Long-term RNA interference from optimized siRNA expression constructs in adult mice

Area of Science:

• Molecular Biology
• Gene Regulation
• Biotechnology

Background:

• DNA constructs for small interfering RNA (siRNA) expression enable sustained gene knockdown in mammalian cells, surpassing synthetic siRNAs.
• Understanding the comparative efficacy and longevity of different siRNA expression vector designs is crucial for advancing RNA interference (RNAi) therapies.

Purpose of the Study:

• To compare the efficacy and longevity of target gene knockdown using various DNA constructs for siRNA expression in adult mice.
• To evaluate the impact of construct type (plasmid, linear PCR, dumbbell-ended PCR) and promoter choice (H1 vs. U6) on RNAi efficiency and duration.

Main Methods:

• Hydrodynamic delivery of different DNA constructs (plasmids, linear PCR, dumbbell-ended PCR) encoding siRNAs into adult mice.
• Assessment of target gene knockdown efficacy and duration using different siRNA expression cassettes and promoters.
• Comparison of construct performance in vivo (mice) versus in vitro (cultured cells).

Main Results:

• Plasmid-based siRNA expression constructs demonstrated superior target gene knockdown efficacy compared to basic PCR constructs.
• Efficacy of PCR constructs was enhanced by terminal extensions and significantly improved by dumbbell ends.
• U6 promoter-driven constructs were more effective in mice than H1 promoter constructs, though both performed similarly in cell culture.
• Sustained target gene knockdown, lasting at least 20 weeks, was observed with both plasmid and PCR-based siRNA expression cassettes.

Conclusions:

• Dumbbell-ended PCR constructs and plasmid vectors are effective for long-term gene knockdown in vivo.
• Promoter selection (U6 preferred over H1 in mice) is critical for optimizing RNAi efficacy in vivo.
• These findings provide valuable insights for designing efficient and durable RNAi vectors for therapeutic applications.