US 5,792,462 AGranted

Alphavirus RNA replicon systems

AssigneesThe University of North Carolina at Chapel Hill, The United States of America as represented by the Secretary of the Army

InventorsRobert E. Johnston, Nancy L. Davis, Jonathan F. Smith, Peter Pushko, Michael Parker, George Ludwig

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Abstract

The present invention provides a helper cell for expressing an infectious, replication defective, alphavirus particle in an alphavirus-permissive cell. The helper cell includes (a) a first helper RNA encoding (i) at least one alphavirus structural protein, and (ii) not encoding at least one alphavirus structural protein; and (b) a second helper RNA separate from the first helper RNA, the second helper RNA (i) not encoding the alphavirus structural protein encoded by the first helper RNA, and (ii) encoding the at least alphavirus one structural protein not encoded by the first helper RNA, such that all of the alphavirus structural proteins assemble together into alphavirus particles in the cell. Preferably, the helper cell also includes a replicon RNA encoding an alphavirus packaging sequence and an inserted heterogeneous RNA.

Claims27

Dependent claims

A helper cell for producing an infectious, defective alphavirus particle, comprising, in an alphavirus-permissivecell:

(a) an alphavirus replicon RNA, wherein the alphavirus is selected from the group consisting of Sindbis virus, Semliki Forest virus, Venezuelan Equine Encephalitis Virus, and South African Arbovirus No. 86;
wherein the replicon RNA comprises the alphavirus packaging signal, a heterologous RNA sequence, and a sequence encoding at least one of the alphavirus structural proteins, wherein the replicon RNA furthermore lacks a sequence encoding at least one of the alphavirus structural proteins;
and
(b) at least one separate helper RNA encoding the structural protein(s) absent from the replicon RNA, said helper RNA(s) lacking the alphavirus packaging signal;
wherein the combined expression of the replicon RNA and the helper RNA(s) produces an assembled alphavirus particle which comprises a heterologous RNA sequence, is able to infect a cell, and is unable to complete viral replication in the absence of helper RNA due to the absence of the structural protein coding sequence in the packaged replicon.

2 more elements

A helper cell for producing an infectious, defective alphavirus particle, comprising an alphavirus-permissive cell transfected with RNAs comprising:

(a) an alphavirus replicon RNA, wherein the alphavirus is selected from the group consisting of Venezuelan Equine Encephalitis Virus and South African Arbovirus No. 86;
wherein the replicon RNA comprises the alphavirus packaging signal, a heterologous RNA sequence, wherein the replicon RNA furthermore lacks sequences encoding alphavirus structural proteins;
and
(b) at least a first and second helper RNAs separate from said replicon RNA and separate from each other, said first and second helper RNAs encoding the structural proteins absent from the replicon RNA;
with said first helper RNA encoding at least one alphavirus structural protein and not encoding at least one other alphavirus structural protein;
with said second helper RNA not encoding said at least one alphavirus structural protein encoded by said first helper RNA and encoding said at least one other alphavirus structural protein not encoded by said first helper RNA;
and with said first and second helper RNAs lacking the alphavirus packaging signal;
wherein the combined expression of the replicon RNA and the helper RNAs produces an assembled alphavirus particle which comprises a heterologous RNA sequence, is able to infect a cell, and is unable to complete viral replication in the absence of the helper RNAs due to the absence of the structural protein coding sequence in the packaged replicon.

5 more elements

Dependencies are inferred from the claim text.