U.S. patent application number 12/273377 was filed with the patent office on 2009-03-12 for treatment of muscular dystrophy with cord blood cells.
This patent application is currently assigned to Viacell, inc.. Invention is credited to Robert H. Brown, JR., Seth P. Finklestein, Morey Kraus.
Application Number | 20090068156 12/273377 |
Document ID | / |
Family ID | 23188804 |
Filed Date | 2009-03-12 |
United States Patent
Application |
20090068156 |
Kind Code |
A1 |
Brown, JR.; Robert H. ; et
al. |
March 12, 2009 |
TREATMENT OF MUSCULAR DYSTROPHY WITH CORD BLOOD CELLS
Abstract
The invention features methods for treating a patient suffering
from muscular dystrophy by administration of umbilical cord blood
cells, e.g., by IV infusion.
Inventors: |
Brown, JR.; Robert H.;
(Needham, MA) ; Finklestein; Seth P.; (Needham,
MA) ; Kraus; Morey; (Jefferson, MA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
Viacell, inc.
Worcester
MA
|
Family ID: |
23188804 |
Appl. No.: |
12/273377 |
Filed: |
November 18, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10201508 |
Jul 23, 2002 |
7452529 |
|
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12273377 |
|
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60307227 |
Jul 23, 2001 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61K 35/51 20130101;
A61P 21/04 20180101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/12 20060101
A61K035/12 |
Claims
1. A method for treating a patient suffering from muscular
dystrophy comprising injecting human umbilical cord blood (UCB)
cells into the muscle of said patient.
2-3. (canceled)
4. The method of claim 1, wherein prior to said injecting, said UCB
cells are treated to increase their number.
5-6. (canceled)
7. The method of claim 1, wherein said injecting results in
engraftment of UCB cells into said patient's muscle, whereby said
engrafted UCB cells express dystrophin and treat said patient by
regenerating muscle mass.
8. A method for treating a patient suffering from muscular
dystrophy comprising administering umbilical cord blood (UCB) cells
to said patient following bone marrow ablation, wherein said UCB
cells reconstitute the immune system of said patient and treat the
patient's muscular dystrophy.
9. The method of claim 8, wherein said administering comprises
injecting said UCB cells into said patient's blood stream.
10. The method of claim 8, wherein said administering comprises
injecting said UCB cells into said patient's muscle.
11. The method of claim 8, wherein prior to said administering said
UCB cells are treated to increase their number.
12. The method of claim 8, wherein said administering results in
engraftment of UCB cells into said patient's muscle, whereby said
engrafted UCB cells express dystrophin and treat said patient by
regenerating muscle mass.
13. The method of claim 8 further comprising culturing said UCB
cells in vitro to produce an expanded population of cells and
repeatedly administering said expanded population of cells to said
patient to treat the patient's muscular dystrophy.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of
U.S. provisional patent application No. 60/307,227, filed Jul. 23,
2001.
BACKGROUND OF THE INVENTION
[0002] Muscular dystrophy represents a family of inherited diseases
of the muscles. Some forms affect children (e.g., Duchenne
dystrophy) and are lethal within two to three decades. Other forms
present in adult life and are more slowly progressive. The genes
for several dystrophies have been identified, including Duchenne
dystrophy (caused by mutations in the dystrophin gene) and the
teenage and adult onset Miyoshi dystrophy or its variant, limb
girdle dystrophy 2B or LGMD-2B (caused by mutations in the
dysferlin gene). These are "loss of function" mutations that
prevent expression of the relevant protein in muscle and thereby
cause muscle dysfunction. Mouse models for these mutations exist,
either arising spontaneously in nature or generated by inactivation
or deletion of the relevant genes. These models are useful for
testing therapies that might replace the missing protein in muscle
and restore normal muscle function.
[0003] Differentiated muscle is composed of multi-nucleated cells
or myofibers that have an extraordinary capacity to regenerate.
This regenerative capacity exists because muscle possesses
primitive muscle precursor cells (muscle stem cells and somewhat
more mature cells known as "satellite cells"). These cells lie
dormant in muscle and can be activated to make new mononucleated
muscle cells (myoblasts) that can adhere to one another and fuse to
make new, multi-nucleated myotubes, as well as the more mature
muscle cells (that are again multinucleated). Because myofibers
arise from the fusion of individual myoblasts, a protein made by
one muscle cell is readily accessible to be shared with neighboring
muscle cells lacking that protein if the two cells fuse into the
same myotube.
[0004] Inherent in this concept of myoblast fusion and muscle
regeneration is the possibility of cell therapy of muscle diseases.
The fusion of a myoblast capable of making a muscular dystrophy
protein with muscle cells that lack the protein should correct the
deficiency in the resulting myotube. That is, the normal nucleus in
the normal myoblasts replaces a gene missing in the dystrophic
muscle cells thus achieving gene and protein replacement through
cell therapy.
[0005] Partridge and colleagues demonstrated more than a decade ago
that a mixed population of muscle precursor cells capable of making
normal dystrophin protein could fuse into muscle of the mdx mouse
that lacks dystrophin and thereby partially replace the missing
protein (Partridge et al., Nature 337:176-179, 1989). In the
seminal experiments of Partridge, it was not clear precisely what
populations of the muscle precursor cells had the capacity to
achieve this effect. At least six human trials of myoblast therapy
were undertaken in Duchenne and Becker dystrophy patients, using
direct intramuscular injections of myoblasts; that none were
effective might be interpreted to mean that myoblasts were not
sufficiently undifferentiated to participate effectively in muscle
cell therapy. This observation stimulated the search for muscle
stem cells.
[0006] Over the last two years, several muscle biologists have had
encouraging initial success in isolating putative muscle stem
cells. Moreover, these studies have documented not only that the
primitive muscle precursors can fuse into injured muscle to make
new muscle, but also that such stem cells or stem-like cells have
an extraordinary capacity to circulate in the blood and then to
leave the blood to enter sites of focal muscle injury in response
to unidentified myotropic factors. Strikingly, in the last three
years it has become apparent that cells with features of muscle
stem cells may be present in tissues thought previously to be
primarily hematogenic, such as the bone marrow. In 1999, Gussoni
and colleagues in the Kunkel laboratory reported that a population
of primitive cells identified by the presence of a multi-drug
resistance transporter as a "side population"(SP) fraction of cells
in either the bone marrow or muscle itself could be delivered to
dystrophic mdx muscle following tail vein injection (Gussoni et
al., Nature 401:390-394, 1999). That these cells included primitive
stem cells was strongly suggested by the finding that the same
injection could populate muscle tissue with enough normal muscle
cells to restore dystrophin expression in up to 10% of myofibers
and, at the same, repopulate the bone marrow of previously
irradiated recipient mice. Analogous findings were subsequently
reported from the laboratories of Huard (Lee et al., J. Cell Biol.
150:1085-1099, 2000) and Bresolin (Torrente et al., J. Cell Biol.
152:335-348, 2001).
[0007] At this time, one criterion for defining a cell as a muscle
stem cell is the capacity to differentiate to form myoblasts and
thereby augment some aspect of muscle regeneration or repair.
Typically, the expression of a previously missing protein (like
dystrophin) after muscle stem cell infusion provides prima facie
evidence that a muscle stem cell is present. To date, no single set
of molecules have been identified that uniquely define muscle stem
cells. However, an evolving family of surface proteins is being
identified that characterizes different stages of differentiation
in the muscle cell lineage, as summarized in Table 1.
TABLE-US-00001 TABLE 1 Selected Surface Markers Differentially
Expressed on Cells in the Muscle Lineage Satellite MPC/ Marker
MSC/SP cell Myoblast Myotube Sca-1 + - c-kit - (? +) + CD34 + +
CD45 (heme - - lines) Desmin - + bcl-2 ? + Flk-1 - (human)
(VEGFR/kdr) M-cadherin - + Pax-7 ? +/- + c-met receptor ? +
Multidrug res + ? prot MyoD - - + + Myogenin - - + + MRF4 - - + +
NCAM - - + ++ CD43 - - - - CD123 - CD90 (+ on MP) + AC133 -/+ ? MSC
= muscle stem cell; SP = side population cells; MPC = muscle
precursor cells
For example, it is likely that a candidate set of stem cells will
express the surface antigen CD34 and perhaps other primitive cell
surface markers such as AC133, but not lineage markers, such as
c-kit or the hematopoietic marker CD45.
[0008] Umbilical cord blood cells ("UCB" cells) contain
sub-populations with properties of stem cells. Replacement of bone
marrow cells can be accomplished via infusion of human UCB
cells.
SUMMARY OF THE INVENTION
[0009] The invention features a method for treating a patient
suffering from muscular dystrophy by administration of human UCB
cells. The cells can be administered in any suitable manner, e.g.,
by injection directly into the muscle or into the blood stream of
the patient. Following systemic injection, some UCB cells will
migrate to the muscles of the patient and integrate with existing
muscle cells where they will provide missing proteins, thus
treating the disease.
[0010] In an embodiment of the invention, the UCB cells are
treated, prior to administration, to increase their number and to
render the cells enriched in relatively undifferentiated cells.
[0011] The invention also features a cell transplant method for
treating a patient suffering from muscular dystrophy that also
reduces the rejection of the administered cells by the immune
system of the patient treated. The method includes providing a
preparation of cord blood cells, reconstituting the immune system
of the patient using cells of the preparation, and administering
cells of the preparation to treat the patient's muscular dystrophy.
In an embodiment of the cell transplant method, the method further
includes expanding the cells of the preparation in vitro and
repeatedly administering the expanded cells of the preparation to
the patient to treat the patient's muscular dystrophy.
[0012] The invention provides several advantages. For example, it
provides methods for treating a patient diagnosed with or suffering
from muscular dystrophy by administering UCB cells to the patient.
The invention relies on the use of umbilical cord blood which
contains a rich source of pluripotent stem cells, the precursors of
two or more cell types of an organism. Bone marrow from donors has
traditionally been used as a source of stem cells for restoration
of diseased bone marrow, but researchers have demonstrated that
umbilical cord blood provides an excellent alternative source of
pluripotent cells that can be used to restore cells of many
different tissues. The invention also features a method of treating
a patient diagnosed with or suffering from muscular dystrophy in
which the patient is administered UCB cells, but, in an effort to
overcome a rejection of the cells, the patient's immune system is
also reconstituted using the UCB cells (which can be provided by
the patient or by a donor).
[0013] In addition, cord blood stem cells are unique in that they
have been found to be successful in transplant cases when the blood
type is only a near match as opposed to stem cells found in bone
marrow which require an exact match from donor to recipient. There
is also a greater occurrence of graft vs. host disease in bone
marrow transplants than there is in cord blood stem cell
transplants.
DEFINITIONS
[0014] By "umbilical cord blood cells, cord blood cells, or
placental blood cells" we mean the blood that remains in the
umbilical cord and placenta following birth. Like bone marrow, cord
blood has been found to be a rich source of stem cells.
[0015] By "stem cell" or "pluripotent stem cell," which can be used
interchangeably, is meant a cell having the ability to give rise to
two or more cell types of an organism.
DETAILED DESCRIPTION
Mouse Experiments
[0016] Human UCB cells are obtained from the umbilical cord of
newly born infants immediately following the resection of the cord
from the infant. The cells (10,000-1,000,000 cells per mouse) are
suspended in standard pharmaceutical buffer or sterile saline and
placed in a syringe.
[0017] The initial mouse model to be used is a mutant mouse (the
sjl mouse) lacking functional dysferlin; these mice are a naturally
occurring model of Miyoshi muscular dystrophy/LGMD2B. These mice,
described in (Lee et al., J. Cell Biol. 150:1085-1099, 2000), are
available in the laboratory of Robert H. Brown, Jr., of the
Massachusetts General Hospital, Boston, Mass.
[0018] The UCB cells are injected into the bloodstream of the mice
via retro-ocular or tail vein injection. After 80 to 100 days, the
mice are sacrificed to determine whether the UCB infusions have
enhanced muscle regeneration and dysferlin expression. Several
endpoints are used to monitor these outcomes including: (a)
immunostaining for markers of nuclei of human origin, to define the
persistence of the UCB in the muscle tissue; (b) detection in the
UCB cells of markers that confirm that the cells have
differentiated into muscle; and (c) detection of dysferlin, which
the host sjl mice lack. Another method to track the fate of the
injected UCB cells is to label them with one or more fluorescent
DNA- binding dyes prior to infusion; the presence of those dyes in
the nucleus of muscle cells 80 to 100 days after infusion strongly
indicates the cells are derived from UCB cells. It is also possible
to pre-label the donor cells with 5-bromo-2'-deoxy-uridine (BrDU)
or with a nuclear dye such as one of the Hoechst dyes. Functional
studies will also be performed to quantitate motor function in the
animals.
[0019] One or more methods of immunosuppression will be employed to
allow the host mice to accept the incoming UCB cells. This can be
achieved pharmacologically with, for example, cyclopsorin A, or
other immunosuppressant drugs. An alternate approach is to use
near-lethal irradiation. Still a third approach to over-coming the
transplantation barriers posed by the immune system is to use mice
that are immunologically defective (e.g. nu/nu or Rag-null
mice).
[0020] These initial experiments will employ whole populations of
UCB cells. Subsequent experiments will employ subsets of UCB cells,
enriched for various categories of stem cells or muscle precursor
cells. Among the methods of enrichment are those described in
Kraus, U.S. Pat. Nos. 5,925,567 and 5,674,750, hereby incorporated
by reference.
Human Therapy
Systemic Infusions
[0021] The method of the invention can be used to treat any human
patients, whether children or adults, who suffer from one of the
forms of muscular dystrophy.
[0022] UCB cells are prepared as described above, with the cell
number increased proportionally. Preferably, the cells are typed
using the standard six transplantation markers; preferably, the
match is at least 4/6; most preferably, there is a 6/6 match. In
instances in which the match is less than 4/6, however, the
procedure may still be viable, with acceptably mild rejection due
to the naivete of the relatively undifferentiated UCB cells. What
rejection does occur is controlled using the standard methods
described above, e.g., the administration of cyclosporin A.
[0023] If the UCB cells have not been expanded and selected as
described above, the cells can be used in the form in which they
are stored. In instances where the number of cells in a single
stored sample is insufficient, several such samples can be combined
to provide the required number of cells. Further, as is discussed
above in connection with the mouse experiments, rather than using
the whole UCB cell population, sub-populations can be used that are
enriched in muscle stem cells or other precursor cells.
[0024] It is anticipated that human therapy is likely to require
multiple infusions of either the whole UCB cell preparation or
various enriched and expanded sub-populations of the cells. Several
infusions of cells can be administered over time, e.g., one on day
one, a second on day five, and a third on day ten. After the
initial ten day period, there can be a period of time, e.g., two
weeks without cell administration, after which time the ten-day
administration protocol can be repeated.
[0025] Whether administered as a single infusion therapy or
multiple infusion therapies, it is likely that the recipient will
require immunosuppression. The protocols followed for this will
follow the precedents now used in human transplantation for bone
marrow replacement (i.e., cell transplantation), with such agents
as cyclosporin A and FK506.
Intramuscular Injections
[0026] Another possible administration route for UCB cells, or
expanded sub-populations of UCB cells, is via direct injection into
muscle. It is well established that under certain circumstances
direct intramuscular injection of muscle precursor cells can lead
to the incorporation of the donor cells into the host, with partial
replacement of a missing protein (e.g., dystrophin in the mdx
mice). It is also well established that direct myoblast injections
into humans with muscular dystrophy is safe, although such
myoblasts have not been incorporated significantly into the host
muscles. Accordingly, it is believed that sub-populations of UCB
cells that are more primitive than myoblasts will fuse into host
muscle and thereby permit protein replacement and muscle
regeneration. This is inherently less efficient than IV injection
for widespread distribution of the injected cells, and thus would
almost certainly require multiple injections into multiple
muscles.
Immune Reconstitution and Serial Injections by UCB Cells
[0027] As is outlined above, maintenance of the donor UCB cells
within the host muscle is likely to require chronic use of one or
more immunosuppressant regimens. This is analogous to the long-term
use of immunosuppressant drugs or other therapy in patients who
undergo whole organ or cell transplantation.
[0028] The method of using UCB cells proposed herein may circumvent
the chronic use of immunosuppressants. The concept has two
elements. First, if the recipient undergoes ablation of bone marrow
prior to the UCB infusion, it may be possible to replace the
recipient's marrow with the UCB cells. It may be possible to
provide the recipient's own UCB cells. This may overcome the need
for immunosuppression. If the recipient's own UCB cells are not
available, donor UCB cells may be used. The donor UCB cells that
reconstitute the ablated marrow will effectively tolerize to the
host and thereby survive after a period of weeks or months without
on-going immunosuppression. In effect, such a recipient is
chimeric, with a reconstituted immune system predominantly of donor
origin co-existing with host tissues. The second key element in
this reconstitution concept is that subsets of UCB cells have an
extraordinary capacity to proliferate. We will determine whether
the UCB cells used to supply the proteins lacking in the muscles of
the dystrophy patients can be used, in parallel, to generate, in
vitro, large populations of muscle precursor cells. If indeed the
donor UCB cells tolerize to the host and render the host chimeric,
then subsequent infusions of the muscle precursor cells derived
from the same UCB cells should be immunologically acceptable to the
host, even when given in multiple serial infusions. That is, immune
reconstitution by UCB cells may permit subsequent serial infusions
of muscle precursor cells derived initially from the same UCB
donor. In this protocol, once chimerism is established, the
subsequent muscle cell infusions should not require
immunosuppression of the host.
Collection and Expansion of Umbilical Cord Blood Cells
[0029] The UCB cells can be collected by methods known in the art,
see for example Koike et al., Acta Paediatrica Japonica 25:275-282,
1983, and expanded by methods described in, for example, U.S. Pat.
No. 5,674,750, U.S. Pat. No. 5,925,567, and U.S. Pat. No.
6,338,942. In general precursor cells that can be obtained from
umbilical cord blood can be used in accordance with the present
invention. The stem cells can be expanded under cell growth
conditions, i.e., conditions that promote proliferation ("mitotic
activity") of the cells.
Gene Therapy
[0030] Gene therapy can also be used to modify the UCB cells to
provide one or more missing protein(s) that are the basis for the
muscular dystrophy. It is envisioned that UCB cells, which contain
subpopulations of stem cells and can be used to replace bone marrow
cells, can be modified with a corrected gene product and
administered to the patient using one or more of the methods
described above. Delivery of the altered UCB cells would then treat
the patient's muscular dystrophy.
[0031] An exemplary therapeutic gene therapy regimen may include
the steps of obtaining a source of UCB cells from a subject or
donor, UCB cell enrichment in vitro, UCB cell expansion by methods
known in the art, transduction of UCB cells with a vector
containing a gene of interest, and reintroduction into the subject.
Transduction of the UCB cells by gene therapy techniques can be
during or after expansion.
[0032] Several methods are known in the art for altering cells for
use in gene therapy. These methods include cell transduction (see,
e.g., Buetler, Biol. Blood Marrow Transplant 5:273-276, 1999; Dao,
Leukemia 13:1473-1480, 1999; and see generally Morgan et al., Ann.
Rev. Biochem. 62:191-217, 1993; Culver et al., Trends
Genet.10:174-178, 1994; and U.S. Pat. No. 5,399,346 (French et
al.)); the use of viral vectors; and the use of non-viral vectors,
for example, naked DNA delivered via liposomes, receptor-mediated
delivery, calcium phosphate transfection, lipofection,
electroporation, particle bombardment (gene gun), microinjection,
cell fusion, chromosome-mediated gene transfer, microcell-mediated
gene transfer, spheroplast fusion, and pressure-mediated gene
delivery. The technique should provide for the stable transfer of
the gene to the cell, so that the gene is expressible by the cell
and preferably heritable and expressible by its cell progeny.
[0033] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical Pharmacy 12:488-505, 1993; Wu and Wu,
Biotherapy 3:87-95, 1991; Tolstoshev, Ann. Rev. Pharmacol. Toxicol.
32:573-596, 1993; Mulligan, Science 260:926-932, 1993; and Morgan
and Anderson, Ann. Rev. Biochem. 62:191-217, 1993. Methods commonly
known in the art of recombinant DNA technology that can be used are
described in Ausubel et al. (eds.), 1993, Current Protocols in
Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990,
Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY.
Other Embodiments
[0034] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each independent publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0035] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure that come
within known or customary practice within the art to which the
invention pertains and may be applied to the essential features
hereinbefore set forth.
* * * * *