Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
2000-09-06
2004-10-26
Crouch, Deborah (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C424S093100, C424S184100, C435S325000, C435S346000, C435S375000, C435S377000, C800S024000
Reexamination Certificate
active
06808704
ABSTRACT:
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. Pat. No. 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 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, Aug. 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. Surg
. 115: 536-546 (1998). Z&dgr;nd 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 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 maybe 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 mitochondrial compatibility, and in particular, transgenic, developmentally differentiated cells, for immune compatibility in an animal model. Such evaluations will provide important information regarding tie suitability of therapeutic tissues in transplantation, and will provide the foundation for controlling these parameters in order to provide immune compatible tissues.
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 mitochondrial DNA of the donor cell and gain the mitochondrial 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 mitochondrial peptides in mice, e.g., the ND1 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 mitochondrial 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 ND1 peptide renders cells displaying foreign alleles susceptible to lysis by specific cytotoxic T cells (Loveland et al. 1990. 60(6): 97
Cibelli Jose
Lanza Robert
West Michael D.
Advance Cell Technology, Inc.
Chrudimsky Kristin
Crouch Deborah
Merchant & Gould LLP
Ton Thai-an N.
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