Guanidinium-Rich Lipopeptide-Based Nanoparticle Enables Efficient Gene Editing in Skeletal Muscles

Area of Science:

• Biotechnology
• Molecular Biology
• Nanomedicine

Background:

• CRISPR-Cas genome editing offers potential for genetic disease treatment.
• Efficient and safe in vivo delivery of CRISPR components remains a significant hurdle.

Purpose of the Study:

• To develop a lipopeptide-based nanoparticle (LNP) for efficient delivery of CRISPR-Cas9/sgRNA ribonucleoprotein (RNP).
• To evaluate the efficacy and safety of the LNP for in vitro and in vivo genome editing.

Main Methods:

• An artificial lipopeptide (GD-LP) was synthesized, linking a hydrophilic head to a hydrophobic tail via a disulfide bond.
• Self-assembled LNPs formed complexes with RNP (up to 20 wt % loading).
• In vitro and in vivo studies assessed gene editing efficiency, transfection rates, cytotoxicity, and therapeutic effects in a Duchenne muscular dystrophy mouse model.

Main Results:

• RNP-LNP nanocomplexes achieved 72.6% gene editing efficiency in GFP-HEK cells with minimal cytotoxicity.
• LNPs demonstrated superior transfection efficiency for mRNA and plasmid DNA compared to Lipofectamine 2000.
• In vivo studies in mice showed efficient muscular tissue editing and restoration of dystrophin expression in a Duchenne muscular dystrophy model.

Conclusions:

• The developed GD-LP based LNP is a highly efficient and safe delivery vehicle for CRISPR-Cas9 RNP.
• This LNP system holds significant therapeutic potential for treating genetic disorders like Duchenne muscular dystrophy.