Lipid Nanoparticles Enable Efficient In Vivo DNA Knock-In via HITI-Mediated Genome Editing.

Area of Science:

• Molecular Biology
• Gene Therapy
• Nanomedicine

Background:

• In vivo genome editing offers therapeutic promise for monogenic diseases.
• Efficient delivery of genome editing tools is a critical challenge.
• Lipid nanoparticles (LNPs) are a potential delivery system for in vivo applications.

Purpose of the Study:

• To investigate the efficacy of LNPs for in vivo DNA knock-in using the HITI method.
• To compare HITI with traditional homology-directed repair (HDR) for knock-in efficiency.
• To optimize LNP delivery for enhanced genome editing in vivo.

Main Methods:

• In vitro assessment of HITI vs. HDR for knock-in efficiency at the Alb locus in hepatic cells.
• Optimization of LNP composition and administration routes for in vivo delivery.
• Intravenous administration of LNP-encapsulated genome editing components in mice.
• Quantification of GFP knock-in efficiency in mouse livers.

Main Results:

• HITI demonstrated significantly higher in vitro knock-in efficiency than HDR.
• Successful HITI-mediated GFP knock-in (2.1-2.7%) was achieved in mouse livers via intravenous LNP delivery.
• Repeated intravenous dosing doubled liver GFP knock-in efficiency (4.3-7.0%) compared to single dosing.

Conclusions:

• LNPs are effective for in vivo delivery of genome editing components.
• HITI mediated by LNPs shows superior knock-in efficiency compared to HDR.
• Optimized LNP delivery strategies, including repeated dosing, can enhance cumulative genome editing effects for potential therapeutic applications.