Next-generation of targeted AAVP vectors for systemic transgene delivery against cancer

Bacteriophage (phage) have attractive advantages as delivery sys-tems compared to mammalian viruses, but have been consideredpoor vectors because they lack evolved strategies to confrontand overcome mammalian cell barriers to infective agents. Wereasoned that improved efficacy of delivery might be achievedthrough structural modification of the viral capsid to avoid pre-and post-internalization barriers to mammalian cell transduction.We generated multifunctional hybrid AAV/phage (AAVP) particlesto enable simultaneous display of targeting ligands on the phage’sminor pIII proteins and also degradation-resistance motifs on thevery numerous pVIII coat proteins. This genetic strategy of directedevolution, bestows a next-generation of AAVP particles that fea-ture resistance to fibrinogen adsorption or neutralizing antibodies,and ability to escape endolysosomal degradation. This results insuperior gene transfer efficacyin vitroand also in preclinicalmouse models of rodent and human solid tumors. Thus, the uniquefunctions of our next-generation AAVP particles enable improvedtargeted gene delivery to tumor cells

Engineering pseudovirions for large-scale targeted gene transfer and recombinant adeno-associated virus production

Gene transfer is a technology central to the development of gene therapy and expression of proteins and biological products necessary for designing and producing drug compounds or creating new research methodologies. Consequently, the field has experienced a large bottleneck in economic and time costs when viral vectors are involved. Over the past decade, recombinant mammalian viral vectors have been exploited for these purposes; however, the limitations in their native biology and production methods warrant new vector systems to be investigated and developed. In this thesis, we reinvestigate the humble bacteriophage, a prokaryotic virus, as a potential tool to circumvent the cost limitation of eukaryotic viruses. By combining their genome with that of Adeno-associated virus (AAV), a well-characterised mammalian virus, and developing a novel method of expressing these hybird vectors, we were able to overcome many limitations that these viruses have as separate entities. The proposed vector, termed Phagemid Adeno-associated Virion (PAAV), is as efficacious as traditional vectors, while economically costing a fraction of what is demanded by current practice in the field. The PAAV, was constructed by inserting a recombinant AAV genome into a phagemid expression vector that carries no phage structural genes. The particles are packaged using a custom-designed mammalian-targeted helper virus, resulting in vectors that can be easily produced at a minimum of 2-fold higher yield than the current gold-standard phage vector. We demonstrated through transmission electron microscopy and transducing unit assays that PAAV vectors generated by our method is less than half the size of traditional full-length phage vectors, and through vesicular staining we are able to determine that the PAAV is internalised at almost 2-fold higher than the efficiencies observed in the gold-standard phage vector. We further assessed qualitative and quantitative gene expression efficacies by the PAAV bearing GFP or Luciferase transgenes in various tumour cells, which show a dramatic increase in gene expression by up to over 10-fold of the gold-standard. To demonstrate that the PAAV and its derived vectors can be used as an alternative to DNA transfection, a method central to mammalian virus production, we designed and validated two proof-of-concept methods that are able to produce rAAV using PAAV vectors. Phages are harmless viruses with a safety profile founded by their historic use as antibiotic agents. The PAAV vector system utilizes the economic advantages of phage vectors and combines them with the efficacy of mammalian viral transgenes, offering an efficacious alternative vector that is able to transduce mammalian tumour cells. Furthermore, the PAAV system has the potential to replace conventional transfection, thereby addressing a significant bottleneck in translational research in the field. Taken together, the PAAV offers a novel and advantageous alternative platform to conventional viruses for use in therapeutic and industrial applications.Open Acces

Left Cathodal Trans-Cranial Direct Current Stimulation of the Parietal Cortex Leads to an Asymmetrical Modulation of the Vestibular-Ocular Reflex

AbstractMulti-sensory visuo-vestibular cortical areas within the parietal lobe are important for spatial orientation and possibly for descending modulation of the vestibular-ocular reflex (VOR). Functional imaging and lesion studies suggest that vestibular cortical processing is localized primarily in the non-dominant parietal lobe. However, the role of inter-hemispheric parietal balance in vestibular processing is poorly understood. Therefore, we tested whether experimentally induced asymmetries in right versus left parietal excitability would modulate vestibular function. VOR function was assessed in right-handed normal subjects during caloric ear irrigation (30 °C), before and after trans-cranial direct current stimulation (tDCS) was applied bilaterally over the parietal cortex. Bilateral tDCS with the anode over the right and the cathode over the left parietal region resulted in significant asymmetrical modulation of the VOR, with highly suppressed responses during the right caloric irrigation (i.e. rightward slow phase nystagmus). In contrast, we observed no VOR modulation during either cathodal stimulation of the right parietal cortex or SHAM tDCS conditions. Application of unilateral tDCS revealed that the left cathodal stimulation was critical in inducing the observed modulation of the VOR. We show that disruption of parietal inter-hemispheric balance can induce asymmetries in vestibular function. This is the first report using neuromodulation to show right hemisphere dominance for vestibular cortical processing

Bidirectional Modulation of Numerical Magnitude

Numerical cognition is critical for modern life; however, the precise neural mechanisms underpinning numerical magnitude allocation in humans remain obscure. Based upon previous reports demonstrating the close behavioral and neuro-anatomical relationship between number allocation and spatial attention, we hypothesized that these systems would be subject to similar control mechanisms, namely dynamic interhemispheric competition. We employed a physiological paradigm, combining visual and vestibular stimulation, to induce interhemispheric conflict and subsequent unihemispheric inhibition, as confirmed by transcranial direct current stimulation (tDCS). This allowed us to demonstrate the first systematic bidirectional modulation of numerical magnitude toward either higher or lower numbers, independently of either eye movements or spatial attention mediated biases. We incorporated both our findings and those from the most widely accepted theoretical framework for numerical cognition to present a novel unifying computational model that describes how numerical magnitude allocation is subject to dynamic interhemispheric competition. That is, numerical allocation is continually updated in a contextual manner based upon relative magnitude, with the right hemisphere responsible for smaller magnitudes and the left hemisphere for larger magnitudes

Next-generation of targeted AAVP vectors for systemic transgene delivery against cancer

Bacteriophage (phage) have attractive advantages as delivery systems compared with mammalian viruses, but have been considered poor vectors because they lack evolved strategies to confront and overcome mammalian cell barriers to infective agents. We reasoned that improved efficacy of delivery might be achieved through structural modification of the viral capsid to avoid pre- and postinternalization barriers to mammalian cell transduction. We generated multifunctional hybrid adeno-associated virus/phage (AAVP) particles to enable simultaneous display of targeting ligands on the phage's minor pIII proteins and also degradation-resistance motifs on the very numerous pVIII coat proteins. This genetic strategy of directed evolution bestows a next-generation of AAVP particles that feature resistance to fibrinogen adsorption or neutralizing antibodies and ability to escape endolysosomal degradation. This results in superior gene transfer efficacy in vitro and also in preclinical mouse models of rodent and human solid tumors. Thus, the unique functions of our next-generation AAVP particles enable improved targeted gene delivery to tumor cells