JP2003509028A - Method for producing immunocompatible cells and tissues using nuclear transfer technology - Google Patents

Abstract

  (57) [Summary] The present invention relates to methods for making immunocompatible tissues and cells for transplantation and tissue engineering purposes using nuclear transfer and cloning techniques. Also included in the invention are methods for determining the effect on immunocompatibility of the expressed transgene and other genetic manipulations that make up cells and tissues.

Description

Detailed Description of the Invention

This application is US Provisional Application No. 60/152 filed Sep. 7, 1999.
U.S. Patent Application No. 60/1 filed on Sep. 22, 1999 and Sep. 22, 1999.
Claim the benefits of No. 55, 107.

TECHNICAL FIELD The present invention combines the fields of cloning, developmental biology, and tissue engineering to devise immunocompatible tissues and cells for purposes of transplantation. The invention further discloses methods for producing therapeutic cells and tissues for transplantation using nuclear transfer technology, and methods for verifying or assessing immunocompatibility of such tissues.

BACKGROUND OF THE INVENTION [0003] The last decade or so has been characterized by significant advances in the science of cloning, the birth of the cloned sheep, the "Dolly" (Roslin BioMed), the "Mira" (Genzyme Transgenics). ), A triple goat cloned goat and over a dozen cloned cows (ACT) have proved this. The technology that enables cloning has also improved, and it is now possible to clone a mammal using the nucleus from an adult differentiated cell, and the nucleus becomes an oocyte after the nucleus is removed. Once introduced, "reprogramming (repro
Scientists now know to do "granming". US Pat. No. 5,945,577, incorporated herein by reference in its entirety.
See issue.

The fact that embryos as well as embryonic stem cells can be produced using nuclei from adult differentiated cells has interesting implications in the field of organ, cell and tissue transplantation. Thousands of patients now await donors of suitable organs, facing both availability and compatibility issues awaiting translocation. If it is possible to create embryonic stem cells produced from the nucleus of cells taken from a patient in need of transplantation and induce them to differentiate into the cell type required for transplantation, It is possible to eliminate the rejection reaction and the risk of immunosuppressants.

Embryonic stem cells have been induced to differentiate into cells from three different germ layers. For example, Anderson et al. Have reported inner cell mass (IC) from bovine and porcine fibroblasts.
M) and scutella were shown to develop into teratomas containing differentiated cell types derived from ectoderm, mesoderm and endoderm when transplanted under the renal sac of athymic mice. A nimal Repro. Sci. 45: 231-240 (1996). Furthermore,
The signals of development that trigger cell differentiation are beginning to be deciphered. For example, Gou
rdie et al., Embryonic muscle cell impulse generation Perkinje (Purkinje)
It was shown to differentiate into fiber cells. Proc. Natl. Acad. Sci . 9
5: 6815-6818 (June 1998). Furthermore, the University of Medicine and Dentistry of New Jersey (University of Medicine and Dent
Researchers at the Institute of New Jersey (UMDNJ)
Reported transformation of bone marrow cells into neurons (Washington Post, 2000.
(August 15, issue, page A6). Thus, it should be possible to isolate cells that have differentiated from embryonic stem cells or teratomas and induce them to differentiate into specific cell types for use in transplantation.

In addition, it is possible to design a tissue or an organ from differentiated cells and use it for transplantation by using a technique born in the field of tissue engineering. For example, Sh
inoka et al., have synthesized biodegradable (polyglactinopolyglycolic acid pol
The autologous transplantation of a living pulmonary artery was devised by inoculating and culturing the cells on a tubing of γlactinlpolyglycolic acid). L. Tho rac. Cardiovasc. Surg . 115: 536-546 (1998)
). Zund et al. Have shown that endothelin treatment of human fibroblasts prior to inoculation of respirable mesh is useful for producing human tissues such as vessels and heart valves. Eur. J. Cardic-Thorac. Surg . 13: 1
60-164 (1998). Freed et al. Have shown that culturing cells under conditions simulating microgravity is advantageous for producing cartilage and heart tissue. I
n Vitro Cell Dev. Bid. -Animal 33: 381-
385 (May 1997).

However, there are problems in the field of cell development and differentiation, and the field of tissue engineering. For example, the teratomas created by Anderson et al. Were created from spontaneously formed embryos. Thus, the genotype of an embryo is unique to each individual embryo. Such cells are not suitable for transplantation because when transplanted into a donor animal, they, like all allogeneic tissues, still induce transplant rejection. Most autologous tissue engineering, in contrast, has been performed with cells from real recipient animals. Such techniques would not provide suitable transplant organs for patients with defective cells or organs, that is, patients with defects probably due to defective gene expression or expression of mutant genes. Moreover, it would be impossible for a patient's organs to function literally to create new organs using their own cells. Thus, there are many deficiencies in applying the concepts of cell differentiation and development and tissue engineering to the treatment of transplant patients.

SUMMARY OF THE INVENTION The present invention is directed to the uncertainties that still have to be resolved regarding the use of engineered cells and tissues for transplantation. The present invention provides a method for making cloned immunocompatible, developmentally differentiated cells into tissues for transplantation, and methods for using such cells in patients in need of transplantation. Is disclosed. In particular, such tissue can be designed to express a therapeutic protein. Since all tissues and cells for transplantation are made from the same original donor cells by nuclear transfer, all cells of the constructed tissue express the heterologous gene of interest. Therefore, the method of the present invention also provides a valuable new tool for tissue-targeted gene therapy.

The present invention also provides a method of determining whether a particular genetically engineered cell confers an immunocompetent organ for transplantation. For example, the present invention discloses a method for assessing mitochondrial compatibility of cloned cells, and immunocompatibility of transgenically developmentally differentiated cells, especially in animal models. Such assessments provide important information regarding the suitability of therapeutic tissues used for transplantation and provide the basis for controlling these parameters in order to provide immunocompatible tissues. To do.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a method of producing immunocompatible tissue using cloning techniques. Cells and tissues produced by the disclosed methods are also included in the invention, as are stable explants produced by transplantation of engineered tissue. Stable transplants do not inappropriately generate immune or rejection responses when transferred to nuclear donors, or avoid transfer rejection, at least for non-cloned control explants. Defined above as a transplant that provides a substantial improvement.

Cloned cells produced by nuclear transfer are not completely identical to the donor cell or animal, eg, such cells generally lack the donor cell's mitochondrial DNA and acceptor cells. It has acquired the mitochondrial DNA of the oocyte or other cell from which the nuclei have been removed and is generally not produced in the in vivo environment, which is a completely simulated condition that exists during embryogenesis. When these cells are re-implanted into a donor animal, the question arises as to whether they are fully immunocompatible.

For example, ND1 made from the amino terminus of NADH dehydrogenase, and
Mouse mitochondrial peptides, such as the MiHA peptide encoded at the amino terminus of the COI gene, are non-classical MHC class I molecules, such as H-2M3.
has been shown to be present on the surface of cells in combination with beta-2-microglobulin (Vyas et al., 1992, "Biochemical specificity of H-2M3a.
3a) ", J. Immunol. 149 (11): 3605-11; Morse.
1996, "The COI mitochondrial gene encodes a weak histocompatibility antigen expressed by H2-M3 (The COI mitochondria.
l gene encodes a minor histocompatib
ilitity anticipated by H2-M3) ", J
． Immunol. 156 (9): 3301-7). It has also been shown that allelic changes at one residue of the NDI peptide make cells displaying the foreign allele more susceptible to cytolytic effects by T cells with specific cytotoxicity (Loveland). Et al., 1990, 60 (6): 971-80). The mitochondrial peptide involved in rat histocompatibility is not the same as the mouse allelic ND1 peptide, but a similar system has been identified in rat (Dav).
1991, "Generation of T Cells with Cytolytic Specificity for Atypical Antigens I, Rat Mitochondrial Antigen (Generation of T ce
lls with lytic specificity for atypi
cal antigens I.C. A mitochondrial anti
gen in the rat), J. Exp. Med. 173: 823-3
2).

Thus, mitochondrial peptides displayed on the surface of cells can act as histocompatibility antigens, and two separate systems have been identified in mouse and rat, respectively. There is no reason why similar systems do not exist in other mammals. Therefore, exogenous mitochondria are expected to result in the rejection of therapeutic tissue made by nuclear transfer. Instead, we surprisingly find that, in the practice of the method of the invention, cells with allogeneic mitochondria made by nuclear transfer are rejected even when transferred to a nuclear donor. I found that it would not be done.

Despite the fact that cloned tissues with allogeneic mitochondria are not rejected after transplantation, the question of transferability is that such cells transfer the transgene, or It becomes even more acute when some other genetic manipulation is performed to improve, supplement or support the function of explanted tissue. Accordingly, the present invention provides methods and animal models for testing the immunocompatibility of cloned cells or tissues in animal models and optionally enhancing the immunocompatibility of such cells or tissues. Generally such methods are: a. Harvesting cells from a donor animal; b. Transferring the nucleus from the cells to a recipient oocyte or other suitable receptor cell to produce an embryo, optionally introducing therapeutic heterologous DNA; c. Isolating scutellum, inner cell mass and / or stem cells from the embryo; d. Injecting the scutellum and / or stem cells into the donor animal while simultaneously
Or injecting stem cells; and e. Examining the injection site for teratoma formation and subsequent signs of rejection.

For the purposes of the present invention, a teratoma is an ectodermal resulting from cells that have the total potential to form,
It is defined as a group of differentiated cells that include mesoderm or endoderm derivatives. The control scutellum, inner cell mass or stem cell is a test animal (heterogeneous or heterologous DNA
) Was not produced using donor cells of origin, and thus teratomas made from such scutellum or cells are either rejected or never grow in the donor animal. I think that the. Teratomas made with nuclei from donor animals (isogenic) and allogeneic receptor oocytes or other suitable recipient cells are also histocompatibility when used for transplantation. It appears to be rejected due to the presentation of the mitochondrial allele as a sex antigen. Thus, it is truly surprising that such therapeutic tissue does not cause rejection during transplantation.

Donor and control scutellum, inner cell mass and / or stem cells are generally injected intramuscularly, subrenally, subcutaneously or intralumbar fascia. At the place where the teratoma was formed, it is removed and examined for the presence of germ layers, and the germ layers are further isolated for the purpose of detecting specific cell types. The formation of teratomas gives an early indication of immunocompatibility, but creates a specific cell type, especially if the transferred heterologous gene is expressed from a cell type-specific promoter, and the donor animal And reintroduced into to further investigate immunocompatibility. Given that the cloned tissue of the invention with allogeneic mitochondria was not rejected, this system is ideal for testing the effect of transgenes on histocompatibility, whereby the donor Cells from animals are transfected with a heterologous gene prior to nuclear transfer.

In general, the methods of the invention can be practiced with any cell of a donor animal. Examples of suitable cells include immune cells such as B cells, T cells,
Dendritic cells, skin cells such as keratinocytes, epithelial cells, chondrocytes, cumulus cells,
They are cells of various organs including nerve cells, heart cells, esophageal cells, primordial germ cells, liver, stomach, intestines, lungs, kidneys and the like. In general, the most suitable cells are those that are easily grown in tissue culture and can be easily transfected. The cell type into which heterologous DNA is transferred and nuclear transfer is performed is preferably fibroblast.

The animal model may be any animal suitable for forming teratomas and studying immunocompatibility. The preferred animal is ungulate, and the more preferred animal is bovine. Otherwise, the animal is a non-human primate, such as a baboon or cynomolgus monkey. Large animals are preferred because they are more prone to the formation of larger teratomas, thereby providing more cells for immune evaluation and transplantation. Examples of suitable animals include pigs, dogs, horses, buffalo and goats.

Included in the present invention is a method of testing the immunocompatibility of a cloned teratoma in a cross-species animal model. For example, when the nucleus of a donor animal is transplanted into an oocyte or other suitable recipient cell of a (heterologous) recipient of another species. The cloned teratoma with mitochondria from the recipient cells was
Immunocompatibility is tested by injecting the inner cell mass and / or stem cells into the donor animal. Particularly preferred is a cross-species model involving closely related species, where the mitochondrial protein of the recipient cell is expected to function in combination with the donor nucleus.

For example, the report of the New York Times, November 12, 1998 issue (Nich
olas Wade, "Human Cells Return to Embryonic State, Scientists Claim (Huma
n Cells Reverse to Embryo State, Science
The mitochondria of cows cannot be expected to function with human nuclei, whereas the mitochondria of chimpanzees and gorillas are expected to function in human cells, according to the Tists Assert) "). In fact, the website www. globalchange. As described in the com, scientists already chimeric "Jeep (geep)" (a complex of sheep and goats) and "Camas (cama
s) ”(complex of camels and llamas), suggesting that cells and subcellular organs of related species may be functionally compatible (“ Bush, Chimera Telegraph ”, Daily Telegraph, January 22, 1998, p. 27, "Jeep: goat and sheep cross-breeding go.
ats and sheep, "Times, February 27, 1984, p. 71," Meet the Jeep: Some Goats-Partly Sheep (Meet the sheep) "
: Part goat-part sheep) ", Science, 1984.
Year, 5: 6). According to Jakovic et al. (1975, “sequence homology between different eukaryotic mitochondrial DNAs (Sequence hom)).
topology between mitochondrial DNAs of
different eukaryotes) ", Biochem. 14 (10
): 2043-50), the evolutionary spread of mtDNA sequences is believed to occur at a similar rate to that of uniquely-ordered nuclear DNA sequences.

Such cross-species models are particularly relevant in xenotransplantation studies and provide a convenient model for identifying mitochondrial proteins that act as histocompatibility antigens. If the mitochondria of the recipient cells are demonstrated to be immunologically non-functional but functionally compatible with the teratoma, they are displayed at the mitochondrial antigen and the cell surface, but within a single species alleles It would be possible to identify peptides that do not show changes in Such a model is directed towards replacing the relevant mitochondrial antigens of the recipient cells with those from nuclear transfer donors to further enhance the immunocompatibility of cloned cells and tissues for transplantation therapy, It will accelerate the methodology of recombinant DNA.

For example, if a cloned “cross-species” teratoma also shows signs of rejection, according to the present invention, for example, by selecting a recipient cell expressing a compatible mitochondrial antigen, or tissue By substituting compatible epitopes, steps can be taken to ensure that cloned cells and tissues are compatible with nuclear donors. In fact, one group of researchers reported that the endogenous mitochondrial DNA of one Drosophila species was completely replaced with that of another species (Niki et al., 1989, complete mitochondrial DNA in Drosophila). Complete replacement (Complete replace
ment of mitochondrial DNA in Drosoph
ila), Nature, 341 (6242): 551-2). Thus, for any particular nuclear translocation donor, or even for a mixture of mitochondrial phenotypes, i.e., isogenic and allogeneic, or isogenic and cross-species. In contrast, it should be possible to create receptor cells with the desired mitochondrial phenotype.

Mitochondrial genes or DNAs involved in histocompatibility of mitochondrial antigens
Fragments, especially in the cross-species model, can be readily identified by using the method of the invention. For example, isogenic nuclei from a particular mammalian nuclear donor can be transplanted into different allogeneic mitochondrial backgrounds of closely related species, producing antibodies and lymphocytes specific for mitochondrial epitopes. Such cells can be used to immunize nuclear transfer donors for isolation and identification. By comparing the specificities of antibodies and lymphocytes obtained by immunizing nuclear donors, it is possible to identify mitochondrial antigens and epitopes that result in immune recognition and possibly transplant rejection in a cross-species model. Identification of such mitochondrial antigens and epitopes allows exchange of the DNA of the corresponding coding, thus avoiding transplant rejection of cloned tissues made by cross-species nuclear transfer.

Thus, the present invention includes a method of identifying mitochondrial histocompatibility antigens using cross-species nuclear transfer, which method comprises: harvesting cells from a donor mammal; A nucleus is transferred from an animal to at least two recipient oocytes or other suitable recipient cells of a mammalian species other than said nuclear donor to produce an embryo, wherein said at least two recipient cells are , Allogeneic with respect to mitochondrial DNA; isolating scutellum and / or stem cells from the embryo; separately reinjecting the scutellum and / or stem cells into the donor mammal to produce antibodies and / or
Or producing a specific panel of lymphocytes; and comparing a panel of antibodies and / or lymphocytes made in response to the allogeneic mitochondrial background, and recognized by the immune system of the donor mammal Identifying mitochondrial antigens and / or epitopes.

Antibodies and lymphocytes (both helper and cytotoxic T cells and B cells) specific for mitochondrial antigens identified by such a method are also included in the present invention. , And DNA or D encoding them
The same applies to the NA fragment.

The cloned mammal is recloned into nuclear and mitochondrial DNA.
The ability to generate isogenic cloned mammalian lineages for both of the two is due to the simultaneous injection of cross-species cloned cells containing heterologous mitochondria into separate mammals, thereby specific for different mitochondrial backgrounds. It makes it possible to accelerate the search for specific antibodies and lymphocytes. A method of recloning a cloned mammal, based on the observation that nuclear transfer can be used to rejuvenate aged cells, is filed concurrently with this application, commonly assigned, and is incorporated by reference in its entirety. Co-pending Application No. incorporated by No. Of course, by performing nuclear transfer from one donor with multiple oocytes from a single recipient mammal or cell line or other suitable recipient cells, isotopic mitochondrial DNA It is also possible to make a cloned mammal having Thus, the method of the invention also provides for the isolation of a panel of antibodies and / or lymphocytes from another mammal that is isogenic to the nuclear donor, both with respect to nuclear and mitochondrial DNA. It may also be carried out when the scutellum and / or stem cells are injected into.

The invention also includes a method of making a therapeutic cloned tissue for transplantation expressing a heterologous protein. The heterologous DNA used in the method of the present invention may encode a therapeutic protein to be expressed at the transplant receptor, but may also be a reporter gene for the purpose of monitoring gene expression in teratoma. . The reporter gene may be any convenient for monitoring expression of the gene, but is preferably
Green fluorescent protein,
GFP), beta-galactosidase, luciferase, their modified forms, antibiotic resistance markers, or other markers.

The use of tissue-specific promoters or enhancers also provides a means to select for expression of heterologous DNA in the desired tissue type. Alternatively stated, cells may be selected based on the expression characteristics of cell surface markers. For example, hematopoietic stem cells are selected based on the expression of CD34.

Donor cells may also include deletions and insertions into the genome to prevent or improve expression of the original gene, but preferably the donor cell is the recipient of the intended transplant. It replaces the original gene that is transfected, ie mutated or not expressed, with a heterologous gene that encodes a protein that is secreted in and exerts a therapeutic function. If the expressed protein is shown to show an immune response,
The animals used in the animal model to test immunocompatibility are then used to assess the immune response and isolate antibody or cytotoxic T cell clones.

Animal-made teratomas used to test the immunocompatibility of cloned tissues are also useful for studying the molecular signals that control cell differentiation and development. For example, a reporter gene construct designed with putative developmental promoters, enhancers, repressors and other gene control sequences is inserted into the nucleus of the donor prior to nuclear transfer, and then the teratoma is monitored visually or by other means. Then, observe at which stage the expression of the reporter gene is started.

As described above, the differentiated teratoma cells are divided and used to test the immunocompatibility of each particular cell or tissue. Once a particular cell type of interest is identified, it can be used to build tissue using the methods described herein and known to those of skill in the art. The disclosed animal models find particular roles in testing new matrix materials in tissue engineering for immunocompatibility. The preferred engineered tissue of the present invention is selected from the group consisting of smooth muscle, skeletal muscle, cardiac muscle, skin, kidney and nerve tissue.

Thus, the invention also relates to a method of producing immunocompatible tissue for transplantation, which comprises: a. Harvesting donor cells from the recipient of the intended transplantation; b. Transferring a nucleus from the cell to a recipient oocyte or other suitable receptor cell,
Produce an embryo; c. Isolating scutellum, inner cell mass and / or stem cells from the embryo; d. Injecting the scutellum, inner cell mass and / or stem cells into an immunocompromised animal; e. Isolate the resulting teratoma; f. Isolating the type of cells required for transplantation from the teratoma and optionally growing the cells in vitro with growth factors; and g. Constructing a tissue from the cell or combination of cells.

In addition, since the signals of cell differentiation and development are identified, it is possible to generate the desired cell type for tissue engineering and transplantation without forming teratomas in advance. This is because the development of a particular cell type takes place in vitro. In other words, it is now possible, at least in mammals other than humans, to obtain cloned tissue directly from the developing embryo or fetus, rather than creating teratomas. However, if the prestigious British Commission on Science and Ethics came to legislative action, the next step would be to collect humans, the cell types that develop at least during the first two weeks of embryonic development (Weiss, “British Commission”). Promotes approval for human embryo cloning (Br
this panel urging allowing human embr
yo cloning) ", Washington Post, August 17, 2000, A2
(See page 6).

Tissue engineering is accomplished using biodegradable polymers such as those used to construct three-dimensional scaffolds or soluble surgical threads. Such a method is
Many have been reported as patents and non-patent literature by companies such as e Engineering and Organogenesis. As examples of patents or documents in the field of tissue engineering, US Pat. Nos. 5,948,429 and 5,709,93
4, No. 5,983,888, No. 5,891,558, No. 5,709,93
No. 4, No. 5,851,290, No. 5,800,537, No. 5,882,92
9, No. 5,800,537, No. 5,891,558, No. 5,709,93
4, No. 5,891,617, No. 5,518,878, No. 5,766,93
7, No. 5,733,337, No. 5,718,012, No. 5,712,16
No. 3 and No. 5,256,418, all of which are incorporated by reference in their entirety. Also Robert Langer and John Va
A number of patents and publications by canti are cited. Both of them have many achievements in the field of tissue regeneration research. As discussed in many of these patents, it is desirable to include biological agents that promote the development of vascular tissue, namely growth factors and other compounds that promote angiogenesis.

In particular, the produced immunocompatible tissue and cells are useful in a method of providing an immunocompatible implant for a patient in need thereof. Such a method further comprises
In addition to the above steps, it includes implanting the tissue so created into a patient. The fact that the inventors have further surprisingly found that cloned cells containing isogenic nuclear DNA and allogeneic mitochondrial DNA do not induce transplant rejection is due to mitochondrial damage, for example: It has a special relationship to transplants that replace the original cells that suffer from amyotrophic lateral sclerosis (ALS) or Leber's inherited optic nerve injury (LHON). In such a case, the cloned tissue containing isogenic nuclear DNA and allogeneic mitochondrial DNA would be
It does not elicit an immune response and is the most ideal tissue for transplantation. Such tissues not only provide the closest histocompatibility combination, but also achieve mitochondrial gene therapy, in which tissues containing damaged mitochondria are replaced.

For example, Dhaliwal and co-workers have recently found that brain tissue of ALS patients is 30 times more frequent in “common mutations”, ie 4977 in various tissues of patients with mitochondria and other diseases. It was shown that a base pair mutation loss occurs. (
"Elevated levels of mitochondrial DNA deletion mutations in the brains of ALS patients (Mit
Ochondrial DNA deletion mutation lev
els are elevated in ALS brains) ", Mol
． Neurosci. 11 (113): 2507-9). In fact, accumulation of mtDNA ⁴⁹⁷⁷ was found in the brain, heart and muscle of healthy older people, suggesting a contribution to the aging process in these tissues (Soong an).
d Amheim, 1995, Methods Neurosci, 26: 1.
05-28). Thus, cloned tissue with allogeneic "young" mitochondrial DNA offers the advantage of being less age-related mitochondrial mutations than the patient's own cells.

Due to the relative lack of DNA repair mechanisms and histones, mitochondrial DNA is believed to be more susceptible to age-related mutations than genomic DNA (Dhaliwall et al., 2000). ). However, maternally inherited mitochondrial mutations are also present and expressed in specific tissues. This favors the cloning and tissue engineering techniques of the present application.

For example, Leber Hereditary Optic Nerve Injury (LHON) is a rare optic nerve disorder that causes legal blindness in most affected patients.
This is due to mutations in the mitochondrial DNA transmitted to the maternal line, but
The disease generally develops later in life (the sudden loss of vision in the first eye
(Generally occurs at the age of 10-50) (Zickermann, 1998, "Analysis of the pathogenic human mitochondrial mutation ND1 / 3460 and mutations of strictly conserved residues in its vicinity ... (Analysis of the pathogen).
ic human mitochondrial mutation ND1 /
3460, and mutations of strictly conse
rved residues in it's vicinity. ． ． ,), B
iochem. 37 (34): 11792-6 ". Researchers at the Institute of Molecular Ophthalmology at the University of Iowa have developed an improved method for detecting mutations that has been used in the diagnosis of LHON.

The cloning, tissue engineering, and transplantation techniques of the present invention are particularly valuable for replacing diseased tissue associated with mitochondrial mutations, in which case the cloned tissue generally contains isogenic nuclear DNA. Although possessed, mitochondrial DNA is allogeneic. Thus, for example, the nerve tissue created for transplantation into an LHON patient performs gene therapy of mitochondrial DNA while simultaneously replacing the affected optic nerve tissue.

As noted above, the donor cell may be genetically modified prior to nuclear transfer by transfer of at least one heterologous gene or disruption or replacement of at least one original gene. Good. Such modification of the genome may occur when the recipient's own genome undergoing transplantation is unable to express the required protein, or expresses a mutated protein such that the original tissue or cell does not function properly. Especially useful. Alternatively or in addition to this, if prior immunocompatibility testing
For example, if allogeneic or heterologous differences in mitochondrial DNA suggest that some rejection may be involved, donor cells may be treated prior to nuclear transfer.
It may be transfected with genes that express proteins that interfere with or reduce immune rejection.

The method of the present invention is particularly useful, especially when repairing and replacing tissue damaged by an autoimmune disease based on aberrant expression of self peptides. For example, primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease characterized by progressive inflammatory obstruction of the intrahepatic bile duct and eventually progresses to cirrhosis (Melegh et al., 2000,
"Autoantibodies against the subunits of pyruvate dehydrogenase and citrate synthase in pediatric biliary cirrhosis (Autoantibody against
ubunits of private dehydrogenase an
d citrate synthesize in a case of Paed
iatric birary irrhosis) ", Gut 2: 753
-6). The disease is characterized by decreased resistance to autologous mitochondrial proteins and is associated with high concentrations of anti-mitochondrial antibodies, which can be detected by techniques known to those of skill in the art (Leung et al. 1991, "Primary biliary cirrhosis. Use of Designer Recombinant Mitochondrial Antigen in Diagnosis (Use of design)
r recombinant mitochondrial antigens
in the diagnosis of primary diary
cirrhosis) ", Hepatol. 15 (3): 367-72). Liver transplantation has become one of the treatment options for patients with advanced disease (S
Ebagh, 1998, "Histological features predictive of recurrence of primary biliary cirrhosis after liver transplantation."
of recurrence of primary biliary ci
rrhosis after liver transplant) ", Tra
Nspplantation, 65 (10): 1328-33).

PBC anti-mitochondrial antibodies generally recognize a restricted epitope of the E2 subunit of the pyruvate dehydrogenase complex (PDC), which complex:
It is a nuclear-encoded protein that is normally transported to mitochondria and loosely associated with the inner membrane. Although PDC proteins are normally shielded from the immune system, PBC patients have been shown to express PDC-E2 on the surface of bile duct epithelial cells (Joplin et al., 1992, “Primary biliary cirrhosis patients”). Of dihydrolipoamide acetyltransferase (E2) in liver and portal lymph nodes of the rat: an immunohistochemical study (Distribution of di).
hydrolipoamide acetate transferase (E2
) In the liver and portal lymph node
s of patients with primary biliary c
irrhosis: an immunohistochemical stud
y) ", Hepatology, 14: 442-7). Thus, one theory of how PBC develops is that genetic changes in the nucleus, such as mutations in the main sequence that direct E2 to the outer membrane, affect the mitochondria of PDC-E2. Is affecting the transportation of Bjorkland (Bjorkland and
Totterman, 1994, "Is primary biliary cirrhosis an autoimmune disease? (Is
primary birary cirrhosis an autoim
mune disease? ) ", Scand. J. Gastroentero
l. 29 Suppl. 204: 32-9).

Thus, in the case of PBC, the cells produced by nuclear transfer can be corrected for nuclear defects leading to autoimmune disease prior to the production of hepatocytes and tissues for transplantation. That is, the correction is made by replacing the main sequence. As a result, the cloned cells and tissues for transplantation into PBC patients not only provide the most immunocompatible tissue to avoid rejection, but also repair the nuclear genes associated with the autoimmune disease itself, It also carries out gene therapy. The methods of the present invention are equally valuable for transplantation and gene therapy of any tissue having a disease in which the nuclear mutations involved in the disease process have been identified. For example, burns, blood disorders, cancer, chronic pain, diabetes, dwarfism, epilepsy, heart diseases such as myocardial infarction, hemophilia, infertility, kidney disease, liver disease, osteoarthritis, osteoporosis, seizures, Affective disorder, Alzheimer's disease,
Treatments include enzyme deficiency, Huntington's disease, hypocholesterolemia, parathyroid dysfunction, immunodeficiency, Rougerig's disease, macular loss, multiple sclerosis, muscular dystrophy, Parkinson's disease, rheumatoid arthritis and spinal cord injury.

In this regard, the inventors have also found that the cloning method of the invention allows the rejuvenation of senescent cells, whereby all of the above with respect to the genetic age of the cloned tissue. It should be noted that the findings of the above were discovered. US Patent Application No. 09 / The disclosure of the publication, in common with the present invention, reports our surprising observations on rejuvenation of primary cells using nuclear transfer and is incorporated herein in its entirety. The finding that the cloning process rejuvenates aged cells is particularly relevant to designing therapeutic tissues that express one or more heterologous genes or in which one or more genes are knocked out. Because
This is because such tissue can be produced by cloning or recloning primary cells having the same genetic background.

It is also possible to make changes to the mitochondrial DNA of the recipient cells using methods known to those skilled in the art (Wheeler et al., 1997, "Modification of the mouse mitochondrial genome by insertion of an exogenous gene ( Modification
of the mouse mitochondrial genome by
insertion of an exogenous gene) ", Ge
ne 198 (102: 203-9); Yamaoka et al., 2000, "Complete repopulation of mouse mitochondrial DNA-free cells with rat mitochondrial DNA ... (Complete repopulation of mouse m.
itochondrial DNA-less cells with rat
mitochondrial DNA. ． ． ) ”, Genetics 155
(1): 301-7). This may be useful for producing immunocompatible cells or tissues for transplantation, especially when the mitochondrial antigen is displayed by the cloned cells and the cloned cells are re-implanted into the nuclear donor. , Is useful in generating an immune response. Alternatively, if preliminary tests indicate that differences in mitochondrial DNA are involved in transplant rejection, suitable receptor cells may be specifically selected based on mitochondrial compatibility, especially in the case of heterologous mitochondria. .

Although any animal will benefit from the cells and tissues produced by the disclosed methods, the preferred recipient for translocation is human. If the recipient of the intended transplant is a human, a teratoma is formed after nuclear transfer, ie, the transfer of fibroblast nuclei from the human to any of the human recipient oocytes. Because it is the donor (the recipient of the intended translocation) that reprograms the cells for development.
Because it is the genome of. Teratomas made from human nuclear donors and acceptors
It is formed in and isolated from immunocompromised animals such as skid and nude mice.

Teratomas formed as described above are removed and germ layer formation is examined. Such germ layers are subdivided or differentiated until the cell type becomes apparent. The apparent cell type is then used to make tissue for transplantation. Preferably, the tissue is selected from the group of smooth muscle, skeletal muscle, myocardium, skin, kidney and nerve tissue. Also included in the invention are tissues and cells produced by the disclosed methods.

The concept of “therapeutic cloning” in humans refers to the fact that from one cell of a patient, ie, a fibroblast, to the oocyte of the receptor that has removed the nucleus or to the cell of another suitable receptor, the Is to transplant. After reprogramming, the nuclei of the donated somatic cells can regain full forming capacity and initiate the cycle of embryogenesis. Multipotent stem cells derived from the resulting embryo retain the patient's nuclear genome and then cardiomyocytes to replace damaged heart tissue, insulin-producing n-cells in diabetic patients, osteoarthritis Chrondrocyte
te), is induced to differentiate into replacement cells, such as Parkinson's disease-treated dopaminergic neurons.

The method of the present invention eliminates, or at least substantially alleviates, the immune response associated with the transplantation of these various tissues, thus cyclosporine, Imoran, FK-506, Glucose. It excludes the need for immunosuppressive agents such as corticoids and their variants. These immunosuppressive agents carry a wide range of significant complication risks including cancer, infectious diseases, renal disease and osteoporosis. However, at least in some cases it is still preferred to use the anti-rejection drug at least initially. As discussed above, the transplanted cells may not be immunologically identical to the cells of the transplant recipient, but the nucleus of one cell of the recipient still functions as a donor. To do. This is caused by differences in mitochondrial DNA, especially in the case of heterologous mitochondria, or as a result of transferred heterologous DNA, or by differences in antigens due to artificial environments that often affect nuclear transfer. obtain. In particular, such an environment
It is not pseudo-identical to the cellular environment present during embryonic development.

For example, it is known that cells that have been cultured for a long period of time may differ antigenically as a result of culture (a phenomenon called “antigen drift”). Therefore,
Prior to transplantation, cells or tissues are rendered resistant by, for example, treatment with soluble CD40, CD40-ligand antagonists, cold culture, use of antibodies that mask donor antigens, or UV irradiation (eg, islets). Still preferred.

Without limitation, the scope and spirit of the invention is illustrated with reference to the following discussion and examples.

Example 1 This experiment was designed for the purpose of testing the immunocompatibility of cells produced by nuclear transfer in a large pre-symptomatic animal: bovine (Bos taurus).

Three adult Holstein steers, aged about 8 to 10 months (weighing about 500 to 1000 pounds), were fed to Thomas Morris (Maryland).
Purchased from South Deerfield at Massachusetts University
d Ship to Farm (Amherst). In order to collect fibroblasts for nuclear transfer, skin living tissue was obtained from each animal by engraving. Proliferation green fluorescent protein (e
enhanced green fluorescent protein: eG
A plasmid expressing a reporter gene encoding FP) was transferred into the cells, and the transferred cells were selected with neomycin. Purified cells analyzed by PCR and / or FISH have already been analyzed in Nature (1998) Biotech.
nol. 16: 642-646 (incorporated herein by reference) and used for nuclear transfer.

The isolated embryo having at least one cell, or scutella / inner cell mass produced from bovine blastocyst / stem cells, is then applied to the lumbar fascia of a donor steer (2 sites on each animal). Experimental stem cells (of the same animal), 2 sites of experimental scutellum (of the same animal), 2 sites of inner cell mass, and 4 sites of control stem cells (of different animals). Two months later the muscles are examined for teratomas. All identified tumors will be removed and histologically analyzed.

This operation is performed on standing animals using IV with 20 mg xylazine / 8 mg butorphanol tartrate administered into the tail vein. Clip the lumbar fascia area,
2% lidocaine is administered surgically by local anesthesia throughout the lumbar region. Animals were treated with antibiotics (cefilofer hydrochloride 50 mg /
cc, 1 cc / 100 pounds) should be administered. Flunixin meglumine capsules, 1 cc / 10 immediately after surgery to control the pain and swelling of the surgical site
A single injection of 0 pounds is given intramuscularly or subrenally. If teratoma formation is not found in the lumbar fascia, analyze elsewhere, eg subcutaneously.

In contrast to “different animal” stem cells, “identical animal” stem cells survive in the host (nuclear donor) animal, or the T cell response or cytotoxicity against external mitochondrial peptides or It is expected to survive more actively or longer depending on other immune responses. In addition, it is expected that all cells from three germ layers, namely ectoderm, mesoderm and endoderm, will be found in the "identical animal" teratoma.

Example 2 This example was designed to test teratoma formation in an immunocompromised animal model. This example presents SCID produced by nuclear transfer from a patient in need of transplantation to produce and isolate differentiated cells and to engineer the constructed tissue for transplantation. It relates to methods of growing in mice or other immunocompromised animals.

ES cells transfected with GFP were derived from adult Holstein steers (two different ES cell lines were derived from each animal). ICM was derived from 12 day old blastocysts.

Cell Preparation and Injection Procedure: Cells were crushed (all into pieces of about 100 cells or less) and loaded into a 1 ml volume syringe at 200 microliters each, preferably 100 microliters each.

The ICM was mechanically isolated and loaded into a 1 ml volume syringe in 100 to 150 microliter increments.

[0061]   Cells were stored in HECM liver at room temperature.

A 22 gauge needle was used for the injection operation. Cells were injected into the skeletal muscle of the hind legs of SCID mice.

[0063]

Bovine stem cells and ICM injected into skeletal muscle of SCID mice were harvested after 7-8 weeks (although cells can be left longer or removed in a shorter period). Mice injected with ES cells (mouse numbers 7 and 9)
Small nodular lesions were identified in 2 animals.

Comprehensive Examination: A milky white nodule with a size of 2 × 2 mm was taken from the right hind leg near the sciatic nerve of mouse number 7. This is ES22. C. Corresponding to the injection of 3 plates.
A 1 × 1 mm size milky white nodule was identified in the muscle tissue of mouse # 9. This is ES25. F. Corresponding to the injection of 3 plates.

Histological analysis: Mouse No. 7: Histological sections of teratomas were taken for hematoxylin and eosin
& E), safranin-O, and immunocytochemistry with cytokeratin (AE1 / AE3) and alpha smooth muscle actin antibodies.

H & E: The injected cells formed round tissue mass within skeletal muscle tissue. Teratomas have four different sized compartments with a central cell debris.
ents). Tissue formation was observed on the walls of each compartment (data not shown). Among teratomas, epithelial cells (circular nuclei) and stromal cells (spindle-shaped nuclei)
Was observed (data not shown). There was no evidence of cartilage, bone or adipose tissue.

Safranin-O: A negative staining test was obtained, suggesting no cartilage tissue formation.

Immunocytochemistry with AE1 / AE3 Antibodies: Teratoma sections showed positive results in a staining test for epithelial cells (data not shown).

Immunocytochemistry with alpha smooth actin antibody: Small islets that were positive in the muscle tissue staining test were found within the teratoma (data not shown). The removed tissue showed epithelial, smooth muscle and stromal tissue elements. In the teratoma, cartilage,
No bone or adipose tissue was identified.

Mouse # 9: Histological analysis of the removed nodules showed skeletal muscle mass. No other tissue formation was observed by microscopic examination.

Example 3 In order to realize all the potential of therapeutic cloning, it may be important to reconstruct more complex tissues and organs in vitro. Although cloning appears to eliminate or significantly reduce the most important problem-immunobicompatibility-the task of binding cells to produce or regenerate functional structures remains.

For example, myocardial infarction is one of the most common diagnoses occurring among hospitalized patients in Western European countries. Infusion of individual or small groups of cardiomyocytes aids in the treatment of some local infarctions, but at a higher risk of scarring, cardiac rupture and other complications, more advanced ischemic In patients with disabilities, it does not seem worth it. Tissue engineering offers the potential to organize cells into three-dimensional myocardial “patches” that can be used to repair damaged heart areas. For myocardium and other relatively simple tissues such as skin and vascular substitutes, tissue engineering may include seeding cells onto a polymeric skeleton or sheet. The production of more complex vital organs, including the whole kidney, liver or even the heart, requires the assembly of different cell types and substances whose combinations are very complex.

To construct tissue for use in animal models, bovine cell mass / blastoderm / stem cells are produced and injected into the hindlimb muscle of nude or SCID mice as described above. Teratomas obtained 7-8 weeks after injection are removed and various cell types are isolated and cultured in medium. A number of tissues have been cloned, including smooth and / or skeletal muscle, sheets or "patches" of cardiomyocytes, elastic cartilage, skin (including placement of hair follicles), and kidneys, including miniature kidneys that secrete urine. Can be produced from the cells. These tissue / organ constructs are then reimplanted into the original adult animal from which the living tissue of the donor cells was collected.

The following data show that isogenic for nuclear DNA, mitochondrial D
It is shown that allogeneic tissues for NA form stable transplants and do not elicit an immune response in nuclear transfer hosts. This supports the utility of such cloned cells and tissues for many medical applications and is observed in response to mitochondrial histocompatibility antigens in different species of mice. Given the response of cytotoxic T cells to mitochondrial antigens, this is quite surprising.

Cell Culture and Inoculation Cells from bovine kidney, heart, skeletal muscle, cartilage and skin were harvested from cloned allogeneic (control) 40-day-old fetuses and grown separately in vitro. did.

Kidney: Kidney tissue was cut into small pieces (1 mm ² ) with a sharp tendon clip. A fragment of kidney tissue was digested with collagenase dispase (1 mg / ml). Cells were harvested, washed with phosphate buffered saline and fixed in culture dishes. DMEM, HEPES
3.1 g / l, Pen / Strep (5 ml / 500 ml), L-glutamine 1
Cells were grown in medium consisting of 46 mg / L and 10% FBS (Sigma, St. Louis, MO).

Muscle: Myocardial and skeletal muscle, Dulbecco's, supplemented with 10% fetal calf serum.
Modified Eagle's Medium (DMEM, HyClon
e Laboratories, Logan, Utah). Cells were incubated in a humidified room containing 5% CO ₂ and maintained at 37 ° C. The two muscle cell types were expanded separately to obtain the desired cell number. Cells were trypsinized, harvested, washed, and counted for inoculation.

Polymer: A non-woven sheet of polyglycolic acid polymer (1 × 2 cm) was used as the cell delivery vehicle. The polymer mesh has a diameter of 15 μm and a distance between fibers of 0-2.
It had a porosity of 95%. This scaffold was designed to hydrolyze in 8 to 12 weeks. The polymer was sterilized in ethylene oxide and placed under sterilizing conditions until the cells were shipped.

Implantation athymic mice: Cardiomyocytes, skeletal muscle cells and chondrocytes inoculated into a polymer scaffold were tested to determine if cells from fetal bovine tissue would form tissue in vivo. Athymic mice were transplanted into the dorsal subcutaneous space. Mice were sacrificed 1 week, 1 month and 3 months after transplantation and subjected to analysis (n = 4).

Steers: Each cell type was inoculated separately on polyglycolic acid polymer (1 × 2 cm) at a concentration of 50 × 10 ⁶ cells / cm ³ (n = 4 per cell type). The cell-polymer scaffold was transplanted into the flank subcutaneous space of the same cell-cloned steer. Cells from a control (nuclear xenogeneic) fetus were transplanted into the opposite flank of the steer. After 6 weeks all implants were removed and submitted for analysis.

Analysis Implantation in athymic mice: 5 μ sections of formalin-fixed paraffin-embedded tissue were cut and stained with hematoxylin and eosin (H & E). Immunocytochemical analysis was performed with specific antibodies to identify the cell type of the removed tissue. Aldehyde lignite (
histochemical analysis using fuschin-alcian blue, monoclonal anti-collagen II (Chemicon, St. Louis,
MO) was used to identify the structure of the cartilage created. Monoclonal sarcomeric tropomyosin (Sigma, St. Louis,
MO) and Troponin I (Chemicon, Temecula, CA) antibodies were used to detect skeletal and myocardial fibers, respectively. Immunolabeling was performed using the avidin-biotin detection system. Counterstaining of the sections was performed using methyl green.

Implantation into steers: Immunocytochemical and histological analysis: 5 μ sections of formalin-fixed paraffin-embedded tissue were cut and stained with hematoxylin and eosin (H & E). Immunocytochemical analysis was performed with specific antibodies to identify the cell type of the removed tissue. Histochemical analysis using periodate Schiff reagent (Sigma, St. Louis, MO), and polyclonal anti-alkaline phosphatase and anti-osteopontin (Chemi).
Con, Inc., Temecula, CA)
Kidney cells were identified. Monoclonal sarcomeric tropomyosin (Sigma,
St. Louis, MO) and Troponin I (Chemicon, Temec)
ura, CA) was used to detect skeletal and myocardial fibers, respectively. Aldehyde Brownin-Alcian Blue and Monoclonal Anti-Collagen II (Che
micon, St. (Louis, MO) was used to stain cartilage tissue implants. To identify keratinocytes, anti-cytokeratin 5/6, AE1 / AE
3 was used. Bronchial cilia antibodies were used to detect airway epithelium. Anti-CD6 antibody was used to identify immunized T and B cells. Immunolabeling was performed using the avidin-biotin detection system. Counterstaining of the sections was performed using methyl green.

Results Confluently grown cells were transplanted into animals with polymer scaffolds and removed without complications. At the time of removal, the transplants each maintained their original size with no evidence of fibrosis.

Grafts recovered from steers: Histochemical and immunocytochemical analysis: The results of histological examination show extensive angiogenesis throughout the graft, with polynuclear giant cells surrounding the polymer fibers. Existence was recognized. However, there were more inflammatory cells throughout the control allogeneic scaffold. The results of the histocellular stochastic analysis of transplanted tissues (ie kidney, skeletal muscle, heart, chondrocytes and keratinocytes) showed that the lymphocyte infiltration of control transplants / constituents was statistically relative to the cloned tissue type. (P <0.05;Student's t-test) increase (data not shown). This data suggests that the control transplant pair is undergoing early transplant rejection.

Engineered Kidney Tissue: Histologically glomerular-like structures were found in the removed scaffolds (data not shown). Histochemical analysis using periodate Schiff reagent identified renal tubular cells (data not shown). Immunocytochemical studies using alkaline phosphatase antibody confirmed the presence of basal tubular cells. Results of studies with osteopontin antibodies were negative in bovine tissue systems.

Produced Muscle Tissue: The explanted myocardial and skeletal muscle cell implants in each case showed spatially oriented muscle fibers (data not shown). Immunocytochemical studies with tropomyosin antibody identified skeletal muscle fibers in the construct (data not shown). Staining of myocardial fibers with anti-troponin I was positive (data not shown).

To prove that the cloned tissue mtDNA was from the recipient oocyte, the sequences of the nuclear donor and cloned embryo mtDNA were examined.
Sequence data confirmed that the mtDNAs were indeed different, especially in the d-loop region with four different corresponding nucleotides in the cloned tissue compared to the nuclear donor.

Example 4 The above results demonstrate that nuclear transfer to an allogeneic background is capable of producing cloned tissue for transplantation, and culturing cloned teratomas or germ cells. It suggests that the differentiated cells and tissues isolated or constructed from can be reimplanted into donor animals without evidence of significant rejection. To further confirm that nuclear transfer technology has the potential to rule out immune responses involved in cell and organ transplantation despite mitochondrial mismatches,
We next perform transplants between fully grown clones with different mitochondrial backgrounds.

For these experiments, two groups of animals to test reciprocal skin grafts were: (1) 4 cloned cows of Trans Ova (animal CL53-8,
CL53-9, CL53-10 and CL53-11), and (2) 5 cloned goats of LSU were prepared. To perform the test, a mutual skin graft (about 2-3 cm in diameter) is exchanged between the two groups of animals. Autologous transplants serve as a positive control, whereas transplants from genetically unrelated animals serve as a negative control.
Monitor the transplants for signs of immune rejection and remove them if they begin to necrotic and the site shows plaques. If rejection is noted, then a second set of transplants is transplanted and the results confirmed, but in this case accelerated rejection should result.

All cloned cows and all cloned goats have the same nuclear genome. However, since mtDNA is transmitted by maternal inheritance, the animal is in fact expected to be a genetic chimera with mitochondria from different oocytes (this has already been described for many cloned animals). ing)
. MtDNA from all individuals involved so that the polymorphisms that "distinguish" these panels of animals can be immediately associated with survival / rejection of skin grafts.
Experiments to obtain the sequence of If so, an in vitro assay is performed to identify the target peptide and isolate the relevant mtDNA encoding that peptide.

Experiments aimed at determining polymorphisms in mitochondrial DNA also
In general, it will reveal information about the level of mtDNA chimerism. For example, if the sequence of mtDNA is clarified, the region of the highest polymorphism,
The most likely is the D-loop, but is selected to amplify and clone this chromosomal segment. The sequence of a range of clones is then examined to determine the extent of change in this region. The sequence of mtDNA from a sufficient number of nuclear clones that are allogeneic to mitochondria can be used to determine an accurate prediction of the level of chimerism. Blood samples will also be collected at regular intervals to perform various immunological assays. For histocompatibility, standard MLC and CML will be performed with allogeneic cells in the panel.

Discussion As demonstrated by the data presented above, the present invention illustrates that it is possible to obtain cloned differentiated cells and tissues for purposes of tissue engineering and transplantation. It is a thing. The present invention also provides for stable transplantation using nuclear transfer-generated cloned cells with allogeneic mitochondria, despite the fact that transplant rejection is expected due to exogenous mitochondrial peptides. It illustrates what can be achieved. Mta in mice
In view of the system and similar systems identified in the rat, it is surprising that bovine tissue produced using this method is not rejected when reimplanted into a nuclear donor.

There may be several reasons why there is no transplant rejection in the cloned tissues of the invention. Without being bound to any particular theory, one hypothesis is that particular MHC molecules in rodents that provide Mtf, MiHA and other mitochondrial antigens have evolved from higher mammals. In fact
H-2M3a, a mouse class I molecule that provides 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. Inheritance in: A model transplant antigen (maternally tran)
smitted antigen of mice: a model tran
splanation antigen) ", Annu. Rev. Immun
ol. 1991; 9: 351-72). Although many genes in this region of the chromosome are conserved between mouse and human, for example, the MHC class I genes in this region appear to diverge and evolve independently from species (Jones).
, 1999, "MHC class I and non-class I organization of MHC class I and non-class I genes in the mouse base H2-M region.
gene organization in the proximal
H2-M region of the mouse) ", Immunogen
etics 49 (3): 183-95). In fact, the H2-M region has many L1 repeats and, according to one hypothesis, is associated with evolutionary flexibility (Yoshino et al., 1
997, "Genomic evolution of the distal MHC class I region on mouse chromosome 17 (Gen
mic evolution of the digital MHC cla
ss I region on mouse chromesome 17) "
, Hereditas 127 (1-2): 141-8).

The other hypothesis is that, perhaps, additional mechanisms have evolved in higher mammals, which is particularly the case in many aging but otherwise healthy human tissues. Modulates the immune response to mitochondrial antigens in the context of MHC, as seen by the inclusion of age-related mitochondrial mutations (Soong and Amheim, 1995). The ongoing experiments described in Example 4 are particularly useful in identifying polymorphisms that are present in a fixed proportion of mtDNA, and in displaying and recognizing abnormalities to mitochondrial antigens. In the meantime, it appears to serve as a useful model system for identifying changes that occur in mtDNA.

In any event, regardless of what was known or understood about the histocompatibility of rodent mitochondria prior to the present invention, the therapeutic cloning described herein. The results achieved with bovine tissue can be expected to be extendable to other ungulates and higher mammals. Thus, the present invention confirms the therapeutic utility of cloned tissue generated by nuclear transfer in the context of transplantation. Further, by providing a model for testing the immunocompatibility of allogeneic and heterologous mitochondrial proteins in an isogenic nuclear background, the present invention resides within and among mammals. , Lays the way to deciphering the immune regulatory system that contributes to mitochondrial stability and distinct evolution of species.

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Claims (55)

[Claims] 1. A method of testing the immunocompatibility of a cloned cell or tissue in an animal model, which comprises: a. Harvesting cells from a donor animal; b. Transferring the nucleus from the cell to a recipient oocyte or other suitable receptor cell to produce an embryo; c. Isolating an embryo having at least one cell, a scutellum and / or a stem cell from the embryo; d. Injecting the embryo, scutellum and / or stem cells into the donor animal at the same time as control scutellum and / or stem cells; and e. Examining the formation of teratomas at the injection site. 2. The method of claim 1, wherein the heterologous gene is transferred to the cells from the donor animal prior to nuclear transfer. 3. The method of claim 1, wherein the donor and control scutellum and / or stem cells are injected subcutaneously or into the lumbar fascia. 4. The method according to claim 1, wherein when the teratoma is formed, it is removed and examined for the presence of germ layers. 5. The method according to claim 4, wherein when the germ layer is formed, it is separated for the purpose of detecting or isolating a specific cell type. 6. The method according to claim 1, wherein the cells collected from the donor animal are fibroblasts. 7. The heterologous gene is a green fluorescent protein (green flu).
or report protein: GFP), β-galactosidase, and luciferase.
The method described. 8. The method of claim 2, wherein said heterologous gene encodes a secreted protein. 9. The method of claim 8, wherein the protein produces an immune response. 10. The method of claim 8, wherein the protein is a therapeutic protein. 11. The method of claim 5, wherein the germ layer cells are further used in assays for assessing potential developmental signals that control cell differentiation. 12. The method of claim 5, wherein at least one type of cell found in the germ layer is used to create tissue. 13. The method of claim 12, wherein the produced tissue is reimplanted into a donor animal to test immunocompatibility. 14. The method of claim 12, wherein the produced tissue is selected from the group consisting of smooth muscle, skeletal muscle, cardiac muscle, skin, kidney and nerve tissue. 15. A method of producing immunocompatible tissue for transplantation comprising: a. Harvesting donor cells from the recipient of the intended transplant; b. Transferring the nucleus from the cell to a recipient oocyte or other suitable receptor cell to produce an embryo or fetus; c. Isolating the type of cells required for transplantation from the embryo or fetus; and d. Producing tissue from the cells. 16. Between said steps (c) and (d), as an additional step: i. Isolating scutellum and / or stem cells from the embryo; ii. Injecting the scutellum and / or stem cells into an immunocompromised animal; iii. Isolate the resulting teratomas; iv. 16. A method according to claim 15, comprising isolating from the teratoma the type of cells required for transplantation, wherein the teratoma cells are used to produce the immunocompatible tissue. 17. The method of claim 15, wherein the tissue contains cells containing isogenic nuclear DNA and allogenic mitochondrial DNA. 18. The method of claim 15, wherein the tissue contains cells that contain isogenic nuclear DNA and a mixture of heterogeneous and isogenic mitochondrial DNA. 19. The method of claim 15, wherein the tissue is selected from the group consisting of smooth muscle, skeletal muscle, myocardium, skin, kidney and nerve tissue. 20. A method of providing an immunocompatible implant to a patient in need of transplantation comprising: a. Collecting donor cells from the patient; b. Transferring the nucleus from the cell to a recipient oocyte or other suitable receptor cell to produce an embryo; c. Isolating scutellum and / or stem cells from the embryo; d. Injecting the scutellum and / or stem cells into an immunocompromised animal to form a teratoma; e. Isolate the resulting teratoma; f. Isolating the type of cells required for transplantation from the teratoma; g. Creating tissue from the cells; and h. Transplanting the produced tissue into the patient. 21. The method of claim 20, wherein the immunocompromised animal is a skid or nude mouse. 22. The method of claim 20, wherein the donor cells harvested from the recipient of the intended transplant are fibroblasts. 23. The method of claim 20, wherein the produced tissue is selected from the group consisting of smooth muscle, skeletal muscle, cardiac muscle, skin, kidney and nerve tissue. 24. The method of claim 20, wherein the produced tissue comprises cells having isogenic nuclear DNA and allogenic mitochondrial DNA. 25. Tissue produced by the method of claim 20. 26. An isolated tissue produced by the method of claim 20. 27. The method of claim 1, wherein the animal is a ungulate. 28. The method of claim 27, wherein the ungulate is bovine. 29. The method of claim 15, wherein the animal is a ungulate. 30. The method of claim 29, wherein the ungulate is bovine. 31. The method of claim 16, wherein the animal is a ungulate. 32. The method of claim 31, wherein the ungulate is bovine. 33. The method of claim 20, wherein the intended recipient of the transplant is a human. 34. The method of claim 16, wherein the patient is a human. 35. The donor cell is genetically altered prior to nuclear transfer.
The method according to claim 16. 36. The method of claim 35, wherein said genetic alteration comprises transfer of at least one heterologous gene. 37. The method of claim 35, wherein said genetic alteration comprises disruption of at least one native gene. 38. An animal containing at least one teratoma produced from cloned cells. 39. The animal of claim 38, wherein the animal is a ungulate. 40. The animal of claim 39, wherein the ungulate is bovine. 41. The animal of claim 38, wherein the at least one teratoma is located in the lumbar fascia. 42. The animal of claim 38, wherein the teratoma is not rejected by the animal's immune system. 43. The animal of claim 42, wherein the teratoma comprises a cloned cell having isogenic nuclear DNA and allogenic mitochondrial DNA. 44. A teratoma isolated from the animal of claim 38. 45. The teratoma of claim 44, wherein the teratoma contains cells derived from all three germ layers. 46. The teratoma of claim 44, wherein the teratoma is derived from cloned ungulate cells. 47. The teratoma of claim 46, wherein the teratoma is derived from cloned bovine cells. 48. The teratoma of claim 48, wherein the teratoma comprises a cloned cell having isogenic nuclear DNA and allogeneic mitochondrial DNA, or a mixture of allogeneic and isogenic mitochondrial DNA. . 49. Isogenic nuclear DNA and allogenic mitochondrial D
Stable transplant with NA. 50. The transplant according to claim 49, wherein the cells of the transplant are produced by nuclear transfer from isogenic somatic cells to allogeneic receptor cells. 51. The implant of claim 49, wherein the tissue is selected from the group consisting of kidney, heart muscle and skeletal muscle. 52. A method of identifying mitochondrial histocompatibility antigens using cross-species nuclear transfer comprising: a. Harvesting cells from a donor mammal; b. Transferring a nucleus from the donor mammal to at least two recipient oocytes or other suitable recipient cells of a mammalian species other than the nuclear donor to produce an embryo, wherein
The at least two receptor cells are allogeneic with respect to mitochondrial DNA; c. Isolating an embryo having at least one cell, a scutellum and / or a stem cell from the embryo; d. Reinjecting the embryo, scutellum and / or stem cells separately into the donor mammal to produce a specific panel of antibodies and / or lymphocytes; and e. Comparing a panel of antibodies and / or lymphocytes produced in response to the allogeneic mitochondrial background to identify mitochondrial antigens and / or epitopes recognized by the immune system of the donor mammal; Including the method. 53. The embryo, scutellum and / or stem cell are nuclear and mitochondrial D
53. The method of claim 52, wherein the method is injected into another mammal that is isogenic to the nuclear donor for both NAs. 54. An antibody specific for the mitochondrial antigen identified in the method of claim 52. 55. A lymphocyte specific for the mitochondrial antigen identified in the method of claim 52.