Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR GENERATING IMMUNE-COMPATIBLE CELLS AND
TISSUES USING NUCLEAR TRANSFER TECHNIQUES
This application claims the benefit of U.S. Provisional Patent Application No.
60/152,354 filed September 7, 1999 and U.S. Provisional Patent Application No.
60/155,107 filed September 22, 1999.
Field of Invention
The present invention combines the fields of cloning, developmental biology
and tissue engineering to devise immune compatible tissues and cells for the
purpose
of transplantation. In addition, the invention discloses methods of generating
therapeutic cells and tissues for transplantation using nuclear transfer
techniques, and
methods of verifying or evaluating the immune compatibility of such tissues.
Background of Invention
The past decade has been characterized by significant advances in the science
of cloning, and has witnessed the birth of a cloned sheep, i.e. "Dolly"
(Roslin Bio-
Med), a trio of cloned goats named "Mira" (Genzyme Transgenics) and over a
dozen
cloned cattle (ACT). The technology which enables cloning has also advanced
such
that a mammal may now be cloned using the nucleus from an adult,
differentiated
cell, which scientists now know undergoes "reprogramming" when it is
introduced
into an enucleated oocyte. See U.S. Patent 5,945,577, herein incorporated by
reference in its entirety.
The fact that an embryo and embryonic stem cells may be generated using the
nucleus from an adult differentiated cell has exciting implications for the
fields of
organ, cell and tissue transplantation. There are currently thousands of
patients
waiting for a suitable organ donor, and face problems of both availability and
incompatibility in their wait for a transplant. If embryonic stem cells
generated from
the nucleus of a cell taken from a patient in need of a transplant could be
made and
induced to differentiate into the cell type required in the transplant, then
the problem
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of transplantation rejection and the dangers of immunosuppressive drugs could
be
precluded.
Embryonic stem cells have been induced to develop into cells from the three
different germ layers. For instance, Anderson et al. demonstrated that inner
cell
masses (ICM) and embryonic discs from bovine and porcine blastocysts will
develop
into teratomas containing differentiated cell types from ectodermal,
mesodermal and
endodermal origins when transplanted under the kidney capsule of athymic mice.
Animal Repro. Sci. 45: 231-240 (1996). Furthermore, the developmental signals
that trigger cell differentiation are beginning to be deciphered. For
instance, Gourdie
et al. demonstrated the differentiation of embryonic myocytes into impulse-
conducting Purkinje fiber cells. Proc Natl Acad Sci USA 95: 6815-6818 (June,
1998). Further, researchers at the University of Medicine and Dentistry of New
Jersey (UMDNJ) have recently reported the transformation of bone marrow cells
into
nerve cells (Washington Post, August 15, 2000, p. A6). Thus, it should be
possible to
isolate differentiated cells from embryonic stem cells or teratomas, and
induce their
differentiation into particular cell types for use in transplantation.
In addition, by using techniques evolving in the field of tissue engineering,
tissues and organs could be designed from the differentiated cells, which
could be
used for transplantation. For instance, Shinoka et al. have designed viable
pulmonary
artery autografts by seeding cells in culture onto synthetic biodegradble
(polyglactinlpolglycolic acid) tubular scaffolds. I Thorac Cardiovasc. Sure.
115:
536-546 (1998). Zund et al. demonstrated that seeding of human fibroblasts
followed by endothelial cells on resorbable mesh is helpful for creation of
human
tissues such as vessels or cardiac valves. Eur J Cardic-Thorac. Surg. 13: 160-
164
(1998). Freed et al. have shown that culturing cells under conditions of
simulated
microgravity is advantageous for the engineering of cartilage and heart
tissue. In
Vitro Cell Dev. Bid. --Animal 33: 38 1-385 (May, 1997).
However, the fields relating to cell development and differentiation, and
tissue
engineering have deficiencies. For instance, the teratomas created by Anderson
et al.
were created from naturally-formed embryos. Thus, the genotype of the embryos
will
be unique to the individual embryos. Such cells are not appropriate for
transplantation, because they would still induce transplant rejection just as
any
allogeneic tissue when transplanted into a donor animal. Most autograft tissue
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engineering studies, in contrast, have been performed using cells from the
actual
recipient animal. Such a technique will not provide suitable transplant organs
to those
patients whose cells or organs are deficient, i.e., perhaps for lack of gene
expression,
or due to expression of a mutant gene. Moreover, for a patient whose organ has
literally shut down, it will not be possible to engineer a new organ from the
patient's
own cells. Thus, there are many deficiencies to be overcome in applying the
concepts
of cellular differentiation and development and tissue engineering to the
treatment of
transplant patients.
Summary of Invention
The present invention addresses the uncertainties still to be overcome in the
use of engineered cells and tissues for transplantation. The invention
discloses
methods of engineering cloned, immune compatible, developmentally
differentiated
cells into tissues for transplantation, and methods of using such tissues to
treat a
patient in need of a transplant. In particular, such tissues may be designed
to express
a therapeutic protein. Because the tissues and cells for transplantation are
all
generated from the same original donor cell through nuclear transfer, all the
cells of
the engineered tissue will express the heterologous gene of interest. The
methods of
the invention therefore additionally provide an invaluable alternative to
tissue-
targeted gene therapy.
The present invention also provides methods for determining whether
particular genetically engineered cells will provide immune compatible organs
for
transplantation. For instance, the present invention discloses methods of
evaluating
cloned cells for mitochondria) compatibility, and in particular, transgenic,
developmentally differentiated cells, for immune compatibility in an animal
model.
Such evaluations will provide important information regarding the suitability
of
therapeutic tissues in transplantation, and will provide the foundation for
controlling
these parameters in order to provide immune compatible tissues.
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Detailed Description
The present invention is directed to methods of producing of immune
compatible tissues using cloning technology. The cells and engineered tissues
produced by the disclosed methods are also encompassed in the present
invention, as
are the stable grafts produced by transplantation of the engineered tissues. A
stable
graft is defined as a graft that does not illicit an immune response or
rejection when
transplanted into a nuclear donor, or at least provides a substantial
improvement in
avoiding graft rejection over non-cloned control transplanted tissue.
Because cloned cells generated by nuclear transfer are not completely
identical
with the donor cell or animal, e.g., they typically lack the mitochondria) DNA
of the
donor cell and gain the mitochondria) DNA of the recipient enucleated oocyte
or other
cell and typically are not produced in an in vivo environment that will
perfectly mimic
conditions present during embryogenesis, the question is raised as to whether
such
cells will be entirely immune-compatible when they are transplanted back into
the
donor animal.
For instance, it has been demonstrated that mitochondria) peptides in mice,
e.g., the ND 1 peptide from the amino terminus of NADH dehydrogenase and the
MiHA peptide encoded by the amino terminus of the COI gene, are presented at
the
cell surface by non-classical MHC class I molecules, e.g., H-2M3a, in
combination
with beta-2-microglobulin (Vyas et al., 1992, "Biochemical specificity of H-
2M3a . .
.," J. Immunol. 149(11):3605-11; Morse et al., 1996, "The COI mitochondria)
gene
encodes a minor histocompatibility antigen presented by H2-M3," J. Immunol.
156(9): 3301-7). It has also been shown that allelic variation at a single
residue in the
ND 1 peptide renders cells displaying foreign alleles susceptible to lysis by
specific
cytotoxic T cells (Loveland et al. 1990. 60(6): 971-80). A similar system has
been
identified in rats, although the mitochondria) peptide which is responsible
for
histocompatibility in the rat is not the same as the allelic ND1 peptide from
mice
(Davies et al., 1991, "Generation of T cells with lytic specificity for
atypical antigens.
I. A mitochondria) antigen in the rat," J. Exp. Med. 173: 823-32).
Thus, mitochondria) peptides displayed at the cell surface can serve as
histocompatibiliy antigens, seeing as two separate systems have been
identified in
mice and rats, respectively. There is no reason to believe that similar
systems would
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not be present in other mammals. Therefore, foreign mitochondria would be
expected
to result in the rejection of therapeutic tissue generated by nuclear transfer
technology. Instead, using the methods of the present invention, the present
inventors
have surprisingly found in performing the methods of the present invention
that
nuclear transfer generated cells having allogeneic mitochondria are not
rejected when
transplanted into the nuclear donor.
Despite the fact that cloned tissues having allogeneic mitochondria were not
rejected after transplant, the question of transplant compatibility becomes
even more
relevant when such cells are transfected with a transgene or undergo some
other
genetic manipulation in order to modify, supplement or bolster the function of
the
transplanted tissue. Accordingly, the present invention provides methods and
animal
models for testing the immune compatibility of cloned cells or tissues in an
animal
model, and for enhancing the immune compatibility of such cells or tissues as
needed.
Generally, such methods comprise:
I S a. obtaining a cell from a donor animal;
b. transferring the nucleus from said cell into a recipient oocyte or
other suitable recipient cell to generate an embryo and
optionally introducing a therapeutic heterologous DNA;
c. isolating an embryonic disc, inner cell mass, and/or stem cell
from said embryo;
d. injecting said disc and/or stem cell into said donor animal at the
same time as control embryonic disc and/or stem cell; and
e. examining the injection sites for teratoma formation, and signs
of subsequent rejection.
For the purposes of the present invention, a teratoma is defined as a group of
differentiated cells containing derivatives of mesoderm, endoderm, or ectoderm
resulting from totipotent cells. A control embryonic disc, inner cell mass, or
stem cell
is one which was not generated using a donor cell from the test animal
(allogeneic or
xenogeneic nuclear DNA), and therefore, the teratoma thus generated from such
disc
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or cell is expected to be rejected in the donor animal, or alternatively, may
never
develop at all. Teratomas generated using a nucleus from the donor animal
(isogenic)
and an allogeneic recipient oocyte or other suitable recipient cell would also
be
expected to be rejected when used in transplantation due to presentation of
mitochondria) alleles as histocompatibility antigens. Thus, the fact that such
therapeutic tissues do not lead to transplant rejection is truly surprising
indeed.
Donor and control embryonic discs, inner cell mass, and/or stem cells are
generally injected intramuscularly, introduced under the renal capsule,
subcutaneously
or into the paralumbar fascia. Where a teratoma is formed, it is removed and
examined for the presence of germ layers, which may further be separated for
the
purpose of detecting or isolating specific cell types. While teratoma
formation may
give an initial indication of immune compatibility, specific cell types may be
generated and re-introduced into the donor animal to further test immune
compatibility, particularly where the transfected heterologous gene is
expressed from
a cell-type specific promoter. Given that the cloned tissues of the present
invention
having allogeneic mitochondria were not rejected, this system is ideal for
testing the
affect of a transgene on tissue compatibility whereby the cell from said donor
animal
is transfected with a heterologous gene prior to nuclear transfer.
Generally, the methods of the invention may be performed using any cell from
the donor animal. Suitable cells include by way of example immune cells such
as B
cells, T cells, dendritic cells, skin cells such as keratinocytes, epithelial
cells,
chondrocytes, cumulus cells, neural cells, cardiac cells, esophagial cells,
primordial
germ cells, cells of various organs including the liver, stomach, intestines,
lung,
kidneys, etc. In general, the most appropriate cells are easily propagatable
in tissue
culture and can be easily transfected. Preferably, cell types for transfecting
heterologous DNA and performing nuclear transfer are fibroblasts.
The animal model may be any animal suitable for generating teratomas and
studying immune compatibility. A preferred animal is an ungulate, and more
preferred is a bovine. Alternatively, the animal may be a non-human primate,
e.g., a
baboon or cynomolgus monkey. Large animals are preferred because they may give
rise to larger teratomas, thereby providing more cells for immunological
evaluation
and for transplantation. Suitable animals include by way of example pigs,
dogs,
horses, buffalo and goats.
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Also included in the present invention are methods of testing the immune
compatibility of cloned teratomas in cross-species animal models, e.g., where
the
nucleus of the donor species is inserted into a recipient oocyte or other
suitable
recipient cell of another species (xenogeneic). Cloned teratomas having the
mitochondria from the recipient cell may then be tested for immune
compatibility by
injecting an embryonic disc, inner cell mass, and/or stem cell into the donor
animal.
Particularly preferred are cross-species models involving closely related
species,
where the mitochondria) proteins of the recipient cell would be expected to
function
in combination with the donor nucleus.
For instance, according to a report in the New York Times on November 12,
1998 (Nicholas Wade, "Human Cells Revert to Embryo State, Scientists Assert"),
although cow mitochondria would not be expected to work with a human nucleus,
the
mitochondria of chimpanzees and gorillas would be expected to be functional in
human cells. In fact, as noted on the website www~lobalchan eg com, scientists
have
already made chimeric "geep" (combined sheep and goat), and "camas" (combined
camels and lamas), suggesting that the cells and cellular organelles of
closely related
species would be functionally compatible (see also "Bush telegraph on
chimeras,"
The Daily Telegraph, January 22, 1998, p. 27; "It's a geep: cross-breeding
goats and
sheep," Time, February 27, 1984, p. 71; "Meet the geep: part goat - part
sheep,"
i n e, May 1984, 5: 6). According to Jakovcic et al. (1975, "Sequence homology
between mitochondria) DNAs of different eukaryotes," Biochem. 14(10): 2043-
50),
evolutionary divergence of mtDNA sequences appears to have occurred at rates
similar to that for unique sequence nuclear DNA.
Such cross-species models have particular relevance to the study of
xenotransplantation, and would provide a convenient model for identifying the
mitochondria) proteins that serve as histocompatibility antigens. If the
mitochondria
of the recipient cell prove to be functionally, but not immunologically,
compatible
using the teratoma model, it will be possible to identify mitochondria)
antigens and
peptides which are displayed on the cell surface but may not exhibit allelic
variation
within a single species. Such a model will facilitate recombinant DNA
methodology
geared toward replacing the relevant mitochondria) antigens in the recipient
cell with
those from the nuclear transfer donor, in order to further enhance the immune
compatibility of the cloned cells and tissues for transplantation therapy.
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For instance, if cloned "cross-species" teratomas also show signs of
rejection,
steps may be taken in accordance with the invention to ensure that cloned
cells and
tissues are compatible with the nuclear donor, for instance, by selecting
recipient cells
which express compatible mitochondria) antigens, or by replacing the
histocompatible
mitochondria) epitopes. In fact, one group of researchers has reported
complete
replacement of endogenous mitochondria) DNA in one Drosophila species with the
mitochondria) DNA of another (Niki et al. 1989. Complete replacement of
mitochondria) DNA in Drosophila. Nature 341(6242): 551-2. Thus, it should be
possible to engineer recipient cells that have a desired mitochondria)
phenotype for
any particular nuclear transfer donor, or even a mixture of mitochondria)
phenotypes,
i.e., isogenic and allogeneic, or isogeneic and cross-species.
Mitochondria) genes or DNA segments responsible for mitochondria) antigen
histocompatibility, particularly in cross-species models, may be readily
identified
using the methods of the present invention. For instance, isogenic nuclei from
a
designated mammalian nuclear donor can be transferred into different
allogeneic
mitochondria) backgrounds of a closely related species, and such cells may be
used to
immunize the nuclear transfer donor in order to isolate and identify
antibodies and
lymphocytes specific for mitochondria) epitopes. By comparing the
specificities of
the panels of antibodies and lymphocytes achieved by immunizing the nuclear
donor,
it is possible to identify mitochondria) antigens and epitopes that result in
immune
recognition and possibly graft rejection in cross-species models.
Identification of
such mitochondria) antigens and epitopes will allow replacement of the
corresponding
encoding DNA, such that transplant rejection of cross-species nuclear transfer
generated cloned tissues may be avoided.
Thus, the present invention includes methods of identifying mitochondria)
histocomptibility antigens using cross-species nuclear transfer, comprising:
obtaining cells from a donor mammal;
transfernng nuclei from said donor mammal into at least two recipient oocytes
or other suitable recipient cells of a mammalian species other than said
nuclear donor
to generate embryos, wherein said at least two recipient cells are allogeneic
with
regard to mitochondria) DNA;
isolating embryonic discs and/or stem cells from said embryos;
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injecting said discs and/or stem cells separately back into said donor mammal
as to generate a specific panel of antibodies and/or lymphocytes; and
comparing panels of antibodies and/or lymphocytes generated in response to
said allogeneic mitochondria) backgrounds in order to identify mitochondria)
antigens
and/or epitopes that are recognized by the immune system of said donor mammal.
Antibodies and lymphocytes (both helper and cytotoxic T-cells and B-cells)
specific for the mitochondria) antigens identified in such methods are also
encompassed by the present invention, as are the mitochondria) peptides,
antigens,
and DNAs or DNA fragments encoding the same.
The ability to re-clone cloned mammals and generate a line of cloned
mammals that are isogenic for both nuclear and mitochondria) DNA allows for
concurrent injection of the cross-species cloned cells containing allogeneic
mitochondria into separate mammals, thereby facilitating the retrieval of
panels of
antibodies and lymphocytes specific for different mitochondria) backgrounds.
Methods of recloning cloned mammals based on the observation that nuclear
transfer
can be used to rejuvenate senescent cells are disclosed in commonly assigned,
copending Application Serial No. , filed concurrently herewith and
incorporated by reference in its entirety. Of course, it is also possible to
generate
cloned mammals having isogenic mitochondria) DNA by performing nuclear
transfer
from a single donor using multiple oocytes or other suitable recipient cells
from a
single recipient mammal or cell line. Thus the methods of the present
invention may
also be performed wherein said discs and/or stem cells are injected into
separate
mammals which are isogenic to the nuclear donor with respect to both nuclear
and
mitochondria) DNA in order to isolate panels of antibodies and/or lymphocytes.
The present invention also encompasses methods of generating therapeutic
cloned tissue for transplant which express a heterologous protein. The
heterologous
DNAs to be used in the methods of the present invention may encode a
therapeutic
protein to be expressed in a transplant recipient, but may also be a reporter
gene for
the purpose of monitoring gene expression in the teratoma. The reporter gene
may be
any which is convenient for monitoring gene expression, but is preferably
selected
from the group consisting of green flourescent protein (GFP), beta-
galactosidase,
luciferase, variants thereof, antibiotic resistance markers, or other markers.
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Also, the use of tissue-specific promoters or tissue specific enhancers
provides
a means of selecting for expression of heterologous DNAs in desired tissue
types.
Alternatively, the cells may be selected based on the expression
characteristics of cell
surface markers. For example, hematopoietic stem cells may be selected based
on
CD34 expression.
While the donor cell may also contain deletions and insertions into the genome
that disrupt or modify the expression of native genes, preferably the donor
cell is
transfected with a heterologous gene that encodes a protein that is secreted
and
performs a therapeutic function in the intended transplant recipient, i.e.,
replaces a
native gene which is mutated, or is not expressed. Where it is found that the
expressed protein generates an immune response, the animal used in the animal
model
to test immune compatibility may then be used for the evaluation of the immune
response and the isolation of antibodies or cytotoxic T cell clones.
The teratomas generated in animals used to test immune compatibility of
cloned tissues will also be useful for the study of molecular signals that
control cell
differentiation and development. For instance, reporter gene constructs
designed with
putative developmental promoters, enhancers, repressors or other gene control
sequences may be inserted into the donor nucleus prior to nuclear transfer,
and the
teratomas may then be monitored visually or by other means to see at which
stage
reporter gene expression is turned on.
As described above, the differentiated teratoma cells may be separated and
used to individually test the immune compatibility of a particular cell or
tissue. Once
a particular cell type of interest is identified, it may be used to engineer a
tissue using
the methods described herein and known in the art. The animal models disclosed
find
particular use in testing new matrix materials in tissue engineering for
immune
compatibility. Preferred engineered tissues of the present invention are
selected from
the group consisting of smooth muscle, skeletal muscle, cardiac muscle, skin,
kidney
and nervous tissue.
Thus, the present invention also concerns methods of generating immune
compatible tissues for transplantation, comprising:
obtaining a donor cell from an intended transplant recipient;
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b. transferring the nucleus from said cell into a recipient oocyte or
other suitable recipient cell to generate an embryo;
c. isolating an embryonic disc, inner cell mass, and/or stem cell
from said embryo;
d. injecting said disc, inner cell mass, and/or stem cell into an
immune compromised animal;
e. isolating the resulting teratoma;
f. isolating from the teratoma a cell of the type required for
transplantation, and optionally expanding said cells in vitro
using a growth factor; and
g. engineering a tissue from said cells or combinations of cells.
It will also be possible as the signals for cell differentiation and
development
are identified to produce the desired cell types for tissue engineering and
transplantation without prior teratoma formation, because the development of
particular cell types will be directed in vitro. Alternatively, at least with
mammals
other than humans, it is currently possible to acquire cloned tissues directly
from
growing embryos or fetuses rather than generate a teratoma. Humans may be
next,
however, at least for harvesting cell types that develop during the first two
weeks of
embryogenesis, if a recent recommendation by a high ranking British science
and
ethics commission results in legislation (see Weiss, "British panel urges
allowing
human embryo cloning," The Washington Post, page A26, August 17, 2000).
Tissue engineering may be effected, e.g., using three-dimensional scaffolds or
biodegradable polymers such as are used in the construction of dissolvable
sutures.
Such methods have been well reported in the patent and non-patent literature
by
companies such as Tissue Engineering, Inc. and Organogenesis. Examples of
patents
and references in the area of tissue engineering include U.S. Patent
5,948,429,
5,709,934, 5,983,888, 5,891,558, 5,709,934, 5,851,290, 5,800,537, 5,882,929,
5,800,537, 5,891,558, 5,709,934, 5,891,617, 5,518,878, 5,766,937, 5,733,337,
5,718,012, 5,712,163, and 5,256,418, all of which are incorporated by
reference in
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their entirety. Also, reference is made to numerous patents and literature
references
by Robert Langer and John Vacanti, who are both prolific in the area of tissue
regeneration research. As discussed in many of there patents, it may be
desirable to
include biologicals that facilitate blood tissue development, i.e., growth
factors and
other compounds that promote angiogensis.
In particular, the immune-compatible tissues and cells generated are useful in
methods of providing a patient in need of a transplant with an immune-
compatible
transplant. Such a method further comprises, in addition to the above steps,
transplanting said engineered tissue into a patient. The fact that the present
inventors
have surprisingly found that cloned cells containing isogenic nuclear DNA and
allogeneic mitochondria) DNA do not induce transplant rejection has particular
relevance for transplants which replace native cells suffering from
mitochondria)
damage, for instance as in amythrophic lateral sclerosis (ALS), or Leber's
hereditary
optic neuropathy (LHON). In such cases, cloned tissue having isogenic nuclear
DNA
and allogeneic mitochondria) DNA that does not induce an immune reaction is
the
most ideal tissue for transplantation in that such tissue not only provides
the closest
histocompatibility match, but it also effectuates mitochondria) gene therapy
in that
tissue containing damaged mitochondria is replaced.
For instance, Dhaliwal and colleagues recently demonstrated that brain tissue
from patients with ALS had a thirty-fold higher incidence of the "common
mutation,"
which is a 4977 base-pair mutation deletion observed in various tissues of
patients
with mitochondria) and other disorders ("Mitochondria) DNA deletion mutation
levels
are elevated in ALS brains," Mol. Neurosci. 11(113): 2507-9). In fact, an
accumulation of mtDNA49~7 has been observed in the brains, hearts and muscles
of
healthy older individuals suggesting a contribution to the aging process in
these
tissues (Soong and Amheim, 1995, Methods Neurosci. 26: 105-28). Thus, cloned
tissues having allogeneic "young" mitochondria) DNA might provide an advantage
over the patient's own cells by virtue of the absence of age-related
mitochondria)
mutations.
Mitochondria) DNA is believed to be more susceptible to age-related
mutations than is genomic DNA because of the relative lack of DNA repair
mechanisms and histones (Dhaliwal et al. 2000). However, there are also
hereditary
mitochondria) mutations transmitted maternally that manifest themselves in
particular
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tissues, that would benefit by the cloning and tissue engineering techniques
in the
present application.
For instance, Leber's hereditary optic neuropathy (LHON) is a rare disorder of
the optic nerve that causes legal blindness in most patients that it affects.
It is caused
by a mutation in the mitochondria) DNA that is passed maternally, however, the
disease typically manifests itself later in life (sudden loss of vision in the
first eye
typically occurs at the age of 10-50) (Zickermann et al., 1998, "Analysis of
the
pathogenic human mitochondria) mutation ND1/3460, and mutations of strictly
conserved residues in its vicinity . . .," Biochem. 37(34): 11792-6).
Researchers at the
Molecular Ophthalmology Laboratory at the University of Iowa have developed an
improved method for detecting the mutation, which is used to diagnose LHON.
The cloning, tissue engineering, and transplantation techniques of the present
invention will be especially valuable for replacing diseased tissue linked to
mitochondria) mutations in that the cloned tissues will typically possess
isogenic
nuclear DNA, but allogeneic mitochondria) DNA. Therefore, for instance,
engineered
nervous tissue for transplantation into LHON patients will effectuate gene
therapy of
the mitochondria) DNA while at the same time, replace the diseased optic nerve
tissue.
As described above, said donor cell may be genetically altered prior to
nuclear
transfer by the transfection of at least one heterologous gene, or the
disruption or
replacement of at least one native gene. Such a genomic modification is
particularly
useful where the transplant recipient's own genome fails to express a required
protein,
or expresses a mutated protein such that the original tissue or organ failed
to function
properly. Alternatively or additionally, if prior tests of immune
compatibility suggest
some rejection is anticipated, e.g., due to allogeneic or xenogeneic
differences in
mitochondria) DNA, the donor cell may be transfected with genes expressing
proteins
that deter or decrease immune rejection prior to nuclear transfer.
The methods of the present invention are particularly useful for repairing and
replacing tissues damage by autoimmune diseases due to the aberrant expression
of
self peptides. For instance, primary biliary cirrhosis (PBC) is a chronic
autoimmune
liver disease characterized by progressive inflammatory obliteration of the
intrahepatic bile ducts ultimately leading to cirrhosis (Melegh et al., 2000,
"Autoantibodies against subunits of pyruvate dehydrogenase and citrate
synthase in a
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case of paediatric biliary cirrhosis," Gut 2: 753-6). The disease is
characterized by
decreased tolerance to self mitochondria) proteins, and is associated with
high titers of
anti-mitochondria) antibodies, which may be detected using techniques known in
the
art (Leung et al., 1991, "Use of designer recombinant mitochondria) antigens
in the
diagnosis of primary biliary cirrhosis," Hepatol. 15(3): 367-72). Liver
transplantation
has become the treatment of choice with patients with advanced disease (Sebagh
et
al., 1998, "Histological features predictive of recurrence of primary biliary
cirrhosis
after liver transplant," Transplantation 65(10): 1328-33).
The anti-mitochondria) antibodies in PBC typically recognize a restricted
epitope on the E2 subunit of the pyruvate dehydrogenase complex (PDC), which
is a
nuclear encoded protein which is normally transported into the mitochondria
and
loosely associated with the inner membrane. Although the PDC protein is
normally
shielded from the immune system, patients having PBC have been shown to
express
PDC-E2 on the surface of biliary epithelial cells (Joplin et al., 1992,
"Distribution of
dihydrolipoamide acetyltransferase (E2) in the liver and portal lymph nodes of
patients with primary biliary cirrhosis: an immunohistochemical study,"
Hepatology
14: 442-7). Thus, one theory as to how PBC is initiated is that a nuclear
genetic
alteration affects the transport of PDC-E2 to the mitochondria, i.e., such as
mutations
in the leader sequence that direct E2 to the outer membrane (Bjorkland and
Totterman, 1994, "Is primary biliary cirrhosis an autoimmune disease?" Scand.
J.
Gastroenterol. 29 Suppl. 204: 32-9).
Thus, in the case of PBC, the nuclear transfer-generated cells can be
corrected
for the nuclear defects that lead to the autoimmune disease prior to
generation of the
liver cells and tissues for transplantation, i.e., by replacing the mutated
leader
sequence. As a result, the cloned cells and tissues used for transplantation
into a PBC
patient would not only provide the closest immune compatible tissue to avoid
rejection, but also effectuate gene therapy which repairs a nuclear gene
linked to the
autoimmune disease itself. The methods of the present invention are equally as
valuable for the transplantation and gene therapy of any diseased tissue where
the
nuclear mutations associated with the disease process have been identified,
e.g., for
the treatment of burns, blood disorders, cancer, chronic pain, diabetes,
dwarfism,
epilepsy, heart disease such as myocardial infarction, hemophilic,
infertility, kidney
disease, liver disease, osteoarthritis, osteoporosis, stroke, affective
disorders,
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Alzheimer's disease, enzymatic defects, Huntington's disease,
hypocholesterolemine,
hypoparathyroidase, immunodeficiencies, Lou Gehrig's disease, macular
degeneration, multiple sclerosis, muscular dystrophy, Parkinson's disease,
rheumatoid
arthritis, and spinal cord injuries.
In this regard, it is pertinent to note that the present inventors have also
discovered that the cloning procedures of the present invention enables the
rejuvenation of senescent cells, thereby foregoing any concerns regarding the
genetic
age of cloned tissues. The disclosure of U.S. application Serial No. 09/ ,
which is co-owned with the present application, reports the inventors'
surprising
observations relating to the rejuvenation of primary cells using nuclear
transfer, and is
herein incorporated in its entirety. The finding that the cloning process
rejuvenates
older cells is particularly relevant for designing therapeutic tissues
expressing more
than one heterologous gene, or having more than one gene knocked out, because
such
tissues can be generated by cloning and re-cloning primary cells of the same
genetic
background.
It is also possible to effectuate changes to the mitochondria) DNA of the
recipient cell using techniques known in the art (see Wheeler et al. 1997.
Modification
of the mouse mitochondria) genome by insertion of an exogenous gene. Gene
198(1-
2): 203-9; Yamaoka et al. 2000. Complete repopulation of mouse mitochondria)
DNA-less cells with rat mitochondria) DNA . . . Genetics 155(1): 301-7). This
may
be helpful for generating immune compatible cells and tissues for
transplantation,
particularly in the case where mitochondria) antigens are displayed by the
cloned
cells, and generate an immune response when the cloned cells are transplanted
back
into the nuclear donor. Alternatively, if pretesting shows that transplant
rejection due
to mitochondria) DNA differences is anticipated, particularly in the case of
xenogeneic mitochondria, the suitable recipient cell may be particularly
selected
based on mitochondria) compatibility.
Although any animal may benefit from the cells and tissues generated by the
disclosed methods, a preferred transplant recipient is a human. When the
intended
transplant recipient is a human, teratomas may be formed following nuclear
transfer,
i.e., of a fibroblast nucleus, from said human into any human recipient
oocyte,
because it is the genome of the donor (intended transplant recipient) that
reprograms
the cell for development. Teratomas generated from human nuclear donors and
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recipients may be formed in and isolated from an immune compromised animal,
such
as a skid or nude mouse.
As described above, the teratomas generated may be removed and examined
for the formation of germ layers, and such germ layers may be further
separated or
differentiated into distinct cell types. Distinct cell types may then be used
to engineer
tissues for transplantation. Preferably, said tissues are selected from the
group
consisting of smooth muscle, skeletal muscle, cardiac muscle, skin, kidney and
nervous tissue. Also encompassed are the tissues and cells generated by the
disclosed
methods.
The concept of human "therapeutic cloning" is to transfer the nucleus from
one of the patient's cells, i.e., a fibroblast cell, into an enucleated
recipient oocyte or
other suitable recipient cell. After reprogramming, the donated somatic
nucleus
regains its totipotency and is able to initiate a round of embryonic
development.
Pluripotent stem cells derived from the resulting embryo carry the nuclear
genome of
the patient, and are then induced to differentiate into replacement cells,
such as
cardiomyocytes to replace damaged heart tissue, insulin-producing n-cells for
patients
with diabetes, chrondrocytes for osteoarthritis, or dopaminergic neurons to
treat
Parkinson's disease.
The methods of the invention should eliminate or at least substantially
alleviate the immune responses associated with transplantation of these
various
tissues, and therefore abrogate the requirement for immunosuppressive drugs,
such as
cyclosporine, imoran, FK-506, glucocorticoids, and variants thereof, which
carry the
risk of a wide variety of serious complications, including cancer, infection,
renal
failure and osteoporosis. However, at least in some instances, it may still be
advisable
to utilize anti-rejection agents at least initially. As discussed above, the
transplanted
cells may not be immunologically identical to the transplant recipient's
cells, even
though the nucleus of one of the recipient's cells served as the donor. This
could be
caused by mitochondria) DNA differences particularly in the case of xenogeneic
mitochondria, or antigenic differences that may result from transfected
heterologous
DNA or because of the artificial environment used to affect nuclear transfer.
In
particular, such an environment does not mimic identically the cellular
environment
that exits during embryonic development.
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For example, it is known that cells cultured for prolonged periods may be
antigenically different as a result of culturing (a phenomenon known as
"antigenic
drift"). Therefore, it may still be desirable to tolerize the cells or tissues
prior to
transplantation, e.g. by treatment with soluble CD40, CD40-ligand antagonists,
low
temperature culture, use of antibodies that mask donor antigens, or by
expression of
UV light (e.g., islets).
Although not limiting, the scope and spirit of the invention are illustrated
by
reference to the following discussion and examples.
EXAMPLE I
This experiment was designed to test the immune compatibility of a nuclear
transfer-generated cells in a pre-clinical large animal model: cattle (Bos
taurus).
Three adult Holstein steers approximately 8-10 months old (weighing
approximately 500-1000 Ibs) were purchased from Thomas Morns, Inc., Maryland,
and shipped to the South Deerfield Farm at the University of Massachusetts,
Amherst.
1 S To obtain fibroblasts for nuclear transfer, skin biopsies were obtained
from each of
the animals by ear notch. A plasmid which expresses a reporter gene encoding
enhanced green fluorescent protein (eGFP) was transfected into the cells, and
transfected cells were selected with neomycin. Purified cells, analyzed by PCR
and/
or FISH, were used for nuclear transfer as described previously in Nature
(1998)
Biotechnol. 16: 642-646, herein incorporated by reference.
Isolated embryos having at least one cell, or embryonic discs/ inner cell mass
or stem cells generated from bovine blastocysts/ stem cells are then injected
into the
paralumbar fascia of the donor steers (two sites with experimental (same
animal) stem
cells, two sites with experimental (same animal) embryonic discs, two sites
with inner
cell mass, and four sites with control (different animal) stem cells, per
animal). After
two months, the muscle is examined for teratoma formation. Any tumors
identified
are removed for histological analysis.
The procedure is performed on the standing animal using 20 mg Xylazine/ 8
mg Butorphanol Tatrate administered IV in the tail vein. The paralumbar fascia
area
is clipped and surgically prepared, using 100 ml of 2% Lidocaine as a local
anesthetic
administered as a paralumbar block. The animals should be given antibiotics
for three
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days post-surgically as a precautionary measure (Cefilofur Hcl 50 mg/cc @
lcc/100
pounds). Immediately following surgery a single injection intramuscularly or
under
the kidney capsule of Flunixin Meglumine @ lcc/100 pounds may be given to
control
pain and swelling at the surgical site. If teratoma formation does not occur
at the
paralumbar fascia, other sites may be analyzed, i.e,. subcutaneously.
It is expected that "same animal" stem cells will survive in the recipient
(donor of nucleus) animal in contrast to "different animal" stem cells, or
survive at
least better or longer depending on the cytotoxic T cell response or other
immune
reaction to foreign mitochondria) peptides. Furthermore, it is expected that
cells from
all three germ layers, i.e., ectoderm, mesoderm, and endoderm, will be
observed in
"same animal" teratomas.
EXAMPLE 2
This example was designed to test teratoma formation in an immune-
compromised animal model. This example is relevant to the methods whereby
cloned, nuclear transfer-generated cells from a patient in need of a
transplant may be
grown in a SCID mouse or other immune-compromised animal in order to generate
differentiated cells for isolation and design of engineered tissues for
transplant.
ES cells transfected with GFP were derived from two adult Holstein steers
(two different ES cell lines were derived from each animal). ICMs were derived
from
12-day-old blastocysts.
Cell preparation and injection procedure:
Cells were cut into pieces (sections of no more than about 100 cells each) and
loaded into a 1 ml syringe, no more than 200 microliters each, and preferably
100
microliters.
ICMS were mechanically isolated and loaded into a 1-ml syringe 100 to 150
microliters.
Cells were kept at room temperature in HECM-Hepes.
Twenty-two-gauge needles were used for injection procedures. Cells were
injected into the skeletal muscle of the hind leg of SCID mice.
Mou Treatment Amount I Observatio
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se # ns
1 ICM day 14 from cow 6 100
#
25
2 ICM day 14 from cow 9 3 ICMs
#22 were found
left
inside the
syringe
3 Monkey cross-species 90 -
(into bovine) 4-8 cell
embryos
4 ES 22.B One plate (30 -
mm)
ES 22.B Three plates -
6 ES 22.C One plate
7 ES 22.C Three plates -
8 ES 25.E One plate -
9 ES 25.E Three plates -
ES 25.F One plate -
right
10 ES 25.F Three plates -
left
Bovine stem cells and ICMs that were injected into the skeletal muscle of the
SCID mice were retrieved after 7-8 weeks (although it is possible to let the
cells go
longer, or remove them sooner). A small nodular lesion was identified in two
of the
mice which received ES cell injections (mice #s 7 and 9).
Gross Examination:
A 2X2 mm sized milky white nodule was retrieved from the right hind leg
near the sciatic nerve of mouse #7. This corresponds with the injection of
three plates
of ES 22.C. A 1X 1 mm sized milky white nodule was identified within the
muscle
10 tissue of mouse # 9 which corresponds with the injection of the three
plates of ES
25.F.
Histologic Analysis:
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Mouse #7: Histologic sections of the teratoma were analyzed with
hematoxylin and eosin (H&E), safranin-O and immunocytochemistry using
cytokeratin (AE1/AE3) and alpha smooth muscle actin antibodies.
H&E: The injected cells formed a round tissue mass within the skeletal muscle
tissue. The teratoma consisted of four different sized compartments with
cellular debris in the center. Tissue formation was noted on the wall of each
compartment (data not shown). Epithelial (round nuclei) and stromal cells
(spindle-
shaped nuclei) were observed in the teratoma tissue (data not shown). There
was no
evidence of cartilage, bone or adipose tissue.
Safranin-O: Negative staining was obtained, which indicates the
absence of cartilage tissue formation.
Immunocytochemistry with AE1/AE3 antibodies: The teratoma
section showed positively stained epithelial cells (data not shown).
Immunocytochemistry with alpha smooth actin antibodies: Small
islands of positively stained muscle tissue was observed within the teratoma
(data not
shown). The retrieved tissue demonstrated epithelial, smooth muscle and
stromal
tissue components. Cartilage, bone and adipose tissues were not identified in
the
teratoma.
Mouse # 9: Histologic analysis on the retrieved nodule demonstrated a
skeletal muscle mass. Microscopic examination showed that no other tissues
formed.
EXAMPLE 3
To realize the full potential of therapeutic cloning, it will be important to
reconstitute more complex tissues and organs in vitro. Although cloning would
eliminate or greatly alleviate the most critical problem--immune compatibility-
-there
is still the task of putting the cells together to create or recreate
functional structures.
For example, myocardial infarction is one of the most common diagnoses
occurring in hospitalized patients in western countries. While injection of
individual
or small groups of cardiomyocytes could aid in the treatment of some localized
infarcts, this approach is unlikely to be of value in patients with more
extended
ischemic injury, where the risk of scar formation, cardiac rupture and other
complications is much greater. Tissue engineering offers the possibility of
organizing
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the cells into three-dimensional myocardial "patches" which could be used to
repair
the damaged portions of the heart. For myocardium and other relatively simple
tissues, such as skin and blood vessel substitutes, this may involve seeding
cells onto
masses or sheets of polymeric scaffold. Creating more complex, vital organs,
such as
the kidney, liver, or even an entire heart will require assembling different
cell types
and materials in greater combinatorial complexity.
To engineer tissues for use in the animal model, bovine inner cell
mass/embryonic discs/stem cells may be generated as described above, and
injected
into the rear leg muscles of nude or SCID mice. Seven to eight weeks after
injection,
the resulting teratomas are removed and various cell types are isolated and
grown in
culture. A number of tissues may be generated from the cloned cells, including
smooth and/or skeletal muscle, sheets or "patches" of cardiomyocytes, elastic
cartilage, skin, (including the placement of hair follicles), and kidney,
including
miniature kidneys that excrete urine. These tissues/ organ constructs are then
transplanted back into the original adult animal from which the donor cell
biopsy was
obtained.
The following data demonstrates that tissues isogenic for nuclear DNA and
allogeneic for mitochondria) DNA form stable transplantation grafts that do
not illicit
an immune response in the nuclear transfer host. This supports the utility of
such
cloned cells and tissues for many medical applications, which is quite
surprising given
the cytotoxic T cell response to mitochondria) antigens observed in different
species
of mice in response to mitochondria) histocompatibility antigens.
CELL CULTURE AND SEEDING
Cells from bovine kidney, heart, skeletal muscle, cartilage and skin were
harvested from cloned and allogenic (control) 40 day old fetuses, and expanded
separately in vitro.
Kidney:
The kidney tissue was cut into small pieces (1 mm') using sharp tenotomy
scissors. The kidney tissue fragments were digested using collagenase dispase
(1
mg/ml) at 37° C for 30 minutes. The recovered cells were washed with
phosphate
buffered saline and plated in culture dishes. The cells were grown in medium
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consisting of DMEM, HEPES 3.1 g/1, Pen/Strep (5 m1/500 ml), L-glutamine 146
mg/L and FBS 10% (Sigma, St. Louis, MO).
Muscle:
Cardiac and skeletal muscle cells were processed by the tissue explant
technique using Dulbecco's Modified Eagle's Medium (DMEM; HyClone
Laboratories, Inc., Logan, Utah) supplemented with 10% fetal calf serum. The
cells
were incubated in a humidified atmosphere chamber containing 5% COZ and
maintained at 37°C. Both muscle cell types were expanded separately
until desired
cell numbers were obtained. The cells were trypsinized, collected, washed and
counted for seeding.
Polymers:
Unwoven sheets of polyglycolic acid polymers ( 1 x 2 cm) were used as cell
delivery vehicles. The polymer meshes were composed of fibers of 15 ~m in
diameter and an interfiber distance between 0 - 200 um with 95% porosity. The
scaffold was designed to degrade via hydrolysis in 8-12 weeks. The polymers
were
sterilized in ethylene oxide and placed under sterile conditions until cell
delivery.
IMPLANTATION
Athymic Mice:
To determine whether cells obtained from fetal bovine tissue would form
tissue in vivo, cardiomyocytes, skeletal muscle cells and chondrocytes seeded
on
polymer scaffolds were implanted in the dorsal subcutaneous space of athymic
mice.
The animals were sacrificed at 1 week, 1 month and 3 months after implantation
for
analyses (n=4).
Steer:
Each cell type was seeded separately onto polyglycolic acid polymers (1 x 2
cm) at a concentration of 50 x 106 cells/cm3 (n=4 per cell types). The cell-
polymer
scaffolds were implanted into the flank subcutaneous space of the same steer
from
which the cells were cloned. The cells obtained from the control (nuclear
allogeneic)
fetuses were implanted on the contralateral flank of the steer. All implants
were
retrieved after 6 weeks for analysis.
ANALYSES
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Implantation in Athymic Mice:
Five micron sections of formalin fixed paraffin embedded tissue were cut and
stained with hematoxylin and eosin (H&E). Immunocytochemical analyses were
performed using specific antibodies in order to identify the cell type of the
retrieved
tissues. Histochemical analyses using aldehyde fuschin-alcian blue, and
immunocytochemical studies using monoclonal anti-collagen II (Chemicon, St.
Louis,
MO) were used to identify the engineered cartilage structures. Monoclonal
sarcomeric tropomyosin (Sigma, St. Louis, MO) and troponin I (Chemicon,
Temecula, CA) antibodies were used to detect skeletal and cardiac muscle
fibers,
respectively. Immunolabeling was performed using he avidin-biotin detection
system. Sections were counterstained with methyl green.
Implantation in the Steer:
Immunocytochemical and histological analyses:
Five micron sections of formalin fixed paraffin embedded tissue were
cut and stained with hematoxylin and eosin (H&E). Immunocytochemical analyses
were performed using specific antibodies in order to identify the cell type of
the
retrieved tissues. Histochemical analysis using Periodic Acid Schiff (Sigma,
St.
Louis, MO), and immunocytochemical studies using polyclonal anti-alkaline
phosphatase and anti-osteopontin (Chemicon, Temecula, CA) were used to
identify
renal cells. Monoclonal sarcomeric tropomyosin (Sigma, St. Louis ,MO) and
troponin I (Chemicon, Temecula, CA) antibodies were used to detect skeletal
and
cardiac muscle fibers, respectively. Aldehyde fuschin-alcian blue and
monoclonal
anti-collagen II (Chemicon, St. Louis, MO) were used to stain cartilage tissue
implants. Anti-cytokeratins 5/6, AE1/AE3 were employed in order to identify
keratinocytes. Bronchial ciliary antibodies were used in order to detect
respiratory
epithelium. Anti-CD6 antibodies were used in order to identify immune T and B
cells. Immunolabeling was performed using the avidin-biotin detection system.
Sections were counterstained with methyl green.
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RESULTS
The cells grew to confluence, were implanted in the animals with the polymer
scaffolds, and retrieved without complications. At retrieval, the implants
maintained
their initial size without any evidence of fibrosis.
Implants retrieved from the steer:
Histochemical and Immunocytochemical Analyses:
Histological examination demonstrated extensive vascularization
throughout the implants and the presence of multinucleated giant cells were
observed
surrounding the polymer fibers. However, higher number of inflammatory cells
were
present throughout the control allogeneic scaffolds. Histomorphomeric analysis
of
the explanted tissue (i.e., kidney, skeletal, heart, chondrocytes and
keratinocytes)
indicated that there was a statistically significant (p<0.05; student's t-
test) increase in
lymphocytic infiltration of the control implants/constructs (non-cloned)
versus the
cloned tissue types (data not shown). This data suggests that the control
grafts were
undergoing early graft rejection.
Engineered Kidney Tissue:
Histologically, glomeruli-like structures were observed in the retrieved
scaffolds (data not shown). Histochemical analyses using periodic acid schiff
identified renal tubular cells (data not shown). Immunocytochemical studies
with
alkaline phosphatases antibodies confirmed the presence of proximal tubular
cells.
Studies using osteopontin antibodies were negative in the bovine tissue
system.
Engineered Muscle Tissue:
Retrieved cardiac and skeletal muscle cell implants showed spatially
oriented muscle fibers in each instance (data not shown). Immunocytochemical
analysis using tropomyosin antibodies identified skeletal muscle fibers within
the
construct (data not shown). Anti-troponin I stained cardiac muscle fibers
positively
(data not shown).
To prove that the mtDNA of the cloned tissues was from the recipient oocyte,
the mtDNA of the nuclear donor and that of the cloned embryo were sequenced.
Sequence data confirmed that the mtDNAs were indeed different, particularly in
the
d-loop region where there were four different corresponding nucleotides in the
cloned
tissues in comparison with the nuclear donor.
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EXAMPLE 4
The above results suggest that it is possible to generate cloned tissues for
transplantation by nuclear transfer into an allogeneic background, and that
differentiated cells and tissues isolated or constructed from cloned teratomas
or
cultures of embryonic cells can be transplanted back into the donor animal
without
significant signs of rejection. To further confirm that nuclear transfer
technology has
the potential to eliminate the immune responses associated with the
transplantation of
cells and organs despite mitochondria) mismatch, the inventors will next
perform
transplants between full grown clones having different mitochondria)
backgrounds.
For these experiments, two groups of animals were assembled to test
reciprocal skin grafts: (1) four cloned cows (animals CL53-8, CL53-9, CL53-10,
and
CL53-11) at Trans Ova and (2) five cloned goats at LSU. To perform the test,
reciprocal skin grafts (approximately 2-3 cm diameter) are exchanged between
the
two groups of animals. Self grafts will serve as positive controls, whereas
grafts from
genetically unrelated animals will serve as negative controls. The grafts are
monitored
for signs of immune rejection, and will be removed if and when they become
necrotic
and the sites patched. If rejection is observed, second-set grafts would then
be
transplanted in order to confirm the results, which should be rejected in an
accelerated
fashion.
All of the cloned cows and all of the cloned goats carry the same nuclear
genome. However, since mtDNA is transmitted by maternal inheritance, we
predict
the animals are in fact genetic chimeras with different oocyte-derived
mitochondria
(this has already been documented in a number of cloned animals). Experiments
are
underway to obtain the sequence of the mtDNAs from all involved individuals,
so that
polymorphisms which "segregate" in these panels of animals may be readily
correlated with the survival/rejection of skin grafts. If there is a
correlation, in vitro
assays will be performed to identify target peptides, and the associated
mtDNAs
which encode the peptides will be isolated.
The experiments aimed at determining mitochondria) DNA polymorphisms
will also reveal information about chimerism levels in mtDNA in general. For
instance, once the sequences of the mtDNAs are known, a region of maximal
polymorphism will be selected, most likely the D-loop, and this segment will
be
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amplified and cloned. A range of clones may then be sequenced to the determine
extent of variation in this region. With the mtDNA sequences from a sufficient
number of nuclear clones that are allogeneic for mitochondria, an accurate
estimate of
the levels of chimerism may be determined. Blood samples will also be
collected at
intervals to carry out various immunological assays. For histocompatibility,
standard
MLCs and CMLs will be run within the panels and with allogeneic cells.
DISCUSSION
As shown by the data provided above, the present invention demonstrates that
it is possible to obtain cloned differentiated cells and tissues for the
purpose of tissue
engineering and transplantation. The present invention also demonstrates that
stable
grafts can be achieved with nuclear transfer-generated cloned cells having
allogeneic
mitochondria, despite the fact that transplantation rejection would be
expected due to
foreign mitochondria) peptides. In view of the Mta system in mice and similar
systems identified in rats, it is surprising that the bovine tissues
engineered using the
present methods were not rejected when they were transplanted back into the
nuclear
donor.
There could be several reasons why transplant rejection was not observed with
the cloned tissues of the present invention. Without being bound by any
particular
theory, one hypothesis is that the particular MHC molecules in rodents that
present
the Mtf , MiHA and other mitochondria) antigens have evolved out of higher
mammals. Indeed, H-2M3a, the mouse class I molecule that presents the Mtf and
MiHA peptides, is encoded by the M3 gene at the telomeric end of the H-2
complex
on mouse chromosome 17 (Fischer Lindahl et al., "Maternally transmitted
antigen of
mice: a model transplantation antigen," Annu. Rev. Immunol. 1991;9:351-72).
Although many genes in this area of the chromosome are conserved between mouse
and human, for instance, the MHC class I genes in this region appear to have
diverged
and evolved independently between species (Jones et al., 1999, "MHC class I
and
non-class I gene organization in the proximal H2-M region of the mouse,"
Immuno eg netics 49(3): 183-95). In fact, the H2-M region is rich in LI
repeats, which
some have hypothesized is associated with evolutionary flexibility (Yoshino et
al.,
1997, "Genomic evolution of the distal MHC class I region on mouse chromosome
17," H r i a 127(1-2): 141-8).
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Alternatively, perhaps additional mechanisms evolved in higher mammals
which regulate the immune reaction to mitochondria) antigens in the context of
MHC,
particularly seeing as many aging but otherwise healthy tissues in humans have
been
shown to contain mitochondria) age-related mutations (Soong and Amheim, 1995).
The ongoing experiments described in Example 4 will be especially useful for
identifying the polymorphims that exist in a given population of mtDNAs, and
may
serve as a useful model system for identifying the changes that occur in
mtDNAs over
time that may lead to the aberrant display and recognition of mitochondria)
antigens.
In any case, despite what was known and understood about rodent
mitochondria) histocompatibility prior to the present invention, the results
achieved
with the therapeutic cloned bovine tissues described herein would predictably
translate to other ungulates and higher mammals. Thus, the present invention
confirms the therapeutic utility of nuclear transfer-generated cloned tissues
in the
context of transplantation. Further, by providing a model for testing the
immune
compatibility of allogeneic and xenogeneic mitochondria) proteins in an
isogenic
nuclear background, the present invention paves the way for deciphering the
immune
regulatory systems that exist in and between mammals, which contribute to
mitochondria) stability and the separate evolution of species.
27
