Engineering Botulinum Neurotoxin C1 as a Molecular Vehicle for Intra-Neuronal Drug Delivery

Edwin J Vazquez-Cintron,Konstantin Ichtchenko,Phillip H Beske,Philip A Band,Charles B Shoemaker,Christopher A Angeles,Bao Q Tran,Luis Tenezaca,Jonathan M Oyler,Patrick M Mcnutt,Jean Mukherjee,Aurelia Syngkon,Elliot J Glotfelty,Suzanne R Kalb

doi:10.1038/srep42923

Abstract

Botulinum neurotoxin (BoNT) binds to and internalizes its light chain into presynaptic compartments with exquisite specificity. While the native toxin is extremely lethal, bioengineering of BoNT has the potential to eliminate toxicity without disrupting neuron-specific targeting, thereby creating a molecular vehicle capable of delivering therapeutic cargo into the neuronal cytosol. Building upon previous work, we have developed an atoxic derivative (ad) of BoNT/C1 through rationally designed amino acid substitutions in the metalloprotease domain of wild type (wt) BoNT/C1. To test if BoNT/C1 ad retains neuron-specific targeting without concomitant toxic host responses, we evaluated the localization, activity, and toxicity of BoNT/C1 ad in vitro and in vivo. In neuronal cultures, BoNT/C1 ad light chain is rapidly internalized into presynaptic compartments, but does not cleave SNARE proteins nor impair spontaneous neurotransmitter release. In mice, systemic administration resulted in the specific co-localization of BoNT/C1 ad with diaphragmatic motor nerve terminals. The mouse LD50 of BoNT/C1 ad is 5 mg/kg, with transient neurological symptoms emerging at sub-lethal doses. Given the low toxicity and highly specific neuron-targeting properties of BoNT/C1 ad, these data suggest that BoNT/C1 ad can be useful as a molecular vehicle for drug delivery to the neuronal cytoplasm.

Highlights

Delivery of biological moieties to the neuronal cytosol provides a means of treating neurological conditions that have been difficult to address by conventional therapies
We found differences in potencies as high as 31-fold among the wt Botulinum neurotoxin (BoNT)/C1 preparations used in our study
The three amino acid residues selected for mutation in BoNT/C1 ad are 100% conserved among seven different BoNT LC serotypes; these amino acids were selected based on similar mutations described in our previous work with wt BoNT/A15,7,8

Summary

Introduction

We have previously described genetic constructs and expression systems that enable facile design and production of atoxic recombinant derivatives of BoNT serotype A1 (BoNT/A1 ad) that retain the structural and trafficking properties of wt BoNT/A15,6 One such derivative, BoNT/A1 ad was developed and described as a “Trojan horse” prototype molecular vehicle for delivering drugs to the neuronal cytoplasm. We describe the design, expression, purification, and functional evaluation of a second-generation neuron-specific delivery vehicle composed of an atoxic BoNT derivative with reduced toxicity that circumvents the limitations of the BoNT/A1 ad. This new delivery vehicle is a derivative of BoNT serotype C1 (BoNT/C1 ad) that was rendered atoxic by three substitutions in the LC. The extremely low in vivo toxicity of BoNT/C1 ad and its neuron-targeting properties suggest that it will be useful as a molecular vehicle for drug delivery to the neuronal cytoplasm

Methods

Results

Conclusion