U.S. patent application number 15/253353 was filed with the patent office on 2017-01-19 for genetically modified mesenchymal stem cells that express an exogenous cytotoxic protein.
The applicant listed for this patent is APCETH GMBH & CO. KG. Invention is credited to Peter J. NELSON.
Application Number | 20170015976 15/253353 |
Document ID | / |
Family ID | 42270435 |
Filed Date | 2017-01-19 |
United States Patent
Application |
20170015976 |
Kind Code |
A1 |
NELSON; Peter J. |
January 19, 2017 |
GENETICALLY MODIFIED MESENCHYMAL STEM CELLS THAT EXPRESS AN
EXOGENOUS CYTOTOXIC PROTEIN
Abstract
This invention provides a method for treating a subject
afflicted with a tumor using genetically modified mesenchymal stem
cells, wherein each genetically modified mesenchymal stem cell
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 mesenchymal
stem cells come into proximity with the tumor's stromal tissue.
This invention further provides genetically modified mesenchymal
stem cells for use in this method.
Inventors: |
NELSON; Peter J.; (Munich,
DE) |
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Applicant: |
Name |
City |
State |
Country |
Type |
APCETH GMBH & CO. KG |
Munich |
|
DE |
|
|
Family ID: |
42270435 |
Appl. No.: |
15/253353 |
Filed: |
August 31, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13264084 |
Dec 23, 2011 |
9434925 |
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PCT/EP2010/054844 |
Apr 13, 2010 |
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15253353 |
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61168787 |
Apr 13, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2830/002 20130101;
C12N 15/86 20130101; A61P 35/00 20180101; C12N 2510/00 20130101;
C12N 5/0663 20130101; A61K 35/28 20130101; C12N 2740/15043
20130101; A61K 2035/124 20130101; A61P 35/04 20180101 |
International
Class: |
C12N 5/0775 20060101
C12N005/0775; A61K 35/28 20060101 A61K035/28; C12N 15/86 20060101
C12N015/86 |
Claims
1. A method for treating a subject afflicted with a tumor
comprising introducing into the subject's bloodstream a
therapeutically effective number of genetically modified
mesenchymal stem cells, wherein each genetically modified
mesenchymal stem cell comprises an exogenous nucleic acid
comprising (i) a cytotoxic protein-encoding region operably linked
to (ii) a promoter or promoter/enhancer combination, wherein the
promoter or promoter/enhancer combination is inducible by
inflammatory mediators.
2. The method of claim 1, wherein the promoter or promoter/enhancer
combination is inducible by cytokines.
3. The method of claim 1, wherein the inflammatory mediator is
selected from the group consisting of TNF-alpha, IFN-gamma, IL-6,
TGF-beta, IL-10, M-CSF and IL1.beta..
4. The method of claim 1, wherein the promoter or promoter/enhancer
combination comprises an NF-k.beta.-responsive element or a
Smad-binding element.
5. The method of claim 1, wherein the promoter is the RANTES
promoter.
6. The method of claim 1, wherein the subject is human.
7. The method of claim 1, wherein the genetically modified
mesenchymal stem cells are CD34.sup.- stem cells.
8. The method of claim 1, wherein the genetically modified
mesenchymal stem cells are allogenic with respect to the
subject.
9. The method of claim 1, wherein the genetically modified
mesenchymal stem cells are autologous with respect to the
subject.
10. The method of claim 1, wherein the tumor is selected from the
group consisting of a prostate tumor, a breast 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,
a glioblastoma 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.
11. The method of claim 1, wherein the cytotoxic protein is Herpes
simplex viral thymidine kinase, and the subject is treated with
ganciclovir in a manner permitting the Herpes simplex viral
thymidine kinase to render the ganciclovir cytotoxic.
12. The method of claim 1, wherein the therapeutically effective
number of genetically modified mesenchymal stem cells is from
1.times.10.sup.5 to 1.times.10.sup.9 cells/kg body weight.
13. The method of claim 1, wherein the therapeutically effective
number of genetically modified mesenchymal stem cells is from
1.times.10.sup.6 to 1.times.10.sup.8 cells/kg body weight.
14. A genetically modified mesenchymal stem cell comprising an
exogenous nucleic acid comprising (i) a cytotoxic protein-encoding
region operably linked to (ii) a promoter or promoter/enhancer
combination, wherein the promoter or promoter/enhancer combination
is inducible by inflammatory mediators.
15. The genetically modified mesenchymal stem cell of claim 14,
wherein the promoter or promoter/enhancer combination is inducible
by cytokines.
16. The genetically modified mesenchymal stem cell of claim 14,
wherein the inflammatory mediator is selected from the group
consisting of TNF-alpha, IFN-gamma, IL-6, TGF-beta, IL-10, M-CSF
and IL1.beta..
17. The genetically modified mesenchymal stem cell of claim 14,
wherein the promoter or promoter/enhancer combination comprises an
NF-k.beta.-responsive element or a Smad-binding element.
18. The genetically modified mesenchymal stem cell of claim 14,
wherein the promoter is the RANTES promoter.
19. The genetically modified mesenchymal stem cell of claim 14,
wherein the stem cell is a human stem cell.
20. The genetically modified mesenchymal stem cell of claim 14,
wherein the stem cell is a CD34.sup.- stem cell.
21. The genetically modified mesenchymal stem cell of claim 14,
further comprising a (iii) selection marker gene operably linked to
(iv) a constitutive promoter or promoter/enhancer combination.
22. The genetically modified mesenchymal stem cell of claim 21,
wherein the cytotoxic protein-encoding region operably linked to
the promoter or promoter/enhancer combination and the selection
marker gene operably linked to the constitutive promoter or
promoter/enhancer combination are a part of a proviral sequence
integrated into the stem cell genome.
23. The genetically modified mesenchymal stem cell of claim 22,
wherein the proviral sequence is a lentiviral, alpha-retroviral or
gamma-retroviral sequence.
24. The genetically modified mesenchymal stem cell of claim 14,
wherein the cytotoxic protein is Herpes simplex viral thymidine
kinase or cytosine deaminase.
25. A retroviral packaging cell comprising: a. a retroviral vector
including (i) a cytotoxic protein-encoding region operably linked
to (ii) a promoter or promoter/enhancer combination, wherein the
promoter or promoter/enhancer combination is inducible by
inflammatory mediators, and b. a gene encoding a viral surface
protein providing a tropism for mesenchymal or CD34- stem
cells.
26. The retroviral packaging cell according to claim 25, wherein
the promoter or promoter/enhancer combination is inducible by
cytokines.
27. The retroviral packaging cell according to claim 25, wherein
the inflammatory mediator is selected from the group consisting of
TNF-alpha, IFN-gamma, IL-6, TGF-beta, IL-10, M-CSF and
IL1.beta..
28. The retroviral packaging cell according to claim 25, wherein
the promoter or promoter/enhancer combination comprises an
NF-k.beta.-responsive element or a Smad-binding element.
29. The retroviral packaging cell according to claim 25, wherein
the promoter is the RANTES promoter.
Description
[0001] Throughout this application, various publications are cited.
The disclosure of these publications is hereby incorporated by
reference into this application to describe more fully the state of
the art to which this invention pertains.
BACKGROUND OF THE INVENTION
[0002] An emerging paradigm suggests that malignant cells exist in
a complex cellular and extracellular microenvironment that
significantly influences the initiation and maintenance of the
malignant phenotype..sup.1,2 Solid tumors can be seen to be
composed of the malignant cell as well as the supporting cells that
comprise the stroma including fibroblasts, endothelium, pericytes,
lymphatics and generally, a mononuclear infiltrate..sup.2-6 These
stromal cells are so vital to the survival of the tumor that they
have become an important target for chemotherapeutic
intervention.
[0003] Mesenchymal stem cells (MSCs) are pluripotent progenitor
cells that contribute to the maintenance and regeneration of
diverse tissues.sup.7,8. MSCs can be found in many tissues where
they serve as local sources of stem cells, such as bone marrow,
blood or different sources of mesenchymal tissue. MSCs contribute
to tissue remodelling after injury or during chronic inflammation.
The damaged tissue is thought to release specific endocrins that
then lead to the mobilization of multi-potent MSCs and their
subsequent recruitment to the site of injury. MSCs are also
strongly attracted to tumor stroma. MSCs infused into the blood in
experimental animals localize to malignancies. Once there, MSCs may
contribute to diverse cell types that comprise tumor stroma
including tumor vasculature and stromal fibroblasts
[0004] In breast cancer, Karnoub et al. recently showed that MSCs
at the tumor site release a small protein, the chemokine CCL5. CCL5
can act as a chemoattractant for diverse stromal cells.sup.9,10 and
its expression is associated with increased tumour
neovascularization. In addition, CCL5 may contribute to cancer
growth and metastasis through the recruitment of a number of
stromal cell types to sites of primary tumor
growth..sup.9,11,12
[0005] It has recently been shown that the CD34.sup.- subpopulation
of MSC progenitors undergo recruitment to the growing tumor and
contribute to tumor neoangiogenesis through their differentiation
into new vascular endothelial cells (ECs) or pericytes..sup.13-15
Importantly, these and related direct effects can be observed
following the direct injection of MSCs into the peripheral
circulation..sup.16
[0006] There exists an unmet need for stem cell-based therapies
that employ cytotoxic protein expression based on proximity with
tumor stromal tissue per se for cytotoxic protein expression,
rather than cytotoxic protein expression based on proximity with
tumor tissue undergoing angiogenesis. This need is particularly
acute regarding treatment of metastatic tumors that have not yet
undergone angiogenesis.
SUMMARY OF THE INVENTION
[0007] One embodiment of 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 mesenchymal stem cells, wherein each
genetically modified mesenchymal stem cell 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 mesenchymal stem cells come into
proximity with the tumor's stromal tissue.
[0008] Another embodiment of the invention also provides
genetically modified mesenchymal stem cells for use in any of the
methods for treating a subject afflicted with a tumor disclosed in
this patent application.
[0009] This invention also provides a method for treating a human
subject afflicted with a pancreatic tumor comprising introducing
into the subject's bloodstream from about 1.times.10.sup.5 to about
1.times.10.sup.9 cells/kg body weight of genetically modified
CD34.sup.- stem cells, wherein (a) each genetically modified
CD34.sup.- stem cell contains an exogenous nucleic acid comprising
(i) a Herpes simplex viral thymidine kinase-encoding region
operably linked to (ii) a RANTES promoter, (b) the subject is
treated with ganciclovir in a manner permitting the Herpes simplex
viral thymidine kinase to render the ganciclovir cytotoxic, and (c)
the introduction of the genetically modified mesenchymal stem cells
is not preceded, accompanied or followed by myeloablation.
[0010] This invention further provides a genetically modified
mesenchymal stem cell comprising 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 mesenchymal stem cell comes into proximity with a tumor's
stromal tissue.
[0011] Yet a further embodiment of the invention is directed to a
retroviral packaging cell comprising: [0012] a retroviral vector
including (i) a cytotoxic protein-encoding region operably linked
to (ii) a promoter or promoter/enhancer combination, inducible by
inflammatory mediators, and [0013] a gene encoding a viral surface
protein providing a tropism for mesenchymal or CD34- stem cells.
[0014] Further the retroviral packaging cell also comprises genes
encoding structural proteins and enzymes for the production of
pseudotyped virions from the packaging cell.
[0015] Finally, this invention provides a genetically modified
human CD34.sup.- stem cell comprising an exogenous nucleic acid
comprising (i) a Herpes simplex viral thymidine kinase-encoding
region operably linked to (ii) a RANTES promoter.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1
(A) With a 1 cm incision at the left flank, the pancreas was
exposed. A 1 ml syringe was used to inject 150,000 Panc02
pancreatic cancer cells into the pancreas. (B) Two weeks following
the procedure, all mice grew palpable tumors and were randomized
into the respective experimental groups.
[0017] FIG. 2
Modified Boyden Chamber assays were used to evaluate the migratory
response of C57B16 MSC to conditioned media derived from Panc02
cells. The results show a dose-dependent induced migration of the
MSC to the Panc02-derived growth media.
[0018] FIG. 3
The ability of MSCs (engineered to constitutively express eGFP
under control of the CMV promoter) to infiltrate implanted Panc02
tumor was examined. Three times a week, 500,000 cells were injected
intravenously into mice with growing pancreatic tumors. After five
weeks, the mice were sacrificed and the tumors removed and analyzed
for the expression of GFP. (A) Results show a strong GFP expression
associated with the tumor. (B) The adoptive transfer of MSC was
found to increase growth of the implanted tumor over the course of
the experiment.
[0019] FIG. 4
The CCL5 promoter provides increased selectivity of reporter gene
expression to MSC targeting the Panc02 tumor. MSCs were engineered
to express either RFP or eGFP under the control of the CCL5
promoter. Following injection of 500,000 cells per week over three
weeks, the animals were sacrificed and the tumor examined for
reporter gene expression by fluorescence microscopy. Both eGFP (A)
and RFP- (B) reporter genes driven by the CCL5 promoter showed
expression within the tumor environment. More precise tumor
morphology was observed by immunohistochemistry on fixed tissue
sections using an RFP specific polyclonal antibody (C and D). (C)
Results show a focal expression of RFP in stromal regions of the
tumor (100.times.). (D+E) Similar tumor samples at a higher
magnification (200.times., 500.times. respectively) show extensive
infiltration of MSC showing expression of the RFP reporter gene in
the tumor environment.
[0020] FIG. 5
Application of MSC engineered to express HSV-TK under the control
of the CCL5 promoter, in conjunction with GVC as a therapeutic
modality for pancreatic carcinoma. (A) Overview of the construct
used to engineer the MSC to express the HSV-TK suicide gene. (B)
For a therapeutic regimen, 500,000 CCL5-TK engineered MSCs were
injected intravenously. The cells were given three days to undergo
recruitment to the growing tumor and activate expression of the TK
gene. The mice received once-daily intravenous injections of 7.5 mg
GVC for four days, followed by one day of rest. The mice were then
again injected with the engineered stem cells, and the cycle was
repeated for the duration of the experiment. After 3 cycles (36
days after tumor induction or 21 days after first MSC injection)
the animals were sacrificed and tumor growth was evaluated. (C)
Examples of tumors excised from animals treated with vehicle
controls, engineered MSC controls (RFP, eGFP) and
HSV-TK/GCV-treatment. (D) HSV-TK/GCV treatment resulted in a
significant reduction in tumor growth over the course of the
experiment.
[0021] FIG. 6
Schematic overview of the transgene cassette with several examples
of genetic elements. An inducible promoter (pInd) is linked to a
cytotoxic protein-encoding region. A second constitutive cellular
or viral promoter (pConst) is linked to a selection marker gene.
The two units may be separated by an insulator sequence (+/-
insulator).
[0022] FIG. 7
Overview of retroviral vectors carrying the transgene cassette for
the genetic modification of MSC depicted in FIG. 6. Vector
production will be driven from the promoter activity of the
U3-region in the 5' long terminal repeat (LTR). The 5' U3-region
may be replaced by other viral promoters. The packaging signal
.PSI. facilitates the incapsidation of the vector genome into
vector particles. For the production lentiviral particles
additional elements like rev responsive element (RRE) and the
central poly purine tract (cPPT) are necessary. The optional
addition of the woodchuck hepatitis post-transcriptional regulatory
element (wPRE) will help to increase vector titer and transgene
expression. An inducible promoter (pInd) is linked to the cytotoxic
protein-encoding region. A second constitutive cellular or viral
promoter (pConst) is linked to a selection marker gene. The two
units may be separated by an insulator sequence (+/-
insulator).
[0023] FIG. 8
Plasmid map of lentiviral vector for the expression of HSV tk under
the control of the RANTES promoter.
[0024] FIG. 9
RANTES mRNA expression after induction of MSCs with TNF.alpha. (10
ng/ml) and IFN.gamma. (10 ng/ml). Relative RANTES mRNA amounts were
calculated using the .DELTA..DELTA.CT method with the 0h,
non-induced sample as normalization value. Mean values from two
biological samples are shown.
[0025] FIG. 10
Treatment of hMSCs with ganciclovir leads to specific cell death
after induction of the RANTES promoter with TNF.alpha. and
IFN.gamma.. Representative microscopic picture of hMSCs that were
not treated (A), induced with TNF.alpha. and IFN.gamma. (B),
treated only with ganciclovir (C) or first induced with TNF.alpha.
and IFN.gamma. and then treated with ganciclovir (D). 100.times.
magnification.
DETAILED DESCRIPTION OF THE INVENTION
Terms
[0026] In this application, certain terms are used which shall have
the meanings set forth as follows.
[0027] 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.
[0028] As used herein, a cell is "autologous" with respect to a
subject if it or its precursor cells are from that same
subject.
[0029] As used herein, "CD34.sup.- stem cell" shall mean a stem
cell lacking CD34 on its surface. CD34.sup.- 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, Apr. 25 [Epub ahead
of print].
[0030] As used herein, "cytotoxic protein" shall mean a protein
that, 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.
[0031] 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.
[0032] As used herein, a stem cell is "genetically modified" if
either it or any of its precursor cells 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).
[0033] As used herein, "introducing" stem cells into a 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 over time
(e.g., daily, every three days, weekly, bi-weekly, monthly,
quarterly, half-yearly or yearly) may be necessary in some
instances. Such administering is also preferably performed using an
admixture of stem cells and a pharmaceutically 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 (34). Preservatives and other additives may
also be present, such as, for example, antimicrobials,
antioxidants, chelating agents, inert gases, and the like.
[0034] "Mesenchymal stem cells" (also referred to as "MSCs") can
give rise to connective tissue, bone, cartilage, and cells in the
circulatory and lymphatic systems. Mesenchymal stem cells are found
in the mesenchyme, the part of the embryonic mesoderm that consists
of loosely packed, fusiform or stellate unspecialized cells. As
used herein, mesenchymal stem cells include, without limitation,
CD34.sup.- stem cells.
[0035] In one embodiment of the invention, the MSCs are fibroblast
like plastic adherent cells defined as multipotent mesenchymal
stromal cells in Horwitz et al..sup.49 and also include CD34-
cells. For the avoidance of any doubt, the term "multipotent
mesenchymal stromal cells" also includes a subpopulation of
mesenchymal stem cells and their precursors, which subpopulation is
made up of pluripotent self-renewing cells capable of
differentiation into specific, multiple cell types in vivo.
[0036] 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 (33).
[0037] 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).
[0038] 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 under appropriate
circumstances.
[0039] 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.
[0040] 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.
[0041] The term "RANTES" promoter is used herein synonymously with
the term "CCL5" promoter.
[0042] As used herein, a cytotoxic protein is "selectively
expressed" when a genetically modified mesenchymal stem cell
encoding same comes into proximity with tumor stromal tissue, 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.
[0043] As used herein, "subject" shall mean any animal, such as a
human, non-human primate, mouse, rat, guinea pig or rabbit.
[0044] 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.
[0045] As used herein, "tumor" shall include, without limitation, 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. These include primary
tumors as well as metastatic tumors (both vascularized and
non-vascularized).
[0046] 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.
[0047] As used herein the term "tumor's stromal tissue" includes
the connective, structural tissue around and in proximity to a
tumor, which comprises various cells such as
fibroblasts/myofibroblasts, glial, epithelial, fat, vascular,
smooth muscle, and immune cells. Tumor stroma also provides the
extracellular matrix and extracellular molecules.sup.53.
[0048] As used herein the term "inflammatory mediators" includes
immune modulatory molecules that act at the site of tissue damage
and tumor growth, and mediate a pro- and anti-inflammatory response
to the tissue damage and tumor growth. Non-limiting examples of
inflammatory mediators are cytokines, and eicosanoides. Tumor cells
and surrounding stromal cells are known to produce numerous
pro-Inflammatory mediators like pro-inflammatory cytokines and
proteases that coordinate inflammatory reactions. Examples for such
mediators are TNF-alpha and IL-6. Additionally, tumor cells and
stromal cells are able to suppress immune responses through
secretion of anti-inflammatory molecules, like TGF-beta, IL-10 and
M-CSF, which e.g inhibit dendritic cell maturation.sup.54.
Embodiments of the Invention
[0049] 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 mesenchymal stem cells, wherein each genetically modified
mesenchymal stem cell 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
mesenchymal stem cells come into proximity with the tumor's stromal
tissue.
[0050] The treated subject can be any animal, and is preferably a
human. The treated tumor can be primary or metastatic, and can be
vascularized or not vascularized. Preferably, the tumor is
metastatic and not vascularized. The tumor can be, for example, a
prostate tumor, a breast 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. Preferably, the tumor
is a pancreatic tumor. In another preferred embodiment, the stromal
tissue of the treated tumor comprises fibroblast-like cells.
[0051] Mesenchymal stem cells can be isolated from various sources:
e.g. Bone marrow, umbilical cord blood, (mobilized) peripheral
blood and adipose tissue.sup.49. In a further preferred embodiment,
the genetically modified mesenchymal stem cells are CD34.sup.- stem
cells. Additionally, the genetically modified mesenchymal stem
cells can be allogenic, autologous or xenogenic with respect to the
subject. In the instant methods employing genetically modified 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.
[0052] In this invention, the introduction of the genetically
modified mesenchymal stem cells is preferably not preceded,
accompanied or followed by myeloablation.
[0053] Another embodiment of the invention provides mesenchymal or
CD34- stem cells for use in any of the methods for treating a
subject afflicted with a tumor. In particular the mesenchymal or
CD34- stem cells can include a promoter or promoter/enhancer
combination, which is inducible by inflammatory mediators and which
controls the transcription of the cytotoxic protein-encoding
region. These inflammatory mediators can be released by the tumor's
stromal tissue so that the expression of the cytotoxic protein in
the mesenchymal stem cells is induced when the stem cells come into
proximity with the tumor's stromal tissue. The inflammatory
mediators can for example be cytokines, such as TNF.alpha. or
IFN.gamma.. In particular the promoter can be the RANTES promoter,
which can inter alia be induced by TNF.alpha. or IFN.gamma..sup.35.
The RANTES promoter also could be induced by differentiative
signals in the context of the differentiation of MSCs. Further
examples of promoters, which are inducible by pro-inflammation
mediators are the NF-kB-responsive element.sup.36 and in general
promoters, which can be induced by IL1.beta. or TNF
.alpha..sup.37.
[0054] Additionally, promoters activated by anti-inflammatory
mediators (e.g. TGF-beta) can be used to achieve a targeted
expression the cytotoxic protein in the mesenchymal stem cells.
Examples are promoters which contain Smad-binding
elements.sup.55.
[0055] Using promoters, which are inducible by inflammation
mediators, enables a selective treatment of tumors, which have not
yet undergone angiogenesis.
[0056] The stem cells for use in any of the therapeutic methods of
the invention can further comprise a (iii) selection marker gene
operably linked to (iv) a constitutive promoter or
promoter/enhancer combination. The selection marker gene can
comprise an antibiotic resistance gene, such as a gene conferring
resistance to Puromycin, Neomycin or Ouabain.sup.38. The antibiotic
resistance genes can be used in order to select for genetically
modified mesenchymal stem cells under the presence of the
antibiotic and to suppress unmodified mesenchymal stem cells, which
should not be used in tumor treatment due to their tumor promoting
potential. Additional or as an alternative to an antibiotic
resistance gene a gene encoding a surface marker protein can be
used, which is only expressed on the surface of genetically
modified mesenchymal stem cells. Non-limiting examples for surface
marker proteins are splice variants of CD34 and CD24.sup.39-40.
Magnetic beads harboring specific antibodies recognizing the
surface marker proteins can be used in order to select for the
genetically modified mesenchymal stem cells.sup.41.
[0057] The constitutive promoter controlling the transcription of
the selection marker gene can be a variety of different promoters
such as pCAG.sup.42, EF1.alpha..sup.43, PGK.sup.42, CMV and
SFFV.sup.42.
[0058] In addition, the stem cells according to various embodiments
of the invention can comprise an (v) insulator sequence located
between the cytotoxic protein-encoding region and the selection
marker gene. The insulator sequence can ensure that the
constitutive promoter of the selection marker gene does not
influence and "turn on" the transcription of the cytotoxic
protein-encoding region in the absence of any inflammatory
mediators.sup.44.
[0059] Preferably, the cytotoxic protein-encoding region operably
linked to the promoter or promoter/enhancer combination and also
the selection marker gene operably linked to the constitutive
promoter or promoter/enhancer combination are integrated into the
stem cell genome. This enables the production of genetically
modified mesenchymal stems cells, which are more stable than
genetically modified stem cells harboring an extrachromosomal
vector.
[0060] In a further embodiment of the invention, the genetically
modified stem cells further comprise a proviral sequence integrated
into the stem cell genome, wherein the cytotoxic protein-encoding
region operably linked to the promoter or promoter/enhancer
combination and the selection marker gene operably linked to the
constitutive promoter or promoter/enhancer combination are part of
the proviral sequence. In particular viruses from the family of
Retroviridae can be used in order to stable genetically modify
mesenchymal stem cells. Examples for retroviruses are lentiviruses,
alpha-retroviruses or gamma-retroviruses.
[0061] In the following, retroviral vectors including a transgene
cassette having two functional units will be described (FIG. 6).
The two functional units are:
1. In vitro selection: A constitutive promoter (e.g. CAG-promoter)
of cellular or viral origin linked to a selection marker gene like
an antibiotic resistance gene (e.g. puromycin-resistance gene) or
surface marker gene suitable for magnetic bead-separation (e.g.
CD34). 2. By-stander killing: An inducible tumor-specific promoter
of cellular or viral origin (e.g. RANTES promoter) linked to
cytotoxic protein-encoding region (e.g. coding for Herpes simplex
Thymidine kinase)
[0062] Optionally, the functional units may be separated by an
insulator sequence to prevent promoter interference between the two
promoters.
[0063] The above described transgene cassette will be integrated
into various viral and non-viral vector systems for delivery and
stable expression in MSC, such as viral vector systems derived from
retroviruses.
[0064] The retroviral vectors for the genetic modification of MSC
will be derived from alpha-, gamma-retroviruses or (human and
non-human) lentiviruses. The retroviral vector systems include the
transfer vector backbone, which carries the transgene of interest,
i. e. the cytotoxic protein-encoding region and all sequence
elements necessary for the reverse transcription and integration of
the vector DNA, but is devoid of most or all viral genes, such as
gag- pol- and env-genes. The newest generation of retroviral
vectors will carry special safety modifications: In these so called
self-inactivating (SIN) vectors the 3' U3-region is partially or
completely removed to shut down viral promoter activity and to
prevent transactivation of neighboring genes in the host cell
genome.sup.50.
[0065] For the production of vector particles a variable number of
helper plasmids is needed which provide the structural proteins,
enzyme and envelope proteins in trans.sup.50. Viral particles can
be produced carrying foreign envelope glycoproteins. This process
is called pseudotyping. It allows altering the tropism of the
vector particles and in some cases enhances vector titer.
[0066] The above described transgene cassette will be inserted into
a SIN-retroviral vector backbone using standard molecular biology
methods and produce viral particles according to standard
methods.sup.50.
[0067] FIG. 7 gives an exemplary overview of alpha- and
gamma-retroviral and lentiviral vector constructs, which will carry
the mentioned transgene depicted in FIG. 6.
[0068] It is planned to pseudotype the vector particles with the
glycoprotein of the Lymphocytic choriomeningitis virus (LCMV GP),
vesicular stomatitis virus (VSV-g), RD114-TR or gibbon ape leukemia
virus (GALV env).
[0069] Another alternative method for the genetic modification of
mesenchymal stem cells is by chemical (e.g. Lipofectamin) or
physical (e.g. electroporation) transfection. Afterwards
transfected cells can be selected as described above to select
stably transfected MSC, where the transgene cassette has integrated
by chance into the mesenchymal stem cell genome.
[0070] Yet another alternative for the genetic modification of MSCs
is by using non-viral vector systems derived from transposons.
After flanking of the above described expression cassette with
terminal inverted repeats, the construct can be transferred into
MSC via transfection. If a Transposase like Sleeping Beauty.sup.51
or Piggybac.sup.52 is expressed in trans during the transfection,
the expression cassette will be stably integrated into the genome
of the MSC.
[0071] The genetically modified mesenchymal stem cells according to
some embodiments of the invention can be prepared by transduction
of native mesenchymal stem cells with pseudotyped virions,
expressing foreign glycoproteins on their surface, which alter the
tropism and often the titer of the virion.
[0072] The pseudotyped virions can be generated by the help of
retroviral packaging cells comprising: [0073] a retroviral vector
including (i) a cytotoxic protein-encoding region operably linked
to (ii) a promoter or promoter/enhancer combination, inducible by
inflammatory mediators, and [0074] a gene encoding a viral surface
protein providing a tropism for mesenchymal or CD34- stem cells.
[0075] Further the retroviral packaging cell also comprises genes
encoding structural proteins and enzymes for the production of
pseudotyped virions from the packaging cell, such as the gag-, pol-
and env-genes, which enable the assembly of the pseudotyped
virions.
[0076] Preferably these genes encoding structural proteins and
enzymes are located on a different vector than the gene encoding a
viral surface protein providing a tropism for mesenchymal or CD34-
stem cells in order to produce virions, which are infectious but
not capable of replication. These pseudotyped virions harbor the
cytotoxic protein-encoding region operably linked to (ii) a
promoter or promoter/enhancer combination and express the viral
surface protein. Normally the supernatant of the retroviral
packaging cells containing the pseudotyped virions will be used for
the transduction of the mesenchymal stem cells.
[0077] The gene encoding a viral surface protein providing a
tropism for mesenchymal or CD34- stem cells (pseudotyping) can be a
large variety of glycoproteins conferring a broad host tropism such
as the Lymphocytic choriomeningitis virus (LCMV GP).sup.45,
vesicular stomatitis virus (VSV-g).sup.45, RD114-TR.sup.46 or
gibbon ape leukemia virus (GALV env).sup.47.
[0078] As already mentioned above, the promoter or
promoter/enhancer combination can be inducible by cytokines or
other inflammatory mediators. In a preferred embodiment the
promoter is the RANTES promoter. The retroviral packaging cell can
also include further genes and promoters
[0079] In another particular embodiment of the invention, the
cytotoxic protein ideally is Herpes simplex viral thymidine kinase,
and the subject ideally is treated with ganciclovir in a manner
permitting the Herpes simplex viral thymidine kinase to render the
ganciclovir cytotoxic. Ganciclovir and its methods of use are well
known in the art. Another possibility is the use of cytosine
deaminase as a cytotoxic protein, which converts 5-fluorocytosine
to the toxic compound 5-fluorouracil.sup.48.
[0080] In this invention, the therapeutically effective number of
genetically modified mesenchymal stem cells includes, without
limitation, the following amounts and ranges of amounts: (i) from
about 1.times.10.sup.5 to about 1.times.10.sup.9 cells/kg body
weight; (ii) from about 1.times.10.sup.6 to about 1.times.10.sup.8
cells/kg body weight; (iii) from about 5.times.10.sup.6 to about
2.times.10.sup.7 cells/kg body weight; (iv) from about
5.times.10.sup.6 to about 1.times.10.sup.7 cells/kg body weight;
(v) from about 1.times.10.sup.7 to about 2.times.10.sup.7 cells/kg
body weight; (vi) from about 7.times.10.sup.6 to about
9.times.10.sup.6 cells/kg body weight; (vii) about 1.times.10.sup.5
cells/kg body weight; (viii) about 1.times.10.sup.6 cells/kg body
weight; (ix) about 5.times.10.sup.6 cells/kg body weight; (x) about
1.times.10.sup.7 cells/kg body weight; (xi) about 6.times.10.sup.6
cells/kg body weight; (xii) about 7.times.10.sup.6 cells/kg body
weight; (xiii) about 8.times.10.sup.6 cells/kg body weight; and
(ix) about 9.times.10.sup.6 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 MSCs.
[0081] This invention also provides a method for treating a human
subject afflicted with a pancreatic tumor comprising introducing
into the subject's bloodstream from about 5.times.10.sup.6 to about
2.times.10.sup.7 cells/kg body weight of genetically modified
CD34.sup.- stem cells, wherein (a) each genetically modified
CD34.sup.- stem cell contains an exogenous nucleic acid comprising
(i) a Herpes simplex viral thymidine kinase-encoding region
operably linked to (ii) a RANTES promoter, (b) the subject is
treated with ganciclovir in a manner permitting the Herpes simplex
viral thymidine kinase to render the ganciclovir cytotoxic, and (c)
the introduction of the genetically modified mesenchymal stem cells
is not preceded, accompanied or followed by myeloablation. In this
method, the tumor is preferably metastatic, and can be vascularized
or not. Additionally, the genetically modified mesenchymal stem
cells can be allogenic, autologous or xenogenic with respect to the
subject.
[0082] This invention further provides a genetically modified
mesenchymal stem cell comprising 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 mesenchymal stem cell comes into proximity with a tumor's
stromal tissue. Preferably, the mesenchymal stem cell is a human
CD34.sup.- stem cell. Also preferred is an exogenous nucleic acid
comprising a RANTES promoter, wherein the cytotoxic protein is
Herpes simplex viral thymidine kinase.
[0083] Finally, this invention provides a genetically modified
human CD34.sup.- stem cell comprising an exogenous nucleic acid
comprising (i) a Herpes simplex viral thymidine kinase-encoding
region operably linked to (ii) a RANTES promoter.
[0084] The various proteins and regulatory sequences used in this
invention can be readily obtained by one skilled in the art. For
example, the RANTES promoter is disclosed in (22) and can be
obtained by the use of ordinary skill. 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).
[0085] 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
Synopsis
[0086] Objective:
[0087] To analyze the efficacy of engineered mesenchymal stem
cell-based therapy directed towards pancreatic tumor stroma.
[0088] Summary Background Data:
[0089] Mesenchymal stem cells (MSC) are actively recruited to tumor
stroma where they enhance tumor growth and metastases. Upregulation
of chemotactic cytokine CCL5 by MSCs within the tumor stroma has
been shown to play a central role in this process. Murine MSCs were
engineered to express reporter genes or therapeutic genes under
control of the CCL5 promoter and adoptively transferred into mice
with growing pancreatic tumors. The effect on tumor growth and
metastases was then evaluated.
[0090] Methods:
[0091] MSCs isolated from bone marrow of C57/B16 p53(-/-) mice were
stably transfected with Red Fluorescent Protein (RFP), enhanced
Green Fluorescent Protein (eGFP) or Herpes simplex virus (HSV)
thymidine kinase (tk) gene driven by the RANTES promoter. MSCs were
intravenously applied once per week over 3 weeks to mice carrying
an orthotopic, syngeneic pancreatic Panc02 tumor.
[0092] Results:
[0093] eGFP and RFP signals driven by the CCL5 promoter were
detected by florescence in treated pancreatic tumor samples. The
HSV-tk therapy group treated i.p. with the prodrug ganciclovir
(GCV) 5-7 days after stem cell application lead to a 50% reduction
of primary pancreatic tumor growth (p<0,0003, student's t-test)
and reduced liver metastases (30% vs. 100%).
[0094] Conclusions:
[0095] The active homing of MSCs into primary pancreatic tumor
stroma and activation of the RANTES promoter was verified using
eGFP- and RFP-reporter genes. In the presence of ganciclovir,
HSV-tk-transfected MSCs led to a significant reduction of primary
pancreatic tumor growth and incidence of metastases.
Materials and Methods
Mesenchymal Stem Cells
[0096] Mesenchymal stem cells were isolated from the bone marrow of
C57BL/6 mice homozygous for the targeted deletion of p53 as
described..sup.15 The cells grew adherently and continuously in
cell culture. After subcloning, single cell clones were selected
and characterized..sup.15 The cells were transfected with red
fluorescent protein (RFP), green fluorescent protein (eGFP) or HSV
thymidine kinase linked to the CCL5 promoter. The sequence of the
promoter used -972 of the upstream region and the complete 5'
untranslated region..sup.18 In addition, all vectors contained a
CMV-controlled Bsr2 Blasticidin resistance gene. Blasticidin was
used to select for transfected cells at a concentration of 5
.mu.g/ml. Prior to injection into the mice, the cells were detached
from the culture flasks, washed twice with PBS, and re-suspended in
PBS.
Modified Boyden Chamber Assay
[0097] Directed MSC migration in vitro was performed using a
modified Boyden chamber assay..sup.15 Stem cells migrated against
descending concentrations of supernatant of Panc02 syngenic
pancreatic carcinoma cells.
Orthotopic Pancreatic Carcinoma Model
[0098] C57BL6 mice were obtained from Jackson Labs. All animal
experiments were conducted with appropriate permission from the
animal rights commission of the state of Bavaria. Two-month-old to
three-month-old C57BL/6 mice with an average weight of
approximately 20 g were used for implantation of the Panc02
(syngenic to C57BL/6 mice) pancreatic tumor..sup.19 The mice were
anesthetized using Ketamine (100 mg per kg body weight), Xylazine
(5 mg per kg body weight) and Atropine. The operation site on the
left flank of the mice was shaved and prepared in a sterile manner.
With a 1 cm incision at the left flank, the pancreas was exposed. A
calibrated push-button device (Hamilton Syringe Company, USA) and a
1 ml syringe with a 30 G needle (both BD Biosciences, Spain) was
used to inject 150,000 Panc02 pancreatic cancer cells in a 40 .mu.l
PBS solution into the pancreas. Caution was taken to ensure that no
pancreatic cancer cells were disseminated into the peritoneum. To
this end, a Q-tip was pressed lightly on the injection site for one
minute after the needle was pulled out of the pancreas. Following
the injection of the tumor cells the peritoneum and skin were
closed with interrupted sutures of 4-0 prolene (Braun AG, Germany).
Two weeks following the procedure, all mice grew palpable tumors
and were randomized into the respective experimental groups. Group
A received no stem cells or Ganciclovir injections, group B
received unmodified stem cells, group C received C57BL/6
p53-/-CCL5/HSV-tk+ mesenchymal stem cells and GCV injections, group
D received C57BL/6 p53-/-CCL5/RFP+ mesenchymal stem cells and group
E received C57BL/6 p53-/-CCL5/eGFP mesenchymal stem cells. All stem
cell injections were dosed at 0.5.times.10.sup.6 cells per week and
administered via the tail vein. GCV (Cymeven.RTM., Roche, Germany)
injections of group C were at a dose of 1.5 mg and were applied
i.p. on days 5 to 7 following the stem cell injections. All mice
were sacrificed after three cycles of treatment and the tumors were
isolated and weighed. Testing for statistic significance was
achieved with an unpaired two-tailed t-test for independent
samples.
Fluorescence Microscopy
[0099] Tissue samples of the pancreatic tumors were embedded in
Tissue-Tek O.C.T. (Miles Inc. USA) and snap-frozen in liquid
nitrogen. Cryosections of 5 .mu.m thickness were obtained. DAPI
nuclear staining solution (Vectashield "Hard set mounting medium
with Dapi", USA) was added to the sections and they were
immediately assessed for GFP or RFP fluorescence signals. Photos
were obtained using the AxioCam MR microscope camera and pictures
of different fluorescence channels were overlaid using the software
Photoshop.RTM. (Adobe, USA).
MSC Injection and Ganciclovir Treatment
[0100] Before injection, the cells were counted and diluted to a
final concentration of 1.times.10.sup.6 cells/ml PBS. Ganciclovir
(GCV) (Cymeven; Roche) was dissolved in H.sub.2O (aqua ad
iniectabilia) to a concentration of 10 mg/ml. Cell suspensions were
administered with a 26 G needle via the tail vein, drugs via
intraperitoneal injections. The treatment started on day 1, with
injection of 0.5 ml cells (500,000 cells). On days 5 through 7,
Ganciclovir was applied in a daily dose of 60 .mu.g/g BW, e.g. 150
.mu.l for a mouse with 25 g BW. After day 7, treatment cycles were
repeated until dissection. During the treatment tumor progression
and behaviour were recorded.
Tissue and Tumor Preparation
[0101] After dissection, all of the tumors were prepared
separately. One third of each tumor was formaldehyde-fixed and
embedded in paraffin wax while an additional third was
flash-frozen. The last third was conserved in RNAlater solution
(Ambion) accordingly to the manufacturer's instructions for later
RNA isolation and intended qRT-PCR analysis.
Immunohistochemistry
[0102] Immunohistochemistry was performed on 5 .mu.m sections, as
previously described..sup.20 As the primary antibodies, the
polyclonal rabbit anti-RFP antibody (Mbl Medical and Biological
Laboratories, Japan) was diluted 1:50 in blocking solution
(milk+superblock). As the secondary antibody, a polyclonal
biotinylated goat anti-rabbit (Linaris, Wertheim-Bettingen,
Germany) antibody was diluted 1:300 in milk.
Results
Recruitment of MSCs to Pancreatic Tumors
[0103] A syngeneic, orthotopic murine pancreatic tumor was
previously established in a C57Bl/6 mouse background..sup.31,32
Tumor cells implanted under the capsule of the pancreas grew and
showed a profound tumor vasculature (FIG. 1). CD34.sup.- MSCs
isolated from bone marrow of P53(-/-) were used to evaluate the
efficacy of engineered MSCs for targeting tumor
stroma..sup.15,16,21
[0104] To first evaluate the general tropism of the MSC for the
Panc02 tumor, modified Boyden chamber assays were use to study the
induced migration of the C57B16 MSC towards tumor-derived factors.
The results show a dose-dependant migration of the MSC in response
to increasing levels of conditioned tumor growth media (FIG.
2).
[0105] The cells were then engineered with a plasmid containing
green fluorescent protein (GFP) under control of the CMV promoter.
Once a week, 500,000 cells were injected intravenously into mice
with growing pancreatic tumors. After five weeks, the mice were
sacrificed and the tumors removed and analyzed for expression of
GFP. Results show a strong GFP expression associated with the tumor
(FIG. 3A). The cells were also found to migrate to secondary
spleen, lymph nodes, thymus, skin and gut (data not shown and
(15)). This demonstrates a homing of the systemically injected stem
cells to the growing tumor. The i.v. injection of MSC into C57B16
mice with growing pancreatic tumors also resulted in a significant
increase in tumor growth (FIG. 3B).
[0106] The effect of i.v.-applied MSC metastases to liver, spleen,
and peritoneum was also evaluated. Application of MSCs was found to
significantly increase metatases to the peritoneum (Table 1).
The CCL5 Promoter Drives Reporter Gene Expression in Tumor
Stroma
[0107] We then explored the possibility of driving a more
controlled expression of reporter genes in the context of MSC
recruitment to tumor stroma using the CCL5 promoter. To this end,
the P53(-/-) MSC C57/B16 cell line was engineered with a RFP and
eGFP reporter genes under the control of the CCL5
promoter..sup.18,22 The CCL5 promoter is active in diverse tissue
types generally in the context of tissue stress or
damage..sup.23-27 The immediate -972 upstream nucleotides and the
complete 5' untranslated region to the start of translation were
cloned upstream of eGFP or RFP in a vector.
[0108] The resultant CCL5-eGFP or RFP stably engineered MSCs showed
weak but detectable levels of expression of the reporter via FACS
(data not shown). The cells (500,000) were then injected into the
peripheral circulation of mice with growing pancreatic tumors every
eight days for 21 days.
[0109] Three weeks later the mice were sacrificed and the tumor and
surrounding tissue was analyzed for RFP and eGFP reporter gene
expression by flourescence microscopy and immunohistochemistry. The
results showed expression of RFP and GFP fluorescence in the
growing tumor (FIGS. 4A and B). To examine the expression RFP in
tissue samples with better morphology, formaldehyde fixed samples
were tested for RFP protein expression by immunohistochemistry. RFP
expressing MSC were detected throughout the tumor stroma (FIGS. 4C,
D and E).
The Use of HSV-Tk as a Therapeutic Modality in the Engineered
MSCs
[0110] In the next phase of the experiment, delivery of therapeutic
genes using the CCL5 promoter was examined. To this end, the gene
for Herpes simplex thymidine kinase (HSV-Tk).sup.28 was cloned
behind the CCL5 promoter (FIG. 5A).
[0111] After 0.5.times.10.sup.6 CCL5-tk engineered MSCs were
injected, the cells were given three days to undergo recruitment to
the growing tumor stroma, undergo differentiation and subsequent
expression of the tk gene. The mice then received a course of
treatment consisting of once-daily intraperitoneal injections of
1.5 mg GVC for three days. The mice were again injected with the
engineered stem cells, and the cycle was repeated for the duration
of the experiment (FIG. 5B). After 36 days, the animals were
sacrificed and tumor growth was evaluated (FIGS. 5C and D). Results
showed a significant decrease in tumor volume in the group of mice
that received the therapeutic CCL5/HSV-Tk stem cell construct with
GCV in comparison to control animals with tumors that received no
treatment or control MSC (CCL5-RFP MSC and CCL5-eGFP MSC). FIG. 5C
shows representative tumors excised upon completion of the
experiment. The weights of the tumors show a statistically
significant decrease in tumor weight as compared to untreated or
treated control animals.
[0112] As an additional parameter, liver, spleen, and peritoneum
were analyzed for metastases in the context of treatment. While the
administration of MSC increased the number of metastases in the
peritoneum, treatment with GCV resulted in a significant reduction
of metastases in spleen and liver (two-tailed Fisher's exact test)
(Table 2). Metastases were evaluated by inspection of spleen,
liver, and peritoneum in the situs and palpation of the respective
organs.
DISCUSSION
[0113] Mesenchymal stem cells are actively recruited to tumor
stroma where they contribute to diverse aspects of tumor growth.
MSC can function as progenitor cells for tumor vessels and also
appear to contribute to the generation of stromal-fibroblast-like
cells. The specific influence of tumor-associated stromal cells on
tumor growth and on the potential to metastasize is an issue of
current research. In preclinical studies of a mamma-carcinoma
model, it was shown that mesenchymal stem cells (MSC) within the
tumor stroma produce increased levels of the cytokine CCL5. The
secretion of CCL5 leads to a higher incidence of lung
metastases.
[0114] CCL5 secretion is also differentially regulated in
pancreatic periacinar myofibroblasts, suggesting a role for these
cells in mediating the infiltration and accumulation of
inflammatory cells in the pancreas..sup.29 Among patients with
pancreatic cancer, pancreatitis has been significantly associated
with polymorphism in the CCL5 promoter..sup.30 The work herein
evaluates the use of engineered MSCs as a therapeutic vehicle for
the selective delivery of a suicide gene in the context of tumor
stroma on primary tumor growth as well as metastases.
[0115] MSCs were engineered to express the herpes simplex virus
(HSV) thymidine kinase (TK) under the control of the CCL5 promoter
for tissue specific expression. MSCs were transfected using
herpes-simplex-virus-thymidine kinase (tk) and under the control of
the CCL5 promoter for a more tissue specific gene expression. Tk
phosphorylates ganciclovir (GCV), generating a toxin that kills the
transfected cells and nearby tumor cells via a bystander effect.
HSV-TK gene therapy with ganciclovir forms the basis of a
widely-used strategy for suicide gene therapy..sup.17
[0116] As solid tumors exert a strong homing drive on circulating
progenitors, the tumor environment is efficiently targeted using
this approach. This targeting of the vehicle stem cells in addition
to the tissue-specific gene expression driven by the CCL5 promoter
of the suicide gene leads to both high efficacy and a low side
effect profile. Furthermore, bone marrow-derived MSC could be
obtained from the cancer patients themselves. This would allow for
the specific delivery of suicide genes through easy intravenous
administration without the need for myeloablation and a bone marrow
transplant.
[0117] Pancreatic cancer treatment strategies based on preclinical
research have not succeeded in significantly extending patient
survival. At the time of diagnosis, only 20% of the patients
suffering from pancreatic cancer present with localized disease
amenable to surgery. Forty percent of the patients present with
locally advanced (and therefore, unresectable) disease, and another
40% already suffer from distant metastases. The pancreatic tumor
model demonstrated that MSCs play important role in pancreatic
carcinoma. The cells actively seek the tumor, as illustrated via
migration assays as well as systemic injections of CCL5/RFP MSC.
The systemically injected stem cells were found exclusively within
the tumor. The ensuing reduction in tumor size and reduced
peritoneal carcinosis are promising for the clinical application of
a patient-tailored combined stem cell/suicide gene therapy.
[0118] Engineered stem cells that are recruited to other tissue
niches do not undergo the same program of differentiation, and
therefore do not express the therapeutic gene. This approach allows
a significant degree of control of the selective expression of the
therapeutic gene within a defined microenvironment.
[0119] Linking stem cell therapy with selective gene therapy
enhances the therapeutic options for the regeneration or
replacement of diseased or missing cells, as well as for tumor
destruction. Here, it is shown that genetically modified stem cells
can serve as a vehicle for the transport of tissue-specific gene
therapy to tumors and that MSC engineered with the CCL5-promoter
can drive tk expression.
TABLE-US-00001 TABLE 1 Group Control (n = 5) native mSC (n = 3)
Metastases Metastases p-value Origin Spleen 5 (100%) 3 (100%) 1.00
Liver 3 (60%) 3 (100%) 0.46 Peritoneum 0 (0%) 3 (100%) 0.018*
[0120] The effect of MSC treatment on the development of
metastases. Comparison of vehicle control and animals treated with
native MSC (MSC given over a period of three weeks with 500,000
cells weekly). Examination was done by inspection and palpation
after 36 days of tumor growth. Numbers express animals with
metastases. Significance was tested by two-tailed Fisher's exact
test.
TABLE-US-00002 TABLE 2 Group Control (n = 5) RaPro/RFP (n = 9)
RaPro/eGFP (n = 6) RaPro/Tk + GCV (n = 10) Metastases Metastases
p-value Metastases p-value Metastases p-value Origin Spleen 5
(100%) 8 (89%) 1.00 6 (100%) 1.00 3 (30%) 0.026* Liver 3 (60%)
.sup. 4 (44.4%) 1.00 2 (33%) 0.57 0 (0%) 0.022* Peritoneum 0 (0%) 7
(78%) 0.021* 4 (67%) 0.06 0 (0%) 1.00
[0121] Table 2. The distribution of tumor metastases. Vehicle
Control animals and GFP and RFP reporter gene transfected control
MSCs were compared to animals treated with the suicide gene
therapy. Examination by inspection and palpation after 36 days of
tumor growth. Numbers express animals with metastases. Significance
was tested by two-tailed Fisher's exact test. All MSC were given
over a period of three weeks with 500,000 cells weekly.
In Vitro Experiments for Stable Transduction of Human MSCs and In
Vitro Assay for Induction of HSV Tk Expression
Generation of Genetically Modified Human MSCs
[0122] Human MSCs were incubated over night with replication
deficient lentiviruses carrying the vector pLenti6 TK RANTES
harboring the HSV tk gene under the control of the RANTES promoter
and the blasticidin resistance gene under the control of the SV40
promoter (multiplicity of infection (MOI): 10) (see FIG. 8).
[0123] After overnight incubation the virus containing media was
removed and replaced with fresh media. The next day blasticidin (6
.mu.g/ml) was added to the cells to select for genetically modified
MSCs. Culture media, including blasticidin was changed every 3 to 4
days for a minimum of 6 days.
In Vitro Induction of the RANTES Promoter
[0124] It was demonstrated that a combination of the cytokines
TNF.alpha. (10 ng/ml) and IFN.gamma. (10 ng/ml) leads to an
induction of the RANTES promoter in human umbilical vascular
endothelial cells (HUVECS).sup.35.
[0125] In our experiments we wanted to demonstrate that the same
combination of cytokines also leads to an induction of the
endogenous RANTES promoter in MSCs. We wanted to use this assay to
be able to induce the expression of the exogenous HSV tk in vitro.
Genetically modified MSCs were cultivated for up to 48 h with
TNF.alpha. and IFN.gamma. or without the cytokines and isolated
whole RNA after 0, 24 and 48 h. The RNA (600 ng) was reverse
transcribed to cDNA which, in turn, was used in qRT-PCR reactions
to quantify endogenous RANTES expression with the LighCycler system
(Roche, Primer: for: CCT CAT TGC TAC TGC CCT CT; rev: GGT GTG GTG
TCC GAG GAA TA; Universal Probe 16). To assure that same amounts of
RNA were used from different samples a housekeeping gene (actin)
was used as reference gene (Universal ProbeLibrary Human ACTB Gene
Assay, Roche) and relative amounts were calculated using the
.DELTA..DELTA.CT method.
[0126] As shown in FIG. 9, we were able to detect a pronounced
increase of endogenous RANTES mRNA 24 and 48 h after induction of
MSCs with TNF.alpha. (10 ng/ml) and IFN.gamma. (10 ng/ml). These
findings demonstrate that RANTES expression is induced by
TNF.alpha. and IFN.gamma. not only in HUVECs but also in human
MSCs.
Specific Cell Death of Induced Genetically Modified MSCs after
Ganciclovir Treatment
[0127] After we could demonstrate the inducibility of the
endogenous RANTES promoter we went on to investigate if it was
possible to induce the expression of the HSV tk which was under the
control of the exogenous RANTES promoter and as a consequence
promote cell death after treatment of the induced cells with
ganciclovir. Genetically modified cells (50000 cells in per 6 well)
that were generated as described above were treated for 9 days with
TNF.alpha. (10 ng/ml) and IFN.gamma. (10 ng/ml) with addition of
fresh cytokines every 3 days. Subsequently the cells were incubated
for 3 days with 100 .mu.M ganciclovir.
[0128] The results clearly demonstrated that genetically modified
MSCs which were induced with TNF.alpha. and IFN.gamma. and
subsequently treated with ganciclovir were not surviving (FIG. 10
D). In contrast to cells that were only induced with TNF.alpha. and
IFN.gamma. (FIG. 10 B) or treated with ganciclovir (FIG. 10 C).
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