Muscular dystrophies (MD) collectively represent a group of genetically inherited disorders characterised by significant muscle wastage leading to an increasing level of disability. The disease is often evident at birth or soon after, and is progressive in nature. Eventually MD affects the heart and muscles involved in breathing, and at this point the disease becomes life threatening rather than life limiting. At present, there are no effective curative therapies for MD, and existing treatments are aimed at manging the symptoms rather than curing the disease.
The genetic nature of the disease has meant that MD has been considered a potential candidate for gene therapy for well over two decades. A major issue in delivering effective gene therapies for MD has been that the genes mutated in this disease are too big to be packaged within the most effective gene therapy agents – viruses. Alternative strategies based on “microdystrophin” (small versions of the MD genes) and “exon skipping” technologies have demonstrated limited efficacy in pre-clinical models.
In this month’s edition of Nature Medicine, Kemaladewi et al. describe the use of a combined AAV9/CRISPR-Cas9 genome editing strategy for treatment of a specific form of MD, termed congenital muscular dystrophy type 1A (MCD1A) (Kemaladewi et al. 2017). This paper builds on a raft of positive findings in the CRISPR-Cas9 genome editing field over the last 5 years. Using an adeno-associated viral vector (renowned in the field for the small transgene insertion potential) and carefully designed guide RNA inserts designed to modify intronic sections of the MCD1A gene.
These intronic regions do not actively code for the MCD1A mRNA or protein, but rather are involved in processing the transcribed mRNA template which is subsequently translated into protein. It is estimated that in ~40% cases MCD1A associated MD, the cause is related to this incorrect processing (also known as splicing) of the mRNA transcript.
In this manuscript, the authors engineer the AAV vector to express guide RNAs to repair this splicing function, and demonstrate efficacy of this approach in several mouse models of the disease. Most promisingly, they demonstrate that treatment of neonatal (2 day old) mice with this version of MD with an intravenous administration of a high dose of the optimised AAV “cocktail” was able to repair a significant percentage of the MCD1A transcript and produce wild type protein expression 30 weeks after treatment.
The authors state that following treatment “..the degree of paralysis in the hind limbs and mobility of the treated mice were markedly improved…” and that this “demonstrates that early restoration of Lama2 expression in both peripheral nerves and skeletal muscles results in near-complete amelioration of both phenotypes”.
Whilst it remains to be seen whether these medicines, and the huge doses potentially required to scale up from mouse to man, can be translated into meaningful clinical benefit for patients with these specific splice form variants of MD. The technology and the study are extremely elegant, and the findings give great hope to the field. The authors also report no obvious “off-target” mutations in other genes – an important consideration for such technologies.
An estimated ~10% of the ~80,000 mutations reported in the human genome database have been shown to affect mRNA splicing, and this technology may have wide ranging implications across a diverse variety of genetic disorders. The data presented in this paper add further evidence of the immense potential of the tiny AAV virus to fix complex genetic disorders- potentiated when combined with the powerful CRIPSR/Cas9 system. It certainly seems to be the boom time to be working in “little fixes”.
Kemaladewi, D. U., et al. (2017). “Correction of a splicing defect in a mouse model of congenital muscular dystrophy type 1A using a homology-directed-repair-independent mechanism.” Nat Med 23(8): 984-989.