Artificial alphavirus-derived RNA replicon expression systems

Inventors

Chahal, Jasdave S • McPartlan, Justine S

Assignees

Tiba Biotech LLC

Abstract

Synthetic alphavirus-derived replicon expression systems comprising nucleic acid sequences encoding at least one modified nonstructural protein, and synthetic nucleic acid sequences encoding at least one heterologous protein are described. Methods of producing at least one heterologous protein in a cell, or of inducing an immune response in a subject by administering and/or expressing the synthetic alphavirus-derived replicon expression systems are provided.

Core Innovation

The invention provides a synthetic alphavirus-derived replicon nucleic acid molecule designed to retain replicative function while incorporating engineered sequence changes. A first nucleic acid encodes alphavirus nonstructural proteins nsP1, nsP2, nsP3, and nsP4 and includes at least one silent mutation introduced within a defined nucleotide region from nt 6381 to nt 7083. The first nucleic acid further includes a polynucleotide sequence with at least 90% identity to a reference sequence set forth in SEQ ID NO: 18.

The invention also includes a second nucleic acid comprising a modified subgenomic open reading frame (ORF). The encoded payload supports early-phase expression of heterologous proteins by positioning a transgene fusion to an nsP4 C-terminus and maintaining or reinstating an intact subgenomic promoter (SGP). In particular, the document describes duplicated SGP region strategies and codon changes upstream of the promoter to avoid repeat-driven instability, and describes disabling or altering SGP homology by mutations.

Across the disclosed constructs and examples, the modified replicon is implemented as synthetic alphavirus-derived replicon RNA that expresses reporter genes and functional heterologous proteins. The disclosed examples include cis expression of functional protease (FMDV 3Cpro) and immunomodulatory STING (constitutively active), and demonstrate reduced innate immune activation and negligible cytotoxicity in normal human fibroblasts while enhancing interferon (IFN) signaling and inhibiting tumor cell growth in melanoma (B16) and epithelial tumors (TC-1). The document further describes expression systems, compositions, and methods of producing heterologous proteins and inducing immune responses, including vaccine and therapeutic formulations and encapsulation options.

Claims Coverage

The independent claim in the provided partial content is clm-00001. The claim is directed to a synthetic alphavirus-derived replicon nucleic acid molecule having two nucleic-acid components (encoding nsP1–nsP4 with a silent-mutation region and including a modified subgenomic ORF), together with sequence-identity constraints, and it is further refined by multiple dependent claims that narrow the payload and reference identity targets.

The claim set centers on engineering an alphavirus replicon such that the nsP1–nsP4-encoding component contains silent mutations in nt 6381–7083 while maintaining at least 90% identity to a reference sequence, and coupling this with a modified subgenomic ORF that provides the expression payload. Dependent claim refinements (within the partial content provided) further specify particular payload proteins (e.g., STING, 3C protease, O1 Manisa P1) and additional nsP4 identity constraints to specific SEQ IDs, and expand coverage to an expression system including a host cell.

Stated Advantages

Documented Applications