This revised Exploratory/Developmental (R21) proposal describes innovative research approaches to eliminate the immune response to botulinum neurotoxin type A (BoNT/A) therapy in dystonia patients. The immunogenicity of BoNT/A gives rise to neutralizing antibodies that reduce or eliminate its therapeutic benefits. The proposed research stems from a recognition that development of protein therapeutics in general must be driven by insights into drug-induced immune responses. A generalizable strategy for de- immunization of functional therapeutics will eliminate or at least reduce this major obstacle to the rapid translation of """"""""genes to drugs"""""""". This novel approach combines cutting-edge bioinformatics tools with classic immunological assays to create an improved BoNT/A that escapes immunological detection much in the same way as tumor cells and viruses. The target will be the T cell dependent mechanism for antibody generation, which is mediated by peptide-loaded class II MHC-T cell receptor interactions. The primary goals of the project are to use epitope mapping algorithms to computationally identify BoNT/A- derived T cell epitopes. Subsequent verification will be performed experimentally using MHC binding assays and T cell stimulation studies of blood drawn from BoNT/A exposed patients. Immunodominant epitopes will be targeted for modification to reduce their immunogenicity with the aim of preserving the overall fold of the toxin. Computational methods will be used to verify reduced immunogenicity of the modified toxin, and homology modeling will be used to determine the structural effects of these changes. The de-immunized peptide sequences will be assayed for MHC binding and in vitro and in vivo (mouse) T cell stimulation. Public Health Relatedness: Knowledge of the elements of botulinum neurotoxin type A that give rise to its immunogenicity will contribute to the development of improved dystonia therapy. ? ? ?
