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Clostridial neurotoxins as a drug delivery vehicle targeting nervous system
Biochimie 92 (2010) 1252e1259
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Review
Clostridial neurotoxins as a drug delivery vehicle targeting nervous system Bal Ram Singh a, *, Nagarajan Thirunavukkarasu a, Koyel Ghosal a, Easwaran Ravichandran a, Roshan Kukreja a, Shuowei Cai a, Peng Zhang b, *, Radharaman Ray c, *, Prabhati Ray b, * a
National Botulinum Research Center, and Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, Dartmouth, MA 02747, USA Walter Reed Army Institute of Research, Silver Spring, Maryland, USA c United States Army Medical Research Institute of Chemical Defense, APG, Maryland, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2009 Accepted 9 March 2010 Available online 24 March 2010
Several neuronal disorders require drug treatment using drug delivery systems for specific delivery of the drugs for the targeted tissues, both at the peripheral and central nervous system levels. We describe a review of information currently available on the potential use of appropriate domains of clostridial neurotoxins, tetanus and botulinum, for effective drug delivery to neuronal systems. While both tetanus and botulinum neurotoxins are capable of delivering drugs the neuronal cells, tetanus neurotoxin is limited in clinical use because of general immunization of population against tetanus. Botulinum neurotoxin which is also being used as a therapeutic reagent has strong potential for drug delivery to nervous tissues. Ó 2010 Published by Elsevier Masson SAS.
Keywords: Botulinum neurotoxins Drug delivery vehicles Neuronal disorders Non-viral drug delivery
1. Introduction Drug delivery to neuronal cells is an unmet need, while neurological disorders affect around one billion people worldwide, and is the cause of 12% of total deaths [1]; with more than 20 million people in the United States alone, accounting for over $400 billion annually for their treatment and prolonged care [2]. Over 12 million people around the world suffer from Alzheimer’s disease [3] and stroke is the third leading cause of death in the western world [4]. Despite aggressive research, patients suffering from fatal and/or debilitating CNS diseases and neurodegenerative disorders far outnumber those dying of all types of systemic cancer or heart disease [5]. With the increase in the world population of aged people, there are several age-related diseases, most notably neuronal disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), epilepsy, multiple Sclerosis (MS), Huntington’s disease (HD) and Lou Gehrig’s disease (or amyotrophic lateral sclerosis, ALS). These are potentially debilitating, life threatening and more importantly affect the quality of life. Another disabling condition which mostly victimizes young healthy people in their ripe productive years with devastating social emotional psychological and life style effects is spinal cord injury. Neurological conditions such as AD, PD, HD epilepsy, or stroke are normally caused by biochemical malfunctioning within neuronal cells of central and/or peripheral nervous system. Also worldwide, an * Corresponding authors. Tel.: þ1 508 999 8588; fax: þ1 508 999 8451. E-mail address: [email protected] (B.R. Singh). 0300-9084/$ e see front matter Ó 2010 Published by Elsevier Masson SAS. doi:10.1016/j.biochi.2010.03.005
estimated 2.5 million people live with spinal cord injury (SCI), with more than 130,000 new injuries reported each year [6]. SCI significantly affect the quality of life, life expectancy, and economic burden, with considerable costs associated with primary care and loss of income. Therapeutic options for these diseases which are progressive are very limited. The clinical failure of potentially effective therapeutics is often not due to lack of potency but rather to the shortcomings in the drug delivery methods [5]. While understanding the molecular basis of these diseases is allowing design and development of effective drugs, their delivery into nerves, particularly those of the central nervous system is a major impediment in the development of effective neurotherapeutics. We have recently developed a nontoxic version of the world’s most toxic neurotoxin, the botulinum neurotoxin (BoNT), which could be utilized as drug delivery vehicle (DDV) to target nerves due to their highly specific and selective binding to neurons. We review here the characteristics of the BoNTbased targeted drug delivery system for neuronal cells which could benefit an array of neurological disorders. 2. Limitations of present drug delivery strategies and other alternatives Although the therapeutic potential of several neurological drugs and targets is currently being studied and established in many cases, there are only limited opportunities considering suitable methods and strategies to deliver them, since neuronal cells are highly polarized cells with somal, axonal, and dendritic domains,
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having distinct membrane compositions. Some limitations and issues with the existing methods are discussed below: a) Non-Specific Delivery and toxicity: In many neurological conditions, targeting neuronal cells is essentially important to reduce toxicity of the drug or to augment the desired therapeutic effect. For e.g., in case of spinal cord injury, neuronal regeneration in vivo, does not rely only on neuronal properties. All three major types of glial cells have an impact on neuronal function under normal conditions, and in determining the success of repair mechanisms in pathological circumstances of CNS. However, this synergistic relationship could be perturbed when the drug also exerts its activity in cells like astrocytes and other glial cells of CNS. Therefore, there is critical need to develop neuron selective drug delivery system. Indeed, it is increasingly recognized to incorporate neuron-targeting ligands for better transfection efficiencies, and reduce toxicity due to widespread entry into off-target cells, in the development of non-viral mediated neuronal drug delivery system [7]. b) Route of delivery: Techniques employing molecular therapies for several CNS disorders include intracerebroventricular, intrathecal or intraspinal injection, continuous infusion or topical application of a drug carrier saturated with the molecule of interest, although minimally invasive or traumatic routes are preferable. Among less invasive routes, intramuscular administration using retrograde transporting carriers are of extreme interest for use in neurotherapeutics associated with CNS pathologies [7]. c) Limitations in other viral and non-viral choice of carriers: Several viral and non-viral carrier based gene delivery methods have been described in literature as promising and clinically feasible for CNS neuronal delivery. However, only few have been successfully applied towards animal models of neurological disease or injury [7]. In vivo gene therapy has been tested in models of SCI using viruses, including herpes simplex virus (HSV), adenovirus, Adeno-Associated Virus (AAV), lentivirus and Moloney leukaemia virus. Injection of HSV into mammalian tissue elicited a local immune response and in case of AAV and the clinical trials was delayed due to production of neutralizing antibodies in humans possibly by previous exposure, and also often encounters safety issues [8]. Currently existing non-viral carrier approaches for neuronal gene delivery suffer from low transfection efficiencies and/or toxicity issues compared to that of neurotropic viral deliveries, as the former can associate with most cell types via non-specific binding to the cell surface. Their internalization also varies depending on the neuronal cell type, size, charge, surface composition, the delivery site, and the specific vehicle formulation. Delivering sufficient quantities to have an effect at the targeted site is often difficult. Limitations in controlling the expression levels or durations of the therapeutic transgene, resulting in suboptimal dosing or over dosing had been indicated in different studies [8]. Polyethylenimine (PEI)/DNA polyplexes, and cationic lipid based lipoplex-mediated deliveries, which were tested on neurons, were shown to aggregate in biological fluids [9]; PEI even exhibited cellular toxicity, and reduced uptake in neuron-like cells as compared to that of undifferentiated cells. Polyplexes and lipoplexes when delivered to neurite terminals undergo internalization in the vesicles that are part of endo-/lysosomal pathway [7] and their inability to escape from the acidification of endosomal compartments in neurons partially explains their poor transfection efficiencies compared to viruses [10]. Therefore, is a great necessity to develop an efficient, safe neuronal delivery system to augment neurotherapeutics in general
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and henceforth the utility of neurotoxin based drug delivery strategies in fulfilling some of these requirements in neurotherapeutics will be discussed. 3. Drug delivery potentials of clostridial neurotoxins Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT), though belong to the same class of proteins capable of selectively targeting neuronal cells and have similar modular structures [11e13], tetanus toxin fragments have been extensively studied in drug delivery. Using the B-IIb fragment of tetanus toxin (involved in recognition by the nerve-cell endings) conjugated with, glucose oxidase as marker (150 kDa), the conjugates were taken up by axon terminals and conveyed retrogradely to the spinal-cord motoneurons after intramuscular injections in mice models [14]. Non-toxic TeNT C fragment fused to a human Cu/Zn superoxide dismutase, improved the delivery and bioavailability of cargo to the CNS neurons following intracerebroventricular (i.c.v) administration [15]. Neuronal targeting of Cardiotrophin-1 by coupling with TeNT C fragment promoted motoneuron survival in a dose-dependent manner [16], suggesting its effective delivery. A glial cell line-derived neurotrophic factor (GDNF):TeNT fragment C protein conjugate improved delivery of GDNF to spinal cord motor neurons in mice upon intramuscular administration [17]. These modalities suggest potential prototype of TeNT as a non-viral vehicle for treating spinal cord injury, amyotrophic lateral sclerosis (ALS) or Alzheimer’s disease, and provide minimally invasive tools, which otherwise require extensive and invasive instrumentation for delivering drugs to multiple regions of the affected CNS. It is fairly recognized that non-toxic, neuron-binding TeNT fragment C could be a potential non-viral neuronal delivery system [7]. However, its translation into clinical practice for human therapy is not feasible due to general vaccination in most of the population with tetanus toxoid, and in patients wound injuries, including SCI during their clinical care, since the antibodies would potentially intervene the efficacy as a delivery vehicle. TeNT fragment C mutants lacking immunodominant epitopes has to be explored [18], which may not be a straight forward approach, either. However, by using non-toxic derivatives of BoNT/A, which share similar principles of neuronal binding, internalization and trafficking in many ways, our goals are achievable. Unlike TeNT, there is very less concern of pre-existing immunity against BoNT/A, as botulism is an extremely rare disease and also only occupational workers are vaccinated. Therapeutic doses used for neuromuscular disorder treatments are extremely low, thereby avoiding systemic immune response. Since the structure of BoNT/A is compatible to deliver therapeutic cargoes, BoNT mediated strategy could efficiently deliver drugs to the cytoplasm through its capability to escape across the acidified endosomal vesicles unlike other non-viral strategies, wherein by lack of such mechanisms drugs enter endo-/lysosomal pathway resulting poor transfection efficiencies [7]. Likewise, its retrograde axonal transport and ability to undergo neuronal transcytosis towards second order neurons could be an asset as non-viral drug carrier delivery for several neurological disorders. BoNT based drug delivery would also be useful in circumstances targeting molecules intrinsic to neurons to modulate neuronal properties during chronic neurological conditions that could not be augmented by treating neuronal growth factors, due to lack of appropriate growth factor receptors, like in case of SCI [6]. 4. Structure, function and internalization of botulinum neurotoxin type A (BoNT/A) The family of clostridial neurotoxins (BoNT and TeNT) belong to “AeB” group of toxin by their principal mode of action comprising
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Fig. 1. Schematic diagram of BoNT/A domains. Letters in the boxes specify amino acid residues in single letter code and its position. Yellow, red and green represent the Heavy Chain (HC); both yellow (Hc2 sub-domain) and red (Hc1 sub-domain) represent the Receptor Binding Domain (RBD or Hc) and green represent the Translocation domain (TD or HN). Blue represent the Light Chain (LC).
an enzymatically active component, ‘A’ and cell binding component, ‘B’. BoNTs possess an enzymatically active, 50 kDa light chain (LC) and a 100 kDa heavy chain (HC), linked through a disulfide bond (Fig. 1). HC is composed of two 50 kDa domains; the N-terminal half (HN) involved in translocation, and the C-terminal half (HC) involved in cellular binding. The LC is confined to catalysis only [11,12,19]. As part of their physiological mode of action, BoNTs are generally known to bind to presynaptic cholinergic nerve cells at the peripheral neuromuscular junctions (NMJ) and block acetylcholine release causing flaccid muscle paralysis. The steps involved in cell intoxication can be divided as follows [20]: binding, internalization, membrane translocation and inhibition of neurotransmitter release. Seven different serotypes of BoNTs produced by Clostridium botulinum, designated as A to G, are known to exist, with BoNT/A being the most toxic and the most frequently encountered. BoNT serotype A (BoNT/A) selectively binds to receptors on the surface of presynaptic membrane through the HC, and is internalized via receptor mediated endocytosis. A 25 kDa C-terminal sub-domain HC2 of BoNT/A HC (Figs. 1 and 2), binds to the luminal domain of synaptic vesicle (SV) glycoproteins, SV2A, SV2B and SV2C (isoforms), albeit with highest affinity to the SV2C
isoform, as one of the receptors which are exposed during synaptic vesicle exocytosis [21], while polysialogangliosides, being the other low-affinity receptor facilitating its binding. With gene knockout experiments it was also demonstrated that the SV isoforms seem to also complement their binding, and associated toxicity. Upon acidification of endosomes, HN domain of HC forms a pore or transmembrane channel to translocate the BoNT/A LC into neuronal cytoplasm. The N-terminus of the HN domain, known as the ‘belt’ (Fig. 2) wraps the LC, and has been implicated as a regulatory loop for membrane interaction during translocation [22]. The interchain disulfide bond between the HC and LC is then reduced by the high reduction potential of the cytosol. The presence of the disulfide bond is shown to play a role in the translocation process [11]. There is also a high likelihood that the host cytosolic proteins or translocation protein complexes could play a critical role in the LC translocation process [23], but such mechanisms are not yet characterized in case of any BoNT serotypes. The BoNT/A LC released in the cytoplasm cleaves SNAP-25 (Synaptosome Associated Protein of MW 25 kDa), a soluble NSF-attachment-protein receptor (SNARE) protein in the vesicle recycling machinery, by its zinc dependant endopeptidase activity, thus intervening the process of synaptic vesicle docking and fusion (exocytosis) involved in acetylcholine release at NMJ [11]. BoNTs are known to bind to the inner surface of SV through their protein and ganglioside receptors during SV recycling, and are internalized by vesicle endocytosis [24]. BoNT molecules are taken up preferentially by hyperactive terminals, and indeed, nerve stimulation accelerates BoNT poisoning [25]. LC of other serotypes also blocks neurotransmitter release by endopeptidase activity against other components of SNARE proteins like VAMP and syntaxin [11]. 5. Peripheral and CNS effects of clostridial toxins and their trafficking
Fig. 2. Crystal structure of BoNT/A. Yellow, red and green represent the Heavy Chain (HC); both yellow (Hc2 sub-domain) and red (Hc1 sub-domain) represent the Receptor Binding Domain (RBD or Hc) and green represent the Translocation domain (TD or HN). Blue represent the Light Chain (LC).
BoNTs are known to act on peripheral motor nerve terminals at the local NMJ, after systemic intoxication, to block acetylcholine resulting in flaccid muscle paralysis, the hallmark of botulism disease [11]. But in case of peripheral intoxication of TeNT, a different clinical condition known as tetanus is caused. The difference primarily stems from its distinct trafficking abilities after binding at NMJ following peripheral administration. TeNT molecules after binding and internalization at NMJ are then sorted to retrograde pathway to reach the soma of motor neuron (MN) and transcytosed to inhibitory interneurons of the spinal cord which make synaptic contact with MN [26] via a process likely to involve SV uptake [27]. The blockade of inhibitory circuits in the spinal cord is by cleaving their target SNAREs, VAMP/synaptobrevin [26].
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Interestingly, the lumen of these retrograde compartments where TeNT Hc sort in MN, display a neutral pH. It was proposed that initial sorting in non-acidic, neutral pH compartments in MN might be essential for undergoing retrograde transport and transcytosis to inhibitory neurons, which would otherwise trigger translocation process in MN in case of acidification. Thus TeNT causes spastic paralysis, the hallmark of tetanus disease. Botulism on the other hand involves blockade of acetylcholine release limitedly at the peripheral nerve terminals of NMJ. However, BoNTs, in general and BoNT/A in particular, have been suggested to exert/induce distant biological effects on CNS. Since direct crossing of an intact blood brain barrier (BBB) by BoNTs and TeNT at specific sites do not seem to occur because of their molecular size exclusion and extremely low therapeutic doses, it becomes evident that this could occur by indirect mechanisms, dictated by plastic rearrangements subsequent to denervation or alterations in sensory inputs [18]. In fact, therapeutic injections of BoNT into affected muscles to treat cervical dystonia in human patients relieved them from pain, not by improving muscle contraction, but possibly by such indirect mechanisms [28]. Additionally, studies have also indicated that therapeutic injection of BoNT through peripheral administration induces distant spinal and cortical effects [28]. However, BoNTs are also known to exert their biological effects in vitro directly with central neurons. Entry of BoNTs in central neurons also depends on SV recycling, and leads to cleavage of SNAP-25, blockade of neurotransmitter release in synaptosomes of brain and spinal cords, as well as from brain primary nerve cell cultures [29e32]. In vitro and in vivo animal studies also show that besides its ability to alter glutamate, noradrenaline, dopamine, and glycine transmission, BoNTs may change electrophysiological properties, differentiation, and survival of central neurons [28]. Notably, BoNT/A is also efficiently internalized by the inhibitory terminals of GABAergic neurons, but these terminals are relatively resistant to BoNT/A action, due to lower expression of SNAP-25 [18]. These studies suggest that in addition to indirect mechanisms, distant central effects of BoNT upon its peripheral administration could also be partly explained by direct mechanisms. Several evidences highlight direct mechanism of BoNTs ability to undergo intra-axonal retrograde transport and neuronal transcytosis, like TeNT. Earlier it was found that the toxin was transported to the ventral roots and adjacent spinal cord segments upon intramuscular injection of BoNT/A in cat [29]. Furthermore, BoNT was observable within the axoplasm of myelinated axons in mice upon peripheral injection [25]. In addition to the local effects at the intoxicated synapses, a significant fraction of active BoNT/A is transported intact to the cell soma with involvement of microtubules and released to second order CNS and motoneurons by neuronal transcytosis without being degraded, wherein it cleaves its substrate SNAP-25 [33]. There is compelling evidence that the fraction of BoNT/A that spreads retrogradely in different systems inducing distant effects, depends upon the dose of injected toxin [25]. Other factors that could influence retrograde transport of BoNT/A include length of axons, size of the muscle and density of innervation [25,28]. It is also reported that BoNT/B serotype exhibits retrograde axonal transport, while BoNT/E was not detectable at least in case of hippocampal neurons [25]. Although mechanisms involved in retrograde transport and neuronal transcytosis of BoNT serotypes remain undefined, it is hypothesized that the BoNT/A cargoes could load onto the axonal transport machinery during slow acidification of their endosomal vesicles or by their known association with fibroblast growth factor (FGF) receptor-3 which undergo axonal transport in neurons [25]. In view of some evidences with respect to TeNT, the Hc domain of TeNT (receptor binding domain) seems to be sufficient to enter into retrograde compartments with same transport features as those of full-length
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TeNT [26]. BoNT might also involve similar non-acidic compartments in their retrograde transport which could be determined by their Hc binding domains and expression levels of relevant receptors in the neuronal terminals [33], and may employ a similar uptake mechanism at afferent synapses, as well. 6. Structure of BoNT as an ideal drug delivery vehicle It is very evident that the structural organization of BoNT/A into distinct domains which also shows functionally distinct features, provide distinct opportunities to create chimeric protein fusions of therapeutic value by protein engineering through recombinant DNA technology. Earlier, chemically coupled LHN/A (LC and the HN)nerve growth factor conjugate (NGF-LHN/A) when applied to PC12 cells, significantly inhibited neurotransmitter release and cleaved the type A toxin substrate [34]. Replacement of the native BoNT/A HC with an alternative targeting ligand, wheat germ agglutinin (WGA-LHN/A conjugate) enabled the delivery of endopeptidase domain that cleaved SNAP-25, inhibited neurotransmitter release in cultured neuronal cell types, and also insulin release in pancreatic B-cell line which is normally resistant to BoNT/A [35]. Also chemical conjugates prepared between Erythrina cristagalli lectin and LHN/A had been successfully extended to the development of novel analgesic agents by testing its in vivo efficacy in primary nociceptive afferent neurons of pain models [36]. Similarly, by tailoring the C-terminal HC (Hc) portions of BoNT/A and/E were swapped to create novel toxin chimera, “AE” and “EA” with differing pharmacological properties [37]. This suggests that there is remarkable flexibility to engineer the domains and sub-domains of BoNTs and could be developed them into a drug delivery vehicle to carry different cargoes for to treat many neurological disorders as well. Therapeutic proteins or peptides could be either delivered using full-length catalytically deactivated toxins or heavy chain based chimeric fusions replacing the endopeptidase domain. In such a design (Figs. 1 and 2) the intact receptor binding domain (HC2) would confer neurospecific targeting, while intact translocation domain (HN) containing the belt region, would preserve the competence for channel formation and physical disassociation of the cargo into the neuronal cytosol (when cleaved by host-cellular proteases or by trypsin in vitro). Other structural aspects that ensure the formation of disulfide bridge between the cargo and heavy chain could also be included for efficient internalization and release of the cargo, when the therapeutic cargoes could be expressed as chimeric protein fusions. Catalytically inactive versions derived from full-length neurotoxins had been long viewed as a potential alternative to develop them as DDVs. It is noteworthy that several cargo proteins (luciferase, GFP, or Dihydrofolate reductase) attached to the amino terminus of the full-length botulinum neurotoxin serotype-D has been delivered in neuronal cells without any replacement of its light chain [38]. For therapeutic drug delivery, the light chain is needed to be rendered inactive or replaced. To meet such requirement we have recently developed a catalytically inactive full-length version of rBoNT/A achieved by mutating two amino acid residues [39]. 7. Specific internalization of deactivated recombinant BoNT/A (DrBoNT/A) in human neuroblastoma SH-SY5Y cells We have tested the selective entry of the catalytically deactivated recombinant BoNT/A (DrBoNT/A), into neuronal and non-neuronal cells, using human SH-SY5Y neuroblastoma, rhabdomyosarcoma cells, respectively. Cells treated with fluorescent dye labeled
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Fig. 3. Binding and internalization of DrBoNT/A-Alexa-488 in human neuroblastoma SH-SY5Y and rhabdomyosarcoma cells. Cells were incubated for 2h at 37 C with Alexa-488 labeled DrBoNT/A. Specific labeling of plasma membrane was carried out as per the manufacturer’s instructions by counter staining the cells with Wheat Germ Agglutinin (WGA)Alexa 594 (red). Merged images were obtained from the treatments for SH-SY5Y (Panel A) and human rhabdomyosarcoma cells (Panel B) for further examination under confocal microscope.
DrBoNT/A was examined under laser scanning confocal microscope to study its neuronal specificity and internalization. As shown in Fig. 3, two major observations were made: (i) BoNT/ A selectively binds in neuronal cells compared to rhabdomyosarcoma muscle cells, as revealed by substantially stronger labeling of neuronal cells; (ii) in SH-SY5Y (panel A) the internalization of Alexa-488 labeled DrBoNT/A (green) inside the cell membrane compartment, counterstained by the WGA-Alexa 594 (red) was observed while in the human rhabdomyosarcoma (panel B) the labeled DrBoNT/A is mostly localized to the cell surface without being internalized. Such selectivity is highly useful for neuronal targeting and drug delivery. 8. BoNT/A heavy chain (HCA) based drug delivery Besides using full-length deactivated toxins as delivery vehicle it is also possible to use HCA based drug delivery and neuronal targeting. A notable feature of the BoNT HC being a specific delivery vehicle is its ability to deliver cargo other than its own LC, as demonstrated by heterologous LC delivery [40]. A related TeNT HC has been shown to deliver an entirely different protein (gelonin) into the cytosol [41] and several passenger proteins into lower motor neurons [15,17]. Furthermore, it is also possible to utilize the cysteine sulfhydryl groups based cross-linking chemistry to deliver several peptide based or chemical drugs as well.
linked by a disulfide bond to a drug stimulant, Oregon green 488 (OG488) labeled 10 kDa dextran (Fig. 4). The PDPH linker is bound to one of four possible cysteineÓ sulfhydryl groups on the BoNT/A rHCA (Fig. 4). It is shown attached to Cys-454, which normally participates in the disulfide linkage with the LC. Cy3 and Oregon green 488 are bound to O-amino groups of lysine in the rHCA and dextran, respectively. The dextran is conjugated to the rHCA by a CeN bond in one of the glucose residues. In a functional DDV, multiple drug molecules could be attached to dextran carrier for neuronal delivery.
10. Demonstration of drug-cargo release from DDV via a similar route as BoNT/A To determine the efficacy of delivering the therapeutic compound, we measured the separation of the drug carrier from DDV by Confocal microscopy (Fig. 5). 3 week old mouse spinal cord neuron cultures were treated for 16 h with 200 nM fluorescently labeled DDV at 37 C and then labeled with antiendosome antibody. The confocal image analysis indicated that about 40% of drug carrier components were separated from DDV and diffused into cytosol from endosome in 3 weeks culture. Results also revealed that the separation of the drug from DDV, as well as neuronal function of glycine release, is cell maturation dependent.
9. Development of BoNT/A e heavy chain as a generic drug delivery vehicle Besides developing catalytically deactivated non-toxic form of BoNT/A, we also tested an alternative drug delivery approach, based on heavy chain of BoNT/A system that excludes its native light chain cargo. In our experiments we have recently demonstrated that BoNT/A HC delivered a dextran-dye cargo specifically to the neuronal cytosol [32] as a generic DDV, utilizing the potential of recombinant BoNT/A heavy chain (rHCA), to specifically bind to the presynaptic nerve terminals, and be internalized via endocytosis. The DDV construct was a modification developed by Goodnough et al. [42], consisting of a targeting molecule, Cy3 labeled rHCA,
11. Toxicity assays of catalytically deactivated recombinant BoNT/A and rHCA In order to evaluate the utility of catalytically deactivated BoNT/ A (DrBoNT/A) as a drug delivery vehicle, we assessed their in vivo toxicity by mouse bioassay. Mice injected with 25 pg of BoNT/A (1MLD) did not survive more than 10 h, while all mice survived throughout the observation period even up to 1ug doses (about 100,000 LD50 dose equivalent of BoNT/A) of DrBoNT/A and rHCA (Fig. 6). This suggests the utility of our catalytically deactivated recombinant BoNT/A, and rHCA as a safe drug delivery vehicle (DDV).
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Fig. 4. Schematic representation of the DDV construct with Oregon green dye (Zhang et al., 2009).
12. Trafficking potentials of BoNT/A in drug delivery BoNTs conceivably undergo limited retrograde transport and transynaptic movements compared to that of TeNT [43]. Even limited trafficking of BoNT to the CNS will be an asset in drug
delivery. Availability of catalytically deactivated full-length mutants of BoNT (DrBoNT/A) or HCA, makes it possible to administer BoNT derived drug carriers at much higher concentration than the therapeutic doses of wild type BoNT usually administered in clinics. This would be an important consideration to utilize the
Fig. 5. Confocal images showing separation of the drug cargo from DDV in mouse spinal cord cells. A, red-rHCA: fluorescence elicited at an excitation wavelength of 543 nm; B, green-OG488-dextran: fluorescence elicited at an excitation wavelength of 488 nm; C, bright blue-Alexa 633-endosomes: fluorescence elicited at an excitation wavelength of 632 nm; D, overlay of red and green showing either co-localization (orange) or separation of rHCA and dextran; E, overlay of red and blue showing either the localization (magenta) of rHCA in the endosomes as believed or its release into the cytosol; F, overlay of green and blue showing either localization (light blue or greenish blue) of dextran in the endosomes or its release into the cytosol. Micrographs were obtained on a Bio-Rad 2000 laser confocal microscope using a 100 oil immersion objective. (adopted from Zhang et al., 2009).
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1 µg
Survivial Time (h)
100
1 µg
80 60 40 20
25 pg
magnetic stimulation changes after EDB injections e cortical excitability changes. On the question of whether the animal models used in Caleo studies as well as those carried out previously are appropriate and applicable to human conditions, there are several unsettled issues [45]. (a) Rat and mouse model e differences in response to BoNT/A by these species and in comparison to man; Rat vibrissal pad model e appears to be little used in BoNT/A research and there is a potential for “special” response to BoNT/A. (b) Doses used were high in comparison to human dosage - 60e600 times higher doses than clinical applications. (c) an assumption made e the cleavage product of BoNT/A action has equivalent “transmission” to BoNT/A itself.
0 Fig. 6. In vivo toxicity mouse bioassay for DrBoNT/A and rHCA (Yang et al., 2008). Six weeks old Swiss Webster female mice (n ¼ 5) via was administered intraperitonially with 25 pg (1MLD) BoNT/A, and 1 mg (about 100,000 LD50 dose equivalent of BoNT/A) DrBoNT/A (filled bar) or rHCA (dash bar) for a 96 h observation period.
retrograde and trans-synaptic movement of the BoNT-drug chimera across the nerve to distant sites even when given through minimally invasive intramuscular route of administration. Dose optimization for such circumstances could be however optimized as needed for the clinical set-up. This way the potential of BoNT to deliver drugs for several neurological conditions, exploiting its retrograde transport and transcytosis is yet to be fully tested. Furthermore, understanding the intracellular trafficking of different serotypes of BoNT would be important not only for the expanding potential of BoNTs in clinical therapy, but also for harnessing its utility in drug delivery to treat many other neurological disorders. Our demonstration of BoNT/A heavy chain based drug delivery system [32] provides the proof of principle, which could be further exploited in delivering therapeutic drugs for many neurological conditions including CNS disorders. In addition, availability of non-toxic variants or derivatives of BoNTs would also provide an effective tool to further study their trafficking across the peripheral as well as CNS nerve terminals.
14. Conclusions Drug delivery vehicle to nerves is a need that has drawn attention from biomedical scientists for several decades. BoNTs provide a natural source of delivery vehicle evolved in a billion year old organism, which targets the nervous system specifically and effectively. It is already an FDA approved drug candidate, making it more amenable for clinically approved products. Our demonstration of BoNT/A heavy chain based drug delivery system [32] provides the proof of principle, which could to be further exploited in delivering therapeutic drugs for many neurological conditions. Development of non-toxic variants or derivatives of BoNTs would also pave way as an effective tool to further study their trafficking across the peripheral as well as CNS nerve terminals. BoNT based drug delivery could be also exploited to deliver therapeutic cargoes to treat many CNS disorders upon peripheral administration and can be used as a potential alternative for tetanus based drug delivery.
Acknowledgement This work was supported by the DoD/Army Contract W81XWH08-P-0705 and DARPA Grant GRANT W911NF-07-1-0623.
References 13. Challenges of the BoNT trafficking into central nervous system Retrograde transport of BoNTs could conceivably be a challenging issue, even though such a property could be used to deliver drugs to the CNS. The idea of BoNT entering the CNS, particularly the brain, could have negative repercussions to the commercial use of the toxin for medical and cosmetic uses currently being practiced [31,44,45], especially for patients who use it as cosmetics. This could have significant repercussion to the business, as cosmetic use corresponds to about half of the market. At the Dartmouth Symposium in 2008, Dr. Caleo presented his findings [45], and several issues were clarified. First of all, Caleo himself pointed out that the dose used in the experiments and the animal model employed is not directly relevant to the clinical use of BoNT products in humans. On the question of whether BoNT/A has an effect on the central nervous system, there are several previous reports which have suggested such an effect; Habermann [29], who reported detection in spinal cord after intramuscular injection; Black & Dolly [46], who reported minimal uptake at central nerve terminals; Lange et al. [47], who demonstrated jitter in distal limb after CD (cervical dystonia) treatment; Byrnes et al. [48], who reported changes in corticomotor map topography after writer’s cramp treatment; and Kim et al. [49], who showed transcranial
[1] T. Dua, M.G. Cumbrere, C. Mathers, S. Saxena, Neurological Disorders: Public Health Challenges. World Health Organization, Geneva, Switzerland, 2006, p. 27e39. [2] M.S. Soichet, S.R. Winn, Cell delivery to the central nervous system. Adv. Drug Deliv. Rev. 42 (2000) 81e102. [3] M. Citron, Strategies for disease modifications in Alzheimer’s disease. Nat. Rev. Neurosci. 5 (2004) 677e685. [4] E.H. Lo, T. Dalkara, M.A. Moskowitz, Mechanisms, challenges and opportunities in stroke. Nat. Rev. Neurosci. 4 (2003) 399e414. [5] A. Misra, S. Ganesh, A. Shahiwala, S.P. Shah, Drug delivery to the central nervous system: a review. J. Pharm. Pharmaceut. Sci. 6 (2003) 252e273. [6] S. Thuret, L.D.F. Moon, F.H. Gage, Therapeutic interventions after spinal cord injury. Nat. Rev. Neurosci. 7 (2006) 628e643. [7] J.M. Bergen, S.H. Pun, Analysis of the intracellular barriers encountered by nonviral gene carriers in a model of spatially controlled delivery to neurons. J. Gene Med. 10 (2008) 187e197. [8] J.M. Bergen, I.K. Park, P.J. Horner, S.H. Pun, Nonviral approaches for neuronal delivery of nucleic acids. Pharm. Res. 25 (2008) 983e998. [9] M. Berry, L. Barrett, L. Seymour, A. Baird, A. Logan, Gene therapy for central nervous system repair. Curr. Opin. Mol. Ther. 3 (2001) 338e349. [10] J.S. Suk, J. Suh, S.K. Lai, J. Hanes, Quantifying the intracellular transport of viral and nonviral gene vectors in primary neurons. Exp. Biol. Med. 232 (2007) 461e469. [11] B.R. Singh, Botulinum neurotoxin structure, engineering, and novel cellular trafficking and targeting. Neurotox. Res. 9 (2006) 73e92. [12] B.R. Singh, Intimate details of the most poisonous poison. Nat. Struct. Biol. 7 (2000) 617e619. [13] P. Bernard, M. Popoff, J. Molgó, How do the Botulinum Neurotoxins block neurotransmitter release: from botulism to the molecular mechanism of action. Botulinum J. 1 (2008) 14e87.
B.R. Singh et al. / Biochimie 92 (2010) 1252e1259 [14] P. Beaude, A. Delacour, B. Bizzini, D. Domuado, M.H. Remy, Retrograde axonal transport of an exogenous enzyme covalently linked to B-IIb fragment of tetanus toxin. Biochem. J. 271 (1990) 87e91. [15] S.C. Benn, I. Ay, E. Bastia, R. Chian, S.A. Celia, R.B. Pepinsky, P.S. Fishman, R.H. Brown Jr., J.W. Francis, Tetanus toxin fragment C fusion facilitates protein delivery to CNS neurons from cerebrospinal fluid in mice. J. Neurochem. 95 (2005) 1118e1131. [16] T. Bordet, L.C. Ptakhine, F. Fauchereau, G. Friocourt, A. Kahn, G. Haase, Neuronal targeting of cardiotrophin-1 by coupling with tetanus toxin C fragment. Mol. Cell. Neurosci. 17 (2001) 842e854. [17] K.E. Larsen, S.C. Benn, I. Ay, R.J. Chian, S.A. Celia, M.P. Remington, M. Bejarano, M. Liu, J. Ross, P. Carmillo, D. Sah, K.A. Phillips, D. Sulzer, R.B. Pepinsky, P.S. Fishman, R.H. Brown Jr., J.W. Francis, A glial cell line-derived neurotrophic factor (GDNF): tetanus toxin fragment C protein conjugate improves delivery of GDNF to spinal cord motor neurons in mice. Brain Res. 1120 (2006) 1e12. [18] M. Caleo, G. Schiavo, Central effects of tetanus and botulinum neurotoxins. Toxicon 54 (2009) 593e599. [19] C. Montecucco, G. Schiavo, Structure and function of tetanus and botulinum neurotoxins. Quart. Rev. Biophys. 28 (1995) 423e472. [20] C. Grumelli, C. Verderio, D. Pozzi, O. Rossetto, C. Montecucco, M. Matteoli, Internalization and mechanism of action of clostridial toxins in neurons. Neurotoxicology 26 (2005) 761e767. [21] M. Dong, F. Yeh, W.H. Tepp, C. Dean, E.A. Johnson, R. Janz, E.R. Chapman, SV2 is the protein receptor for botulinum neurotoxin A. Science 312 (2006) 592e596. [22] M. Galloux, H. Vitrac, C. Montagner, S. Raffestin, M.R. Popoff, A. Chenal, V. Forge, D. Gillet, Membrane interaction of botulinum neurotoxin A T domain: the belt region is a regulatory loop for membrane interaction. J. Biol. Chem. 283 (2008) 27668e27676. [23] R. Ratts, C. Trujillo, A. Bharti, J. VanderSpek, R. Harrison, J.R. Murphy, A conserved motif in transmembrane helix 1 of diphtheria toxin mediates catalytic domain delivery to the cytosol. Proc. Natl. Acad. Sci. U S A 102 (2005) 15635e15640. [24] C. Verderio, O. Rossetto, C. Grumelli, C. Frassoni, C. Montecucco, M. Matteoli, Entering neurons: botulinum toxins and synaptic vesicle recycling. EMBO Rep. 7 (2006) 995e999. [25] M. Caleo, F. Antonucci, L. Restani, R. Mazzocchio, A reappraisal of the central effects of botulinum neurotoxin type A: by what mechanism? J. Neurochem. 109 (2009) 15e24. [26] G. Lalli, S. Bohnert, K. Deinhardt, C. Verastegui, G. Schiavo, The journey of tetanus and botulinum neurotoxins in neurons. Trends. Microbiol. 11 (2003) 431e437. [27] M. Matteoli, C. Verderio, O. Rossetto, N. Iezzi, S. Coco, G. Schiavo, C. Montecucco, Synaptic vesicle endocytosis mediates the entry of tetanus neurotoxin into hippocampal neurons. Proc. Natl. Acad. Sci. U S A 93 (1996) 13310e13315. [28] A. Curra, A. Berardelli, Do the unintended actions of botulinum toxin at distant sites have clinical implications? Neurology 72 (2009) 1095e1099. [29] E. Habermann, 125 I-labeled neurotoxin from Clostridium botulinum A: preparation, binding to synaptosomes and ascent to the spinal cord. Naunyn Schmiedeberg’s Arch. Pharmacol. 281 (1974) 47e56. [30] H. Bigalke, I. Heller, B. Bizzini, E. Habermann, Tetanus toxin and botulinum A toxin inhibit release and uptake of various transmitters, as studied with particulate preparations from rat brain and spinal cord. Naunyn Schmiedeberg’s Arch. Pharmacol. 316 (1981) 244e251. [31] S.K. Sharma, B.R. Singh, Botulinum toxin in neurological diseases. Saudi Arab. J. Rehab. 10 (2004) 111e117. [32] P. Zhang, R. Ray, B.R. Singh, D. Li, M. Adler, P. Ray, An efficient drug delivery vehicle for botulism countermeasure. BMC Pharmacol. 9 (2009) 12. [33] F. Antonucci, C. Rossi, L. Gianfranceschi, O. Rossetto, M. Caleo, Long-distance retrograde effects of botulinum neurotoxin A. J. Neurosci. 28 (2008) 3689e3696.
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[34] J.A. Chaddock, J.R. Purkiss, M.J. Duggan, C.P. Quinn, C.C. Shone, K.A. Foster, A conjugate composed of nerve growth factor coupled to a nontoxic derivative of Clostridium botulinum neurotoxin type A can inhibit neurotransmitter release in vitro. Growth Factors 18 (2000a) 147e155. [35] J.A. Chaddock, J.R. Purkis, L.M. Friis, M.J. Broadbridge, M.J. Duggan, S.J. Fooks, C.C. Shone, C.P. Quinn, K.A. Foster, Inhibition of vesicular secretion in both neuronal and non-neuronal cells by a retargeted endopeptidase derivative of Clostridium botulinum neurotoxin type A. Infect. Immun. 68 (2000b) 2587e2593. [36] J.A. Chaddock, M.J. Duggan, C.P. Quinn, J.R. Purkiss, F.C.G. Alexander, S. Doward, S.J. Fooks, L. Friis, Y. Hall, E.R. Kirby, N.J. Leeds, H.J. Moulsdale, A. Dickenson, G.M. Green, W. Rahman, R. Suzuki, C.C. Shone, K.A. Foster, Retargeted clostridial endopeptidases: inhibition of nociceptive neurotransmitter release in vitro, and antinociceptive activity in in vivo models of pain. Mov. Disord. 19 (Suppl. 8) (2004) S42eS47. [37] J. Wang, J. Meng, G.W. Lawrence, T.H. Zurawski, A. Sasse, M.O. Bodeker, M.A. Gilmore, E. Fernández-Salas, J. Francis, L.E. Steward, K.R. Aoki, J.O. Dolly, Novel chimeras of botulinum neurotoxins A and E unveil contributions from the binding, translocation, and protease domains to their functional characteristics. J. Biol. Chem. 283 (2008) 16993e17002. [38] S. Bade, A. Rummel, C. Reisinger, T. Karnath, G. A-Hilger, H. Bigalke, T. Binz, Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates. J. Neurochem. 91 (2004) 461e472. [39] W. Yang, P. Lindo, S. Riding, T.W. Chang, S. Cai, T. Van, R. Kukreja, Y. Zhou, K. Vasa, B.R. Singh, Expression, purification and comparative characterization of deactivated recombinant botulinum neurotoxin type A. Botulinum J. 1 (2008) 219e241. [40] S. Bandyopadhyay, A.W. Clark, B.R. Das Gupta, V. Sathyamoorthy, Role of the heavy and light chains of botulinum neurotoxin in neuromuscular paralysis. J. Biol. Chem. 262 (1987) 2660e2663. [41] S.R. Johnstone, L.M. Morrice, S. V-Heyningen, The heavy chain of tetanus toxin can mediate the entry of cytotoxic gelonin into intact cells. FEBS Lett. 265 (1990) 101e103. [42] M.C. Goodnough, G. Oyler, P.S. Fishman, E.A. Johnson, E.A. Neale, J.E. Keller, W.H. Tepp, M. Clark, S. Hartz, M. Adler, Development of a delivery vehicle for intracellular transport of botulinum neurotoxin antagonists. FEBS Lett. 513 (2002) 163e168. [43] B.M. Kuehn, Studies, reports say botulinum toxins may have effects beyond injection site. JAMA 299 (2008) 2261e2263. [44] S.S. Arnon, R. Schechter, T.V. Inglesby, D.A. Henderson, J.G. Bartlett, M.S. Ascher, E. Eitzen, A.D. Fine, J. Hauer, M. Layton, S. Lillibridge, M.T. Osterholm, T. O’Toole, G. Parker, T.M. Perl, P.K. Russell, D.L. Swerdlow, K. Tonat, Botulinum toxin as a biological weapon: medical and public health management. JAMA 285 (2001) 1059e1070. [45] B.R. Singh, The second annual Dartmouth Botulinum Research Symposium discusses the effect of botulinum neurotoxin on the central nervous system. Botulinum J. 1 (2009) 340e346. [46] J.D. Black, J.O. Dolly, Selective location of acceptors for botulinum neurotoxin A in the central and peripheral nervous systems. Neuroscience 23 (1987) 767e779. [47] D.J. Lange, M. Rubin, P.E. Greene, U.J. Kang, C.B. Moskowitz, M.F. Brin, R.E. Lovelace, S. Fahn, Distant effects of locally injected botulinum toxin: a double-blind study of single fiber EMG changes. Muscle Nerve 14 (1991) 672e675. [48] M.L. Byrnes, G.W. Thickbroom, S.A. Wilson, P. Sacco, J.M. Shipman, R. Stell, F.L. Mastaglia, The corticomotor representation of upper limb muscles in writer’s cramp and changes following Botulinum toxin injection. Brain 121 (1998) 977e988. [49] D.Y. Kim, B.M. Oh, N.J. Paik, Central effect of Botulinum toxin type A in humans. Int. J. Neurosci. 116 (2006) 667e680.
Contents lists available at ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Review
Clostridial neurotoxins as a drug delivery vehicle targeting nervous system Bal Ram Singh a, *, Nagarajan Thirunavukkarasu a, Koyel Ghosal a, Easwaran Ravichandran a, Roshan Kukreja a, Shuowei Cai a, Peng Zhang b, *, Radharaman Ray c, *, Prabhati Ray b, * a
National Botulinum Research Center, and Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, Dartmouth, MA 02747, USA Walter Reed Army Institute of Research, Silver Spring, Maryland, USA c United States Army Medical Research Institute of Chemical Defense, APG, Maryland, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2009 Accepted 9 March 2010 Available online 24 March 2010
Several neuronal disorders require drug treatment using drug delivery systems for specific delivery of the drugs for the targeted tissues, both at the peripheral and central nervous system levels. We describe a review of information currently available on the potential use of appropriate domains of clostridial neurotoxins, tetanus and botulinum, for effective drug delivery to neuronal systems. While both tetanus and botulinum neurotoxins are capable of delivering drugs the neuronal cells, tetanus neurotoxin is limited in clinical use because of general immunization of population against tetanus. Botulinum neurotoxin which is also being used as a therapeutic reagent has strong potential for drug delivery to nervous tissues. Ó 2010 Published by Elsevier Masson SAS.
Keywords: Botulinum neurotoxins Drug delivery vehicles Neuronal disorders Non-viral drug delivery
1. Introduction Drug delivery to neuronal cells is an unmet need, while neurological disorders affect around one billion people worldwide, and is the cause of 12% of total deaths [1]; with more than 20 million people in the United States alone, accounting for over $400 billion annually for their treatment and prolonged care [2]. Over 12 million people around the world suffer from Alzheimer’s disease [3] and stroke is the third leading cause of death in the western world [4]. Despite aggressive research, patients suffering from fatal and/or debilitating CNS diseases and neurodegenerative disorders far outnumber those dying of all types of systemic cancer or heart disease [5]. With the increase in the world population of aged people, there are several age-related diseases, most notably neuronal disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), epilepsy, multiple Sclerosis (MS), Huntington’s disease (HD) and Lou Gehrig’s disease (or amyotrophic lateral sclerosis, ALS). These are potentially debilitating, life threatening and more importantly affect the quality of life. Another disabling condition which mostly victimizes young healthy people in their ripe productive years with devastating social emotional psychological and life style effects is spinal cord injury. Neurological conditions such as AD, PD, HD epilepsy, or stroke are normally caused by biochemical malfunctioning within neuronal cells of central and/or peripheral nervous system. Also worldwide, an * Corresponding authors. Tel.: þ1 508 999 8588; fax: þ1 508 999 8451. E-mail address: [email protected] (B.R. Singh). 0300-9084/$ e see front matter Ó 2010 Published by Elsevier Masson SAS. doi:10.1016/j.biochi.2010.03.005
estimated 2.5 million people live with spinal cord injury (SCI), with more than 130,000 new injuries reported each year [6]. SCI significantly affect the quality of life, life expectancy, and economic burden, with considerable costs associated with primary care and loss of income. Therapeutic options for these diseases which are progressive are very limited. The clinical failure of potentially effective therapeutics is often not due to lack of potency but rather to the shortcomings in the drug delivery methods [5]. While understanding the molecular basis of these diseases is allowing design and development of effective drugs, their delivery into nerves, particularly those of the central nervous system is a major impediment in the development of effective neurotherapeutics. We have recently developed a nontoxic version of the world’s most toxic neurotoxin, the botulinum neurotoxin (BoNT), which could be utilized as drug delivery vehicle (DDV) to target nerves due to their highly specific and selective binding to neurons. We review here the characteristics of the BoNTbased targeted drug delivery system for neuronal cells which could benefit an array of neurological disorders. 2. Limitations of present drug delivery strategies and other alternatives Although the therapeutic potential of several neurological drugs and targets is currently being studied and established in many cases, there are only limited opportunities considering suitable methods and strategies to deliver them, since neuronal cells are highly polarized cells with somal, axonal, and dendritic domains,
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having distinct membrane compositions. Some limitations and issues with the existing methods are discussed below: a) Non-Specific Delivery and toxicity: In many neurological conditions, targeting neuronal cells is essentially important to reduce toxicity of the drug or to augment the desired therapeutic effect. For e.g., in case of spinal cord injury, neuronal regeneration in vivo, does not rely only on neuronal properties. All three major types of glial cells have an impact on neuronal function under normal conditions, and in determining the success of repair mechanisms in pathological circumstances of CNS. However, this synergistic relationship could be perturbed when the drug also exerts its activity in cells like astrocytes and other glial cells of CNS. Therefore, there is critical need to develop neuron selective drug delivery system. Indeed, it is increasingly recognized to incorporate neuron-targeting ligands for better transfection efficiencies, and reduce toxicity due to widespread entry into off-target cells, in the development of non-viral mediated neuronal drug delivery system [7]. b) Route of delivery: Techniques employing molecular therapies for several CNS disorders include intracerebroventricular, intrathecal or intraspinal injection, continuous infusion or topical application of a drug carrier saturated with the molecule of interest, although minimally invasive or traumatic routes are preferable. Among less invasive routes, intramuscular administration using retrograde transporting carriers are of extreme interest for use in neurotherapeutics associated with CNS pathologies [7]. c) Limitations in other viral and non-viral choice of carriers: Several viral and non-viral carrier based gene delivery methods have been described in literature as promising and clinically feasible for CNS neuronal delivery. However, only few have been successfully applied towards animal models of neurological disease or injury [7]. In vivo gene therapy has been tested in models of SCI using viruses, including herpes simplex virus (HSV), adenovirus, Adeno-Associated Virus (AAV), lentivirus and Moloney leukaemia virus. Injection of HSV into mammalian tissue elicited a local immune response and in case of AAV and the clinical trials was delayed due to production of neutralizing antibodies in humans possibly by previous exposure, and also often encounters safety issues [8]. Currently existing non-viral carrier approaches for neuronal gene delivery suffer from low transfection efficiencies and/or toxicity issues compared to that of neurotropic viral deliveries, as the former can associate with most cell types via non-specific binding to the cell surface. Their internalization also varies depending on the neuronal cell type, size, charge, surface composition, the delivery site, and the specific vehicle formulation. Delivering sufficient quantities to have an effect at the targeted site is often difficult. Limitations in controlling the expression levels or durations of the therapeutic transgene, resulting in suboptimal dosing or over dosing had been indicated in different studies [8]. Polyethylenimine (PEI)/DNA polyplexes, and cationic lipid based lipoplex-mediated deliveries, which were tested on neurons, were shown to aggregate in biological fluids [9]; PEI even exhibited cellular toxicity, and reduced uptake in neuron-like cells as compared to that of undifferentiated cells. Polyplexes and lipoplexes when delivered to neurite terminals undergo internalization in the vesicles that are part of endo-/lysosomal pathway [7] and their inability to escape from the acidification of endosomal compartments in neurons partially explains their poor transfection efficiencies compared to viruses [10]. Therefore, is a great necessity to develop an efficient, safe neuronal delivery system to augment neurotherapeutics in general
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and henceforth the utility of neurotoxin based drug delivery strategies in fulfilling some of these requirements in neurotherapeutics will be discussed. 3. Drug delivery potentials of clostridial neurotoxins Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT), though belong to the same class of proteins capable of selectively targeting neuronal cells and have similar modular structures [11e13], tetanus toxin fragments have been extensively studied in drug delivery. Using the B-IIb fragment of tetanus toxin (involved in recognition by the nerve-cell endings) conjugated with, glucose oxidase as marker (150 kDa), the conjugates were taken up by axon terminals and conveyed retrogradely to the spinal-cord motoneurons after intramuscular injections in mice models [14]. Non-toxic TeNT C fragment fused to a human Cu/Zn superoxide dismutase, improved the delivery and bioavailability of cargo to the CNS neurons following intracerebroventricular (i.c.v) administration [15]. Neuronal targeting of Cardiotrophin-1 by coupling with TeNT C fragment promoted motoneuron survival in a dose-dependent manner [16], suggesting its effective delivery. A glial cell line-derived neurotrophic factor (GDNF):TeNT fragment C protein conjugate improved delivery of GDNF to spinal cord motor neurons in mice upon intramuscular administration [17]. These modalities suggest potential prototype of TeNT as a non-viral vehicle for treating spinal cord injury, amyotrophic lateral sclerosis (ALS) or Alzheimer’s disease, and provide minimally invasive tools, which otherwise require extensive and invasive instrumentation for delivering drugs to multiple regions of the affected CNS. It is fairly recognized that non-toxic, neuron-binding TeNT fragment C could be a potential non-viral neuronal delivery system [7]. However, its translation into clinical practice for human therapy is not feasible due to general vaccination in most of the population with tetanus toxoid, and in patients wound injuries, including SCI during their clinical care, since the antibodies would potentially intervene the efficacy as a delivery vehicle. TeNT fragment C mutants lacking immunodominant epitopes has to be explored [18], which may not be a straight forward approach, either. However, by using non-toxic derivatives of BoNT/A, which share similar principles of neuronal binding, internalization and trafficking in many ways, our goals are achievable. Unlike TeNT, there is very less concern of pre-existing immunity against BoNT/A, as botulism is an extremely rare disease and also only occupational workers are vaccinated. Therapeutic doses used for neuromuscular disorder treatments are extremely low, thereby avoiding systemic immune response. Since the structure of BoNT/A is compatible to deliver therapeutic cargoes, BoNT mediated strategy could efficiently deliver drugs to the cytoplasm through its capability to escape across the acidified endosomal vesicles unlike other non-viral strategies, wherein by lack of such mechanisms drugs enter endo-/lysosomal pathway resulting poor transfection efficiencies [7]. Likewise, its retrograde axonal transport and ability to undergo neuronal transcytosis towards second order neurons could be an asset as non-viral drug carrier delivery for several neurological disorders. BoNT based drug delivery would also be useful in circumstances targeting molecules intrinsic to neurons to modulate neuronal properties during chronic neurological conditions that could not be augmented by treating neuronal growth factors, due to lack of appropriate growth factor receptors, like in case of SCI [6]. 4. Structure, function and internalization of botulinum neurotoxin type A (BoNT/A) The family of clostridial neurotoxins (BoNT and TeNT) belong to “AeB” group of toxin by their principal mode of action comprising
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Fig. 1. Schematic diagram of BoNT/A domains. Letters in the boxes specify amino acid residues in single letter code and its position. Yellow, red and green represent the Heavy Chain (HC); both yellow (Hc2 sub-domain) and red (Hc1 sub-domain) represent the Receptor Binding Domain (RBD or Hc) and green represent the Translocation domain (TD or HN). Blue represent the Light Chain (LC).
an enzymatically active component, ‘A’ and cell binding component, ‘B’. BoNTs possess an enzymatically active, 50 kDa light chain (LC) and a 100 kDa heavy chain (HC), linked through a disulfide bond (Fig. 1). HC is composed of two 50 kDa domains; the N-terminal half (HN) involved in translocation, and the C-terminal half (HC) involved in cellular binding. The LC is confined to catalysis only [11,12,19]. As part of their physiological mode of action, BoNTs are generally known to bind to presynaptic cholinergic nerve cells at the peripheral neuromuscular junctions (NMJ) and block acetylcholine release causing flaccid muscle paralysis. The steps involved in cell intoxication can be divided as follows [20]: binding, internalization, membrane translocation and inhibition of neurotransmitter release. Seven different serotypes of BoNTs produced by Clostridium botulinum, designated as A to G, are known to exist, with BoNT/A being the most toxic and the most frequently encountered. BoNT serotype A (BoNT/A) selectively binds to receptors on the surface of presynaptic membrane through the HC, and is internalized via receptor mediated endocytosis. A 25 kDa C-terminal sub-domain HC2 of BoNT/A HC (Figs. 1 and 2), binds to the luminal domain of synaptic vesicle (SV) glycoproteins, SV2A, SV2B and SV2C (isoforms), albeit with highest affinity to the SV2C
isoform, as one of the receptors which are exposed during synaptic vesicle exocytosis [21], while polysialogangliosides, being the other low-affinity receptor facilitating its binding. With gene knockout experiments it was also demonstrated that the SV isoforms seem to also complement their binding, and associated toxicity. Upon acidification of endosomes, HN domain of HC forms a pore or transmembrane channel to translocate the BoNT/A LC into neuronal cytoplasm. The N-terminus of the HN domain, known as the ‘belt’ (Fig. 2) wraps the LC, and has been implicated as a regulatory loop for membrane interaction during translocation [22]. The interchain disulfide bond between the HC and LC is then reduced by the high reduction potential of the cytosol. The presence of the disulfide bond is shown to play a role in the translocation process [11]. There is also a high likelihood that the host cytosolic proteins or translocation protein complexes could play a critical role in the LC translocation process [23], but such mechanisms are not yet characterized in case of any BoNT serotypes. The BoNT/A LC released in the cytoplasm cleaves SNAP-25 (Synaptosome Associated Protein of MW 25 kDa), a soluble NSF-attachment-protein receptor (SNARE) protein in the vesicle recycling machinery, by its zinc dependant endopeptidase activity, thus intervening the process of synaptic vesicle docking and fusion (exocytosis) involved in acetylcholine release at NMJ [11]. BoNTs are known to bind to the inner surface of SV through their protein and ganglioside receptors during SV recycling, and are internalized by vesicle endocytosis [24]. BoNT molecules are taken up preferentially by hyperactive terminals, and indeed, nerve stimulation accelerates BoNT poisoning [25]. LC of other serotypes also blocks neurotransmitter release by endopeptidase activity against other components of SNARE proteins like VAMP and syntaxin [11]. 5. Peripheral and CNS effects of clostridial toxins and their trafficking
Fig. 2. Crystal structure of BoNT/A. Yellow, red and green represent the Heavy Chain (HC); both yellow (Hc2 sub-domain) and red (Hc1 sub-domain) represent the Receptor Binding Domain (RBD or Hc) and green represent the Translocation domain (TD or HN). Blue represent the Light Chain (LC).
BoNTs are known to act on peripheral motor nerve terminals at the local NMJ, after systemic intoxication, to block acetylcholine resulting in flaccid muscle paralysis, the hallmark of botulism disease [11]. But in case of peripheral intoxication of TeNT, a different clinical condition known as tetanus is caused. The difference primarily stems from its distinct trafficking abilities after binding at NMJ following peripheral administration. TeNT molecules after binding and internalization at NMJ are then sorted to retrograde pathway to reach the soma of motor neuron (MN) and transcytosed to inhibitory interneurons of the spinal cord which make synaptic contact with MN [26] via a process likely to involve SV uptake [27]. The blockade of inhibitory circuits in the spinal cord is by cleaving their target SNAREs, VAMP/synaptobrevin [26].
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Interestingly, the lumen of these retrograde compartments where TeNT Hc sort in MN, display a neutral pH. It was proposed that initial sorting in non-acidic, neutral pH compartments in MN might be essential for undergoing retrograde transport and transcytosis to inhibitory neurons, which would otherwise trigger translocation process in MN in case of acidification. Thus TeNT causes spastic paralysis, the hallmark of tetanus disease. Botulism on the other hand involves blockade of acetylcholine release limitedly at the peripheral nerve terminals of NMJ. However, BoNTs, in general and BoNT/A in particular, have been suggested to exert/induce distant biological effects on CNS. Since direct crossing of an intact blood brain barrier (BBB) by BoNTs and TeNT at specific sites do not seem to occur because of their molecular size exclusion and extremely low therapeutic doses, it becomes evident that this could occur by indirect mechanisms, dictated by plastic rearrangements subsequent to denervation or alterations in sensory inputs [18]. In fact, therapeutic injections of BoNT into affected muscles to treat cervical dystonia in human patients relieved them from pain, not by improving muscle contraction, but possibly by such indirect mechanisms [28]. Additionally, studies have also indicated that therapeutic injection of BoNT through peripheral administration induces distant spinal and cortical effects [28]. However, BoNTs are also known to exert their biological effects in vitro directly with central neurons. Entry of BoNTs in central neurons also depends on SV recycling, and leads to cleavage of SNAP-25, blockade of neurotransmitter release in synaptosomes of brain and spinal cords, as well as from brain primary nerve cell cultures [29e32]. In vitro and in vivo animal studies also show that besides its ability to alter glutamate, noradrenaline, dopamine, and glycine transmission, BoNTs may change electrophysiological properties, differentiation, and survival of central neurons [28]. Notably, BoNT/A is also efficiently internalized by the inhibitory terminals of GABAergic neurons, but these terminals are relatively resistant to BoNT/A action, due to lower expression of SNAP-25 [18]. These studies suggest that in addition to indirect mechanisms, distant central effects of BoNT upon its peripheral administration could also be partly explained by direct mechanisms. Several evidences highlight direct mechanism of BoNTs ability to undergo intra-axonal retrograde transport and neuronal transcytosis, like TeNT. Earlier it was found that the toxin was transported to the ventral roots and adjacent spinal cord segments upon intramuscular injection of BoNT/A in cat [29]. Furthermore, BoNT was observable within the axoplasm of myelinated axons in mice upon peripheral injection [25]. In addition to the local effects at the intoxicated synapses, a significant fraction of active BoNT/A is transported intact to the cell soma with involvement of microtubules and released to second order CNS and motoneurons by neuronal transcytosis without being degraded, wherein it cleaves its substrate SNAP-25 [33]. There is compelling evidence that the fraction of BoNT/A that spreads retrogradely in different systems inducing distant effects, depends upon the dose of injected toxin [25]. Other factors that could influence retrograde transport of BoNT/A include length of axons, size of the muscle and density of innervation [25,28]. It is also reported that BoNT/B serotype exhibits retrograde axonal transport, while BoNT/E was not detectable at least in case of hippocampal neurons [25]. Although mechanisms involved in retrograde transport and neuronal transcytosis of BoNT serotypes remain undefined, it is hypothesized that the BoNT/A cargoes could load onto the axonal transport machinery during slow acidification of their endosomal vesicles or by their known association with fibroblast growth factor (FGF) receptor-3 which undergo axonal transport in neurons [25]. In view of some evidences with respect to TeNT, the Hc domain of TeNT (receptor binding domain) seems to be sufficient to enter into retrograde compartments with same transport features as those of full-length
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TeNT [26]. BoNT might also involve similar non-acidic compartments in their retrograde transport which could be determined by their Hc binding domains and expression levels of relevant receptors in the neuronal terminals [33], and may employ a similar uptake mechanism at afferent synapses, as well. 6. Structure of BoNT as an ideal drug delivery vehicle It is very evident that the structural organization of BoNT/A into distinct domains which also shows functionally distinct features, provide distinct opportunities to create chimeric protein fusions of therapeutic value by protein engineering through recombinant DNA technology. Earlier, chemically coupled LHN/A (LC and the HN)nerve growth factor conjugate (NGF-LHN/A) when applied to PC12 cells, significantly inhibited neurotransmitter release and cleaved the type A toxin substrate [34]. Replacement of the native BoNT/A HC with an alternative targeting ligand, wheat germ agglutinin (WGA-LHN/A conjugate) enabled the delivery of endopeptidase domain that cleaved SNAP-25, inhibited neurotransmitter release in cultured neuronal cell types, and also insulin release in pancreatic B-cell line which is normally resistant to BoNT/A [35]. Also chemical conjugates prepared between Erythrina cristagalli lectin and LHN/A had been successfully extended to the development of novel analgesic agents by testing its in vivo efficacy in primary nociceptive afferent neurons of pain models [36]. Similarly, by tailoring the C-terminal HC (Hc) portions of BoNT/A and/E were swapped to create novel toxin chimera, “AE” and “EA” with differing pharmacological properties [37]. This suggests that there is remarkable flexibility to engineer the domains and sub-domains of BoNTs and could be developed them into a drug delivery vehicle to carry different cargoes for to treat many neurological disorders as well. Therapeutic proteins or peptides could be either delivered using full-length catalytically deactivated toxins or heavy chain based chimeric fusions replacing the endopeptidase domain. In such a design (Figs. 1 and 2) the intact receptor binding domain (HC2) would confer neurospecific targeting, while intact translocation domain (HN) containing the belt region, would preserve the competence for channel formation and physical disassociation of the cargo into the neuronal cytosol (when cleaved by host-cellular proteases or by trypsin in vitro). Other structural aspects that ensure the formation of disulfide bridge between the cargo and heavy chain could also be included for efficient internalization and release of the cargo, when the therapeutic cargoes could be expressed as chimeric protein fusions. Catalytically inactive versions derived from full-length neurotoxins had been long viewed as a potential alternative to develop them as DDVs. It is noteworthy that several cargo proteins (luciferase, GFP, or Dihydrofolate reductase) attached to the amino terminus of the full-length botulinum neurotoxin serotype-D has been delivered in neuronal cells without any replacement of its light chain [38]. For therapeutic drug delivery, the light chain is needed to be rendered inactive or replaced. To meet such requirement we have recently developed a catalytically inactive full-length version of rBoNT/A achieved by mutating two amino acid residues [39]. 7. Specific internalization of deactivated recombinant BoNT/A (DrBoNT/A) in human neuroblastoma SH-SY5Y cells We have tested the selective entry of the catalytically deactivated recombinant BoNT/A (DrBoNT/A), into neuronal and non-neuronal cells, using human SH-SY5Y neuroblastoma, rhabdomyosarcoma cells, respectively. Cells treated with fluorescent dye labeled
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Fig. 3. Binding and internalization of DrBoNT/A-Alexa-488 in human neuroblastoma SH-SY5Y and rhabdomyosarcoma cells. Cells were incubated for 2h at 37 C with Alexa-488 labeled DrBoNT/A. Specific labeling of plasma membrane was carried out as per the manufacturer’s instructions by counter staining the cells with Wheat Germ Agglutinin (WGA)Alexa 594 (red). Merged images were obtained from the treatments for SH-SY5Y (Panel A) and human rhabdomyosarcoma cells (Panel B) for further examination under confocal microscope.
DrBoNT/A was examined under laser scanning confocal microscope to study its neuronal specificity and internalization. As shown in Fig. 3, two major observations were made: (i) BoNT/ A selectively binds in neuronal cells compared to rhabdomyosarcoma muscle cells, as revealed by substantially stronger labeling of neuronal cells; (ii) in SH-SY5Y (panel A) the internalization of Alexa-488 labeled DrBoNT/A (green) inside the cell membrane compartment, counterstained by the WGA-Alexa 594 (red) was observed while in the human rhabdomyosarcoma (panel B) the labeled DrBoNT/A is mostly localized to the cell surface without being internalized. Such selectivity is highly useful for neuronal targeting and drug delivery. 8. BoNT/A heavy chain (HCA) based drug delivery Besides using full-length deactivated toxins as delivery vehicle it is also possible to use HCA based drug delivery and neuronal targeting. A notable feature of the BoNT HC being a specific delivery vehicle is its ability to deliver cargo other than its own LC, as demonstrated by heterologous LC delivery [40]. A related TeNT HC has been shown to deliver an entirely different protein (gelonin) into the cytosol [41] and several passenger proteins into lower motor neurons [15,17]. Furthermore, it is also possible to utilize the cysteine sulfhydryl groups based cross-linking chemistry to deliver several peptide based or chemical drugs as well.
linked by a disulfide bond to a drug stimulant, Oregon green 488 (OG488) labeled 10 kDa dextran (Fig. 4). The PDPH linker is bound to one of four possible cysteineÓ sulfhydryl groups on the BoNT/A rHCA (Fig. 4). It is shown attached to Cys-454, which normally participates in the disulfide linkage with the LC. Cy3 and Oregon green 488 are bound to O-amino groups of lysine in the rHCA and dextran, respectively. The dextran is conjugated to the rHCA by a CeN bond in one of the glucose residues. In a functional DDV, multiple drug molecules could be attached to dextran carrier for neuronal delivery.
10. Demonstration of drug-cargo release from DDV via a similar route as BoNT/A To determine the efficacy of delivering the therapeutic compound, we measured the separation of the drug carrier from DDV by Confocal microscopy (Fig. 5). 3 week old mouse spinal cord neuron cultures were treated for 16 h with 200 nM fluorescently labeled DDV at 37 C and then labeled with antiendosome antibody. The confocal image analysis indicated that about 40% of drug carrier components were separated from DDV and diffused into cytosol from endosome in 3 weeks culture. Results also revealed that the separation of the drug from DDV, as well as neuronal function of glycine release, is cell maturation dependent.
9. Development of BoNT/A e heavy chain as a generic drug delivery vehicle Besides developing catalytically deactivated non-toxic form of BoNT/A, we also tested an alternative drug delivery approach, based on heavy chain of BoNT/A system that excludes its native light chain cargo. In our experiments we have recently demonstrated that BoNT/A HC delivered a dextran-dye cargo specifically to the neuronal cytosol [32] as a generic DDV, utilizing the potential of recombinant BoNT/A heavy chain (rHCA), to specifically bind to the presynaptic nerve terminals, and be internalized via endocytosis. The DDV construct was a modification developed by Goodnough et al. [42], consisting of a targeting molecule, Cy3 labeled rHCA,
11. Toxicity assays of catalytically deactivated recombinant BoNT/A and rHCA In order to evaluate the utility of catalytically deactivated BoNT/ A (DrBoNT/A) as a drug delivery vehicle, we assessed their in vivo toxicity by mouse bioassay. Mice injected with 25 pg of BoNT/A (1MLD) did not survive more than 10 h, while all mice survived throughout the observation period even up to 1ug doses (about 100,000 LD50 dose equivalent of BoNT/A) of DrBoNT/A and rHCA (Fig. 6). This suggests the utility of our catalytically deactivated recombinant BoNT/A, and rHCA as a safe drug delivery vehicle (DDV).
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Fig. 4. Schematic representation of the DDV construct with Oregon green dye (Zhang et al., 2009).
12. Trafficking potentials of BoNT/A in drug delivery BoNTs conceivably undergo limited retrograde transport and transynaptic movements compared to that of TeNT [43]. Even limited trafficking of BoNT to the CNS will be an asset in drug
delivery. Availability of catalytically deactivated full-length mutants of BoNT (DrBoNT/A) or HCA, makes it possible to administer BoNT derived drug carriers at much higher concentration than the therapeutic doses of wild type BoNT usually administered in clinics. This would be an important consideration to utilize the
Fig. 5. Confocal images showing separation of the drug cargo from DDV in mouse spinal cord cells. A, red-rHCA: fluorescence elicited at an excitation wavelength of 543 nm; B, green-OG488-dextran: fluorescence elicited at an excitation wavelength of 488 nm; C, bright blue-Alexa 633-endosomes: fluorescence elicited at an excitation wavelength of 632 nm; D, overlay of red and green showing either co-localization (orange) or separation of rHCA and dextran; E, overlay of red and blue showing either the localization (magenta) of rHCA in the endosomes as believed or its release into the cytosol; F, overlay of green and blue showing either localization (light blue or greenish blue) of dextran in the endosomes or its release into the cytosol. Micrographs were obtained on a Bio-Rad 2000 laser confocal microscope using a 100 oil immersion objective. (adopted from Zhang et al., 2009).
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1 µg
Survivial Time (h)
100
1 µg
80 60 40 20
25 pg
magnetic stimulation changes after EDB injections e cortical excitability changes. On the question of whether the animal models used in Caleo studies as well as those carried out previously are appropriate and applicable to human conditions, there are several unsettled issues [45]. (a) Rat and mouse model e differences in response to BoNT/A by these species and in comparison to man; Rat vibrissal pad model e appears to be little used in BoNT/A research and there is a potential for “special” response to BoNT/A. (b) Doses used were high in comparison to human dosage - 60e600 times higher doses than clinical applications. (c) an assumption made e the cleavage product of BoNT/A action has equivalent “transmission” to BoNT/A itself.
0 Fig. 6. In vivo toxicity mouse bioassay for DrBoNT/A and rHCA (Yang et al., 2008). Six weeks old Swiss Webster female mice (n ¼ 5) via was administered intraperitonially with 25 pg (1MLD) BoNT/A, and 1 mg (about 100,000 LD50 dose equivalent of BoNT/A) DrBoNT/A (filled bar) or rHCA (dash bar) for a 96 h observation period.
retrograde and trans-synaptic movement of the BoNT-drug chimera across the nerve to distant sites even when given through minimally invasive intramuscular route of administration. Dose optimization for such circumstances could be however optimized as needed for the clinical set-up. This way the potential of BoNT to deliver drugs for several neurological conditions, exploiting its retrograde transport and transcytosis is yet to be fully tested. Furthermore, understanding the intracellular trafficking of different serotypes of BoNT would be important not only for the expanding potential of BoNTs in clinical therapy, but also for harnessing its utility in drug delivery to treat many other neurological disorders. Our demonstration of BoNT/A heavy chain based drug delivery system [32] provides the proof of principle, which could be further exploited in delivering therapeutic drugs for many neurological conditions including CNS disorders. In addition, availability of non-toxic variants or derivatives of BoNTs would also provide an effective tool to further study their trafficking across the peripheral as well as CNS nerve terminals.
14. Conclusions Drug delivery vehicle to nerves is a need that has drawn attention from biomedical scientists for several decades. BoNTs provide a natural source of delivery vehicle evolved in a billion year old organism, which targets the nervous system specifically and effectively. It is already an FDA approved drug candidate, making it more amenable for clinically approved products. Our demonstration of BoNT/A heavy chain based drug delivery system [32] provides the proof of principle, which could to be further exploited in delivering therapeutic drugs for many neurological conditions. Development of non-toxic variants or derivatives of BoNTs would also pave way as an effective tool to further study their trafficking across the peripheral as well as CNS nerve terminals. BoNT based drug delivery could be also exploited to deliver therapeutic cargoes to treat many CNS disorders upon peripheral administration and can be used as a potential alternative for tetanus based drug delivery.
Acknowledgement This work was supported by the DoD/Army Contract W81XWH08-P-0705 and DARPA Grant GRANT W911NF-07-1-0623.
References 13. Challenges of the BoNT trafficking into central nervous system Retrograde transport of BoNTs could conceivably be a challenging issue, even though such a property could be used to deliver drugs to the CNS. The idea of BoNT entering the CNS, particularly the brain, could have negative repercussions to the commercial use of the toxin for medical and cosmetic uses currently being practiced [31,44,45], especially for patients who use it as cosmetics. This could have significant repercussion to the business, as cosmetic use corresponds to about half of the market. At the Dartmouth Symposium in 2008, Dr. Caleo presented his findings [45], and several issues were clarified. First of all, Caleo himself pointed out that the dose used in the experiments and the animal model employed is not directly relevant to the clinical use of BoNT products in humans. On the question of whether BoNT/A has an effect on the central nervous system, there are several previous reports which have suggested such an effect; Habermann [29], who reported detection in spinal cord after intramuscular injection; Black & Dolly [46], who reported minimal uptake at central nerve terminals; Lange et al. [47], who demonstrated jitter in distal limb after CD (cervical dystonia) treatment; Byrnes et al. [48], who reported changes in corticomotor map topography after writer’s cramp treatment; and Kim et al. [49], who showed transcranial
[1] T. Dua, M.G. Cumbrere, C. Mathers, S. Saxena, Neurological Disorders: Public Health Challenges. World Health Organization, Geneva, Switzerland, 2006, p. 27e39. [2] M.S. Soichet, S.R. Winn, Cell delivery to the central nervous system. Adv. Drug Deliv. Rev. 42 (2000) 81e102. [3] M. Citron, Strategies for disease modifications in Alzheimer’s disease. Nat. Rev. Neurosci. 5 (2004) 677e685. [4] E.H. Lo, T. Dalkara, M.A. Moskowitz, Mechanisms, challenges and opportunities in stroke. Nat. Rev. Neurosci. 4 (2003) 399e414. [5] A. Misra, S. Ganesh, A. Shahiwala, S.P. Shah, Drug delivery to the central nervous system: a review. J. Pharm. Pharmaceut. Sci. 6 (2003) 252e273. [6] S. Thuret, L.D.F. Moon, F.H. Gage, Therapeutic interventions after spinal cord injury. Nat. Rev. Neurosci. 7 (2006) 628e643. [7] J.M. Bergen, S.H. Pun, Analysis of the intracellular barriers encountered by nonviral gene carriers in a model of spatially controlled delivery to neurons. J. Gene Med. 10 (2008) 187e197. [8] J.M. Bergen, I.K. Park, P.J. Horner, S.H. Pun, Nonviral approaches for neuronal delivery of nucleic acids. Pharm. Res. 25 (2008) 983e998. [9] M. Berry, L. Barrett, L. Seymour, A. Baird, A. Logan, Gene therapy for central nervous system repair. Curr. Opin. Mol. Ther. 3 (2001) 338e349. [10] J.S. Suk, J. Suh, S.K. Lai, J. Hanes, Quantifying the intracellular transport of viral and nonviral gene vectors in primary neurons. Exp. Biol. Med. 232 (2007) 461e469. [11] B.R. Singh, Botulinum neurotoxin structure, engineering, and novel cellular trafficking and targeting. Neurotox. Res. 9 (2006) 73e92. [12] B.R. Singh, Intimate details of the most poisonous poison. Nat. Struct. Biol. 7 (2000) 617e619. [13] P. Bernard, M. Popoff, J. Molgó, How do the Botulinum Neurotoxins block neurotransmitter release: from botulism to the molecular mechanism of action. Botulinum J. 1 (2008) 14e87.
B.R. Singh et al. / Biochimie 92 (2010) 1252e1259 [14] P. Beaude, A. Delacour, B. Bizzini, D. Domuado, M.H. Remy, Retrograde axonal transport of an exogenous enzyme covalently linked to B-IIb fragment of tetanus toxin. Biochem. J. 271 (1990) 87e91. [15] S.C. Benn, I. Ay, E. Bastia, R. Chian, S.A. Celia, R.B. Pepinsky, P.S. Fishman, R.H. Brown Jr., J.W. Francis, Tetanus toxin fragment C fusion facilitates protein delivery to CNS neurons from cerebrospinal fluid in mice. J. Neurochem. 95 (2005) 1118e1131. [16] T. Bordet, L.C. Ptakhine, F. Fauchereau, G. Friocourt, A. Kahn, G. Haase, Neuronal targeting of cardiotrophin-1 by coupling with tetanus toxin C fragment. Mol. Cell. Neurosci. 17 (2001) 842e854. [17] K.E. Larsen, S.C. Benn, I. Ay, R.J. Chian, S.A. Celia, M.P. Remington, M. Bejarano, M. Liu, J. Ross, P. Carmillo, D. Sah, K.A. Phillips, D. Sulzer, R.B. Pepinsky, P.S. Fishman, R.H. Brown Jr., J.W. Francis, A glial cell line-derived neurotrophic factor (GDNF): tetanus toxin fragment C protein conjugate improves delivery of GDNF to spinal cord motor neurons in mice. Brain Res. 1120 (2006) 1e12. [18] M. Caleo, G. Schiavo, Central effects of tetanus and botulinum neurotoxins. Toxicon 54 (2009) 593e599. [19] C. Montecucco, G. Schiavo, Structure and function of tetanus and botulinum neurotoxins. Quart. Rev. Biophys. 28 (1995) 423e472. [20] C. Grumelli, C. Verderio, D. Pozzi, O. Rossetto, C. Montecucco, M. Matteoli, Internalization and mechanism of action of clostridial toxins in neurons. Neurotoxicology 26 (2005) 761e767. [21] M. Dong, F. Yeh, W.H. Tepp, C. Dean, E.A. Johnson, R. Janz, E.R. Chapman, SV2 is the protein receptor for botulinum neurotoxin A. Science 312 (2006) 592e596. [22] M. Galloux, H. Vitrac, C. Montagner, S. Raffestin, M.R. Popoff, A. Chenal, V. Forge, D. Gillet, Membrane interaction of botulinum neurotoxin A T domain: the belt region is a regulatory loop for membrane interaction. J. Biol. Chem. 283 (2008) 27668e27676. [23] R. Ratts, C. Trujillo, A. Bharti, J. VanderSpek, R. Harrison, J.R. Murphy, A conserved motif in transmembrane helix 1 of diphtheria toxin mediates catalytic domain delivery to the cytosol. Proc. Natl. Acad. Sci. U S A 102 (2005) 15635e15640. [24] C. Verderio, O. Rossetto, C. Grumelli, C. Frassoni, C. Montecucco, M. Matteoli, Entering neurons: botulinum toxins and synaptic vesicle recycling. EMBO Rep. 7 (2006) 995e999. [25] M. Caleo, F. Antonucci, L. Restani, R. Mazzocchio, A reappraisal of the central effects of botulinum neurotoxin type A: by what mechanism? J. Neurochem. 109 (2009) 15e24. [26] G. Lalli, S. Bohnert, K. Deinhardt, C. Verastegui, G. Schiavo, The journey of tetanus and botulinum neurotoxins in neurons. Trends. Microbiol. 11 (2003) 431e437. [27] M. Matteoli, C. Verderio, O. Rossetto, N. Iezzi, S. Coco, G. Schiavo, C. Montecucco, Synaptic vesicle endocytosis mediates the entry of tetanus neurotoxin into hippocampal neurons. Proc. Natl. Acad. Sci. U S A 93 (1996) 13310e13315. [28] A. Curra, A. Berardelli, Do the unintended actions of botulinum toxin at distant sites have clinical implications? Neurology 72 (2009) 1095e1099. [29] E. Habermann, 125 I-labeled neurotoxin from Clostridium botulinum A: preparation, binding to synaptosomes and ascent to the spinal cord. Naunyn Schmiedeberg’s Arch. Pharmacol. 281 (1974) 47e56. [30] H. Bigalke, I. Heller, B. Bizzini, E. Habermann, Tetanus toxin and botulinum A toxin inhibit release and uptake of various transmitters, as studied with particulate preparations from rat brain and spinal cord. Naunyn Schmiedeberg’s Arch. Pharmacol. 316 (1981) 244e251. [31] S.K. Sharma, B.R. Singh, Botulinum toxin in neurological diseases. Saudi Arab. J. Rehab. 10 (2004) 111e117. [32] P. Zhang, R. Ray, B.R. Singh, D. Li, M. Adler, P. Ray, An efficient drug delivery vehicle for botulism countermeasure. BMC Pharmacol. 9 (2009) 12. [33] F. Antonucci, C. Rossi, L. Gianfranceschi, O. Rossetto, M. Caleo, Long-distance retrograde effects of botulinum neurotoxin A. J. Neurosci. 28 (2008) 3689e3696.
1259
[34] J.A. Chaddock, J.R. Purkiss, M.J. Duggan, C.P. Quinn, C.C. Shone, K.A. Foster, A conjugate composed of nerve growth factor coupled to a nontoxic derivative of Clostridium botulinum neurotoxin type A can inhibit neurotransmitter release in vitro. Growth Factors 18 (2000a) 147e155. [35] J.A. Chaddock, J.R. Purkis, L.M. Friis, M.J. Broadbridge, M.J. Duggan, S.J. Fooks, C.C. Shone, C.P. Quinn, K.A. Foster, Inhibition of vesicular secretion in both neuronal and non-neuronal cells by a retargeted endopeptidase derivative of Clostridium botulinum neurotoxin type A. Infect. Immun. 68 (2000b) 2587e2593. [36] J.A. Chaddock, M.J. Duggan, C.P. Quinn, J.R. Purkiss, F.C.G. Alexander, S. Doward, S.J. Fooks, L. Friis, Y. Hall, E.R. Kirby, N.J. Leeds, H.J. Moulsdale, A. Dickenson, G.M. Green, W. Rahman, R. Suzuki, C.C. Shone, K.A. Foster, Retargeted clostridial endopeptidases: inhibition of nociceptive neurotransmitter release in vitro, and antinociceptive activity in in vivo models of pain. Mov. Disord. 19 (Suppl. 8) (2004) S42eS47. [37] J. Wang, J. Meng, G.W. Lawrence, T.H. Zurawski, A. Sasse, M.O. Bodeker, M.A. Gilmore, E. Fernández-Salas, J. Francis, L.E. Steward, K.R. Aoki, J.O. Dolly, Novel chimeras of botulinum neurotoxins A and E unveil contributions from the binding, translocation, and protease domains to their functional characteristics. J. Biol. Chem. 283 (2008) 16993e17002. [38] S. Bade, A. Rummel, C. Reisinger, T. Karnath, G. A-Hilger, H. Bigalke, T. Binz, Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates. J. Neurochem. 91 (2004) 461e472. [39] W. Yang, P. Lindo, S. Riding, T.W. Chang, S. Cai, T. Van, R. Kukreja, Y. Zhou, K. Vasa, B.R. Singh, Expression, purification and comparative characterization of deactivated recombinant botulinum neurotoxin type A. Botulinum J. 1 (2008) 219e241. [40] S. Bandyopadhyay, A.W. Clark, B.R. Das Gupta, V. Sathyamoorthy, Role of the heavy and light chains of botulinum neurotoxin in neuromuscular paralysis. J. Biol. Chem. 262 (1987) 2660e2663. [41] S.R. Johnstone, L.M. Morrice, S. V-Heyningen, The heavy chain of tetanus toxin can mediate the entry of cytotoxic gelonin into intact cells. FEBS Lett. 265 (1990) 101e103. [42] M.C. Goodnough, G. Oyler, P.S. Fishman, E.A. Johnson, E.A. Neale, J.E. Keller, W.H. Tepp, M. Clark, S. Hartz, M. Adler, Development of a delivery vehicle for intracellular transport of botulinum neurotoxin antagonists. FEBS Lett. 513 (2002) 163e168. [43] B.M. Kuehn, Studies, reports say botulinum toxins may have effects beyond injection site. JAMA 299 (2008) 2261e2263. [44] S.S. Arnon, R. Schechter, T.V. Inglesby, D.A. Henderson, J.G. Bartlett, M.S. Ascher, E. Eitzen, A.D. Fine, J. Hauer, M. Layton, S. Lillibridge, M.T. Osterholm, T. O’Toole, G. Parker, T.M. Perl, P.K. Russell, D.L. Swerdlow, K. Tonat, Botulinum toxin as a biological weapon: medical and public health management. JAMA 285 (2001) 1059e1070. [45] B.R. Singh, The second annual Dartmouth Botulinum Research Symposium discusses the effect of botulinum neurotoxin on the central nervous system. Botulinum J. 1 (2009) 340e346. [46] J.D. Black, J.O. Dolly, Selective location of acceptors for botulinum neurotoxin A in the central and peripheral nervous systems. Neuroscience 23 (1987) 767e779. [47] D.J. Lange, M. Rubin, P.E. Greene, U.J. Kang, C.B. Moskowitz, M.F. Brin, R.E. Lovelace, S. Fahn, Distant effects of locally injected botulinum toxin: a double-blind study of single fiber EMG changes. Muscle Nerve 14 (1991) 672e675. [48] M.L. Byrnes, G.W. Thickbroom, S.A. Wilson, P. Sacco, J.M. Shipman, R. Stell, F.L. Mastaglia, The corticomotor representation of upper limb muscles in writer’s cramp and changes following Botulinum toxin injection. Brain 121 (1998) 977e988. [49] D.Y. Kim, B.M. Oh, N.J. Paik, Central effect of Botulinum toxin type A in humans. Int. J. Neurosci. 116 (2006) 667e680.