Non-visual exploration of novel objects increases the levels of plasticity factors in the rat primary visual cortex.

BACKGROUND: Historically, the primary sensory areas of the cerebral cortex have been exclusively associated with the processing of a single sensory modality. Yet the presence of tactile responses in the primary visual (V1) cortex has challenged this view, leading to the notion that primary sensory areas engage in cross-modal processing, and that the associated circuitry is modifiable by such activity. To explore this notion, here we assessed whether the exploration of novel objects in the dark induces the activation of plasticity markers in the V1 cortex of rats. METHODS: Adult rats were allowed to freely explore for 20 min a completely dark box with four novel objects of different shapes and textures. Animals were euthanized either 1 (n = 5) or 3 h (n = 5) after exploration. A control group (n = 5) was placed for 20 min in the same environment, but without the objects. Frontal sections of the brains were submitted to immunohistochemistry to measure protein levels of egr-1 and c-fos, and phosphorylated calcium-dependent kinase (pCaKMII) in V1 cortex. RESULTS: The amount of neurons labeled with monoclonal antibodies against c-fos, egr-1 or pCaKMII increased significantly in V1 cortex after one hour of exploration in the dark. Three hours after exploration, the number of labeled neurons decreased to basal levels. CONCLUSIONS: Our results suggest that non-visual exploration induces the activation of immediate-early genes in V1 cortex, which is suggestive of cross-modal processing in this area. Besides, the increase in the number of neurons labeled with pCaKMII may signal a condition promoting synaptic plasticity.

Viral Vector-Mediated Antisense Therapy for Genetic Diseases.

RNA plays complex roles in normal health and disease and is becoming an important target for therapeutic intervention; accordingly, therapeutic strategies that modulate RNA function have gained great interest over the past decade. Antisense oligonucleotides (AOs) are perhaps the most promising strategy to modulate RNA expression through a variety of post binding events such as gene silencing through degradative or non-degradative mechanisms, or splicing modulation which has recently demonstrated promising results. However, AO technology still faces issues like poor cellular-uptake, low efficacy in target tissues and relatively rapid clearance from the circulation which means repeated injections are essential to complete therapeutic efficacy. To overcome these limitations, viral vectors encoding small nuclear RNAs have been engineered to shuttle antisense sequences into cells, allowing appropriate subcellular localization with pre-mRNAs and permanent correction. In this review, we outline the different strategies for antisense therapy mediated by viral vectors and provide examples of each approach. We also address the advantages and limitations of viral vector use, with an emphasis on their clinical application.

Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system.

Recombinant adeno-associated viruses (rAAV) are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology.

Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors.

Recombinant adeno-associated viruses (rAAV) are promising candidates for gene therapy approaches. The last two decades were particularly fruitful in terms of processes applied in the production and purification of this type of gene transfer vectors. This rapid technological evolution led to better yields and higher levels of vector purity. Recently, some reports showed that rAAV produced by transient tri-transfection method in adherent human embryonic kidney 293 cells can be harvested directly from supernatant, leading to easier and faster purification compared to classical virus extraction from cell pellets. Here, we compare these approaches with new vector recovery method using small quantity of detergent at the initial clarification step to treat the whole transfected cell culture. Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity. Moreover, this approach leads to the reduction of the total process duration. Finally, the vectors maintain their functionality, showing unexpected higher in vitro and in vivo transduction efficacies. This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality, and scalability.