Large scale production of packaged alphavirus replicons

Chemistry: molecular biology and microbiology – Vector – per se

Reexamination Certificate

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C435S236000, C435S239000, C435S455000, C435S456000, C435S457000, C424S093200, C424S199100

Reexamination Certificate

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06943015

ABSTRACT:
Large-scale packaging of alphavirus replicons is accomplished by co-transfecting host cells with three RNA molecules: (1) an alphavirus replicon comprising a sequence encoding a heterologous protein; (2) a helper RNA encoding a capsid protein and (3) a helper RNA encoding two alphavirus glycoproteins. The helper RNAs contain cis-acting elements that allow efficient replication of the helper RNAs and their packaging into viral particles as well as packaging of replicon genomes. These populations of viral particles can be propagated at high titers in cell culture by infecting cells at high multiplicity. Propagation of packaged replicons at an escalating scale is useful for large-scale production of recombinant proteins and/or vaccine.

REFERENCES:
Frolov, I. et al., “Selection of RNA Replicons Capable of Persistent Noncytopathic Replication of Mammalian Celss”, 1999, J. Virol. vol. 73: pp. 3854-3865.
Polo, J.M. et al., “Stable alphavirus packaging cell lines for Sindbis virus- and Semliki Forest virus-derived vectors”, 1999, PNAS, vol. 96: pp. 4598-4603.
Smerdou, C. et al., “Two-Helper RNA System for Production of Recombinant Semliki Forest Virus Particles”, J. Virol., vol. 73: pp. 1092-1098.

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