Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1997-01-21
2003-04-01
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S004000, C435S006120, C435S320100, C435S325000, C435S350000, C435S351000, C435S352000, C435S366000
Reexamination Certificate
active
06541197
ABSTRACT:
BACKGROUND OF THE INVENTION
Gene therapy involves the transfer of therapeutic genes into living cells. The potential clinical applications of gene therapy are numerous and include the treatment of a wide variety of diseases, such as those resulting from genetic defects as well as cancer and diseases caused by viral infections, such as AIDS. A number of human genetic diseases that result from a lesion in a single gene have been proposed as candidates for gene therapy. These include bone marrow disorders, erythroid cell defects, metabolic disorders resulting from defects in liver enzymes, and diseases of the central nervous system.
For some of these diseases, the introduction of a functional homolog of the defective gene and the production of even small amounts of the missing gene product would have a beneficial effect. For example, 10-20% production of the normal levels of Factor IX can alleviate severe hemophilia B. Yao, et al. (1991)
B. Proc. Natl. Acad. Sci.
88:8101-8105.
Since gene therapy evolved in the early 70's there have been several clinical trials involving retroviral transfer of either therapeutic genes or suicide genes. Gene transfer of ADA gene to correct for a genetic defect, was the first gene therapy trial which began in 1990. Culver, K., et al, (1990). Transfer of a suicide gene into brain tumors followed in 1992. Culver, K., et al. (1992)
Science
256:1550-1552. The gene transfer vehicle in both of these trials is a disabled retrovirus. Retroviral vectors are designed to transfer the gene of interest into target cells which must be undergoing cell division.
ADA is a rare genetic immunodeficiency disease caused when a defect occurs in both copies of the ADA gene. Children affected by this disease may have a severe combined, immunodeficiency (SCID) which could lead to death by common infections in their first months of life. Ex vivo gene transfer of the ADA gene into patients' T lymphocytes resulted in a beneficial therapy for the children in the 1990 trial. However, treatments must be repeated often to maintain sufficient levels of ADA in the bloodstream. In brain tumor trials, in situ gene transfer of the suicide gene, the HsTk gene, followed by ganciclovir treatment was used to eradicate the tumors. Although only a small portion of the tumor cells are transduced using this method, a “bystander” effect is hypothesized to help spread the killing.
In the above-described systems the therapeutic impact of gene therapy is at a minimum. Thus, there is a need to improve the efficiency of gene transfer. Currently, researchers are experimenting with alternative methods to increase transduction efficiency. However, there is a need for a quick and efficient marker gene to assess the results.
Murine retroviral vectors have emerged in the past several years as the most common vehicle to deliver marker genes. Other viral vectors such as adenoviruses, herpes viruses, adeno-associated viruses, and non-viral methods such as plasmids have also been used for gene transfer. Gene transfer systems often include markers such as &bgr;-galactosidase, luciferase, chloramphenicol acetyltransferase, and alkaline phosphatase. Detection of these markers involve either cell fixation that kills the cells and the addition of a substrate or antibody mediated detection. These methods are often time consuming and are prone to endogenous high background.
Another group of gene transfer markers convey drug resistance and thus allow positive selection of transfected cells through selection of resistant colonies. Although drug selectable markers allow the detection of living cells by expressing the transgene, they require that the cells survive in a toxic environment over a long period of time. Also, the neomycin-resistance gene, which confers resistance to the neomycin analog G418, has been shown to have deleterious effects upon the expression of other genes in retroviral vectors. Emerman, M., et al. (1986)
Nucleic Acids Res.
14, 9381-9396.
A novel marker gene is now available that will alleviate these cumbersome and time consuming steps for detecting gene transfer. The Green Fluorescent Protein (GFP) is a vibrant green bioluminescent marker which offers outstanding properties. The gene has been sequenced, humanized and is commercially available through several sources, however there has been much difficulty in finding a suitable transformation vehicle that will give stable expression in mammalian cells.
It is therefore a primary objective of the present invention to provide a gene transfer marker that overcomes the deficiencies of currently available gene transfer markers as described above.
It is another objective of the present invention to provide a gene transfer marker that provides rapid identification of gene transfer in living mammalian cells.
It is a further objective of the present invention to provide a gene transfer marker that can be easily visualized.
It is yet a further objective of the present invention to provide a gene transfer marker that is stable and is effectively and efficiently transferred into living cells.
These and other objectives will become apparent from the following description.
SUMMARY OF THE INVENTION
The present invention describes the cloning and characterization of amphotropic retroviral vectors capable of demonstrating efficient, stable transfer of humanized, red shifted GFP (hRGFP) gene into mammalian cells. Living cells transfected and/or transduced with hRGFP have a stable, bright green fluorescence after excitation with blue light.
The inventors have generated transformation vehicles containing a gene for an improved, humanized and red-shifted version of the Aequorea victoria green fluorescent protein (hRGFP) from various viral vectors. The hRGFP gene has been used to produce amphotropic vector producer cell lines that demonstrate vibrant green fluorescence after excitation with blue light. These vehicles represent a substantial improvement over currently available gene transfer marking systems. Bright, long-term expression of the hRGFP gene in living eukaryotic cells will advance the study of gene transfer, gene expression, and gene product function in vitro and in vivo, particularly for human gene therapy applications.
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Cody, C., “Chemical Structure of Hexapeptide Chromophore of the Aequorea Green-Fluorescent Protein”,Biochemistry, 1993, 32:1212-1218.
Inouye, S., “Aequorea Green Fluorescent Protein Expression of the Gene and Fluorescence Characteristics of the Recombinant Protein”, FEBS Lett. 341 (1994) 277-280.
Prasher, D., (1992) “Primary Structure of theAequorea victoriaGreen-Fluorescent Protein”,Gene, 111:229-233, Elsevier Science Publishers.
Perozzo, M., “X-Ray Diffraction and Time-Resolved Fluorescence Analyses of Aequorea Green Fluorescent Protein Crystals”,The J. of Biological Chemistry, 263(16) 7713-7716 (1988).
Ward, W., “Reversible Denaturation of Aequorea Green-Fluorescent Protein: Physical Separation and Characterization of the Renatured Protein”,Biochemistry, 21(19) 4535-4540 (1982).
Deschamps, J., “Rapid Purification of Recombinant Green Fluorescent Protein Using the Hydrophobic Properties of a
Levy John P.
Link, Jr. Charles J.
Seregina Tatiana
Wang Suming
Human Gene Therapy Research Institute
McKee Voorhees & Sease, P.L.C.
Slobodyansky Elizabeth
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