Chimeric alphavirus replicon particles
AssigneeNovartis Vaccines and Diagnostics, Inc.
InventorsJohn M. Polo, Silvia Perri, Kent Thudium
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Abstract
Chimeric alphaviruses and alphavirus replicon particles are provided including methods of making and using same. Specifically, alphavirus particles are provided having nucleic acid molecules derived from one or more alphaviruses and structural proteins (capsid and/or envelope) from at least two or more alphaviruses. Methods of making, using, and therapeutic preparations containing the chimeric alphavirus particle, are disclosed.
Claims48
A chimeric alphavirus particle, comprising:
- RNA derived from one or more alphaviruses;
- and
- structural proteins, wherein at least one of said structural proteins is derived from two or more alphaviruses.
A chimeric alphavirus particle comprising
- RNA encoding one or more nonstructural proteins derived from a first alphavirus and a packaging signal derived from a second alphavirus different from said first alphavirus, wherein said packaging signal is inserted into a site selected from the group consisting of the junction of nsP3 with nsP4, following the open reading frame of nsP4, and a deletion in a nonstructural protein gene;
- a capsid protein derived from said second alphavirus;
- and
- an envelope protein derived from an alphavirus different from said first alphavirus.
An alphavirus replicon RNA comprising a 5′ sequence required for nonstructural protein-mediated amplification, sequences encoding biologically active alphavirus nonstructural proteins, an alphavirus subgenomic promoter, a non-alphavirus heterologous sequence, and a 3′ sequence required for nonstructural protein-mediated amplification, wherein the sequence encoding at least one of said nonstructural proteins is derived from a Biosafety Level 3 (BSL-3) alphavirus and wherein the sequences of said replicon RNA exhibit sequence identity to at least one third but no more than two-thirds of a genome of a BSL-3 alphavirus.
A method for producing alphavirus replicon particles, comprising introducing into a host cell:
- a) an alphavirus replicon RNA derived from one or more alphaviruses, further containing one or more heterologous sequence(s);
- and
- b) at least one separate defective helper RNA(s) encoding structural protein(s) absent from the replicon RNA, wherein at least one of said structural proteins is derived from two or more alphaviruses,
- wherein alphavirus replicon particles are produced.
A method for producing alphavirus replicon particles, comprising introducing into a host cell:
- a) an alphavirus replicon RNA encoding one or more nonstructural proteins from a first alphavirus, a packaging signal derived from a second alphavirus, and one or more heterologous sequence(s), wherein said packaging signal is inserted into a site selected from the group consisting of the junction of nsP3 with nsP4, following the nsP4 open reading frame, and a deletion in a nonstructural protein gene;
- and
- b) at least one separate defective helper RNA(s) encoding structural protein(s) absent from the replicon RNA, wherein at least one of said structural proteins is a capsid protein derived from said second alphavirus, and at least one of said structural proteins is an envelope protein derived from an alphavirus different from said first alphavirus,
- wherein alphavirus replicon particles are produced.
An alphavirus packaging cell line comprising one or more structural protein expression cassettes comprising sequences encoding one or more structural proteins, wherein at least one of said structural proteins is derived from two or more alphaviruses.
A method for producing alphavirus replicon particles, comprising introducing into a permissible cell,
- (a) an alphavirus replicon RNA comprising control elements and polypeptide-encoding sequences encoding (i) biologically active alphavirus nonstructural proteins and (ii) a heterologous protein, and
- (b) one or more defective helper RNA(s) comprising control elements and polypeptide-encoding sequences encoding at least one alphavirus structural protein, wherein said control elements comprise, in 5′ to 3′ order, a 5′ sequence required for nonstructural protein-mediated amplification, a means for expressing the polypeptide-encoding sequences, and a 3′ sequence required for nonstructural protein-mediated amplification, and further wherein one or more of said RNA replicon control elements are different than said defective helper RNA control elements;
- and
- incubating said cell under suitable conditions for a time sufficient to permit production of replicon particles.
Dependencies are inferred from the claim text.