Duchenne Muscular Dystrophy and CRISPR Technology

Duchenne Muscular Dystrophy (DMD) is a disease of progressive muscle weakness and inflammation that leads to loss of mobility. People with DMD have a mutation in the dystrophin gene which renders them unable to make dystrophin protein, a critical protein for muscle function. For each person with DMD, a single mutation in this one gene is what separates them from having healthy muscles. Dystrophin gene mutations are like errors in the instructions for making dystrophin protein. Correcting those errors at the DNA level may be possible using gene editing technology called CRISPR-Cas9, which is showing promising results in preclinical research.

Approved exon-skipping therapeutics using antisense oligonucleotides (ASOs) can partially restore the ability of individuals with DMD to produce functional dystrophin protein. These exon-skipping ASO therapies are not curative and require ongoing treatments. The gene editing technology CRISPR has the potential to more permanently correct dystrophin gene mutations in people with DMD. CRISPR therapies for Duchenne are not yet available to patients but genetic diseases like DMD, caused by a single gene mutation are ideal candidates for CRISPR technology.

What is CRISPR?

The gene editing technology called CRISPR or CRISPR-Cas9 was developed from parts of the immune system in bacteria which protects them from viruses. When viruses infect bacteria, bacteria capture tiny segments of virus DNA and keep them within their DNA as a record so they can remember those viruses in case they attack again. The collection of virus DNA segments stored in bacteria are called CRISPR arrays. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. The CRISPR array consists of repeating DNA segments separated by sequences called spacers, which is where bacteria insert the small segments of virus DNA that serve as their record.

Once bacteria carry a piece of the virus, it is like having a photo on file of a criminal. When that type of virus returns, the bacteria’s immune system will recognize and remove the viral DNA. CRISPR arrays are used to make copies called CRISPR RNA which work together with CRISPR-associated (Cas) DNA enzymes that act like scissors to excise DNA sequences that match the record of the offending virus. Instead of searching for and eliminating viral sequences, scientists have modified this system to search for and remove DNA errors. This clever adaptation earned the pioneers of CRISPR technology the Nobel Prize in chemistry in 2020.

Removing or skipping exons with CRISPR

The coding regions of genes are organized into segments of DNA called exons, with intervening sequences, called introns in between. When cells produce protein, they first make a copy of the gene, in RNA form, which is processed to remove introns and join exons together. An exon of the dystrophin gene containing a mutation could be targeted and permanently removed using CRISPR. This would give a result similar to exon-skipping ASO therapies but in a more permanent way. Exon-skipping ASO treatments do not remove the mutant exon in the DNA but instead, alter RNA processing. CRISPR can be used to remove a mutation-containing exon or duplication at the DNA level. Both types of treatment resulted in a shortened form of dystrophin is partially functional and better than no dystrophin at all. Whereas exon-skipping ASO treatments require multiple treatments, exon-skipping with CRISPR has the potential to be “one and done”.

CRISPR therapeutics can be programmed to find and remove different dystrophin mutations, by swapping the target sequence, called guide RNA. CRISPR approaches that cause skipping of exons apply to more than 70% of DMD patients. In these patients removing the problematic part of the gene can correct the reading frame, changing out-of-frame mutations to an in-frame alternative. This can be achieved either by deleting an exon or exons with CRISPR or by making a small change in the exon DNA sequence that designates where exons join, the exon splice site. The type of CRISPR system used to target exon splice sites may be easier to deliver and may have less risk of causing unwanted DNA mutations compared to CRISPR therapies that remove an exon.

Replacing mutations with the correct DNA sequence

In addition to the “find and remove” function, CRISPR Cas9 systems can also be used for “find and replace” like in a word processor. Dystrophin mutations can be removed and replaced with the correct DNA sequence when a template DNA sequence is provided. In theory, this approach could allow fully functional dystrophin protein to be produced. However, there is a limit to the length of the template DNA sequence introduced into muscle cells. It is not possible to replace large deletions that span multiple exons using CRISPR.

Point mutations are very small mutations that account for 25-35% of DMD cases and these can potentially be corrected using a form of CRISPR Cas9 called base editing. Base editing CRISPR swaps a single DNA base for another using a modified system considered to be safer because no DNA cutting occurs. Nonsense mutations are point mutations where a single DNA base is changed to cause an errant “stop” signal in the protein-building instructions. CRISPR-based editing can be used to correct nonsense mutations or to alter a splice site.

Challenges to overcome

While Duchenne could theoretically be cured with CRISPR there are still obstacles to overcome. CRISPR only gains access to and corrects a proportion of muscle cells. It is not known what percentage of muscle cells need to be corrected to have a therapeutic benefit in humans. It is encouraging that some estimates suggest that people with DMD would greatly benefit from even a 15% improvement in dystrophin levels.

Maintaining long-term benefits is also likely to be a challenge because of the natural turnover of skeletal muscle cells. Replacement muscle cells grow from precursor cells called satellite cells that are usually not edited by CRISPR-Cas9. Over time the edited muscle cells may be diluted by non-edited muscle cells. Since people develop antibodies against AAV and CRISPR-Cas9, a second dose of the same treatment would not be effective.

There is a possibility of CRISPR editing to produce unwanted DNA changes through off-target effects. CRISPR-Cas9 systems are often delivered into animal or patient-derived cell lines in the laboratory using adeno-associated viruses (AAV). AAV used for transferring genetic material into cells is derived from natural AAV, which does not appear to cause disease. AAV is considered low risk because on its own it cannot make more copies of itself inside the body like other viruses do. However, AAV may have a risk of causing cancer or toxicity. A person’s immune reaction to AAV may cause health complications or prevent the therapy from being effective.

CRISPR corrects dystrophin mutations in the laboratory

Researchers at the University of Texas Southwestern Medical Center in partnership with a therapeutics company have shown promising results for CRISPR-Cas9 with guide RNA directed at correcting dystrophin exon 44 mutations and creating in-frame dystrophin. The CRISPR therapy proved successful in cultured cells from patients with Duchenne and in mice that carry the same DMD mutations.

At Duke University, CRISPR-Cas9 was designed to delete the dystrophin exon with a nonsense mutation in a DMD mouse model. This mouse model, called mdx mice, do not produce any functional dystrophin but the CRISPR therapy caused partially functional dystrophin to be produced which improved muscle function. A similar CRISPR-Cas9 approach showed promising results in a beagle model of DMD.

Researchers in Germany tested AAV CRISPR-Cas9 in a pig model of DMD. Removal of exon 51 with their system restored dystrophin expression and the animals showed improvements in the functioning of skeletal and heart muscle. At UT Southwestern, scientists have shown marked improvements in the ability of cultured heart cells from DMD patients to beat after CRISPR-Cas9 editing.

The invention of CRISPR-Cas9 technology has brought many new Duchenne muscular dystrophy CRISPR-Cas9 treatments into the research pipeline. There is hope that CRISPR technology can fix the root cause of DMD. More research is needed to understand potential risks, improve efficiency and ensure that CRISPR-Cas9 therapeutic benefits will be long-term.

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