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;
• wherein the replicon does not encode at least protein C.

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 at least a portion of an Old World alphavirus HVD comprising a repeat selected from the group consisting of:
• an FGDF/FGDF (SEQ ID NO:
• 20) repeat, an FGSF/FGSF (SEQ ID NO:
• 21) repeat, an FGDF/FGSF (SEQ ID NO:
• 22) repeat, and an FGSF/FGDF (SEQ ID NO:
• 23) repeat;
• and further wherein the repeat sequences are separated by at least 10 but not more than 25 amino acids;
• wherein the HVD comprises the Old World alphavirus HVD comprising said portion of the Old World alphavirus HVD, or the HVD comprises a portion of an HVD from a New World alphavirus and said portion of an HVD from the Old World alphavirus;
• 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);
• and
• wherein the replicon does not encode at least protein C.