Recombinant mutants of rhabdovirus and methods of use thereof
A rhabdovirus and cytopathic technology, applied in the direction of viruses, vectors, metabolic diseases, etc., can solve the problems that wild-type tropism is not easy to overcome
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Embodiment 1
[0216] Example 1: Mutations in the M protein of VSV produce infectious, non-cytopathic viruses
[0217] Materials and methods
[0218] Site-directed mutagenesis of VSV expressing the GFP gene was performed. BHK-21 cells were then infected with the mutated VSV. Virus particles were concentrated and viral RNA was isolated from cell culture supernatants by ultracentrifugation. Full-length cDNAs of NCP-12 variants were obtained using reverse transcription-PCR. The M gene and cDNA were subjected to automated sequence analysis.
[0219] Cultured infected BHK-21 (MOI 10) cells and cell infectivity and morphology were determined by fluorescence microscopy at the indicated times. Round cells were aspirated from the culture and the culture was washed several times with gentle aspiration, then incubated and checked periodically. After 7 days, GFP-positive cells were detected in the culture, indicating infection, and the culture supernatant was harvested and aliqu...
Embodiment 2
[0228] Example 2: Mutations in the M protein of VSV do not affect cell tropism
[0229] Materials and methods:
[0230] The following cell types were treated with rVSV / M at a multiplicity of infection of 10 NCP12.1 Infection: BHK, CV-1, Vero or HeLa cells. Cells were incubated at 37°C for 12 or 24 hours, fixed in 3% paraformaldehyde and washed twice with phosphate buffered saline (PBS) containing 50 mM glycine. Cells were then examined for GFP expression by fluorescence microscopy (Zeiss Axiophot, West Germany) and morphology was assessed by phase contrast microscopy.
[0231] result
[0232] To determine non-cytopathic rVSV / M NCP12.1Whether cell tropism was altered, BHK, CV-1, Vero and HeLa cells were infected at a multiplicity of infection of 10 (FIG. 7), and infection was determined as a function of GFP expression. Regardless of cell type, rVSV / M NCP12.1 Both were able to infect and replicate intracellularly without any si...
Embodiment 3
[0233] Example 3: Development of Advanced Vectors for Gene Therapy Applications
[0234] Materials and methods
[0235] Recombinant VSV M mutants were generated as described above. Mutated viruses were grown and recovered during infection of BHK-21 cells by co-expression with plasmids expressing N, P and L proteins. The mutated virus expresses M NCP12.1 proliferate in cells. Supernatants from cells infected with rVSV-ΔM (VSV replicon) were pre-treated with 5 μg of pc-M 24 h before NCP12.1 Plasmid-transfected cells. Cells were fixed 24 hours after infection and probed with an N-specific monoclonal antibody labeled with a rhodamine-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc.).
[0236] result
[0237] Previous attempts to recover mutated or missing M protein VSV ([Delta]M-VSV) failed, presumably because the toxic effect of the M protein kills the cells thereby limiting the amount of M ...
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