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CRISPR mediated upregulation of B4galnt2 as a potential treatment for Duchenne Muscular Dystrophy

R Javed(1) J T March(1) N Lu-Nguyen(1) J Selvakumaran(1) L Popplewell(2) A Malerba(1)

1:Royal Holloway, University of London; 2:National Horizons Centre, Teesside University

Duchenne Muscular Dystrophy (DMD) is a rare X-linked muscular disease affecting about 1:5000 new born males. It is caused by mutations on the dystrophin gene leading to a truncated protein that is degraded. DMD primarily affects skeletal and cardiac muscles. The initial signs start with difficulty in movement and inability to climb stairs which eventually lead to loss of cardiac and respiratory functions. Genetic engineering particularly the bacterial CRISPR-Cas9 system has advanced significantly in the last few years providing a platform for genome editing and regulation of gene expression. This project aims to use one of the CRISPR applications (nuclease deactivated Cas9 or dCas9) to upregulate the expression of B4galnt2. This gene encodes for an enzyme (β-1,4-N-acetyl galactosaminyl transferase-2) responsible for the glycosylation of proteins such as α-dystroglycan, laminin-α2, integrin and others which are a part of dystrophin-glycoprotein complex(DGC) present at the muscle sarcolemma. DGC is responsible for maintaining the structural integrity of muscle fibres which is crucial in DMD. Absence of B4galnt2 has shown to increase inflammation and muscle pathology in case of an injury. We hypothesise that the activation of this gene can, at least partially, compensate for the absence of dystrophin function and increase muscle functionality in vivo. To access the effect of B4galnt2 upregulation we designed RNA guides and used dCas9 in mouse myoblasts. Our data suggests that B4galnt2 can be successfully upregulated with this approach paving the way for more studies in human cells and finally in a mouse model of DMD.

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