Will Muscular Dystrophy Patients Walk Again?

Novel research using the CRISPR-gene editing tool introduces a one-size-fits-all therapeutic model for patients with Congenital Muscular Dystrophy.

Daniel Abd Assamad, Navneet Aujla, and Talia Silver

Dr. Ronald Cohn, of SickKids Hospital in Toronto, has taken a monumental step towards a new form of therapy that could revolutionize how we treat inherited diseases. Since its discovery in 2011, the sensationalized gene editing technology, CRISPR-Cas9, has been used to alter the DNA of animal models and human cells¹. Although there are major ethical implications to editing germline cells, researchers are refining the CRISPR-Cas9 system to safely edit mutations in somatic cells, with hopes to alleviate the symptoms of some inherited disorders. Dr. Cohn is focusing his research on developing CRISPR-cas9 treatments for inherited conditions like Congenital Muscular Dystrophy (CMD) along with a handful of other disorders.

CMD is a category of muscular dystrophies, characterized by muscle weakness starting in infancy and results in decreased life-expectancy. Currently, CMD is thought to occur in approximately 1/100,000 individuals worldwide and there are no known treatments, with the exception of physiotherapy to reduce the effects of muscle weakness². “They’re born in pretty bad shape,” says Dr. Cohn, “and the longer [the muscle weakness] goes on, the more impact it has.” CMD is formally diagnosed when fibrotic lesions are identified in a muscle biopsy². These visible lesions are a result of a deeper molecular defect in the structure of the muscle. In a healthy individual, the LAMA2 gene codes for a protein called laminin α2, which acts within a larger complex of proteins to enable muscle contraction.When the LAMA2 gene carries a mutation, it creates a dysfunctional laminin α2 protein, resulting in a sub-type of CMD – type 1A (MDC1A)³. The muscle weakness typical of MDC1A is a direct consequence of the disrupted muscular mechanics at a molecular level. The MDC1A muscle is like a bicycle with a broken chain, when the various parts are not connected, the whole machine cannot move (Figure 1). Therefore, considering the inherent cause of MDC1A at the microscopic level, Dr. Cohn’s group decided to investigate gene-editing to correct the root of the problem.

CRISPR-Cas9 is the most accurate gene editing technique available today. The researcher will design a guide sequence that helps bind the Cas9 enzyme (the molecular “scissors”) to the gene of interest, after which the Cas9 enzyme cuts the DNA at that specific location. After the DNA is cut, fragments of DNA can be deleted or added to make changes to the gene¹. There is widespread consensus in the scientific community that currently, CRISPR-Cas9 should only be used to modify somatic cells, so that in the event that off-target effects take place, they cannot be passed down through the individual’s germline cells to their offspring.

CRISPR-Cas9 is an attractive therapeutic technology, but its use would be impractical in cases of CMD. Presently, there are over 350 known mutation in the LAMA2 gene, therefore each patient would require individualized therapy to correct the mutation causing CMD³. In a paper currently under review for publishing, Dr. Cohn’s group used a modified version of CRISPR-Cas9 called CRISPR-deactivated Cas9 (CRISPR-dCas9), where the “scissors” in the system are inactivated (Figure 2). CRISPR-dCas9 differs from CRISPR-Cas9 in that it does not introduce any breaks in the DNA but is still able to preferentially bind to DNA at a specific gene in order to regulate expression³. Such an approach circumvents the heterogeneity present within cases of CMD, allowing for treatment. They used dCas9 to target a sister gene of LAMA2 known as LAMA1.

LAMA1 is believed to be a disease modifying gene for CMD and encodes for laminin α1 which has a similar structure and function to laminin α23. Dr. Cohn’s group previously demonstrated in a 2017 paper4, that overexpression of the LAMA1 gene is able to restore muscle wasting and paralysis in MDC1A mouse models, alleviating the predominant symptoms of CMD. Upregulation of LAMA1 gene expression leads to an increase of laminin α1 protein that can compensate for the lack of laminin α2, due to the similarities between the proteins5. At the molecular level, laminin α1 can supplement for the role of laminin α2, resulting in functional muscle mechanics. This makes LAMA1 a viable target for therapeutic intervention. In their treatment, dCas9 fused with a transcriptional activator targeting LAMA1, increases laminin α1 levels providing a therapy without needing to correct the underlying disease mutation3. “This is a mutation independent approach, a one-for-all in theory … it’s a very attractive approach!” stated Dr. Cohn.

To test the efficacy of this design, Dr. Cohn’s group injected the system into mice with CMD (Figure 3). As is standard in most current gene therapies, the system was delivered using a viral vector, known as adeno-associated virus (AAV9) that is directed to muscle cells. Scientists have taken advantage of the innate ability of viruses to deliver their own DNA into human cells, by eliminating the bulk of the viral genome and manipulating it to deliver biologically engineered material instead6. The result is a virtually empty and harmless virus, which can easily be packed with CRISPR-dCas9. Two groups of mice were injected with the AAV9 capsules: 1) 2-day old mice without CMD symptoms, and 2) 3-week old mice displaying CMD symptoms. In both cases, there were genetically-identical control mice with CMD, that were not given the CRISPR-dCas9 treatment.

When LAMA1 was upregulated in the test mice in comparison to the control group, it resulted in decreased muscle fibrosis and improved muscle function. Dr. Cohn’s group demonstrated that the symptoms of CMD could be prevented in 2-day old neonatal mice that lacked a normal LAMA2 gene. Then, Dr. Cohn described the most impressive finding of his paper. “We injected [the system] at a time where we knew that there was already existing fibrosis, existing hind limb paralysis, and that was reversed.” He believes this was the most important finding because, “there’s a general notion that once you have a muscle that is completely fibrotic, there is nothing you can do.” These results demonstrate that in the early stages of the disease, there is a possibility of reducing the amount of fibrotic tissue that has already developed with the use of this CRISPR design – this has major implications for improving patient outcomes.

Scientists have come a long way in understanding disease variability and the contribution of genetic factors to the development of disease. The identification of genes with similar functions, like LAMA1 and LAMA2 in this case, allows for the development of new targets for therapy⁷. This is an entirely new, mutation-independent approach for the use of CRISPR-Cas9. While CMD is a very specific and rare disease, CMD patients are not the only ones who will benefit from the exploration of dCas9 as an avenue of treatment. The modification of the expression of LAMA1 can restore the phenotype of CMD; this can be seen as a proof-of-concept. It demonstrates that regulating the expression of genes with similar functions can ameliorate symptoms of a disease or prevent the disease development altogether. As such, CRISPR-dCas9 can be used as a therapeutic tool for a variety of cases in which the mutation underlying disease is highly complex.

While the results for therapeutic use of CRIPSR-dCas9 are promising, we now have to optimize it for clinical implementation in humans. One improvement being explored is changing the method of delivery. The current method, like in other gene therapies, uses a viral vector to deliver the CRISPR-dCas9 system. This method has proved to work efficiently in the lab,and has a history of being clinically safe; nonetheless it does have some inherent disadvantages⁸. One is that while it allows the CRISPR system to be delivered into every cell, the body develops an immunity to the empty virus. As a result, delivery of subsequent vectors would not be effective, and so this treatment can only be administered once in a lifetime. As CRISPR therapeutics are not yet a reality, it is unknown if multiple shots would need to be administered, so the potential to develop immunity to the virus may inhibit the efficacy of the treatment.  Another disadvantage is the concern that these viral vectors will elicit “off-target” effects by randomly integrating the material into nearby regions of the genome, thereby disrupting the function of an important gene⁸. The chances of this occurring are extremely low with AAV9, but rare incidents have been recorded⁹. The risk of off-target consequences of gene editing can become a major concern when editing germline cells.

Dr. Cohn shared his perspective about the recently born germline-edited human twins in China that shook the scientific world. The CRISPR system was used to correct a gene related to HIV susceptibility¹⁰. This work was condemned by scientists globally but remains as a crucial lifelong experiment as the health of these twins will be monitored for unintended consequences¹⁰. With germline editing, any changes made are heritable. “Let’s say there are off-target effects,” commented Dr. Cohn, “they are going to be healthy and protected from HIV but have a mutation somewhere else which can be passed down for generations to come.” When it comes to somatic editing the risks are nowhere near as consequential.

The public response to the birth of CRISPR-edited humans is the fear that it opened a can of worms for creating designer babies¹¹. Dr. Cohn offered his thoughts on the topic, “Thanks to evolution, things like intelligence or hair colour or athletic bodies are multifactorial.” The fact that the area’s most people would want to improve are dependent on multiple genes and regions across the genome, precisely orchestrated development and epigenetic controls, makes the practicality of designing humans to our specifications nearly impossible. “It’s much more complicated than people think it is,” says Dr. Cohn. Furthermore, he believes that since there is such a fundamental difference between germline editing and somatic editing, the reputation of CRISPR has not been tarnished. Somatic gene modulation using CRISPR is an actionable strategy to alleviate the effects of genetic diseases like CMD, while limiting the potential off-target effects of CRISPR.

Dr. Cohn’s team is proud to have been able to demonstrate their work as a proof of concept that can ultimately be applied to other disorders. They are now working on developing their design into a treatment and are investigating the best ways to administer this treatment clinically. To circumvent the difficulties in using a viral vector like AAV9 to deliver the editing system, they are considering the use of synthetic nanoparticles. With nanoparticles, the body’s immune system should not develop resistance as nanoparticles do not present with antigens and as a result do not elicit an immune response, thereby enabling the treatment to be given as required in multiple doses over time. Now that Dr. Cohn’s lab has essentially completed the research and development aspects of this therapy, it is already halfway through the typical pipeline that a treatment follows before coming to market. Practically speaking, the release of a gene-editing drug that can prevent muscular dystrophy in a person destined to develop the disease is not all that far away. Dr. Cohn concluded, “I think it’s the best time to be in genetics. We are moving towards whole genome sequencing as a standard clinical test, which will improve our diagnostic grades, and with CRISPR technology now available, we have the conceptual ability to begin to think how we can actually fix some of these diseases. That’s what we’re going to be thinking about in the next 15-20 years.”

References

1. Hsu, P., Lander, E. & Zhang, F. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell 157, 1262-1278 (2014).
2. Falsaperla, R. et al. Congenital muscular dystrophy: from muscle to brain. Italian journal of pediatrics 42, 78 (2016).
3. Kemaladewi, D. et al. A mutation-independent approach via transcriptional upregulation of a disease modifier gene rescues muscular dystrophy in vivo. bioRxiv, 286500 (2018).
4. Kemaladewi, D. et al. Correction of a splicing defect in a mouse model of congenital muscular dystrophy type 1A using a homology-directed-repair-independent mechanism. Nature Medicine 23, 984 (2017).
5. Gawlik, K. Laminin 1 chain reduces muscular dystrophy in laminin 2 chain deficient mice. Human Molecular Genetics 13, 1775-1784 (2004).
6. Thomas, C. E., Ehrhardt, A. & Kay, M. A. Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Gen.4, 346-358 (2003).
7. Cutting, G. R. Modifier genes in Mendelian disorders: the example of cystic fibrosis. Annals of the New York Academy of Sciences 1214, 57-69 (2010).
8. Chandler, R. J., Sands, M. S. & Venditti, C. P. Recombinant Adeno-Associated Viral Integration and Genotoxicity: Insights from Animal Models. Hum. Gene Ther.28, 314-322 (2017).
9. Mingozzi, F. & High, K. A. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat. Rev. Genet.12, 341-355 (2011).
10. Wang, C.et al. Gene-edited babies: Chinese Academy of Medical Sciences’ response and action. Lancet 393, 25-26 (2019).
11. Peters, T. Flashing the yellow traffic light: Choices forced upon us by gene editing technologies. Theology Sci.17, 79-89 (2019).

Image Credits

1. SickKids (n.d.). Projects – Muscular Dystrophies. [image] Available at: https://lab.research.sickkids.ca/cohn/projects/ [Accessed 5 Apr. 2019].
2. Nature (2016). Hacking CRISPR. [image] Available at: https://www.nature.com/news/crispr-gene-editing-is-just-the-beginning-1.19510 [Accessed 5 Apr. 2019].
3. The Scientist (2018). Putting Exomes to Work. [image] Available at: https://www.the-scientist.com/modus-operandi/putting-exosomes-to-work-64684 [Accessed 5 Apr. 2019].