Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1998-12-22
2002-12-24
Reynolds, Deborah J. (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C435S235100, C435S239000, C435S320100, C435S440000, C435S455000
Reexamination Certificate
active
06497873
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a recombinant Rhabdovirus that expresses at least a fusion protein which facilitates fusion and entry of the recombinant Rhabdovirus into a cell target. This invention includes a recombinant Vesicular Stomatitis Virus (VSV) which expresses a fusion protein on the surface of the VSV particle. These recombinant Rhabdoviruses which express a fusion protein can be used to study function and specificity of proteins not naturally found on the Rhabdovirus being engineered, and as a method of targeting abnormal and diseased cells (e.g., virus infected cells or cancer cells) for diagnostic and therapeutic purposes. This invention also discloses methods of producing recombinant Rhabdoviruses.
BACKGROUND OF THE INVENTION
A. Using Viruses to Target Cells
Viruses have been engineered in the last decade to target cells, mainly for purposes of gene therapy. Gene therapy involves the delivery of a gene, often a diseased cell, and usually involves insertion of the gene into the genome of the host cell. Viruses from the families of Adenoviridae, Parvoviridae and Retroviridae have successfully been engineered not only to insert genes to cell genomes, but also to deliver the gene to specific cells or tissues.
To deliver viruses to specific cells, the virus must be able to infect that cell type. Viruses cannot typically infect all cells or even all organisms. The ability of a virus to infect a cell is based on the “tropism” the virus has for the host organism and the cells of that organism. For a virus to be able to infect a cell, the cell must have a receptor for a virus protein which allows the virus to recognize and bind to the cellular receptor, whereupon it enters the cell either via endocytosis, phagocytosis or macropinocytosis. Upon entry into the cell, the virus begins replicating. To infect cells for which the virus does not have a tissue tropism, the virus must be engineered to recognize and bind to a receptor on the cell or tissue of interest. Even then, a virus may still not be able to replicate, as it may require additional cellular factors not produced in that cell.
Gene therapy viral vectors typically do not kill or lyse the cells they target. Viral vectors used for gene therapy are engineered to deliver therapeutically effective DNAs with relative safety, like a drug (see for example, D. T. Curiel et al., U.S. Pat. No. 5,547,932). Some of these vectors are capable of replicating upon infection, but only in the targeted cells (F. McCormick, U.S. Pat. No. 5,677,178). Other gene therapy vectors are engineered such that they are unable to replicate. Non-replicating gene therapy vectors are usually produced using helper plasmids (see for example, G. Natsoulis, U.S. Pat. No. 5,622,856; M. Mamounas, U.S. Pat. No. 5,646,034) or packaging cells that confer genetic elements missing in the virus genome.
Gene therapy vectors have also encountered problems with overcoming the wild-type tropisms natural to the viral vector being utilized. Pseudotype viruses were created to overcome this by engineering a virus genome to contain the DNA encoding an envelope protein from another virus, even from a different virus family or genus, that would be capable of infecting the tissue or cell target. In recent years, many gene therapy patents have been issued describing adenovirus vectors (M. Cotten et al., U.S. Pat. No. 5,693,509); adeno-associated virus vectors (J. S. Lebkowski et al., U.S. Pat. No. 5,589,377); retrovirus vectors (B. O. Palsson et al., U.S. Pat. No. 5,616,487); vectors containing chimeric fusion glycoproteins (S. Kayman et al., U.S. Pat. No. 5,643,756); vectors that contain an antibody to a virus coat protein (Cotten et al.); viruses have been engineered to allow study of human immunodeficiency type 1 (HIV-1) in monkeys, a species that normally cannot be infected by HIV-1, by creating hybrid viruses (J. Sodroski et al., U.S. Pat. No. 5,654,195); and pseudotype retrovirus vectors which contain the G protein of Vesicular Stomatitis Virus (VSV) (J. C. Burns et al., U.S. Pat. Nos. 5,512,421 and 5,670,354). Some of these gene therapy vectors use methods which attempt to overcome some aspects of the tropism related problems encountered, while maintaining the efficacy of the vector for use in gene therapy.
Virus delivery vehicles have also been created for transient gene therapy, wherein expression of the gene delivered to the cell is transient and not permanent (I. H. Maxwell et al., U.S. Pat. No. 5,585,254). Vectors have been created that selectively express certain toxin-encoding genes, such as the gene for diphtheria toxin (U.S. Pat. No. 5,585,254.). Viral vectors also can be engineered to make the host cells they infect more immunogenic (U.S. Pat. No. 5,580,564).
B. Using Rhabdoviruses to Target Cells
Both Vesicular Stomatitis Virus (VSV) and Rabies Virus (RV) are members of the Rhabdoviridae family. VSV belongs to the Vesiculovirus genus, while RV belongs to the Lyssavirus genus. All members of the Rhabdovirus family possess lipid membrane envelopes which comprise the surface of the Rhabdovirus virion.
(1) Rabies Virus
A recent paper by T. Mebatsion et al. described the engineering of a Rabies Virus (RV) such that either the entire G protein was deleted or only a small portion of the G protein was expressed. This recombinant RV was further engineered such that either CD4 or CD4 and the CXCR4 co-receptor also were expressed on the envelope of the recombinant RV virion (T. Mebatsion et al., (1997)
Cell
90: 941-951). Characterization of and experiments with these engineered RV pseudotype viruses demonstrated that a CD4/CXCR4 construct, which also contained the tail of the G protein, could infect cells expressing the HIV-1 envelope protein, gp120. A drawback of this viral system was that effective incorporation of the non-RV proteins (e.g., CD4 and CXCR4) only occurred when at least the tail of the G protein (a 44 amino acid cytoplasmic domain) was expressed on the virion in the form of a chimera fused to either CD4 (RV-CD4) or CXCR4 (RV-CXCR4). A recombinant RV expressing only the RV-CD4 and the truncated G protein chimera cDNA did not contain detectable amounts of CD4 in the virion. However, a recombinant RV expressing both RV-CD4 and RV-CXCR4 yielded a virus particle with both the CD4 and CXCR4 proteins in the virus envelope (T. Mebatsion et al., 1997). The authors' conclusion was that CD4-derived proteins are incorporated only in the form of a complex with a heterologous “carrier” protein. The carrier protein in the RV construct is the CXCR4 co-receptor.
(2) Vesicular Stomatitis Virus
CD4 also has been expressed in VSV particles along with all five VSV gene products: N, P, M, G and L (Schnell et al., (1996)
Proc. Nat'l Acad. Sci. USA
93: 11359-11365). A more recent publication by Schnell et al., demonstrated that both CD4 and a co-receptor protein, such as CXCR4, can be expressed in virus particles even if the entire gene encoding the G protein is deleted (&Dgr;G) (Schnell et al., (1997)
Cell
90: 849-857). This CD4/CXCR4 recombinant was produced by utilizing a complementing plasmid containing DNA encoding the G protein. The gene encoding CXCR4 was then placed downstream of the gene encoding CD4 in the &Dgr;G recombinant VSV. Levels of the &Dgr;G-CD4 virus were 25% of the levels reported for the CD4 construct which contained the G protein (Schnell et al., 1997). However, CD4 was incorporated in the recombinant virion with the same efficiency as other VSV proteins despite the absence of a G protein. When comparing the ability of the &Dgr;G-CD4 construct to infect HIV-1 infected Jurkat cells to the VSV &Dgr;G construct containing both CD4 and CXCR4 (&Dgr;G-CD4/CXCR4), the &Dgr;G-CD4 construct infected the cells at 10% the rate of the &Dgr;G-CD4/CXCR4 VSV recombinant. Moreover, the &Dgr;G-CD4/CXCR4 was able to reduce the number of HIV-1 positive cells. These constructs were demonstrated to be capable of entering and propagating in cells infected with HIV-1 or that express the HIV-1 envelope protein (Schnell et al., 1997).
Althoug
Robison Clinton S.
Whitt Michael A.
Chen Shin-Lin
Cohen Mark S.
Eitan Pearl Latzer & Cohen-Zedek
Reynolds Deborah J.
The University of Tennessee Research Corporation
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