Tyrosine triple mutated AAV2-BDNF gene therapy in a rat model of transient IOP elevation.

PURPOSE: We examined the neuroprotective effects of exogenous brain-derived neurotrophic factor (BDNF), which provides protection to retinal ganglion cells (RGCs) in rodents, in a model of transient intraocular pressure (IOP) elevation using a mutant (triple Y-F) self-complementary adeno-associated virus type 2 vector encoding BDNF (tm-scAAV2-BDNF). METHODS: The tm-scAAV2-BDNF or control vector encoding green fluorescent protein (GFP; tm-scAAV2-GFP) was intravitreally administered to rats, which were then divided into four groups: control, ischemia/reperfusion (I/R) injury only, I/R injury with tm-scAAV2-GFP, and tm-scAAV2-BDNF. I/R injury was then induced by transiently increasing IOP, after which the rats were euthanized to measure the inner retinal thickness and cell counts in the RGC layer. RESULTS: Intravitreous injection of tm-scAAV2-BDNF resulted in high levels of BDNF expression in the neural retina. Histological analysis showed that the inner retinal thickness and cell numbers in the RGC layer were preserved after transient IOP elevation in eyes treated with tm-scAAV2-BDNF but not in the other I/R groups. Significantly reduced glial fibrillary acidic protein (GFAP) immunostaining after I/R injury in the rats that received tm-scAAV2-BDNF indicated reduced retinal stress, and electroretinogram (ERG) analysis confirmed preservation of retinal function in the tm-scAAV2-BDNF group. CONCLUSIONS: These results demonstrate the feasibility and effectiveness of neuroprotective gene therapy using tm-scAAV2-BDNF to protect the inner retina from transiently high intraocular pressure. An in vivo gene therapeutic approach to the clinical management of retinal diseases in conditions such as glaucoma, retinal artery occlusion, hypertensive retinopathy, and diabetic retinopathy thus appears feasible.

Treatment of hypophosphatasia by muscle-directed expression of bone-targeted alkaline phosphatase via self-complementary AAV8 vector.

Hypophosphatasia (HPP) is an inherited disease caused by genetic mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). This results in defects in bone and tooth mineralization. We recently demonstrated that TNALP-deficient (Akp2 (-/-) ) mice, which mimic the phenotype of the severe infantile form of HPP, can be treated by intravenous injection of a recombinant adeno-associated virus (rAAV) expressing bone-targeted TNALP with deca-aspartates at the C-terminus (TNALP-D10) driven by the tissue-nonspecific CAG promoter. To develop a safer and more clinically applicable transduction strategy for HPP gene therapy, we constructed a self-complementary type 8 AAV (scAAV8) vector that expresses TNALP-D10 via the muscle creatine kinase (MCK) promoter (scAAV8-MCK-TNALP-D10) and examined the efficacy of muscle-directed gene therapy. When scAAV8-MCK-TNALP-D10 was injected into the bilateral quadriceps of neonatal Akp2 (-/-) mice, the treated mice grew well and survived for more than 3 months, with a healthy appearance and normal locomotion. Improved bone architecture, but limited elongation of the long bone, was demonstrated on X-ray images. Micro-CT analysis showed hypomineralization and abnormal architecture of the trabecular bone in the epiphysis. These results suggest that rAAV-mediated, muscle-specific expression of TNALP-D10 represents a safe and practical option to treat the severe infantile form of HPP.

Evaluation of Rhubarb using antioxidative activity as an index of pharmacological usefulness.

We attempted to estimate the pharmacological activity by measuring the concentrations of a composition ingredient using a multivariate statistical analysis method. Medicinal herb of Rhubarb has been many largely unrecognized biochemical and pharmacological effect components. Therefore, we attempted to estimate the antioxidative activity of Rhubarb on low-density lipoprotein (LDL) of its components. Thirty specimens of Rhubarb from various origins were used, chose nine components of anthraquinones, two components of anthrones, two components of flavan-3-ols, one component of procyanidin, one component of naphthalene, two components of phenylbutanones and one component of stilbene. Quantitative analysis of 18 components was performed with high-performance liquid chromatography (HPLC) and antioxidative activities were measured with plasma taken from spontaneous familial hypercholesterolemia model rabbits. There was considerable variation among the specimens in the concentration of components and antioxidative activities on LDL. As a result of multiple regression analysis, significant multiple correlation coefficient for antioxidative activities on LDL (R=0.914, P<0.01) was found in relation to the concentrations of five components: aloe-emodin, chrysophanol, emodin 1-O-beta-D-glucoside, lindleyin and 6-hydroxymusizin 8-O-beta-D-glucoside. Three of the five components were not active in promoting antioxidative activity and there was no significant correlation between the concentrations of the most active component and the activity. We consider this a useful method for selecting of Rhubarb and propose a new scientific approach for the selection of natural medicines.