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

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.

Novel Botulinum Neurotoxins: Exploring Underneath the Iceberg Tip

“…Translocation of protein cargoes inside neuronal cytosol was efficient, with the only constraint that the cargo must be able to undergo unfolding [ 140 ]. Similar examples have been also provided by others [ 141 , 142 , 143 , 144 ]. Alternatively, specific fragments of BoNTs can be produced and rejoined by using a “protein-stapling” technology to combine distinct parts of different BoNTs (and possibly other targeting proteins), still maintaining enzymatic activity of the L chain [ 145 , 146 ].…”

Small molecule metalloprotease inhibitor with in vitro, ex vivo and in vivo efficacy against botulinum neurotoxin serotype A

“…This realization has led to consideration of alternative strategies for treatment of botulism. Among the promising emergent candidates, two are especially noteworthy: 1) atoxic BoNT constructs (Vazquez-Cintron et al, 2017) capable of intracellular delivery of a broad range of inhibitors (e.g., highly potent camelid antibodies that target the active site of the LC (Tremblay et al, 2010); 2) selective inhibitors for the deubiquitinating enzyme VCIP135/VCPIP1, which is largely responsible for the neuronal persistence of LC/A (Tsai et al, 2017). It is envisioned that an effective strategy for future treatment of BoNT/A intoxication will consist of one or more SMIs and/or potent camelid antibodies delivered by recombinant atoxic BoNTs to effectively inhibit LC/A and deubiquitinase inhibitors to accelerate its degradation.…”

Recombinant Botulinum Neurotoxin Hc Subunit (BoNT Hc) and Catalytically Inactive Clostridium botulinum Holoproteins (ciBoNT HPs) as Vaccine Candidates for the Prevention of Botulism

“…Advances in rapid gene synthesis and targeted mutagenesis techniques have enabled the production of complete BoNT holoproteins with mutations designed to render the protein nontoxic. Several of these nontoxic holoproteins have been investigated for use in trafficking studies [ 20 ], as possible targeting and delivery vehicles for drugs [ 21 , 22 ], and as vaccines [ 23 , 24 , 25 , 26 ]. We previously reported on the production and immunological assessment of a recombinant, catalytically inactive BoNT/A1 holoprotein vaccine (ciBoNT/A1 HP) which was found to have greater potency than recombinant protein antigens representing any other /A1 subtype domain, or combination of domains, against a challenge of both parental toxin and two dissimilar toxin subtypes (Webb 2009).…”