Global diffuse distribution in the brain and efficient gene delivery to the dorsal root ganglia by intrathecal injection of adeno-associated viral vector serotype 1.

BACKGROUND: The success of gene therapy for inherited neurodegenerative diseases such as metachromatic leukodystrophy (MLD) depends on the development of efficient gene delivery throughout the brain guarded by the blood-brain barrier and achieves distribution of the deficient enzyme throughout the brain. Direct injection of viral vector into the brain parenchyma is too invasive and may not be sufficient to treat the entire brain. As an alternative approach, we examined the feasibility of intrathecal (IT) injection of adeno-associated viral vector serotype 1 (AAV1). METHODS: AAV1 vector expressing arylsulfatase A (ASA) and green fluorescence protein (GFP) was intrathecally injected into ASA knockout MLD model mice. Expression of GFP was assessed by fluorescence microscopy and immunohistochemical methods, whereas the concentration of ASA was determined by a quantitative enzyme-linked immunosorbent assay. RESULTS: Broad distribution of GFP expression was seen throughout the brain after IT injection of AAV1 vector. In addition, a large number of nerve fibers in the dorsal spinal cord and many neural cell bodies in the dorsal root ganglia were efficiently transduced. Widespread distribution of ASA activity and a significant reduction of sulfatide content were confirmed in treated MLD model mice. CONCLUSIONS: IT injection of AAV1 vector is a useful and non-invasive method for widespread gene delivery to the brain and dorsal root ganglia.

AAV1 mediated co-expression of formylglycine-generating enzyme and arylsulfatase a efficiently corrects sulfatide storage in a mouse model of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA) and is characterized by deposition of sulfatide in all organs, particularly the nervous system. Recently, formylglycine-generating enzyme (FGE) was found to be essential for activation of sulfatases. This study examined the utility of FGE co-expression in AAV type 1 vector (AAV1)-mediated gene therapy of ASA knockout (MLD) mice. AAV1-ASA alone or AAV1-ASA and AAV1-FGE were co-injected into a single site of the hippocampus. Enzyme assay and immunohistochemical analysis showed that ASA was detected not only in the injected hemisphere but also in the non-injected hemisphere by 7 months after injection. Level of ASA activity and extent of ASA distribution were significantly enhanced by co-introduction of AAV1-FGE. Marked reductions in sulfatide levels were observed throughout the entire brain. The unexpectedly widespread distribution of ASA may be due to a combination of diffusion in extracellular spaces, transport through axons, and circulation in cerebrospinal fluid. The rotarod test revealed improvement of neurological functions. These results demonstrate that direct injection of AAV1 vectors expressing ASA and FGE represents a highly promising approach with significant implications for the development of clinical protocols for MLD gene therapy.

Correlation between dry eye and rheumatoid arthritis activity.

PURPOSE: To evaluate the incidence of dry eye in rheumatoid arthritis (RA) patients with or without Sjögren syndrome (SS), and to investigate the correlation between dry eye and RA activity. DESIGN: Prospective case-control study. METHODS: In 72 RA patients, the severity of dry eye was assessed by the Schirmer test, tear break-up time, rose bengal staining, and fluorescein staining. The RA activity was evaluated by the Lansbury index (LI), which is based on the duration of morning stiffness, erythrocyte sedimentation rate (ESR), grip strength, and joint score. RESULTS: Ten percent of patients met the Japanese criteria for SS. No difference in dry eye tests or LI was observed between SS patients and non-SS patients. Even in the non-SS group, 90% of patients were diagnosed with probable dry eye. In SS patients, positive correlations were observed between LI and Schirmer test (P = .048), ESR and Schirmer test (P = .035), ESR and rose bengal staining (P = .001), and grip strength and rose bengal staining (P = .047). No such correlations were observed in the non-SS patients. CONCLUSIONS: Dry eye is common in RA patients, including those without SS. We found that there was a correlation between LI and Schirmer test in RA patients with SS, but no correlation when the entire group was analyzed. Dry eye always should be taken into consideration regardless of the RA activity, because the severity of dry eye is independent of RA activity.

Systemic cancer gene therapy using adeno-associated virus type 1 vector expressing MDA-7/IL24.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL24), selectively induces apoptosis in cancer cells without harming normal cells. It also exerts immunomodulatory and antiangiogenic effects, as well as potent antitumor bystander effects, making it an ideal candidate for a new anticancer gene therapy. Here, we examined the feasibility of adeno-associated virus type 1 (AAV1) vector-mediated systemic gene therapy using mda-7/IL24. In vitro studies showed that medium conditioned by AAV1-mda7-transducedC2C12 cells induces tumor cell-specific apoptosis and inhibits angiogenesis in a human umbilical vein endothelial cell tube formation assay. To assess the in vivo effects of AAV1-mediated systemic delivery of MDA-7/IL24, we generated a subcutaneous tumor model by injecting Ehrlich ascites tumor cells into the dorsum of DDY mice. A single intravenous injection of AAV1-mda7 (2.0 x 10(11) viral genomes) significantly inhibited tumor growth. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical analyses showed significant induction of tumor-cell-specific apoptosis and reduction of microvessel formation within the tumors, and there was a significant increase in survival among the AAV1-mda7-treated mice. These results clearly demonstrate that continuous systemic delivery of MDA-7/IL24 can serve as an effective treatment for cancer. Thus, AAV1 vector-mediated systemic delivery of MDA-7/IL24 represents a potentially important new approach to anticancer therapy.