Gold nanoparticles effectively deliver CRISPR to mouse models of DMD

CRISPR-Cas9 has emerged in recent years as a promising weapon against genetic disease. But it comes with some safety concerns, namely, off-target gene editing that may bring about harmful side effects such as cancer. A team at UC Berkeley is investigating the possibility that nanoparticles can address these problems.

The three components of CRISPR—the Cas9 enzyme, a guide RNA and donor DNA—are delivered using a viral vector, most commonly an adeno-associated virus (AAV). While this is the most advanced method of CRISPR delivery, it allows the Cas9 enzyme to persist in the cells, as there is no way to control Cas9 expression once the enzyme is in the cells, said Niren Murthy, a professor of bioengineering at UC Berkeley.

So, even after editing is complete, the Cas9 will be “chewing up” parts of the genome, which can cause mutations, Murthy said. Additionally, AAVs are too small to fit all three CRISPR components, so at least two viruses must be used. A large dose of viruses—higher than what is clinically recommended—must be injected in order to get the intended effect, he said.

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Murthy worked on a study investigating the use of a nonviral, nanoparticle delivery vehicle for CRISPR, developed by GenEdit. The technology, dubbed CRISPR-Gold, is made up of gold nanoparticles combined with DNA. This is complexed with all three CRISPR components, as well as a polymer that helps the nanoparticle penetrate into cells.

The researchers delivered CRISPR-Gold to mouse models of Duchenne muscular dystrophy via intramuscular injection. They injected the three CRISPR components without a delivery vehicle into another group of mice, which served as a negative control. They chose Duchenne because it lacks effective treatments and because it affects skeletal muscle, an area into which gene therapy can be easily injected, said GenEdit CEO Kunwoo Lee.

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Two weeks after a single injection, CRISPR-Gold corrected the mutated dystrophin gene and restored the expression of the dystrophin protein in the mice’s muscle tissue with minimal off-target effects, according to the study. Specifically, the treatment corrected 5.4% of the dystrophin gene in the mice, a promising result, the team believes. In additional experiments, mice treated with CRISPR-Gold showed better strength and agility than mice treated in the negative control group. The findings appear in the journal Nature Biomedical Engineering.

Other research on safer CRISPR focuses on using proteins as an “off switch” for gene editing. Scientists from the University of Toronto and the University of Massachusetts identified three families of proteins that bind to the Cas9 enzyme, halting its activity. And a team from UC Berkeley and UC San Francisco applied an anti-CRISPR protein to a CRISPR-Cas9 molecule that reduced off-target effects fourfold without compromising the desired gene editing.

While Sarepta’s Duchenne drug earned FDA approval last year, it is only effective in a narrow group of patients. CRISPR could become a treatment for the approximately 30% of Duchenne patients whose disease is caused by single-base mutations or small deletions, the Berkeley researchers said.