Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to ...their widespread use in the neuroscience laboratory is the cost, labor, skill and time-intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when the vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared with standard AAV. Here, we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood-brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV-transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing three-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex vivo serial two-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable the widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant.
Correction to: Gene Therapy (2016) 23, 380-392; doi:10.1038/gt.2016.11 The initial Figure 2a was erroneously generated from a file from a mouse injected with conventional AAV9-GFP and not ...exo-AAV9-GFP, as described in the manuscript. We have therefore reformatted this specific panel, and corrected Figure 2a and the figure legend accordingly, now showing an image of the GFP signal detected by 2-photon microscopy after intravenous injection of exo-AAV9-GFP in a mouse.
Conversion of soluble peptides and proteins into amyloid fibrils and/or intermediate oligomers is believed to be the central event in the pathogenesis of most human neurodegenerative diseases, ...including Parkinson’s disease (PD). Here we describe the modulating effect of filamentous phages on aggregation of α-synuclein (AS)
in vitro and in a PD cellular model. Filamentous phages, well understood at both structural and genetic levels, have a nanotubular appearance, showing conformational similarities to amyloid fibrils. Since filamentous phages can infect only bacteria and have no tropism to mammalian cells, we utilized the f88 system to present a peptide containing a cyclic RGD (arg-gly-asp), which enabled phage internalization into the cells. Detection of intracellular AS oligomers, in differentiated SH-SY5Y cells, stably transfected with wild type AS gene, was performed using Western blot and ELISA measurements. Data presented here show reduced levels of AS soluble aggregates in phage treated cells compared to non-treated cells, suggesting new therapeutics for PD.
The tumor suppressor gene p53 is mutated in more than half of human tumors. One important characteristic of p53 mutants is their accumulation in the nucleus of cancer cells. Thus, reactivation of ...mutant p53 proteins may trigger massive apoptosis in tumor cells. Pharmacologic methods are currently under development to induce mutant p53 proteins to resume their wild-type function. We have identified a human single-chain Fv fragment, designated as transcriptional transactivation and apoptosis restoring (TAR1), which specifically and with high affinity binds to mutant p53 and restores its wild-type active conformation. Binding of TAR1 to mutant p53 induced transcriptional transactivation of p53 target genes and down-regulation of mutant p53 transcriptional target genes. TAR1 treatment induced apoptosis in a variety of cell lines endogenously expressing p53 carrying different point mutations DNA contact or structural p53 mutants. Moreover, in an animal model of mice carrying human xenografts, TAR1 induced tumor regression with no apparent deleterious side effects. Thus, it may be considered as a potential candidate for anticancer treatment, targeting tumors with mutant p53.
This paper describes crack growth processes in carbon fibre and glass fibre-epoxy laminates under stress and quantifies by physical modelling the accumulation of damage with load cycling. Cracking ...mechanisms are observed directly and identified by in-situ dynamic scanning electron microscopy. Armed with this information, particular attention is given to the differences between these cracking processes and the way they interact, their growth-rate, and their effect on the damage-state of the structure with variations in laminate geometry, with changes in magnitude of applied stress, and with fatigue cycles. Particular attention is given to the internal damage state variable approach to physical modelling.
In 2003, The John A. Hartford Foundation Institute for Geriatric Nursing, New York University Division of Nursing, convened an expert panel to explore the potential for developing recommendations for ...the caseloads of advanced practice nurses (APNs) in nursing homes and to provide substantive and detailed strategies to strengthen the use of APNs in nursing homes. The panel, consisting of nationally recognized experts in geriatric practice, education, research, public policy, and long‐term care, developed six recommendations related to caseloads for APNs in nursing homes. The recommendations address educational preparation of APNs; average reimbursable APN visits per day; factors affecting APNs caseload parameters, including provider characteristics, practice models, resident acuity, and facility factors; changes in Medicare reimbursement to acknowledge nonbillable time spent in resident care; and technical assistance to promote a climate conducive to APN practice in nursing homes. Detailed research findings and clinical expertise underpin each recommendation. These recommendations provide practitioners, payers, regulators, and consumers with a rationale and details of current advanced practice nursing models and caseload parameters, preferred geriatric education, reimbursement strategies, and a range of technical assistance necessary to strengthen, enhance, and increase APNs' participation in the care of nursing home residents.
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