Scientists have successfully employed gene editing to inactivate PCSK9 in macaque monkeys, according to a new study in Nature Biotechnology. PCSK9 is an antagonist of the LDL receptor, and PCSK9 inhibitors are monoclonal antibodies that reduce cholesterol levels.
“To begin to address the paucity of in vivo genome editing data in animal models other than mice, we studied genome editing in macaques using adeno-associated virus (AAV) serotype 8 vectors, which have shown success in gene therapy clinical studies,” wrote the authors, led by Lili Wang, PhD, Gene Therapy Program, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia. “We report here that pairing an engineered meganuclease with AAV8 is an effective strategy for genome editing in the primate liver.”
Previous studies have demonstrated the ability of disrupting PCSK9 in mouse livers with CRISPR-Cas9 delivered by either viral or non-viral vectors.
Meganucleases are homing endonucleases that target DNA sequences for genomic editing. The meganuclease used in the current study targets a specific DNA sequence in the PCSK9 gene and forms abnormal breaks in DNA. When DNA repair machinery kicks in to fix these breaks, other fragments of DNA are then inserted or deleted, thus interfering with PCSK9 function.
In the current study, investigators administered a single injection of engineered meganuclease delivered by AAV8 (AAV8-M2PCSK9) in six macaques. They found that a moderate dose yielded a 59% reduction in serum PCSK9 and a 34% drop in LDL.
Unlike gene therapy, in which stable transgene expression is the goal, successful gene editing needs only transient expression. The team found that there was a peak of vector genomes and transgene expression at the first liver biopsy (days 17/18) followed a reduction to nearly undetectable levels at second liver biopsy (day 128/129). This rapid decline was followed by stable populations of genome-edited hepatocytes.
“To account for the pronounced loss of transgene expression in this setting, we believe the vector genomes must have been destabilized and transcription of the expression cassette extinguished,” the authors wrote, “This may explain the well-described discrepancy in AAV liver transduction between mice and primates, which could reflect a difference in stability rather than efficiency.”
Investigators observed immune toxicity in the current study, which is unsurprising because the meganuclease used was Chlamydomonas reinhardtii, which is foreign to the body. All macaques exhibited a transient increase in liver enzymes secondary to T cell formation.
The first-generation meganuclease was not only highly active against the target but also against various off-target sites. Consequently, the team went on to develop a second-generation meganuclease that exhibited reduced off-target cleavages.
The researchers noted that although off-target cleavage with the first-generation meganuclease did not yield any notable safety issues, the sample size of the study was small and the follow-up period was short.
Overall, the authors suggested the results of this study could benefit some patients with hypercholesterolemia who are unable to tolerate repeated injections of PCSK9 inhibitors. Furthermore, because gene editing changes hepatocytes, one injection would last much longer. Nevertheless, more research needs to be done.
“We demonstrated sustained and therapeutically beneficial editing in non-human primate liver with an engineered meganuclease delivered by AAV8,” concluded the researchers. “Our studies provide a data set on in vivo genome editing efficacy, immune toxicity, and safety.”
This study was funded by Penn Medicine and Precision Biosciences.