Gene Editing Technology in Mice Shows Promises as Approach for DMD

Rare Daily Staff

Scientists at the University of California, Berkeley have developed a way to deliver gene-editing technology inside cells, and have demonstrated in mice that the technology can repair the mutation that causes Duchenne muscular dystrophy, a progressive and fatal muscle disease.

The researchers showed that a new delivery system for CRISPR-Cas9 gene editing dubbed “CRISPR-Gold,” so-called because it makes use of gold nanoparticles, was able through a single injection in mice with DMD to produce an 18-times higher correction rate of the faulty gene and a two-fold increase in strength and agility test compared to mice in a control groups.

“CRISPR-Gold and, more broadly, CRISPR-nanoparticles, open a new way for safer, accurately controlled delivery of gene-editing tools,” said Irina Conboy, a Berkeley professor of bioengineering whose lab co-led the study. “Ultimately, these techniques could be developed into a new medicine for Duchenne muscular dystrophy and a number of other genetic diseases.”

Since its development, CRISPR-Cas9 has been hailed as accurate and inexpensive means of gene editing with the potential to revolutionize the treatment of genetic diseases. One of the challenges, though, has been delivery of CRISPR-Cas9 into the body’s cells where it needs to act. Researchers have relied on viruses to do this, but that approach carries complications.

In the new study, published October 2 in the journal Nature Biomedical Engineering, research lead by the labs Conboy and Berkeley bioengineering Professor Niren Murthy, demonstrated that their CRISPR-Gold approach can deliver Cas9—the protein that binds and cuts DNA—along with guide RNA and donor DNA into the cells of a living organism to fix a gene mutation.

“CRISPR-Gold is the first example of a delivery vehicle that can deliver all of the CRISPR components needed to correct gene mutations, without the use of viruses,” Murthy said.

CRISPR-Gold repairs DNA mutations through a process called homology-directed repair, a natural process to repair damaged DNA within the cells. Scientists have struggled to develop homology-directed repair-based therapeutics because they require activity at the same place and time as Cas9 protein, an RNA guide that recognizes the mutation and donor DNA to correct the mutation.

To overcome these challenges, the Berkeley scientists created a delivery vessel that binds all of these components together, and then releases them when the vessel is inside a wide variety of cell types, triggering homology directed repair. CRISPR-Gold’s gold nanoparticles coat the donor DNA and also bind Cas9. When injected into mice, their cells recognize a marker in CRISPR-Gold and then import the delivery vessel. Then, through a series of cellular mechanisms, CRISPR-Gold is released into the cells’ cytoplasm and breaks apart, rapidly releasing Cas9 and donor DNA.

A single injection of CRISPR-Gold into muscle tissue of mice that model Duchenne muscular dystrophy restored 5.4 percent of the dystrophin gene, which causes the disease, to the normal sequence. This correction rate was approximately 18 times higher than in mice treated with Cas9 and donor DNA by themselves, which experienced only a 0.3 percent correction rate.

The study authors note that CRISPR-Gold restored the normal sequence of dystrophin, which is a significant improvement over previously published approaches that only removed the faulty part of the gene, making it shorter and converting one disease into another, milder disease.

CRISPR-Gold was also able to reduce tissue fibrosis, a hallmark of diseases where muscles do not function properly, and enhanced strength and agility in mice with Duchenne muscular dystrophy. CRISPR-Gold-treated mice showed a two-fold increase in hanging time in a common test for mouse strength and agility, compared to mice injected with a control.

The study found that CRISPR-Gold’s approach to Cas9 protein delivery is safer than viral delivery of CRISPR, which, in addition to toxicity, amplifies the side effects of Cas9 through continuous expression of the DNA-cutting enzyme. When the research team tested CRISPR-Gold’s gene-editing capability in mice, they found that CRISPR-Gold efficiently corrected the DNA mutation that causes Duchenne muscular dystrophy, with minimal collateral DNA damage.

The study was funded by the National Institutes of Health, the W.M. Keck Foundation, the Moore Foundation, the Li Ka Shing Foundation, Calico, Packer, Roger’s and SENS, and the Center of Innovation Program of the Japan Science and Technology Agency.

A clinical trial will be needed to determine whether CRISPR-Gold is an effective treatment for genetic diseases in humans. Study co-authors Kunwoo Lee and Hyo Min Park have formed a start-up company, GenEdit (Murthy has an ownership stake in GenEdit), which is focused on translating the CRISPR-Gold technology into humans.

October 23, 2017