Alphavirus-based replicons for administration of biotherapeutics

An RNA replicon comprising

• (i) an RNA sequence encoding a heterologous protein or peptide;
• (ii) RNA sequences encoding amino acid sequences derived from New World alphavirus nonstructural proteins nsP1, nsP2, and nsP4;
• and
• (iii) an RNA sequence encoding an amino acid sequence derived from an Old World alphavirus nsP3 protein, wherein the first 1-6 amino acids on the N-terminal and/or C-terminal side of the nsP3 protein are derived from a New World alphavirus sequence.

An alphavirus replicon RNA, comprising:

• in order from 5′ to 3′ end,
• (i) an alphavirus 5′ untranslated sequence for directing replication of the alphavirus replicon;
• (ii) an RNA sequence encoding alphaviral nonstructural proteins nsP1, nsP2, nsP3, and nsP4;
• (iii) an alphavirus subgenomic promoter sequence;
• (iv) an RNA sequence encoding one or more heterologous proteins or peptides;
• and
• (v) an alphavirus 3′ untranslated sequence;
• wherein the nsP1, nsP2 and nsP4 are from one or more New World alphaviruses,
• wherein the nsP3 comprises, in order from amino-terminus to carboxyl terminus:
• an alphavirus nsP3 macro domain, an alphavirus nsP3 central domain, and an alphavirus nsP3 hypervariable domain (HVD),
• wherein the macro domain and the central domain are from one or more New World alphaviruses and/or Old World alphaviruses,
• wherein the HVD comprises an HVD of an Old World alphavirus, or the HVD comprises a portion of an HVD from a New World alphavirus and a portion of an HVD from an Old World alphavirus;
• and
• wherein the New World alphavirus is selected from the group consisting of:
• a Venezuelan equine encephalitis virus (VEEV), a western equine encephalitis virus (WEEV), and an eastern equine encephalitis virus (EEEV);
• and the Old World alphavirus is selected from the group consisting of:
• Sindbis virus (SINV), Chickungunya virus (CHIKV), Semliki Forest Virus (SFV), Ross River Virus (RRV), Sagiyama virus (SAGV), Getah virus (GETV), Middleburg virus (MIDV), Bebaru virus (BEBV), O'nyong nyong virus (ONNV), Ndumu (NDUV), and Barmah Forest virus (BFV).