Duchenne muscular dystrophy is an X-linked developmental disorder caused by mutations in the dystrophin (Dys) gene leading to progressive muscular weakness and premature death due to respiratory and/or cardiac failure. RGX-202 is a recombinant adeno-associated virus of serotype 8 (AAV8) with an optimized human microdystrophin (microDys) transgene and a promoter designed to increase expression in muscle (Spc5-12). Quantitation of microDys levels in skeletal and cardiac muscles in subjects dosed with RGX-202 will be an important factor to understand treatment effect of the gene therapy.
A capillary-based western method utilizing JESS automation (by ProteinSimple) has been developed and validated to quantify both microDys and Dys over a large calibration range in tissue lysates from various species to support our gene therapy program. The automated JESS system offers advantages over traditional Western Blot such as quick run-time, no blotting, multiplexing for loading control, and use of very low amounts of samples (100-fold less) and antibodies (500 times less).
A recombinant microDys protein was generated as a reference standard which allows for direct quantitation of RGX-202 transgene product. The calibration curve range for microDys protein was 4.0- 200 ng/mg in monkey method and 5.0-160 ng/mg in mouse method. The methods were validated in mouse and monkey tissues following the principles outlined in the Bioanalytical Method Validation guidance by FDA. The sensitivity of the monkey tissue method was demonstrated to be 4.0 ng of microDys/mg of total tissue lysates with an overall accuracy and precision of within ±30% and mouse tissue method was 5.0 ng of microDys/mg of total tissue lysates with an accuracy and precision of within ±20%. Specificity for dystrophin detection in monkey tissues was also confirmed by various commercially available antibodies. Overall, results show that capillary-based western methods are sensitive, specific and robust. The assays have successfully been used to measure microDys levels in support of various pre-clinical studies.