High-Level Expression of Alkaline Phosphatase by Adeno-Associated Virus Vector Ameliorates Pathological Bone Structure in a Hypophosphatasia Mouse Model.

Hypophosphatasia (HPP) is a systemic skeletal disease caused by mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). We recently reported that survival of HPP model mice can be prolonged using an adeno-associated virus (AAV) vector expressing bone-targeted TNALP with deca-aspartate at the C terminus (TNALP-D10); however, abnormal bone structure and hypomineralization remained in the treated mice. Here, to develop a more effective and clinically applicable approach, we assessed whether transfection with TNALP-D10 expressing virus vector at a higher dose than previously used would ameliorate bone structure defects. We constructed a self-complementary AAV8 vector expressing TNALP driven by the chicken beta-actin (CBA) promoter (scAAV8-CB-TNALP-D10). The vector was injected into both quadriceps femoris muscles of newborn HPP mice at a dose of 4.5 × 1012 vector genome (v.g.)/body, resulting in 20 U/mL of serum ALP activity. The 4.5 × 1012 v.g./body-treated HPP mice grew normally and displayed improved bone structure at the knee joints in X-ray images. Micro-CT analysis showed normal trabecular bone structure and mineralization. The mechanical properties of the femur were also recovered. Histological analysis of the femurs demonstrated that ALP replacement levels were sufficient to promote normal, growth plate cartilage arrangement. These results suggest that AAV vector-mediated high-dose TNALP-D10 therapy is a promising option for improving the quality of life (QOL) of patients with the infantile form of HPP.

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.

Intraperitoneal AAV9-shRNA inhibits target expression in neonatal skeletal and cardiac muscles.

Systemic injections of AAV vectors generally transduce to the liver more effectively than to cardiac and skeletal muscles. The short hairpin RNA (shRNA)-expressing AAV9 (shRNA-AAV9) can also reduce target gene expression in the liver, but not enough in cardiac or skeletal muscles. Higher doses of shRNA-AAV9 required for inhibiting target genes in cardiac and skeletal muscles often results in shRNA-related toxicity including microRNA oversaturation that can induce fetal liver failure. In this study, we injected high-dose shRNA-AAV9 to neonates and efficiently silenced genes in cardiac and skeletal muscles without inducing liver toxicity. This is because AAV is most likely diluted or degraded in the liver than in cardiac or skeletal muscle during cell division after birth. We report that this systemically injected shRNA-AAV method does not induce any major side effects, such as liver dysfunction, and the dose of shRNA-AAV is sufficient for gene silencing in skeletal and cardiac muscle tissues. This novel method may be useful for generating gene knockdown in skeletal and cardiac mouse tissues, thus providing mouse models useful for analyzing diseases caused by loss-of-function of target genes.