Transduction of Brain Dopamine Neurons by Adenoviral Vectors Is Modulated by CAR Expression: Rationale for Tropism Modified Vectors in PD Gene Therapy

Gene-based therapy is a new paradigm for the treatment of Parkinson disease (PD) and offers considerable promise for precise targeting and flexibility to impact multiple pathobiological processes for which small molecule agents are not available. Some success has been achieved utilizing adeno-associated virus for this approach, but it is likely that the characteristics of this vector system will ultimately create barriers to progress in clinical therapy. Adenovirus (Ad) vector overcomes limitations in payload size and targeting. The cellular tropism of Ad serotype 5 (Ad5)-based vectors is regulated by the Ad attachment protein binding to its primary cellular receptor, the coxsackie and adenovirus receptor (CAR). Many clinically relevant tissues are refractory to Ad5 infection due to negligible CAR levels but can be targeted by tropism-modified, CAR-independent forms of Ad. Our objective was to evaluate the role of CAR protein in transduction of dopamine (DA) neurons in vivo.Ad5 was delivered to the substantia nigra (SN) in wild type (wt) and CAR transgenic animals. Cellular tropism was assessed by immunohistochemistry (IHC) in the SN and striatal terminals. CAR expression was assessed by western blot and IHC. We found in wt animals, Ad5 results in robust transgene expression in astrocytes and other non-neuronal cells but poor infection of DA neurons. In contrast, in transgenic animals, Ad5 infects SNc neurons resulting in expression of transduced protein in their striatal terminals. Western blot showed low CAR expression in the ventral midbrain of wt animals compared to transgenic animals. Interestingly, hCAR protein localizes with markers of post-synaptic structures, suggesting synapses are the point of entry into dopaminergic neurons in transgenic animals.These findings demonstrate that CAR deficiency limits infection of wild type DA neurons by Ad5 and provide a rationale for the development of tropism-modified, CAR-independent Ad-vectors for use in gene therapy of human PD

Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells)

In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described

Submillimeter superconducting integrated receivers: Fabrication and yield

Fabrication procedure and yield analysis of superconducting integrated receivers is reported. These chip receivers, apart from the quasi-optical SIS mixers, contain internal local oscillators and associated rf and de interfaces. Due to both complexity and design requirements of the integrated circuit, certain restrictions are applied to the standard Nb/Al/AlxOy/Nb SNEAP process. To obtain accurate area for micron-size SIS junctions and thickness for multi-layer SiO2 insulation, a few solutions and modifications were developed. The possibility of transfering this fabrication process worldwide has been proven experimentally

Forward and backward waves in Cherenkov flux-flow oscillators

Josephson flux-flow oscillators (FFOs) have been used as an on-chip local oscillator at frequencies up to 650 GHz. An autonomous FFO linewidth of about 1 MHz was measured in the resonant regime at V-b <950 mu V for niobium-aluminium oxide-niobium tunnel junctions, while considerably larger values were reported at higher voltages. To overcome this fundamental linewidth broadening we propose an on-chip Cherenkov radiation Aux-flow oscillator (CRFFO). It consists of a long Josephson junction and a superconducting slow-wave transmission line that modifies significantly the junction dispersion relation. Two superconductor-insulator-superconductor junction detectors are connected to both the long Josephson junction and the slow-wave line to determine the available microwave power. The power is measured at different CRFFO biasing conditions. Both a forward wave and a backward wave oscillation regime are observed. An FFO and a CRFFO with the same junction parameters are compared

Niobium tunnel junctions with multi-layered electrodes

The current-voltage characteristics of the niobium - aluminum oxide - niobium tunnel junctions have been studied systematically and are compared with numerical simulations based on the microscopic theory of the proximity effect. The thickness of the base niobium layer is varied from 35 to 500 mn while the thickness of the aluminum layer is kept constant (about 9 mm). In a separate series of experiments the aluminum thickness is varied from 2 to 30 mn for two fixed thickness of the base electrode: 50 and 200 mn. The appropriate conditions for a full suppression of the so called "knee" structure at the gap voltage in the current-voltage characteristic are experimentally determined and theoretically interpreted in the framework of the microscopic theory. The influence of the additional aluminum layer in a composite base electrode on the properties of the tunnel junction have been studied in dependence on the aluminum thickness and distance of this layer from the barrier. The obtained results demonstrate that the current-voltage characteristics of tunnel junction can be engineering by an appropriate layer thickness of compound base electrode

Design and fabrication of Cherenkov flux-flow oscillator

The Josephson Flux-Flow Oscillator (FFO) has been used as an on chip local oscillator at frequencies up to 650 GHz. The FFO linewidth of about 1 MHz was measured in the resonant regime at V <915 mu V for niobium - aluminum oxide - niobium tunnel junctions, while considerably larger values were reported at higher voltages. To overcome this fundamental linewidth broadening we propose a novel on chip Cherenkov radiation flux-flow oscillator (CRFFO). It consists of a long Josephson junction and a superconducting slow wave transmission line that modifies essentially the junction dispersion relation. Two SIS detectors are connected both to the long Josephson junction and the transmission line to evaluate available microwave power. The output power coming both from the long junction and the transmission line is estimated at different bias conditions

The influence of LO power heating of the tunnel junction on the performance of THz SIS mixers

We describe the performance of a superconductor- insulator-superconductor (SIS) mixer operating in the frequency range of 780-950 GHz. Unlike most SIS mixers, the tunnel junction employs two different superconductors, a niobium nitride top and a niobium bottom electrode sandwiching an aluminum nitride barrier layer, fabricated on a niobium titanium nitride ground plane. The mixer was tested in a pulse tube cryostat, with all the optical components, in the signal path, mounted inside the vacuum environment to avoid attenuation of the RF signal as it propagates from the hot/cold loads to the mixer. With this setup, we have measured an RF-corrected noise temperature of ~220 K. In this article, we focus on investigating the influence of local oscillator (LO) power heating on the performance of the terahertz mixer. The increase in the junction's physical temperature can be observed experimentally by noting the suppression of the gap voltage in the pumped current-voltage (I-V ) curve as the LO pumping level is increased. Similar observation has already been reported, and attempts were made to estimate the effective temperature of the device using equations of heat transfer between the mixer chip layers. Here, we present an experimental method of quantifying this effect by recovering the effective temperature of the junction through comparing the pumped I-V curves at different pumping levels and fixed bath temperature, with the unpumped I-V curves obtained at varying bath temperatures. We also estimate, for the first time, the effect of heating on the noise temperature as a function of bath temperature and frequency. We show that for typical experimental parameters, the LO heating can increase the double-sideband receiver noise temperature by as much as 20%, and that in the frequency range of the measurements, the effective temperature of the junction at fixed LO power increases linearly with frequency at a rate of 0.5 K/100 GHz