Gene Therapy for Huntington's Disease Using Targeted Endonucleases.

Area of Science:

• Genetics
• Neuroscience
• Molecular Biology

Background:

• Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by CAG repeat expansion in the huntingtin gene (HTT).
• The resulting mutant huntingtin protein is toxic to neurons, leading to progressive cell death.
• Gene therapy offers a promising avenue for treating monogenic diseases like HD.

Purpose of the Study:

• To develop and validate a CRISPR-Cas9n-based gene editing protocol for inactivating the HTT gene in Huntington's disease models.
• To assess the efficiency of HTT gene knockout regardless of CAG repeat length.

Main Methods:

• Utilized a modified Cas9 nickase (Cas9n) and two single-guide RNAs (sgRNAs) to flank and excise the CAG repeat tract in the HTT gene.
• Employed HD patient-derived fibroblasts electroporated with plasmid vectors expressing CRISPR-Cas9n tools.
• Confirmed HTT gene inactivation using Western blotting analysis.

Main Results:

• Demonstrated efficient inactivation of the HTT gene using the CRISPR-Cas9n system.
• Showed that the gene knockout was effective irrespective of the length of the CAG repeat expansion.
• Excision of the CAG repeat tract led to a frameshift mutation and premature translation termination.

Conclusions:

• The developed CRISPR-Cas9n protocol effectively inactivates the HTT gene, offering a potential therapeutic strategy for Huntington's disease.
• This method can be adapted for creating precise genetic modifications, such as generating isogenic cellular models for further HD research.
• The study highlights the potential of programmable endonucleases in targeting and correcting genetic defects in monogenic disorders.