Article

Targeted delivery into motor nerve terminals of inhibitors for SNARE-cleaving proteases via liposomes coupled to an atoxic botulinum neurotoxin

International Centre for Neurotherapeutics, Dublin City University, Dublin, Ireland.
FEBS Journal (Impact Factor: 3.99). 05/2012; 279(14):2555-67. DOI: 10.1111/j.1742-4658.2012.08638.x
Source: PubMed

ABSTRACT A targeted drug carrier (TDC) is described for transferring functional proteins or peptides into motor nerve terminals, a pivotal locus for therapeutics to treat neuromuscular disorders. It exploits the pronounced selectivity of botulinum neurotoxin type B (BoNT/B) for interacting with acceptors on these cholinergic nerve endings and becoming internalized. The gene encoding an innocuous BoNT/B protease-inactive mutant (BoTIM) was fused to that for core streptavidin, expressed in Escherichia coli and the purified protein was conjugated to surface-biotinylated liposomes. Such decorated liposomes, loaded with fluorescein as traceable cargo, acquired pronounced specificity for motor nerve terminals in isolated mouse hemidiaphragms and facilitated the intraneuronal transfer of the fluor, as revealed by confocal microscopy. Delivery of the protease light chain of botulinum neurotoxin type A (BoNT/A) via this TDC accelerated the onset of neuromuscular paralysis, indicative of improved translocation of this enzyme into the presynaptic cytosol with subsequent proteolytic inactivation of synaptosomal-associated protein of molecular mass 25 kDa (SNAP-25), an exocytotic soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) essential for neurotransmitter release. BoTIM-coupled liposomes, loaded with peptide inhibitors of proteases, yielded considerable attenuation of the neuroparalytic effects of BoNT/A or BoNT/F as a result of their cytosolic transfer, the first in situ demonstration of the ability of designer antiproteases to suppress the symptoms of botulism ex vivo. Delivery of the BoNT/A inhibitor by liposomes targeted with the full-length BoTIM proved more effective than that mediated by its C-terminal neuroacceptor-binding domain. This demonstrated versatility of TDC for nonviral cargo transfer into cholinergic nerve endings has unveiled its potential for direct delivery of functional targets into motor nerve endings.

0 Followers
 · 
122 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Botulism is a severe neuroparalytic disease caused by the toxins produced from several Clostridium species. Botulinum neurotoxins (BoNTs) cause flaccid paralysis by inducing a blockade at voluntary motor and autonomic cholinergic junctions that, if not treated, can be fatal. Vaccination to elicit protective circulating antibodies that bind, neutralize and clear toxins before they can be internalized and affect cholinergic neurons remains the most effective form of protection against BoNT. A pentavalent BoNT toxoid vaccine administered in the USA under an Investigational New Drug protocol to at-risk workers was discontinued by the CDC in 2011 due to diminished potency and reactogenic effects. Subsequent research efforts have primarily focused on recombinant protein antigens. This review focuses on the development of a recombinant bivalent vaccine (rBV A/B) composed of purified recombinant BoNT/A and BoNT/B receptor-binding domain proteins, as well as presenting a summary of progress and issues associated with alternative vaccines currently being developed against botulism.
    Expert Review of Vaccines 05/2013; 12(5):481-92. DOI:10.1586/erv.13.37 · 4.22 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Delivering therapeutic cargos to specific cell types in vivo poses many technical challenges. There is currently a plethora of drug leads and therapies against numerous diseases, ranging from small molecule compounds to nucleic acids to peptides to proteins with varying binding or enzymatic functions. Many of these candidate therapies have documented potential for mitigating or reversing disease symptoms, if only a means for gaining access to the intracellular target were available. Recent advances in our understanding of the biology of cellular uptake and transport processes and the mode of action of bacterial protein toxins have accelerated the development of toxin-based cargo-delivery vehicle platforms. This review provides an updated survey of the status of available platforms for targeted delivery of therapeutic cargos, outlining various strategies that have been used to deliver different types of cargo into cells. Particular emphasis is placed on the application of toxin-based approaches, examining critical issues that have hampered realization of post-intoxication antitoxins against botulism.
    Journal of Applied Statistics 10/2014; DOI:10.2174/1568026614666141022094517 · 0.45 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Treatment of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, is hampered by its complex aetiology and lack of efficient means for targeted transfer of therapeutics into motoneurons. Objective: Engineering of a versatile motoneuron targeting adapter - a full-length atoxic tetanus toxin fused to core-streptavidin (CS-TeTIM) - for retro-axonal transduction of viral vectors. Validation of the targeting efficiency of CS-TeTIM in vivo, by expression of green fluorescence protein (GFP) reporter in motoneurons of pre- and symptomatic ALS-like SOD1(G93A) mice, and comparison with age-matched controls. Appraisal of lentiviral transduction with CS-TeTIM relative to: (1) a HC binding fragment of tetanus toxin CS-TeTx(HC), (2) rabies glycoprotein (RG), and (3) a CS-TeTIM-RG dual targeting approach. Methods: CS-TeTIM and CS-TeTx(HC) were engineered using recombinant technology and site-directed mutagenesis. Biotinylated vectors, pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) or RG, were linked to these adaptors and injected intra-peritoneally (i.p) into pre- (12 weeks old), symptomatic SOD1(G93A) (22 weeks old) or wild type control mice, followed by monitoring of GFP expression in the spinal cord and supra-spinal motor structures with quantitative PCR and immuno-histochemistry. Results: Transcripts were detected in the spinal cord and supra-spinal motor structures of all mice 2 weeks after receiving a single i.p. injection, although in symptomatic SOD1(G93A) animals reporter RNA levels were lower compared to pre-symptomatic and wild-type controls irrespective of the targeting approach. GFP transduction with CS-TeTIM proved more efficient than CS-TeTx(HC) across all groups while CS-TeTIM-RG dual-targeted vectors yielded the highest transcript numbers. Importantly, in both wild-type and pre-symptomatic SOD1(G93A) mice strong co-labelling of choline-acetyltransferase (ChAT) and GFP was visualised in neurons of the brain stem and spinal cord. Conclusions: CS-TeTIM, a versatile adaptor protein for targeted lentiviral transduction of motoneurons has been engineered and its competence assessed relative to CS-TeTx(HC) and RG. Evidence has been provided that highlights the potential usefulness of this novel recombinant tool for basic research with implications for improved transfer of therapeutic candidates into motoneurons for the amelioration of ALS and related diseases.
    Molecular Pharmaceutics 09/2013; 10(11). DOI:10.1021/mp400247t · 4.79 Impact Factor

Full-text (2 Sources)

Download
10 Downloads
Available from
Sep 2, 2014