In vitro evolution of enhanced RNA replicons for immunotherapy

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.

An Update on Self-Amplifying mRNA Vaccine Development

“…saRNA vaccines have been primarily investigated for active vaccination strategies for prevention of infectious diseases, wherein the host’s cells produce a pathogenic antigen encoded in saRNA to induce a humoral and cellular immune response. saRNA encoding viral glycoproteins are the most predominant application, although this has recently been expanded to include bacterial infections ( Chlamydia trachomatis [ 57 ], Group A and B Streptococci [ 58 ]), parasites ( Toxoplasma gondii [ 59 , 60 ]) and cancer (colon carcinoma, [ 61 , 62 ] melanoma [ 62 ]). A more novel approach to saRNA antigen design includes encoding monoclonal antibodies for passive vaccination [ 63 ].…”

The Potential of Self-amplifying RNA Vaccines for Infectious Diseases and COVID-19

“…This could be achieved by co-administration of compounds able to block IFN responses, like for example vaccinia virus immune evasion proteins [80]. A different approach to boost saRNA vaccines has been based on in vitro evolution of RNA replicons in IFN-competent cells [119]. This strategy led to the identification of six mutations in the VEE nonstructural proteins (nsPs) that promoted subgenomic RNA expression.…”

A new generation of vaccines based on alphavirus self-amplifying RNA

“… Immune responses, unaffected by TLR-agonists [ 36 ] pABOL HA Protection from influenza virus challenge [ 40 ] CNE VEEV TC-83 Full protection against VEEV challenge [ 30 •• ] cVEEV Viral ag. Rodents/NHP Specific T cell and antibody responses [ 24 ] HA Mouse/Ferret Protection against heterologous virus challenge [ 26 ] NP+GM-CSF Mouse Enhanced APC recruitment & virus protection [ 27 • ] Mannose-LNP IM/ID HA Enhanced APC uptake & immune responses [ 31 ] VEEVm LNP IT IL-2 Enhanced expression & antitumor effects [ 44 ] a N.I., not indicated; ta, trans -amplifying RNA; SFV, Semliki Forest virus; VEEV, Venezuelan equine encephalitis virus; cVEEV chimeric VEEV-Sindbis RNA vector; VEEVm, mutant VEEV. b Nak, naked; EP, electroporation; inh, inhi...…”

“…This could be achieved by co-administration of compounds able to block IFN responses, like for example vaccinia virus immune evasion proteins [ 43 ]. A different approach to boost saRNA vaccines has been based on in vitro evolution of RNA replicons in IFN-competent cells [ 44 ]. This strategy led to the identification of six mutations in VEEV nonstructural proteins (nsPs) that promoted subgenomic RNA expression.…”