A drug-tunable Flt23k gene therapy for controlled intervention in retinal neovascularization.

Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.

Surfactin structures at interfaces and in solution: the effect of pH and cations.

We have investigated the interfacial and bulk phase structures of surfactin at different pH and in the presence of mono/divalent cations using neutron scattering techniques. Neutron reflectivity profiles were recorded at the air/water and sapphire/water interfaces as a function of pH and ionic strength. The air/water results show that surfactin has a hydrophobic ball-like structure and that changes in pH and cations lead to changes in the area per molecule and hydrophilicity of surfactin. However the adsorption of surfactin on the hydrophilic sapphire/water interface is highly pH dependent because of electrostatic interactions with the surface. The bulk phase structures were characterized by small angle neutron scattering (SANS) and are more sensitive to pH and cation than the interfacial structure. At high pH surfactin forms micellar structures with low aggregation numbers, but at low pH values the bulk phase structure becomes rod-like at pH 6.5 and lamellar at pH 5.5. The addition of cations in the subphase tends to neutralize the two acidic groups of the peptide ring and the neutralization seems to be more complete for divalent cations than for monovalent cations.