U.S. patent application number 14/707739 was filed with the patent office on 2016-05-19 for cd34 stem cell-related methods and compositions.
This patent application is currently assigned to APCETH GMBH & CO. KG. The applicant listed for this patent is Ralf Huss, Peter J. Nelson, Matthias C. Raggi, Manfred J. Stangl. Invention is credited to Ralf Huss, Peter J. Nelson, Matthias C. Raggi, Manfred J. Stangl.
Application Number | 20160136207 14/707739 |
Document ID | / |
Family ID | 40093981 |
Filed Date | 2016-05-19 |
United States Patent
Application |
20160136207 |
Kind Code |
A1 |
Huss; Ralf ; et al. |
May 19, 2016 |
CD34 STEM CELL-RELATED METHODS AND COMPOSITIONS
Abstract
This invention provides novel stem cell-based methods for
treating a number of conditions. These methods employ CD34 stem
cells, and have a tremendous advantage in that they do not require
myeloablation in the subject being treated. The CD34 stem cells
used in the instant methods can be genetically, modified or not,
depending on the disorder treated.
Inventors: |
Huss; Ralf; (Waakirchen,
DE) ; Nelson; Peter J.; (Munich, DE) ; Raggi;
Matthias C.; (Munchen, DE) ; Stangl; Manfred J.;
(Sauerlach, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Huss; Ralf
Nelson; Peter J.
Raggi; Matthias C.
Stangl; Manfred J. |
Waakirchen
Munich
Munchen
Sauerlach |
|
DE
DE
DE
DE |
|
|
Assignee: |
APCETH GMBH & CO. KG
Munich
DE
|
Family ID: |
40093981 |
Appl. No.: |
14/707739 |
Filed: |
May 8, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13179374 |
Jul 8, 2011 |
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14707739 |
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12154059 |
May 20, 2008 |
7998472 |
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13179374 |
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60931622 |
May 24, 2007 |
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61003050 |
Nov 14, 2007 |
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Current U.S.
Class: |
424/93.21 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 2035/124 20130101; A61K 48/00 20130101; A61K 35/12 20130101;
A61P 9/00 20180101; A61K 38/1709 20130101; A61K 38/1866 20130101;
A61P 21/04 20180101; A61P 35/02 20180101; A61P 41/00 20180101; A61K
35/545 20130101; A61K 38/18 20130101; A61K 35/28 20130101; A61P
1/00 20180101; A61P 3/10 20180101; A61P 21/00 20180101; C07K 14/49
20130101; A61P 35/04 20180101; C12N 5/0663 20130101; A61P 9/10
20180101; A61K 2035/122 20130101; A61P 9/14 20180101; A61P 1/04
20180101; A61P 17/02 20180101; A61P 35/00 20180101 |
International
Class: |
A61K 35/545 20060101
A61K035/545; A61K 38/18 20060101 A61K038/18 |
Claims
1. A method for treating a diabetic subject or a pre-diabetic
subject comprising introducing into the subject's bloodstream a
therapeutically effective number of genetically modified CD34- stem
cells, wherein (a) each of the genetically modified CD34- stem
cells contains an exogenous nucleic acid comprising (i) a region
encoding a protein which enhances endothelial cell growth, which
region is operably linked to (ii) an endothelium-specific promoter
or promoter/enhancer combination, and (b) the introduction of the
genetically modified CD34- stem cells is not preceded, accompanied
or followed by myeloablation.
2. The method of claim 1, wherein the subject is human.
3. The method of claim 2, wherein the subject is pre-diabetic.
4. The method of claim 3, wherein the pre-diabetic subject is
pre-diabetic for type I diabetes.
5. The method of claim 3, wherein the pre-diabetic subject is
pre-diabetic for type II diabetes.
6. The method of claim 2, wherein the subject is diabetic.
7. The method of claim 6, wherein the diabetic subject is afflicted
with type I diabetes.
8. The method of claim 6, wherein the diabetic subject is afflicted
with type II diabetes.
9. The method of claim 2, wherein the promoter/enhancer combination
is the Tie2 promoter/enhancer and the protein which enhances
endothelial cell growth is a vascular endothelial growth factor
(VEGF) associated with angiogenesis.
10. The method of claim 2, wherein the genetically modified CD34-
stem cells are allogenic with respect to the subject.
11. The method of claim 2, wherein the genetically modified CD34-
stem cells are autologous with respect to the subject.
12. The method of claim 2, wherein the therapeutically effective
number of genetically modified CD34- stem cells is from about
1.times.10.sup.3 to about 1.times.10.sup.7 cells/kg body weight.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/179,374 filed Jul. 8, 2011, which is a continuation of U.S.
application Ser. No. 12/154,059 filed May 20, 2008, which claims
priority from U.S. Provisional Patent Application Ser. No.
60/931,622 filed May 24, 2007 and U.S. Provisional Patent
Application Ser. No. 61/003,050 filed Nov. 14, 2007.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] Throughout this application, various publications are cited.
The disclosure of these publications, as well as of the
above-identified provisional applications, is hereby incorporated
by reference into this application to describe more fully the state
of the art to which this invention pertains.
[0003] Stem cells mediate the reproduction and transmission of
genetic information to subsequent cellular generations. They can
self-renew and generate differentiated progeny. In recent years
progress has been made in our understanding of the molecular
mechanisms that underlie the interactions between stem cells and
their tissue niches. This has led to a better understanding of the
molecular regulatory mechanisms at work in stem cells.
[0004] While gene therapy is still an experimental approach, the
technology holds promise for making an impact on human health. The
scope and definition of gene therapy has changed and expanded over
the past few years. In addition to correcting inherited genetic
disorders such as cystic fibrosis, hemophilia and others entities,
gene therapy approaches are also being developed to combat acquired
diseases such as cancer, AIDS, chronic vascular ischemia,
osteoarthritis, diabetes, Parkinson's and Alzheimer's disease.
[0005] At present, germ line gene therapy is not being contemplated
due to its complex technical nature and ethical considerations.
However, somatic cell gene therapy exclusively for the benefit of
one individual (that cannot be passed on to succeeding generations)
is a major focus of stem cell research. It has taken over 15 years
of effort from the initial description of successful gene transfer
into murine hematopoietic stem cells, to the first unambiguously
successful clinical trials in patients born with x-linked combined
immunodeficiency (SCID) and adenosine deaminase deficiency
(ADA)-deficiency (Aiuti et al., 2002; Cavazzana-Calvo et al., 2000;
Gaspar et al., 2004). Many aspects of stem cell therapy are being
explored. For example, retroviral vectors have been used in many
settings for the transfer of genes into stem cells to repair
mutated or incomplete genes. These include severe combined immune
deficiencies, Fanconi anemia and other hemoglobinopathies (Herzog
et al., 2006).
[0006] A central issue in stem cell engineering is the specific
methodology used to introduce therapeutic genes into the progenitor
cells. Because retroviruses tend to insert into active genes (it is
thought that condensed chromatin opens up in these regions), it has
been suggested that their use may also increase the risk of cancer
(Young et al., 2006), because the insertion of retroviral vectors
proximal to genes involved in cell proliferation could in theory
generate a precursor cancer stem cell. However, the overall risk of
this type of event is difficult to establish. There are now many
examples of complete success achieved in patients with chronic
granulomatous disease (CGD) where NADPH oxidase activity was
restored following the infusion of genetically altered blood stem
cells (Barese et al., 2004).
[0007] The minimal requirement for productive gene therapy is the
sustained production of the therapeutic gene product in the correct
biological context with minimal harmful side effects. To achieve
this end, the application of stem cells in genetic therapy will
require the development of new strategies for modulating
therapeutic gene expression, as well as methods for the efficient
delivery of foreign genes into stem cells. The selective control of
therapeutic gene expression by differentiating stem cells within a
defined tissue environment is an important goal in stem cell
engineering. This approach could, for example, help in the control
of stem cell differentiation into specific lineages, the
maintenance of their undifferentiated state for later
transplantation, proliferation, and the regulation of expression of
therapeutic genes such as suicide genes, cytokines or growth
factors in defined tissue environments.
SUMMARY OF THE INVENTION
[0008] This invention provides a method for treating a subject
afflicted with a gastrointestinal disorder comprising introducing
into the subject's bloodstream a therapeutically effective number
of CD34 stem cells, wherein (a) the CD34 stem cells are not
genetically modified, (b) the introduction of the CD34 stem cells
is not preceded, accompanied or followed by myeloablation, and (c)
the gastrointestinal disorder is characterized by a need for cell
proliferation in the gastrointestinal endothelium.
[0009] This invention also provides a method for treating a
diabetic subject or a pre-diabetic subject comprising introducing
into the subject's bloodstream a therapeutically effective number
of CD34 stem cells, wherein (a) the CD34 stem cells are not
genetically modified, and (b) the introduction of the CD34 stem
cells is not preceded, accompanied or followed by
myeloablation.
[0010] This invention further provides a method for treating a
subject afflicted with muscular dystrophy comprising introducing
into the subject's bloodstream a therapeutically effective number
of non-autologous CD34 stem cells, wherein (a) the CD34 stem cells
are not genetically modified, and (b) the introduction of the CD34
stem cells is not preceded, accompanied or followed by
myeloablation.
[0011] This invention further provides a method for improving
microcirculation and/or acute wound healing in a subject who is
about to undergo, is undergoing or has undergone surgery comprising
introducing into the subject's bloodstream a therapeutically
effective number of CD34 stem cells, wherein (a) the CD34 stem
cells are introduced into the subject's bloodstream immediately
prior to, during, and/or immediately following surgery, (b) the
CD34 stem cells are not genetically modified, and (c) the
introduction of the CD34 stem cells is not preceded, accompanied or
followed by myeloablation.
[0012] This invention further provides a method for improving
microcirculation and/or acute wound healing in a subject who is
about to undergo, is undergoing or has undergone a physical trauma
comprising introducing into the subject's bloodstream a
therapeutically effective number of CD34 stem cells, wherein (a)
the CD34 stem cells are introduced into the subject's bloodstream
immediately prior to, during, and/or immediately following the
physical trauma, (b) the CD34 stem cells are not genetically
modified, and (c) the introduction of the CD34 stem cells is not
preceded, accompanied or followed by myeloablation.
[0013] This invention provides a method for treating a subject
afflicted with a tumor comprising introducing into the subject's
bloodstream a therapeutically effective number of genetically
modified CD34 stem cells, wherein (a) each of the genetically
modified CD34 stem cells contains an exogenous nucleic acid
comprising (i) a cytotoxic protein-encoding region operably linked
to (ii) a promoter or promoter/enhancer combination, whereby the
cytotoxic protein is selectively expressed when the genetically
modified CD34 stem cells come into proximity with, and
differentiate in proximity with, tumor tissue undergoing
angiogenesis, and (b) the introduction of the genetically modified
CD34 stem cells is not preceded, accompanied or followed by
myeloablation.
[0014] This invention further provides a method for treating a
subject afflicted with a gastrointestinal disorder comprising
introducing into the subject's bloodstream a therapeutically
effective number of genetically modified CD34 stem cells, wherein
(a) each of the genetically modified CD34 stem cells contains an
exogenous nucleic acid comprising (i) a region encoding a protein
which enhances endothelial cell growth, which region is operably
linked to (ii) an endothelium-specific promoter or
promoter/enhancer combination, (b) the introduction of the
genetically modified CD34 stem cells is not preceded, accompanied
or followed by myeloablation, and (c) the gastrointestinal disorder
is characterized by a need for cell proliferation in the
gastrointestinal endothelium.
[0015] This invention further provides a method for treating a
diabetic subject or a pre-diabetic subject comprising introducing
into the subject's bloodstream a therapeutically effective number
of genetically modified CD34 stem cells, wherein (a) each of the
genetically modified CD34 stem cells contains an exogenous nucleic
acid comprising (i) a region encoding a protein which enhances
endothelial cell growth, which region is operably linked to (ii) an
endothelium-specific promoter or promoter/enhancer combination, and
(b) the introduction of the genetically modified CD34 stem cells is
not preceded, accompanied or followed by myeloablation.
[0016] This invention further provides a method for treating a
subject afflicted with muscular dystrophy comprising introducing
into the subject's bloodstream a therapeutically effective number
of genetically modified CD34 stem cells, wherein (a) each of the
genetically modified CD34 stem cells contains an exogenous nucleic
acid comprising (i) a region encoding a protein which is absent
from or under-expressed in the subject's muscle cells or whose
overexpression in the subject's muscle cells is desired, which
region is operably linked to (ii) a muscle-specific promoter or
muscle-specific promoter/enhancer combination, and (b) the
introduction of the genetically modified CD34 stem cells is not
preceded, accompanied or followed by myeloablation.
[0017] This invention further provides a method for improving
microcirculation and/or acute wound healing in a subject who is
about to undergo, is undergoing or has undergone surgery comprising
introducing into the subject's bloodstream a therapeutically
effective number of genetically modified CD34 stem cells, wherein
(a) each of the genetically modified CD34 stem cells contains an
exogenous nucleic acid comprising (i) a region encoding a protein
which enhances endothelial cell growth, which region is operably
linked to (ii) an endothelium-specific promoter or
promoter/enhancer combination, and (b) the introduction of the
genetically modified CD34 stem cells is not preceded, accompanied
or followed by myeloablation.
[0018] Finally, this invention provides a method for improving
microcirculation and/or acute wound healing in a subject who is
about to undergo, is undergoing or has undergone a physical trauma
comprising introducing into the subject's bloodstream a
therapeutically effective number of genetically modified CD34 stem
cells, wherein (a) each of the genetically modified CD34 stem cells
contains an exogenous nucleic acid comprising (i) a region encoding
a protein which enhances endothelial cell growth, which region is
operably linked to (ii) an endothelium-specific promoter or
promoter/enhancer combination, and (b) the introduction of the
genetically modified CD34 stem cells is not preceded, accompanied
or followed by myeloablation.
[0019] Other objects and features of the present invention will
become apparent from the following detailed description considered
in conjunction with the accompanying drawings. It is to be
understood, however, that the drawings are designed solely for
purposes of illustration and not as a definition of the limits of
the invention, for which reference should be made to the appended
claims. It should be further understood that the drawings are not
necessarily drawn to scale and that, unless otherwise indicated,
they are merely intended to conceptually illustrate the structures
and procedures described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] In the drawings:
[0021] FIG. 1
[0022] Top row: expression of insulin in a normal murine
.beta.-islet before (left) and after alloxan (ALX) treatment
(right) with almost complete insulin depletion. Bottom row: size of
a normal insulin producing .beta.-islet at different magnification
(20.times.; 40.times.); the treatment of insulin-depleted
.beta.-islet after ALX treatment with CD34-negative stem cells (SC)
completely restores insulin production with signs of hypertrophy
(20.times. and 40.times.).
[0023] FIG. 2
[0024] Isolated cells from a murine pancreas after ALX-induced
diabetes and SC restoration of insulin production. The transplanted
CD34-negative stem cells were marked with a constitutively
expressed green fluorescence protein, and the insulin-producing
cells showed a red fluorescence. There was no co-expression of both
markers, suggesting that the transplanted stem cells do not express
insulin by themselves but rather facilitate the endogenous
regeneration.
[0025] FIG. 3
[0026] Left: Blood glucose levels of mice after ALX treatment
without SC transplantation (top), with SC transplantation and no
correction of the blood glucose level (middle) and mice with a
normalized blood glucose level after SC transplantation (bottom).
Right: Only mice that revealed the presence of stem cells in their
pancreas (homogenized for FACS analysis and detection of green
fluorescence) (E3; red circle) showed a normalized blood glucose
level, suggesting the pivotal role of transplanted cells in the
correction of insulin production.
[0027] FIG. 4
[0028] Schematic presentation of the development of an epithelial
malignancy from an in-situ carcinoma to invasive cancer and the
connection to the endogenous blood vessel system. CD34-negative
stem cells home to a site of neo-angiogenesis as demonstrated here
and can therefore be utilized as a Trojan Horse to deliver
cytotoxic or immune modulatory agents.
[0029] FIG. 5
[0030] Detection RFP-positive cells in mammary tumors. Tie2-RFP
transfected stem cells differentiate to endothelial and transcribe
the RFP. (A) Counterstained with DAPI. (B) RFP-positive cells
forming vessels.
[0031] FIG. 6
[0032] Reduced tumor progression under GCV treatment. (A) Stem
cell-GCV application protocol. The cell suspension (day 0) and the
GCV-solution (day 5-8) respectively were applied as shown. Increase
of bodyweight during the treatment of mice reflected the total
tumor load as all breasts were involved. Body weight was measured
on day 0 and 5 of each cycle of therapy and the day of dissection.
(B) Groups of mice, treatment starting in week 22, average showing
standard deviation. Mice were sorted in one treatment group and two
control groups. First control group received 1.times.PBS instead of
a stem cell suspension and no drug-injection (dashed line); the
second control group received a stem cell suspension transfected
with Tie2-RFP but no GCV (dotted line). The treatment group
received stem cells and GCV as shown in A, (solid line). (C) Groups
of treatment starting in week 18, average and standard
deviation.
[0033] FIG. 7
[0034] Age at dissection. Controls vs. treatment group of mice
starting treatment at week 22. Note the significant difference in
time to reach similar tumor sizes (see table 1) and the extended
life span of mice after successful treatment with MSC TK-vehicles
and GCV.
[0035] FIG. 8
[0036] Tumor regrowth model: Primary breast tumor was resected at
18 weeks and MSC/tk treatment was initiated during regrowth of the
tumor.
[0037] FIG. 9
[0038] MSC-expressing green florescent protein (GFP) home to the
growing pancreatic tumor. In parallel experiments, MSC engineered
to express red florescent protein (RFP) under the control of the
Tie2 promoter/enhancer show a directed expression in the tumor
vasculature. Top row: MSC engineered to express GFP under the
control of the CMV promoter home to the tumor following i.v.
injection. Bottom row: MSC engineered to express RFP under the
control of the Tie2.
[0039] FIG. 10
[0040] The effect of the tk/GCV treatment was then assessed after
the injection of the C57B1/6 MSC (Tie2-tk) cells.
[0041] The treatment regimen was essentially as described for the
breast cancer study. 500,000 cells were injected on day one,
followed by three days where the cells were allowed to be recruited
to the growing tumor and to differentiate into endothelial-like
Tie2 expressing cells thus expressing the TK suicide gene. The mice
were then treated for four days with GVC. After one day of rest,
the cycle repeated for the duration of the experiment.
[0042] FIG. 11
[0043] Figure shows an additional example of the effect of
treatment of the orthotopic pancreatic tumor with therapeutic stem
cells together with GCV. A dramatic reduction in tumor size (50%)
as well as reduced peritoneal carcinosis was seen in comparison to
the untreated group.
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
Terms
[0044] In this application, certain terms are used which shall have
the meanings set forth as follows.
[0045] As used herein, "acute wound healing" shall include, without
limitation, a cellular and molecular process which is activated to
repair tissue from the moment of injury under the control of
biological and mechanical signals. Successful acute wound healing
occurs when a dynamic balance is met between the loads placed
across a provisional matrix and the feedback of repair cells.
[0046] As used herein, a cell is "allogenic" with respect to a
subject if it or any of its precursor cells are from another
subject of the same species.
[0047] As used herein, a cell is "autologous" with respect to a
subject if it or its precursor cells are from that same
subject.
[0048] As used herein, "CD34 stem cell" shall mean a stem cell
lacking CD34 on its surface. CD34 stem cells, and methods for
isolating same, are described, for example, in Lange C. et al.,
Accelerated and safe expansion of human mesenchymal stromal cells
in animal serum-free medium for transplantation and regenerative
medicine. J. Cell Physiol. 2007, April 25 [Epub ahead of
print].
[0049] As used herein, "cell proliferation" shall mean the
division, growth in size and/or differentiation of cells.
[0050] As used herein, "cytotoxic protein" shall mean a protein
which, when present in, on and/or in proximity with a cell, causes
that cell's death directly and/or indirectly. Cytotoxic proteins
include, for example, suicide proteins (e.g. HSV-tk) and apoptosis
inducers. Cytotoxic genes include null genes, siRNA or miRNA for
gene knockdown (e.g. CCR5-/-). A number of suicide gene systems
have been identified, including the herpes simplex virus thymidine
kinase gene, the cytosine deaminase gene, the varicella-zoster
virus thymidine kinase gene, the nitroreductase gene, the
Escherichia coli gpt gene, and the E. coli Deo gene. Cytosine
deaminase; Cytochrome P450; Purine nucleoside phosphorylase;
Carboxypeptidase G2; Nitroreductase. As detailed in: Yazawa K,
Fisher W E, Brunicardi F C: Current progress in suicide gene
therapy for cancer. World J Surg. 2002 July; 26(7):783-9. Cytotoxic
factors include the following: (i) homing factors such as
chemokines and mucin chemokine GPI fusions (chemokine derived
agents can be used to facilitate the directed recruitment of
engineered stem cells, see, e.g., PCT International Application No.
PCT/EP2006/011508, regarding mucin fusions anchored with GPI); (ii)
viral antigens (measles, chicken pox) as cytotoxic proteins; and
(iii) Her2/neu antigens which can be presented on the surfaces of
engineered stem cells, followed by administration of her-2/neu
antibody, and CamPath.RTM. (Alemtuzumab) directed against a CD52
epitope.
[0051] As used herein, "endothelial cell" shall include, without
limitation, a cell that forms the inner lining of the intima in
blood vessels during or after a process called angiogenesis. The
factors controlling this process are called angiogenic factors.
Endothelial cells also act with circulating blood cells by means of
receptor-ligand interactions.
[0052] As used herein, an "endothelium-specific promoter or
promoter/enhancer combination" is a promoter or promoter/enhancer
combination, respectively, which when in an endothelial cell in or
in proximity with endothelial cells, causes expression of an
operably linked encoding region more than it would in any other
milieu in the subject.
[0053] As used herein, a nucleic acid is "exogenous" with respect
to a cell if it has been artificially introduced into that cell or
any of that cell's precursor cells.
[0054] As used herein, "gastrointestinal disorder" shall mean any
disorder of the stomach, small intestine and/or large
intestine.
[0055] As used herein, a stem cell is "genetically modified" if
either it or any of its precursor ceils have had nucleic acid
artificially introduced thereinto. Methods for generating
genetically modified stem cells include the use of viral or
non-viral gene transfer (e.g., plasmid transfer, phage integrase,
transposons, AdV, AAV and Lentivirus).
[0056] As used herein, "immediately prior to" an event includes,
for example, within 5, 10 or 30 minutes prior to, or 1, 2, 6, 12 or
24 hours prior to the event. "Immediately following" an event
includes, for example, within 5, 10 or 30 minutes after, or 1, 2,
6, 12 or 24 hours after to the event.
[0057] As used herein, "integration" of a nucleic acid into a cell
can be transient or stable.
[0058] As used herein, "introducing" CD34 stem cells "into the
subject's bloodstream" shall include, without limitation,
introducing such cells into one of the subject's veins or arteries
via injection. Such administering can also be performed, for
example, once, a plurality of times, and/or over one or more
extended periods. A single injection is preferred, but repeated
injections overtime (e.g., quarterly, half-yearly or yearly) may be
necessary in some instances. Such administering is also preferably
performed using an admixture of CD34 stem cells and a
pharmaceutical acceptable carrier. Pharmaceutically acceptable
carriers are well known to those skilled in the art and include,
but are not limited to, 0.01-0.1 M and preferably 0.05 M phosphate
buffer or 0.8% saline. Additionally, such pharmaceutically
acceptable carriers can be aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Aqueous
carriers include water, alcoholic/aqueous solutions, emulsions and
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's and fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers such as Ringer's dextrose, those based on
Ringer's dextrose, and the like. Fluids used commonly for i.v.
administration are found, for example, in Remington: The Science
and Practice of Pharmacy, 20.sup.th Ed., p. 808, Lippincott
Williams & Wilkins (2000). Preservatives and other additives
may also be present, such as, for example, antimicrobials,
antioxidants, chelating agents, inert gases, and the like.
[0059] As used herein, "microcirculation" shall include, without
limitation, the flow of blood from aterioles to capillaries or
sinusoids to venules. Under certain circumstances, the term
microcirculation is also applied to lymphatic vessels.
[0060] As used herein, "myeloablation" shall mean the severe or
complete depletion of bone marrow cells caused by, for example, the
administration of high doses of chemotherapy or radiation therapy.
Myeloablation is a standard procedure and is described, for
example, in Deeg H J, Klingemann H G, Philips G L, A Guide to Bone
Marrow Transplantation. Springer-Verlag Berlin Heidelberg 1992.
[0061] As used herein, a stem cell is "not genetically modified" if
neither it nor any of its precursor cells have had nucleic acid
artificially introduced thereinto.
[0062] As used herein, "nucleic acid" shall mean any nucleic acid
molecule, including, without limitation, DNA, RNA and hybrids
thereof. The nucleic acid bases that form nucleic acid molecules
can be the bases A, C, G, T and U, as well as derivatives thereof.
Derivatives of these bases are well known in the art, and are
exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer
Catalogue 1996-1997,
Roche Molecular Systems, Inc., Branchburg, N.J., USA).
[0063] As used herein, a cytotoxic protein-encoding nucleic acid
region is "operably linked" to a promoter or promoter/enhancer
combination if such promoter or promoter/enhancer combination
causes the expression of the cytotoxic protein.
[0064] As used herein, a "polypeptide" means a polymer of amino
acid residues. A "peptide" typically refers to a shorter
polypeptide (e.g., 10 amino acid residues), and a "protein"
typically refers to a longer polypeptide (e.g., 200 amino acid
residues). The
amino acid residues can be naturally occurring or chemical
analogues thereof. Polypeptides can also include modifications such
as glycosylation, lipid attachment, sulfation, hydroxylation, and
ADP-ribosylation.
[0065] As used herein, a "prediabetic" subject includes, without
limitation, a subject who has the complex of symptoms that indicate
he will likely develop insulin-dependent diabetes. Prediabetic
subjects have a higher-than-normal insulin levels.
[0066] As used herein, a "promoter" includes, without limitation,
endothelin-1 promoter, pre-proendothelin-1 promoter, myoD promoter,
NeuroD promoter, CD20 promoter, insulin promoter, Pdx-1 promoter,
VEGF promoter, VEGF-R promoter, SCL promoter, Seal promoter,
BDNF(-R) promoter, NGF(-R) promoter and EGF-R promoter.
[0067] As used herein, "promoter/enhancers" include, without
limitation, Tie2 promoter enhancer, and Flk1 promoter and intronic
enhancer.
[0068] As used herein, in "proximity with" a tissue includes, for
example, within 1 mm of the tissue, within 0.5 mm of the tissue and
within 0.25 mm of the tissue.
[0069] As used herein, a cytotoxic protein is "selectively
expressed" when a genetically modified CD34 stem cell encoding same
comes into proximity with, and differentiates in proximity with,
tumor tissue undergoing angiogenesis, if the cytotoxic protein is
expressed in that milieu more than it is expressed in any other
milieu in the subject. Preferably, the cytotoxic protein is
expressed in that milieu at least 10 times more than it is
expressed in any other milieu in the subject.
[0070] As used herein, "subject" shall mean any animal, such as a
human, non-human primate, mouse, rat, guinea pig or rabbit.
[0071] As used herein, a "therapeutically effective number of CD34
stem cells" includes, without limitation, the following amounts and
ranges of amounts: (i) from about 1.times.10.sup.2 to about
1.times.10.sup.8 cells/kg body weight; (ii) from about
1.times.10.sup.3 to about 1.times.10.sup.7 cells/kg body weight;
(iii) from about 1.times.10.sup.4 to about 1.times.10.sup.6
cells/kg body weight; (iv) from about 1.times.10.sup.4 to about
1.times.10.sup.5 cells/kg body weight; (v) from about
1.times.10.sup.5 to about 1.times.10.sup.6 cells/kg body weight;
(vi) from about 5.times.10.sup.4 to about 0.5.times.10.sup.5
cells/kg body weight; (vii) about 1.times.10.sup.3 cells/kg body
weight; (viii) about 1.times.10.sup.4 cells/kg body weight; (ix)
about 5.times.10.sup.4 cells/kg body weight; (x) about
1.times.10.sup.5 cells/kg body weight; (xi) about 5.times.10.sup.5
cells/kg body weight; (xii) about 1.times.10.sup.6 cells/kg body
weight; and (xiii) about 1.times.10.sup.7 cells/kg body weight.
Human body weights envisioned include, without limitation, about 50
kg, about 60 kg; about 70 kg; about 80 kg, about 90 kg; and about
100 kg. These numbers are based on pre-clinical animal experiments
and standard protocols from the transplantation of CD34+
hematopoietic stem cells. Mononuclear cells (including CD34.sup.+
cells) usually contain between 1:23,000 to 1:300,000 CD34
cells.
[0072] As used herein, "treating" a subject afflicted with a
disorder shall mean slowing, stopping or reversing the disorder's
progression. In the preferred embodiment, treating a subject
afflicted with a disorder means reversing the disorder's
progression, ideally to the point of eliminating the disorder
itself. As used herein, ameliorating a disorder and treating a
disorder are equivalent.
[0073] As used herein, "tumor" shall include, without limitation, a
vascularized tumor such as a prostate tumor, a pancreatic tumor, a
squamous cell carcinoma, a breast tumor, a melanoma, a basal cell
carcinoma, a hepatocellular carcinoma, [0074] testicular cancer, a
neuroblastoma, a glioma or a malignant astrocytic tumor such as
glioblastma multiforme, a colorectal tumor, an endometrial
carcinoma, a lung carcinoma, an ovarian tumor, a cervical tumor, an
osteosarcoma, a rhabdo/leiomyosarcoma, a synovial sarcoma, an
angiosarcoma, an Ewing sarcoma/PNET and a malignant lymphoma. These
include primary tumors as well as metastatic diseases.
[0075] As used herein, a cell is "xenogenic" with respect to a
subject if it or any of its precursor cells are from another
subject of a different species.
EMBODIMENTS OF THE INVENTION
[0076] This invention provides novel stem cell-based methods for
treating certain conditions. These methods employ CD34 stem cells,
as opposed to later stage stem cells, and have a tremendous
advantage in that they do not require myeloablation for the subject
being treated. The CD34 stem cells used in the instant methods can
be genetically modified or not, depending on the disorder treated.
The instant methods are detailed below, beginning with methods
employing non-genetically modified CD34 stem cells and followed by
methods employing genetically modified CD34 stem cells.
[0077] Specifically, this invention provides a method for treating
a subject afflicted with a gastrointestinal disorder comprising
introducing into the subject's bloodstream a therapeutically
effective number of CD34 stem cells, wherein (a) the CD34 stem
cells are not genetically modified, (b) the introduction of the
CD34 stem cells is not preceded, accompanied or followed by
myeloablation, and (c) the gastrointestinal disorder is
characterized by a need for cell proliferation in the
gastrointestinal endothelium.
[0078] In this method, the gastrointestinal disorder includes,
without limitation, colitis, ulcerative colitis, inflammatory bowel
disorder, Crohn's disease, colitis due to acute and chronic
intestinal ischemia, celiac disease, Whipple disease, or
Graft-versus-Host disease after stem cell transplantation.
[0079] This invention also provides a method for treating a
diabetic subject or a pre-diabetic subject comprising introducing
into the subject's bloodstream a therapeutically effective number
of CD34 stem cells, wherein (a) the CD34 stem cells are not
genetically modified, and (b) the introduction of the CD34 stem
cells is not preceded, accompanied or followed by
myeloablation.
[0080] In one embodiment of this method, the subject is
pre-diabetic, either for type I diabetes or type II diabetes. In
another embodiment, the subject is diabetic, afflicted either with
type I diabetes or type II diabetes.
[0081] This invention further provides a method for treating a
subject afflicted with muscular dystrophy comprising introducing
into the subject's bloodstream a therapeutically effective number
of non-autologous CD34 stem cells, wherein (a) the CD34 stem cells
are not genetically modified, and (b) the introduction of the CD34
stem cells is not preceded, accompanied or followed by
myeloablation.
[0082] In the preferred embodiment of this method, the subject is
afflicted with Duchenne or Becker's muscular dystrophy, and the
CD34 stem cells are allogenic with respect to the subject.
[0083] This invention further provides a method for improving
microcirculation and/or acute wound healing in a subject who is
about to undergo, is undergoing or has undergone surgery comprising
introducing into the subject's bloodstream a therapeutically
effective number of CD34 stem cells, wherein (a) the CD34 stem
cells are introduced into the subject's bloodstream immediately
prior to, during, and/or immediately following surgery, (b) the
CD34 stem cells are not genetically modified, and (c) the
introduction of the CD34 stem cells is not preceded, accompanied or
followed by myeloablation.
[0084] This method is appropriate for any type of surgery
including, without limitation, abdominal surgery, thoracic surgery,
neurosurgery, plastic surgery or trauma surgery. Additionally, the
surgery can be laproscopic surgery or open surgery.
[0085] This invention further provides a method for improving
microcirculation and/or acute wound healing in a subject who is
about to undergo, is undergoing or has undergone a physical trauma
comprising introducing into the subject's bloodstream a
therapeutically effective number of CD34 stem cells, wherein (a)
the CD34 stem cells are introduced into the subject's bloodstream
immediately prior to, during, and/or immediately following the
physical trauma, (b) the CD34 stem cells are not genetically
modified, and (c) the introduction of the CD34 stem cells is not
preceded, accompanied or followed by myeloablation.
[0086] This method is appropriate for any type of physical trauma.
Specifically envisioned are (i) childbirth, wherein the CD34 stem
cells are introduced into the subject's bloodstream immediately
prior to, during or immediately following the event, (ii) a flesh
wound caused by a violent act, wherein the CD34 stem cells are
introduced into the subject's bloodstream immediately following the
physical trauma, and (iii) a burn wound, wherein the CD34 stem
cells are introduced into the subject's bloodstream immediately
following the physical trauma.
[0087] In the above methods employing non-genetically modified CD34
stem cells, the subject treated can be any subject. In the
preferred embodiment, the subject is human. Furthermore, in the
subject methods employing non-genetically modified CD34 stem cells,
the CD34 stem cells can be allogenic, autologous or xenogenic with
respect to the subject, unless stated or implied otherwise.
[0088] This invention provides a method for treating a subject
afflicted with a tumor comprising introducing into the subject's
bloodstream a therapeutically effective number of genetically
modified CD34 stem cells, wherein (a) each of the genetically
modified CD34 stem cells contains an exogenous nucleic acid
comprising (i) a cytotoxic protein-encoding region operably linked
to (ii) a promoter or promoter/enhancer combination, whereby the
cytotoxic protein is selectively expressed when the genetically
modified CD34 stem cells come into proximity with, and
differentiate in proximity with, tumor tissue undergoing
angiogenesis, and (b) the introduction of the genetically modified
CD34 stem cells is not preceded, accompanied or followed by
myeloablation.
[0089] All tumor types are envisioned for this method including,
for example, a prostate tumor, a pancreatic tumor, a squamous cell
carcinoma, a breast tumor, a melanoma, a basal cell carcinoma, a
hepatocellular carcinoma, testicular cancer, a neuroblastoma, a
glioma or a malignant astrocytic tumor such as glioblastma
multiforme, a colorectal tumor, an endometrial carcinoma, a lung
carcinoma, an ovarian tumor, a cervical tumor, an osteosarcoma, a
rhabdo/leiomyosarcoma, a synovial sarcoma, an angiosarcoma, an
Ewing sarcoma/PNET and a malignant lymphoma.
[0090] Numerous promoter/enhancer combinations and cytotoxic
proteins are also envisioned for this method. In one embodiment,
the promoter/enhancer combination is the Tie2 promoter/enhancer,
the cytotoxic protein is Herpes simplex viral thymidine kinase, and
the subject is treated with Ganciclovir.RTM. in a manner permitting
the Herpes simplex viral thymidine kinase to render the
Ganciclovir.RTM. cytotoxic. Ganciclovir.RTM. and its methods of use
are well known in the art.
[0091] This invention further provides a method for treating a
subject afflicted with a gastrointestinal disorder comprising
introducing into the subject's bloodstream a therapeutically
effective number of genetically modified CD34 stem cells, wherein
(a) each of the genetically modified CD34 stem cells contains an
exogenous nucleic acid comprising (i) a region encoding a protein
which enhances endothelial cell growth, which region is operably
linked to (ii) an endothelium-specific promoter or
promoter/enhancer combination, (b) the introduction of the
genetically modified CD34 stem cells is not preceded, accompanied
or followed by myeloablation, and (c) the gastrointestinal disorder
is characterized by a need for cell proliferation in the
gastrointestinal endothelium.
[0092] In this method, the gastrointestinal disorder is preferably
colitis, ulcerative colitis, inflammatory bowel disorder or Crohn's
disease.
[0093] Numerous promoter/enhancer combinations and endothelial cell
growth-enhancing proteins are envisioned for this method. In one
embodiment, the promoter/enhancer combination is the Tie2
promoter/enhancer and the protein which enhances endothelial cell
growth is a vascular endothelial growth factor (VEGF). Other
angiogenic factors in addition to VEGF are also envisioned, such as
HIF-1a and Carboanhydrase IX.
[0094] This invention further provides a method for treating a
diabetic subject or a pre-diabetic subject comprising introducing
into the subject's bloodstream a therapeutically effective number
of genetically modified CD34 stem cells, wherein (a) each of the
genetically modified CD34 stem cells contains an exogenous nucleic
acid comprising (i) a region encoding a protein which enhances
endothelial cell growth, which region is operably linked to (ii) an
endothelium-specific promoter or promoter/enhancer combination, and
(b) the introduction of the genetically modified CD34 stem cells is
not preceded, accompanied or followed by myeloablation.
[0095] In one embodiment of this method, the subject is
pre-diabetic, either for type I diabetes or type II diabetes. In
another embodiment, the subject is diabetic, afflicted either with
type I diabetes or type II diabetes.
[0096] Numerous promoter/enhancer combinations and endothelial cell
growth-enhancing proteins are envisioned for this method. In one
embodiment, the promoter/enhancer combination is the Tie2
promoter/enhancer and the protein which enhances endothelial cell
growth is a vascular endothelial growth factor (VEGF) associated
with angiogenesis.
[0097] This invention further provides a method for treating a
subject afflicted with muscular dystrophy comprising introducing
into the subject's bloodstream a therapeutically effective number
of genetically modified CD34 stem cells, wherein (a) each of the
genetically modified CD34 stem cells contains an exogenous nucleic
acid comprising (i) a region encoding a protein which is absent
from or under-expressed in the subject's muscle cells or whose
overexpression in the subject's muscle cells is desired, which
region is operably linked to (ii) a muscle-specific promoter or
muscle-specific promoter/enhancer combination, and (b) the
introduction of the genetically modified CD34 stem cells is not
preceded, accompanied or followed by myeloablation.
[0098] In the preferred embodiment of this method, the subject is
afflicted with Duchenne or Becker's muscular dystrophy, and the
CD34 stem cells are allogenic or autologous with respect to the
subject.
[0099] Numerous muscle-specific promoter/enhancer combinations are
envisioned for this method. In one embodiment, the muscle-specific
promoter/enhancer combination is the MyoD promoter/enhancer. In the
preferred embodiment of Duchenne muscular dystrophy, the protein
absent from the subject's muscle cells is dystrophin.
[0100] This invention further provides a method for improving
microcirculation and/or acute wound healing in a subject who is
about to undergo, is undergoing or has undergone surgery comprising
introducing into the subject's bloodstream a
therapeutically effective number of genetically modified CD34 stem
cells, wherein (a) each of the genetically modified CD34 stem cells
contains an exogenous nucleic acid comprising (i) a region encoding
a protein which enhances endothelial cell growth, which region is
operably linked to (ii) an endothelium-specific promoter or
promoter/enhancer combination, and (b) the introduction of the
genetically modified CD34 stem cells is not preceded, accompanied
or followed by myeloablation.
[0101] This method is appropriate for any type of surgery
including, without limitation, abdominal surgery, thoracic surgery,
neurosurgery or plastic surgery. Additionally, the surgery can be
laproscopic surgery or open surgery.
[0102] Numerous promoter/enhancer combinations and endothelial cell
growth-enhancing proteins are envisioned for this method. In one
embodiment, the promoter/enhancer combination is the Tie2
promoter/enhancer and the protein which enhances endothelial cell
growth is a vascular endothelial growth factor (VEGF) associated
with angiogenesis.
[0103] Finally, this invention provides a method for improving
microcirculation and/or acute wound healing in a subject who is
about to undergo, is undergoing or has undergone a physical trauma
comprising introducing into the subject's bloodstream a
therapeutically effective number of genetically modified CD34 stem
cells, wherein (a) each of the genetically modified CD34 stem cells
contains an exogenous nucleic acid comprising (i) a region encoding
a protein which enhances endothelial cell growth, which region is
operably linked to (ii) an endothelium-specific promoter or
promoter/enhancer combination, and (b) the introduction of the
genetically modified CD34 stem cells is not preceded, accompanied
or followed by myeloablation.
[0104] This method is appropriate for any type of physical trauma.
Specifically envisioned are (i) childbirth, (ii) a flesh wound
caused by a violent act, wherein the CD34 stem cells are introduced
into the subject's bloodstream immediately following the physical
trauma, and (iii) a burn wound, wherein the CD34 stem cells are
introduced into the subject's bloodstream immediately following the
physical trauma.
[0105] Numerous promoter/enhancer combinations and endothelial cell
growth-enhancing proteins are envisioned for this method. In one
embodiment, the promoter/enhancer combination is the Tie2
promoter/enhancer and the protein which enhances endothelial cell
growth is a vascular endothelial growth factor (VEGF) associated
with angiogenesis.
[0106] In the above methods employing genetically modified CD34
stem cells, the subject treated can be any subject. In the
preferred embodiment, the subject is human. Furthermore, in the
subject methods employing genetically modified CD34 stem cells, the
CD34 stem cells can be allogenic, autologous or xenogenic with
respect to the subject, unless stated or implied otherwise.
[0107] In the instant methods employing genetically modified CD34
stem cells, the exogenous genes are expressed, i.e., "turned on",
when the stem cells (i) come into proximity with the appropriate
cells in target tissue, (ii) differentiate, and/or (iii) fuse with
the appropriate cells in target tissue.
[0108] The various proteins and regulatory sequences used in this
invention can be readily obtained by one skilled in the art. For
example, endothelial cell specificity of the Tie2 promoter enhancer
is shown in Schlaeger T M, Bartunkova S, Lawitts J A, Teichmann G,
Risau W, Deutsch U, Sato T N. Uniform
vascular-endothelial-cell-specific gene expression in both
embryonic and adult transgenic mice. Proc Natl Acad Sci USA. 1997
94:3058-63. The HSV TK--V00467 Herpes gene can be used for
thymidine kinase (ATP:thymidine 5' phosphotransferase, e.c.
2.7.1.21) (type 1 strain CL101).
[0109] This invention will be better understood by reference to the
Experimental Details which follow, but those skilled in the art
will readily appreciate that the specific experiments detailed are
only illustrative of the invention as described more fully in the
claims which follow thereafter.
Experimental Details
Part I
Genetically Engineered Transgenic CD34-Negative Stem Cells for
Therapeutic Gene Delivery
Synopsis
[0110] Stem cell and gene therapy approaches hold much hope for the
development of new tools to treat many life-threatening diseases.
The linking of stem cell therapy with selective gene therapy
enhances therapeutic options for the regeneration or replacement of
diseased or missing cells. Tissue-specific gene expression in the
context of differentiation of CD34-negative, in-vitro adherent
growing stem cells are used to generate transgenic CD34-negative
progenitor cells, which will lead to selectivity of cells and
inducibility of gene expression also for safety reasons. Viral and
non-viral genes delivering technologies are detailed as are
techniques for the modulation of gene expression in the context of
stem cell recruitment and differentiation. Potential clinical
applications for this new therapeutic strategy are described,
bringing the transgenic progenitor cells to the cancer or site of
tissue regeneration to induce antitumor therapy or promote tissue
remodeling and wound healing. Transgenic progenitor cells serve as
potent gene delivery vehicle.
Stem Cells as Gene Delivery Vehicles
[0111] Stem cells offer the potential to provide cellular therapies
for diseases that are refractory to other treatments. For each type
of stem cell the ultimate goal is the same: the cell should express
a specific repertoire of genes, thereby modifying cell identity to
maintain, replace, or rescue a particular tissue. To help support
differentiation in the specific tissue environment attempts are
being made to modify the "nuclear programming" of stem cells.
[0112] Multipotent stem cells, mesenchymal stem cells and
multipotent adult progenitor cells (MAPCs) represent promising stem
cell populations as they are capable of differentiating along
different lineages. They represent the "cellular engines" that
drive the renewal of adult mammalian tissues. These cells divide
continuously throughout life to produce new progeny cells that
undergo a program of differentiation and maturation to replace
older expired tissue cells. The same cell turnover program is
thought in some cases to provide a source of cells for the repair
and regeneration of adult tissues. The regenerative potential of
the different stem cell types underlies the current interest in
adapting these cells for applications in cell replacement
therapy.
[0113] Potential sources of stem cells for therapy include bone
marrow, peripheral blood, CNS, liver, pancreas, muscle, skin lung,
intestine heart and fat (Koerbling M, Estrov Z, Adult stem cells
for tissue repair-anew therapeutic concept?) NEJM 2003 349:
570-582). For clinical application the sources of stem cells should
be easily accessible and readily harvested with minimal risk to the
patient and provide abundant cells. In this regard fat tissue
represents a promising tissue source. (Adipose derived stem cells
for the regeneration of damaged tissue Parker M, Adam K, Expert
Opion Biol Therap, 2006, 567-568) Adipose derived stem cells and
bone marrow derived stem cells share similar growth kinetics,
characteristics regarding cell senescence, gene transduction
efficiency, CD surface marker expression and gene transcription
profiles (Cells Tissues Organs. 2003; 174(3): 101-9. Comparison of
multi-lineage cells from human adipose tissue and bone marrow. De
Ugarte D A, Morizono K, Elbarbary A, Alfonso Z, Zuk P A, Zhu M,
Dragoo J L, Ashjian P, Thomas B, Benhaim P, Chen I, Fraser J,
Hedrick M H, Mol Biol Cell. 2002 December; 13(12):4279-95. Human
adipose tissue is a source of multipotent stem cells. Zuk P A, Zhu
M, Ashjian P, De Ugarte D A, Huang J I, Mizuno H, Alfonso Z C,
Fraser J K, Benhaim P, Hedrick M H).
[0114] Stem cells derived from different sources are also being
evaluated as potential vehicles for cell and gene specific therapy
against disease. Their high self-renewal potential makes them
promising candidates for the restoration or replacement
of organ systems and/or the delivery of gene products. While
progenitor cells may show good proliferation and differentiation
potential in vitro, their biological properties in vivo remain to
be defined. Stem cells expanded in vitro represent heterogeneous
populations that include multiple generations of mesenchymal
(stromal) cell progeny, which lack the expression of most
differentiation markers like CD34. These populations may have
retained a limited proliferation potential and responsiveness for
terminal differentiation and maturation along mesenchymal and
non-mesenchymal lineages. Hopefully in the future better markers
for multipotent stem cell populations will improve the ability to
distinguish these stem cells from other progenitor cell
populations.
Tissue-Specific Promoters Used to Deliver Therapeutic Gene
Expression in the Correct Biological Context.
[0115] Stem-cell-mediated therapy ultimately entails nuclear
reprogramming--the alteration of gene expression patterns unique to
cell types in diverse tissues and organs. In a number of inherited
stem cell diseases, a genetic defect imparts a survival
disadvantage to the affected stem cell population. In these
diseases, the transplantation of a "corrected" stem cell population
is thought to undergo spontaneous in vivo selection in the absence
of any exogenously applied selective pressure. For example, in
X-linked SCID the introduction of a therapeutic transgene confers a
continuous proliferation and survival advantage to the transduced
cell population (Neff et al., 2006). However, similar in vivo
selection effects are usually not directly possible in a majority
of diseases. In settings where an over expression of the
therapeutic gene does not confer a survival advantage, a second
selectable gene, ideally under pharmacological regulation, can be
incorporated into the vector. (Tirana and Kim, 2005). Systems that
allow for pharmacologically regulated selection include reversible
forced protein-protein interaction using so called "chemical
inducers of dimerization" (CIDs). These systems rely on two
components. The first is a ligand or drug, and the second is a
fusion protein that combines a ligand-binding protein domain and an
effector domain (usually the intracellular proportion of a growth
factor receptor). The effector domain is activated by drug binding
leading to protein dimerization. The signaling fusion thus serves
as a switch that is turned on in the presence of the CID and off
following withdrawal of the CID. The incorporation of systems like
this into a stem cell population can allow a drug-dependent control
of proliferation of the transduced cell population (Neff et al.,
2006; Neff and Blau, 2001).
[0116] The use of stem cells as delivery vehicles for therapeutic
genes can be seen to offer a series of advantages. Stem cells are
often actively recruited to damaged tissues where they undergo
differentiation during tissue repair. For example CD34+ bone
marrow-derived progenitor cells contribute to tissue repair by
differentiating into endothelial cells, vascular smooth muscle
cells, hematopoietic cells, and possibly other cell types. However,
the mechanisms by which circulating progenitor cells home to
remodeling tissues remain unclear. Jin et al. have demonstrated
that integrin .alpha.4.beta.1 (VLA-4) can promote the homing of
circulating progenitor cells to the .alpha.4.beta.1 ligands VCAM
and cellular fibronectin expressed on actively remodeling
neovasculature. Progenitor cells which express integrin
.alpha.4.beta.1 were shown to home to sites of active tumor
neovascularization but not to normal tissues. Antagonists of
integrin .alpha.4.beta.1, but not other integrins, blocked the
adhesion of these cells to endothelia and outgrowth into
differentiated cell types. (Jin et al, 2006)
[0117] In addition to integrins, chemokines and their receptors
also appear to play central roles in the tissue-specific homing of
stem cells. On the basis of their chemokine receptor expression
profile, CD34 MSCs were predicted to home to secondary lymphatic
organs (CCR7), skin (CCR4, CCR10), small intestine (CCR10), and
salivary glands (CCR10). After transiently labeling CD34- MSC with
CMFDA or stably introducing green fluorescent protein (GFP)
expression plasmids, the cells were injected into syngeneic healthy
mice and the tissue distribution of the cells determined one three
and seven days later. Interestingly, the stem cells did not home
back to the bone marrow but were found to migrate to secondary
lymphatic organs, salivary glands, intestine and skin in accordance
with their chemokine receptor expression profile.
[0118] Given that stem cells can show a selective migration to
different tissue microenvironments in normal as well as diseased
settings, the use of tissue-specific promoters linked to the
differentiation pathway initiated in the recruited stem cell could
in theory be used to drive the selective expression of therapeutic
genes only within a defined biologic context. Stem cells that are
recruited to other tissue niches, but do not undergo the same
program of differentiation, should not express the therapeutic
gene. This approach allows a significant degree of potential
control for the selective expression of the therapeutic gene within
a defined microenvironment and has been successfully applied to
regulate therapeutic gene expression during neovascularization.
[0119] A large number of promoters have been characterized for
their tissue-specific expression. A good source for this
information can be found in the transgene literature, or for
example in the various databases that list tissue-specific promoter
activity for the expression of the CRE transgenes used to drive
tissue-specific CRE/Lox targeted gene deletion models in mouse (for
example: http://www.mshri.on.ca/nagy/Cre-works.htm). Promoters can
be introduced that are selectively regulated in the context of
inflammation or neovascularization. In this regard the
Tie2-promoter, Flk1 promoter and intronic enhancer, endothelin-1
promoter and the pre-proendothelin-1 promoter have been studied for
endothelial specific expression (Huss et al., 2004). The
application of specific reporter genes and new imaging techniques
can be used to define the tissue-specific expression of the
candidate promoter within the context of stem cell transplantation.
Other options regarding the delivery of genes include the
application of an internal ribosome entry site (IRES) signal for
the expression of multiple genes from a single promoter (Jackson,
2005), for example, a therapeutic gene in conjunction with a
reporter gene can be used to better follow the distribution of
expression of the therapeutic gene in an experimental context.
[0120] Importantly, many promoters can show "leakage" of expression
in other tissue types or a low level basal expression in the
engineered cells. Promoter engineering is a new technology that can
allow one to "tune" promoter specificity to limit cross tissue
activity thus allowing a more restrictive expression to specific
cell types (Fessele et al., 2002; Werner et al., 2003).
Gene Delivery Methods
[0121] The various gene delivery methods currently being applied to
stem cell engineering include viral and non viral vectors, as well
as biological or chemical methods of transfection. The methods can
yield either stable or transient gene expression in the system
used.
Viral Gene Delivery Systems
[0122] Because of their high efficiency of transfection,
genetically modified viruses have been widely applied for the
delivery of genes into stem cells.
DNA Virus Vectors
[0123] (i) Adenovirus
[0124] Adenoviruses are double stranded, nonenveloped and
icosahedral viruses containing a 36 kb viral genome (Kojaoghlanian
et al., 2003). Their genes are divided into early (E1A, E1B, E2,
E3, E4), delayed (IX, IVa2) and major late (L1, L2, L3, L4, L5)
genes depending on whether their expression occurs before or after
DNA replication. To date, 51 human adenovirus serotypes have been
described which can infect and replicate in a wide range of organs.
The viruses are classified into the following subgroups: A--induces
tumor with high frequency and short latency, B--are weakly
oncogenic, and C--are non-oncogenic (Cao et al., 2004;
Kojaoghlanian et al., 2003).
[0125] These viruses have been used to generate a series of vectors
for gene transfer cellular engineering. The initial generation of
adenovirus vectors were produced by deleting the E1 gene (required
for viral replication) generating a vector with a 4 kb cloning
capacity. An additional deletion of E3 (responsible for host immune
response) allowed an 8 kb cloning capacity (Bett et al., 1994;
Danthinne and Imperiale, 2000; Danthinne and Werth, 2000). The
second generation of vectors was produced by deleting the E2 region
(required for viral replication) and/or the E4 region
(participating in inhibition of host cell apoptosis) in conjunction
with E1 or E3 deletions. The resultant vectors have a cloning
capacity of 10-13 kb (Armentano et al., 1995). The third "gutted"
generation of vectors was produced by deletion of the entire viral
sequence with the exception of the inverted terminal repeats (ITRs)
and the cis acting packaging signals. These vectors have a cloning
capacity of 25 kb (Kochanek et al., 2001) and have retained their
high transfection efficiency both in quiescent and dividing
cells.
[0126] Importantly, the adenovirus vectors do not normally
integrate into the genome of the host cell, but they have shown
efficacy for transient gene delivery into adult stem cells. These
vectors have a series of advantages and disadvantages. An important
advantage is that they can be amplified at high titers and can
infect a wide range of cells (Benihoud et al., 1999; Kanerva and
Hemminki, 2005). The vectors are generally easy to handle due to
their stability in various storing conditions. Adenovirus type 5
(Ad5) has been successfully used in delivering genes in human and
mouse stem cells (Smith-Arica et al., 2003). The lack of adenovirus
integration into host cell genetic material can in many instances
be seen as a disadvantage, as its use allows only transient
expression of the therapeutic gene.
[0127] For example in a study evaluating the capacity of
mesenchymal stem cells to undergo chondrogenesis when TGF-beta1 and
bone morphogencic protein-2 (BMP-2) were delivered by
adenoviral-mediated expression, the chondrogenesis was found to
closely correlated with the level and duration of the transiently
expressed proteins. Transgene expression in all aggregates was
highly transient, showing a marked decrease after 7 days.
Chondrogenesis was inhibited in aggregates modified to express
>100 ng/ml TGF-beta1 or BMP-2; however, this was partly due to
the inhibitory effect of exposure to high adenoviral loads (Mol
Ther. 2005 August; 12(2):219-28. Gene-induced chondrogenesis of
primary mesenchymal stem cells in vitro. Palmer G D, Steinert A,
Pascher A, Gouze E, Gouze J N, Betz O, Johnstone B, Evans C H,
Ghivizzani S C.) In a second model using rat adipose derived stem
cells transduced with adenovirus carrying the recombinant human
bone morphogenic protein-7 (BMP-7) gene showed promising results
for an autologous source of stem cells for BMP gene therapy.
However, activity assessed by measuring alkaline phosphatase in
vitro was transient and peaked on day 8. Thus the results were
similar to those found in the chondrogenesis model (Cytotherapy.
2005; 7(3):273-81).
[0128] Thus for therapies or experiments that do not require stable
gene expression adenovirus vectors may be a good option. An
additional important problem in using adenovirus vectors is that
they can elicit a strong immune response directed against the
engineered cells upon transfer into the host. Clearly this may be
important issue when considering the application of engineered
cells in a therapeutic setting (J. N. Glasgow et al.,
Transductional and transcriptional targeting of adenovirus for
clinical applications. Curr Gene Ther. 2004 March; 4(1): 1-14). In
vitro and in vivo induction of bone formation based on ex vivo gene
therapy using rat adipose-derived adult stem cells expressing
BMP-7, Yang M, Ma Q J, Dang G T, Ma K, Chen P, Zhou C Y.)
[0129] Adenovirus vectors based on Ad type 5 have been shown to
efficiently and transiently introduce an exogenous gene via the
primary receptor, coxsackievirus, and adenovirus receptor (CAR).
However, some kinds of stem cells, such as MSC and hematopoietic
stem cells, apparently cannot be efficiently transduced with
conventional adenovirus vectors based on Ad serotype 5 (Ad5),
because of the lack of CAR expression. To overcome this problem,
fiber-modified adenovirus vectors and an adenovirus vector based on
another serotype of adenovirus have been developed. (Mol Pharm.
2006 March-April; 3(2):95-103. Adenovirus vector-mediated gene
transfer into stem cells. Kawabata K, Sakurai F, Koizumi N,
Hayakawa T, Mizuguchi H. Laboratory of Gene Transfer and
Regulation, National Institute of Biomedical Innovation, Osaka
5670085, Japan.) [0130] (ii) Adeno-Associated Virus
[0131] Adeno-Associated viruses (AAV) are ubiquitous,
noncytopathic, replication-incompetent members of ssDNA animal
virus of parvoviridae family (G. Gao et al., New recombinant
serotypes of AAV vectors. Curr Gene Ther. 2005 June; 5(3):285-97).
AAV is a small icosahedral virus with a 4.7 kb genome. These
viruses have a characteristic termini consisting of palindromic
repeats that fold into a hairpin. They replicate with the help of
helper virus, which are usually one of the many serotypes of
adenovirus. In the absence of helper virus they integrate into the
human genome at a specific locus (AAVS1) on chromosome 19 and
persist in latent form until helper virus infection occurs
(Atchison et al., 1965, 1966). AAV can transduce cell types from
different species including mouse, rat and monkey. Among the
serotypes, AAV2 is the most studied and widely applied as a gene
delivery vector. Its genome encodes two large opening reading
frames (ORFs) rep and cap. The rep gene encodes four proteins Rep
78, Rep 68, Rep 52 and Rep 40 which play important roles in various
stages of the viral life cycle (e.g. DNA replication,
transcriptional control, site specific integration, accumulation of
single stranded genome used for viral packaging). The cap gene
encodes three viral capsid proteins VP1, VP2, VP3 (Becerra et al.,
1988; Buning et al., 2003). The genomic 3' end serves as the primer
for the second strand synthesis and has terminal resolution sites
(TRS) which serve as the integration sequence for the virus as the
sequence is identical to the sequence on chromosome 19 (Young and
Samulski, 2001; Young et al., 2000).
[0132] These viruses are similar to adenoviruses in that they are
able to infect a wide range of dividing and non-dividing cells.
Unlike adenovirus, they have the ability to integrate into the host
genome at a specific site in the human genome. Unfortunately, due
to their rather bulky genome, the AAV vectors have a limited
capacity for the transfer of foreign gene inserts (Wu and Ataai,
2000).
RNA Virus Vectors
[0133] (i) Retroviruses
[0134] Retroviral genomes consist of two identical copies of single
stranded positive sense RNAs, 7-10 kb in length coding for three
genes; gag, pol and env, flanked by long terminal repeats (LTR) (Yu
and Schaffer, 2005). The gag gene encodes the core protein capsid
containing matrix and nucleocapsid elements that are cleavage
products of the gag precursor protein. The pol gene codes for the
viral protease, reverse transcriptase and integrase enzymes derived
from gag-pol precursor gene. The env gene encodes the envelop
glycoprotein which mediates viral entry. An important feature of
the retroviral genome is the presence of LTRs at each end of the
genome. These sequences facilitate the initiation of viral DNA
synthesis, moderate integration of the proviral DNA into the host
genome, and act as promoters in regulation of viral gene
transcription. Retroviruses are subdivided into three general
groups: the oncoretroviruses (Maloney Murine Leukemia Virus,
MoMLV), the lentiviruses (HIV), and the spumaviruses (foamy virus)
(Trobridge et al., 2002).
[0135] Retroviral based vectors are the most commonly used
integrating vectors for gene therapy. These vectors generally have
a cloning capacity of approximately 8 kb and are generated by a
complete deletion of the viral sequence with the exception of the
LTRs and the cis acting packaging signals.
[0136] The retroviral vectors integrate at random sites in the
genome. The problems associated with this include potential
insertional mutagenesis, and potential oncogenic activity driven
from the LTR. The U3 region of the LTR harbors promoter and
enhancer elements, hence this region when deleted from the vector
leads to a self-inactivating vector where LTR driven transcription
is prevented. An internal promoter can then be used to drive
expression of the transgene.
[0137] The initial studies of stem cell gene transfer in mice
raised the hope that gene transfer into humans would be equally as
efficient (O'Connor and Crystal, 2006). Unfortunately gene transfer
using available retroviral vector systems to transfect
multi-lineage long-term repopulating stem cells is still
significantly more efficient in the mouse. The reduced efficacy of
gene transfer in humans, as well as the uncontrolled integration of
the retroviral vector represents important hurdles for the
application of these vectors as a treatment modality in the context
of stem cell engineering.
[0138] (ii) Lentivirus
[0139] Lentiviruses are members of Retroviridae family of viruses
(M. Schen et al., Gene transfer into hematopoietic stem cells using
lentiviral vectors. Curr Gene Ther. 2002 February; 2(1):45-55.)
They have a more complex genome and replication cycle as compared
to the oncoretroviruses (Beyer et al., 2002). They differ from
simpler retroviruses in that they possess additional regulatory
genes and elements, such as the tat gene, which mediates the
transactivation of viral transcription (Sodroski et al., 1996) and
rev, which mediates nuclear export of unspliced viral RNA (Cochrane
et al., 1990; Emerman and Temin, 1986).
[0140] Lentivirus vectors are derived from the human
immunodeficiency virus (HIV-1) by removing the genes necessary for
viral replication rendering the virus inert. Although they are
devoid of replication genes, the vector can still efficiently
integrate into the host genome allowing stable expression of the
transgene. These vectors have the additional advantage of a low
cytotoxicity and an ability to infect diverse cell types.
Lentiviral vectors have also been developed from Simian, Equine and
Feline origin but the vectors derived from Human Immunodeficiency
Virus (HIV) are the most common (Young et al., 2006).
[0141] Lentivirus vectors are generated by deletion of the entire
viral sequence with the exception of the LTRs and cis acting
packaging signals. The resultant vectors have a cloning capacity of
about 8 kb. One distinguishing feature of these vectors from
retroviral vectors is their ability to transduce dividing and
non-dividing cells as well as terminally differentiated cells
(Kosaka et al., 2004). The lentiviral delivery system is capable of
high infection rates in human mesenchymal and embryonic stem cells.
In a study by Clements et al., the lentiviral backbone was modified
to express mono- and bi-cistronic transgenes and was also used to
deliver short hairpin ribonucleic acid for specific silencing of
gene expression in human stem cells. (Tissue Eng. 2006 July; 12(7):
1741-51. Lentiviral manipulation of gene expression in human adult
and embryonic stem cells. Clements M O, Godfrey A, Crossley J,
Wilson S J, Takeuchi Y, Boshoff C.)
Table 1 summarizes the viral vectors described above.
TABLE-US-00001 Insert Vector genome Inflammatory Vector capacity
(kb) Tropism form Expression potential Efficiency Enveloped
Retrovirus 8 Dividing cells Integrated Stable Low High only
Lentivirus 8 Dividing and Integrated Stable Low High non-dividing
Non-enveloped Adeno-associated <5 Dividing and Episomal and
Stable Low High virus non-dividing integrated Adenovirus 4-25
Dividing and Episomal Transient High High non-dividing
Non-Viral Gene Delivery Systems
[0142] (i) Methods for the Facilitated Integration of Genes
[0143] In addition to the viral based vectors discussed above,
other vector systems that lack viral sequence are currently under
development. The alternative strategies include conventional
plasmid transfer and the application of targeted gene integration
through the use of integrase or transposase technologies. These
represent important new approaches for vector integration and have
the advantage of being both efficient, and often site specific in
their integration. Currently three recombinase systems are
available for genetic engineering: cre recombinase from phage P1
(Lakso et al., 1992; Orban et al., 1992), FLP (flippase) from yeast
2 micron plasmid (Dymecki, 1996; Rodriguez et al., 2000), and an
integrase isolated from streptomyses phage .uparw.:C31 (Ginsburg
and Calos, 2005). Each of these recombinases recognize specific
target integration sites. Cre and FLP recombinase catalyze
integration at a 34 bp palindromic sequence called lox P (locus for
crossover) and FRT (FLP recombinase target) respectively. Phage
integrase catalyzes site-specific, unidirectional recombination
between two short att recognition sites in mammalian genomes.
Recombination results in integration when the att sites are present
on two different DNA molecules and deletion or inversion when the
att sites are on the same molecule. It has been found to function
in tissue culture cells (in vitro) as well as in mice (in
vivo).
[0144] The Sleeping Beauty (SB) transposon is comprised of two
inverted terminal repeats of 340 base pairs each (Izsvak et al.,
2000). This system directs the precise transfer of specific
constructs from a donor plasmid into a mammalian chromosome. The
excision and integration of the transpo son from a plasmid vector
into a chromosomal site is mediated by the SB transposase, which
can be delivered to cells as either in a cis or trans manner
(Kaminski et al., 2002). A gene in a chromosomally integrated
transposon can be expressed over the lifetime of a cell. SB
transposons integrate randomly at TA-dinucleotide base pairs
although the flanking sequences can influence integration. While
the results to date do not suggest that random insertions of SB
transposons represent the same level of risks seen with viral
vectors, more data are required before the system can be safely
applied to human trials.
Physical Methods to Introduce Vectors into Cell
[0145] (i) Electroporation
[0146] Electroporation relies on the use of brief, high voltage
electric pulses which create transient pores in the membrane by
overcoming its capacitance. One advantage of this method is that it
can be utilized for both stable and transient gene expression in
most cell types. The technology relies on the relatively weak
nature of the hydrophobic and hydrophilic interactions in the
phospholipid membrane and its ability to recover its original state
after the disturbance. Once the membrane is permeabilized, polar
molecules can be delivered into the cell with high efficiency.
Large charged molecules like DNA and RNA move into the cell through
a process driven by their electrophoretic gradient. The amplitude
of the pulse governs the total area that would be permeabilized on
the cell surface and the duration of the pulse determines the
extent of permeabilization (Gabriel and Teissie, 1997). The
permeabilized state of the cell depends on the strength of the
pulses. Strong pulses can lead to irreversible permeabilization,
irreparable damage to the cell and ultimately cell death. For this
reason electroporation is probably the harshest of gene delivery
methods and it generally requires greater quantities of DNA and
cells. The effectiveness of this method depends on many crucial
factors like the size of the cell, replication and temperature
during the application of pulse (Rols and Teissie, 1990).
[0147] The most advantageous feature of this technique is that DNA
can be transferred directly into the nucleus increasing its
likelihood of being integrated into the host genome. Even cells
difficult to transfect can be stably transfected using this method
(Aluigi et al., 2005; Zernecke et al., 2003). Modification of the
transfection procedure used during electroporation has led to the
development of an efficient gene transfer method called
nucleofection. The Nucleofector.TM. technology, is a non-viral
electroporation-based gene transfer technique that has been proven
to be an efficient tool for transfecting hard-to-transfect cell
lines and primary cells including MSC (Michela Aluigi, Stem Cells
Vol. 24, No. 2, February 2006, pp. 454-461).
Biomolecule-Based Methods
[0148] (i) Protein Transduction Domains (PTD)
[0149] PTD are short peptides that are transported into the cell
without the use of the endocytotic pathway or protein channels. The
mechanism involved in their entry is not well understood, but it
can occur even at low temperature (Derossi et al. 1996). The two
most commonly used naturally occurring PTDs are the
trans-activating activator of transcription domain (TAT) of human
immunodeficiency virus and the homeodomain of Antennapedia
transcription factor. In addition to these naturally occurring
PTDs, there are a number of artificial peptides that have the
ability to spontaneously cross the cell membrane (Joliot and
Prochiantz, 2004). These peptides can be covalently linked to the
pseudo-peptide backbone of PNA (peptide nucleic acids) to help
deliver them into the cell.
[0150] (ii) Liposomes
[0151] Liposomes are synthetic vesicles that resemble the cell
membrane. When lipid molecules are agitated with water they
spontaneously form spherical double membrane compartments
surrounding an aqueous center forming liposomes. They can fuse with
cells and allow the transfer of "packaged" material into the cell.
Liposomes have been successfully used to deliver genes, drugs,
reporter proteins and other biomolecules into cells (Felnerova et
al., 2004). The advantage of liposomes is that they are made of
natural biomolecules (lipids) and are nonimmunogenic.
[0152] Diverse hydrophilic molecules can be incorporated into them
during formation. For example, when lipids with positively charged
head group are mixed with recombinant DNA they can form lipoplexes
in which the negatively charged DNA is complexed with the positive
head groups of lipid molecules. These complexes can then enter the
cell through the endocytotic pathway and deliver the DNA into
lysosomal compartments. The DNA molecules can escape this
compartment with the help of dioleoylethanolamine (DOPE) and are
transported into the nucleus where they can be transcribed
(Tranchant et al., 2004).
[0153] Despite their simplicity, liposomes suffer from low
efficiency of transfection because they are rapidly cleared by the
reticuloendothelial system due to adsorption of plasma proteins.
Many methods of stabilizing liposomes have been used including
modification of the liposomal surface with oligosaccharides,
thereby sterically stabilizing the liposomes (Xu et al., 2002).
[0154] (iii) Immunoliposomes
[0155] Immunoliposomes are liposomes with specific antibodies
inserted into their membranes. The antibodies bind selectively to
specific surface molecules on the target cell to facilitate uptake.
The surface molecules targeted by the antibodies are those that are
preferably internalized by the cells so that upon binding, the
whole complex is taken up. This approach increases the efficiency
of transfection by enhancing the intracellular release of liposomal
components. These antibodies can be inserted in the liposomal
surface through various lipid anchors or attached at the terminus
of polyethylene glycol grafted onto the liposomal surface. In
addition to providing specificity to gene delivery, the antibodies
can also provide a protective covering to the liposomes that helps
to limit their degradation after uptake by endogenous RNAses or
proteinases (Bendas, 2001). To further prevent degradation of
liposomes and their contents in the lysosomal compartment, pH
sensitive immunoliposomes can be employed (Torchilin, 2006). These
liposomes enhance the release of liposomal content into the cytosol
by fusing with the endosomal membrane within the organelle as they
become destabilized and prone to fusion at acidic pH.
[0156] In general non-viral gene delivery systems have not been as
widely applied as a means of gene delivery into stem cells as viral
gene delivery systems. However, promising results were demonstrated
in a study looking at the transfection viability, proliferation and
differentiation of adult neural stem/progenitor cells into the
three neural lineages neurons. Non-viral, non-liposomal gene
delivery systems (ExGen500 and FuGene6) had a transfection
efficiency of between 16% (ExGen500) and 11% (FuGene6) of cells.
FuGene6-treated cells did not differ from untransfected cells in
their viability or rate of proliferation, whereas these
characteristics were significantly reduced following ExGen500
transfection. Importantly, neither agent affected the pattern of
differentiation following transfection. Both agents could be used
to genetically label cells, and track their differentiation into
the three neural lineages, after grafting onto ex vivo organotypic
hippocampal slice cultures (J Gene Med. 2006 January; 8(1):72-81.
Efficient non-viral transfection of adult neural stem/progenitor
cells, without affecting viability, proliferation or
differentiation. Tinsley R B, Faijerson J, Eriksson P S).
[0157] (iv) Polymer-Based Methods
[0158] The protonated e-amino groups of poly L-lysine (PLL)
interact with the negatively charged DNA molecules to form
complexes that can be used for gene delivery. These complexes can
be rather unstable and showed a tendency to aggregate (Kwoh et al.,
1999). The conjugation of polyethylene glycol (PEG) was found to
lead to an increased stability of the complexes (Lee et al., 2005,
Harada-Shiba et al., 2002). To confer a degree of
tissue-specificity, targeting molecules such as tissue-specific
antibodies have also been employed (Trubetskoy et al., 1992, Suh et
al., 2001).
[0159] An additional gene carrier that has been used for
transfecting cells is polyethylenimine (PEI) which also forms
complexes with DNA. Due to the presence of amines with different
pKa values, it has the ability to escape the endosomal compartment
(Boussif et al., 1995). PEG grafted onto PEI complexes was found to
reduce the cytotoxicity and aggregation of these complexes. This
can also be used in combination with conjugated antibodies to
confer tissue-specificity (Mishra et al., 2004, Shi et al., 2003,
Chiu et al., 2004, Merdan et al., 2003).
Implications for Medicine
[0160] Stem cells not only have the ability to differentiate into
diverse tissues, but due to their inherent ability to home to
damaged tissue, they have the potential to deliver the expression
of therapeutic genes to specific tissue environments. Through the
use of molecular engineering approaches, stem cells can be used as
vehicles to selectively express genes in areas of defects or need,
thereby releasing the therapeutic product of the transfection only
where it is required. Diseases where genetically engineered stem
cells might play a role in future are those where a protein or an
entire enzyme is missing or nonfunctional or where certain factors
provide improved function in a specific tissue.
[0161] A series of studies using stem cells in therapeutic settings
have been already been conducted for the treatment of a diverse
range of diseases that include cancer, neurodegenerative disorders
such as Parkinson's disease or Alzheimer's disease, ischemic
disease of the heart, and muscle dystrophies.
[0162] The transfer of drug resistance genes into hematopoietic
stem cells shows promise for the treatment of a variety of
inherited diseases. These include; X-linked severe combined immune
deficiency, adenosine deaminase deficiency, thalassemia.
[0163] The combined stem cell and gene therapy approach has the
potential for being tailored for acquired disorders such as breast
cancer, lymphomas, brain tumors, and testicular cancer. In this
regard, studies using the combined approach for the treatment of
cancer have been initiated. These studies range from improving the
drug resistance of transplanted hematopoietic stem cells to using
genetically modified stem cells to target cancer.
[0164] Drug resistance genes have been transferred into
hematopoietic stem cells for providing myeloprotection against
chemotherapy-induced myelosuppression or for selecting
hematopoietic stem cells that are concomitantly transduced with
another gene for correction of an inherited disorder. (Cancer Gene
Ther., 2005 November; 12(11):849-63. Hematopoietic stem cell gene
therapy with drug resistance genes: an update. Budak-Alpdogan T,
Banerjee D, Bertino J R).
[0165] Examples of using stem cells to target cancer include the
enhancement of bystander effect-mediated gene therapy using
genetically engineered neural stem cells for the treatment of
gliomas, and using hematopoietic stem cells carrying the gene of
ribonuclease inhibitor to target the vasculature of melanomas.
[0166] An additional approach for cancer makes use of the ability
of stem cells to be recruited to tumor vasculature and to
differentiate into endothelial-like cells. Depending upon the tumor
type, approximately 30% of new vascular endothelial cells in tumors
can be derived from bone marrow progenitors (Hammerling and Ganss,
2006). Thus, the use of genetically modified progenitor cells
recruited from the peripheral circulation may represent a potential
vehicle for gene therapy of tumors (Reyes et al., 2002). The Herpes
simplex virus 1 (HSV) thymidine kinase (tk) suicide gene together
with Ganciclovir.RTM. (GCV) have been successfully used for the in
vivo treatment of various solid tumors (Dancer et al., 2003;
Pasanen et al., 2003). The selective expression of HSV-tk by
endothelial cells during neovascularization in combination with tk
modification of GCV leads to a lethal environment for proliferating
cells. A series of promoters have been identified that are induced
during neovascularization allowing the selective activation of the
suicide gene following the recruitment and differentiation of
engineered precursor cells.
[0167] A "bystander effect" is described as the ability of cells to
mediate cell damage to distant cells. In a recent study by Li et
al. the bystander effect of neural stem cells transduced with the
HSV-tk gene (NSCtk) on rat glioma cells was examined. Intracranial
co-implantation experiments in athymic nude mice or Sprague-Dawley
rats, showed that the animals co-implanted with NSCtk and glioma
cells and then treated with Ganciclovir.RTM. (GCV) showed no
intracranial tumors and survived more than 100 days, while those
treated with physiological saline (PS) died of tumor progression.
(Cancer Gene Ther., 2005 July; 12(7):600-7. Bystander
effect-mediated gene therapy of gliomas using genetically
engineered neural stem cells. Li S, Tokuyama T, Yamamoto J, Koide
M, Yokota N, Namba H).
[0168] Human ribonuclease inhibitor (hRI) can inhibit the activity
of pancreatic RNase (RNase A) and it has been suggested that RI may
act as a latent antiangiogenic agent. Fu et al. examined the
feasibility of transfecting the RI gene into murine hematopoietic
cells and then inducing expression to block angiogenesis in solid
tumors. RI from human placenta was cloned and inserted into the
retroviral vector pLNCX. Murine bone marrow hematopoietic cells
were then infected with the pLNCX-RI retroviral vector. Infected
cells were then injected into lethally irradiated mice. After
administration of hematopoietic cells carrying the RI gene, the
mice were implanted with B16 melanomas and the tumor was grown for
21 days. Tumors from the control groups became large and well
vascularized. In contrast, tumors from mice treated with
hematopoietic cells carrying the RI gene were small and possessed a
relatively low density of blood vessels. (Cancer Gene Ther. 2005
March; 12(3):268-75. Anti-tumor effect of hematopoietic cells
carrying the gene of ribonuclease inhibitor. Fu P, Chen J, Tian Y,
Watkins T, Cui X, Zhao B.)
[0169] Many studies that focus on Parkinson's disease use either
cell transplantation or gene therapy (Gene Ther., 2003 September;
10(20): 1721-7. Gene therapy progress and prospects: Parkinson's
disease. Burton E A, Glorioso J C, Fink D J). However, few studies
to date have combined the two approaches. Liu et al. used bone
marrow derived stromal cells to deliver therapeutic genes to the
brain. The authors used an adeno-associated virus (AAV) vector to
deliver tyrosine hydroxylase (TH) gene to bone marrow stromal
cells. MSCs expressing TH gene were then transplanted into the
striatum of Parkinson's disease rat. The gene expression efficiency
was found to be approximately 75%. Functional improvement in the
diseased rats was detected after TH-engineered marrow stromal cells
engraftment. Histological examination showed that the TH gene was
expressed around the transplantation points, and that the dopamine
levels in the lesioned striatum of the rats were higher than in
controls. Functional improvement of the animals was observed (Brain
Res Brain Res Protoc., 2005 May; 15(1):46-51. Epub 2005, April 22.
Therapeutic benefit of TH-engineered mesenchymal stem cells for
Parkinson's disease. Lu L, Zhao C, Liu Y, Sun X, Duan C, Ji M, Zhao
H, Xu Q, Yang H.)
[0170] Ischemic cardiovascular disease is an additional target for
engineered stem cell therapy. Chen et al., used purified CD34(+)
cells obtained from human umbilical cord blood, transfected with
human angiopoietin-1 (Ang1) and VEGF(165) genes using an AAV
vector. The engineered cells were injected together with VEGF
intramyocardially at the left anterior free wall, which led to
decreased infarct size, and significantly increased capillary
density after treatment, as well as improved long term cardiac
performance measured using echocardiography 4 weeks after
myocardial infarction. (Eur J Clin Invest., 2005 November;
35(11):677-86. Combined cord blood stem cells and gene therapy
enhances angiogenesis and improves cardiac performance in mouse
after acute myocardial infarction. Chen H K, Hung H F, Shyu K G,
Wang B W, Sheu J R, Liang Y J, Chang C C, Kuan P.)
[0171] The muscle dystrophies represent a heterogeneous group of
neuromuscular disorders characterized by progressive muscle
wasting. To date no adequate treatment modality exists for these
patients. Adult stem cell populations, including MSC, as well as
embryonic stem cells have been evaluated for their ability to
correct the dystrophic phenotype. To date, the described methods
have not shown much promise. The reasons described for failure
exemplifies the difficulties researchers encounter when using
genetically modified stem cells: the underlying mechanism
responsible for a myogenic potential in stem cells has not yet been
fully elucidated, homing of the donor population to the muscle is
often inadequate, and poorly understood immune responses in the
recipient can lead to limited treatment success (Stem cell based
therapies to treat muscular dystrophies. Price, Kuroda, Rudnicki)
One approach used for the treatment of Duchenne muscular dystrophy
(DMD) utilizes autologous cell transplantation of myogenic stem
cells that have been transduced with a therapeutic expression
cassette. Development of this method has been hampered by a series
of problems including; a low frequency of cellular engraftment,
difficulty in tracing transplanted cells, rapid loss of autologous
cells carrying marker genes, and difficulty in introducing the
stable transfer of the large dystrophin gene into myogenic stem
cells.
[0172] A mini Dys-GFP fusion gene was engineered by replacing the
dystrophin C-terminal domain (DeltaCT) with an eGFP coding sequence
and removing much of the dystrophin central rod domain
(DeltaH2-R19). In a transgenic mdx(4Cv) mouse expressing the
miniDys-GFP fusion protein under the control of a skeletal
muscle-specific promoter, the green fusion protein localized on the
sarcolemma, where it assembled the dystrophin-glycoprotein complex
and prevented the development of dystrophy in transgenic mdx(4Cv)
muscles. (Hum Mol Genet., 2006 May 15; 15(10):1610-22. Epub 2006
April 4. A highly functional mini-dystrophin/GFP fusion gene for
cell and gene therapy studies of Duchenne muscular dystrophy. Li S,
Kimura E, Ng R, Fall B M, Meuse L, Reyes M, Faulkner J A,
Chamberlain J S.)
[0173] Wiskott-Aldrich-Syndrome is characterized by
thrombocytopenia, dysregulation and propensity towards lymphoma
development later in life and represents a potential target for
engineered stem cell therapy (Dupre et al., 2006). Fanconi anemia,
is considered a "stem cell disease" and has been the subject of
intensive research for treatment using gene therapy. This disease
represents the best-characterized congenital defect of
hematopoietic stem cells. It is a rare hereditary disease
characterized by bone marrow failure and developmental anomalies; a
high incidence of myelodysplasia, acute nonlymphocytic leukemia,
and solid tumors. The genetic basis for Fanconi anemia lies in
selective mutations in any one of the known Fanconi anemia genes,
making this disease a candidate for gene therapy. But the disease
is complex as at least 12 genetic subtypes have been described
(FA-A, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M) and all, with
the exception of FA-I have been linked to a distinct gene. Most FA
proteins form a complex that activates the FANCD2 protein via
monoubiquitination, while FANCJ and FANCD1/BRCA2 function
downstream of this step. The FA proteins typically lack functional
domains, except for FANCJ/BRIP1 and FANCM, which are DNA helicases,
and FANCL, which is probably an E3 ubiquitin conjugating enzyme.
Based on the hypersensitivity to cross-linking agents, the FA
proteins are thought to function in the repair of DNA interstrand
cross-links, which block the progression of DNA replication forks.
(Cell Oncol. 2006; 28(1-2):3-29. The Fanconi anemia pathway of
genomic maintenance. Levitus M. Joenje H, de Winter J P.
[0174] Additional inherited stem cell defects that are potential
candidates for gene therapy include amegakaryocytic
thrombocytopenia, dyskeratosis congenity and Shwachman-Diamond
syndrome. Thalassemias and Sickle Cell disease belong to the group
of hereditary hemolytic anemias that represent the most common
inherited diseases worldwide and thus are important candidates for
stem cell gene therapy (Persons and Tisdale, 2004).
[0175] A good example using genetically modified mesenchymal stem
cells in a clinical setting is the correction of the genetic
mutation in the bridled bone disease osteogenesis imperfecta.
Osteogenesis imperfecta causes fragile bones due to mutations in
the collagen-1-encoding genes, COLIA1 or COLIA2. Chamberlain et al.
obtained mesenchymal stem cells (MSCs) from the bones of
osteogenesis imperfecta patients and identified point mutations in
the COLIA1 gene (Chamberlain et al., 2004). MSCs were successfully
infected with an adeno-associated virus to target and deactivate
the mutated COLIA1 gene. The corrected MSCs were then transplanted
into immunodeficient mice and damaged cells demonstrated improved
stability and collagen processing.
EXAMPLES
Example 1
[0176] Multipotent adult stem cells are isolated from the bone
marrow and other sources of a patient or donor using adherent
growth in-vitro to determine cell activity and biological function.
At this in-vitro stage the adherent growing cell do not express the
"stem cell marker" CD34 and are therefore considered CD34-negative
during in-vitro culturing. At this stage, CD34-negative, adherent
growing stem cells are transient or stably transfected by viral or
non-viral technologies and expanded selectively in-vitro before
in-vivo application. For the generation of transgenic CD34-negative
progenitor cells two promoters are used for selection and
organ/target-specific inducibility of the therapeutic gene. The
gene transfer system is chosen based on their transfection and
integration (if desired) combined with adhesion selectivity. As
this example, the tie2-promotor enhancer is driving the HSV-TK
gene, which is expressed only in the context of endothelial
differentiation, which happens during tumor neo-angiogenesis. While
circulating endogenous as well as systemically administered stem
cells are recruited physiologically to the site of tumor growth to
participate in the tumor neo-angiogenesis (independently whether it
is the primary tumor site or metastasis), the stem cells
differentiate into tumor endothelial cells. During this process of
organ-specific differentiation, the stem cells express the HSV-TK
gene driven by the angiogenesis-related tie2 activation. Now the
prodrug Ganciclovir.RTM. can be given to the patient and is
converted by the HSV-TK into the cytotoxic substance at the site of
tumor angiogenesis. This approach has been successfully shown in
pre-clinical models for breast cancer, metastatic colo-rectal
cancer, pancreas carcinoma and glioblastoma. An application can be
envisioned for any (malignant) neoplasia that relies on tumor
neo-angiogenesis. This approach aims at the disruption of tumor
angiogenesis.
Example 2
[0177] As in EXAMPLE 1, but instead of expressing HSV-TK,
expressing clotting substances as cytotoxic proteins.
Example 3
[0178] Angiogenesis is also a pivotal biological process in tissue
remodeling and wound healing. This does not only apply for lesions
of the skin or mucosa but also for other tissues, like the lack of
insulin-producing beta-cells in the pancreas, leading to
Insulin-dependent Diabetes mellitus (IDDM). The systemic
application of transgenic CD34-negative progenitor cells can also
induce the activation of otherwise quiescent islet progenitor cells
in the pancreas, replenishing the endocrine pancreas and correcting
the state of hyperglycemia in IDDM patients. The tie-2 enhancer
promoter activates the gene for vasoactive substances like VEGF
promoting tissue remodeling and wound healing.
Example 4
[0179] As in EXAMPLE 3, but in combination with the transplantation
of allogeneic islet cells if endogenous regeneration is not
sufficient anymore.
Example 5
[0180] As in EXAMPLE 1, but transgenic cells with enhanced homing
capabilities to the site of tumor growth, tissue remodeling or
wound healing applying chemokine biology. CD34-negative, adherent
growing stem cells are engineered using GPI-mucin-chemokines. These
agents will allow the selective expression of specific chemokines
linked to the mucin-domain taken either from CX3CL1 or CXC16 fused
to a GPI anchor. The expression of these chemokine-mucin agents
will recruit complementary leukocytes expressing the chemokine
receptor. For example, CXCL10-mucin-GPI expression under the
control of the tie2 promoter enhancer in the context of tumor
therapy will facilitate the recruitment of effector T cells into
the tumor environment. This will act as an adjuvant for tumor
immune therapy. The same approach could also be used in tissue
remodeling to facilitate the parallel recruitment of select
leukocyte populations.
Example 6
[0181] As in EXAMPLE 1, but genetically engineering CD34-negative
stem cells that can modulate the inflammatory environment, e.g. in
autoimmune disorders like chronic-inflammatory bowel disease or
graft-versus-host disease after allogeneic bone marrow/stem cell
transplantation. This can also be facilitated by the site-specific
expression of anti-inflammatory substances like interleukins
(IL-10).
Example 7
[0182] As in EXAMPLE 1, but with the site-specific expression of
common viral antigens e.g. which induce an internal vaccination
boost at the site of tumor growth, e.g. measles or chicken pox.
Example 8
[0183] As in EXAMPLE 1, but the therapeutic gene activation is
suppressed by genes of an early developmental stage (e.g. Noggin),
which eventually becomes down regulated during the differentiation
of the transgenic progenitor cells in mature tissue at the site of
the tumor or tissue remodeling/regeneration.
Part II
Breast and Pancreatic Tumor Models
Synopsis
[0184] Tumor angiogenesis represents a promising target for the
selective delivery of cancer therapeutics. Bone marrow-derived
mesenchymal stem cells were developed to selectively target
exogenous genes to tumor angiogenesis environments. The results of
these experiments show that exogenously added MSC home to tumors
where they undergo differentiation. Genes such as the RFP reporter
gene as well as the suicide gene HSV-tk are selectively expressed
during differentiation under control of the Tie2 promoter/enhancer.
The administration of the pro-drug Ganciclovir.RTM. in concert with
tk expression effectively targets the tumor and results in the
suppression of tumor growth.
Endogenous Mouse Breast Cancer Model
[0185] A previously established murine breast cancer model was used
by Dr. Christoph Klein to study the use of engineered MSC in tumor
angiongenesis. This model is broadly applicable to human breast
cancer. In this model, transgenic mice carrying the activated rat
c-neu oncogene under transcriptional control of the MMTV promoter
have been backcrossed to BALB/c mice with the aim of developing a
broadly applicable model for cancer therapy. Female HER-2/neu
(neu-N) transgenic mice, which express the nontransforming rat
proto-oncogene, develop spontaneous focal mammary adeno-carcinomas
beginning at 5-6 months of age. The development and histology of
these tumors bear resemblance to what is seen in patients with
breast cancer.
[0186] Expression of RFP and GFP Genes in imMSC Under Control of
Endothelial Specific Promoters in the Context of Tumor
Angiogenesis
[0187] To assess the control of tissue specific expression gene
expression in imMSC in the context of tumor angiogenesis, and to
follow the distribution of imMSCs over longer time periods
microscopically, red and green fluorescent protein (RFP, GFP) genes
have been cloned into modified expression vectors to detect in
vivo.
[0188] Mice
[0189] Female HER-2/neu (neu-N) transgenic mice expressing the
nontransforming rat proto-oncogene, are known to develop
spontaneous focal mammary adeno-carcinomas at 5-6 months of age.
BALB-neuT transgenic mice were maintained in accordance with the
Agreement to the European Union Guidelines. Mice were screened for
hemizygosity (neuT+/neuT-). Mammary glands of Balb-neuT female mice
were inspected twice a week and arising tumors were measured.
[0190] The genetically modified cells were injected into the breast
cancer model mice following surgical resection of the primary
tumor. As the residual tumor grew back, the exogenously added imMSC
provided precursor cells for neovascularization. The Tie-2-RFP
(endothelial specific promoter driving red florescence protein)
stably transfected imMSCs were found to readily home to the tumor,
differentiate to endothelial cells, and express the RFP reporter
gene (FIG. 5). In these experiments the integration of the
mesenchymal cells into the primary mammary tumors of Balb-neuT mice
was evaluated. After three applications of RFP-transfected,
RFP-expressing cells could be detected in vessel-like regions in
all sections. The results demonstrate that the cells home to sites
of neovascularization and express marker genes through the
activated Tie2 promoter/enhancer.
[0191] Inhibiting Tumor Growth by Targeting a Suicide Gene in the
Endothelium
[0192] The protocol was then altered to evaluate the effect of the
suicide gene HSV-tk. The tk gene product in combination with the
prodrug Ganciclovir.RTM. (GCV) produces a potent toxin which
affects replicative cells. ImMSCs were stably transfected with
plasmids carrying the herpes simplex virus-thymidine kinase (tk)
gene driven by the vascular endothelial Tie2 promoter/enhancer. To
this end, the murine model of breast cancer angiogenesis was used
again to evaluate the engineered MSC line in anti-angiogenesis
therapy.
Approach I. Injection of Engineered MSC and Ganciclovir.RTM.
Treatment in the Phase of Exponential Tumor Growth at the Age of 18
or 22 Weeks
[0193] The Treatment of Balb-neuT trsg
[0194] Mice started on day 0 (week 22), with injection of 0.2 ml
cells (500,000 cells) and 0.2 ml PBS as control. On days 5 to 8,
Ganciclovir.RTM. was applied in a daily dose of 30 .mu.g/g BW, e.g.
100 .mu.l for a mouse with 21 g BW. After day 9, mice treatment
cycles were repeated until dissection. During the treatment tumor
progression, bodyweight (measured on day 0 and 6 of each therapy
cycle) and behavior were recorded.
[0195] To get an overall impression of the effect of the treatment
with Tie2-Tk-as-transfected cells and GCV respectively compared
with both control groups, the macroscopic value of bodyweight was
recorded during treatment until dissection. Measure points were
days 0 and 5 of each cycle of therapy and the day of dissection.
The experiments included one treatment and two control groups of
mice and started at two different time points, 18 and 22 weeks of
age.
TABLE-US-00002 TABLE 2 Data of treatment groups after dissection,
including bodyweight, absolute and relative tumor load. Amount
Absolute Relative Treatment of Bodyweight tumorload tumorload group
mice [g] SD [g] SD [g] SD PBS-22- 2 32.6 0.8 8.5 1 0.265 0.035
RFP-22- 3 29.4 3 8.3 0.9 0.287 0.032 Tk-as/GCV-22- 3 31.8 1.1 8.1
0.9 0.257 0.021
Approach II. Evaluation of Therapy in the Context of Tumor Regrowth
Following Surgical Resection
[0196] In patients following the surgical removal of a tumor,
residual tumor that is missed during surgery often grows out,
leading to a reoccurrence of cancer. To test the efficacy of the
engineered MSC/tk therapy in this context, the breast tissue from
Balb/c neu-N transgenic mice was resected at 18 weeks of age,
leading to a delayed onset of primary tumor. Following surgery, the
mice were treated with the MSC-tk and GCV regimen as described
above. The treatment resulted in a dramatic reduction in tumor
growth in the treated mice (FIG. 8).
[0197] Pancreatic Tumor Model
[0198] An orthotopic pancreatic carcinoma model was then developed
in C57BI/6 mice to assess the efficacy of the MSC-based therapy in
a different tumor system. The system had been previously
established by Christiane Bruns and Claudius Conrad (Surgery
Department, LMU). In this model, Panc02 pancreatic carcinoma cells
syngeneic to C57BI/6 mice were injected subcapsularly in a region
of the pancreas just beneath the spleen to create primary
pancreatic tumors. Constructs with GFP under the control of the CMV
promoter, and RFP and tk under the control of Tie2
promoter/enhancer, were introduced into the MSC isolated from
C57BI/6 mice. The transfected stem cells were given systemically
via i.v. injections. In a preliminary experiment, MSC engineered to
constitutively express GFP (under control of the CMV promoter) were
injected into mice with growing tumors. The cells were found to
efficiently home to the tumors (FIG. 9).
[0199] In parallel experiments, mice with growing tumors were
injected with the Tie2-RFP engineered MSC. After five days, the
animals were sacrificed and the tumors were examined for expression
of RFP. The results show a strong upregulation of RFP in the
context of tumor (FIG. 9). RFP was not detected in other organs
(spleen, lymph nodes and thymus).
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