Engineering gene expression dynamics via self-amplifying RNA with drug-responsive non-structural proteins

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Immunology

Background:

• Gene therapies require precise control over therapeutic payload expression for safety and efficacy.
• Alphavirus-derived self-amplifying RNAs (saRNAs) offer a platform for sustained RNA expression.
• Drug-regulatable systems are crucial for managing gene expression in therapeutic applications.

Purpose of the Study:

• To design and validate a drug-regulatable expression system based on saRNAs.
• To achieve trimethoprim (TMP)-mediated control over saRNA self-amplification and payload expression.
• To evaluate the therapeutic potential of this system in vivo for immunotherapy.

Main Methods:

• Engineered saRNAs by fusing TMP-responsive degradation domains (DDs) to non-structural proteins (nsPs) and payload.
• Screened nsP-DD fusions to identify optimal constructs for TMP-regulated expression.
• Assessed RNA expression dynamics in response to oral TMP administration in mice.
• Implemented the system for controlled expression of an HIV antigen to evaluate immune responses.

Main Results:

• Identified an optimal saRNA design with DDs on nsP2, nsP3, and payload, yielding high TMP-induced expression and low basal expression.
• Demonstrated dynamic and tunable gene expression patterns in mice following oral TMP administration.
• Showcased enhanced germinal center responses, crucial for B cell maturation, upon escalating TMP treatment for HIV antigen expression.

Conclusions:

• Developed a novel, drug-regulated RNA technology using saRNAs controlled by TMP.
• This system provides precise control over gene expression, with potential applications in vaccines, immunotherapies, and gene therapies.
• The technology enables dynamic control of therapeutic payloads, offering improved safety and efficacy profiles.