In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates

Link to paper

Summary

1. The paper demonstrates the successful in vivo delivery of CRISPR adenine base editors using lipid nanoparticles (LNPs) to modify disease-related genes in cynomolgus monkeys (Macaca fascicularis).
2. CRISPR base editors efficiently and precisely modified the PCSK9 gene in the liver, resulting in significant reductions in PCSK9 and low-density lipoprotein cholesterol (LDL-C) levels.
3. The reduction in PCSK9 and LDL-C levels remained stable for at least 8 months after a single-dose treatment, supporting the potential for a “once-and-done” approach.
4. The study establishes proof-of-concept for the efficient in vivo delivery of base editors to (non-human) primate livers, a crucial step for developing these editors for human disease treatment.
5. Base-editing demonstrated advantages over standard gene-editing nucleases in terms of precision, efficiency, and potential for correcting disease-causing single-nucleotide mutations.

The Paper

Background

what is the gap in knowledge this research is trying to address?

• Gap in knowledge regarding efficient in vivo delivery of CRISPR adenine base editors to nonhuman primates
  • There are many examples of in vivo editing of target genes using CRISPR-Cas9 nucleases and CRISPR base editors in rodent models but not primates
  • Transition from rodent models ⇒ nonhuman primates is critical before considering in vivo administration of gene editors to human patients in clinical trials
• PCSK9 gene is targeted for in vivo gene editing due to its relevance to familial hypercholesterolaemia, and the study aims to explore the potential of a ‘once-and-done’ approach for reducing LDL-C levels compared to existing therapies

Hypothesis

• CRISPR adenine base editors delivered in vivo using LNPs can efficiently and precisely modify disease-related genes, specifically the PCSK9 gene, in cynomolgus monkeys

Experiments

1. Base Editing in Hepatocytes In Vitro:
   • Identified 20 gRNAs targeting PCSK9 splice sites and selected PCSK9-1 with high editing activity and orthogonality
   • Formulated LNPs containing ABE8.8 mRNA and PCSK9-1 gRNA for efficient delivery to human and cynomolgus monkey hepatocytes
   • Achieved over 60% base editing of the PCSK9 splice site in primary human hepatocytes and observed similar editing in cynomolgus monkey hepatocytes
2. Base Editing in Mice:
   • Formulated LNPs containing ABE8.8 mRNA and PCSK9-1m gRNA for intravenous infusion in mice
   • Observed approximately 70% liver base editing at various doses, indicating saturation editing of hepatocytes
3. Base Editing in Cynomolgus Monkeys:
   • Administered LNPs to cynomolgus monkeys via intravenous infusion at different doses
   • Observed over 50% mean base editing frequencies in short-term studies, with reductions in PCSK9 and LDL cholesterol
   • Long-term study with a higher dose (3.0 mg kg−1) to assess durability and tolerability
4. Assessment of Off-Target Editing:
   • Used oligonucleotide enrichment and sequencing to evaluate off-target editing in cynomolgus monkey hepatocytes and liver samples
   • Identified minimal off-target editing at specific sites, demonstrating the precision of base editing

Observations and Conclusions

• Adenine base editing provide highly effective in knocking down PCSK9 gene function in cynomolgus monkeys, achieving > 60% editing
• Reductions in PCSK9 in the blood and LDL-C levels remained stable for at least 8 months, which surpasses the effects of current cholesterol-lowering drugs
• LNPs with base editors showed fewer off target effects compared to AAV-delivered meganucleases in previous studies

Iteration / Future Directions

• Further evaluation of the risks associated with in vivo base editing is needed before advancing to human studies
• Future research should explore the application of base editing to other therapeutic target genes and its use in different organs (in addition to the liver)

Other Notes

Things I had to look up

• Concomitant := naturally accompanying
• PCSK9 gene: provides instructions for making a protein (PCSK9 protein) that helps regulate the amount of cholesterol in the bloodstream (source)
  • PCSK9 protein: breaks down low-density lipoprotein receptors before they reach the cell surface, so more cholesterol can remain in the bloodstream
• Meganuclease := endonucleases that recognize large DNA target sites (source)