Reliable Resolution of Full-Length Huntingtin Alleles by Quantitative Immunoblotting.

Background Therapeutics that lower mutant huntingtin (mHTT) have shown promise in preclinical studies and are in clinical development for the treatment of Huntington's disease (HD). Multiple assays have been developed that either quantify mHTT or total HTT but may not accurately measure levels of wild type HTT (wtHTT) in biological samples. Objective To optimize a method that can be used to resolve, quantify and directly compare levels of full length wtHTT and mHTT in HD samples. Methods We provide a detailed quantitative immunoblotting protocol to reproducibly resolve full length wtHTT and mHTT in multiple HD mouse and patient samples. Results We show that this assay can be modified, depending on the sample, to resolve wtHTT and mHTT with a wide range of polyglutamine differences (ΔQs 22-179). We also demonstrate that this method can be used to quantify allele-selective lowering of mHTT using an antisense oligonucleotide in HD patient-derived cells. Conclusion This quantitative immunoblotting method can be used to reliably resolve full length HTT alleles with ΔQs≥22 and allows for direct comparison of wtHTT and mHTT levels in HD samples.