The human genome contains approximately 20,000 protein-coding genes. Of these, dystrophin is the third largest; spanning ~2.3 megabases of DNA, it would cover the distance between London and Cambridge if the whole genome was unravelled and stretched the length of the UK.
Historically, the size of the dystrophin gene has been a problem for gene therapy groups trying to develop new treatments for Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder caused by mutations in the dystrophin gene (www.musculardystrophyuk.org/DMD/information). Comprising 79 separate DNA subunits (or exons), dystrophin acts as an elastic linker for muscle cells, supporting muscle fibers during contraction and extension. Without this elastic linker protein, muscle cells become stressed and are gradually replaced by fat cells and fibrous tissue. Over time, this process causes the muscle dysfunction that is a hallmark of DMD. Affecting 1 in 3500 males, DMD ultimately causes death by respiratory or cardiac failure.
Adeno-associated viruses (or AAVs) can safely deliver therapeutic genes to muscle cells, making them ideal delivery vectors for genetic conditions such as DMD. Unfortunately, however, AAVs cannot package long DNA sequences. In a Science paper published last month (Amoasii et al. 2018), the Olson group solve this conundrum by using a gene editing approach, restoring dystrophin levels in a canine model of DMD by coupling nature’s molecular scissors with an AAV that has evolved to preferentially infect muscle tissues.
The CRISPR/Cas9 system uses short RNA sequences (sgRNA) to ‘guide’ molecular Cas9 scissors to edit specific regions of the genome, helped by the host DNA repair machinery. In an earlier study (Amoasii et al. 2017), Olson hypothesised that CRISPR/Cas9 could be used to edit a region close to exon 51 of the mutated dystrophin gene. He reasoned that this would cause the cellular machinery responsible for producing proteins to ‘skip’ exon 51, generating shortened but still functional dystrophin proteins. Moreover, the small size of the CRISPR/Cas9 components would render them amenable to packaging in AAVs. Harnessing the power of AAV9, which preferentially transduces skeletal and heart muscle cells, Olson’s team was able to show restoration of dystrophin protein expression following CRISPR/Cas9 genome editing in DMD mice.
The recent Science paper follows on from the 2017 study (Amoasii et al. 2017), moving this promising therapeutic approach one step closer to clinical translation. To start with, AAV9 vectors were engineered to carry the CRISPR/Cas9 components and a short gene promoter sequence that confers additional muscle specificity. In collaboration with the Piercy lab at the Royal Veterinary College in London, these vectors were then injected into the muscles of DMD dogs. Promisingly, muscle biopsies taken 6 weeks later showed a high frequency of ‘exon skipping’ events, with restoration of dystrophin expression to ~60% of normal levels in injected tissues.
Localised therapy would clearly be beneficial for conditions affecting isolated tissues. However the holy grail of gene therapy for generalised disorders such as DMD is to effectively treat all affected muscle groups in one go. Building on the intramuscular administration experiments, two DMD dogs were injected with different doses of CRISPR/Cas9 AAVs via the intravenous route.
The high-dose treatment proved a resounding success; analysis of muscle tissues from these dogs two months after treatment showed widespread restoration of dystrophin expression, from the tricep and bicep muscles to the heart, diaphragm and tongue. Blood tests found no evidence of any systemic side-effects, while a video displayed at the Cold Springs Harbor 2018 CRISPR-Cas meeting showed a DMD dog able to walk and jump normally following CRISPR/Cas9 therapy.
Although these results underscore the great promise of CRISPR/Cas9 therapy for DMD, a note of caution must be sounded. Only a very small number of young DMD dogs were treated in the latest study (Amoasii et al. 2018) and they were only followed up for a short period of time. For it to be truly transformational, the therapy will need to have beneficial effects that are both widespread and long-lasting.
Off-target or excessive genome editing is a particular concern for CRISPR/Cas9, with recent studies in stem cells identifying problematic rearrangements and deletions caused by the DNA repair machinery ‘over-editing’ genes (Kosicki et al. 2018). The identification of antibodies in humans that could neutralise Cas9 has also recently caused a stir, although the prevalence of these antibodies has yet to be fully established (Charlesworth et al. 2018).
A potential solution to both these problems would be to use newer and safer Cas9 variants, although these are still in development and have yet to be fully tested in vivo (Vakulskas et al. 2018). Nevertheless, it is safe to say that CRISPR/Cas9 has opened up a new horizon for the treatment of devastating genetic conditions such as DMD. The first CRISPR clinical trial is slated to begin in Europe later this year, using an ex vivo approach to correct the blood cells of beta-thalassemia patients (https://www.clinicaltrialsregister.eu/ctr-search/trial/2017-003351-38/DE). The gene and cell therapy community will follow the progress of this trial with bated breath.
Amoasii, L., et al. (2018). “Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.” Science.
Amoasii, L., et al. (2017). “Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy.” Sci Transl Med 9(418).
Charlesworth, C. T., et al. (2018). “Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans.” bioRxiv.
Kosicki, M., et al. (2018). “Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.” Nat Biotechnol 36(8): 765-771.
Vakulskas, C. A., et al. (2018). “A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells.“ Nat Med 24(8): 1216-1224.