Structural Components for Amplification of Positive and Negative Strand VEEV Splitzicons

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Replicon RNA is an advanced nucleic acid technology for vaccines and therapeutics, offering higher protein expression than messenger RNA.
• Replicon RNA utilizes alphaviral non-structural proteins for self-amplification, driving increased therapeutic and vaccine development.

Purpose of the Study:

• To introduce RNA splitzicons, a split replicon system for RNA technology.
• To investigate the essential structural components for positive and negative strand RNA replicon amplification.
• To demonstrate the potential of split replicons for screening and developing novel RNA-based therapeutics.

Main Methods:

• Designed and constructed positive and negative strand RNA splitzicons encoding firefly luciferase.
• Assessed amplification efficiency by varying structural components like 5' UTR, CSE, SGP, and IRES.
• Evaluated NSP-mediated amplification across multiple cell types and over 72 hours.

Main Results:

• Both positive and negative strand splitzicons demonstrated self-amplification when paired with NSP constructs.
• Specific components (CSE, SGP, UTR) were crucial for positive strand amplification, while SGP was key for the negative strand.
• Splitzicons showed dose-dependent protein expression and sustained amplification exceeding 100,000-fold over 72 hours.

Conclusions:

• RNA splitzicons represent a new approach to RNA replicon technology, enabling efficient self-amplification.
• This system facilitates the screening of critical components for RNA replicon amplification.
• The findings pave the way for developing next-generation RNA vaccines and therapeutics with enhanced expression and stability.