Bioengineered Hybrid Rep 2/6 Gene Improves Encapsulation of a Single-Stranded Expression Cassette into AAV6 Vectors.

The production of clinical-grade recombinant adeno-associated viral (AAV) vectors for gene therapy trials remains a major hurdle in the further advancement of the gene therapy field. During the past decades, AAV research has been predominantly focused on the development of new capsid modifications, vector-associated immunogenicity, and the scale-up vector production. However, limited studies have examined the possibility to manipulate non-structural components of AAV such as the Rep genes. Historically, naturally isolated, or recombinant library-derived AAV capsids have been produced using the AAV serotype 2 Rep gene to package ITR2-flanked vector genomes. In the current study, we mutated four variable amino acids in the conservative part of the binding domain in AAV serotype 6 Rep to generate a Rep2/6 hybrid gene. This newly generated Rep2/6 hybrid had improved packaging ability over wild-type Rep6. AAV vectors produced with Rep2/6 exhibited similar in vivo activity as standard AAV6 vectors. Furthermore, we show that this Rep2/6 hybrid also improves full/empty capsid ratios, suggesting that Rep bioengineering can be used to improve the ratio of fully encapsulated AAV vectors during upstream manufacturing processes.

High-Performance Photocatalytic H2 Production Using a Binary Cu/TiO2/SrTiO3 Heterojunction.

Cu/TiO2/SrTiO3 hybrid structures have been synthesized by the simple impregnation method from Cu/TiO2 and SrTiO3 systems. The structural and surface characterization stated that Cu/TiO2/SrTiO3 composites form an effective covering of SrTiO3 by Cu/TiO2. The heterostructured catalysts lead to an outstanding improved photoactivity for hydrogen production from methanol photoreforming that would be related with the efficient separation of charge pairs favored by the Cu/TiO2/SrTiO3 heterojunction. The best photoproduction is attained for the 30 wt % SrTiO3 heterojunction showing 81.7 mmol/g H2 after 6 h (leading to an apparent quantum yield of ca 1%), 1.7 times higher than that of bare Cu/TiO2.

Almond Shell as a Microporous Carbon Source for Sustainable Cathodes in Lithium⁻Sulfur Batteries.

A microporous carbon derived from biomass (almond shells) and activated with phosphoric acid was analysed as a cathodic matrix in Li⁻S batteries. By studying the parameters of the carbonization process of this biomass residue, certain conditions were determined to obtain a high surface area of carbon (967 m² g-1) and high porosity (0.49 cm³ g-1). This carbon was capable of accommodating up to 60% by weight of sulfur, infiltrated by the disulphide method. The C⁻S composite released an initial specific capacity of 915 mAh g-1 in the Li⁻S cell at a current density of 100 mA g-1 with a high retention capacity of 760 mAh g-1 after 100 cycles and a coulombic efficiency close to 100%. The good performance of the composite was also observed under higher current rates (up to 1000 mA g-1). The overall electrochemical behaviour of this microporous carbon acting as a sulfur host reinforces the possibility of using biomass residues as sustainable sources of materials for energy storage.

Clinical and Histopathological Amelioration of Experimental Autoimmune Encephalomyelitis by AAV Vectors Expressing a Soluble Interleukin-23 Receptor.

The role of the T helper (Th)17 pathway has been clearly demonstrated in the onset and progression of autoimmune diseases, where interleukin (IL)-23 is a key molecule in maintaining the response mediated by Th17 cells. As a consequence, recent strategies based on blocking the interaction between IL-23 and its receptor (IL-23R), for example the anti-p19 antibody tildrakizumab, have been developed to regulate the Th17 pathway from the initial stages of the disease. Here, a soluble (s)IL-23R cDNA was cloned in expression plasmids and viral vectors. The clinical efficacy of sIL-23R was evaluated in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis mice intravenously injected with a single dose of adeno-associated virus AAV8-sIL-23R vectors. Cytokine secretion was determined by multiplex assay, while histopathological analysis of the central nervous system was performed to study demyelination, inflammatory infiltration, and microglia and astroglia activation. We observed that administration of adeno-associated vector 8 encoding sIL-23R was associated with a significant disease improvement, including delay in the onset of the clinical signs; slower progress of the disease; interference with IL-23-mediated signal transducer and activator of transcription response by inhibiting of signal transducer and activator of transcription 3 phosphorylation; reduced demyelination and infiltration in the central nervous system; and lower astrocyte and microglia activation. Our results suggest that the use of vectors carrying sIL-23R to block the IL-23/IL-23R interaction may be a new therapeutic strategy for the treatment of multiple sclerosis.

Production of chimeric adenovirus.

The use of chimeric pseudotyped vectors is a common way to modify the adenoviral tropism by replacing the fiber protein. In this chapter the procedure to generate a chimeric adenovirus pre-stock from a plasmid containing the adenoviral genome is described. Also, the chimeric adenovirus replicative cycle to increase the yield in further productions is determined. Finally, two different protocols, in culture plates and in suspension cultures, to produce the virus at large scale are also detailed.