A Novel Self-Amplifying mRNA with Decreased Cytotoxicity and Enhanced Protein Expression by Macrodomain Mutations

Area of Science:

• Molecular Biology
• Immunology
• Vaccine Technology

Background:

• Self-amplifying mRNA (saRNA) offers dose-sparing advantages over conventional non-replicating mRNA (nrmRNA) due to its self-replication mechanism.
• Optimizing saRNA expression efficiency is crucial for its broader application in vaccines and therapeutics.
• saRNA's replication can trigger innate immune responses via double-stranded RNA (dsRNA) sensors, potentially limiting its efficacy and causing cytotoxicity.

Purpose of the Study:

• To investigate the impact of mutations in saRNA's nonstructural protein (NSP) on its replication, immunogenicity, and payload expression.
• To identify strategies for reducing saRNA-induced innate immune responses and enhancing protein translation efficiency.

Main Methods:

• In vitro adaptive passaging of saRNA under interferon pressure to select for mutations.
• Characterization of mutations in the NSP3 macrodomain (MD) for their effect on enzyme activity and saRNA properties.
• Transcriptome sequencing to analyze the activation of dsRNA sensors and immune responses.
• In vitro and in vivo assessment of protein expression, translation inhibition, and cell apoptosis.

Main Results:

• Two stable mutations (Q48P and I113F) in the NSP3 macrodomain attenuated its MAR hydrolysis activity.
• Mutant saRNA exhibited decreased replication but significantly increased payload expression compared to wild-type saRNA (wt saRNA).
• Mutant saRNA showed diminished activation of dsRNA sensors, leading to a reduced innate immune response.
• Less translation inhibition and apoptosis were observed with mutant saRNA, resulting in higher protein expression both in vitro and in vivo.

Conclusions:

• Genetic modification of saRNA, specifically targeting NSP3, can reduce replication-dependent dsRNA-induced innate immune responses.
• These modifications enhance payload translation efficiency and mitigate saRNA-induced cytotoxicity.
• This strategy represents a promising approach for optimizing saRNA technology for improved therapeutic and vaccine applications.