An RNA Interference/Adeno-Associated Virus Vector-Based Combinatorial Gene Therapy Approach Against Hepatitis E Virus

Area of Science:

• Virology
• Molecular Biology
• Gene Therapy

Background:

• Hepatitis E virus (HEV) poses a significant global health challenge.
• Current therapeutic strategies for HEV infection are limited.
• Developing effective antiviral treatments is crucial.

Purpose of the Study:

• To engineer adeno-associated viral vectors (AAV6) for delivering short hairpin RNAs (shRNAs) targeting HEV.
• To down-regulate HEV replication in vivo using RNA interference (RNAi).
• To assess the efficacy of multiplexed shRNAs against HEV genotype 3 (GT3).

Main Methods:

• Design and construction of 20 shRNAs targeting HEV GT3 genome.
• Utilizing a selectable HEV GT3 reporter replicon for screening.
• Delivery of shRNAs via AAV6 transduction in cell-based systems.
• Evaluation of HEV replication inhibition in full-length HEV infection models.

Main Results:

• Three shRNAs demonstrated significant inhibition of HEV replication.
• HEV replication reduced by up to 95% within 72 hours post-transduction.
• Potent shRNAs targeted the methyltransferase domain, ORF junction, and 3' end of ORF2.
• Multiplexing shRNAs enhanced inhibition efficacy and duration (up to 21 days).

Conclusions:

• Combination of RNA interference and AAV gene therapy shows potential for HEV suppression.
• Multiplexed shRNA strategy can overcome viral escape mutations.
• The AAV vector-based approach is potentially translatable to clinical settings due to AAV's safety profile.