Alphavirus RNA replicon systems

A population comprising infectious, defective, alphavirus particles produced by a method comprising:

• (a) providing a helper cell for producing an infectious, defective alphavirus particle, comprising in an alphavirus-permissive cell:
• (i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal, one or more heterologous RNA sequence(s), and a sequence encoding at least one alphavirus structural protein, and wherein the replicon RNA furthermore lacks a sequence encoding at least one alphavirus structural protein;
• and
• (ii) one or more separate helper RNA(s) encoding the alphavirus structural protein(s) absent from the replicon RNA, wherein at least one of said separate helper RNA(s) lacks an alphavirus packaging signal;
• wherein the combined expression of the alphavirus replicon RNA and the separate helper RNA(s) produces an assembled alphavirus particle which comprises one or more heterologous RNA sequence(s), is able to infect a cell, and is unable to complete viral replication;
• and further wherein the population contains no replication-competent alphavirus particles as determined by passage on permissive cells in culture;
• (b) producing the alphavirus particles in the helper cell;
• and
• (c) collecting the alphavirus particles from the helper cell.

A population of infectious, defective, alphavirus particles produced by a method comprising:

• (a) providing a helper cell for producing an infectious, defective alphavirus particle, comprising in an alphavirus-permissive cell:
• (i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal, one or more heterologous RNA sequence(s), and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
• (ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein;
• and
• (iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
• and with at least one of said helper RNAs lacking an alphavirus packaging signal;
• wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which comprises one or more heterologous RNA sequence(s), is able to infect a cell, and is unable to complete viral replication, and further wherein the population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;
• and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;
• (b) producing the alphavirus particles in the helper cell;
• and
• (c) collecting the alphavirus particles from the helper cells.