A single dose of gene therapy based on CRISPR-Cas9 may successfully treat Duchenne muscular dystrophy (DMD) in the long term or even permanently, a preclinical study in mice shows.

The findings shed light on safety factors that may affect the long-term efficacy of this gene therapy, and provide new evidence on the issues that still prevent this approach from achieving its full potential to treat DMD patients.

The study, “ Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy,” was published in the journal Nature Medicine.

DMD is a genetic disease, caused by different types of mutations in the DMD gene, which provides cells with instructions to produce a protein called dystrophin. This protein is important to strengthen and protect muscles from damage when they contract and relax.

Because a single gene is the cause of the disease, DMD stands as an ideal target for gene therapy. In fact, there have been recent studies reporting encouraging results after testing gene therapy in preclinical models of DMD, such as in patient cells cultured in the lab or in animal models of the disease. Positive results have also been found in clinical trials. However, the long-term efficacy and safety of these treatments have not been fully determined.

Duke University researchers, led by associate professor Charles Gersbach, PhD, have been working on potential gene therapies for DMD since 2009, and they were among the first to use a recently developed gene editing tool called CRISPR-Cas9 as a means to treat DMD.

More recently, they have been focused on understanding if a single gene therapy treatment would genetically correct dystrophin-deficient muscle cells for the long term.

The team designed a strategy that uses CRISPR-Cas9 to cut the mutated portion of the DMD gene, leaving the cells’ natural DNA repair mechanism to join the remaining “healthy” parts of the gene. The result is a shortened but corrected version of the gene that’s able to produce a functional dystrophin protein.

“As we continue to work to develop CRISPR-based genetic therapies, it is critical to test our assumptions and rigorously assess all aspects of this approach,” Gersbach said in a university news release written by Ken Kingery.

Cas9 is an enzyme of bacterial origin that will work as the molecular “scissors” to cut the DMD gene sequence. Patients may develop an immune response against Cas9, given its bacterial nature, which will compromise the safety and efficacy of the treatment.

In fact, some patients were reported to have preexisting immunity against Cas9, probably because they were once exposed to Cas9-carrying bacteria.

Earlier studies demonstrated that the approach employed by Gersbach’s team could effectively recover dystrophin levels and improve muscle strength in mice over eight weeks.

“It is widely believed that gene editing leads to permanent gene correction,” he said. But, until now, no study has assessed if the treatment lasts long-term or if it is permanent.

To address this issue, the team injected a single dose of CRISPR-based therapy into both adult and newborn mice that lacked dystrophin protein, mimicking DMD human disease. CRISPR-Cas9 was delivered using a safe and non-active adeno-associated viral vector.

The researchers confirmed that this strategy could promote the synthesis of a working version of dystrophin protein in the muscles of mice. This positive result was noted at eight weeks after the treatment and lasted for at least one year (the total study duration). The genetic correction remained stable and, in some cases, got stronger with time.

Still, the team reported that some adult mice developed an immune response against AAV-CRISPR, but without evident or significant toxicity. This immune-related reaction could be prevented if newborn DMD mice were treated with immunosuppressive agents during gene therapy administration. Therefore, the researchers believe that delivering the CRISPR-Cas9 therapy in the clinic to infants may prevent or control a future unwanted immune response.

While these findings are encouraging, the immune system of a mouse often works differently from that of a human, so scientists and clinicians should remain careful when translating these results into human studies, the researchers said.

The team also found that CRISPR-Cas9 not only promoted the desired “cut and paste” effect, but also induced additional unintended edits in the DMD gene. Many of these additional alterations are of concern, as in this case “the dystrophin gene is already defective,” Christopher E. Nelson, PhD, postdoctoral researcher and lead author of the study, said.

Although prior studies have demonstrated that this genetic editing approach has minimal off-target effects, unwanted events may occur in other sites of the genome (all DNA content), the researchers noted.

“Moving forward, this phenomenon needs to be monitored carefully and better understood. Methods that avoid these alternative edits and increase the frequency of the intended edit will be important to maximizing the potential of genome editing to treat disease,” Gersbach said.

This study demonstrates “the potential of AAV-CRISPR for permanent genome corrections” and highlights the need for further studies in larger animal models to enable translation of this technology to treat human genetic diseases, the researchers said.