Recombinant Rhabdovirus containing a heterologous fusion protein

a fusion protein and rhabdovirus technology, applied in the field of rhabdovirus recombinant, can solve the problems of virus not being able to replicate, gene therapy vectors that have encountered problems such as overcoming wild-type tropisms, effective incorporation of non-rvs, etc., and achieve the effect of facilitating the fusion of the lipid envelop

Inactive Publication Date: 2003-07-24
TENNESSEE RESEARCH CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

0020] The present invention relates to recombinant or genetically engineered Rhabdoviruses that express a heterologous "F Protein" (as defined herein) to facilitate fusion of the lipid envelope of the recombinant virus to the cell membrane of a target cell. Such constructs overcome the limitations of systems known in the art that require specific co-receptors or exhibit specific target cell tropisms.

Problems solved by technology

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 vectors have also encountered problems with overcoming the wild-type tropisms natural to the viral vector being utilized.
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 (V-CD4) or CXCR4 (RV-CXCR4).
A drawback of using the recombinant Rhabdoviruses as described by Mebatsion et al.

Method used

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  • Recombinant Rhabdovirus containing a heterologous fusion protein
  • Recombinant Rhabdovirus containing a heterologous fusion protein
  • Recombinant Rhabdovirus containing a heterologous fusion protein

Examples

Experimental program
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Effect test

example 1

Creation of a Modified Rhabdovirus

[0107] Construction of recombinant VSV cDNAs. A diagram of the method used, in this example, to construct a recombinant VSV that expresses the F protein of paramyxovirus SV5 is shown in FIG. 5. Full-length VSV cDNAs encoding G proteins with truncated cytoplasmic domains were generated by replacing the gene encoding the wild-type G protein in pVSVFL(+).sub.I / NIG (N. Lawson et al., Proc. Nat'l Acad. Sci. USA (1995) 92: 4477-4481). Another version of CT-1 was also constructed, which contained three consecutive stop codons, and which had the remaining portion of the cytoplasmic tail coding region to the NheI site deleted. This mutant is called CT-1TS (CT-1 Triple Stop) and was generated using standard PCR-mediated mutagenesis methods with a sense-strand primer (MW-36) complementary to nucleotides 975-997 (see the sequence for the complete genome of VSV-Indiana, GenBank # J02428), which are 5' to the KpnI site in the G protein cDNA, and an antisense olig...

example 2

In vitro Administration of Recombinant VSV Containing a Fusion Protein to Cells

[0113] The filtrate was then applied to cells in 10 cm dishes. After 18 to 24 hr, the cells were examined for cytopathic effects (CPE). Supernatants from cultures exhibiting CPE were titered, and individual plaques were purified. Plaque purified isolates were used to infect BHK cells at a m.o.i. of one for the production of stocks of the tail mutant viruses. Direct sequencing of viral RNA isolated from each of the mutants was performed to ascertain whether the recovered virus contained the appropriate mutations.

[0114] One-half of each filtered supernatant from the VSV-HAGS / minivirus technique was used to infect fresh cells. Successful recoveries were indicated when cultures showed the typical cytopathic effects indicative of a normal VSV infection 18 to 24 hr post infection. The supernatants from those cultures were then titered and stocks of the co-complementing viruses were produced by infecting fresh c...

example 3

Administration of Modified VSV to Cells in vivo

[0115] Recombinant VSV particles prepared by either of the methods described above are inoculated into a subject infected with HIV-1. The concentration of the virus to be administered to a subject may vary, but the titer of the virus to be administered preferably should be in the order of about 10.sup.6 to 10.sup.12 IU / ml. The amount of the virus used may be greater if the patient has a relatively high number of infected cells. The viruses harvested as described in Example 1 would e resuspended in an appropriate carrier or excipient for purposes of injection.

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Abstract

This invention relates to a composition comprising a recombinant or genetically engineered Rhabdovirus that expresses a Fusion Protein, such as the F protein of the Paramyxovirus SV5 strain. This recombinant Rhabdovirus may express other non-Rhabdovirus attachment proteins and/or an enhancer protein. The invention also relates to methods of making recombinant Rhabdoviruses which express an F Protein. These recombinant compositions can be used for purposes of research, as well as for diagnostic and therapeutic compositions for treatment of diseases.

Description

II. FIELD OF THE INVENTION[0002] 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.III. BACKGROUND OF THE INVENTION[0003] A. Using Viruses to Target Cells[0004] 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...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/76A61K48/00A61P31/00A61P33/00A61P35/00C12N15/09C07K14/115C07K14/145C12N7/00C12N15/40C12N15/86
CPCC12N2760/20222C12N2760/20243C12N2810/855C12N2810/859C12N2760/18022C07K14/005C07K2319/00C12N15/86C12N2840/20A61P31/00A61P33/00A61P35/00
Inventor WHITT, MICHAEL A.ROBISON, CLINTON S.
Owner TENNESSEE RESEARCH CORPORATION
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