D002 Development of a new bioanalytical method for selective measurement of wild-type Huntingtin in human cerebrospinal fluid

<h3></h3> The past decade has seen the development of several novel ligand-binding immunoassays for measuring huntingtin protein (HTT) that have been successfully used in preclinical cellular and animal model studies, as well as in Huntington’s disease (HD) individual biosamples. Specifically, we have developed and validated ultrasensitive immunoassays for the measurement of either polyglutamine-expanded (mutant) HTT protein (mHTT) or total (polyglutamine length-independent) HTT protein, both to low femtomolar sensitivity range allowing for measurements of HTT protein in HD individual cerebrospinal fluid samples (CSF). To enable therapeutic programs that selectively target mHTT, it is crucial to quantify both the mHTT and the non-expanded wild-type HTT (wtHTT) proteins to determine the pharmacodynamic effect of such drug candidates. However, due to the lack of antibodies that selectively recognize the wtHTT protein, its selective measurement has been difficult to achieve. In this current work, we adapted and optimized a method initially published by Fodale, et al. (2022) to quantify wtHTT protein in human CSF that have been cleared of mHTT by employing selective immunodepletion, followed by performing the clinically validated polyglutamine length-independent HTT SMCxPRO immunoassay (CHDI_HTT_145; 2B7/D7F7) for measuring the remaining wtHTT. We performed a survey of magnetic particles and mHTT-directed monoclonal antibody (mAb) combinations to achieve an effective mHTT selective depletion (&gt; 90%) protocol. Critical parameters, including incubation buffer, time, temperature, and antibody-magnetic particle working dilution were varied to attain selective and reproducible immunodepletion of mHTT and accurate quantitation of wtHTT. The next steps will be the performance evaluation of the assay method in human CSF samples from healthy and HD individuals, followed by technical assay validation. We believe this method will be a useful tool to facilitate the development of allele-selective mHTT lowering therapies.