U.S. patent application number 13/403816 was filed with the patent office on 2012-08-23 for isolated stromal cells and methods of using the same.
Invention is credited to Jose Caro, Joseph Kulkosky, Alexey Laptev, Dennis B. Leeper, Michael D. O'Hara, Ruth F. Pereira, Donald Phinney, Darwin J. Prockop.
Application Number | 20120213752 13/403816 |
Document ID | / |
Family ID | 29255138 |
Filed Date | 2012-08-23 |
United States Patent
Application |
20120213752 |
Kind Code |
A1 |
Prockop; Darwin J. ; et
al. |
August 23, 2012 |
ISOLATED STROMAL CELLS AND METHODS OF USING THE SAME
Abstract
Isolated stromal cells, containers comprising isolated stromal
cells transfected with exogenous DNA, and methods of treating
patients suffering from diseases characterized by a bone cartilage
or lung defect are disclosed. The methods comprise obtaining a bone
marrow sample from a donor, isolating stromal cells from the
sample, and administering the isolated stromal cells to the
patient.
Inventors: |
Prockop; Darwin J.;
(Philadelphia, PA) ; Pereira; Ruth F.; (Lansdowne,
PA) ; Leeper; Dennis B.; (Wynnewood, PA) ;
O'Hara; Michael D.; (Wyncote, PA) ; Kulkosky;
Joseph; (Philadelphia, PA) ; Phinney; Donald;
(Maple Glen, PA) ; Laptev; Alexey; (Philadelphia,
PA) ; Caro; Jose; (Gladwyn, PA) |
Family ID: |
29255138 |
Appl. No.: |
13/403816 |
Filed: |
February 23, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12043903 |
Mar 6, 2008 |
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13403816 |
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10423232 |
Apr 25, 2003 |
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12043903 |
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08913918 |
Dec 8, 1997 |
6974571 |
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10423232 |
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PCT/US1996/004407 |
Mar 28, 1996 |
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08913918 |
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08412066 |
Mar 28, 1995 |
5716616 |
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PCT/US1996/004407 |
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60006627 |
Nov 13, 1995 |
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Current U.S.
Class: |
424/93.21 ;
424/93.7; 435/174; 435/325 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 9/00 20180101; C12N 2502/1394 20130101; A61K 48/005 20130101;
C07K 14/4702 20130101; A61K 2035/124 20130101; A61K 2035/128
20130101; C12N 5/0663 20130101; A61K 48/0025 20130101; C07K 14/61
20130101; A61P 19/00 20180101; A61P 11/00 20180101; C07K 14/78
20130101; C12N 2799/027 20130101; A61K 48/00 20130101; C07K 14/745
20130101; A61K 35/28 20130101; A61P 35/00 20180101; A61P 19/04
20180101 |
Class at
Publication: |
424/93.21 ;
435/174; 424/93.7; 435/325 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 5/10 20060101 C12N005/10; A61P 11/00 20060101
A61P011/00; C12N 11/00 20060101 C12N011/00; A61P 19/04 20060101
A61P019/04 |
Claims
1. A method of treating patient who is suffering from a disease,
disorder or condition characterized by a bone cartilage or lung
defect comprising the steps of: a) obtaining a bone marrow sample
from a donor who is not suffering from a disease, disorder or
condition characterized by a bone, cartilage or lung defect and who
is syngeneic with said patient; b) isolating stromal cells from
said sample; and, c) administering said isolated stromal cells by
intravenous infusion to said patient.
2. The method of claim 1 wherein said patent undergoes bone marrow
ablation prior to administration of isolated stromal cells.
3. The method of claim 2 wherein said stromal cells are
administered by intravenous infusion to said patient together with
hematopoeitic precursor cells from a bone marrow sample from a
donor who is not suffering from a disease, disorder or condition
characterized by a bone cartilage or lung defect and who is
syngeneic with said patient.
4. The method of claim 2 wherein said stromal cells are
administered by intravenous infusion to said patient free from
hematopoeitic precursor cells.
5. The method of claim 1 wherein prior to administering said
stromal cells, said stromal cells are transfected with a gene
construct that comprises a herpes thymidine kinase gene, wherein
said gene is operably linked to regulatory sequences and is
expressed by said stromal cells.
6. The method of claim 1 wherein said disease, disorder or
condition is characterized by a defect in said patient's bone.
7. The method of claim 6 wherein said disease, disorder or
condition is osteogenesis imperfecta or osteoporosis.
8. The method of claim 1 wherein said disease, disorder or
condition is characterized by a defect in said patient's
cartilage.
9. The method of claim 8 wherein said disease, disorder or
condition is chondrodysplasia or osteoarthritis.
10. The method of claim 1 wherein said disease, disorder or
condition is characterized by defect in said patient's lungs.
11. The method of claim 10 wherein said disease, disorder or
condition characterized is cystic fibrosis.
12. A method of treating patient who suffering from a disease,
disorder or condition characterized by a mutated, non-functioning
or under-expressed gene which results in a defect in the bone,
cartilage or lungs of said patient comprising the steps of: a)
obtaining a bone marrow sample from said patient; b) isolating
stromal cells from said sample; c) transfecting said stromal cells
with a normal copy of said mutated, non-functioning or
under-expressed gene wherein said copy of said gene is operably
linked to functional regulatory elements; and d) administering said
transfected stromal cells to said patient by intravenous
infusion.
13. The method of claim 12 wherein said patent undergoes bone
marrow ablation prior to administration of stromal cells.
14. The method of claim 13 wherein said stromal cells are
administered by intravenous infusion to said patient together with
hematopoietic precursor cells from said sample.
15. The method of claim 12 wherein prior to administering said
stromal cells, said stromal cells are transfected with a gene
construct that comprises a herpes thymidine kinase gene, wherein
said gene is operably linked to regulatory sequences and is
expressed by said stromal cells.
16. The method of claim 12 wherein said disease, disorder or
condition is characterized by a defect in said patient's bone.
17. The method of claim 16 wherein said disease, disorder or
condition is osteogenesis imperfecta and said gene encodes type I
procollagen or type I collagen.
18. The method of claim 12 wherein said disease, disorder or
condition is characterized by a defect in said patient's
cartilage.
19. The method of claim 18 wherein said disease, disorder or
condition is chondrodysplasia and said gene encodes type II
procollagen or type II collagen.
20. The method of claim 12 wherein said disease, disorder or
condition is characterized by defect in said patient's lungs.
21. The method of claim 20 wherein said disease, disorder or
condition characterized is cystic fibrosis and said gene is a
cystic fibrosis gene.
22. An implant device comprising: a container having at least one
membrane surface stromal cells that comprise a gene construct, said
gene construct comprising a nucleotide sequence that encodes a
beneficial protein operably linked to regulatory elements which
function in said stromal cell.
23. The implant device of claim 22 wherein said membrane has a pore
size of 0.3 microns.
24. The implant device of claim 22 having a membrane surface area
of at least 100 mm.sup.2.
25. The implant device of claim 22 comprising 10.sup.4 to 10.sup.11
stromal cells.
26. The implant device of claim 22 comprising 10.sup.4 to 10.sup.8
stromal cells.
27. The implant device of claim 22 wherein said beneficial protein
is selected form the group consisting of human growth hormone,
obesity factor and human Factor VIII.
28. A method of treating an individual with a disease, disorder or
condition which can be treated with a beneficial protein comprising
the step of introducing into such an individual, immunologically
isolated stromal cells that comprise a gene construct, said gene
construct comprising a nucleotide sequence that encodes a
beneficial protein operably linked to regulatory elements which
function in said stromal cell.
29. The method of claim 28 wherein said disease, disorder or
condition which can be treated with a beneficial protein is a
disease, disorder or conditions characterized by a gene defect.
30. The method of claim 29 wherein said beneficial protein is
selected from the group consisting of human growth hormone and
human Factor VIII.
31. The method of claim 28 wherein said immunologically isolated
stromal cells are within an implant device that comprises said
stromal cells and a container having at least one membrane
surface.
32. The method of claim 31 wherein said membrane of said implant
device has a pore size of 0.3 microns.
33. The method of claim 31 wherein said implant device has a
membrane surface area of at least 100 mm.sup.2.
34. The method of claim 31 wherein said implant device comprises
10.sup.4 to 10.sup.11 stromal cells.
35. The method of claim 31 wherein said implant device comprises
10.sup.4 to 10.sup.8 stromal cells.
36. The method of claim 31 wherein said implant device is implanted
into said individual subcutaneously.
37. Immunologically isolated stromal cells that comprise a gene
construct, said gene construct comprising a nucleotide sequence
that encodes a beneficial protein operably linked to regulatory
elements which function in said stromal cell.
38. The immunologically isolated stromal cells of claim 37 wherein
said stromal cells are microencapsulated.
39. A method of treating patient who is suffering from a disease,
disorder or condition characterized by a bone cartilage or lung
defect comprising the steps of: a) obtaining a bone marrow sample
from a donor who is not suffering from a disease, disorder or
condition characterized by a bone or cartilage defect and who is
syngeneic with said patient; and, b) administering a
therapeutically effective amount of said bone marrow by intravenous
infusion to said patient.
40. The method of claim 39 wherein said patent undergoes bone
marrow ablation prior to administration of isolated stromal
cells.
41. The method of claim 39 wherein said disease, disorder or
condition is characterized by a defect in said patient's bone.
42. The method of claim 41 wherein said disease, disorder or
condition is osteogenesis imperfecta.
43. The method of claim 39 wherein said disease, disorder or
condition is characterized by a defect in said patient's
cartilage.
44. The method of claim 43 wherein said disease, disorder or
condition is chondrodysplasia.
45. A method of treating patient who suffering from a disease,
disorder or condition characterized by a mutated, non-functioning
or under-expressed gene which results in a defect in the bone,
cartilage or lungs of said patient comprising the steps of: a)
obtaining a bone marrow sample from said patient; b) isolating
stromal cells from said sample; c) culturing said stromal cells
under conditions which result in replication of said stromal cells
into an expanded culture of stromal cells; and d) administering
stromal cells of said expanded culture of stromal cells to said
patient by intravenous infusion.
46. The method of claim 45 wherein said patent undergoes bone
marrow ablation prior to administration of stromal cells.
47. The method of claim 46 wherein said stromal cells are
administered by intravenous infusion to said patient together with
hematopoietic precursor cells from said sample.
48. The method of claim 46 wherein said stromal cells are
administered by intravenous infusion to said patient free from
precursor cells from said sample.
49. The method of claim 45 wherein said disease, disorder or
condition is characterized by a defect in said patient's bone.
50. The method of claim 49 wherein said disease, disorder or
condition is osteogenesis imperfecta or osteoporosis.
51. The method of claim 45 wherein said disease, disorder or
condition is characterized by a defect in said patient's
cartilage.
52. The method of claim 46 wherein said disease, disorder or
condition is chondrodysplasia or osteoarthritis.
53. The method of claim 45 wherein said disease, disorder or
condition is characterized by defect in said patient's lungs.
54. The method of claim 53 wherein said disease, disorder or
condition characterized is cystic fibrosis and said gene is a
cystic fibrosis gene.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions comprising
isolated stromal cells, to containers which comprise isolated
stromal cells transfected with exogenous DNA, to methods of
treating individuals suffering from diseases associated with bone
and cartilage, and to methods of using stromal cells in the
treatment of individuals who have diseases associated with bone,
cartilage or lung tissue, diseases associated with dermis, blood
vessels, heart and kidney tissue, diseases associated with genetic
defects and/or diseases or conditions that can be treated by
administering or delivering beneficial proteins.
BACKGROUND OF THE INVENTION
[0002] In addition to hematopoietic stem cells, bone marrow
contains "stromal cells" which are mesenchymal precursor cells
(Friedenstein, A, J. et al., Exp. Hemet. 4:267-274 (1976) which is
incorporated herein by reference) that are characterized by their
adherence properties when bone marrow cells are removed and put on
to plastic dishes. Within about four hours, stromal cells adhere to
the plastic and can thus be isolated by removing non-adhered cells
form the dishes. These bone marrow cells that tightly adhere to
plastic have been studied extensively (Castro-Malaspina, H. et al.,
Blood 56:289-301 (1980); Piersma, A. H. et al., Exp. Hematol
13:237-243 (1985); Simmons, P. J. and Torok-Storb, B., Blood
78:55-62 (1991); Beresford, J. N. et al., J. Cell. Sci. 102:341-351
(1992); Liesveld, J. L. et al., Blood 73:1794-1800 (1989);
Liesveld, J. L. et al., Exp. Hematot 19:63-70 (1990); and Bennett,
J. H. et al., J. Call. Sci. 99:131-139 (1991)) which are
incorporated herein by reference. As used herein, the term
"adherent cells" is meant to refer to stromal cells and the term
"non-adherent cells" is meant to refer to hematopoietic precursor
cells.
[0003] Stromal cells are believed to participate in the creation of
the microenvironment with the bone marrow in vivo. When isolated,
stromal cells are initially quiescent but eventually begin dividing
so that they can be cultured in vitro. Expanded numbers of stromal
cells can be established and maintained. Stromal cells have been
used to generate colonies of fibroblastic adipocytic and osteogenic
cells when cultured under appropriate condition. If the adherent
cells are cultured in the presence of hydrocortisone or other
selective conditions populations enriched for hematopoietic
precursors or osteogenic cells are obtained (Carter, R. F. et al.,
Blood 79:356-364 (1992) and Bienzle, D. et al., Proc. Natl. Acad.
Sci. USA, 91:350-354 (1994)) which are incorporated herein by
reference.
[0004] There are several examples of the use of stromal cells.
European Patent EP 0,381,490, which is incorporated herein by
reference, discloses gene therapy using stromal cells. In
particular, a method of treating hemophilia is disclosed. Stromal
cells have been used to produce fibrous tissue, bone or cartilage
when implanted into selective tissues in vivo (Ohgushi, H. et al.,
Acte. Orthop. Scand. 60:334-339 (1989); Nakahara, H. et al.,
Orthop. Res. 9:465-476 (1991); Niedzwiedski, T. et al.,
Biomaterials 14:115-121 (1993); and Wakitani, S. et al., J. Bone
& Surg. 76A:579-592 (1994)). In some reports, stromal cells
were used to generate bone or cartilage in vivo when implanted
subcutaneously with a porous ceramic (Ohgushi, H. et al. Acta.
Orthop. Scand. 60:334-339 (1989)), intraperitoneally in a diffusion
chamber (Nakahara, H. et al. J. Orthop. Res. 9:465-476 (1991)),
percutaneously into a surgically induced bone defect (Niedzwiedski,
T. et al. Biomaterials. 14:115-121 (1993)), or transplanted within
a collagen gel to repair a surgical defect in a joint cartilage
(Wakitani, S. et al. J. Bone & Surg. 76A:579-592 (1994)).
Piersma, A. H. et al. Brit. J. Hematol. 54:285-290 (1983) disclose
that after intravenous bone marrow transplantation, the fibroblast
colony-forming cells which make up the hemopoietic stroma lodge and
remain in the host bone marrow. Stewart et al. (Blood 81:2566-2571
(1993)) recently observed that unusually large and repeated
administrations of whole marrow cells produced long-term
engraftment of hematopoietic precursors into mice that had not
undergone marrow ablation. Also, Bienzle et al. (Proc. Natl. Acad.
Sci. USA, 91:350-354 (1994)) successfully used long-term bone
marrow cultures as donor cells to permanently populate
hematopoietic cells in dogs without marrow ablation. In some
reports, stromal cells were used either as cells that established a
microenvironment for the culture of hematopoietic precursors
(Anklesaria, PNAS USA 84:7681-7685 (1987)) or as a source of an
enriched population of hematopoietic stem cells (Kiefer, Blood
78(10):2577-2582 (1991)).
SUMMARY OF THE INVENTION
[0005] One aspect of the present invention relates to methods of
treating patients who are suffering from a disease, disorder or
condition characterized by a bone, cartilage or lung defect or
diseases disorder or condition characterized by defects in dermis,
blood vessels, heart or kidney. The method comprises the steps of:
obtaining a bone marrow sample from a normal, matched, syngeneic
donor; isolating adherent cells from the sample; and administering
the isolated adherent cells to the patient by intravenous infusion.
Another aspect of the present invention relates to methods of
treating patients who are suffering from a disease, disorder or
condition that characterized by a mutated, non-functioning or
under-expressed gene which results in a defect in the patient's
bones, cartilage or lungs or dermis, blood vessels, heart or
kidney. The method comprises the steps of obtaining a bone marrow
sample from the patient or a matched syngeneic donor, isolating
adherent cells from the sample, transfecting said adherent cells
with a normal copy of said mutated, non-functioning or
under-expressed gene that is operably linked to functional
regulatory elements, and administering the transfected adherent
cells to the patient by intravenously.
[0006] The present invention relates to implant devices that
comprise a container having at least one membrane surface and
stromal cells. The stromal cells comprise a gene construct, which
includes a nucleotide sequence that encodes a beneficial protein
operably linked to regulatory elements which function in stromal
cells.
[0007] The invention relates to methods of treating individuals who
have diseases, disorders or conditions which can be treated with a
beneficial protein, including diseases, disorders or conditions
characterized by genetic defects. The methods comprise the step of
introducing into such individuals, immunologically isolated stromal
cells that comprise a gene construct. The gene construct comprises
a nucleotide sequence that encodes a beneficial protein operably
linked to regulatory elements which function in stromal cells.
[0008] The invention relates to stromal cells that comprise a gene
construct and that are administered in a manner that physically
isolates them from the host's immune system. The gene construct
comprises a nucleotide sequence that encodes a beneficial protein
operably linked to regulatory elements which function in stromal
cells.
BRIEF DESCRIPTION OF THE FIGURE
[0009] FIG. 1 shows a schematic of the retroviral constructs
pCMV-lac Z, pCOL1-lac Z, and pCOL2-lac Z. The cassettes of the gene
constructs are: LTR-Neo-promoter-Lac Z-LTR.
[0010] FIG. 2 is a schematic illustration of a diffusion
chamber.
DETAILED DESCRIPTION OF THE INVENTION
[0011] As used herein, "stromal cells", "colony forming
fibroblasts", "marrow stromal cells", "adherent cells" and "MSCs"
are used interchangeably and meant to refer to the small fraction
of cells in bone marrow which can serve as stem-cell-like
precursors of osteocytes, chondrocytes, and adipocytes and which
can be isolated from bone marrow by their ability adhere to plastic
dishes. Stromal cells may be derived form any animal. In some
embodiments, stromal cells are derived from primates, preferably
humans.
[0012] As used herein, "diseases, disorders and conditions
characterized by a gene defect" is meant to refer to diseases,
disorders and conditions in which defective genes and/or
insufficient gene expression is causally linked to the disease or
symptoms. Individual who have any of several well known diseases,
disorders and conditions characterized by a gene defect can be
identified by those having ordinary skill in the art. Examples of
diseases, disorders and conditions characterized by a gene defect
include, but are not limited to, growth hormone deficiency,
diabetes, adenine deaminase deficiency, hemophilia A and hemophilia
B. The methods and means for diagnosing each such condition is well
known.
[0013] As used herein, the term "disease, disorder or condition
characterized by a bone, cartilage or lung defect" is meant to
refer to diseases, disorders and conditions which are caused by a
genetic mutation in a gene that is expressed by bone cells, cells
which make cartilage or lung cells such that one of the effects of
such a mutation is manifested by abnormal structure and/or function
of the bone, cartilage and lungs respectively.
[0014] As used herein, the term "disease, disorder or condition
characterized by a defect in the dermis, blood vessels, heart and
kidney" is meant to refer to diseases, disorders and conditions
which are caused by a genetic mutation in a gene that is expressed
by cells of the dermis, blood vessels, heart and kidney such that
one of the effects of such a mutation is manifested by abnormal
structure and/or function of the dermis, blood vessels, heart and
kidney respectively. Examples of diseases, disorders and conditions
characterized by a defect in the dermis includes burns, bed sores
and diabetic ulcers.
[0015] As used herein, "diseases, disorders and conditions
characterized by a gene defect of a gene which encodes a secreted
protein" is meant to refer to diseases, disorders and conditions
characterized by a gene defect in which the gene that is defective
genes or insufficiently expressed encodes a protein that is
normally secreted.
[0016] As used herein, "diseases, disorders and conditions which
can be treated with beneficial proteins" is meant to refer to
diseases, disorders and conditions that can be treated or prevented
by the presence of a protein which alleviates, reduces, prevents or
causes to be alleviated, reduced or prevented, the causes and/or
symptoms that characterize the disease, disorder or condition.
Diseases, disorders and conditions which can be treated by with
proteins includes diseases, disorders and conditions characterized
by a gene defect as well as those which are not characterized by a
gene defect but which nonetheless can be treated or prevented by
the presence of a protein which alleviates, reduces, prevents or
causes to be alleviated, reduced or prevented, the causes and/or
symptoms that characterize the disease, disorder or condition.
[0017] As used herein, "immunologically isolated", "immunologically
protected", "immunologically neutralized", and "a manner that
physically isolates them from the recipient's immune system" are
meant to refer to the encapsulation, containment or other physical
separation of an implanted cell from the body into which it is
implanted such that the cell is not exposed to and cannot be
eliminated by the immune system of the body such that cells which
are immunologically isolated are administered in a manner that
physically isolates them from the recipient's immune system.
Examples of immunological isolation means include, but are not
limited by, well known technologies and devices such as
microencapsulation, biocompatible matrices, diffusion chambers,
implantable cartridges, implant devices with membrane assemblies
and other containers with membranes. It is preferred that cells are
immunologically isolated by maintaining them with implant
devices.
[0018] As used herein, "beneficial protein" and "heterologous
protein" are interchangeable and are meant to refer to 1) proteins
which can compensate for the protein encoded by defective genes
and/or insufficient gene expression that is causally linked to the
disease or symptoms in diseases, disorders and conditions
characterized by a gene defect and 2) proteins whose presence
alleviates, reduces, prevents or causes to be alleviated, reduced
or prevented, the causes and/or symptoms that characterize
diseases, disorders and conditions which can be treated with
beneficial proteins:
[0019] As used herein, "gene construct" is meant to refer to
foreign recombinant nucleic acid molecules which include coding
sequences that encode beneficial proteins operably linked to
regulatory elements sufficient for expression of the coding
sequence in stromal cells.
[0020] As used herein, "foreign recombinant nucleic acid molecules"
is meant to refer to recombinant nucleic acid molecules which
either are not present in stromal cells or are not expressed as
proteins in sufficiently high levels in stromal cells until they
are introduced into the cell by means such as but not limited to
classical transfection (CaPO.sub.4 or DEAE dextran),
electroporation, microinjection, liposome-mediated transfer,
chemical-mediated transfer, ligand mediated transfer or recombinant
viral vector transfer.
[0021] As used herein, "heterologous gene" is meant to refer to the
coding sequence of the gene construct.
[0022] As used herein, the terms "exogenous genetic material" and
"exogenous gene" are used interchangeably and meant to refer to
genomic DNA, cDNA, synthetic DNA and RNA, mRNA and antisense DNA
and RNA which is introduced into the stromal cell. The exogenous
genetic material may be heterologous or an additional copy or
copies of genetic material normally found in the individual or
animal. When cells are used as a component of a pharmaceutical
composition in a method for treating human diseases, conditions or
disorders, the exogenous genetic material that is used to transform
the cells may encode proteins selected as therapeutics used to
treat the individual and/or to make the cells more amenable to
transplantation.
[0023] As used herein, "transfected stromal cells" is meant to
refer to stromal cells to which a gene construct has been provided
through any technology used to introduce foreign nucleic acid
molecules into cells such as, but not limited to, classical
transfection (CaPO.sub.4 or DEAE dextran), electroporation,
microinjection, liposome-mediated transfer, chemical-mediated
transfer, ligand mediated transfer or recombinant viral vector
transfer.
[0024] Some aspects of the present invention arise from the
discovery that some stromal cells which are introduced into a
patient develop into bone, cartilage and lung while others remain
precursors cells which throw off daughter cells that develop into
bone, cartilage and lung. This discovery allows for the successful
treatment of individuals suffering from diseases, conditions and
disorders associated with defects in bone cartilage or lung cells
by either providing such individuals with stromal cells from a
normal, matched syngeneic donor or by isolating stromal cells from
the patient, culturing them and genetically modifying them to
correct whatever genetic defect is responsible for the diseases,
conditions and disorders associated with defects in bone, cartilage
or lung cells. Similarly, it is believed that stromal cells will
also develop into cells of the dermis, blood vessels, heart and
kidneys, or throw off daughter cells that will do so.
[0025] The discovery that isolated, cultured stromal cells
repopulate tissue, particularly bone, cartilage and lung tissue,
when administered into the bloodstream of an individual makes them
suited for treating individuals suffering from diseases, conditions
and disorders associated with defects in bone, cartilage or lung
cells. The discovery that some isolated, cultured stromal cells,
when administered into the blood stream of an individual, act as
precursor cells which produce daughter cells that then mature into
differentiated cells makes the invention particularly useful
because it allows for the long term and continued presence of
donated normal or genetically modified cells without the need for
continuous readministration of cells. Similarly, the development of
stromal cells into cells of the dermis, blood vessels, heart and
kidneys, or throw off daughter cells allows for the treatment of
diseases effecting those tissues by similar means.
[0026] Accordingly, stromal cells from a matched donor may be
administered intravenously to individuals suffering from diseases
involving bone, cartilage or lung cells, or dermis, blood vessel,
heart or kidney cells, in order to augment or replace the
individual's bone, cartilage or lung cells, or dermis, blood
vessel, heart or kidney cells. Stromal cells from a matched donor
may be administered intravenously to individuals suffering from
diseases associated with defective gene expression in bone,
cartilage or lung cells, or dermis, blood vessel, heart or kidney
cells, in order to replace the individual's bone, cartilage or lung
cells, or dermis, blood vessel, heart or kidney cells, that don't
express or under express a normal gene and/or express a mutated
scene. Stromal cells may also be transfected with heterologous
genes in gene therapy protocols. According to such aspects of the
invention, matched donor stromal cells or stromal cells from an
individual may be removed and genetically altered prior to
reintroducing the cells into the individual. The cells may be
genetically altered to introduce a gene whose expression has
therapeutic effect on the individual. According to some aspects of
the invention, stromal cells from an individual may be genetically
altered to replace a defective gene and/or to introduce a gene
whose expression has therapeutic effect on the individual.
[0027] In some aspects of the invention, individuals suffering from
diseases and disorders that affect bone and that are characterized
by a genetic defect may be treated by supplementing, augmenting
and/or replacing defective or deficient bone cells with cells that
correctly express a normal gene. The cells may be derived from
stromal cells of a normal matched donor or stromal cells from the
individual to be treated. If derived from the individual to be
treated, the cells may be genetically modified to correct the
defect. An example of a disease or disorder that affects bone and
that is characterized by a genetic defect is osteogenesis
imperfecta. Another example of a disease or disorder that affects
bone and that is characterized by a genetic defect is osteoporosis.
Osteoporosis is frequently regarded as a multifactorial disease to
which environmental factors such as diet and exercise contribute.
However, studies of disease in twins, large families and large
populations demonstrate that many individuals develop the disease
primarily because of a genetic defect (see Morrison at al. Nature
367:284-287 (1994)). Individuals suffering from osteogenesis
imperfecta may be administered stromal cells from a normal matched
donor which replace the bone cells in the individual which have a
mutated collagen gene. In such embodiments, the normal cells will
compensate for the defective cells. In some embodiments, the normal
cells may be prepared from the individual's own stromal cells,
since cells with a mutated collagenous defect have a growth
disadvantage compared to normal cells when grown in culture.
Therefore, if stromal cells from an individual with osteogenesis
imperfecta are grown as culture, they will gradually become
enriched for normal cells. This embodiment will be particularly
effective if the individual is a mosaic for the mutated collagen so
that some of his or her cells contained the mutated collagen gene
and others do not. In an alternative embodiment, stromal cells are
isolated from an individual suffering from osteogenesis imperfecta
and a normal gene for collagen I is inserted into the isolated
stromal cells. The transfected cells are then reintroduced into the
individual. A few individuals suffering from osteoporosis also have
mutations in one of the two genes for collagen I and the same
embodiments, will compensate for the defective cells. In most
individuals with osteoporosis, the genes at fault are still unknown
but are likely to be identified soon. In such individuals, normal
cells will compensate for the defect. Also, when the genes at fault
are identified and isolated, an alternative embodiment will be to
isolate stromal cells from the individual, insert normal copy or
copies of the mutated gene, and reintroduce the cells to the
individual.
[0028] In some aspects of the invention, individuals suffering from
diseases and disorders that affect cartilage and that are
characterized by a genetic defect can be treated by supplementing,
augmenting and/or replacing defective cells with cells that
correctly express a normal gene. The cells may be derived from
stromal cells of a normal matched donor or stromal cells from the
individual to be treated. If derived from the individual to be
treated, the cells may be genetically modified to correct the
defect. An example of a disease or disorder that affects cartilage
and that is characterized by a genetic defect is chondrodysplasia
which cause severe dwarfism, severe problems with joints and
related problems. Individuals suffering from chondrodysplasia may
be administered stromal cells from a normal matched donor which
replace the cells that produce cartilage in the individual which
have a mutated collagen gene. In such embodiments, the normal cells
will compensate for the defective cells. In an alternative
embodiment, stromal cells are isolated from an individual suffering
from chondrodysplasia and a normal gene for collagen II is inserted
into the isolated stromal cells. The transfected cells are then
reintroduced into the individual. The embodiment with the collagen
II gene will be useful for the 20% to 90% of individuals with
various types of severe chondrodysplasia. The remaining individuals
with chondrodysplasia have mutations in other collagen genes
(collagen X and X1), in other genes (fibroblast growth factor
receptor 3), and in still unidentified genes. In such individuals,
normal cells will compensate for the defective cells. Also, an
alternative embodiment will be to isolate stromal cells from the
individual, insert a normal copy or copies of the mutated gene, and
reintroduce the cells to the individual. Another example of a
disease or disorder that affects cartilage is osteoarthritis.
Osteoarthritis is a heterogeneous disease both in terms of etiology
and manifestations. Some individuals develop the degeneration of
cartilage in joints that characterize osteoarthritis because of
trauma or the late sequelae of infections. A few individuals
develop osteoarthritis in multiple joints because of mutations in
the gene for collagen II similar to the mutations in the gene that
cause chondrodysplasia. Such individuals may or may not show signs
of a mild chondrodysplasia. The cause of osteoarthritis in other
individuals is unknown, but studies in large families suggest that
the disease is inherited and therefore caused mutations is still
unidentified genes. Therefore the same embodiments that will be
useful to compensate for mutated genes in individuals with
chondrodysplasia will also be useful for many individuals with
osteoarthritis.
[0029] In some aspects of the invention, individuals suffering from
diseases and disorders that affect the lungs and that are
characterized by a genetic defect can be treated by supplementing,
augmenting and/or replacing defective cells with cells that
correctly express a normal gene. The cells may be derived from
stromal cells of a normal matched donor or stromal cells from the
individual to be treated. If derived from the individual to be
treated, the cells may be genetically modified to correct the
defect. An example of a disease or disorder that affects the lungs
and that is characterized by a genetic defect is cystic fibrosis.
Another example of a disease or disorder that affects the lungs and
that is characterized by a genetic defect is a deficiency of
.alpha.1-antitrypsin. Individuals suffering from cystic fibrosis
may be administered stromal cells from a normal-matched donor which
have a norma cystic fibrosis to replace or supplement the lungs
cells in the individual which have a mutated cystic fibrosis gene.
In such embodiments, the normal cells will compensate for the
defective cells. In an alternative embodiment, stromal cells are
isolated from an individual suffering from cystic fibrosis and a
normal cystic fibrosis gene is inserted into the isolated stromal
cells. The transfected cells are then reintroduced into the
individual.
[0030] In some aspects, individuals suffering from diseases of the
bone, cartilage and lungs as well as those suffering from diseases
of the dermis, blood vessels, heart and kidneys can be treated by
isolated stromal cells, expanding their number and systemically
administering the expanded, rejuvenated stromal cells. Some of the
rejuvenated stromal cells will develop into normal bone, cartilage,
lung, dermis, blood vessel, heart or kidney cells. Normal stromal
cells expand more quickly the defective ones and the expanded
rejuvenated population will reflect a greater proportion of normal
cells. Table 1 describes media useful to culture expanded
rejuvenated cultures of isolated stromal cells.
[0031] In addition to replacing cells that are defective with
repaired cells or normal cells from matched donors, the invention
may also be used to express desired proteins that are secreted.
That is, stromal cells may be isolated, furnished with a gene for a
desired protein and introduced into an individual within whom the
desired protein would be produced and exert or otherwise yield a
therapeutic effect. This aspect of the invention relates to gene
therapy in which therapeutic proteins are administered to an
individual.
[0032] According to some aspects of the present invention,
immunologically isolated transfected stromal cells are used as cell
therapeutics to treat diseases, disorders and conditions
characterized by a gene defect and/or diseases, disorders and
conditions which can be treated with proteins. In particular, gene
constructs that comprise heterologous genes which encode beneficial
proteins are introduced into stromal cells. The transfected stromal
cells are then immunologically isolated and implanted into an
individual who will benefit when the protein is expressed and
secreted by the cell into the body.
[0033] Immunologically isolated stromal cells are particularly
useful in cell therapeutic compositions, because in addition to
being suitable hosts for expressing heterologous genes and
producing heterologous proteins, stromal cells perform favorably
when they are immunologically isolated. Immunologically isolated
stromal cells have a very high viability when implanted in
locations that lack a direct vascular blood supply. Moreover,
stromal cells can be easily and readily obtained, they rapidly
expand in culture making them a good source of an adequate supply
of useful cells for immunologically isolated cell therapeutics.
[0034] According to the present invention, gene constructs which
comprise nucleotide sequences that encode heterologous proteins are
introduced into stromal cells. That is, the cells are genetically
altered to introduce a gene whose expression has therapeutic effect
on the individual. According to some aspects of the invention,
stromal cells from an individual or from another individual or from
a non-human animal may be genetically altered to replace a
defective gene and/or to introduce a gene whose expression has
therapeutic effect on the individual.
[0035] According to the present invention, stromal cells are useful
to prepare transfected cells that can be immunologically isolated
and express heterologous beneficial genes provides the means to
correct genetic defects and/or to produce therapeutic proteins.
Stromal cells may be isolated with relative ease and isolated
stromal cells may be cultured to increase the number of cells
available. Stromal cells can be transfected, immunologically
isolated and implanted with a high degree of viability into
locations that lack direct blood supply such as subcutaneous
locations. In some embodiments, stromal cells may immortalized such
as by SV40 virus or proteins with transforming properties.
[0036] In some aspects of the invention, individuals suffering from
genetic diseases and disorders may be treated by supplementing,
augmenting and/or replacing defective or deficient genes by
providing immunologically isolated stromal cells containing gene
constructs that include normal, functioning copies of the deficient
gene. This aspect of the invention relates to gene therapy in which
the individual is provided with genes for which they are deficient
in presence and/or function. The genes provided in the cell
therapeutic compensate for the defective gene of the individual.
Such genes preferably encode proteins that are secreted.
[0037] The stromal cells are transfected and immunologically
isolated. In some embodiments, stromal cells are transfected with
genes for which the individual to be treated suffers from a
complete absence of a non-mutated copy of the gene, or suffers from
an absence or insufficient expression of a non-mutated form of the
protein. Stromal cells are transfected with a non-mutated copy of
the gene in an expressible form. That the protein encoded by the
transfected gene will be expressed by the stromal cells, preferably
as a secreted protein. Examples of diseases, conditions or
disorders in which defective genes or insufficient gene expression
is causally linked to the disease or symptoms include, but are not
limited to, growth hormone deficiency, diabetes, adenine deaminase
deficiency, hemophilia A and hemophilia B. Other genetic diseases
which may be treated using methods of the invention include:
.alpha..sub.1-antitrypsin deficiency, Fabray disease, familial
hypercholesterolemia, Gaucher's disease, Lesch-Nyhan Syndrome,
Maple syrup urine disease, Ornithine transcarbamylase deficiency,
phenylketonuria, Sandhoff disease, Tay-Sachs disease and von
Willebrand disease. By introducing, normal genes in expressible
form which encode, growth hormone, insulin, adenine deaminase or an
appropriate blood clotting factor, individuals suffering from
growth hormone deficiency, diabetes, adenine deaminase deficiency,
and hemophilia, respectively, can be provided the means to
compensate for genetic defects and eliminate, alleviate or reduce
some or all of the symptoms associated with such diseases. Tables
IV and V contain partial lists of diseases, conditions and
disorders which can be treated using the present invention.
[0038] In addition to replacing genes that are defective with
functional genes, the invention may also be used to express desired
secreted proteins which exert a biologically active therapeutic or
prophylactic effect. Such proteins are preferably secreted by the
cells. That is, stromal cells may be isolated, furnished with a
gene for a desired protein, immunologically isolated and introduced
into an individual within whom the desired protein would be
produced and exert or otherwise yield a therapeutic effect. This
aspect of the invention relates to gene therapy in which
therapeutic proteins are administered to an individual. According
to these aspects of the invention, the isolated stromal cells are
vectors for introducing therapeutic genes into the individual as
well as hosts for such genes when the cells are administered to the
individual.
[0039] In such embodiments, stromal cells are transfected with
genes that encode proteins which will have a therapeutic effect
when expressed in the individual to be treated. Rather than
administering the therapeutic protein directly and at a series of
time intervals, the present invention provides a means of
administering a therapeutic protein continuously by administering
cells which produce the protein. Stromal cells are transfected with
a gene that encodes the protein in an expressible form. That is,
the protein encoded by the transfected gene will be expressed by
the stromal cells, preferably as a secreted protein. Examples of
therapeutic proteins include, but are not limited to, obesity
factor (Considine, R. V. et al., J. Clin. Invest., 1995, 95,
2986-2988; Arner, P., N. Engl. J. Med., 1995, 333, 382; Emorine, L.
et al., Trends Pharmacol. Sci., 1994, 15, 3; Flier, J. S., Cell,
1995, 80, 15; Lowell, E. B., et al., J. Clin. Invest., 1995, 95,
923; and Rink, T. J. et al., Nature, 1994, 372, 406)
granulocyte-macrophage colony stimulating factor, granulocyte
colony stimulating factor, erythropoietin, interleukin-2 and
interleukin-1 receptor antagonist protein. Tables IV and V contain
partial lists of diseases, conditions and disorders which can be
treated using the present invention contains the names of the
proteins which can be delivered via gene construct according to the
invention.
[0040] In all cases in which a gene construct is transfected into a
stromal cell, the heterologous gene is operably linked to
regulatory sequences required to achieve expression of the gene in
the stromal cell. Such regulatory sequences include a promoter and
a polyadenylation signal.
[0041] The gene construct is preferably provided as en expression
vector which includes the coding sequence for a heterologous
protein operably linked to essential regulatory sequences such that
when the vector is transfected into the cell, the coding sequence
will be expressed by the cell. The coding sequence is operably
linked to the regulatory elements necessary for expression of that
sequence in the cells. The nucleotide sequence that encodes the
protein may be cDNA, genomic DNA, synthesized DNA or a hybrid
thereof or an RNA molecule such as mRNA.
[0042] The gene construct includes the nucleotide sequence encoding
the beneficial protein operably linked to the regulatory elements
may remain present in the cell as a functioning cytoplasmic
molecule, a functioning episomal molecule or it may integrate into
the cell's chromosomal DNA. Exogenous genetic material may be
introduced into cells where it remains as separate genetic material
in the form of a plasmid. Alternatively, linear DNA which can
integrate into the chromosome may be introduced into the cell. When
introducing DNA into the cell, reagents which promote DNA
integration into chromosomes may be added. DNA sequences which are
useful to promote integration may also be included in the DNA
molecule. Alternatively, RNA may be introduced into the cell.
[0043] The regulatory elements necessary for gene expression
include: a promoter, an initiation codon, a stop codon, and a
polyadenylation signal. It is necessary that these elements be
operable in the stromal cells or in cells that arise from the
stromal cells after infusion into an individual. Moreover, it is
necessary that these elements be operably linked to the nucleotide
sequence that encodes the protein such that the nucleotide sequence
can be expressed in the stromal cells and thus the protein can be
produced. Initiation codons and stop codon are generally considered
to be part of a nucleotide sequence that encodes the protein.
However, it is necessary that these elements are functional in the
stromal cells or cells that arise from stromal cells. Similarly,
promoters and polyadenylation signals used must be functional
within the stromal cells or cells that arise from stromal cells.
Examples of promoters useful to practice the present invention
include but are not limited to promoters that are active in many
cells such as the cytomegalic virus promoter, SV40 promoters and
retroviral promoters. Other examples of promoters useful to
practice the present invention include but are not limited to
tissue-specific promoters, i.e. promoters that function in some
tissues but not in others; also, promoters of genes normally
expressed in stromal cells with or without specific or general
enhancer sequences. In some embodiments, promoters are used which
constitutively express genes in stromal cells with or without
enhancer sequences. Enhancer sequences are provided in such
embodiments when appropriate or desirable
[0044] According to some embodiments, desired genes for
transfection into stromal cells are operably linked to the human
procollagen I promoter, human procollagen II promoter, and the
human procollagen III promoter. In some embodiments, the genes are
linked to relatively short 5'-fragments from either the COL1A1 or
COL2A1 gene which comprise the promoter together with the some of
the 5' translated and/or untranslated sequences of the gene. In
some embodiments, the gene to be transfected is operably linked to
a sequence that contains a 1.9 kb SphI-HindIII fragment from the
5'-end of the human COL1A1. The fragment contains from -476 bp to
+1440 bp of COL1A1 gene and, therefore, includes the promoter (476
bp), exon 1 (222 bp) and most of the intron 1 (1223 bp of a total
of 1453 bp). In some embodiments, the gene to be transfected is
operably linked to a sequence that contains a fragment from the
5'-end of the human COL2A1. In some embodiments, the fragment
contains -4.0 kb of the COL2A1 promoter and the complete COL2A1
gene the one or more exons and introns sequentially from axon 1 to
exon 15 and intron 1 to intron 14. Some constructs may be designed
as taught in co-pending U.S. Ser. No. 08/184,260 filed Jan. 18,
1994 entitled "Methods of targeting DNA insertion into genome",
which is incorporated herein by reference.
[0045] Examples of polyadenylation signals useful to practice the
present invention includes but is not limited to human collagen I
polyadenylation signal, human collagen II polyadenylation signal,
and SV40 polyadenylation signal.
[0046] In order for exogenous genetic material in an expression
vector to be expressed, the regulatory elements must be operably
linked to the nucleotide sequence that encodes the protein. In
order to maximize protein production, regulatory sequences may be
selected which are well suited for gene expression in the desired
cells. Moreover, codons may be selected which are most efficiently
transcribed in the cell. One having ordinary skill in the art can
produce exogenous genetic material as expression vectors which are
functional in the desired cells.
[0047] It is also contemplated that regulatory elements may be
selected to provide tissue specific expression of the protein.
Thus, for example, specific promoters may be provided such that the
heterologous gene will only be expressed in tissue where the
immunologically isolated stromal cells are implanted.
[0048] The heterologous protein preferably includes a signal
sequence which directs the transport and secretion of the
heterologous protein in the stromal cell. The signal sequences is
generally processed and removed upon secretion of the mature
protein from the cell.
[0049] In addition to providing cells with genetic material that
either 1) corrects genetic defects in the cells, 2) encodes
proteins which are otherwise not present in sufficient quantities
and/or functional condition so that the genetic material corrects
genetic defects of the individual, and/or 3) encodes proteins which
are useful as therapeutics in the treatment or prevention of a
particular disease condition or disorder or symptoms associated
therewith, genetic material may also be introduced into the stromal
cells used in the present invention to provide a means for
selectively terminating such cells should such termination become
desirable. Such means for targeting cells for destruction may be
introduced into stromal cells which are to be otherwise genetically
modified as well as those to which no other exogenous genetic
material is to be introduced.
[0050] According to the invention, isolated stromal cells are
furnished with genetic material which renders them specifically
susceptible to destruction. For example, the stromal cells may be
provided with genes that encode a receptor that can be specifically
targeted with a cytotoxic agent. An expressible form of a gene that
can be used to induce selective cell death can introduced into the
cells. In such a system, cells expressing the protein encoded by
the gene are susceptible to targeted killing under specific
conditions or in the presence or absence of specific agents. For
example, an expressible form of a herpes virus thymidine kinase
(herpes tk) gene can be introduced into the cells and used to
induce selective cell death. When the exogenous genetic material
that includes (herpes tk) gene is introduced into the individual,
herpes tk will be produced. If it is desirable or necessary to kill
the implanted cells, the drug gangcyclovir can be administered to
the individual and that drug will cause the selective killing of
any cell producing herpes tk. Thus, a system can be provided which
allows for the selective destruction of implanted cells.
[0051] Those having ordinary skill in the art can identify
individuals suffering from genetic diseases such as those listed in
Tables IV and V including growth hormone deficiency, diabetes,
adenine deaminase deficiency and hemophilia, routinely using
standard diagnostic procedures.
[0052] Stromal cells may be obtained by removing bone marrow cells
from a donor and placing the cells in a sterile container with a
plastic surface or other appropriate surface that the cells come
into contact with. The stromal cells will adhere to the plastic
surface within 30 minutes to about 3 days. After at least 30
minutes, preferably about four hours, the non-adhered cells may be
removed and discarded. The adhered cells are stromal cells which
are initially non-dividing. After about 2-4 days however the cells
begin to proliferate and can be cultured to increase their numbers
using standard cell culture techniques.
[0053] According to preferred embodiments, stromal cells are
cultured in medium supplemented with 2-20% fetal calf serum or
serum-free medium with or without additional supplements.
[0054] Isolated stromal cells may be transfected using well known
techniques readily available to those having ordinary skill in the
art. Foreign genes may be introduced into stromal bells by standard
methods are employed for introducing gene constructs into cell
which will express the proteins encoded by the genes. In some
embodiments, cells are transfected by calcium phosphate
precipitation transfection, DEAE dextran transfection,
electroporation, microinjection, liposome-mediated transfer,
chemical-mediated transfer, ligand mediated transfer or recombinant
viral vector transfer.
[0055] In some embodiments, recombinant adenovirus vectors are used
to introduce DNA with desired sequences into the stromal cell. In
some embodiments, recombinant retrovirus vectors are used to
introduce DNA with desired sequences into the stromal cell. In some
embodiments, standard CaPO.sub.4, DEAE dextran or lipid carrier
mediated transfection techniques are employed to incorporate
desired DNA into dividing cells. Standard antibiotic resistance
selection techniques can be used to identify and select transfected
cells. In some embodiments, DNA is introduced directly into cells
by microinjection. Similarly, well known electroporation or
particle bombardment techniques can be used to introduce foreign
DNA into isolated stromal cells. A second gene is usually
co-transfected or linked to the therapeutic gene. The second gene
is frequently a selectable antibiotic-resistance gene. Transfected
cells can be selected by growing the cells in an antibiotic that
will kill cells that do not take up the selectable gene. In most
cases where the two genes are unlinked and co-transfected, the
cells that survive the antibiotic treatment have both genes in them
and express both of them.
[0056] After isolating the stromal cells, the cells can be
administered upon isolation or after they have been cultured.
Isolated stromal cells administered upon isolation are administered
within about one hour after isolation. Generally, stromal cells may
be administration immediately upon isolation in situations in which
the donor is large and the recipient is an infant. It is preferred
that stromal cells are cultured prior to administrations. Isolated
stromal cells can be cultured from 1 hour to over a year. In some
preferred embodiments, the isolated stromal are cultured prior to
administration for a period of time sufficient to allow them to
convert from non-cycling to replicating cells. In some embodiments,
the isolated stromal cells are cultured for 3-30 days, preferably
5-14 days, more preferably 7-10 days. In some embodiments, the
isolated stromal cells are cultured for 4 weeks to a year,
preferably 6 weeks to 10 months, more preferably 3-6 months.
[0057] If the cells are transfected, either 1) isolated,
non-cycling stromal cells are first transfected and then
administered as non-cycling cells, 2) isolated, non-cycling stromal
cells are first transfected, then cultured for a period of time
sufficient to convert from non-cycling to replicating cells and
then administered, 3) isolated, non-cycling stromal cells are first
cultured for, a period of time sufficient to convert from
non-cycling to replicating cells, then transfected, and then
administered, or 4) isolated, non-cycling stromal cells are first
cultured for a period of tithe sufficient to convert from
non-cycling to replicating cells, then transfected, then cultured
and then administered. In some embodiments, stromal cells are
isolated, transfected and immediately administered. It is preferred
that stromal cells are cultured prior to transfection and/or
administrations. Isolated stromal cells can be cultured from
cultured for 3-30 days, in some embodiments 5-14 days, in some
embodiments 7-10 days prior to transfection. Transfected stromal
cells can be cultured from cultured for 3-30 days, in some
embodiments 5-14 days, in some embodiments 7-10 days prior to
administration. Isolated stromal cells can be cultured from
cultured for 3-30 days, in some embodiments 5-14 days, in some
embodiments 7-10 days prior to transfection and upon transfection,
additionally cultured for 3-30 days, in some embodiments 5-14 days,
in some embodiments 7-10 days prior to administration. In some
embodiments, the isolated stromal cells are cultured for 4 weeks to
a year, in some embodiments 6 weeks to 10 months, in some
embodiments 3-6 months prior to transfection. Transfected stromal
cells can be cultured for 4 weeks to a year, in some embodiments 6
weeks to 10 months, in some embodiments 3-6 months prior to
administration. In some embodiments, the isolated stromal cells are
cultured for 4 weeks to a year, in some embodiments 6 weeks to 10
months, in some embodiments 3-6 months prior to transfection and
upon transfection, further cultured for 4 weeks to a year, in some
embodiments 6 weeks to months, in some embodiments 3-6 months prior
to administration.
[0058] Isolated stromal cells may be transfected using well known
techniques readily available to those having ordinary skill in the
art. In some embodiments, recombinant adenovirus vectors are used
to introduce DNA with desired sequences into the stromal cell. In
some embodiments, standard CaPO.sub.4, DEAE dextran or lipid
carrier mediated transfection techniques are employed to
incorporate desired DNA into dividing cells. Standard antibiotic
resistance selection techniques can be used to identify and select
transfected cells. In some embodiments, DNA is introduced directly
into cells by microinjection. Similarly, well known electroporation
or particle bombardment techniques can be used to introduce foreign
DNA into isolated stromal cells.
[0059] For administration of stromal cells, the isolated stromal
cells are removed from culture dishes, washed with saline,
centrifuged to a pellet and resuspended in a glucose solution which
is infused into the patient. In some embodiments, bone marrow
ablation is undertaken prior to infusion in order to make space in
the bone for introduced cells. Bone marrow ablation may be
accomplished by X-radiating the individual to be treated,
administering drugs such as cyclophosphamide or by a combination of
X-radiation and drug administration. In some embodiments, bone
marrow ablation is produced by administration of radioisotopes
known to kill metastatic bone cells such as, for example,
radioactive strontium, .sup.135Samarium or .sup.166Holmium (see
Applebaum, F. R. et al. 1992 Blood 80(6):1608-1613, which is
incorporated herein by reference).
[0060] If bone marrow ablation precedes administration of stromal
cells, the administration of stromal cells must be accompanied by
the administration of non-adherent cells which comprise blood cell
precursors necessary for survival. Such non-adherent cells may be
saved from the same sample used as starting materials in the
isolation of stromal cells and stored or they can be derived from a
different sample. In some preferred embodiments, the non-adherent
cells are provided by the recipient/patient. Prior to procedures
which generate bone marrow ablation, a sample of the
patient/recipients bone marrow is obtained and stored. The entire
sample may be used or the non-adherent cells may be isolated and
used to administer in conjunction with isolated stromal cells.
Non-adherent cells administered in conjunction with administration
of stromal cells may be administered separately before or after
stromal cell administration or may be mixed with isolated stromal
cells prior to administration.
[0061] Bone marrow ablation is optional. In some embodiments,
partial but not complete bone marrow ablation is produced prior to
administration of stromal cells. In some embodiments, stromal cells
are administered without any bone marrow ablation.
[0062] Between 10.sup.7 and 10.sup.13 cells per 100 kg person are
administered per infusion. In some embodiments, between about
1-5.times.10.sup.8 and 1-5.times.10.sup.12 cells are infused
intravenously per 100 kg person. In some embodiments, between about
1.times.10.sup.9 and 5.times.10.sup.11 cells are infused
intravenously per 100 kg person. In some embodiments,
4.times.10.sup.9 cells are infused per 100 kg person. In some
embodiments, 2.times.10.sup.11 cells are infused per 100 kg
person.
[0063] In some embodiments, a single administration of cells is
provided. In some embodiments, multiple administrations are
provided. In some embodiments, multiple administrations are
provided over the course of 3-7 consecutive days. In some
embodiments, 3-7 administrations are provided over the course of
3-7 consecutive days. In some embodiments, 5 administrations are
provided over the course of 5 consecutive days.
[0064] In some embodiments, a single administration of between
10.sup.7 and 10.sup.13 cells per 100 kg person is provided. In some
embodiments, a single administration of between about
1-5.times.10.sup.9 and 1-5.times.10.sup.12 cells per 100 kg person
is provided. In some embodiments, a single administration of
between about 1.times.10.sup.9 and 5.times.10.sup.11 cells per 100
kg person is provided. In some embodiments, a single administration
of 4.times.10.sup.9 cells per 100 kg person is provided. In some
embodiments, a single administration of 2.times.10.sup.11 cells per
100 kg person is provided.
[0065] In some embodiments, multiple administrations of between
10.sup.7 and 10.sup.13 cells per 100 kg person are provided, In
some embodiments, multiple administrations of between about
1-5.times.10.sup.8 and 1-5.times.10.sup.12 cells per 100 kg person
are provided. In some embodiments, multiple administrations of
between about 1.times.10.sup.9 and 5.times.10.sup.11 cells per 100
kg person are provided over the course of 3-7 consecutive days. In
some embodiments, multiple administrations of 4.times.10.sup.9
cells per 100 kg person are provided over the course of 3-7
consecutive days. In some embodiments, multiple administrations of
2.times.10.sup.11 cells per 100 kg person are provided over the
course of 3-7 consecutive days. In some embodiments, 5
administrations of 3-5.times.10.sup.9 cells are provided over the
course of 5 consecutive days. In some embodiments, 5
administrations of 4.times.10.sup.9 cells are provided over the
course of 5 consecutive days. In some embodiments, 5
administrations of 1-3.times.10.sup.11 cells are provided over the
course of 5 consecutive days. In some embodiments, 5
administrations of 2.times.10.sup.11 cells are provided over the
course of 5 consecutive days.
[0066] Stromal cells in diffusion chambers are described in
Benayahu, D. et al. (1989) J. Cell Physiol. 140:1-7 and Mardon, H.
J. et al. (1987) Cell Tissue Res. 250:157-165, which are both
incorporated herein by reference.
[0067] After introducing the gene construct into the stromal cells,
the cells can be immunologically isolated immediately or after they
have been cultured. Stromal cells can be implanted after they are
immunologically isolated. Stromal cells may be immunologically
isolated by any number of well known methods using readily
available starting materials and/or devices. Stromal cells may be
microencapsulated using many such microencapsulation protocols
including those disclosed, for example, in U.S. Pat. No. 4,391,909,
U.S. Pat. No. 4,806,355, U.S. Pat. No. 4,942,129, and U.S. Pat. No.
5,334,640.
[0068] Stromal cells may be administered in chambers with
diffusible membranes or encapsulated in microbeads. In another
embodiment, the stromal cells are contained in hollow fibers such
as those available from Amicon, Inc. (Beverly Mass.). These fibers
are used for example to make cartridges for dialysis. One end can
be pulled out from under the skin and reduced in size if dosages of
the protein made by the cells are to be reduced. The surface area
of the fibers is very high. Further, cells in the fiber can be
flushed out and replaced periodically. Hollow fibers are described
on pages 50-51 of Amicon, Inc. Publication No. 323 which is
incorporated herein by reference.
[0069] Similarly, incorporation of transfected stromal cells in
biocompatible matrices will allow for secretion of beneficial
protein to the individual while maintaining the cells in an
immunologically isolated condition. Examples of biocompatible
matrices are disclosed, for example, in U.S. Pat. No. 4,902,295 and
U.S. Pat. No. 4,997,443. In some embodiments, transfected stromal
cells are immunologically isolated by encasing them within tissue
implant systems that are membrane assemblies. That is, cells are
maintained in containers that include at least one porous membrane.
The cells within the membrane assembly are immunologically isolated
while beneficial proteins may be made available to the individuals
by passing through the membrane. Implant devices which are membrane
assemblies, include, but are not limited to, those described in
U.S. Pat. No. 5,314,471 and U.S. Pat. No. 5,344,454. According to
one embodiment of the invention, an implant device is provided
which comprises two ring assembles. Each ring assembly comprises a
circular plastic ring and a 0.3 micron millipore membrane covering
the area of the circle. Transfected stromal cells are disposed
between the two ring assembly which are connected to each other at
the circumference. The constructed implant device is preferably
implanted subcutaneously.
[0070] In some preferred implant devices, 10.sup.4 to 10.sup.11
cells are provided.
[0071] Immunologically isolated cells may be implanted
subcutaneously or intraperitoneally. Alternatively, they may be
attached or otherwise implanted adjacent to organs and tissues to
which the beneficial protein is preferably delivered. In preferred
embodiments, implant devices are implanted subcutaneously or
intraperitoneally.
[0072] The invention is particularly useful to treat those
diseases, disorder and conditions which require relatively small
quantities of protein, most preferably secreted protein. Examples
include growth factor, obesity factor and factor IX which each
require very small amount of protein in order to function. Tables
IV and V contain partial lists of diseases, conditions and
disorders which can be treated using the present invention.
[0073] It is preferred that stromal cells are cultured prior to
immunological isolation. Stromal cells can be cultured from 1 hour
to over a year. In some preferred embodiments, the stromal cells
are cultured for a period of time sufficient to allow them to
convert from non-cycling to replicating cells. In some embodiments,
the stromal cells are cultured for 3-30 days, preferably 5-14 days,
more preferably 7-10 days. In some embodiments, the stromal cells
are cultured for 4 weeks to a year, preferably 6 weeks to 10
months, more preferably 3-6 months.
[0074] In preferred embodiments, cells are either 1) isolated,
non-cycling stromal cells that are first transfected and then
immunologically isolated, then implanted as non-cycling cells, 2)
isolated, non-cycling stromal cells that are first transfected,
then cultured for a period of time sufficient to convert from
non-cycling to replicating cells, then immunologically isolated and
then implanted, 3) isolated, non-cycling stromal cells that are
first cultured for a period of time sufficient to convert from
non-cycling to replicating cells, then transfected, then
immunologically isolated and then implanted, or 4) isolated,
non-cycling stromal cells that are first cultured for a period of
time sufficient to convert from non-cycling to replicating cells,
then transfected, then cultured, then immunologically isolated and
then implanted. In some embodiments, stromal cells are isolated,
transfected, immunologically isolated and implanted. It is
preferred that stromal cells are cultured prior to and after
transfection. Isolated stromal cells can be cultured from cultured
for 3-30 days, in some embodiments 5-14 days, in some embodiments
7-10 days prior to transfection. Transfected stromal cells can be
cultured from cultured for 3-30 days, in some embodiments 5-14
days, in some embodiments 7-10 days prior to administration.
Isolated stromal cells can be cultured from cultured for 3-30 days,
in some embodiments 5-14 days, in some embodiments 7-10 days prior
to transfection and upon transfection, additionally cultured for
3-30 days, in some embodiments 5-14 days, in some embodiments 7-10
days prior to administration. In some embodiments, the isolated
stromal cells are cultured for 4 weeks to a year, in some
embodiments 6 weeks to 10 months, in some embodiments 3-6 months
prior to transfection. Transfected stromal cells can be cultured
for 4 weeks to a year, in some embodiments 6 weeks to 10 months, in
some embodiments 3-6 months prior to implantation. In some
embodiments, the isolated stromal cells are cultured for 4 weeks to
a year, in some embodiments 6 weeks to 10 months, in some
embodiments 3-6 months prior to transfection and upon transfection,
further cultured for 4 weeks to a year, in some embodiments 6 weeks
to 10 months, in some embodiments 3-6 months prior to
implantation.
[0075] Another aspect of the present invention relates to methods
of treating patients who are suffering from a disease, disorder or
condition characterized by a bone or cartilage defect. The method
comprises the steps of identifying an individual with a bone or
cartilage defect, obtaining a bone marrow sample from a normal,
matched, syngeneic donor, and, administering said bone marrow
sample to the patient by intravenous infusion.
[0076] As stated above, the bone marrow sample for transplantation
may be derived from a matched donor. Those having ordinary skill in
the art can readily identify matched donors using standard
techniques and criteria.
[0077] Bone marrow samples for transplantation may be obtained from
matched donors by standard techniques. In some embodiments, bone
marrow ablation is undertaken prior to infusion in order to make
space in the bone for introduced cells. Bone marrow ablation may be
accomplished by X-radiating the individual to be treated,
administering drugs such as cyclophosphamide or by a combination of
X-radiation and drug administration. In some embodiments, bone
marrow ablation is produced by administration of radioisotopes
known to kill metastatic bone cells such as, for example,
radioactive strontium, .sup.135Samarium or .sup.166Holmium (see
Applebaum, F. R. et al. 1992 Blood 80(6):1608-1613, which is
incorporated herein by reference).
[0078] Bone marrow ablation is optional. In some embodiments,
partial but not complete bone marrow ablation is produced prior to
bone marrow transplantation. In some embodiments, bone marrow is
administered without any bone marrow ablation.
[0079] It is preferred that the number of cells used in bone marrow
transplantation for treating bone and cartilage disease exceed that
which is normally used for other treatments. Between 3 to 10 times
the normal bone marrow dosage per 100 kg person are administered
per infusion.
[0080] In some embodiments, a single administration of cells is
provided. In some embodiments, multiple administrations are
provided. In some embodiments, multiple administrations are
provided over the course of 3-7 consecutive days. In some
embodiments, 3-7 administrations are provided over the course of
3-7 consecutive days. In some embodiments, 5 administrations are
provided over the course of 5 consecutive days.
[0081] In some embodiments, a single administration of between
10.sup.7 and 10.sup.13 cells per 100 kg person is provided, In some
embodiments, a single administration of between about
1-5.times.10.sup.8 and 1-5.times.10.sup.12 cells per 100 kg person
is provided. In some embodiments, a single administration of
between about 1.times.10.sup.9 and 5.times.10.sup.11 cells per 100
kg person is provided. In some embodiments, a single administration
of 4.times.10.sup.9 cells per 100 kg person is provided. In some
embodiments, a single administration of 2.times.10.sup.11 cells per
100 kg person is provided.
[0082] In some embodiments, multiple administrations of between 10
and 10 cells per 100 kg person are provided, In some embodiments,
multiple administrations of between about 1-5.times.10.sup.8 and
1-5.times.10.sup.12 cells per 100 kg person are provided. In some
embodiments, multiple administrations of between about
1.times.10.sup.9 and 5.times.10.sup.1i cells per 100 kg person are
provided over the course of 3-7 consecutive days. In some
embodiments, multiple administrations of 4.times.10.sup.8 cells per
100 kg person are provided over the course of 3-7 consecutive days.
In some embodiments, multiple administrations of 2.times.10.sup.11
cells per 100 kg person are provided over the course of 3-7
consecutive days. In some embodiments, 5 administrations of
3-5.times.10.sup.8 cells are provided over the course of 5
consecutive days. In some embodiments, 5 administrations of
4.times.10.sup.1 cells are provided over the course of 5
consecutive days. In some embodiments, 5 administrations of
1-3.times.10.sup.11 cells are provided over the course of 5
consecutive days. In some embodiments, 5 administrations of
2.times.10.sup.11 cells are provided over the course of 5
consecutive days.
EXAMPLES
Example 1
[0083] Cells from a transgenic mouse line that expresses a human
mini-gene for collagen I in a tissue-specific manner were used to
see whether precursor mesenchymal cells from marrow that are
expanded in culture can serve as long-term precursors of bone and
other connective tissues after intravenous infusion into irradiated
mice. The marker gene consisted of an internally deleted mini-gene
for the human pro.alpha.1(I) chain of procollagen I that caused
synthesis of shortened pro.alpha.1(I) chains (Khillan, J. S. et
al., J. Biol. Chem. 266:23373-23379 (1991); Pereira, R. et al., J.
Clin. Invest. 91:709-716 (1983); and Sokolov, B. P. et al.,
Biochemistry 32:9242-9249 (1993)) which are incorporated herein by
reference. Cells expressing the gene were obtained from a line of
transgenic mice in which the copy number of the human mini-gene
relative to the endogenous mouse gene was about 100 to 1, and the
steady-state levels of mRNA from the human mini-gene relative to
mRNA from the endogenous mouse gene was about 0.5:1 in most
tissues.
[0084] Donor cells from marrow partially enriched for mesenchymal
precursors were prepared by standard protocols (Friedenstein, A. J.
et al., Exp. Hemet. 4:267-274 (1976); Castro-Malaspina, H. et al.,
Blood 56:289-301 (1980); Piersma, A. H. et al., Exp. Hematol
13:237-243 (1985); Simmons, P. J. and Torok-Storb, B., Blood
78:55-62 (1991); Beresford, J. N. et al., J. Cell. Sci. 102:341-351
(1992); Liesveld, J. L. et al., Blood 73:1794-1800 (1989);
Liesveld, J. L. et al., Exp. Hematot 19:63-70 (1990); and Bennett,
J. H. et al J. Call. Sci. 99:131-139 (1991)) which are incorporated
herein by reference. Briefly, the ends of long bones from the
transgenic mice were cut, and the marrow was extracted with a
pressurized syringe filled with .alpha.-MEM (Sigma) containing 10%
fetal bovine serum (Atlanta Biologicals). About 10.sup.7 nucleated
cells were plated onto 175 cm.sup.2 plastic culture flasks in 25 ml
of .alpha.-MEM containing 10% fetal bovine serum. After 4 h, the
non-adherent cells were discarded by replacing the medium. Foci
containing two to four fibroblast-like cells appeared in 2 to 3
days, and the foci grew to near-confluent colonies in about 1 wk.
The yield was about 10.sup.7 cells per flask after trypsin
digestion. By phase-contrast microscopy, most of the cells were
fibroblast-like, but a few macrophages, and adipocytes were also
seen.
[0085] About 10.sup.5 of the cultured adherent cells were mixed
with 6.times.10.sup.5 non-adherent cells obtained by incubation of
marrow from normal mice for 4 h on 175 cm.sup.2 flasks under the
same conditions used for the initial isolation of the adherent
cells. The mixture of about 7.times.10.sup.5 cells in 0.2 to 0.4 ml
of .alpha.-MEM and 10% fetal bovine serum was injected into the
tail vein of each recipient mouse.
[0086] Eight-week old mice from the same inbred FVB/N line were
prepared to receive the donor cells by irradiation with a
.sup.137Cu irradiator (Atomic Energy of Canada, Ltd.). The unit had
a dose rate of 116 cG/min with a parallel opposed beam
configuration. Each animal received 9.0 Gy in two fractions with a
4 h interval (4.5 Gy+4.5 Gy) (O'Hara, M. D. et al., Exp. Hemat
19:878-881 (1991)). One to 2 h after the second radiation fraction,
the mixture of marked adherent cells and normal non-adherent cells
was injected intravenously. Control irradiated mice that did not
receive a cell infusion died after 10 to 13 days of marrow
failure.
[0087] To follow the fate of the donor cells, two PCR assays for
the human COL1A1 mini-gene and the mouse endogenous COL1A1 gene
were developed. With a two-primer assay, the values for the ratio
of the human to mouse gene were linear over a range of 10.sup.-4 to
about 10.sup.+1 and, therefore, of about 10.sup.-6 to 10.sup.-1
donor cells per recipient cell. With the three-primer assay, the
values were linear over a range of about 10.sup.+1 to 10.sup.+2
and, therefore, about 10-.sup.5 to 1 donor cell per recipient
call.
[0088] Assays of irradiated mice after one day indicated only trace
amounts of the donor cells in marrow, spleen, bone, lung or brain
(Table 1). Slightly higher levels were seen at seven days. At 30
days and 150 days, progeny of the donor cells accounted for 2.0 to
12% of the cells in marrow, spleen, bone and lung (Table 1). At 150
days, they also accounted for 1.5 to 5.0% of the cells in xiphoid
cartilage that was dissected free of any mineralized or fibrous
tissue under a microscope. Although the mean values appeared to
show a decrease between 1 and 5 months, there was no statistically
significant decrease in the combined values for marrow, spleen,
bone and lung between these two time periods (Table 1). Assays of
non-irradiated mice revealed only very low levels of the donor
cells at the same time points (<0.0001 to 0.05%). PCR in situ
assay of tissue sections of lung demonstrated that progeny of the
donor cells were evenly distributed in the parenchyma of both
alveoli and bronchi.
[0089] To confirm that progeny of the donor calls were present in
cartilage, chondrocytes were isolated from xiphoid and articular
cartilage by digestion at 37.degree. C. overnight with 0.5 mg/ml
bacterial collagenase (Sigma) in DMEM. PCR assays indicated that
progeny of the donor cells accounted for 2.5% of the isolated
chondrocytes.
[0090] To determine whether the donor cells became functional
mesenchymal cells in the tissues they populated, tissues from the
recipient mice were assayed by RT-PCR for expression of the human
mini-gene for collagen I contained in the donor cells. In three
mice assayed at 150 days, the mini-gene was expressed in bone), a
tissue in which over half the protein synthesized in collagen I.
The expression in bone was confirmed by a similar assay on bone
cells isolated from femur and cultured for 1 wk. Expression of the
mini-gene for collagen I was more variable in marrow, spleen and
lung, tissues in which the rate of collagen I synthesis is less
than in bone. As expected, the mini-gene was not expressed in
cartilage, a tissue in which about half the protein is synthesized
in collagen II but in which there is no synthesis of collagen I.
The mini-gene for collagen I was also not expressed in cultures of
chondrocytes from the recipient mice that contained the marker gene
and that synthesize collagen II but not collagen I.
[0091] Earlier reports have shown that assays of the cells with
cytochemical markers or for mRNAs indicated that the cells
synthesized collagen I, collagen III, fibronectin, alkaline
phosphatase and osteopontin, but did not have features
characteristic of macrophages, granulocytes, T lymphocytes, B
lymphocytes or endothelial cells. The results here demonstrate that
after intravenous injection into irradiated mice, the expanded
cultures of adherent cells efficiently populate several connective
tissues. The results also demonstrate that the cells serve as true
precursor cells for these tissues, since they expressed the marker
gene for collagen I in a tissue-specific manner, and they were
diffusely incorporated into the mesenchymal parenchyma of lung.
Example 2
Conditions for Isolation and Culture of MSCs
[0092] Conditions for culture of MSCs so that they expand but
retain the stem-cell-like phenotype have been studied. Table I
shows that co-culture of MSCs with pieces of bone increased the
number of cells obtained after 1 wk. At the same time, co-culturing
with bone decreased the alkaline phosphatase (APase) levels in the
cells, an observation suggesting that the cells did not
differentiate into osteoblasts. Also, there was a decrease in the
levels of tartrate-resistant acid phosphatase (TRAP), an
observation suggesting that the cells did not differentiate into
osteoclasts. Similar effects were observed with secondary cultures
of the MSCs. Therefore, the results suggest that co-culturing with
pieces of bone may provide improved conditions for expansion of
MSCs. Also, the medium of cultured bone pieces may be an important
source of cytokines and growth factors for expansion of MSCs in
culture.
[0093] In related experiments, it has been found that secondary
cultures of MSCs can be maintained for long periods of time MSCs
can be passed in culture for over 4 months by trypsinization and
re-plating. The cells are remarkably stable in stationary phase
cultures. In one experiment, stationary cultures remained viable
for over 4 months with re-feeding about once per wk. In another
experiment, the cells remained viable when, through an oversight,
they were left in an incubator without re-feeding for 1 month.
Stable Transfection of MSCs with a Retrovirus Vector.
[0094] To obtain virus for infection of MSCs, the LNCZ retroviral
vector (Miller, A. D. and Rosman, G. J. 41989) BioTechniques 7,
980-990 which is incorporated herein by reference) was modified so
that the promoter for cytomegalovirus (pCMV) drove expression of
the lacZ gene (FIG. 1). The vector was stably transfected into an
amphotropic murine packaging cell line (PA317). Constitutive virus
producer clones were isolated by G418 selection, and supernatant
from the clones was used for infection of MSCs. Primary cultures
MSCs (3 days old) were infected for three successive days with
fresh supernatant from the producer line with the highest titer.
Staining of the cells 5 days later indicated that about 15-20% of
the cells typically expressed the lacZ gene. Several cultures of
the infected cells were placed under selection with G418 (0.4 4
.mu.g/ml active concentration) for 5 days. Most of the cells
recovered continued to express the lacZ gene. Modifications of LNCZ
were also constructed so that expression of the lacZ gene is driven
by the promoter of the COL1 A1 gene and the promoter of the COL2 A1
gene (pCOL2A1). Expression of the lacZ gene was successfully
obtained in primary cultures of MSCs with both constructs.
Replacement of Bone Cells in Transgenic Mice with Normal MSCs.
[0095] MSCs from normal mice were infused into the transgenic mice
that expressed high levels of the mutated COL1 A1 gene (Tables II
and III). One month after the infusion of normal MSCs into the
osteoimperfecta (OI) mice (Table II), progeny of the donor cells
accounted for 10 to 45% of the bone cells in recipient mice that
had been irradiated with a maximally tolerated dose of X-ray (700
centi-Gray or cGy). Similar values were obtained with mice
irradiated with one-half of the maximally tolerated dose of X-ray
(350 cGy). However, reducing the dose to one-quarter (175 cGy)
reduced the values in four mice to 0%, 5%, 10% and 40%. Similar
results were obtained when OI transgenic mice were infused with
large numbers of whole marrow cells from which MSCs were not
removed (Table III).
[0096] In five recipient mice in which the synthesis of
pro.alpha.1(1) chains was examined (Tables II and III), the
replacement of the recipient's bone cells by normal donor MSCs was
accompanied by an increase in the ratio of normal pro.alpha.1(1)
chains to mutated pro.alpha.1(1) chains in bone. Hence, the
replacement by normal cells was accompanied by the expected changes
at the protein level.
Example 3
Long-Term Expression of the Human Genes for hGH, Factor Ix or Ob in
Stably Transfected MSCs
[0097] MSCs are isolated from mice and cultured under the
conditions described in Pereira, B. F., et al. (1995) Proc. Natl.
Acad. Sci. USA 92, 4857-4861, which is incorporated herein by
reference. MSCs are infected with retroviral vectors or transfected
with naked DNA to obtain clones that express the genes for human
growth hormone (hGH), the human obesity protein (Ob), or the gene
for human factor IX. Because a lacZ gene has been successfully
introduced into mouse MSCs with a retroviral vector, variants of
the same vector are used. At the same time, MSCs are stably
transfected with electroporation (Andreason, G. L and Evans, G. A.
(1988) BioTechniques 6, 650-660 and Toneguzzo, F., et al. (1986)
Mol. Call. Biol. 6, 703-706, which are incorporated herein by
reference), lipofectamine and nuclear injection (Mercer, W. E., et
al. (1992) In: Antisense Strategies, Ann. N.Y. Acad. Sci. Biol.
660, 209-218, which is incorporated herein by reference) so that
larger endogenous genes can be used. Further, some of the potential
disadvantages of retroviruses are avoided using alternative
introduction methodology.
[0098] Standard conditions for isolation and culture are: Whole
marrow is obtained from the tibias and femurs of 6- to 10-wk old
FVB/N mice by cutting the ends of the bones and extruding the
marrow with a syringe that contains 1 to 2 ml of ice-cold
.alpha.MEM and 10.sup.9; fetal bovine serum (FBS). The pooled
marrow cells are dispersed by gentle shaking and counted in an
automatic counter (Coulter model ZM). From 5.times.10.sup.6 to
5.times.10.sup.7 nucleated cells in 25 ml of .alpha.-MEM and 10%
FBS are plated onto 75-cm.sup.2 culture flasks. After 4 h or 3
days, the non-adherent Cells are removed by replacing the medium.
The adherent cells are expanded as primary cultures for 10 to 12
days with a re-feeding about every 4 days. The cells are recovered
by digestion with 0.25% trypsin and 1 to 5 mM EDTA for 5 min at
37.degree. C. followed by gentle scraping. The cells are diluted
with .alpha.-MEM with 10% FBS and replated at a density of from
3.times.10.sup.6 to 1.times.10.sup.5 cells per 9.5 cm.sup.2 in
6-well plates. Under these conditions, the doubling time of the
cells is 19 to 22 hours. The secondary cultures are re-fed about
every 4 days, and passed by trypsinization and replating under the
same conditions.
Preparation of Gene Constructs
[0099] The retrovirus vector LNCX is used As the parent construct.
Convenient cloning sites in the construct are used to prepare the
modified constructs pRSV-lacZ, pCMV-lacZ, pCOL1/LacZ and pCOL2-lacZ
(FIG. 1). The pCOL1 promoter is a 1.4 kb fragment that contains 476
bp of the promoter, the first exon and most of the first intron of
the human COL1A1 gene. The promoter has been shown in transgenic
mice to express a promoterless form of the COL2A1 gene in a highly
tissue specific and developmental specific manner (Sokolov, B. P.,
et al. (1995) J. Biol. Chem. 270, 9622-9629 which is incorporated
herein by reference). The COL2A1 promoter is a 1 kb fragment from
the human COL2A1 gene (Ala-Kokko, L., et al. (1991) J. Biol. Chem.
266, 14175-14178 which is incorporated herein by reference) that
confers tissue-specificity of expression (Bradham, D. M., et al.
(1994) J. Cell Physiol. 158, 61-68 which is incorporated herein by
reference). The lacZ gene is replaced with the hGH gene (Nichols
Laboratories); the OB gene (Considine, R. V., et al. (1995) J.
Cain. Invest. 95, 2986-2988, which is incorporated herein by
reference) or the human factor IX gene (Genetic Therapy, Inc.)
Use of the Retrovirus Vector.
[0100] Retrovirus Producer Cell Lines.
[0101] To establish producer cell lines, amphotrophic retrovirus
packaging murine cells PA317 were used. The cells were transfected
at 20% confluency in 100 mm dishes by the calcium phosphate
precipitation procedure (Promega) using 15 .mu.g of plasmid DNA
that was linearized by digestion with ScaI that cuts in the pBR322
region of the retrovirus vector. One day post-transfection G418
(GIBCO/BRL) was added to the medium at an active concentration of 1
mg/ml. Neomycin-resistant colonies appeared at 7 to 10 days of
selection and were isolated by cloning with mechanical rings. The
clones were expanded and individual clones were tested for the
ability to express lacZ by direct staining of duplicate wells. The
titer of the virus produced by the positive cells was assayed by
single addition of 50 .mu.l of medium to HT-1080 human tumor cells
grown to 20% confluency in 6-well microliter plates with 3 ml
medium per well and in the presence of 4 .mu.g/ml of polybrene.
[0102] The titer was assayed by determining the number of HT-1080
cells that stained positively for expression of the lacZ gene.
Typically, the titer was 1.times.10.sup.5 to 1.times.10.sup.6.
[0103] Retrovirus Infection of Mouse MSCs.
[0104] Primary cultures of mouse MSCs were prepared as described
above. After 3 days, the non-adherent marrow cells were discarded
and fresh medium added. The cells were then infected with the
retrovirus in the presence of 4 .mu.g/ml of polybrene by addition
of 1/4 vol of fresh supernate medium from stably transfected
producer cells that had the highest titer of virus production. The
infection was repeated on two additional successive days. The cells
were then either stained directly for lacZ expression or divided
into larger dishes and placed under selection with 0.4 mg/ml of
G418 (active concentration). About 15 to 20% of primary cultures
were positive for lacZ and most of the cells that survived G418
selection were positive for lacZ.
Lipofectamine Transfection.
[0105] Primary cultures of MSCs were grown for 10 days in
(.alpha.-MEM containing 10% FES). After trypsinization and light
scraping, the cells were seeded in a 6-well plate at a density of
10.sup.5 cells per well. The cells were grown for 2 days, then
washed 2 times with PBS and incubated with a DNA-lipofectamine
complex. The DNA-lipofectamine complex was prepared as follows: 6
.mu.l of lipofectamine (GIBCO/BRL) were mixed with 1 .mu.g of LINCZ
DNA in 200 .mu.l of .alpha.-MEM, incubated at room temperature for
30 min, and added to one well of a 6-well plate containing MSCs in
800 .mu.l .alpha.-MEM. After 6 h incubation at 37.degree. C., the
DNA-lipofectamine complex was replaced with 2 ml of .alpha.-MEM
containing 10% FBS. The cells were stained for lacZ or placed under
G418 selection after 18 h incubation in FBS-containing medium.
Positive clones were obtained, but they grew slowly, apparently
because the cell density was too low after the G418 selection. To
circumvent this situation, three different strategies can be used:
(a) cells are plated at higher densities; (b) co-culture cell
culture inserts will be placed over surviving clones early in the
selection process and place fresh MSCs or pieces of bone in the
inserts (see Table 1) on a daily basis to provide the necessary
cell factors to stimulate growth; (c) at the time that selection
with G418 has killed many of the non-transfected calls, the
cultures are reseeded with MSCs that have been infected with a
variant of the retrovirus LNCX (FIG. 1) in which the lacZ gene is
replaced with a selectable gene for thymidine kinase. Therefore,
the MSCs stably transfected with retrovirus are used to provide the
necessary cytokines, growth factors, and cell interactions required
for the initial growth of the transfected MSCs during selection in
G418. We can then remove the cells infected with the retrovirus by
negative selection with gangcyclovir.
Delivery Methods
[0106] Nuclear Injections.
[0107] Nuclear injections are highly efficient as a means of
transfecting some cells. Cells were plated in 60-mm dishes
containing a 22.times.22 mm coverslip marked with a circle to
delineate the area for microinjection. Cells were incubated in
medium containing 0.1% CS for 5 days to induce growth arrest before
microinjection. Under these conditions between 8 and 15% of the
cells incorporated [.sup.3H] thymidine during continuous labeling
for 24 h between days 5 and 6. Microinjection was performed using a
Zeiss Axiovert inverted-microscope equipped with an Eppendorf
microinjector and micromanipulator using commercially purchased
glass-capillary femtotips (Eppendorf). All cells within a
delineated area of the coverslip (usually 150-200) were
microinjected into the nucleus with DNA at concentrations ranging
from 0.01-1 0 .mu.g/.mu.l in 10 mM Tris buffer (pH 7.6). The
injected cells will be expanded and assayed as described above.
[0108] Electroporation
[0109] MSCs are treated with 0.25% trypsin and 1 to 5 mM EDTA for 5
min at room temperature and then harvested by scraping. The cells
are pelletted by centrifugation at 4,000.times.g for 10 min, and
then are washed twice by resuspending the pellet in ice cold PBS
(pH 7.4). MSCs are resuspended at 2.times.10.sup.6 cells per 0.8 ml
and aliquoted into an electroporation cuvette (0.4 cm gap). The
cells are incubated 10 min on ice, DNA is added to the suspension
(5-50 .mu.g), and the cells are chilled for an additional 10 min.
The cell suspension is then electroporated using a commercial
instrument (BioRad Gene Pulser; model 1652076) at an empirically
determined field strength which yields the greatest percentage of
cells that retain the exogenously added DNA. To determine the
appropriate field strength for MSCs, titrations have been performed
ranging from 0.25-2.5 kv/cm. Electroporation efficiency was
monitored by introducing a lacZ gene (LNCZ vector) and then
staining cells 48 to 72 h after electroporation. Assays.
[0110] hGH
[0111] Expression of the hGH gene is monitored by assaying medium
from clones of calls grown in 6-well microliter plates with an
enzyme linked immunoabsorbent assay with a Commercially available
kit (GIBCO/BRL). In this assay, 0.1 ml of 2.times. diluent buffer
is added per well of a microliter plate. After 5 min, 0.1 ml of
test sample is added and the plate incubated at 37.degree. C. for
30 min. The wells are washed 5 times and 0.2 ml of primary antibody
added per well. The samples are incubated at 37.degree. C. for 30
min, and washed 5 times. Then 0.2 ml of substrate buffer containing
O-phenylenediamine substrate is added. Samples are incubated at
room temperature for 30 min and the reaction stopped by addition of
0:1 ml of 2 N sulfuric acid. The absorbance of the sample is
assayed at 490 nm.
[0112] Ob Protein.
[0113] Cells are assayed for expression of the OB gene with a
protein radioimmunoassay of cell medium. The primary antibody for
human OB protein was raised in rabbits against recombinant protein
synthesized in an E. coli expression system and purified to
homogeneity. The human protein is highly homologous to the mouse
and, therefore, anti-human antibodies should cross-react with the
mouse protein. If they do not, the short mouse cDNA (619 nt) is
expressed in E. coli, the protein is purified and antibodies are
prepared. Alternatively, synthetic peptides with the mouse sequence
are purchased and use these to prepare antibodies. For the assay,
recombinant human Ob protein was radiolabeled with -.sup.125Iodine
by the Bolton-Hunter method followed by gel filtration purification
using Sephadex G-25. The specific activity obtained was -30
.mu.Ci/.mu.g. Samples of assay (0.2 ml) were preincubated with
primary antiserum (1:2000 dilution) in phosphate buffered saline
containing 0.1% Triton X-100 for 16 h at 4.degree. C. in a total
Volume of 0.4 ml. .sup.125I-Ob protein (-30,000 cpm carried in 100
l) was then added and the incubation continued for an additional 24
h. The bound Ob protein (12.+-.1%; nonspecific binding 1.4.+-.0.1%)
was immunoprecipitated by addition of 0.1 ml sheep anti-rabbit IgG
serum (Antibodies, Inc., Davis, Calif.), 0.1 ml normal rabbit serum
(GIBCO/BRL, Gaithersburg, Md.), and 0.1 ml of 10% polyethylene
glycol. The tubes were centrifuges for 15 min (2200 rpm), and
unbound label decanted and the pellet counted in a Packard 5000
gamma counter (Downers Grove, Ill.). The concentration of Ob
protein in unknown samples was calculated using Rodbard's
unweighted four parametric logistic model. The limit of detection
of this assay is 0.39 ng/ml. The intra-assay variance is 11.6% at
12 ng/ml with an interassay variance of 20.8% at 13.1 ng/ml.
[0114] Human Factor IX.
[0115] Expression of the gene for factor IX will be assayed with a
commercially available ELISA (American Bioproducts Company) under
conditions similar to those used for the hGH assay (above). As
reported by Smith et al. (74), the standard curve ranged from 1-50
ng/ml' and the limit of sensitivity was 1 ng/ml.sup.-1. The assay
did not cross-react with mouse factor IX.
Example 4
Sustained Expression of the Three Genes at Physiologically
Important Levels by Systemic Infusion of Stably Transfected MSCs
into Mice
[0116] Experiments with the OI transgenic mice (Tables II and III)
have demonstrated that cultured MSCs can serve as stem-cell-like
precursors of bone, cartilage and other mesenchymal tissues after
systemic infusion. Therefore, MSCs expressing hGH, the Ob protein
or factor IX are infused into irradiated and nonirradiated mice to
evaluate sustained expression of the genes in vivo.
Infusion of MSCs.
[0117] Initially, MSCs are infused into mice under conditions such
as those described in Table II (3-week old mice: 300 or 700 Gray
irradiation; intraperitoneal injection; 1.times.10.sup.6 MSCs; and
2.times.10.sup.8 whole marrow cells). In addition, intravenous
infusion is compared to intraperitoneal; and lower levels of X-ray
irradiation are employed. Also, the cells are infused 30.degree.
into embryos by Cesarean section. In preliminary trials, 50 .mu.l
of 5.times.10.sup.4ES were injected into the amnion of seven 13-day
embryos; 6 of 7 were delivered as viable pups. Therefore,
intrauterine injection of MSCs is feasible.
Growth Curves.
[0118] Effective in vivo expression of hGH should increase the
growth rate of mice and expression of the Ob protein should induce
starvation. Therefore, the weight and size of the treated mice and
of control littermates are monitored.
Assays for Gene Expression.
[0119] Blood is obtained from the retro-orbital plexus of mice at 1
wk, 1 month, 3 month, 5 month, 10 month, and 20 month after
infusion of the MSCs. hGH and factor IX are assayed by ELISA, and
the Ob protein is assayed with a radioimmune assays. In addition,
if measurable increases in human factor IX are obtained with ELISA,
the procedure described in Smith, T.A.G., et al., (1993) Nature
Genet. 5 397-402, which is incorporated herein by reference, to
assay biologically active human factor IX. In this procedure, human
factor IX was first captured in a microtiter well with the
monoclonal antibody, BGIX1, and then activated by factor X1a. The
active factor IX, in combination with factor VIII, converted factor
X to Xa. Factor Xa cleaved the chromogenic substrate, 52765,
yielding a yellow product. BGIX1-coated microtiter plates and
Factor VIII were purchased from Elcatech, Inc. (Winston-Salem,
N.C.). Factor X1a was purchased from Enzyme Research Labs, Inc.
(South Bend, Ind.). Factor X, phospholipid solution, S-2765, and
the thrombin inhibitor, 1-2581, were purchased from Kabi Pharmacia
Hepar, Inc. (Franklin, Ohio). Four buffers were prepared: A, 50 mM
Tris, 150 mM NaCl, 1% BSA, pH 7.5; B, 150 mM Tris, 5 mM CaCl.sub.21
10 mg/ml.sup.-1 gelatin, pH 7.6; C, 50 mM Tris, 1 0 mM CaCl.sub.2
pH 7.5; D, 50 mM Tris, 150 mM NaCl, pH 8.4. The Factor VIII/X
reaction mix was prepared fresh by mixing equal quantities of the
following stocks: factor VIII, 5 U ml.sup.-1 in buffer A; factor X,
1 U ml.sup.-1 in buffers; 1-2581, 34 .mu.g/ml.sup.-1 in buffer A;
CaCl.sub.2, 25 mM in water; and phospholipid. Plasma samples were
diluted in buffer A and 100 .mu.l were added to each microtiter
well. The plate was incubated for 90 min at room temperature and
then washed five times with buffer B. 100 .mu.l of Factor X1a (2
.mu.g/ml.sup.-1 in buffer C) were added to each well. After 30 min
at 37.degree. C., 100 .mu.l of 52765 (0.5 mM in buffer D) were
added to each well and the plate was incubated for 10 min at room
temperature before the reaction was stopped by adding acetic acid
to a final concentration of 10%. Absorbances at 405 nm were
determined with a Bio-Rad microplate reader. The standard curve,
prepared with dilutions of human normal pooled plasma, was linear
from 3-25 ng/ml.sup.-1. The assay did not cross react with mouse
factor IX. Factor IX, levels of 250 ng per ml or 5% of normal are
generally considered therapeutic and 100 to 150 ng/ml are
considered beneficial.
Example 5
Sustained Expression of the Genes at Physiologically Important
Levels by Placing the MSCs in Subcutaneous Diffusion Chambers
[0120] Cells implanted in subcutaneous diffusion chambers have at
least two distinct advantages for therapy of patients: (a) immune
responses are circumvented; and (b) when implanted in capsules in
mice (Benayahu, D., et al. (1989) J. Cell Physiol. 140, 1-7, which
is incorporated herein by reference), rats (Mardon, et al. (1987)
Cell Tissue Res. 250, 157-165, which is incorporated herein by
reference) or rabbits (Friedenstein, A. J., et al. (1987) Cell
Tissue Kinet. 20, 263-272, which is incorporated herein by
reference), they survive for at least 6 wks (Wakitani, S., et al.
(1994) J. Bone and J.T. Surgery 76A, 579-592, which is incorporated
herein by reference), apparently because they persist as bone,
fibrous tissue or cartilage that does not require vascularization
(Benayahu, D., et al. (1989) Supra, Mardon, H. J., et al. (1987)
Supra, Owen, M. and Friedenstein, A. J. (1988) In: Cell and
Molecular Biology of Invertebrate Hard Tissues, Wiley Chicester,
CIBA Foundation Symposium, 136, 42-60, which is incorporated herein
by reference, and Friedenstein, A. J., et al. (1987) Supra).
Preparation of Chambers.
[0121] Diffusion chambers are assembled from commercially available
components (Millipore Corp.) and used as described in previous
reports (Benayahu, D., et al. (1989) Supra, Mardon, H. J., et al.
(1987) Supra,). Briefly, membrane filters with 0.3 .mu.m pore size
are glued to one side of each of two plastic rings with acryloid
glue. The two rings are then glued together to form a chamber, the
dimensions are 9 mm inner diameter and 2 mm thick with a volume of
about 127 mm.sup.3. From 10.sup.4 to 10.sup.7 MSCs are inoculated
into the chambers through a hole in one ring and the hole sealed
with a tapered plastic plug coated with glue. The chambers are
implanted into mice either subcutaneously on the back or
intraperitoneally under anesthesia. Initially, one or more chambers
are inserted into freshly weaned mice (3 wk). Subsequently,
chambers are inserted in 1 wk old mice. For the experiments with
the 1-wk old mice, smaller chambers are prepared from discs (5 mm,
I.D.) cut from plastic tips for micropipettes.
Assays
[0122] Blood is obtained from the retro-orbital plexus at 1 wk, 1
month, 3 month, 5 month, 10 month and 20 month after implantation
of the chambers. The plasma is assayed for hGH, Ob protein and
factor 1.times. as described above.
Example 6
[0123] Referring to FIG. 2, the diffusion chamber (1) may have a
chamber barrel (3) having two ends, a first end (5) and a second
end (7). The barrel may be comprised of one or more rings secured
together by non-toxic means. The chamber is fitted at each end with
a filter, a first filter (9) and a second filter (11). The filters
are porous to factors such that the factors may pass between the
chamber and the mammal. The filter pores size may be about 0.25
.mu.M or smaller, preferably about 0.1 .mu.m. The filters may be
made of plastic, teflon, polyester, or any inert material which is
strong, flexible and able to withstand chemical treatments. The
filters may be secured in position with rubber gaskets which may
also provide a tighter seal. Optionally, the barrel portion of the
chamber may have an opening (13) which may be covered by a cap (not
shown). The cap may be screw on type of self sealing rubber and
fitted to the opening. Inserting cells into the chamber contents
may thus be performed by accessing the opening by removing the cap
and inserting cells using an ordinary needle and syringe. The
chamber may be made of any substance, such as and not limited to
plastic, teflon, lucite, titanium, or any inert material, which is
non-toxic to, and well tolerated by, mammals. In addition, the
chambers should be able to survive sterilization.
[0124] The chamber may be implanted in the following non-limiting
ways: subcutaneously or intraperitoneally, for example. The chamber
may be removed about 24 to about 30 hours after implantation.
Alternatively, a refillable chamber may be employed such that the
chamber may be re-used for treatments and emptied following
treatments.
TABLE-US-00001 TABLE I Conditions for Growth of Primary and
Secondary Cultures of MSCs. Cells APase.sup.a TRAP.sup.a Culture
per well (mmol (mmol MSCs conditions .times.10.sup.5 min/mg)
min/mg) Primary Standard.sup.a 2.0 426 144 Co-cultured.sup.b 6.41
22.3 102 Co-cultured.sup.c 6.94 42.0 105 (Matrigel) Secondary.sup.d
Standard 1.33 2,052 75 Co-cultured 7.40 362 60.6 Co-cultured 5.08
506 59.2 (Matrigel) .sup.aWhole marrow cells (20 .times. 10.sup.6)
from 6-week old mice were cultured in individual 9.5 cm.sup.2 wells
in 2 ml of 10% FCS and .alpha.-MEM. Non-adherent cells were removed
on day 3 and the incubation continued in fresh medium until day 7.
APase and TRAP was assayed as described in reference 55.
.sup.bCo-cultured with pieces of bone (one-half femur and one-half
tibia) in cell culture inserts (23 mm; 3 .mu.m pore size; Becton
Dickinson). .sup.cSame as .sup.b, with inserts coated with
Matrigel. .sup.dPrimary cultures on day 10 were detached with 0.25%
trypsin and 1 mM EDTA for 5 min at 37.degree. C. followed by gentle
scraping. Cells from one well (2 .times. 10.sup.5) were diluted 1:4
and cultured in 9.5 cm.sup.2 wells for 7 days with changes of
medium on day 3 and day 6. .sup.eAPase (20,54) and TRAP (55)
Activities were per mg total protein.
TABLE-US-00002 TABLE II Experiments with (a) Transgenic Mice as
Recipients; (b) Normal MSCs as Donor Cells; and (c) Decreasing
X-Ray Dose. Decrease Bone replacement in mutated Recipient X-ray
Donor Cells.sup.a at pro.alpha.1 (1) mice (cG) MSCs Whole marrow 1
month (%) chains Transgenic 700 0.7 .times. 10.sup.6 (N).sup.a 15
.times. 10.sup.6 (TG).sup.ab 10 to 45% 26 to 73% (3 wk) (n = 3) (n
= 3) Transgenic 350 1.2 .times. 10.sup.6 (N) 2 .times. 10.sup.6
(TG) 28 to 60% (3 wk) (n = 4) Transgenic 175 1.2 .times. 10.sup.6
(N) 2 .times. 10.sup.6 (TG) 0 to 40% (3 wk) (n = 4) .sup.a(N),
normal; (TG), transgenic. .sup.bMSCs removed from whole marrow
cells before infusion by incubation on plastic culture dish for 4 h
at 37.degree. C.
TABLE-US-00003 TABLE III Experiments with (a) Transgenic Mice as
Recipients; (b) 10-Fold Increase in Whole Marrow Cells as Donor
Cells; and (c) Decreasing X-Ray Dose. Bone Decrease replacement in
mutated Recipient X-ray Donor Cells.sup.a at pro.alpha.1(1) mice
(cG) MSCs Whole marrow 1 month (%) chains Transgenic 700 5 .times.
10.sup.6 (N).sup.a 20 to 38% (3 wk) (n = 3) Transgenic 350 16
.times. 10.sup.6 (N) 50 to 78% 21 to 24% (3 wk) (n = 3) (n = 3) (n
= 2) Transgenic 350 5 .times. 10.sup.6 (N) 22 to 45% (3 wk) (n = 4)
.sup.a(N) whole marrow from normal mice without any treatment to
remove MSCs.
TABLE-US-00004 TABLE IV Complex Genetic or Acquired Diseases
Treatable by Encapsulated Stromal Cells (Partial List). Deficient
or excessive Potential therapeutic Disease metabolite or protein
Gene defect gene in stromal cells Growth hormone Low growth hormone
Growth hormone Growth hormone deficiencies plus in some in some
growth defects Obesity Unknown Unknown Gene for the Ob protein
(decreases appetite) Renal disease Anemia plus Multiple genes
Erythroprotein other changes and acquired forms Diabetes Decreased
insulin Multiple Several genes for insulin synthesis and regulated
release (complex) Atherosclerosis Relatively low HDL levels
Multiple Gene for Apo-A1 (increases HDL in transgenic mice)
Osteoporosis Unknown Multiple Estrogen agonist specific for bone
cells Infectious diseases, Antibodies to including AIDS infectious
agent Autoimmune diseases Antagonists for the immune epitope
TABLE-US-00005 TABLE V Defined Monogenic Diseases Treatable by
Encapsulated Stromal Cells (Partial List). Deficient or excessive
Potential therapeutic Disease metabolite or protein Gene defect
gene in stromal cells Urea cycle defects Glutamine Several
different Glutaminase Branched chain Keto acids of leucine, Three
different Specific decarboxylases organic acidurias isoleucine, and
valine Adenine deaminase Deoxy-adenosine Adenine deaminase Adenine
deaminase deficiency immuno- deficiency Familial lipoprotein
Chylomicrons Lipoprotein lipase Lipoprotein lipase Gaucher Disease
Glucosylceramide Acid .beta.-glucosidase Acid .beta.-glucosidase
(type I) .alpha.1-Antitrypsin Low serum protein .alpha.-Antitrypsin
.alpha.-Antitrypsin Deficiency Galactosemia Increased galactose One
of four enzymes One of four enzymes Hemophilia Decreased factor
VIII or IX Factor VIII or IX Factor VIII or IX
* * * * *