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

Daniel B Drachman,Uma Balasubramanian,Yang Lu,Robert N Adams

doi:10.3390/toxins2122872

Daniel B Drachman, Uma Balasubramanian + Show 2 more

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https://doi.org/10.3390/toxins2122872

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Journal: Toxins	Publication Date: Dec 20, 2010
Citations: 64	License type: CC BY 3.0

Affiliation: Johns Hopkins University

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.

Highlights

Amyotrophic Lateral Sclerosis (ALS) is a universally fatal and remarkably common disease, with a lifetime risk of developing the disease estimated at between 1 in 400 [1] and 1 in 2,000 [2]
We have engineered and expressed fusion proteins consisting of the binding domain of botulinum toxin type A fused to streptavidin (SAv)
We have succeeded in engineering and expressing a heterobifunctional protein, designed to carry biotinylated viral vector cargoes into motor nerves

Summary

Introduction

Amyotrophic Lateral Sclerosis (ALS) is a universally fatal and remarkably common disease, with a lifetime risk of developing the disease estimated at between 1 in 400 [1] and 1 in 2,000 [2]. ALS is due to selective loss of motor neurons, resulting in progressive muscular weakness, denervation atrophy and spasticity. Spinal Muscular Atrophy (SMA) is a recessively inherited disorder that results in loss of motor neurons. In its most severe form, it occurs in infancy, and is the leading genetic cause of infant death, with an estimated incidence of 10–16 in 100,000 live births. SMA causes milder forms of the disease with weakness developing in childhood or later. The carrier frequency of the recessive genetic abnormality in SMA is 1 in 40 to 1 in 50 [3,4]

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