An engineered U7 small nuclear RNA scaffold greatly increases ADAR-mediated programmable RNA base editing.

Area of Science:

• Molecular Biology
• Gene Therapy
• Biochemistry

Background:

• Adenosine Deaminase Acting on RNA (ADAR) enables precise RNA base editing (A-to-G).
• Targeting ADAR activity requires effective in vivo delivery of guide RNAs (gRNAs).
• Current methods face challenges in achieving therapeutic RNA editing efficiencies.

Purpose of the Study:

• To develop an enhanced RNA base editing system using a U7 small nuclear RNA (snRNA) framework.
• To improve the in vivo delivery and efficacy of ADAR-guided RNA editing.
• To establish a universal scaffold for ADAR-based RNA therapies.

Main Methods:

• Embedding guide RNAs (gRNAs) into a U7 snRNA framework.
• Utilizing a 750-plex single-cell mutagenesis screen to optimize the U7 scaffold.
• Employing an optimized scaffold with a synthetic U7 promoter for enhanced editing.
• Systemic delivery via Adeno-Associated Virus (AAV) in a Hurler syndrome mouse model.

Main Results:

• Achieved 76% RNA editing in vitro from a single DNA construct per cell.
• Demonstrated 75% RNA editing in a Hurler syndrome mouse brain after a single AAV injection.
• Improved existing DMD exon-skipping designs by 25-fold in differentiated myoblasts.
• Outperformed circular gRNA approaches in editing efficiency.

Conclusions:

• The engineered U7 framework significantly enhances ADAR-based RNA editing efficacy and in vivo delivery.
• This technology provides a potent and versatile platform for RNA-based gene therapies.
• The U7 scaffold represents a universal solution for ADAR editing and other antisense RNA therapies.