Tyrosine triple mutated AAV2-BDNF gene therapy in an inner retinal injury model induced by intravitreal injection of N-methyl-D-aspartate (NMDA).

Purpose: Glaucoma is a group of chronic optic neuropathies characterized by the degeneration of retinal ganglion cells (RGCs) and their axons, and they ultimately cause blindness. Because neuroprotection using neurotrophic factors against RGC loss has been proven a beneficial strategy, extensive attempts have been made to perform gene transfer of neurotrophic proteins. This study used the inner retinal injury mouse model to evaluate the neuroprotective effect of tyrosine triple mutated and self-complementary adeno-associated virus (AAV) encoding brain-derived neurotrophic factor (BDNF; tm-scAAV2-BDNF). Methods: C57BL/6J mice were intravitreally injected with 1 µl of tm-scAAV2-BDNF and its control AAV at a titer of 6.6 E+13 genome copies/ml. Three weeks later, 1 µl of 2 mM N-methyl-D-aspartate (NMDA) was administered in the same way as the viral injection. Six days after the NMDA injection, we assessed the dark-adapted electroretinography (ERG). Mice were sacrificed at one week after the NMDA injection, followed by RNA quantification, protein detection, and histopathological analysis. Results: The RNA expression of BDNF in retinas treated with tm-scAAV2-BDNF was about 300-fold higher than that of its control AAV. Meanwhile, the expression of recombinant BDNF protein increased in retinas treated with tm-scAAV2-BDNF. In addition, histological analysis revealed that tm-scAAV2-BDNF prevented thinning of the inner retina. Furthermore, b-wave amplitudes of the tm-scAAV2-BDNF group were significantly higher than those of the control vector group. Histopathological and electrophysiological evaluations showed that tm-scAAV2-BDNF treatment offered significant protection against NMDA toxicity. Conclusions: Results showed that tm-scAAV2-BDNF-treated retinas were resistant to NMDA injury, while retinas treated with the control AAV exhibited histopathological and functional changes after the administration of NMDA. These results suggest that tm-scAAV2-BDNF is potentially effective against inner retinal injury, including normal tension glaucoma.

Funiculosin variants and phosphorylated derivatives promote innate immune responses via the Toll-like receptor 4/myeloid differentiation factor-2 complex.

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) complex is essential for LPS recognition and induces innate immune responses against Gram-negative bacteria. As activation of TLR4/MD-2 is also critical for the induction of adaptive immune responses, TLR4/MD-2 agonists have been developed as vaccine adjuvants, but their efficacy has not yet been ascertained. Here, we demonstrate that a funiculosin (FNC) variant, FNC-RED, and FNC-RED and FNC derivatives are agonists for both murine and human TLR4/MD-2. FNC-RED induced nuclear factor-κB (NF-κB) activation via murine TLR4/MD-2, whereas FNC had no TLR4/MD-2 stimulatory activity. Biacore analysis revealed that FNC-RED binds to murine TLR4/MD-2 but not murine radioprotective 105 (RP105)/myeloid differentiation factor-1 (MD-1), another LPS sensor. FNC-RED induced CD14-independent expressions of pro-inflammatory cytokines and co-stimulatory molecules in murine macrophages and dendritic cells. In contrast, FNC-RED stimulation was reduced in CD14-dependent LPS responses, including dimerization and internalization of TLR4/MD-2 and IFN-ß expression. FNC-RED-induced IL-12p40 production from murine dendritic cells was dependent on NF-κB but not MAPK pathway. In addition, fetal bovine serum augmented lipid A-induced NF-κB activation but blocked FNC-RED-mediated responses. Two synthetic phosphate group-containing FNC-RED and FNC derivatives, FNC-RED-P01 and FNC-P01, respectively, activated human TLR4/MD-2, unlike FNC-RED. Finally, computational analysis revealed that this species-specific activation by FNC-RED and FNC-RED-P01 resulted from differences in electrostatic surface potentials between murine and human TLR4/MD-2. We conclude that FNC-RED and its synthetic derivative represent a novel category of murine and human TLR4/MD-2 agonist.

Effect of end groups of poly(n-butyl methacrylate) on its biocompatibility.

Telechelic poly(n-butyl methacrylate)s (PBMAs) with various end groups were prepared using nonionic, anionic, cationic or zwitterionic azo-type radical initiators and cell adhesion onto the surfaces of the polymers was investigated. The tendency for cell adhesion to the polymers differed with and without pretreatment with phosphate-buffered saline (PBS, pH 7.4). The cell adhesion to polymer surfaces without pretreatment was lower than that with pretreatment. The effect of pretreatment with PBS was significant for PBMA with ionic end groups. Furthermore, cell adhesion to the surface of PBMA with zwitterionic end groups was suppressed compared with that to the surfaces of other polymers. It was presumed that positive and negative charges of zwitterionic groups in the same molecule negated each other at pH 7.4 and that the polymers with zwitterionic end groups had no effective charges. The results clearly indicated that biocompatibility of polymers can be changed by the introduction of functional groups at the ends of the polymer chains. Fabrication of functional material surfaces will be anticipated by the similar method in the future.

Anti-biofouling properties of polymers with a carboxybetaine moiety.

The resistance of random copolymers of BMA and CMB against biofouling was evaluated. The amount of proteins adsorbed onto the CMB copolymers was smaller than that onto other polymers (non-ionic polymers and copolymers of ordinary ionic monomers and BMA) and decreased with an increase in the content of CMB residues. Furthermore, there was a dramatic decrease in the number of cells (platelets and fibroblasts) that adhered to the CMB copolymers compared with that to other polymers. In contrast with this, CMB copolymers were slightly perturbative to both complement and coagulation systems. However, the overall results suggest that zwitterionic moieties are effective for making polymer materials biocompatible due to their excellent anti-biofouling property.

Effect of zwitterionic polymers on wound healing.

The effect of a thin film of a zwitterionic random copolymer composed of carboxybetaine [1-carboxy-N,N-dimethyl-N-(2'-methacryloyloxyethyl)methanaminium inner salt] (CMB) and n-butyl methacrylate (BMA), poly(CMB-r-BMA) (CMB, 30 mol%), on the healing of a full-thickness excisional and incisional wound in hairless rats was examined. The poly(CMB-r-BMA) film significantly enhanced wound closure and complete healing of a full-thickness excisional wound compared with the effect of the poly(n-butyl methacrylate) (PBMA) and the poly(ethylene terephthalate) (PET) films. However, the poly(CMB-r-BMA) film did not enhance healing of a full-thickness incisional wound in hairless rats. The amount of proteins adsorbed and that of neutrophiles adhered onto the poly(CMB-r-BMA) film were significantly smaller than those onto the PBMA and PET films. The results suggested that various cells and growth factors in the wound exudate are utilized effectively by covering an excisional wound with the poly(CMB-r-BMA) film, resulting in acceleration of healing. In addition, the poly(CMB-r-BMA) film significantly enhanced healing of a full-thickness excisional wound in hairless rats compared with the effect of Tegaderm as wound dressings. The poly(CMB-r-BMA) film has potential as a new wound dressing.