C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
C12N 15/11 (2006.01) C07K 14/11 (2006.01) C07K 14/16 (2006.01) C12N 7/01 (2006.01) C12N 9/12 (2006.01) C12N 9/24 (2006.01) C12N 15/10 (2006.01) C12N 15/86 (2006.01) A61K 39/00 (2006.01)
Patent
CA 2065245
Recombinant negative strand virus RNA templates which may be used to express heterologous gene products and/or to construct chimeric viruses are described. Influenza viral polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicative that the promoter lay solely within the 15 nucleotide 3' terminus. Sequences not specific for the influenza viral termini were not copied, and, surprisingly, RNAS containing termini identical to those from plus sense cRNA were copied at low levels. The specificity for recognition of the virus-sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap-endonuclease primed and primer-free RNA synthesis from these model templates as well as from genomic length RNAs. This indicated that this reconstituted system has catalytic properties very similar to those of native viral RNPs. High levels of expression of a heterologous gene was obtained using the constructs and methods described.
Krystal Mark
Palese Peter
Parvin Jeffrey D.
Mount Sinai School Of Medicine
Mount Sinai School Of Medicine Of The City University Of New Yor
Osler Hoskin & Harcourt Llp
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