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AssigneesBoehringer Ingelheim International GmbH, Viratherapeutics GmbH
InventorsMeike Dorothee Holm-von Laer, Emmanuel Heilmann, Janine Kimpel, Guido Wollmann, Lisa Egerer, Benedikt Hofer
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The present invention relates to a novel mechanism to control RNA virus replication and gene expression using a conditional protease approach using a specific protease inhibitor for regulation. More specifically it relates to a single-stranded RNA virus, preferably of the order Mononegavirales, comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and/or replication, a protease and a cleavage site for said protease. The protease can be inhibited using a protease inhibitor and hence the protease and the cleavage site for said protease form a regulatable switch. By changing the insertion site of the regulatable switch from an INTRA- to an INTER-molecular location in the at least one protein essential for viral transcription and/or replication, the effect of the protease inhibitor can be altered from an ON switch to an OFF switch. RNA virus may further encode a heterologous protein, the expression of which is then regulated by regulating viral activity. The ON switch may also be used in an RNA virus to directly regulate heterologous protein expression. Further provided are in vivo and in vitro uses of said virus with conditional viral activity or heterologous protein expression.
A single-stranded RNA virus comprising a modified genome of the virus comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and/or replication, a protease and a cleavage site for said protease, wherein
The single-stranded RNA virus of any one of the preceding claims, wherein the protease is the HIV-1 protease, preferably a single chain dimer of the HIV-1 protease, and the protease can be inhibited by a protease inhibitor selected from the group consisting of indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenevir, atazanavir, tipranavir and darunavir.
The single-stranded RNA virus of any one of the preceding claims, wherein at least the cleavage site for said protease and optionally further the protease is located within the intramolecular insertion site of the least one protein essential for viral transcription and/or replication, and wherein
The single-stranded RNA virus of any one of the preceding claims, wherein the single-stranded RNA virus is Vesicular Stomatitis Virus (VSV), the at least one protein essential for viral transcription and/or replication is the P-protein and/or the L-protein and wherein the intramolecular insertion site is
An RNA virus comprising a modified genome of the virus comprising a polynucleotide sequence encoding at least one heterologous protein, a protease and a cleavage site for said protease, wherein the at least one heterologous protein comprises an insert at an intramolecular insertion site comprising at least the cleavage site for said protease and optionally further the protease, preferably wherein the heterologous protein is a therapeutic protein, a reporter or a tumor antigen.
A recombinant VSV L-protein comprising an insert in the loop of the methyltransferase domain of the L-protein corresponding to amino acids 1614 to 1634, preferably to amino acids 1614 to 1629, more preferably to amino acids 1616 to 1625 and more preferably to amino acid 1620 of VSVi L-protein having the amino acid sequence of SEQ ID NO:
A method for controlling RNA virus replication comprising
Dependencies are inferred from the claim text.