repopulation. Repopulation of beta-gal+ve Rosa transplanted
hepatocytes in irradiated anterior lobe as compared to shielded
posterior lobe. 8B. A single liver lobe or a portion of the Right
anterior lobe in C57Bl/6 mice after a regimen of regional/focal
HIR+Ad-HGF.
[0071] FIG. 9. Ex vivo UGT1A1 gene therapy in Gunn rats. A.
Experimental design. B. UGT1A1 immuno-histochemistry demonstrating
progressive repopulation of liver lobes by UGT1A1-transduced
hepatocytes following PH+HIR at pretransplant, 2 weeks, 4 weeks, 8
weeks, and 16 weeks. C. Complete correction of serum
hyperbilirubinemia in Gunn rats after ex vivo gene therapy and
transplantation of autologous, conditionally immortalized
hepatocytes.
[0072] FIG. 10. Transplanted liver stem cells (oval cells, OC)
differentiate into hepatocytes and bile ducts in rat livers treated
with HIR and Ad-HGF. A: y-GGT staining on isolated oval cells
before transplantation, B-D: DPPIV histochemistry of fresh frozen
liver sections of F344 rats treated with HIR and Ad-HGF, 6 weeks
after oval cell transplantation. C, DPPIV+ve bile duct. B and D,
DPPIV+ve hepatocytes.
[0073] FIG. 11. Liver stem cells (oval cells, OC) proliferate and
repopulate irradiated median lobes of rats treated with partial HIR
(median lobe) and Ad-HGF. a-c, Liver repopulation in irradiated
median lobes; d-f, engraftment of oval cells without liver
repopulation in caudate lobes that were shielded and not
irradiated. Note cell-cell competition between non-irradiated OC
and irradiated host hepatocytes.
[0074] FIG. 12. Liver stem cells (oval cells) engraft but fail to
proliferate in rats that received oval cell transplantation after
treatment with either HIR alone (a-c) or Ad-HGF alone (d-f) or oval
cell transplantation without any preparative regimen (g-i).
[0075] FIG. 13. Isolation of EpCAM+ve liver stem cells (oval cells)
with magnetic beads. Immunocytochemistry with EpCAM and CK19.
[0076] FIGS. 14A and 14B. EpCAM+ve liver stem cells proliferate and
repopulate irradiated median lobes of rats treated with partial HIR
(median lobe) and Ad-HGF. DPPIV histochemistry demonstrating
DPPIV+ve transplanted donor hepatocytes in DPPIV-ve host liver.
[0077] FIG. 15. Development of bile duct cancer after
transplantation of liver stern cells (oval cells) in rats that
received partial hepatectomy, Gross (a) anterior view, (b)
posterior view of livers transplanted with liver cancer stem cells.
(c), (d). Microscopic views of recipient liver after HE staining.
(c-10X, d-20X). RAL: right anterior lobe.
[0078] FIG. 16. Development of bile duct cancer after
transplantation of liver stem (oval cells) in rats that received
partial hepatectomy. DPPlV staining of recipient liver five weeks
after partial hepatectomy and oval cell transplantation. Long
arrows: strong DPPIV+ gland-like structure. Short black arrow: weak
DPPPIV-gland-like structure. White arrow: DPPEV-gland-like
structure.
DETAILED DESCRIPTION OF THE INVENTION
[0079] Our experiments demonstrate that parenchymal cell
transplantation is of benefit in ameliorating radiation injury and
other injury to organs. FIG. 1D-E demonstrates parenchymal cell
transplantation can modulate the late effects of RT. Accordingly,
cell transplantation can replace the loss of parenchymal cells due
to radiation or other injury of organs. In our RILD model of PH+HIR
in rats, we observed that adult liver stem cells (a.k.a oval cells)
proliferate extensively and attempt to restore the parenchymal cell
loss caused by PH and HIR. Therefore, it was contemplated that
adult progenitor cells or stem cells can engraft in injured organs,
such as, liver and can differentiate into organ-specific
parenchymal cells. Thus a preparative regimen of focal irradiation,
delivered by SRS or IMRT, can serve to ablate parenchymal cells in
various organs and create a microenvironment that promotes the
engraftment, growth and differentiation of progenitor/stem cell in
vivo. The irradiation is thought to increase the engraftment by
inhibiting the proliferation of endogenous cells, thereby leaving
`room` for the engrafted cells to grow.
[0080] The liver was used as our primary model organ. Adult liver
cells (hepatocytes) have remarkable regenerative potential and have
extensively repopulated rodent livers on serial transplantation
(Overturf, K. et al., Am J Pathol, 151:1273-1280 (1997)). However,
shortage of donor hepatocytes and inability to grow primary
hepatocytes in culture have triggered a search for progenitor cells
that can be grown in culture and cryopreserved in "cell banks" for
future use. Accordingly, in exemplary embodiments where the liver
is the target organ, the ex vivo cells are hepatic progenitor/stem
cells--fetal hepatocytes, amniotic epithelial cells, "oval" cells
and "small" hepatocytes. Fetal hepatocytes can differentiate into
primary hepatocytes after intraportal cell transplantation. It is
contemplated that the amniotic epithelial cells can maintain the
plasticity of pregastrulation embryo cells and have the potential
to differentiate to all three germ layers. Miki et al has recently
demonstrated that amniotic epithelial cells isolated from human
term placenta express surface markers normally present on embryonic
stem and germ cells (Miki, T. et al., Stem Cells, 23:1549-1559
(2005)). In addition, amniotic epithelial cells express the
pluripotent stem cell-specific transcription factors
octamer-binding protein 4 (Oct-4) and nanog and can form spheroids
that retain stem cell characteristics under certain culture
conditions. They further demonstrate that amniotic epithelial cells
do not require other cell-derived feeder layers to maintain Oct-4
expression, do not express telomerase, and are nontumorigenic upon
transplantation. Based on immunohistochemical and genetic analysis,
amniotic epithelial cells have the potential to differentiate to
all three germ layers-endoderm (liver, pancreas), mesoderm
(cardiomyocyte), and ectoderm (neural cells) in vitro. Thus, amnion
derived from term placenta after live birth may be a useful and
noncontroversial source of stem cells for cell transplantation and
regenerative medicine. Accordingly, in one embodiment, the ex vivo
cell is a amniotic epithelial cells which has the potential to
engraft, differentiate and repopulate the host liver and rescue it
from radiation injury.
[0081] Adult hepatic progenitor/stem cells (a.k.a. "oval" cells)
expand during liver injury, such as, in alcoholic liver injury, and
submassive parenchymal necrosis as well as experimental injury
models featuring blocked hepatocyte replication, such as, after
HIR. Oval cells can potentially become either hepatocytes or
biliary epithelial cells and may be critical to liver regeneration,
particularly when hepatocyte replication is impaired. Recently, it
was reported that a TNF family member called TWEAK (TNF-like weak
inducer of apoptosis) stimulates oval cell proliferation in mouse
liver through its receptor Fn14 (Jakubowski, A. et al., J Clin
Invest, 115:2330-2340 (2005)). Accordingly, in one embodiment,
liver is treated with ex vivo "oval" cells and optionally, TWEAK is
the mitogenic agent administered to rescue hepatic radiation injury
by stimulating the proliferation of the transplated "oval"
cells.
[0082] Finally, a "small hepatocyte" (SH) fraction has been
described to have a better replicative potential than adult
parenchymal hepatocytes (PaH) (Tateno, C. et al., Hepatology,
31:65-74 (2000); Katayama, S. et al., Am J Pathol, 158:97-105
(2001)). SH can be isolated from the supernatant or the
nonparenchymal component of collagenase-perfused rat liver. In
experiments, we examined whether SH can repopulate faster than PaH.
Extensive studies have been performed to evaluate the growth
potential of a small hepatocyte population (<16 .mu.m diameter)
(Lemire, J. et al., American Journal of Pathology, 139:535-552
(1991)) isolated from the non-parenchymal fractions of
collagenase-perfused livers (Kubota, H. et al., Proc Natl Acad Sci
USA, 97:12132-12137 (2000); Sigal, S. et al., Hepatology,
19:999-1006 (1994); Sigal, S. et al., Differentiation, 59:35-42
(1995); Taniguchi, H. et al., Cell Transplant, 9:697-700 (2000);
Yin, L. et al., Hepatology, 35:315-324 (2002); Suzuki, A. et al.,
Hepatology, 32:1230-1239 (2000); Fujikawa, T. et al., J Hepatol,
39:162-170 (2003)). These cells could proliferate in cell culture
(Tateno, C. et al., Hepatology, 31:65-74 (2000), Tateno, C. et al.,
Am J Pathol, 149:1593-1605 (1996); Tateno, C. et al., Am J Pathol,
148:383-392 (1996); He, Z. et al., Differentiation, 71:281-290
(2003); Ikeda, S. et al., J Hepatol, 37:7-14 (2002)) and retained
their proliferative capacity following long-term cryopreservation
(Ikeda, S. et al., J Hepatol, 37:7-14 (2002)). It was reported that
these cells have a higher proliferative capacity than adult
parenchymal hepatocytes in the retrorsine+PH model of liver
repopulation Katayama, S. et al., Am J Pathol, 158:97-105 (2001).
In the context of clinical application, livers from adult donors
contain very few oval cells, but a larger number of small
hepatocytes can be isolated from the liver by Percoll gradient
fractionation of liver cells. Our studies indicate that small
hepatocytes may indeed have a better replicative potential than
adult parenchymal hepatocytes (Section c.4.4, FIG. 9). Accordingly,
in one embodiment where the liver is the target organ, the ex vivo
cells are SH cells.
DEFINITIONS
[0083] Unless otherwise stated, the following terms used in the
specification and claims have the meanings given below.
[0084] It is noted here that as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural reference unless the context clearly dictates otherwise.
[0085] Ex vivo cells--A mammalian cell capable of repopulating the
host organ. They can be freshly harvested cells, cultured cells,
and/or genetically transformed to provide a genetic marker, correct
a genetic defect in the ex vivo cell (as in the case of some
autologous ex vivo cells), or to increase or modulate their ability
to proliferate when engrafted or in culture. In some embodiments,
the host organ acts as a culture medium for the ex vivo cells. In
some further embodiments, the cultured ex vivo cells can later be
harvested for study or engraftment to another individual. In some
embodiments, the ex vivo cell is a cell type whose function and/or
number is deficient in the host organ. In some embodiments, the ex
vivo cells is of the same cell type as that of the cell whose
number or function is deficient in the host organ. In some
embodiments, the ex vivo cell is a cell which can differentiate
into such a cell. In some embodiments, the ex vivo cell is a
cultured cell (e.g., one that can be passaged at least 25 or 50
times in culture). The ex vivo cell can be derived from the subject
or another individual or another species. The ex vivo cell can be
cultured to increase its population. In some embodiments, the cell
is treated ex vivo to enhance a biological property (rate of
mitosis, gene therapy) before being administered to the subject or
engrafted onto the host. Ex vivo cells harvested from the host
organ or host may be the subject of gene therapy and subsequently
cultured to expand their population and then engrafted into or upon
the host organ. Accordingly, the ex vivo cells can be adult somatic
cells, adult progenitor cells, adult stem cells, embryonic
progenitor cells, fetal cells, cord blood cells, xenogenic cells,
or embryonic stem cells. The ex vivo cells can be fetal
hepatocytes, amniotic epithelial cells, "oval" cells and "small"
hepatocytes. In still other embodiments, the ex vivo cells are
heterologous to the subject. In still other embodiments, the ex
vivo cells are autologous to the subject. In additional
embodiments, the ex vivo cells are the same cell type as an
endogenous cell type of the organ or heterologous to the organ. In
other embodiments, the ex vivo cells are not an endogenous cell
type of the organ. The ex vivo cells can be cultured prior to
administration. In some embodiments, the ex vivo cells express a
heterologous nucleic acid for gene therapy. In some embodiments,
the grafted cells repopulate the damaged organ with healthy cells
of the same type and/or function as those whose growth was targeted
to be disadvantaged, or disadvantaged, by administration of the
agent that confers a growth disadvantage. In some embodiments, the
cells are bone marrow progenitor cells, embryonic stem cells,
neural progenitor cells, endothelial progenitor cells, progenitor
cells obtained from peripheral blood, cord blood cells, amniotic
epithelial cells, fetal cells, organ-specific stem cells, such as,
oval cells, primary hepatocytes, primary parenchymal cells, human
umbilical microvascular endothelial cells, blood outgrowth
endothelial cells, glia, or human brain microvascular endothelial
cells. In one embodiment, the ex vivo cell line is a highly
differentiated immortalized human hepatocytes cell line (e.g., a
highly differentiated immortalized human hepatocyte line with
simian virus 40 large tumor antigen for liver based cell therapy,
see, Li et al., ASAIO J. 51(3):262-8 (2005). Human progenitor liver
epithelial cells with extensive replication capacity and
differentiation into mature hepatocytes are also suitable (see, Yin
et al., Stem Cells 20:338-346 (2002)). In some instances, an ex
vivo cell (e.g., a stem cell) may fuse with an endogenous
hepatocytes to provide the repopulated cell. The ex vivo cells may
be administered locally or systemically. in some embodiments, the
ex vivo cells are cryopreserved and stored for later use.
[0086] For instance, the ex vivo cells can be tissue stem cells
which have the capacity to self-renew and are capable of producing
progeny in at least two lineages. They can also be capable of
long-term tissue reconstitution and serial transplantability.
[0087] Oval cells are liver stem cells which can either give rise
to bile duct cells (cholangiocytes) or liver cells (hepatocytes).
Whether or not the cells become cholangiocytes or hepatocytes
depends on the pathophysiological circumstances they were grown
under. Oval cells can be cryopreserved, stored and grown later.
[0088] In some embodiments, the ex vivo cells are bone marrow
progenitor cells, embryonic stem cells, neural progenitor cells,
endothelial progenitor cells, progenitor cells obtained from
peripheral blood, cord blood cells, amniotic epithelial cells,
fetal cells, organ-specific stem cells, such as, oval cells,
primary hepatocytes, primary parenchymal cells, human umbilical
microvascular endothelial cells, blood outgrowth endothelial cells,
glia, or human brain microvascular endothelial cells.
[0089] In some embodiments, the ex vivo cell expresses a protein or
polypeptide not normally expressed by the host tissue at the
engraftment site and this protein is used as a label to detect the
ex vivo cells. In some embodiments, the ex vivo cells are modified
ex vivo to express a protein or provide a nucleic acid not normally
expressed by the host or by tissues of the engraftment site or by
the damaged organ or organ having a reduced function. In some
embodiments, the modification introduces a gene which is expressed
in other tissues of the host but which is not normally expressed at
the engraftment site tissue or by the damaged organ. In some
embodiments, the gene expresses creatine kinase and the damaged
organ is the liver. The protein or nucleic acid can serve as a
label by which the ex vivo cells and their progeny can be detected
in the host. In some embodiments, the protein can be a fluorescent
protein not otherwise expressed in the host. Detection of the ex
vivo cells can be used to monitor the progress of the engraftment
and to further adjust the regimens used to administer the agents
which confers a growth disadvantage and/or mitogenic stimulus or to
further administer additional ex vivo cells. A preferred method of
evaluating host organ or engraftment site repopulation is taught by
Landis et al., Hepatology 44:1250-1258 (2006), which is
incorporated by reference in its entirety. This method discloses
the use of magnetic resonance spectroscopic imaging detect ex vivo
cells. Accordingly, in some embodiments, the ex vivo cells are
detected using MRSI to detect substrates or products of reactions
suitable for such imaging methods which are produced by enzymes
which are differentially expressed by the ex vivo cells as opposed
to the engraftment site tissues or damaged organ (e.g., .sup.31P
magnetic resonance spectroscopic imaging of liver engrafted with
cells expressing creatine kinase). In some embodiments, the label
is not otherwise expressed in the host and can be used to target
the ex vivo cell for destruction by use of an antibody directed
toward the protein in the event the ex vivo cells prove harmful to
the host. Preferably, in this embodiment, the protein is expressed
on the cell surface of the ex vivo cell. Methods of genetically
modifying cells or making recombinant cells are well known to
persons of ordinary skill in the art.
[0090] "Endogenous" cells are cells originating from the host and
found in the target organ which may compete with ex vivo cells
following their introduction for repopulating the organ.
[0091] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all. In some embodiments according to the invention, the ex vivo
donor cell has been modified by the introduction of a heterologous
nucleic acid or protein or the alteration of a native nucleic acid
or protein, or that the cell is derived from a cell so
modified.
[0092] "Engraft" means to become established as a living part or
attachment of a host organ. The graft may be orthotopic or
heterotopic to the host organ.
[0093] Proliferation references the rate of cell division or
multiplication and increase of cell number.
[0094] "Autologous" references biological mater derived from
tissues or DNA of the subject or host. The ex vivo cells can be
autologous.
[0095] "Heterologous" references biological matter derived from the
tissues or DNA of a different species or different individual of
the same species as the subject or host (e.g., allogenic or
xenogenic). The ex vivo cells can be heterologous.
[0096] Agents that confer a growth disadvantage do not necessarily
kill the cell, but competitively disadvantage the growth of the
cell with respect to the engrafted ex vivo cells. The disadvantage
is with respect to cellular proliferation rates and/or the ability
of the endogenous cells to compete with the ex vivo cells in
populating a host organ or survive. Such agents can be ionizing
radiation, heat, ultrasound, LASAR, cytotoxic chemicals (preferably
toxic chemicals which are selectively toxic for the endogenous
cells of the target organ, and more particularly, the endogenous
cells of the type to be repopulated. Ultrasound, focused
ultrasound, focused ultrasound targeted by magnetic resonance
imaging of the target site temperature, biological agents
(viruses); cell growth suppressors. The agents may be cytostatic or
cytotoxic. They may be biological agents, nucleic acids (e.g.,
siRNA) proteins (e.g., antibodies or polypeptides), drugs or small
molecules that suppress cell division. Preferably, growth or cell
proliferation of the endogenous cells can be reduced by at least
50%, 75%, 80%, 90%, 95% or 98% (or any range therebetween) of the
rate observed for endogenous cells not so treated. The agents may
be administered locally or systemically.
[0097] Mitogenic composition or mitogenic stimulus refer to subject
matter that stimulates cell division such as a growth factor. One
or more stimuli can be used. The stimulus can be a mitogen,
chemokine, cytokine, hormone or agents which acts similarly on
their receptors (e.g., antibodies). Preferred mitogens may be
tissue specific and/or at least stimulate the ex vivo cell type to
be engrafted. Suitable mitogens include mammalian growth factor
(e.g., HGF, EGF, FGF, VEGF, NGF, 11-6, TNF-alpha, circulating
tumour necrosis factor (CTNF), R-spondin 1, Noggin, and TWEAK,
growth receptor ligand) and c-met activating antibody. EGF promotes
proliferation of mesenchymal, glial and epithelial cells. NGF
promotes neurite outgrowth and neural cell survival. FGF promotes
proliferation of many cells; inhibits some stem cells; induces
mesoderm to form in early embryos. FGF has at least 19 family
members and 4 distinct receptors. Additional suitable mitogens
include PDGF which promotes proliferation of connective tissue,
glial and smooth muscle cells and has two different different
protein chains forming 3 distinct dimer forms; AA, AB and BB;
insulin-like growth factor-I (IGF-I) which promotes proliferation
of many cell types and is related to Insulin-like growth factor-II
(IGF-II) and proinsulin, also called Somatomedin C; and IGF-II
which promotes proliferation of many cell types primarily of fetal
origin. The factors may be wild-type (e.g, human, primate, murine,
rat, etc.) or substantially identical to the wild-type factor.
Preferably, cell growth rates are increased by at least 1.5-fold,
2-fold, 3-fold, 4-fold, or 6-fold (or any range therebetween). The
compositions or stimuli may be administered locally or
systemically. PDGF promotes proliferation of connective tissue,
glial and smooth muscle cells. Two different protein chains form 3
distinct dimer forms; AA, AB and BB. Osteoinductive molecules are
one or more of the following: BMP-1, BMP-2, BMP-3, BMP-4, BMP-5,
BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14,
BMP-15 and BMP-16. The stimulus may be a heterologous gene that
increases their intrinsic proliferation capacity or survival. The
mitogenic stimulus may comprise a biological agent, antibody or
peptide factor that increases the proliferation capacity of the
donor cells or a procedure that deliberately injures the
engraftment site to make "space" for the engrafted ex vivo cells
and/or to provide a compensatory growth signal for the ex vivo
cells.
[0098] "Repopulate" means that the engrafted ex vivo cells grow to
provide or replace at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, or 95% (or any range represented therebetween) of the
cells of the damaged organ. The cells to be replaced may be of the
same tissue type as the engrafted cells. "Repopulate" also
references a cell population which is sufficient in number and
activity to ameliorate a condition associated with the damage to
the organ or improve a homeostasis (e.g., metabolism of glucose,
ammonia, blood lipids, etc.) originally impaired by the damage to
the organ. The repopulation by the ex vivo cells typically can
provide organ specific parenchymal cells. The ex vivo cell can be
homologous or heterologous to the organ.
[0099] "Effective amount" or "effective dose" or "therapeutically
sufficient or effective amount or dose" and the like reference a
dose of an agent, mitogenic compound, or ex vivo cell that produces
the intended effect for which it is administered. The exact dose
will depend on the purpose of the treatment, and can be
ascertainable by one skilled in the art using known techniques. An
intended effect of the agent which confers a growth disadvantage on
the endogenous cells is just that. An intended effect of a mitogen
is to repopulate the target organ with the ex vivo cells. An
intended effect of the ex vivo cell is to restore or maintain a
homeostasis effected by the target organ. Methods of tracing the
origin of cell populations and assessing homeostatis are well known
in the transplantation and clinical arts.
[0100] An "organ" is a group of tissues that perform a specific
function or group of functions. Usually there are main tissues and
their cell types that uniquely predominate for the specific organ
and sporadic tissues and cell types. For example, in the brain the
main tissues are neurons and glia and sporadic tissues include the
blood and endothelium of the blood vessel wall. Organs which may be
treated according to the invention include the lungs, brain, eye,
skin, stomach, spleen, bone, pancreas, kidneys, liver, intestines,
skin, uterus, and bladder, prostate, ear, and testes. An exemplary
organ according to the invention is the liver. Accordingly, the ex
vivo cells can repopulate an organ to provide cells of a desired
type including those of the following tissues: bone, cartilage,
ligament, tendon, heart, liver, kidney, brain, skin, cartilage,
bladder, lung, thymus, thyroid, spinal cord, pancreas, skin, gut,
bowel, blood vessels, bladder, joint cartilage, intervertebral
disc, ligament, tendon, meniscus, or pancreas.
[0101] A "damaged organ" or "organ having a reduced function" is
one which has lost a substantial portion of its biological
functioning and/or mass due to the damage. The damage can be due to
trauma, surgery, infection, cancer, congenital or genetic
condition, or a chemical exposure. The damage may relate to the
ability to provide a needed product (e.g., glucose, insulin,
secretion, signal) or function (e.g., metabolize drugs, motility,
filtration, excretion). In some embodiments, there is a proviso
that the damage to be treated is one which was not itself caused by
exposure to the agent to be used to confer the growth disadvantage.
In other embodiments, there is a proviso that the damaged organ is
not the pancreas and/or bone marrow. One of ordinary skill in the
clinical art can recognize a subject having a damaged organ or an
organ having a reduced function and who, accordingly, would benefit
from treatments according to the invention. Further, one of
ordinary skill in the clinical arts can ascertain when a function
impaired by the damage to the host organ or tissue has been
ameliorated, improved, or restored by use of appropriate clinical
endpoints.
[0102] A "host" or "target" organ is one to be populated or
repopulated with ex vivo cells. It may be the same organ type from
which the ex vivo cells were derived or different. For instance, ex
vivo hepatocytes may be engrafted onto the spleen. In an exemplary
embodiment, the host organ is the liver, and the ex vivo cells are
capable of repopulating the liver with hepatocytes.
[0103] A subject references a mammal and includes human and
non-human animals (e.g., primates, rodents, lagomorphs, pigs). An
exemplary subject is the human.
[0104] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer. Methods for obtaining (e.g., producing,
isolating, purifying, synthesizing, and recombinantly
manufacturing) polypeptides are well known to one of ordinary skill
in the art.
[0105] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0106] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0107] As to "conservatively modified variants" of amino acid
sequences, one of skill will recognize that individual
substitutions, deletions or additions to a nucleic acid, peptide,
polypeptide, or protein sequence which alters, adds or deletes a
single amino acid or a small percentage of amino acids in the
encoded sequence is a "conservatively modified variant" where the
alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0108] The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0109] "Nucleic acid" or "polynucleotide" refers to
deoxyribonucleotides or ribonucleotides and polymers thereof in
either single- or double-stranded form. The term encompasses
nucleic acids containing known nucleotide analogs or modified
backbone residues or linkages, which are synthetic, naturally
occurring, and non-naturally occurring, which have similar binding
properties as the reference nucleic acid, and which are metabolized
in a manner similar to the reference nucleotides. Examples of such
analogs include, without limitation, phosphorothioates,
phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,
2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
[0110] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The
term nucleic acid is used interchangeably with gene, cDNA, mRNA,
oligonucleotide, and polynucleotide.
[0111] Polynucleotides may comprise a native sequence (i.e., an
endogenous sequence that encodes an individual antigen or a portion
thereof) of a protein of interest or may comprise a variant of such
a sequence as set forth above. Polynucleotide variants may contain
one or more substitutions, additions, deletions and/or insertions
such that the biological activity of the encoded chimeric protein
is not diminished, relative to a chimeric protein comprising native
antigens. Variants preferably exhibit at least about 70% identity,
more preferably at least about 80% identity and most preferably at
least about 90%, 95%, 96%, 97%, 98% or 99% identity to a
polynucleotide sequence that encodes a native polypeptide or a
portion thereof.
[0112] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The
term nucleic acid is used interchangeably with gene, cDNA, mRNA,
oligonucleotide, and polynucleotide.
[0113] An "expression cassette" refers to a polynucleotide molecule
comprising expression control sequences operatively linked to
coding sequence(s).
[0114] A "vector" is a replicon in which another polynucleotide
segment is attached, so as to bring about the replication and/or
expression of the attached segment.
[0115] "Control sequence" or "control element" refers to
polynucleotide sequences which are necessary to effect the
expression of coding sequences to which they are ligated. The
nature of such control sequences differs depending upon the host
organism; in prokaryotes, such control sequences generally include
promoter, ribosomal binding site, and terminators; in eukaryotes,
generally, such control sequences include promoters, terminators
and, in some instances, enhancers. The term "control sequences" is
intended to include, at a minimum, all components whose presence is
necessary for expression, and may also include additional
components whose presence is advantageous, for example, leader
sequences.
[0116] "Operably linked" refers to a juxtaposition wherein the
components so described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences.
[0117] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are near each other, and, in the case of
a secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0118] "Conservatively modified variants" also applies to nucleic
acid sequences. With respect to particular nucleic acid sequences,
conservatively modified variants refers to those nucleic acids
which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence with respect to the expression product, but not with
respect to actual probe sequences.
[0119] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, including
siRNA and polypeptides, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues or nucleotides that are the same (i.e., about
60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a
specified region, when compared and aligned for maximum
correspondence over a comparison window or designated region) as
measured using a BLAST or BLAST 2.0 sequence comparison algorithms
with default parameters described below, or by manual alignment and
visual inspection (see, e.g., NCBI web site
http://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are
then said to be "substantially identical." This definition also
refers to, or may be applied to, the compliment of a test sequence.
The definition also includes sequences that have deletions and/or
additions, as well as those that have substitutions. As described
below, the preferred algorithms can account for gaps and the like.
Preferably, identity exists over a region that is at least about 25
amino acids or nucleotides in length, or more preferably over a
region that is 50-100 amino acids or nucleotides in length.
[0120] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Preferably, default program parameters can be used,
or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters.
[0121] A "comparison window," as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to the full length of the
reference sequence, usually about 25 to 100, or 50 to about 150,
more usually about 100 to about 150 in which a sequence may be
compared to a reference sequence of the same number of contiguous
positions after the two sequences are optimally aligned. Methods of
alignment of sequences for comparison are well-known in the art.
Optimal alignment of sequences for comparison can be conducted,
e.g., by the local homology algorithm of Smith & Waterman, Adv.
Appl. Math. 2:482 (1981), by the homology alignment algorithm of
Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search
for similarity method of Pearson & Lipman, Proc. Nat'l. Acad.
Sci. USA 85:2444 (1988), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(eds. 1995 supplement)).
[0122] A preferred example of algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J.
Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0
are used, with the parameters described herein, to determine
percent sequence identity for the nucleic acids and proteins of the
invention. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0123] "Naturally-occurring" as applied to an object refers to the
fact that the object can be found in nature. For example, a
polypeptide or polynucleotide sequence that is present in an
organism (including viruses) that can be isolated from a source in
nature and which has not been intentionally modified by man in the
laboratory is naturally-occurring.
[0124] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal is at least two times background,
preferably 10 times background hybridization. Exemplary stringent
hybridization conditions can be as following: 50% formamide,
5.times.SSC, and 1% SDS, incubating at 42.degree. C., or,
5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash in
0.2.times.SSC, and 0.1% SDS at 65.degree. C.
[0125] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency. Additional guidelines
for determining hybridization parameters are provided in numerous
reference, e.g., and Current Protocols in Molecular Biology, ed.
Ausubel, et al., John Wiley & Sons.
[0126] For PCR, a temperature of about 36.degree. C. is typical for
low stringency amplification, although annealing temperatures may
vary between about 32.degree. C. and 48.degree. C. depending on
primer length. For high stringency PCR amplification, a temperature
of about 62.degree. C. is typical, although high stringency
annealing temperatures can range from about 50.degree. C. to about
65.degree. C., depending on the primer length and specificity.
Typical cycle conditions for both high and low stringency
amplifications include a denaturation phase of 90.degree.
C.-95.degree. C. for 30 sec-2 min., an annealing phase lasting 30
sec.-2 min., and an extension phase of about 72.degree. C. for 1-2
min. Protocols and guidelines for low and high stringency
amplification reactions are provided, e.g., in Innis et al., PCR
Protocols, A Guide to Methods and Applications, Academic Press,
Inc. N.Y. (1990)).
[0127] The term "heterologous" when used with reference to a
protein or a nucleic acid indicates that the protein or the nucleic
acid comprises two or more sequences or subsequences which are not
found in the same relationship to each other in nature. For
instance, the nucleic acid is typically recombinantly produced,
having two or more sequences from unrelated genes arranged to make
a new functional nucleic acid. For example, in one embodiment, the
nucleic acid has a promoter from one gene arranged to direct the
expression of a coding sequence from a different gene. Thus, with
reference to the coding sequence, the promoter is heterologous.
Similarly, a heterologous protein indicates that the protein
comprises two or more subsequences that are not found in the same
relationship to each other in nature (e.g., a fusion protein).
"Heterologous" accordingly includes those proteins and
polynucleotide sequences which are not found in a cell in which
they are introduced. Such proteins can be of mammalian, primate,
human, reptilian, or insect origin.
[0128] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under-expressed or not expressed at
all. Methods of making recombinant cells using vectors, promoters,
and other gene regulatory agents, and nucleic acids encoding
heterologous proteins of interest are well known to persons of
ordinary skill in the art.
[0129] The term "isolated" with regard to polypeptide or peptide
fragment or polynucleotides as used herein refers to a polypeptide
or a peptide fragment or polynucleotide which either has no
naturally-occurring counterpart or has been separated or purified
from components which naturally accompany it, e.g., in normal
tissues such as lung, kidney, or placenta, tumor tissue such as
colon cancer tissue, or body fluids such as blood, serum, or urine.
Typically, the polypeptide or peptide fragment or polynucleotide is
considered "isolated" when it is at least 70%, by dry weight, free
from the proteins and other naturally-occurring organic molecules
with which it is naturally associated. Preferably, a preparation of
a polypeptide (or peptide fragment thereof) or polynucleotide of
the invention is at least 80%, more preferably at least 90% or 95%,
and most preferably at least 99%, by dry weight, the polypeptide
(or the peptide fragment thereof), or polynucleotide, respectively,
of the invention. Thus, for example, a preparation of polypeptide x
is at least 80%, more preferably at least 90%, and most preferably
at least 99%, by dry weight, polypeptide x. Since a polypeptide or
polynucleotide that is chemically synthesized is, by its nature,
separated from the components that naturally accompany it, the
synthetic polypeptide is "isolated."
[0130] Once a recombinant chimeric protein is expressed, it can be
identified by assays based on the physical or functional properties
of the product, including radioactive labeling of the product
followed by analysis by gel electrophoresis, radioimmunoassay,
ELISA, bioassays, etc.
[0131] In some embodiments, the mitogenic stimulus or agent
conferring a growth disadvantage can be an antibody. Antibodies can
also be used to detect or target ex vivo cells engrafted in the
host. The targeting can be for the purpose of killing a cell which
has proved harmful. "Antibody" refers to a polypeptide comprising a
framework region from an immunoglobulin gene or fragments thereof
that specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
Typically, the antigen-binding region of an antibody will be most
critical in specificity and affinity of binding.
[0132] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0133] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially Fab with part of the
hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)).
While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by
using recombinant DNA methodology. Thus, the term antibody, as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., Nature, 348:552-554 (1990)).
[0134] For preparation of antibodies, e.g., recombinant,
monoclonal, or polyclonal antibodies, many technique known in the
art can be used (see, e.g., Kohler & Milstein, Nature,
256:495-497 (1975); Kozbor et al., Immunology Today, 4:72 (1983);
Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology
(1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988);
and Goding, Monoclonal Antibodies: Principles and Practice (2d ed.
1986)). The genes encoding the heavy and light chains of an
antibody of interest can be cloned from a cell, e.g., the genes
encoding a monoclonal antibody can be cloned from a hybridoma and
used to produce a recombinant monoclonal antibody. Gene libraries
encoding heavy and light chains of monoclonal antibodies can also
be made from hybridoma or plasma cells. Random combinations of the
heavy and light chain gene products generate a large pool of
antibodies with different antigenic specificity (see, e.g., Kuby,
Immunology (3.sup.rd ed. 1997)). Techniques for the production of
single chain antibodies or recombinant antibodies (U.S. Pat. No.
4,946,778, U.S. Pat. No. 4,816,567) can be adapted to produce
antibodies to polypeptides of this invention. Also, transgenic
mice, or other organisms such as other mammals, may be used to
express humanized or human antibodies (see, e.g., U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016,
Marks et al., Bio/Technology, 10:779-783 (1992); Lonberg et al.,
Nature, 368:856-859 (1994); Morrison, Nature, 368:812-13 (1994);
Fishwild et al., Nature Biotechnology, 14:845-51 (1996); Neuberger,
Nature Biotechnology, 14:826 (1996); and Lonberg & Huszar,
Intern. Rev. Immunol., 13:65-93 (1995)). Alternatively, phage
display technology can be used to identify antibodies and
heteromeric Fab fragments that specifically bind to selected
antigens (see, e.g., McCafferty et al., Nature, 348:552-554 (1990);
Marks et al., Biotechnology, 10:779-783 (1992)). Antibodies can
also be made bispecific, i.e., able to recognize two different
antigens (see, e.g., WO 93/08829, Traunecker et al., EMBO J.,
10:3655-3659 (1991); and Suresh et al., Methods in Enzymology,
121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two
covalently joined antibodies, or immunotoxins (see, e.g., U.S. Pat.
No. 4,676,980, WO 91/00360; WO 92/200373; and EP 03089).
[0135] Methods for humanizing or primatizing non-human antibodies
are well known in the art. Generally, a humanized antibody has one
or more amino acid residues introduced into it from a source which
is non-human. These non-human amino acid residues are often
referred to as import residues, which are typically taken from an
import variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (see, e.g., Jones et
al., Nature, 321:522-525 (1986); Riechmann et al., Nature,
332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)
and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such humanized
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0136] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0137] In one embodiment, the antibody is conjugated to an
"effector" moiety. The effector moiety can be any number of
molecules, including labeling moieties such as radioactive labels
or fluorescent labels, or can be a therapeutic moiety. In one
aspect the antibody modulates the activity of the protein.
[0138] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein,
often in a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein at least two
times the background and more typically more than 10 to 100 times
background. Specific binding to an antibody under such conditions
requires an antibody that is selected for its specificity for a
particular protein. For example, polyclonal antibodies can be
selected to obtain only those polyclonal antibodies that are
specifically immunoreactive with the selected antigen and not with
other proteins. This selection may be achieved by subtracting out
antibodies that cross-react with other molecules. A variety of
immunoassay formats may be used to select antibodies specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select antibodies
specifically immunoreactive with a protein (see, e.g., Harlow &
Lane, Antibodies, A Laboratory Manual (1988) for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity).
Methods of Conferring a Growth Disadvantage
[0139] Irradiation can be used to confer a growth disadvantage on
endogenous cells of the target organ. For instance, toward the
development of a clinically applicable approach to liver
repopulation, we have explored preparative hepatic irradiation
(HIR) to injure host hepatocytes and prevent them from
proliferating and competing with donor hepatocytes in response to
mitotic stimuli. Preparative irradiation is routinely used for bone
marrow transplantation (Thomas, E. et al., N Engl J Med,
292:832-843 (1975)), but we were the first to use HIR to facilitate
HT in rodent models (Guha, C. et al., Int J Radiat Oncol Biol Phys,
49:451-457 (2001); Guha, C. et al., Artif Organs, 25:522-528
(2001); Guha, C. et al., Hepatology, 36:354-362 (2002); Takahashi,
M. et al., Gene Ther, 10:304-313 (2003); Guha, C. et al., Cancer
Res, 59:5871-5874 (1999)). Ionizing radiation (IR) induces
apoptosis of bone marrow cells and thereby, makes "room" for donor
cell engraftment. Our results demonstrate that HIR in combination
with a compensatory hepatic regenerative stimuli, such as, partial
hepatectomy (PH) (Guha, C. et al., Hepatology, 36:354-362 (2002);
Guha, C. et al., Cancer Res, 59:5871-5874 (1999)), Fas-induced
apoptosis (Takahashi, M. et al., Gene Ther, 10:304-313 (2003)), or
portal vein branch ligation (Deb, N. et al., Hepatology, 34
(4):153A., 34:153A (2001)), or a direct mitotic stimuli, provided
by hepatotropic growth factors, such as, thyroid hormone (Parashar,
B. et al., Hepatology, 32:206A (2000)), or hepatocyte growth factor
(HGF) (Guha, C. et al., Am J Nephrol, 25:161-170 (2005)) induces
extensive repopulation of transplanted hepatocytes in the host
liver. We have demonstrated that HIR, in combination with
proliferative stimuli for hepatocytes, could potentially suppress
host hepatocellular proliferation and induce post-mitotic death,
thus making "room" for donor hepatocytes that preferentially
proliferate and repopulate the irradiated host liver. HIR can be
safely administered in the clinic using stereotactic radiosurgery
(SRS) or 3-D conformal RT (3-D CRT) techniques and can be used as a
preparative regimen for HT. Thus, preparative HIR can facilitate HT
for i) the treatment of inherited liver diseases, ii) ex vivo
hepatic gene therapy, iii) rescuing patients with liver cancer,
following chemotherapy or radiation therapy, or other liver damage;
and iv) expanding human hepatocytes in animal liver for generating
animal models for human-specific infections (Table 1).
[0140] First, although PH provided the mitotic signal in our
initial repopulation experiments, a non-invasive substitute to PH
is more desirable for clinical application. Accordingly, we have
examined whether HIR in combination with hepatic growth factors,
such as, HGF and EGF, could promote repopulation of transplanted
hepatocytes. Use of an activating antibody to the HGF receptor,
c-met, to promote selective proliferation of hepatocytes in the
irradiated liver is also contemplated. Second, radiation-induced
liver injury is a function of mean liver dose and the irradiated
liver volume (Lawrence, T. et al., International Journal of
Radiation Oncology, Biology, Physics, 19:1041-1047 (1990); Dawson,
L. et al., Int J Radiat Oncol Biol Phys, 53:810-821 (2002)) in
humans. A lower dose of HIR, or partial liver irradiation is
desirable for clinical application of HIR. Partial liver
irradiation is well tolerated in cancer patients and with modern
techniques of IMRT, doses higher than 50 Gy to parts of the liver
can safely be offered to patients in the clinic. Our initial
studies demonstrate that HIR administered to the anterior lobes of
the liver can induce selective lobar repopulation of donor cells
(Deb, N. et al., Hepatology, 34 (4):153A., 34:153A (2001)). It is
accordingly contemplated that partial liver irradiation along with
hepatic growth factors can be used in preparative regimens of
HT.
TABLE-US-00001 TABLE 1 The scope of liver cell transplantation and
hepatocyte-based gene therapy A. REPLACEMENT OF A MISSING GENE
PRODUCT Liver manifests disease Genetic Disorders Wilson's disease
Lipidoses, e.g., Niemann-Pick disease Tyrosinemia Type I
Protoporphyria Ornithine transcarbamylase deficiency Extrahepatic
organs manifest disease Metabolic deficiency disorders
Crigler-Najjar Syndrome Familial hypercholesterolemia Defects in
carbohydrate metabolism Coagulation Disorders Hemophilia A &
Factor IX deficiency B. MASSIVE REPLACEMENT OF HOST HEPATOCYTES
THAT EXPRESS AN OFFENDING GENE Primary hyperoxaluria .alpha.-1
antitrypsin deficiency (mutant A1AT) C. REPLACE DISEASED
HEPATOCYTES: AMELIORATION OF RT/DRUG-INDUCED LIVER INJURY "BRIDGE"
TO OLT IN LIVER FAILURE PATIENTS Acute liver failure Chronic viral
hepatitis and cirrhosis Drug or Radiation-induced liver injury in
cancer patients D. EXPRESSION OF GENES THAT ARE NORMALLY NOT
EXPRESSED IN THE LIVER Hormones, e.g., insulin, immunosuppressive
Genes, anti-angiogenic agents, cytokine immunomodulation (for
suppressing tumor growth) E. DEVELOPMENT OF HUMAN-MOUSE LIVER
CHIMERA/GENERATION OF ANIMAL MODELS OF HUMAN-SPECIFIC INFECTIONS
Malaria Viral Hepatitis
[0141] Additionally, the invention provides methods of pretreating
an organ to reduce the growth or proliferation of endogenous cells
of the organ by administering an agent selectively or predominantly
toxic to the endogenous cells of the organ as opposed to other
organs. The toxic effects and target organs for a variety of
chemicals are disclosed in Registry of Toxic Effects of Chemical
Substances (RTECS), RTECS (NIOSH 1980 or later editions, including
1995). RTECS is a database of toxicity information compiled from
the published scientific literature. Prior to 2001, RTECS was
maintained by US National Institute for Occupational Safety and
Health (NIOSH). Now, it is maintained by Elsevier MDL. See, also,
Olson et al. Ed., Poisoning and Drug Overdose, 5th Edition,
published by McGraw Hill/Lange for a listing of toxic agents and
their effects and target organs. For instance, as to the liver,
some halogenated compounds and carcinogens which are selectively or
principally toxic for the liver may be used.
Methods of Engrafting and Repopulating
Ex Vivo Cell Isolation.
[0142] Cells can be isolated with a modified collagenase perfusion
method from a mammalian organ or tissue, as originally described by
Berry and Friend (Takahashi, M. et al., Gene Ther, 10:304-313
(2003)). After dissociation, cells can be filtered through a Dacron
mesh of a dimension corresponding to the cell of interest and then
washed twice at 50.times.g for 1 min each. Cell viability can be
determined by trypan blue dye exclusion. Cells with >90%
viability can be used for transplantation. Ex vivo cells can be
adult somatic cells, adult progenitor cells, adult stem cells,
embryonic progenitor cells, or embryonic stem cells. Sources of
such cells are well known to persons of ordinary skill in the art.
For example, with regard to the liver, hepatocytes can be isolated
with a modified collagenase perfusion method using male F344 rats,
as originally described by Berry and Friend (Takahashi, M. et al.,
Gene Ther, 10:304-313 (2003)). After liver dissociation, cells were
filtered through an 80-.mu.m Dacron mesh and washed twice at
50.times.g for 1 min each. Cell viability can be determined by
trypan blue dye exclusion. Hepatocytes with >90% viability were
used for transplantation (see, also, Guha, C. et al., Cancer
Research 59, 5871-5874, (1999) and, also, Nowak, G., et al., Gut
54:972-979 (2005)).
Administration of Ex Vivo Cells
[0143] Ex vivo cells are introduced into the subject in a number
which depends upon the species and organ to be engrafted and the
extent of the need for such therapy. Ex vivo cells can be injected
directly into the spleen, kidney capsule, or target organ of the
subject as described previously (Guha, C. et al., Artif Organs,
25:522-528 (2001)) or administered intraperitoneally or other site
of engraftment. They may also be administered intravenously or
intraportally (in the case of the liver). For instance, when
administering hepatocytes to the rat, under ether anesthesia, the
spleen can be exposed, and 5.times.10.sup.6 hepatocytes suspended
in 0.5 ml of RPMI 1640 can be injected into the splenic pulp.
Generally, hepatocytes can be administered in suitable media
providing single or divided doses of from 1.times.10.sup.5 to
5.times.10.sup.9 cells per treatment. Total dosages, administered
singly or as a divided dose, may be from 1 from 1.times.10.sup.5 to
5.times.10.sup.10 ex vivo cells/kg of body weight; in some
embodiments the total dosages, administered singly or as a divided
dose, may be from 1 from 1.times.10.sup.6 to 1.times.10.sup.9 ex
vivo cells/kg of body weight; in some embodiments the total
dosages, administered singly or as a divided dose, may be from 1
from 1.times.10.sup.7 to 5.times.10.sup.8 ex vivo cells/kg of body
weight.
Methods of Determining Engraftment
Histological Analysis.
[0144] By methods known to one of ordinary skill in the art,
engrafted ex vivo cells can be distinguished from endogenous cells
by the use of antigenic markers, identifying chromosomal or genomic
nucleic acid sequence differences between the engrafted and
endogenous cells, fluorescent markers such as GFP, or enzymatic
markers such as DPPIV, or according to a functional enzyme present
in the ex vivo cell and deficient in the host endogenous cells.
Sections of an organ can be embedded in OCT, frozen in liquid
nitrogen, and stored at -70.degree. C. or fixed in formalin for
paraffin embedding and standard H&E staining. For the liver,
Reticulin and trichrome stains can be performed in a standard
histopathology laboratory. Ex vivo cells can be distinguished from
endogenous cells by the use of antigenic markers, identifying
chromosomal or genomic nucleic acid sequence differences between
the engrafted and endogenous cells, fluorescent markers such as
GFP, or enzymatic markers such as DPPIV, or according to a
functional enzyme present in the ex vivo cell and deficient in the
host endogenous cells. For instance, in the case of DPPIV marked ex
vivo cells, DPPIV activity in situ can be assessed using
5-.mu.m-thick cryostat sections fixed in chloroform and acetone
(1:1, v/v) for 10 min at 40.degree. C., as described previously
(Guha, C. et al., Cancer Res, 59:5871-5874 (1999)). After air
drying, sections can be incubated for 30 min at room temperature in
a solution containing 0.4 mg
glycyl-L-proline-4-methoxy-2-naphthylamide, along with 1 mg Fast
Blue B salt in PBS (pH 7.4) and the fluorescent reaction products
detected. The reaction can be terminated by washing with water and
sections and counterstaining with hematoxylin.
[0145] A variety of model systems can be used to detect engraftment
and repopulation of donor hepatocytes. For instance, one can use a
mouse model, where transgenic beta-galactosidase
(.beta.-gal)-expressing (Rosa) C57Bl/6 hepatocytes are transplanted
into wild-type C57Bl/6 mice. Or, DPPIV+ve F344 hepatocytes can be
transplanted into congeneic, DPPIV-ve F344 host liver. Since DPPIV
is highly expressed in the bile canalicular domain of the
hepatocytes, the transplanted cells can easily be detected by
enzyme histochemistry. In addition, after characterizing a
noninvasive, robust, preparative regimen of hepatocyte
repopulation, one can further examine its effectiveness in
ameliorating a rodent model of metabolic liver disease, such as the
Gunn rat, which is a model for Crigler-Najjar syndrome. Experiments
can be simultaneously performed in more than one species. Initial
dose of HIR would be 50 Gy, which has been very effective in
inducing donor cell proliferation in our studies and is safe in
rodents. One can perform a dose response study of HIR (e.g., 10,
20, 30 and 50 Gy), in order to identify the lowest dose of HIR that
permits effective donor cell repopulation. To investigate the
nature of radiation injury to the host hepatocytes, experiments can
also be performed with mice that received HIR and HGF without HT.
Animals from various cohorts can be sacrificed at various time
points (1 d, 2 d, 3 d, 1 wk, 3 wk, 6 wk and 12 wk) and liver
sections can be stained with H&E for histopathological
analysis. BrdU and TUNEL staining can be performed for examining
hepatocyte proliferation and apoptosis, respectively.
Organ Function Tests
[0146] In the clinical setting, engraftment can be assessed by
biopsy with analyses as described above. Additionally, clinical
tests can be used to assess the extent to which homeostasis is
supported by the engrafted organ. Improvement in one or more
clinical parameters related to the functioning of a target or
engrafted organ can be used to indirectly assess the efficacy of
the engraftment. Suitable clinical tests will vary with respect to
the organ. With regard to the liver, standard liver function tests
which monitor blood levels of any of alanine aminotransferase;
spartate aminotransferase; alkaline phosphatase;
gamma-glutamyltransferase, bilirubin, or ammonia may be used.
[0147] i) Donor hepatocyte engraftment and repopulation. Donor
cells can be identified by beta-galactosidase and DPP IV
histochemistry in C57Bl/6 mice and DPP IV-ve, F344 rats,
respectively. To demonstrate the engraftment of transplanted cells,
double staining for beta-galactosidase and ATPase in the rosa/B16
model or for DPPIV and ATPase in the DPP IV-ve can be performed in
the F344 model. Donor cell proliferation in DPPIV-ve rats can be
determined by co-localization of DPPIV activity (histochemistry)
and immunostaining for BrdU incorporation in the nuclei, according
to previously published methods (Gupta, S. et al., Proceedings of
the National Academy of Sciences of the United States of America,
92:5860-5864 (1995)).
[0148] ii) Physiological function of repopulated hepatocytes. The
ability of transplanted cells to perform unique hepatocyte
biochemical functions, albumin synthesis (albumin immunostaining),
glucose metabolism (stain for glucose-6-phosphatase) and
gluconeogenesis (glycogen staining) can be examined in fresh frozen
section according to published protocols (Laconi, E. et al., Am J
Pathol, 153:319-329 (1998)). In Gunn rats, amelioration of
hyperbilirubinemia can be used to indicate the degree of
repopulation and the physiological function of the transplanted
hepatocytes. At various time points (1, 2, 3 and 6 months), animals
can be sacrificed and the function of the engrafted normal
hepatocytes can be evaluated by measuring UGT1A1 activity in liver
biopsy specimens, performing immunoblot analysis of UGT1A1 and
immunohistochemistry to detect donor UGT1A1+ve hepatocytes and by
determining the excretion of bilirubin glucuronides in bile.
[0149] iii) Toxicities of the preparative regimens. As described in
our publications (Guha, C. et al., Hepatology, 36:354-362 (2002);
Guha, C. et al., Cancer Res, 59:5871-5874 (1999); Nakatani, T. et
al., J Biol Chem, 277:9562-9569 (2002)), serum albumin,
transferrin, AST, ALT, GGT, GST-pi and alpha-feto protein (AFP) can
be measured to examine the normal physiological functions of the
repopulated hepatocytes and to determine any potential
manifestations of radiation injury and tumorigenesis at the end of
experiments. H & E staining of the liver biopsies can be
performed to examine for histopathological evidence of hepatic
radiation injury and tumorigenesis.
Gene Therapy
[0150] In some embodiments, the ex vivo cell to be engrafted is a
recombinant cell transduced or transformed ex vivo to express a
protein or to have increased ability to compete with endogenous
cells upon engrafting or to grow in culture. In some embodiments,
the protein may function as a marker, to provide a function
deficient in the host organ or to modulate the growth and/or
survival of the ex vivo cell (e.g., may provide a mitogenic stimuli
to growth of the ex vivo cells). The nucleic acid expressing the
genome is operably linked to regulatory elements which may be
introduced or transduced into the cell by conventional means which
are well known in the art. Construction of suitable vectors
containing the desired gene coding and control sequences employs
standard ligation and restriction techniques, which are well
understood in the art (see Maniatis et al., in Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982)).
Isolated plasmids, DNA sequences, or synthesized oligonucleotides
are cleaved, tailored, and religated in the form desired. The
regulatory elements may be in turn regulated by agents which are
subject to manipulation in the host (e.g., heat shock promoters
or
[0151] A variety of viral and non-viral delivery vectors useful to
achieve expression of nucleotide sequences in transduced cells are
known in the art. See, e.g. Boulikas, T in Gene Therapy and
Molecular Biology, Volume 1 (Boulikas, T. Ed.) 1998 Gene Therapy
Press, Palo Alto, Calif. pages 1-172. Methods of non-viral delivery
of nucleic acids include lipofection, microinjection, biolistics,
virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic
acid conjugates, naked DNA, artificial virions, and agent-enhanced
uptake of DNA. Lipofection is described in, e.g., U.S. Pat. No.
5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat. No. 4,897,355)
and lipofection reagents are sold commercially (e.g.,
Transfectam.TM. and Lipofectin.TM.). Cationic and neutral lipids
that are suitable for efficient receptor-recognition lipofection of
polynucleotides include those of Felgner, WO 91/17424, WO 91/16024.
Delivery can be to cells (ex vivo administration) or target tissues
(in vivo administration). A targeting construct for knocking the
desired first gene into another second gene having suitable
expression can be a replacement vector designed to replace the
second gene coding sequences with those of the first (see, Yin et
al, Stem Cells 20:338-346 (2002). As indicated in the Examples,
genes may also be modified using viral vectors as known in the
art.
[0152] Examples of non-viral delivery systems used to introduce a
gene to a target cell include expression plasmids capable of
directing the expression of the protein. Expression plasmids are
autonomously replicating, extrachromosomal circular DNA molecules,
distinct from the normal genome and nonessential for cell survival
under nonselective conditions capable of effecting the expression
of a DNA sequence in the target cell. The expression plasmid may
also contain promoter, enhancer or other sequences aiding
expression of the therapeutic gene and/or secretion can also be
included in the expression vector. Additional genes, such as those
encoding drug resistance, can be included to allow selection or
screening for the presence of the recombinant vector. Such
additional genes can include, for example, genes encoding neomycin
resistance, multi-drug resistance, thymidine kinase,
beta-galactosidase, dihydrofolate reductase (DHFR), and
chloramphenicol acetyl transferase.
[0153] The expression plasmid containing the gene may be
encapsulated in liposomes. Liposomes include emulsions, foams,
micelles, insoluble monolayers, liquid crystals, phospholipid
dispersions, lamellar layers and the like. The delivery of nucleic
acids to cells using liposome carriers is well known in the art. A
variety of methods are available for preparing liposomes, as
described in, e.g., Szoka et al. Ann. Rev. Biophys. Bioeng. 9:467
(1980), Szoka, et al. U.S. Pat. No. 4,394,448 issued Jul. 19, 1983,
as well as U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and
5,019,369. Liposomes useful in the practice of the present
invention may be formed from one or more standard vesicle-forming
lipids, which generally include neutral and negatively charged
phospholipids and a sterol, such as cholesterol. Examples ofsuch
vesicle forming lipids include DC-chol, DOGS, DOTMA, DOPE, DOSPA,
DMRIE, DOPC, DOTAP, DORIE, DMRIE-HP, n-spermidine cholesterol
carbamate and other cationic lipids as disclosed in U.S. Pat. No.
5,650,096. The selection of lipids is generally guided by
consideration of, e.g., liposome size, acid lability and stability
of the liposomes in the blood stream. Additional components may be
added to the liposome formulation to increase serum half-life such
as polyethylene glycol coating (so called "PEG-ylation") as
described in U.S. Pat. No. 5,013,556 issued May 7, 1991 and U.S.
Pat. No. 5,213,804 issued May 25, 1993.
[0154] In order to provide directed delivery of the non-viral gene
to a particular cell, it may be advantageous to incorporate
elements into the non-viral delivery system which facilitate
cellular targeting. For example, a lipid encapsulated expression
plasmid may incorporate modified surface cell receptor ligands to
facilitate targeting.
[0155] In one embodiment of the invention as exemplified herein,
the vector is a viral vector. The terms virus(es) and viral
vector(s) are used interchangeably herein. The viruses useful in
the practice of the present invention include recombinantly
modified enveloped or non-enveloped DNA and RNA viruses, preferably
selected from baculoviridiae, parvoviridiae, picomoviridiae,
herpesveridiae, poxyiridae, adenoviridiae, or picornnaviridiae. The
viral genomes may be modified by conventional recombinant DNA
techniques to provide expression of the gene and may be engineered
to be replication deficient, conditionally replicating or
replication competent. Chimeric viral vectors which exploit
advantageous elements of each of the parent vector properties (See
e.g., Feng, et al. (1997) Nature Biotechnology, 15:866-870) may
also be useful in the practice of the present invention. Minimal
vector systems in which the viral backbone contains only the
sequences needed for packaging of the viral vector and may
optionally include a gene expression cassette may also be employed
in the practice of the present invention. In some instances it may
be advantageous to use vectors derived from different species from
that to be treated which possess favorable pathogenic features such
as avoidance of pre-existing immune response. For example, equine
herpes virus vectors for human gene therapy are described in
WO98/27216 published Aug. 5, 1998. The vectors are described as
useful for the treatment of humans as the equine virus is not
pathogenic to humans. Similarly, ovine adenoviral vectors may be
used in human gene therapy as they are claimed to avoid the
antibodies against the human adenoviral vectors. Such vectors are
described in WO 97/06826 published Apr. 10, 1997.
[0156] Many viruses exhibit the ability to infect a broad range of
cell types. However, in some applications it may be desirable to
infect only a certain subpopulation of cells. Consequently, a
variety of techniques have evolved to facilitate selective or
"targeted" vectors to result in preferential infectivity of the
mature viral particle of a particular cell type. Cell type
specificity or cell type targeting may also be achieved in vectors
derived from viruses having characteristically broad infectivity
such as adenovirus by the modification of the viral envelope
proteins. For example, cell targeting has been achieved with
adenovirus vectors by selective modification of the viral genome
knob and fiber coding sequences to achieve expression of modified
knob and fiber domains having specific interaction with unique cell
surface receptors.
[0157] In one embodiment of the invention as exemplified herein,
the vector is an adenoviral vector. The use of adenoviral vectors
for the delivery of exogenous transgenes are well known in the art.
See e.g., Zhang, W-W. Cancer Gene Therapy, 6:113-138 (1999).
Vectors can also be derived from the adenoviral, adeno-associated
viral and retroviral genomes. In the most preferred practice of the
invention, the vectors are derived from the human adenovirus
genome. The replicative capacity of such vectors may be attenuated
(to the point of being considered "replication deficient") by
modifications or deletions in the E1a and/or E1b coding
regions.
[0158] The viral genome may be modified to include inducible
promoters which achieve replication or expression only under
certain conditions. Examples of inducible promoters are known in
the art (See, e.g. Yoshida and Hamada, Biochem. Biophys. Res.
Comm., 230:426-430 (1997); lida, et al., J. Virol., 70(9):6054-6059
(1996); Hwang, et al., J. Virol, 71(9):7128-7131 (1997); Lee, et
al., Mol. Cell. Biol., 17(9):5097-5105 (1997); and Dreher, et al.,
J. Biol. Chem., 272(46); 29364-29371 (1997). The viruses may also
be designed to be selectively replicating viruses such as those
described in Ramachandra, et al. PCT International Publication No.
WO 00/22137, International Application No. PCT/US99/21452 published
Apr. 20, 2000 and Howe, J., PCT International Publication No. WO
WO0022136, International Application No. PCT/US99/21451 published
Apr. 20, 2000. The virus may also be modified to be attenuated for
replication in certain cell types. For example the adenovirus
dl1520 containing a specific deletion in the E1b55K gene (Barker
and Berk (1987) Virology 156: 107) has been used with therapeutic
effect in human beings. Such vectors are also described in
McCormick (U.S. Pat. No. 5,677,178 issued Oct. 14, 1997) and
McCormick, U.S. Pat. No. 5,846,945 issued Dec. 8, 1998.
Methods of Administration and Pharmaceutical Compositions of Active
Agents
[0159] The pharmaceutically active agents, including but not
limited to pharmaceutical agents which disadvantage the endogenous
cells with respect to the engrafted ex vivo cells, the mitogenic
compositions, and immunosuppressive agents for use according to the
invention can be administered to a subject in accord with known
methods, such as intravenous administration, e.g., as a bolus or by
continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or inhalation routes.
Intravenous or subcutaneous administration of biopolymers is
preferred. The administration may be local or systemic.
[0160] The compositions for administration will commonly comprise
the active agent as described herein dissolved in a
pharmaceutically acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers can be used, e.g., buffered saline
and the like. These solutions are sterile and generally free of
undesirable matter. These compositions may be sterilized by
conventional, well known sterilization techniques. The compositions
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, for example, 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.
[0161] Thus, a typical pharmaceutical composition for intravenous
administration will vary according to the active agent. Actual
methods for preparing parenterally administrable compositions will
be known or apparent to those skilled in the art and are described
in more detail in such publications as Remington: The Science and
Practice of Pharmacy, 20th ed., Lippincott, Williams, and Wilkins,
(2000).
[0162] The pharmaceutical compositions can be administered in a
variety of unit dosage forms depending upon the method of
administration. For example, unit dosage forms suitable for oral
administration include, but are not limited to, powder, tablets,
pills, capsules and lozenges. It is recognized that antibodies when
administered orally, should be protected from digestion. This is
typically accomplished either by complexing the molecules with a
composition to render them resistant to acidic and enzymatic
hydrolysis, or by packaging the molecules in an appropriately
resistant carrier, such as a liposome or a protection barrier.
Means of protecting agents from digestion are well known in the
art.
[0163] Pharmaceutical formulations can be prepared by mixing an
active agent having the desired degree of purity with optional
pharmaceutically acceptable carriers, excipients or stabilizers.
Such formulations can be lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations used.
Acceptable carriers, excipients or stabilizers can be acetate,
phosphate, citrate, and other organic acids; antioxidants (e.g.,
ascorbic acid) preservatives low molecular weight polypeptides;
proteins, such as serum albumin or gelatin, or hydrophilic polymers
such as polyvinylpyllolidone; and amino acids, monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents; and ionic and non-ionic surfactants
(e.g., polysorbate); salt-forming counter-ions such as sodium;
metal complexes (e.g. Zn-protein complexes); and/or non-ionic
surfactants.
[0164] The compositions can be administered in a "therapeutically
effective dose." Single or multiple administrations of the
compositions may be administered depending on the dosage and
frequency as required and tolerated by the subject. A "patient" or
"subject" for the purposes of the present invention includes both
humans and other animals, particularly mammals. Thus the methods
are applicable to both human therapy and veterinary applications.
In the preferred embodiment the patient is a mammal, preferably a
primate, and in the most preferred embodiment the patient is human.
Other known therapies can be used in combination with the methods
of the invention.
[0165] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the packaged
nucleic acid suspended in diluents, such as water, saline or PEG
400; (b) capsules, sachets or tablets, each containing a
predetermined amount of the active ingredient, as liquids, solids,
granules or gelatin; (c) suspensions in an appropriate liquid; and
(d) suitable emulsions. Tablet forms can include one or more of
lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato starch, microcrystalline cellulose, gelatin,
colloidal silicon dioxide, talc, magnesium stearate, stearic acid,
and other excipients, colorants, fillers, binders, diluents,
buffering agents, moistening agents, preservatives, flavoring
agents, dyes, disintegrating agents, and pharmaceutically
compatible carriers. Lozenge forms can comprise the active
ingredient in a flavor, e.g., sucrose, as well as pastilles
comprising the active ingredient in an inert base, such as gelatin
and glycerin or sucrose and acacia emulsions, gels, and the like
containing, in addition to the active ingredient, carriers known in
the art.
[0166] The compositions of the present invention may be sterilized
by conventional, well-known sterilization techniques or may be
produced under sterile conditions. Aqueous solutions can be
packaged for use or filtered under aseptic conditions and
lyophilized, the lyophilized preparation being combined with a
sterile aqueous solution prior to administration. The compositions
can contain pharmaceutically or physiologically acceptable
auxiliary substances as required to approximate physiological
conditions, such as pH adjusting and buffering agents, tonicity
adjusting agents, wetting agents, and the like, e.g., sodium
acetate, sodium lactate, sodium chloride, potassium chloride,
calcium chloride, sorbitan monolaurate, and triethanolamine
oleate.
[0167] The compound of choice, alone or in combination with other
suitable components, can be made into aerosol formulations (i.e.,
they can be "nebulized") to be administered via inhalation. Aerosol
formulations can be placed into pressurized acceptable propellants,
such as dichlorodifluoromethane, propane, nitrogen, and the
like.
[0168] Suitable formulations for rectal administration include, for
example, suppositories, which consist of the packaged nucleic acid
with a suppository base. Suitable suppository bases include natural
or synthetic triglycerides or paraffin hydrocarbons. In addition,
it is also possible to use gelatin rectal capsules which consist of
a combination of the compound of choice with a base, including, for
example, liquid triglycerides, polyethylene glycols, and paraffin
hydrocarbons.
[0169] Formulations suitable for parenteral administration, such
as, for example, by intravenous, intramuscular, intraorgan,
intratumoral, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. In the practice
of this invention, compositions can be administered, for example,
by intravenous infusion, orally, topically, intraperitoneally,
intravesically or intrathecally. Parenteral administration, oral
administration, and intravenous administration are the preferred
methods of administration. The formulations of compounds can be
presented in unit-dose or multi-dose sealed containers, such as
ampules and vials.
[0170] Injection solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously
described.
[0171] The pharmaceutical preparation is preferably in unit dosage
form. In such form the preparation is subdivided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of preparation, such as packeted tablets,
capsules, and powders in vials or ampoules. Also, the unit dosage
form can be a capsule, tablet, cachet, or lozenge itself, or it can
be the appropriate number of any of these in packaged form. The
composition can, if desired, also contain other compatible
therapeutic agents.
[0172] Preferred pharmaceutical preparations deliver one or more
agents.
[0173] In use, the active agent utilized in the methods of the
invention are administered at the initial dosage of about 0.001
mg/kg to about 1000 mg/kg daily. A daily dose range of about 0.01
mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or
about 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50
mg/kg, can be used. The dosages, however, may be varied depending
upon the requirements of the patient, the severity of the condition
being treated, the effect sought, and the compound being employed.
Determination of the proper dosage for a particular situation is
within the skill of the practitioner.
EXAMPLES
[0174] The following examples are offered to illustrate aspects of,
but not to limit, the claimed invention.
Example 1
Hepatocyte Transplantation (HT) Ameliorates Radiation-Induced Liver
Damage (RILD) and Increases Survival After Partial Hepatic
Resection and Irradiation (12)
[0175] Liver cancer is the sixth most common cancer worldwide in
terms of number of cases (626,00/yr) but because of very poor
prognosis, the number of deaths is almost the same as its incidence
(598,000/yr). Besides primary liver cancer, metastatic liver
cancer, arising from abdominal malignancies, remains a vexing and
commonly encountered problem. Although, surgery is the only
curative therapy, most patients with liver tumors are unresectable
and chemotherapy fails to cure patients. This is rather unfortunate
because a significant proportion of these patients have limited
hepatic metastases (oligometastases), without harboring tumor
deposits in extrahepatic sites. Failure to control the hepatic
oligometastases results in eventual systemic progression of the
cancer. In many cancers, such as head and neck, esophagus, lung,
cervix and rectal cancer, radiation therapy (RT).+-.chemotherapy
improves local tumor control and survival of patients. But RT has
been traditionally used in a palliative role because of the
potential for inducing potentially fatal RILD.
[0176] To simulate post-operative RT following resection of hepatic
tumors, we examined RILD in F344 rats after HIR and partial
hepatectomy (PH). HIR/RT induced severe RILD as evidenced by
increased mortality and histopathological changes that included
perivenous lobular collapse (FIG. 1A) with centrizonal steatosis
(FIG. 1B), bile ductular proliferation and activation of liver stem
cells, followed by periportal fibrosis (FIG. 1D-E). Since RT
inhibited hepatic regeneration, we hypothesized that
transplantation of unirradiated hepatocytes, via portal vein or
intrasplenic injection, would result in preferential proliferation
of the donor cells in the partially resected and irradiated host
liver. Transplanted hepatocytes would further provide metabolic
support and ameliorate consequences and mortality associated with
RILD. We tested this hypothesis in F344 rats where dipeptidyl
peptidase-positive (DPPIV+ve) F344 hepatocytes were transplanted
into congeneic, DPPIV-ve F344 hosts after PH+HIR. We demonstrated
that HT ameliorated the histological changes of RILD (FIG. 1C, F)
and increased survival of irradiated F344 rats (FIG. 2). By 12
weeks, donor hepatocytes constituted 76.9.+-.3.9% of the host
liver, in rats in the PH+HIR+HT group (FIG. 3C,D). In contrast,
among the rats that received PH without HIR, the donor cells
accounted for only 12.7.+-.1.8% of the recipient liver hepatocytes
after 12 weeks (p=0.006, t-test) (FIG. 3). This is the first
demonstration of amelioration of RILD by HT and indicates one can
rescue liver function with HT in patients having unresectable liver
cancer after high dose chemo-RT.
Example 2
PH+HIR as a Preparative Regimen for Liver Repopulation by
Transplanted Hepatocytes
Guha, C. et al., Hepatology, 36:354-362 (2002)
[0177] Encouraged by our findings, we examined whether PH+HIR could
be used as a preparative regimen of HT for amelioration of
inherited metabolic liver diseases. Gunn rat is an animal model for
bilirubin-uridinediphosphoglucuronate glucuronosyltransferase
(UGT1A1) deficiency, which causes Crigler-Najjar syndrome type 1 in
humans. UGT1A1 deficiency results in the lack of glucuronidation of
bilirubin, resulting in the accumulation of unconjugated bilirubin
in plasma and consequent bilirubin encephalopathy. We transplanted
congeneic UGT1A1-proficient, Wistar-RHA hepatocytes in jaundiced
Gunn rats, 4 days after PH+HIR. Five months after HT, there was
60-80% repopulation of the host liver by the engrafted UGT1A1+ve,
transplanted hepatocytes (FIG. 4B-D). HPLC of bile collected 5
months after PH+HIR+HT showed complete normalization of the pigment
profile, with excretion of conjugated bile pigments. There was
complete normalization of serum bilirubin levels in the
transplanted Gunn rats, which received PH+HIR+HT. In this group
(n=9), serum bilirubin concentrations declined from initial levels
of 10.4.+-.2.3 mg/dl to completely normal levels (0.64.+-.0.16
mg/dl) by 12 weeks (p=0.00012, t-test). In rats receiving either PH
or HIR alone before HT, serum bilirubin concentrations declined by
25-30% in 28 weeks (Fig FIG. 4E), indicating minimal repopulation.
The complete normalization of serum bilirubin with PH+HIR has never
been seen with current protocols of HT that are available in the
clinic for Crigler-Najjar patients. These results indicate that HT
in combination with a preparative regimen of HIR can be a highly
effective treatment for such patients.
Example 3
Noninvasive Substitutes to PH in the Preparative Regimen of HT
[0178] The success of PH+HIR as a preparative regimen for HT
depends on HIR suppressing the host hepatocellular proliferation
and inducing mitotic catastrophe in host cells, while PH providing
the mitotic stimuli and a selective growth advantage for
transplanted hepatocytes. Although PH provides a very strong
mitotic stimulus to the hepatocytes, it is invasive and is not
desirable in clinical protocols of liver repopulation of
transplanted hepatocytes for patients with metabolic disorders. We,
therefore, examined noninvasive alternatives to PH (Table 2)
(Takahashi, M. et al., Gene Ther, 10:304-313 (2003); Deb, N. et
al., Hepatology, 34 (4):153A., 34:153A (2001); Parashar, B. et al.,
Hepatology, 32:206A (2000); Guha, C. et al., Am J Nephrol,
25:161-170 (2005); Malhi, H. et al., Proc Natl Acad Sci USA,
99:13114-13119 (2002)).
TABLE-US-00002 TABLE 2 HIR-BASED PREPARATIVE REGIMENS OF LIVER
REPOPULATION (SUBSTITUTES TO PH) MECHANISM OF PREPARATIVE REGIMEN
REPOPULATION EFFICACY COMPENSATORY REGENERATIVE STIMULI 1. HIR +
Anti-Fas or FasL Fas-induced apoptosis in Extensive liver
(Takahashi, M. et al., Gene host cells repopulation of Ther, 10:
304-313 (2003)) PVBL-induced apoptosis transplanted hepatocytes. 2.
HIR + Portal vein branch Oxidative injury to host Partial HIR
results in ligation (Deb, N. et al., HIR-induced injury to host
selective repopulation in Hepatology, 34 cells a single lobe of the
liver. (4): 153A., 34: 153A Compensatory regenerative Potential
regimen for (2001)) stimuli Cancer patients. 3. HIR +
Ischemia-reperfusion injury (Malhi, H. et al., Proc Natl Acad Sci
USA, 99: 13114-13119 (2002)) DIRECT HEPATIC MITOGENS 4. HIR +
Thyroid hormone (T3) HIR-induced genotoxic Extensive repopulation.
(Parashar, B. et al., injury to host Partial HIR results in
Hepatology, 32: 206A Hepatic mitogen provides single lobe
repopulation. (2000)) selective growth advantage Potential regimen
for 5. HIR + Adeno-Hepatocyte to transplanted cells patients with
metabolic Growth Factor (HGF) disorders. (Guha, C. et al., Am J
Nephrol, 25: 161-170 (2005))
[0179] Our results demonstrate that HIR in combination with a
variety of mitotic stimuli (Table 2) can stimulate preferential
proliferation of transplanted hepatocytes in rat and mouse livers.
These experiments demonstrated that HIR induces genotoxic injury to
the host hepatocytes, resulting in cell cycle arrest, accelerated
senescence and mitotic catastrophe, in response to a mitotic
stimulus, such as PH or HGF. In contrast to the host hepatocytes,
the nonirradiated transplanted hepatocytes preferentially
proliferated in response to the same mitotic stimulus. The gradual
loss of host hepatocytes over a period of 3-5 months decreases the
risk of sudden liver failure and provides regenerative stimuli to
the transplanted cells over a long period of time. This also gave
the engrafted cells time to proliferate and provide metabolic
support to the diseased liver.
[0180] PH, PVBL and Fas-based strategies can provide compensatory
regenerative stimuli to donor cells, while the host cells are
prevented from dividing by HIR. These methods can find use in
patients with liver cancer where the tumor with adjacent host liver
tissue can be resected or ablated followed by HIR and
transplantation of autologous hepatocytes after purging tumor cells
(analogous with bone marrow rescue). The second method can use
direct hepatic mitogens, such as, T3 and HGF. Accordingly, these
protocols can be applied in patients with inherited or other liver
diseases. Additionally, as shown in Table 2, a variety of
proliferative stimuli can be used in combination with HIR to
promote extensive liver repopulation by the transplanted cells.
Example 4
Focal Liver Irradiation for Selective Lobar Repopulation
[0181] Irradiation to a portion of the whole liver is well
tolerated in cancer patients. Doses higher than 50 Gy to parts of
the liver can safely be offered to patients in the clinic with
modern techniques of 3-D conformal RT or intensity-modulated RT.
This is because clinical hepatic injury induced by irradiation is a
function of dose and the volume of liver that is irradiated.
Clinical trials have demonstrated that RILD or liver failure is not
precipitated even when one-third of the liver receives a RT dose of
70-80 Gy (Dawson, L. et al., Int J Radiat Oncol Biol Phys,
53:810-821 (2002); Dawson, L. et al., Semin Radiat Oncol,
11:240-246 (2001)). We hypothesized that administration of focal
liver irradiation would provide selective growth advantage of the
engrafted hepatocytes, only in irradiated lobes. This would enable
us to repopulate a single lobe of the liver or a portion of a
single lobe by delivering focal high dose HIR. To provide mitotic
signals, a compensatory regenerative signal following right portal
vein branch ligation (PVBL) was used (see, Deb, N. et al.,
Hepatology, 34 (4):153A., 34:153A (2001)) (FIG. 5) and direct
mitogens, Tri-iodo thyronine (T3) (FIG. 6), and HGF (FIG. 7).
PVBL+partial HIR experiments were performed in DPPIV model, while
adeno-HGF+partial HIR was performed in the rosa/C57Bl/6 model. As
seen in FIG. 5B-E, clusters of red DPPIV+ve hepatocytes appeared by
3 weeks. There was gradual repopulation of the anterior irradiated
lobes with near-total replacement of the lobe by 5 months (FIG.
5E). In contrast, the posterior lobes that did not receive HIR
failed to be repopulated by the donor cells (FIG. 5F). The ability
of partial HIR to induce selective lobar repopulation was confirmed
in the rosa model of HT, following administration of adeno-HGF and
HIR (FIG. 8). C57Bl/6 mice received HIR (50 Gy) to the anterior
liver lobes after shielding the caudate and the right posterior
lobe, followed by an intravenous injection of a recombinant
adenoviral vector expressing human HGF (1.times.10.sup.11
particles). Two days after HIR, they received a transplantation of
0.5-1.0 million congenic rosa hepatocytes, there was selective
repopulation of the irradiated anterior lobes (FIG. 8). Results
show that adeno-HGF+HIR enabled near-total repopulation of the host
liver by beta-galactosidase-positive transgenic hepatocytes within
4 months of HT (FIG. 7-8). The ability to selectively repopulate
portions of the liver is unique to HIR-based repopulation
protocols. It is contemplated for most metabolic liver diseases,
functional correction of a fraction of the liver mass (10-25%)
would be sufficient for significant clinical benefit and cure.
Thus, the use of focal HIR to selectively repopulate a portion of
the liver can enhance the safety because the larger portion of the
liver would not be subjected to irradiation.
Example 5
Ex Vivo UGT1A1 Gene Therapy for Gunn Rats Using Preparative
Regimens of Massive Liver Repopulation with Genetically Modified
Hepatocytes
[0182] Having described a safe and reproducible method of massive
hepatic repopulation, we wanted to evaluate whether the liver could
be repopulated by hepatocytes that have been genetically modified
ex vivo. We have used Gunn rat hepatocytes that had been
conditionally immortalized by transduction with a thermolabile SV40
T antigen in our laboratory (Fox, I. et al., Hepatology, 21:837-846
(1995)). These hepatocytes proliferate at 33.degree. C., but at
physiological temperatures (37.degree. C. to 39.degree. C.), the T
antigen is degraded, and the hepatocytes stop proliferating and
exhibit liver-specific functions. The cells were further transduced
using a retroviral vector expressing human UGT1A1 and several
transduced colonies were cloned by serial dilution (Tada, K. et
al., Liver Transpl Surg, 4:78-88 (1998)). We transplanted these
UGT1A1-transduced conditionally immortalized Gunn rat hepatocytes
into autologous Gunn rats after a preparative regimen of PH+HIR.
FIG. 9A, shows the experimental design. Immunohistochemical
staining using anti-UGT1A1 antibodies demonstrate progressive
repopulation of the genetically engineered conditionally
immortalized Gunn rat hepatocytes expressing the human UGT1A1
transgene (FIG. 9B). By 16 weeks, there was near total replacement
of the irradiated host liver. The physiological functioning of the
transplanted cells was evident by the gradual decrease in serum
bilirubin with complete correction of hyperbilirubinemia and
jaundice by 12 weeks (FIG. 9C).
Example 6
Treatment of a Human with Chronic Liver Disease
[0183] Treatment of a human with metabolic or chronic liver failure
as revealed by abnormal liver function tests is first treated with
a dose of x-ray radiation 10 to 30 Gy to one or more lobes of the
liver. The remainder of the exposed subject and liver is shielded
from the radiation. Subsequently, 5.times.10.sup.10 to
5.times.10.sup.12 ex vivo hepatocytes (freshly harvested from a
suitably matched human donor) per kg of body weight are
administered to the patient and the patient is started on a daily
regimen of recombinant human hepatocyte growth factor continuous
infusion over 1 to 3 days, repeated over several weeks and
recombinant epidermal growth factor (1 mg/kg), for two months and
immunosuppressive therapy. The irradiated portion of the liver is
biopsied at 2, 4, and 8 weeks and engrafted hepatocytes are
identified by distinguishing cells according to a difference in
their genomic nucleic acid sequence. Liver function tests are
performed weekly to assess liver function. Over the period of
monitoring the number of engrafted cells as a percent of the
biopsied cells reaches 50% and the liver function tests return to
more normal values.
Example 7
[0184] In this report, we report on the differentiation of oval
cells into adult hepatocytes and bile ducts upon transplantation
into liver that has been treated with a preparative regimen of
hepatic irradiation (HIR) and systemic injection with adenovirus
expressing human hepatocyte growth factor (Ad-HGF). We report that
adult hepatic progenitor cells can engraft, integrate and
proliferate in irradiated rat livers that have also received
adeno-HGF.
Summary of Liver Stem Cell Transplantation Experiments.
[0185] Although adult hepatic progenitor/stem cells (HPC) are
normally not seen in the liver, they expand and regenerate an
injured liver when primary hepatocytes fail to proliferate. The
role of HPC in the repair of radiation-induced liver damage (RILD)
is unknown. The question whether HPCs would proliferate and
compensate for parenchymal cell loss in a rodent model of RILD,
induced by partial hepatectomy (PH) and hepatic irradiation (HIR)
was investigated in this example by examining whether HPC
proliferation is associated with liver regeneration after PH+HIR in
F344 rats. Furthermore, the questions whether HPCs engraft and
preferentially proliferate and repopulate in an irradiated rat
liver following HIR and administration of a hepatic mitotic
stimulus, such as, PH or administration of hepatocyte growth factor
(HGF), was investigated.
[0186] In these experiments, dipeptidyl peptidase IV
(DPPIV)-deficient (DPPIV-ve) F344 rats received PH, followed by HIR
(50Gy) to the anterior liver lobes after exposing the liver by
laparotomy and shielding the posterior liver lobes and other
abdominal organs. Separate cohorts received HIR followed by an
injection of a recombinant adenoviral vector expressing human HGF
(Ad-HGF, 1.times.10.sup.11 particles). One day after HIR,
1.times.10.sup.7 freshly isolated, DPPIV-positive (DPPIV+ve) HPCs
were transplanted into the liver of the DPPIV-ve rats by
intrasplenic injection. HPCs were isolated from congeneic DPPIV+ve
F344 rats that were previously treated either with D-galactosamine
or with 2-acetylaminofluorence and PH to induce hepatic injury.
Since HPCs are smaller in size than adult primary hepatocytes, HPCs
were purified from the liver nonparenchymal cell fraction by
discontinuous Nycodenz gradients or by using epithelial cell
adhesion molecule (EpCAM)-coated magnetic beads for positive
selection. Animals were sacrificed at 2, 6 and 8 weeks after
treatment. Proliferation of HPCs and liver repopulation were
detected by OV-6 immunohistochemistry and DPPIV histochemical
staining.
[0187] With respect to activation and proliferation of oval cells
in PH+HIR-treated animals. H&E staining of frozen liver
sections revealed that the OV-6+ve HPC cells were activated and
proliferated between 2-6 weeks after PH+HIR, indicating that HPC
population participates in liver regeneration following HIR.
Typically, oval cells appeared as small size, with an oval nucleus
and scanty cytoplasm and were predominantly present at the
interface between the portal tracts and the hepatic parenchyma.
They formed tubular structures with poorly defined lumen and no
basement membrane and were Immunoreactive for CK19, OV6 and EpCAM.
The distinct morphology and markers of oval cells were similar to
those described in humans and rodents.
[0188] Hepatic oval cells were found to differentiate into
hepatocytes and bile ducts in irradiated liver. To determine the
repopulation potential of liver stem cells/HPC/oval cells in vivo,
we isolated the oval cell-enriched non-parenchymal cells from rat
livers treated with D-gal and 2-AAF, using the method of
discontinuous Nycodenz density gradient. The non-parenchymal cells
at the interface of 13% and 17% Nycodenz layers were collected. The
number of oval cells was estimated by histochemical staining for
GGT and immunostaining for CK19 and 15-20% of GGT+/CK19+ oval cells
was found at the fraction. To determine the repopulation capacity
of the oval cells in vivo, 1.times.10.sup.7 of freshly isolated
oval enriched non-parenchymal cells were transplanted into the
liver of the DPPIV deficient Fisher rats through intrasplenic
injection. The transplanted DPPIV competent oval cells in the DPPIV
deficient host liver can be easily traced by histochemical staining
or immunostaining for DPPIV. The recipients were treated with HIR
and Ad-HGF prior to cell transplantation. Six weeks after
transplantation, the differentiation and repopulation of
transplanted oval cells in the recipient liver was evaluated. A
significant part of recipient liver, around 40%, was replaced by
transplanted oval cells (FIGS. 10 and 11). The regenerated
hepatocyte nodules showed typical linear bile canalicular staining
pattern for DPPIV. Around the regenerated hepatocyte nodules, DPPIV
positive duct structures were also observed (FIG. 10). CK19
staining co-localized with the diffusely stained DPPIV ducts. These
data demonstrated that transplanted oval cells showed bipotential
characteristic by differentiating into hepatic and biliary lineages
in the irradiated recipient liver.
[0189] HGF was found to stimulate the proliferation and
differentiation of transplanted oval cells in vivo. To define the
condition of oval cell proliferation and differentiation in vivo,
isolated oval cells were transplanted into the liver of normal
DPPIV deficient rats and DPPIV deficient rats pretreated with
Ad-HGF, HIR and HIR/Ad-HGF, respectively. Six weeks after cell
transplantation, the repopulation of the liver was evaluated by
histochemical staining for DPPIV activity. Compared to controls
(Ad-HGF alone or HIR alone), livers of the rats subjected to
HIR/Ad-HGF were significantly replaced by the DPPIV+hepatocytes
after 6 weeks (p<0.01) (FIG. 11). However, transplantation of
oval cells in normal rats, and rats subjected to HIR alone or
Ad-HGF alone yields few scattered small DPPIV+hepatocyte clusters,
which indicating no repopulation (FIG. 12).
[0190] EpCAM+cells showed liver repopulation capacity in the
irradiated host liver. EpCAM+HPCs were isolated from the oval cell
enriched non-parenchyma fraction ( 13/17%) using magnetic beads
positive selection (FIG. 13) and their repopulation potential was
evaluated in DPPIV deficient rats pretreated with HIR/Ad-HGF.
EpCAM+cells were also demonstrated GGT+/CK19+ by histochemical
staining for GGT and immunostaining. Eight weeks after
transplantation, radiation damaged host liver was significantly
replaced by DPPIV positive hepatocytes (FIG. 14). There was
near-total replacement of irradiated hepatic parenchyma by
transplanted HPCs within 6 weeks after transplantation. EpCAM+ve
HPC fraction exhibited a 40-60% repopulation of irradiated liver
lobes by 8 weeks. In contrast, there was engraftment but minimal
repopulation of HPCs in rat livers that received AdHGF alone
(p<0.01) or in liver lobes that were shielded from HIR. H&E
staining demonstrated a normal liver architecture in livers that
received HPC transplantation after HIR.
[0191] Cholangiocarcinoma stem cells can be derived from oval cells
and grown in rat livers. Oval cells are bipotential and are
postulated to originate both hepatocellular or bile duct cancer.
Oval cells that were cultured in vitro were transplanted into rats
that received partial hepatectomy. Mitotic stimuli from partial
hepatectomy resulted in the development of cholangiocarcinoma or
bile duct cancer (FIGS. 15 and 16). Thus these models can be used
to grow cancer stem cells in rat livers treated with HIR/PH.
[0192] Accordingly, amplification of adult HPC population in a
rodent model of RILD has now been demonstrated. Adult HPC
transplantation, combined with a hepatic mitotic stimulus, can
engraft and rapidly regenerate liver tissues following high doses
of HIR. EpCAM+ve HPCs had strong liver repopulation capacity.
Accordingly, EpCAM is contemplated as a cell surface marker for
HPCs which can be used to purify HPC population from donor livers.
As HPCs exhibit bipotential differentiation towards both
hepatocytic and biliary lineages, transplantation of HPCs can
provide a salvage therapy for RILD and for the treatment of
end-stage liver diseases.
[0193] The referenced patents, patent applications, and scientific
literature, including accession numbers to GenBank database
sequences, referred to herein are hereby incorporated by reference
in their entirety. Any conflict between any reference cited herein
and the specific teachings of this specification shall be resolved
in favor of the latter. Likewise, any conflict between an
art-understood definition of a word or phrase and a definition of
the word or phrase as specifically taught in this specification
shall be resolved in favor of the latter.
* * * * *
References