In vitro evolution of enhanced RNA replicons for immunotherapy

Yingzhong Li,Brian Dobosh,Yuan Zhang,Tyler Wagner,Ely Porter,Darrell J. Irvine,Brian Teague,Ron Weiss,Zhijun Su

doi:10.1038/s41598-019-43422-0

Abstract

Self-replicating (replicon) RNA is a promising new platform for gene therapy, but applications are still limited by short persistence of expression in most cell types and low levels of transgene expression in vivo. To address these shortcomings, we developed an in vitro evolution strategy and identified six mutations in nonstructural proteins (nsPs) of Venezuelan equine encephalitis (VEE) replicon that promoted subgenome expression in cells. Two mutations in nsP2 and nsP3 enhanced transgene expression, while three mutations in nsP3 regulated this expression. Replicons containing the most effective mutation combinations showed enhanced duration and cargo gene expression in vivo. In comparison to wildtype replicon, mutants expressing IL-2 injected into murine B16F10 melanoma showed 5.5-fold increase in intratumoral IL-2 and 2.1-fold increase in infiltrating CD8 T cells, resulting in significantly slowed tumor growth. Thus, these mutant replicons may be useful for improving RNA therapeutics for vaccination, cancer immunotherapy, and gene therapy.

Highlights

Nucleic acid therapeutics have the potential to treat or cure many diseases that are difficult to address with more traditional therapies[1]
While many replicon studies are carried out using BHK-21 cells that are deficient in interferon signaling, we employed human Jurkat cells that have a functional interferon pathway for transfection and passage of replicons, in order to potentially select for mutations limiting the host cell interferon response
Alphavirus replicons are of substantial interest as platforms for gene therapy and vaccination, with the ability to encode therapeutic genes or antigens under the subgenomic promoter in place of the structural proteins required for replicon replication

Summary

Introduction

Replicons delivered as engineered viral particles or as synthetic RNA encapsulated in lipid nanoparticles have shown promise as non-viral vaccine vectors[11,12], for expression of therapeutic agents in cancer immunotherapy[13,14], and for correction of genetic defects such as hemophilia[15] Motivated by these promising examples, we began exploring strategies to regulate gene expression using replicons derived from the Venezuelan Equine Encephalitis (VEE) virus[16,17]. Rose et al used infectious Semliki Forest viral particles to evolve replicons through long-term serial infections of in vitro cell cultures, and identified multiple mutations promoting high-titer production of virus-like vesicles from SFV replicons[25] Based on these past successes, we started with non-cytopathic VEE26 and designed a new in vitro evolution strategy that uses interferon (IFN)-competent cells and multiple long duration rounds of replicon enrichment for higher subgenome expression. The mutant replicon sequences identified here improve the utility of replicon RNA as a platform for gene therapy

Methods

Results

Conclusion