Generation of Rhesus Macaque Embryos with Expanded CAG Trinucleotide Repeats in the Huntingtin Gene

Area of Science:

• Genetics
• Neuroscience
• Developmental Biology

Background:

• Huntington's disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the Huntingtin (HTT) gene.
• Misfolded HTT protein accumulation leads to neuronal dysfunction, cell death, and HD pathogenesis.
• A nonhuman primate (NHP) model is crucial for understanding HD and developing therapies due to genetic and physiological similarities to humans.

Purpose of the Study:

• To establish a nonhuman primate (NHP) model for Huntington's disease (HD) research.
• To investigate the efficacy of CRISPR/Cas9 and single-stranded DNA (ssDNA) for introducing expanded CAG repeats into the rhesus macaque HTT gene.

Main Methods:

• CRISPR/Cas9 gene editing was employed to insert expanded CAG repeats into the HTT gene of rhesus macaque embryos.
• Analysis of arrested embryos and biopsied trophectoderm (TE) cells from blastocysts confirmed gene insertion.
• Genotyping and cryopreservation were performed on modified embryos.

Main Results:

• Fifteen percent of the embryos successfully incorporated expanded CAG repeats into the HTT gene.
• Five blastocysts with integrated expanded CAG repeat regions were identified and cryopreserved for NHP HD model production.
• Off-target genetic events were detected in some biopsied samples.

Conclusions:

• The successful generation of NHP embryos with expanded CAG repeats marks a significant step towards establishing an NHP HD model.
• This NHP model will be invaluable for studying HD pathology and advancing the development of novel therapeutic strategies.
• Further research is needed to optimize gene editing efficiency and minimize off-target effects.