Effects of Altering HSPG Binding and Capsid Hydrophilicity on Retinal Transduction by AAV.

Adeno-associated viruses (AAVs) have recently emerged as the leading vector for retinal gene therapy. However, AAV vectors which are capable of achieving clinically relevant levels of transgene expression and widespread retinal transduction are still an unmet need. Using rationally designed AAV2-based capsid variants, we investigate the role of capsid hydrophilicity and hydrophobicity as it relates to retinal transduction.

Novel AAV44.9-Based Vectors Display Exceptional Characteristics for Retinal Gene Therapy.

The majority of inherited retinal diseases (IRDs) are caused by mutations in genes expressed in photoreceptors (PRs). The ideal vector to address these conditions is one that transduces PRs in large areas of retina with the smallest volume/lowest titer possible, and efficiently transduces foveal cones, the cells responsible for acute, daylight vision that are often the only remaining area of functional retina in IRDs. The purpose of our study was to evaluate the retinal tropism and potency of a novel capsid, AAV44.

Development and characterization of "push-pull" sampling device with fast reaction quenching coupled to high-performance liquid chromatography for pharmaceutical process analytical technologies.

A push-pull sampling system interfaced on-line to high-performance liquid chromatography (HPLC) was developed for micro-volume real-time monitoring of reaction mixtures. The device consists of concentric tubes wherein sample was continuously withdrawn through the outer tube and reaction quenchant continuously delivered through a recessed inner tube. The device allowed sampling rates of 0.

Continuous-flow enzyme assay on a microfluidic chip for monitoring glycerol secretion from cultured adipocytes.

A dual-chip microfluidic platform that coupled perfusion of cultured adipocytes with on-line fluorescence-based enzyme assay was developed to monitor glycerol secretions in real time from cultured adipocytes. The perfusion cell chip, which could be reversibly sealed to allow reloading of cells and reuse of the chip, was shown by modeling to generate low shear stress on the cells under study. Effluent from the perfusion chip was pumped into an enzyme assay chip for monitoring of secretion from the cells.

Assay for glucosamine 6-phosphate using a ligand-activated ribozyme with fluorescence resonance energy transfer or CE-laser-induced fluorescence detection.

A naturally occurring aptazyme, the glmS ribozyme, is adapted to an assay for glucosamine 6-phosphate, an effector molecule for the aptazyme. In the assay, binding of analyte allosterically activates aptazyme to cleave a fluorescently labeled oligonucleotide substrate. The extent of reaction, and hence analyte concentration, is detected by either fluorescence resonance energy transfer (FRET) or capillary electrophoresis with laser-induced fluorescence (CE-LIF).

Sampling and electrophoretic analysis of segmented flow streams using virtual walls in a microfluidic device.

A method for sampling and electrophoretic analysis of aqueous plugs segmented in a stream of immiscible oil is described. In the method, an aqueous buffer and oil stream flow parallel to each other to form a stable virtual wall in a microfabricated K-shaped fluidic element. As aqueous sample plugs in the oil stream make contact with the virtual wall, coalescence occurs and sample is electrokinetically transferred to the aqueous stream.

Improved temporal resolution for in vivo microdialysis by using segmented flow.

Microdialysis sampling probes were interfaced to a segmented flow system to improve temporal resolution for monitoring concentration dynamics. Aqueous dialysate was segmented into nanoliter plugs by pumping sample stream into the base of a tee channel structure microfabricated on a PDMS chip that had an immiscible carrier phase (perfluorodecalin) pumped into the cross arm of the tee. Varying the oil flow rate from 0.