Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs.

Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions.

Photoreceptor-targeted gene delivery using intravitreally administered AAV vectors in dogs.

Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, photoreceptor transduction efficiency following intravitreal injection (IVT) has improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Thickness of the inner limiting membrane (ILM) in large animals is thought to impair retinal penetration by AAV. Our study compared two newly developed AAV vectors containing multiple capsid amino acid substitutions following IVT in dogs. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. AAV vectors containing the interphotoreceptor-binding protein (IRBP) promoter drove expression exclusively in rod and cone photoreceptors, with transduction efficiencies of ~4% of cones and 2% of rods. Notably, in the central region containing the cone-rich visual streak, 15.6% of cones were transduced. Significant regional variation existed, with lower transduction efficiencies in the temporal regions of all eyes. This variation did not correlate with ILM thickness. Vectors carrying a cone-specific promoter failed to transduce a quantifiable percentage of cone photoreceptors. The newly developed AAV vectors containing the IRBP promoter were capable of producing photoreceptor-specific transgene expression following IVT in the dog.

Solute washout experiments for characterizing mass transport in hollow fiber immunoisolation membranes.

The transport characteristics of immunoisolation membranes can have a critical effect on the design of hybrid artificial organs and cell therapies. However, it has been difficult to quantitatively evaluate the desired transport properties of different hollow fiber membranes due to bulk mass transfer limitations in the fiber lumen and annular space. An attractive alternative to existing methodologies is to use the rate of solute removal or "washout" from the annular space during constant flow perfusion through the fiber lumen. Experimental washout curves were obtained for glucose and a 10 kD dextran in two different hollow fiber devices. Data were analyzed using a theoretical model which accounts for convective and diffusive transport in the lumen, membrane, and annular space. The model was in good agreement with the experimental results and provided an accurate measure of the effective membrane diffusion coefficient for both small and large solutes. This approach should prove useful in theoretical analyses of solute transport and performance of hollow fiber artificial organs.

Analysis of protein fouling during ultrafiltration using a two-layer membrane model.

Protein fouling can significantly alter both the flux and retention characteristics of ultrafiltration membranes. There has, however, been considerable controversy over the nature of this fouling layer. In this study, hydraulic permeability and dextran sieving data were obtained both before and after albumin adsorption and/or filtration using polyethersulfone ultrafiltration membranes. The dextran molecular weight distributions were analyzed by gel permeation chromatography to evaluate the sieving characteristics over a broad range of solute size. Protein fouling caused a significant reduction in the dextran sieving coefficients, with very different effects seen for the diffusive and convective contributions to dextran transport. The changes in dextran sieving coefficients and diffusive permeabilities were analyzed using a two-layer membrane model in which a distinct protein layer is assumed to form on the upstream surface of the membrane. The data suggest that the protein layer formed during filtration was more tightly packed than that formed by simple static adsorption. Hydrodynamic calculations indicated that the pore size of the protein layer remained relatively constant throughout the adsorption or filtration, but the thickness of this layer increased with increasing exposure time. These results provide important insights into the nature of protein fouling during ultrafiltration and its effects on membrane transport.

A two layer model for the effects of blood contact on membrane transport in artificial organs.

The performance of many artificial organs can be strongly affected by the transport characteristics of the semi-permeable membranes used in these devices, but there is little data on the effects of blood contact on membrane transport properties. Experimental data were obtained for the solute flux through cellulosic, polyacrylonitrile, and polyethersulfone membranes using polydispersed dextrans. Blood contact had a very large effect on diffusive solute transport through the asymmetric polyethersulfone membranes, but only a small effect on diffusion through the symmetric AN69 and Cuprophan membranes. In contrast, blood contact caused a similar reduction in convective solute transport (sieving) through both the polyethersulfone and AN69 membranes. Convective transport through the blood contacted membranes was also dependent on the flow direction, with greater transport obtained when the membrane was oriented with the blood contacted surface downstream. These data were analyzed using a two layer membrane model consisting of an upper layer of blood cells and proteins adsorbed to the surface of the native membrane. This model accurately accounted for the different effects of blood contact on convection and diffusion, as well as the observed asymmetry in convective solute transport. These results have important implications for the analysis of solute transport in artificial organs.

The effect of some divalent cations on extracellular polysaccharide synthesis in Streptococcus salivarius.

The amount of extracellular insoluble polysaccharide produced by Streptococcus salivarius can be effected by some divalent cations. Calcium at concentrations of 1 X 10(-3) and 10(-4) M caused a reduction in polysaccharide synthesis. Magnesium at 1 X 10(-3) M inhibited extracellular polysaccharide production but at 1 X 10(-4) M had little effect. Manganese was without effect on polysaccharide synthesis. Zinc at 1 X 10(-3) and 1 X 10(-4) M caused a substantial increase in extracellular polysaccharide synthesis.