Progress in gene therapy for neurological disorders

Diseases of the nervous system have devastating effects and are widely distributed among the population, being especially prevalent in the elderly. These diseases are often caused by inherited genetic mutations that result in abnormal nervous system development, neurodegeneration, or impaired neuronal function. Other causes of neurological diseases include genetic and epigenetic changes induced by environmental insults, injury, disease-related events or inflammatory processes. Standard medical and surgical practice has not proved effective in curing or treating these diseases, and appropriate pharmaceuticals do not exist or are insufficient to slow disease progression. Gene therapy is emerging as a powerful approach with potential to treat and even cure some of the most common diseases of the nervous system. Gene therapy for neurological diseases has been made possible through progress in understanding the underlying disease mechanisms, particularly those involving sensory neurons, and also by improvement of gene vector design, therapeutic gene selection, and methods of delivery. Progress in the field has renewed our optimism for gene therapy as a treatment modality that can be used by neurologists, ophthalmologists and neurosurgeons. In this Review, we describe the promising gene therapy strategies that have the potential to treat patients with neurological diseases and discuss prospects for future development of gene therapy

On the functional diversity of dynamical behaviour in genetic and metabolic feedback systems

<p>Abstract</p> <p>Background</p> <p>Feedback regulation plays crucial roles in the robust control and maintenance of many cellular systems. Negative feedbacks are found to underline both stable and unstable, often oscillatory, behaviours. We explore the dynamical characteristics of systems with single as well as coupled negative feedback loops using a combined approach of analytical and numerical techniques. Particularly, we emphasise how the loop's characterising factors (strength and cooperativity levels) affect system dynamics and how individual loops interact in the coupled-loop systems.</p> <p>Results</p> <p>We develop an analytical bifurcation analysis based on the stability and the Routh- Hurwitz theorem for a common negative feedback system and a variety of its variants. We demonstrate that different combinations of the feedback strengths of individual loops give rise to different dynamical behaviours. Moreover, incorporating more negative feedback loops always tend to enhance system stability. We show that two mechanisms, in addition to the lengthening of pathway, can lower the Hill coefficient to a biologically plausible level required for sustained oscillations. These include loops coupling and end-product utilisation. We find that the degradation rates solely affect the threshold Hill coefficient for sustained oscillation, while the synthesis rates have more significant roles in determining the threshold feedback strength. Unbalancing the degradation rates between the system species is found as a way to improve stability.</p> <p>Conclusion</p> <p>The analytical methods and insights presented in this study demonstrate that reallocation of the feedback loop may or may not make the system more stable; the specific effect is determined by the degradation rates of the newly inhibited molecular species. As the loop moves closer to the end of the pathway, the minimum Hill coefficient for oscillation is reduced. Furthermore, under general (unequal) values of the degradation rates, system extension becomes more stable only when the added species degrades slower than it is being produced; otherwise the system is more prone to oscillation. The coupling of loops significantly increases the richness of dynamical bifurcation characteristics. The likelihood of having oscillatory behaviour is directly determined by the loops' strength: stronger loops always result in smaller oscillatory regions.</p

Detection of single-molecule H₂O₂ signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes

An emerging concept in cell signalling is the natural role of reactive oxygen species such as hydrogen peroxide (H₂O₂) as beneficial messengers in redox signalling pathways. The nature of H₂O₂ signalling is confounded, however, by difficulties in tracking it in living systems, both spatially and temporally, at low concentrations. Here, we develop an array of fluorescent single-walled carbon nanotubes that can selectively record, in real time, the discrete, stochastic quenching events that occur as H₂O₂molecules are emitted from individual human epidermal carcinoma cells stimulated by epidermal growth factor. We show mathematically that such arrays can distinguish between molecules originating locally on the cell membrane from other contributions. We find that epidermal growth factor induces 2 nmol H₂O₂ locally over a period of 50 min. This platform promises a new approach to understanding the signalling of reactive oxygen species at the cellular level.Arnold and Mabel Beckman Foundation (Beckman Young Investigator award)National Science Foundation (U.S.) ((NSF) Career Award)National Science Foundation (U.S.) (Nanoscale Interdisciplinary Research Team)Czech Republic. Ministry of Education, Youth, and Sports (project no. MSM0021620806)Czech Republic. Ministry of Education, Youth, and Sports (KAN grant no. 400100701

Organization of long-range inputs and outputs of frontal cortex for top-down control

Long-range projections from the frontal cortex are known to modulate sensory processing in multiple modalities. Although the mouse has become an increasingly important animal model for studying the circuit basis of behavior, the functional organization of its frontal cortical long-range connectivity remains poorly characterized. Here we used virus-assisted circuit mapping to identify the brain networks for top-down modulation of visual, somatosensory, and auditory processing. The visual cortex is reciprocally connected to the anterior cingulate area, whereas the somatosensory and auditory cortices are connected to the primary and secondary motor cortices. Anterograde and retrograde tracing identified the cortical and subcortical structures belonging to each network. Furthermore, using novel viral techniques to target subpopulations of frontal neurons projecting to the visual cortex versus the superior colliculus, we identified two distinct subnetworks within the visual network. These findings provide an anatomical foundation for understanding the brain mechanisms underlying top-down control of behavior

Engineering a serum-resistant and thermostable vesicular stomatitis virus G glycoprotein for pseudotyping retroviral and lentiviral vectors

Vesicular stomatitis virus G glycoprotein (VSV-G) is the most widely used envelope protein for retroviral and lentiviral vector pseudotyping; however, serum inactivation of VSV-G pseudotyped vectors is a significant challenge for in vivo gene delivery. To address this problem, we conducted directed evolution of VSV-G to increase its resistance to human serum neutralization. After six selection cycles, numerous common mutations were present. Based on their location within VSV-G, we analyzed whether substitutions in several surface exposed residues could endow viral vectors with higher resistance to serum. S162T, T230N, and T368A mutations enhanced serum resistance, and additionally K66T, T368A, and E380K substitutions increased the thermostability of VSV-G pseudotyped retroviral vectors, an advantageous byproduct of the selection strategy. Analysis of a number of combined mutants revealed that VSV-G harboring T230N + T368A or K66T + S162T + T230N + T368A mutations exhibited both higher in vitro resistance to human serum and higher thermostability, as well as enhanced resistance to rabbit and mouse serum. Finally, lentiviral vectors pseudotyped with these variants were more resistant to human serum in a murine model. These serum-resistant and thermostable VSV-G variants may aid the application of retroviral and lentiviral vectors to gene therapy