Developing Biosensors for High Throughput Drug Screening against Muscular Dystrophy

R. Mahrat, R. Chaturvedi
Calico Biosciences,
United States

Keywords: Biosensor, CRISPER-Cas, Dystrophin, Exon skipping


We at Calcio Biosciences have developed a battery of Biosensors using CRISPR/Cas9 genome editing. We intend to use the developed biosensors for high throughput drug screening against rare genetic diseases like Duchenne Muscular Dystrophy (DMD). Our Biosensors are cell lines with a reporter gene inserted at the 3’ end of the exon mutated DMD gene, which will fluoresce if a drug molecule activates and allows the expression of the DMD gene. Duchenne Muscular Dystrophy (DMD), is a severe, progressive, and X-linked recessive neuromuscular disorder, also known as a muscle-wasting disease affecting 2 in 10,000 live male births. DMD occurs due to mutations in the DMD gene leading to reduced dystrophin production in muscle cells. The exon-skipping mechanism has been demonstrated to be a highly effective therapeutic technique. Three FDA-approved drugs with the exon skipping agent, Golodirsen, Eteplirsen, and Viltolarsen, are also available. However, they are substantially more expensive and thus unaffordable for patients in low- or middle-income countries. This needs a novel perspective drug based on this mechanism must be identified. We have developed biosensors with knock-in of multiple copies of the dystrophin gene with clinically relevant mutations. Each mutated copy of dystrophin is tagged with a fusion fluorescence protein at the 3’ end. We have designated this sensor as tandem Biosensors. This tandem sensor can be used highly through put screening of the library of small molecules library of antisense. If biosensors emit fluorescence by adding a therapeutic molecule, it signifies the activation of an inactivated DMD gene so that the molecule can be regarded as a possible hit. For example, a Tandem Biosensor has four copies of DMD genes arranged sequentially with a mutation in exon 51, 52, 53, and 54, with reporter gene expressing red fluorescent protein (RFP), Yellow (YFP), Cyan (CFP), and far-red (FRRFP), respectively. The fluorescence emissions from the respective fluorescent protein in a Tandem Biosensor indicate the skipping of the respective exon, activating a particular copy of the DMD gene. Overall, Biosensor technology will advance high-throughput drug screening against DMD, offering users a more streamlined and accurate way to monitor the therapeutic potential of a drug candidate. We are currently developing Tandem Biosensors with a maximum of four copies of DMD genes which can be upscaled up to 10 to 15 copies.