CRISPR & Duchenne muscular dystrophy

The success of CRISPR in a model of Duchenne muscular dystrophy

Original story from Duke University

Researchers at Duke University have shown that by using genome editing technology, CRISPR can safely and stably correct a genetic condition such as Duchenne Muscular Dystrophy (DMD). The study appears in the journal Nature Medicine. Links here


CRISPR/Cas9 is, by far, the most effective technique to correct DNA defects. Described as a “molecular toolkit,” this intelligent and sophisticated technique enables researchers to perform cut and paste” operations on DNA.


How does CRISPR work?

This technology works as follows:

  • CRISPR is a type of gene sequence found naturally in specific organisms, including viruses. It can recognize a particular DNA sequence, i.e., find the place on a DNA strand where a disease-causing genetic defect (mutation) lies.
  • Cas9 acts like a pair of scissors. It “cuts” DNA at a specific location, i.e., where an action is needed to repair or eliminate a genetic defect.
  • With this technology, researchers can remove a genetic mutation or insert a fix for a faulty gene sequence.
  • Using standard DNA repair mechanisms, the cell will then naturally reattach the severed DNA strands.


About Gersbach’s latest research

In 2016, Charles Gersbach, a Rooney Family Associate Professor of Biomedical Engineering, was in the first published positive results concerning the CRISPR technique likely to be translated into human therapy. Since then, many other examples have been released, and several genome editing therapies are targeting human diseases that are currently undergoing clinical trials, and others are underway.

Gersbach’s latest research focuses on a mouse model of DMD, that is unable to produce dystrophin.

The genetic cause of DMD

Dystrophin is encoded by a gene containing 79 exons responsible for producing a protein, called dystrophin. If one or more exons are disrupted or removed by a genetic mutation, the chain is not built, causing the muscle to deteriorate slowly. Most patients use a wheelchair before the age of 10 and do not live in their twenties or early thirties.


Gersbach has been working on potential genetic treatments for Duchenne muscular dystrophy since 2009. His lab was one of the first to begin focusing on CRISPR/Cas9. Cas9. CRISPR/Cas9 is used to cut into the dystrophin gene around the exons responsible for the genetic mutation that causes DMD. Then, the body’s natural DNA repair system, picks up the previously cut gene, to create an abbreviated version of the dystrophin gene.


“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. A goal of our experiments was to test some ideas being discussed in the field, which will help us understand the potential of CRISPR to treat genetic diseases in general and Duchenne muscular dystrophy in particular. This includes monitoring the long-term durability of the response in the face of potential immune responses against the bacterial Cas9 protein.” – Charles Gersbach


“It is widely believed that gene editing leads to permanent gene correction. However, it is important to explore theoretical possibilities that could undermine the effects of gene editing, such as losing treated cells or an immune response.”  – Charles Gersbach


The purpose of this new study

The purpose of this new study is to explore the factors that may alter the long-term effects of CRISPR/Cas9-based gene editing.

A single dose of the CRISPR therapy was administered intravenously to adult mice and newborn mice carrying a defective dystrophin gene. The following year, the researchers measured how many muscle cells had been successfully edited and what types of genetic modifications had been made, as well as the possibility of an immune response against the bacterial CRISPR protein, Cas9, which acts as the “scissors” that cuts into the gene.

An immune reaction against CRISPR

Other studies have reported that the mouse immune system can mount a response to Cas9, which could potentially interfere with the benefit of CRISPR therapies. Several groups have also said that some people have preexisting immunity to Cas9 proteins.


“The good news is that even though we observed both antibody and T cell responses to Cas9, neither appeared to result in any toxicity in these mice. The response also did not prevent the therapy’s ability to edit the dystrophin gene and produce long-term protein expression successfully.” – Christopher Nelson, a post-doctoral fellow in Gersbach’s lab.


Some results suggest approaches to face potential challenges. When two-day-old mice without a fully developed immune system are treated intravenously, no immune response is detected. Genome editing by CRISPR has remained stable and, in some cases, even strengthened in one year. We could imagine that administering therapy to infants would be a method of circumventing an undesirable immune response.

The immune system of the mouse works differently from the human immune system. Screening for DMD in newborns is not yet widespread; most Duchenne diagnoses occur when children are three to five years old. The suppression of the immune system during treatment might be an approach.


“We were pleased to observe that all the mice were doing well a year after treatment, but our results show that there needs to be more focus on the immune response as we move toward larger animal models,” – Christopher Nelson


Some studies have shown that CRISPR can cut out genetic sections much more significant than intended or that pieces of DNA can embed at the site of the cut.

To exhaustively map all the changes in the dystrophin gene, Nelson used a DNA sequencing approach. Surprisingly, many types of modifications have been made in addition to the intended removal of the targeted exon, including a high level of DNA sequence insertion.


“None of these edits would necessarily be a cause for concern in this case because the dystrophin gene is already defective. That being said, any unintended results could potentially take away from the efficiency of the gene editing you are trying to achieve, which supports the importance of designing ways to identify and mitigate alternative edits in future studies objectively. 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.”- Christopher Nelson


An encouraging step in the fight against DMD

Duchenne muscular dystrophy (DMD) results from an error in the “writing” of the dystrophin gene. In children with DMD, the dystrophin gene is corrupt—it contains a genetic defect (mutation). The gene is so badly “written” that the cellular machinery cannot accurately “read” the genetic instructions to produce dystrophin—a protein that’s essential for proper neuromuscular function.

Researchers are using CRISPR/Cas9 to remove the corrupt part of the genes (mutations). “Erasing” problem areas on a DNA strand may restore accurate protein decoding. With this technology, DMD researchers have been able to restore the production of dystrophin.

Researchers have found a way to transport this molecular toolkit aboard viruses with a particular attraction to muscle cells. They injected this mixture into mice models that could not synthesize dystrophin. After a few weeks, the rodents’ muscles began to produce dystrophin.

A brilliant idea!



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