Duchenne Muscular dystrophy (DMD) is an X-linked recessive disorder characterised by progressive muscle weakness and shortened life expectancy. DMD and less severe Becker muscular dystrophy (BMD) are caused by mutations/ truncations in the DMD gene, which codes for the protein dystrophin. The severity of the illness is linked to levels of dystrophin in the muscles, and a key therapeutic approach is to attempt to restore dystrophy levels.
Assessing dystrophin levels in model organisms and patients is vital for evaluating new drug therapies. However, current methods such as Western blots and liquid chromatography-mass spectrometry (LC-MS/MS), which use sodium dodecyl sulphate (SDS) gel separation, are limited by the variability and throughput of the gel-based procedure.
To address these challenges, Hendrik Neubert, Joe Palandra and their team at Pfizer’s department of Biomedicine design enlisted the help of Cambridge Research Biochemicals (CRB) to produce a range of anti-peptide antibodies. These custom reagents are vital to developing a peptide immunoaffinity (IA) method linked to LC-MS/MS for dystrophin quantification. CRB has over 40 years’ experience in producing bespoke reagents with high affinity and high specificity which are the requirements for the IA linked LC-MS/MS assay to succeed.
The method can detect dystrophin and mini-dystrophin in skeletal muscle biopsies in model species and clinical samples at levels as low as ~ 1% relative to healthy muscle tissue. Such low detection levels are essential as most patients with DMD produce only trace amounts of the protein. Using this technique, the team showed a dose-dependent increase in mini-dystrophin expression 6 months after an adenovirus-based therapy was given.
Peptide selection was based on several criteria, including specificity to mini or full-length dystrophin, specificity to rat dystrophin and antigenicity to generate specific anti-peptide antibodies.
The team generated an anti-peptide antibody column holding two anti-peptide antibodies against dystrophin LLQV and LEMP peptides. The use of anti-peptide antibodies in this assay was essential to eliminate many of the challenges associated with anti-protein antibodies, including a lack of capture efficiency and protein denaturation during extraction. This method also allows a large number of samples to be run simultaneously (96 total; 60 unknown samples), unlike gel-based assays.
The collaboration between Pfizer, Hendrik Neubert and Cambridge Research Biochemicals has pushed the boundaries of DMD assays. This will provide vital information about the efficacy of the therapy patients are undergoing. With more accurate data, DMD and BMD treatments can be moved forward with continuing teamwork between Cambridge Research Biochemicals and researchers.
Farrokhi et al., (2022). Dystrophin and mini-dystrophin quantification by mass spectrometry in skeletal muscle for gene therapy development in Duchenne muscular dystrophy. Gene Ther., 29: 608. doi: 10.1038/s41434-021-00300-7.
Le Rumeur et al., (2010). Dystrophin: more than just the sum of its parts. Biochim Biophys Acta Proteins Proteom., 1804(9): 1713. doi: 10.1016/j.bbapap.2010.05.001.