Strategies that address the basic problem in Duchenne, the absence of dystrophin, are critically important to patients and families and have attracted considerable attention from drug developers.

We already have two technologies – exon skipping and stop codon read-through therapy – that allow the body to produce nearly full-length dystrophin. These therapies are steps in the right direction, but are not a cure. They also do not work for everyone, so we need other ways of tackling Duchenne.

The potential to correct disease-causing mutations at the DNA level using gene editing has been the subject of many recent headlines. Many of you have commented on this potential promising new technology, and there are of course many questions. This new technology is called CRISPR/Cas9. The technology is an incredible opportunity and, as all promising approaches, brings along challenges that are currently being addressed before we will see clinical trials.

PPMD wanted to help take a deeper dive into gene editing and CRISPR/Cas9 so that we can better understand the potential of this cutting-edge technology.

What is gene editing?

Just as an editor looks for typos in a book, gene editing also tries to identify “typos” in the genetic code or DNA and then fix those mistakes. Our own bodies and other living organisms all have systems in place to find and correct the “typos” in the genetic code. This natural gene editing is always taking place, however we know that it is not fail proof.

While people working in the lab have had ways of finding and trying to fix mistakes in DNA, our technologies to this point have not been very specific and are more difficult to use. This is why using them in humans has never been an option, until now. This new technology, CRISPR/CAS9 system, works very specifically and is much easier to use which is why it is getting so much attention in the press. Cas9 is an enzyme that “cuts” double-stranded DNA just like scissors cut paper. When DNA is cut, this triggers the natural DNA repair mechanisms. If Cas9 was used alone it would cut DNA indiscriminately, and thus be useless as a candidate therapeutic. The CRISPR/Cas9 system, however, uses single guide RNA molecules (or sgRNA) to target Cas9 to a pre-specified location on the DNA, so that the “cut” is applied in order to allow a specific mutation to be corrected. These sgRNA are signals or guides to the Cas9 pointing to the exact location that the Cas9 needs to make a cut.

The challenges currently being addressed for using of the CRISPR/Cas9 platform as a therapy for Duchenne include:

1. the delivery of the reagents to the cell target;
2. the specificity and selectivity of the sgRNA molecules to ensure that only the intended DNA is recognized and targeted;
3. the efficiency of the Cas9 enzyme in achieving the DNA “cut”; and
4. the activation of natural DNA repair mechanisms.

We know through the work done with exon skipping that sections of the dystrophin molecule may be somewhat dispensable and can be removed from mRNA to restore the reading frame. Currently exon skipping technologies target the removal of a single exon (51, 53, 45…). This type of concept also applies to CRISPR/Cas9 targeting of the dystrophin gene, but with CRISPR/Cas9 the target is the DNA, not the RNA. Remember that the DNA is the genetic code we inherit from our parents. The DNA gets transcribed into RNA and then translated into protein.

How can gene editing be applied to Duchenne?

There are three conceivable ways that CRISPR/Cas9 platform could be applied to treating Duchenne:

1. Editing of the dystrophin gene in germ cells or embryos. Editing human germ cells or embryos (“designer babies”) would produce permanent changes in the genome of the individual and their children. An Institute of Medicine (IOM) meeting was held in December, 2015 to discuss ethical issues around use of CRISPR/Cas9 for germ cell/embryo editing. There are important ethical issues to consider around this and it is important for us as a community to start thinking about this.
2. Editing of the dystrophin gene ex vivo (outside the human body), in muscle stem or satellite cells that are then transferred back into the patient. The ‘engineered’ cells that are put into the body are capable of producing dystrophin. Researchers are currently working to scale up production of sufficient numbers of satellite/stem cells for transplantation back into the patient and strategies to ensure their survival and integration into muscle fibers.
3. Introduction of CRISPR/Cas9 reagents directly into the muscles of the Duchenne patient, to edit the dystrophin mutations in vivo (inside the body). Researchers are working to address the specificity of the sgRNA guide molecules and delivery to muscles throughout the body (bioavailability) in sufficient amounts to achieve gene editing which would result in restoring dystrophin.

What gene editing research is being conducted in Duchenne models?

Currently, CRISPR/Cas9 technology is being explored for Duchenne in several academic labs. As of the date of this blog, the labs of Eric Olson (University of Texas Southwest; with an NIH Wellstone Muscular Dystrophy Research Center grant focused on this topic), Charlie Gersbach (Duke University), Ronnie Cohn (Hospital for Sick Children), Renzhi Han (Ohio State University), and Akitsu Hotta (Kyoto Uunivesity) have published peer-reviewed articles on the use of CRISPR/Cas9 in Duchenne cells or animal models.

• Olson’s lab has used CRISPR/Cas9 technology to correct the exon 23 nonsense mutations in dystrophin in the germ cells of mdx mice. They then used these germ cells to produce a series of genetic “mosaic” animals with between 2% and 100% correction of the dystrophin gene, and showed correction of the mdx phenotype in mice with more than 40% gene correction.

• Gersbach, Hotta, and Cohn performed gene editing in cells isolated from Duchenne patients (ex vivo), showing that correction of dystrophin mutations could be performed in isolated human patient cells. These studies provide proof of concept that CRISPR/Cas9 gene editing could provide for corrections that could address a considerable proportion of Duchenne patients (e.g., via deletions of exons 45-55 with a single set of reagents) as well as patients with duplications.

• Han’s lab has achieved gene correction of mdx mouse muscles in vivo, by surgically exposing individual muscles and electroporating (driving the reagents into the muscle cells by passing electrical current) CRISPR/Cas9 reagents into the exposed muscle. Utilizing gene therapy approaches, they also injected CRISPR/Cas9 component encoding AAV into individual mdx mouse muscles, showing dystrophin expression and protection of the injected muscle from stress injury.

Progressing toward a viable gene editing therapy

Gene editing is personalized medicine and may have fewer regulatory hurdles in that a single Cas9 nuclease and two sgRNAs may address mutations in over 60% of Duchenne patients (as opposed to the need to evaluate and seek regulatory approval for drugs targeted to different exons). Researchers are currently exploring issues from the design of the sgRNA guide molecules to confer sufficient specificity/selectivity to be safe and effective to the delivery systems for CRISPR/Cas9 system components to muscle in effective concentrations. From gene therapy approaches, we know the immune response to dystrophins generated through gene editing will have to be considered and managed, as well as any potential immune response to the CRISPR/Cas9 reagents themselves.

If the approach chosen is to edit the dystrophin gene in muscle precursor cells isolated from Duchenne patients, the challenge then includes the same issues to any of the cell-based therapies being explored—delivering those cells back into the body and having them survive and populate muscles and produce new muscle fibers.

There is considerable corporate interest in CRISPR technology among some venture capital and drug development companies, and some of the companies have licensed the technology to begin development in other diseases. A major biotech player in CRISPR/Cas9 technology, Editas Medicine, has mentioned Duchenne among a list of inherited conditions that they may target.

Where are we now?

Overall, the state of the science and the level of corporate interest in CRISPR/Cas9 gene editing for Duchenne can be characterized as early stage. As you heard throughout the month of December, PPMD has a long history of supporting early-stage research at pivotal moments. Gene editing provides us with another, very promising technology with the potential to end Duchenne.

For those interested in more detail, there is a nice summary of gene editing in a recent issue of The New Yorker (“The Gene Hackers”). And the December 31, 2015 article in the NY Times.

Related links

• Duchenne Therapeutic Pipeline
• Expanding the Duchenne Therapeutic Opportunities by Targeting Myost...
• Dystrophin Mutations Affect Not Only Existing Muscle Fibers, but Sa...
• A New Dog Model for Drug Development in Duchenne