Strategy for Treating Motor Neuron Diseases Using a Fusion Protein of Botulinum Toxin Binding Domain and Streptavidin for Viral Vector Access: Work in Progress

Abstract

Although advances in understanding of the pathogenesis of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) have suggested attractive treatment strategies, delivery of agents to motor neurons embedded within the spinal cord is problematic. We have designed a strategy based on the specificity of botulinum toxin, to direct entry of viral vectors carrying candidate therapeutic genes into motor neurons. We have engineered and expressed fusion proteins consisting of the binding domain of botulinum toxin type A fused to streptavidin (SAv). This fusion protein will direct biotinylated viral vectors carrying therapeutic genes into motor nerve terminals where they can enter the acidified endosomal compartments, be released and undergo retrograde transport, to deliver the genes to motor neurons. Both ends of the fusion proteins are shown to be functionally intact. The binding domain end binds to mammalian nerve terminals at neuromuscular junctions, ganglioside GT1b (a target of botulinum toxin), and a variety of neuronal cells including primary chick embryo motor neurons, N2A neuroblastoma cells, NG108-15 cells, but not to NG CR72 cells, which lack complex gangliosides. The streptavidin end binds to biotin, and to a biotinylated Alexa 488 fluorescent tag. Further studies are in progress to evaluate the delivery of genes to motor neurons in vivo, by the use of biotinylated viral vectors.

Affinity biosensors using recombinant native membrane proteins displayed on exosomes: application to botulinum neurotoxin B receptor

“…Alternatively this might be explained by differences in affinity of BoNT subtypes, as it was reported that BoNT/B2 may be more active than BoNT/B1 29 . This finding could be of specific importance since HcB has potential for the development of protective recombinant vaccines against botulism, and to target specific drugs to nerve terminals 35 , 36 .…”