No abstract

The retina is an ideal target for gene therapy because of its easy accessibility and limited immunological response. We previously reported that intravitreally injected adeno-associated virus (AAV) vector transduced the inner retina with high efficiency in a rodent model. In large animals, however, the efficiency of retinal transduction was low, because the vitreous and internal limiting membrane (ILM) acted as barriers to transduction. To overcome these barriers in cynomolgus monkeys, we performed vitrectomy (VIT) and ILM peeling before AAV vector injection. Following intravitreal injection of 50 μL triple-mutated self-complementary AAV serotype 2 vector encoding EGFP, transduction efficiency was analyzed. Little expression of GFP was detected in the control and VIT groups, but in the VIT+ILM group, strong GFP expression was detected within the peeled ILM area. To detect potential adverse effects, we monitored the retinas using color fundus photography, optical coherence tomography, and electroretinography. No serious side effects associated with the pretreatment were observed. These results indicate that surgical ILM peeling before AAV vector administration would be safe and useful for efficient transduction of the nonhuman primate retina and provide therapeutic benefits for the treatment of retinal diseases.

Hypophosphatasia (HPP) is an inherited disease caused by a deficiency of tissue-nonspecific alkaline phosphatase (TNALP). The major symptom of human HPP is hypomineralization, rickets, or osteomalacia, although the clinical severity is highly variable. The phenotypes of TNALP knockout (Akp2

Hypophosphatasia (HPP) is an inherited systemic skeletal disease caused by mutations in the gene encoding the tissue-nonspecific alkaline phosphatase (TNALP) isozyme. The clinical severity of HPP varies widely, with symptoms including rickets and osteomalacia. TNALP knockout (Akp2−/−) mice phenotypically mimic the severe infantile form of HPP; that is, TNALP-deficient mice are born with a normal appearance but die by 20 days of age owing to growth failure, hypomineralization, and epileptic seizures. In this study, a lentiviral vector expressing a bone-targeted form of TNALP was injected into the jugular vein of newborn Akp2−/− mice. We found that alkaline phosphatase activity in the plasma of treated Akp2−/− mice increased and remained at high levels throughout the life of the animals. The treated Akp2−/− mice survived for more than 10 months and demonstrated normal physical activity and a healthy appearance. Epileptic seizures were completely inhibited in the treated Akp2−/− mice, and X-ray examination of the skeleton showed that mineralization was significantly improved by the gene therapy. These results show that severe infantile HPP in TNALP knockout mice can be treated with a single injection of lentiviral vector during the neonatal period. © 2011 American Society for Bone and Mineral Research.

Background-We previously identified INT6/eIF3e as a novel regulator of hypoxia-inducible factor 2␣ (HIF2␣) activity. Small interfering RNA (siRNA)-Int6 adequately stabilized HIF2␣, even under normoxic conditions, and thereby enhanced the expression of several angiogenic factors in vitro, suggesting that siRNA-Int6 may induce angiogenesis in vivo. Methods and Results-We demonstrated a 6-to 8-fold enhanced formation of normal arteries and veins in the subcutaneous regions of adult mice 5 days after a single siRNA-Int6 application. Subcutaneous fibroblasts were identified as the major source of secreted angiogenic factors that led to the formation of functional vessels during Int6 silencing. Fibroblasts transfected ex vivo with siRNA-Int6 induced potent neoangiogenesis when transplanted into a subcutaneous region of nude mice. Application of siRNA-Int6 promoted neoangiogenesis in the area surrounding the injury in wound healing models, including genetically diabetic mice, thereby accelerating the closure of the injury. HIF2␣ accumulation caused by siRNA-Int6 was confirmed as the unequivocal cause of the angiogenesis by an in vivo angiogenesis assay. Further analysis of the Int6 silencing-induced neoangiogenesis revealed that a negative feedback regulation of HIF2␣ stability was caused by HIF2␣-induced transcription of Int6 via hypoxia-response elements in its promoter. Thus, siRNA-Int6 temporarily facilitates an accumulation of HIF2␣ protein, leading to hypoxia-independent transcription of angiogenic factors and concomitant neoangiogenesis. Conclusions-We suggest that the pathway involving INT6/HIF2␣ acts as a hypoxia-independent master switch of functional angiogenesis; therefore, siRNA-Int6 application might be of clinical value in treating ischemic diseases such as heart and brain ischemia, skin injury, and diseases involving obstructed vessels. (Circulation. 2010;122:910-919.)Key Words: angiogenesis Ⅲ hypoxia Ⅲ molecular biology Ⅲ signal transduction Ⅲ siRNA T he process of vessel formation is complex but well coordinated, involving the combined action of numerous growth factors and related signaling pathways. 1 Nevertheless, a single angiogenic factor such as vascular endothelial growth factor (VEGF), 2 fibroblast growth factor (FGF), 3,4 or plateletderived growth factor 5 can induce neoangiogenesis, 6 -8 albeit with incomplete and leaky vessels. 6,9 The transgenic expression of angiopoietin-1 (ANG-1) and VEGF significantly increases both the size and number of blood vessels. 8 These results suggest that several angiogenic factors are essential for the formation of functional vessels and that they must be expressed in a complementary and coordinated manner 10 to strike a balance among many stimulatory and inhibitory signals. 11 Clinical Perspective on p 919The expression of various angiogenic factors such as VEGF, ANG-1, and pleiotrophin 12 is triggered by hypoxia through the action of hypoxia-inducible factors (HIFs); these angiogenic factors play important roles in blood vessel formation and oncogenesis. 13 ...