Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1995-05-18
2003-02-04
Beckerleg, A. M. S. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C435S325000, C435S455000, C536S023100, C800S013000
Reexamination Certificate
active
06514752
ABSTRACT:
INTRODUCTION
1. Technical Field
The field of the subject invention is the generation and use of major histocompatibility complex antigen lacking cells and organs lacking expression of functional major histocompatibility complex (MHC) antigen which may serve as universal donors in cellular and organ therapies including transplantation and to produce chimeric non-human mammals.
2. Background
To protect vertebrates from disease and infection, elaborate protective systems have evolved. In mammals, the immune system serves as the primary defense with many different types of cells and mechanisms to protect the host. A wide variety of hematopoietic cells exists, with the major protective lineages being lymphoid and myeloid.
The immune system, which results from cells of the lymphoid and myeloid lineages is developed in vivo, so as to recognize self from non-self. Those aberrant situations where the immune system attacks self, such as rheumatoid arthritis, lupus erythematosus, and certain forms of diabetes, are evidence of the importance to the host that only foreign agents be attacked. The protective mechanism which protects the host from disease, as a result of invasion of viruses, bacteria, or other pathogens, is also able to recognize cells which come from a different mammalian host, even an allogeneic host.
As part of the system for the self-versus-non-self recognition, the surface membrane protein major histocompatibility complex (MHC) antigens serve an important role. Each host has a personal set of Class I and II MHC antigens, which serve to distinguish that host from other hosts. The T-lymphoid system is predicated upon recognition of the presence of such MHC antigens as self. Where transplantation from another allogeneic host occurs, unless the transplant is matched with the host or the host is immunocompromised, the transplant may be attacked and destroyed by the immune system. When a transplant occurs which includes lymphocytes, monocytes or progenitors thereof, particularly bone marrow, a graft may attack the host as foreign, resulting in graft-versus-host disease.
There are many situations where one may wish to transplant cells into a recipient host where the recipient's cells are missing, damaged or dysfunctional. When the host is immunocompromised, there may be an interest in transfusing specific white cells, particularly T-cells, which may protect the host from various diseases. When the host lacks the ability to raise a defense against a particular disease, there may also be an interest in administering specific T-cells or B-cells or precursors thereof which may supplement the host's compromised immune system. In other cases, where certain cells are lacking, such as islets of Langerhans in the case of diabetes, or cells which secrete dopamine in the case of Parkinson's disease, or bone marrow cells in various hematopoietic diseases, or muscle cells in muscle wasting disease, or retinal epithelial cells in visual disorders, or keratinocytes for burns and non-healing wounds, it would be desirable to be able to provide cells which could fulfill the desired function. In order for the cells to be effective, they must be safe from attack by the host, so that they may function without being destroyed by the immune system. It is therefore of interest to find effective ways to produce cells which may function, proliferate, and differentiate as appropriate, while being safe from attack by a recipient's immune system, for example by the use of gene targeting to inactivate the expression of gene products that cause rejection of the transplanted cells. The same reasons apply to the use of organs for transplantation including but not limited to the heart, lung, liver and kidney.
Homologous recombination permits site-specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the genome. The application of homologous recombination to gene therapy depends on the ability to carry out homologous recombination efficiently in normal diploid somatic cells. Homologous recombination or “gene targeting” in normal, somatic cells for transplantation represents a potentially powerful method for gene therapy; however, with the exception of pluripotent mouse embryonic stem (ES) cells, and continuous cell lines, homologous recombination has not been reported for a well-characterized, non-transformed, i.e., “normal” mammalian somatic cell. In contrast to mouse ES cell lines, normal somatic human cells may have a finite life span in vitro (Hayflick and Moorhead,
Exptl. Cell. Res
. 25:585-621 (1961)). This makes their modification by gene targeting especially challenging, given the low efficiency of this process, i.e., 10
−5
to 10
−8
recombinants/input cell. Moreover, this process is further complicated by the fact that mammalian cells tend to integrate transfected DNA at random sites 100 to 1000 fold more efficiently than at the homologous site.
The present invention discloses methods for targeting non-transformed diploid somatic cells to inactivate genes associated with MHC antigen expression, including the &bgr;
2
-Microglobulin and IFN-&ggr;R genes in cells such as retinal epithelial cells, keratinocytes and myoblasts. These methods provide novel targeting means for inactivating target genes resulting in lack of expression of functional MHC. In a method of the invention for targeting integral membrane proteins, the role of such proteins may be studied, and their expression manipulated, for example membrane proteins that serve as receptors, such as T cell receptors.
There is also substantial interest in being able to study various physiological processes in vivo in an animal model. In many of these situations, one would wish to have a specific gene(s) inactivated or introduced in a site-directed fashion. Where all or a substantial proportion of the cells present in the host would be mutated, the various processes could be studied. In addition, heterozygous hosts having one wild-type gene and one mutated gene could be mated to obtain homozygous hosts, so that all of the cells would have the appropriate modification. Such genetically mutated animals could serve for screening drugs, investigating physiologic processes, developing new products, and the like.
Relevant Literature
A number of papers describe the use of homologous recombination in mammalian cells, including human cells. Illustrative of these papers are Kucherlapati et al.,
Proc. Natl. Acad. Sci. USA
81:3153-3157, 1984; Kucherlapati et al.,
Mol. Cell. Bio
. 5:714-720, 1985; Smithies et al,
Nature
317:230-234, 1985; Wake et al.,
Mol. Cell. Bio
. 8:2080-2089, 1985; Ayares et al.,
Genetics
111:375-388, 1985; Ayares et al.,
Mol. Cell. Bio
. 7:1656-1662, 1986; Song et al.,
Proc. Natl. Acad. Sci. USA
84:6820-6824, 1987; Thomas et al.
Cell
44:419-428, 1986; Thomas and Capecchi,
Cell
51: 503-512, 1987; Nandi et al.,
Proc. Natl. Acad. Sci. USA
85:3845-3849, 1988; and Mansour et al.,
Nature
336:348-352, 1988.
Evans and Kaufman,
Nature
294:146-154, 1981; Doetschman et al.,
Nature
330:576-578, 1987; Thoma and Capecchi,
Cell
51:503-512,4987; Thompson et al.,
Cell
56:316-321, 1989; individually describe various aspects of using homologous recombination to create specific genetic mutations in embryonic stem cells and to transfer these mutations to the germline. The polymerase chain reaction used for screening homologous recombination events is described in Kim and Smithies, Nucleic Acids Res. 16:8887-8903, 1988; and Joyner et al., Nature 338:153-156, 1989. The combination of a mutant polyoma enhancer and a thymidine kinase promoter to drive the neomycin gene has been shown to be active in both embryonic stem cells and EC cells by Thomas and Capecchi, supra, 1987; Nicholas and Berg (1983) in
Teratocarcinoma Stem Cell
, eds. Siver, Martin and Strikland (Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. (pp. 469-497); and Linney and Donerly,
Cell
35:693-699, 1983.
Bare lymphocytes are described in Schuu
Arbones De Rafael Mariona Lourdes
Capon Daniel J.
Dubridge Robert B.
Greenburg Gary
Koller Beverly H.
Beckerleg A. M. S.
Cell Genesys Inc.
Linda Judge Gates & Cooper LLP
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