Developing novel nanobody-based biosensors and therapeutics for Huntington’s Disease

PROJECT SUMMARY Misfolding and aggregation of mutant huntingtin exon1 (mHTTex1) promotes neurodegeneration in Huntington’s disease (HD). The intrinsically disordered mHTTex1 misfolds into a heterogeneous mixture of assemblies, however, how they form, and which conformers mediate neurotoxicity in vivo remain to be identified. This gap in knowledge is due to a lack of molecular diagnostic tools to study the oligomerization pathway of native mHTTex1 in live neurons and to determine how it may induce neurodegeneration. Conformer-specific nanobodies will enable us to perform these studies. Nanobodies are genetically encoded variable heavy domains (VHH), which are naturally produced by camelids and sharks and display potent specificity towards target antigens. This project will combine the experience of Dr. Langen’s lab in generating defined and stable helical oligomers and protofibrils of recombinant mHTTex1, with the that of Dr. Khoshnan’s lab in nanobody technology and its application to studies on the neurobiology of mHTTex1. We recently reported that among the various assemblies of recombinant mHTTex1, protofibrils and their precursors helical oligomers are neuroinvasive since they can penetrate human neurons, propagate, and induce nuclear damage. Towards identifying similar conformers in vivo, we now have isolated llama nanobodies, which bind to helical oligomers, and protofibrils of recombinant mHTTex1. We plan to investigate the binding properties of these nanobodies and develop diagnostic tools like chromobodies by fusing them to GFP or RFP. These biosensors will be used to study the early steps of native mHTTex1 oligomerization in live human neuronal stem-cell derived neurons, and examine whether helical oligomers and/or protofibrils are neurotoxic. Nanobodies, which may inhibit the oligomerization of mHTTex1, will be tested for ability to prevent aggregation and toxicity in the same model. This project has the potential to make a significant contribution to our understanding of mHTTex1 oligomerization and neurotoxicity in a physiologically relevant model, identify therapeutic modalities, and provide a set of novel molecular diagnostic tools for the broader HD research community.