Every 36 seconds a person dies in the United States from heart disease. Elevated levels of low-density lipoprotein cholesterol (LDL-c) in the circulation over long periods promotes plaque formation in the thin vessels that supply blood to the heart and result in heart disease.

Precision BioSciences has announced the publication of preclinical data of a three-year follow-up study showing long-term, stable reduction of LDL-c levels in nonhuman primates (NHPs) through gene silencing using their proprietary ARCUS® genome editing platform.

The study, “Long-term Stable Reduction of Low-density Lipoprotein in Nonhuman Primates Following In Vivo Genome Editing of PCSK9” published in Molecular Therapy, was led by James M. Wilson, MD, PhD, professor of medicine and director of the Penn Gene Therapy Program and the Penn Orphan Disease Center, and Lili Wang, PhD, research director in the Penn Gene Therapy Program and research associate professor of medicine at the Perelman School of Medicine at the University of Pennsylvania.

“Building on the work we previously published in Nature Biotechnology in 2018, which was the first demonstrated use of any gene editing technology to create a clinically relevant reduction of gene expression of the PCSK9 protein in nonhuman primates, these latest preclinical results showed that targeted in vivo gene disruption with ARCUS has had a lasting therapeutic effect after a single dose and provide pivotal data for safety considerations that support advancement towards clinical translation,” says Wilson.

ARCUS is a proprietary genome editing technology developed by scientists at Precision BioSciences that uses sequence-specific DNA mega-nucleases that insert, remove, or repair DNA in living cells and organisms. The technology is based on a naturally occurring genome editing enzyme, I-CreI found in the algae Chlamydomonas reinhardtii.

I-Cre-I includes a built-in safety switch that shuts it off after a single, specific DNA edit reducing the risk of off-target edits.

The researchers delivered a gene encoding an ARCUS nuclease in an adeno-associated virus (AAV) vector to inactivate the PCSK9 gene and inhibit its protein expression.

“These results not only contribute to the growing evidence of gene editing for potential therapeutic use, but specifically showed that ARCUS nuclease gene editing could be a very promising new approach leading to treatments for heart disease patients that do not tolerate commonly used PCSK9 inhibitors,” says Wilson.

Mutations in PCSK9 that are naturally present in some individuals and prevent the expression of the gene, are associated with reduced LDL-c levels and a decreased risk of cardiovascular disease, with no apparent adverse health consequences. This observation makes PCSK9 a compelling therapeutic target for lowering LDL-c levels to render an individual resistant to heart disease.

In liver cells, PCSK9, a protein cleaving enzyme, binds and breaks down LDL-receptors that take up LDL-c and normally reduce circulating LDL-c levels.

The research team monitored NHPs for more than three years and have continued to show a sustained reduction in LDL-c levels and stable gene editing without any obvious adverse effects. After the single treatment, NHPs continue to show reductions of up to 85% in PCSK9 protein levels and a 56% in LDL-c levels.

“To our knowledge, this is the longest duration gene editing data in a large animal model. The data demonstrates that a single administration of an ARCUS nuclease could represent a potential one-time, permanent treatment for familial hypercholesteremia,” said Derek Jantz, PhD, co-author on the paper and chief scientific officer at Precision BioSciences.

“ARCUS has attributes that we believe significantly differentiate it from RNAi or conventional AAV gene therapy approaches, as well as CRISPR gene editing approaches. At more than three years out, we are seeing a stable gene edit that is being inherited by subsequent generations of hepatocytes, and evidence thus far supports that this is a permanent change. We look forward to continued monitoring of these animals and applying these learnings to our other in vivo gene editing programs,” says Jantz.

A unique challenge in translational research of in vivo genome editing is to demonstrate human genome-specificity. The researchers use the ARCUS mega-nuclease to target a sequence conserved between the human and NHP PCSK9 gene. This allows them to evaluate on-target editing and safety in NHPs.

The authors show low-frequency off-target editing remains stable with no apparent changes in the tissue architecture of the liver. Since human genome-specific off-target editing cannot be tested in NHPs, the authors use a chimeric liver humanized mouse model to study on- and off-targeting in primary human hepatocytes in vivo. These mice lack an immune system and that could increase off-target edits. In further studies the team is exploring the possibility of reducing mega-nuclease expression by using a weak promoter to further reduce off-target activity without affecting on target editing.

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