水上 浩明

Background Inflammation plays an important role in the pathogenesis of hypertension and hypertensive organ damage. Interleukin (IL)-10, a pleiotropic anti-inflammatory cytokine, exerts vasculoprotective effects in many animal models. In the present study, we examined the preventive effects of adeno-associated virus (AAV) vector-mediated sustained IL-10 expression against hypertensive heart disease and renal dysfunction in Dahl salt-sensitive rats. Methods We injected the rats intramuscularly with an AAV type I-based vector encoding rat IL-10 or enhanced green fluorescent protein (EGFP) at 5 weeks of age; subsequently, the rats were fed a high-sodium diet from 6 weeks of age. Results Sustained IL-10 expression significantly improved survival rate of Dahl salt-sensitive rats compared with EGFP expression (62.5% versus 0%, p < 0.001); it also caused 26.0% reduction in systolic blood pressure at 15 weeks (p < 0.0001). Echocardiography exhibited a 22.0% reduction in hypertrophy (p < 0.0001) and a 26.3% improvement in fractional shortening (p < 0.0001) of the rat left ventricle in the IL-10 group compared to the EGFP group. IL-10 expression also caused a 21.7% decrease in the heart weight/body weight index and cardiac atrial natriuretic peptide levels. Histopathological studies revealed that IL-10 decreased inflammatory cell infiltration, fibrosis, and transforming growth factor-P, levels in the failing heart. Furthermore, IL-10 expression significantly reduced urine protein excretion with increased glomerular filtration rates. Conclusions This is the first study to demonstrate that the anti-inflammatory cytokine IL-10 has a significant anti-hypertensive effect. AAV vector-mediated IL-10 expression potentially prevents the progression of refractory hypertension and hypertensive organ damage in humans. Copyright (c) 2008 John Wiley & Sons, Ltd.

Background Inflammation plays an important role in the pathogenesis of hypertension and hypertensive organ damage. Interleukin (IL)-10, a pleiotropic anti-inflammatory cytokine, exerts vasculoprotective effects in many animal models. In the present study, we examined the preventive effects of adeno-associated virus (AAV) vector-mediated sustained IL-10 expression against hypertensive heart disease and renal dysfunction in Dahl salt-sensitive rats. Methods We injected the rats intramuscularly with an AAV type I-based vector encoding rat IL-10 or enhanced green fluorescent protein (EGFP) at 5 weeks of age; subsequently, the rats were fed a high-sodium diet from 6 weeks of age. Results Sustained IL-10 expression significantly improved survival rate of Dahl salt-sensitive rats compared with EGFP expression (62.5% versus 0%, p < 0.001); it also caused 26.0% reduction in systolic blood pressure at 15 weeks (p < 0.0001). Echocardiography exhibited a 22.0% reduction in hypertrophy (p < 0.0001) and a 26.3% improvement in fractional shortening (p < 0.0001) of the rat left ventricle in the IL-10 group compared to the EGFP group. IL-10 expression also caused a 21.7% decrease in the heart weight/body weight index and cardiac atrial natriuretic peptide levels. Histopathological studies revealed that IL-10 decreased inflammatory cell infiltration, fibrosis, and transforming growth factor-P, levels in the failing heart. Furthermore, IL-10 expression significantly reduced urine protein excretion with increased glomerular filtration rates. Conclusions This is the first study to demonstrate that the anti-inflammatory cytokine IL-10 has a significant anti-hypertensive effect. AAV vector-mediated IL-10 expression potentially prevents the progression of refractory hypertension and hypertensive organ damage in humans. Copyright (c) 2008 John Wiley & Sons, Ltd.

Since standard aminoglycoside treatment progressively causes hearing disturbance with hair cell degeneration, systemic use of the drugs is limited. Adeno-associated virus (AAV)-based vectors have been of great interest because they mediate stable transgene expression in a variety of postmitotic cells with minimal toxicity. In this study, we investigated the effects of regulated AAV1-mediated glial cell line-derived neurotrophic factor (GDNF) expression in the cochlea on aminoglycoside-induced damage. AAV1-based vectors encoding GDNF or vectors encoding GDNF with an rtTA2s- S2 Tet- on regulation system were directly microinjected into the rat cochleae through the round window at 5 x 10(10) genome copies/body. Seven days after the virus injection, a dose of 333 mg/kg of kanamycin was subcutaneously given twice daily for 12 consecutive days. GDNF expression in the cochlea was confirmed and successfully modulated by the Tet-on system. Monitoring of the auditory brain stem response revealed an improvement of cochlear function after GDNF transduction over the frequencies tested. Damaged spiral ganglion cells and hair cells were significantly reduced by GDNF expression. Our results suggest that AAV1-mediated expression of GDNF using a regulatedexpression system in the cochlea is a promising strategy to protect the cochlea from aminoglycoside-induced damage.

The main mode of progression of ovarian cancer is peritoneal dissemination, and its inhibition may lead to improved outcome. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) reportedly inhibits the proliferation, migration, and invasion of cancer cells. The purpose of this study is to explore the possibility of PTEN gene therapy for ovarian cancer. We transfected the ovarian cancer cell line SHIN-3 [vascular endothelial growth factor (VEGF) - hypersecretory cell line] with PTEN or luciferase (LUC) - expressing plasmid. After selection, PTEN-overexpressing cells (SHIN-3/PTEN) and control cells (SHIN-3/LUC) were obtained. SHIN-3/PTEN implanted s.c. into nude mice was examined for the change in tumor diameter and the number of new blood vessels. Mice with peritoneally disseminated tumors created by i.p. inoculation of the same cells were examined for changes in body weight and abdominal circumference and for survival time. The growth of s.c. SHIN-3/PTEN was significantly lower than that of control (P < 0.001). Compared with controls, mice with i.p. inoculated SHIN-3/PTEN showed significantly smaller increases in the body weight and abdominal circumference (P < 0.01) and a significantly longer survival time (P < 0.05). VEGF concentration in the supernatant of SHIN-3/PTEN was about half that of controls (P < 0.05). The number of new blood vessels in SHIN-3/PTEN was significantly smaller than that in controls (P < 0.001). Overexpression of PTEN suppressed tumor growth and peritoneal dissemination of VEGF-hypersecretory ovarian cancer cells and prolonged the survival time of the mice with peritoneal disseminated tumor. PTEN gene therapy could have therapeutic potential for ovarian cancer and exerts some of this effect by inhibiting angiogenesis.

Since standard aminoglycoside treatment progressively causes hearing disturbance with hair cell degeneration, systemic use of the drugs is limited. Adeno-associated virus (AAV)-based vectors have been of great interest because they mediate stable transgene expression in a variety of postmitotic cells with minimal toxicity. In this study, we investigated the effects of regulated AAV1-mediated glial cell line-derived neurotrophic factor (GDNF) expression in the cochlea on aminoglycoside-induced damage. AAV1-based vectors encoding GDNF or vectors encoding GDNF with an rtTA2s- S2 Tet- on regulation system were directly microinjected into the rat cochleae through the round window at 5 x 10(10) genome copies/body. Seven days after the virus injection, a dose of 333 mg/kg of kanamycin was subcutaneously given twice daily for 12 consecutive days. GDNF expression in the cochlea was confirmed and successfully modulated by the Tet-on system. Monitoring of the auditory brain stem response revealed an improvement of cochlear function after GDNF transduction over the frequencies tested. Damaged spiral ganglion cells and hair cells were significantly reduced by GDNF expression. Our results suggest that AAV1-mediated expression of GDNF using a regulatedexpression system in the cochlea is a promising strategy to protect the cochlea from aminoglycoside-induced damage.

Purpose Adeno-associated virus (AAV) vectors can achieve long-term gene expression and are now feasible for use in human gene therapy. We constructed hepatocyte growth factor (HGF) expressing AAV (AAV5-HGF) and examined its effect in two mouse hepatic fibrosis models. Methods A model of hepatic fibrosis was established by carbon tetrachloride (CCl4) administration in Balb/c mice. After the establishment of liver fibrosis, AAV5-HGF was injected once into the portal vein. Mice were killed 3, 6, 9, and 12 weeks after injection. Another model was established by bile duct ligation (BDL). Seven weeks after AAV5-HGF injection, mice underwent BDL, and were then killed 2 weeks after BDL. Results Mice that received AAV5-HGF achieved stable HGF expression both in the serum and liver for at least 12 weeks. In both models, significant improvement of the liver fibrosis was found in all mice receiving AAV5-HGF based on Azan-Mallory staining. Suppression of hepatic stellate cells (HSC) was confirmed by immunohistochemistry. Fibrogenic markers were significantly suppressed and collagenase activity increased in the livers of mice receiving AAV5-HGF. Conclusions A single injection of AAV vector containing HGF gene achieved long-term expression of HGF and resulted in resolution of mouse liver fibrosis. HGF gene therapy mediated by AAV is feasible for the treatment of liver fibrosis.

The main mode of progression of ovarian cancer is peritoneal dissemination, and its inhibition may lead to improved outcome. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) reportedly inhibits the proliferation, migration, and invasion of cancer cells. The purpose of this study is to explore the possibility of PTEN gene therapy for ovarian cancer. We transfected the ovarian cancer cell line SHIN-3 [vascular endothelial growth factor (VEGF) - hypersecretory cell line] with PTEN or luciferase (LUC) - expressing plasmid. After selection, PTEN-overexpressing cells (SHIN-3/PTEN) and control cells (SHIN-3/LUC) were obtained. SHIN-3/PTEN implanted s.c. into nude mice was examined for the change in tumor diameter and the number of new blood vessels. Mice with peritoneally disseminated tumors created by i.p. inoculation of the same cells were examined for changes in body weight and abdominal circumference and for survival time. The growth of s.c. SHIN-3/PTEN was significantly lower than that of control (P < 0.001). Compared with controls, mice with i.p. inoculated SHIN-3/PTEN showed significantly smaller increases in the body weight and abdominal circumference (P < 0.01) and a significantly longer survival time (P < 0.05). VEGF concentration in the supernatant of SHIN-3/PTEN was about half that of controls (P < 0.05). The number of new blood vessels in SHIN-3/PTEN was significantly smaller than that in controls (P < 0.001). Overexpression of PTEN suppressed tumor growth and peritoneal dissemination of VEGF-hypersecretory ovarian cancer cells and prolonged the survival time of the mice with peritoneal disseminated tumor. PTEN gene therapy could have therapeutic potential for ovarian cancer and exerts some of this effect by inhibiting angiogenesis.

Pulmonary arterial hypertension (PAH) is a fatal disease associated with inflammation and pathological remodeling of the pulmonary artery (PA). Interleukin (IL)-10 is a pleiotropic antiinflammatory cytokine with vasculoprotective properties. Here, we report the preventive effects of IL-10 on monocrotaline-induced PAH. Three-week-old Wistar rats were intramuscularly injected with an adeno-associated virus serotype 1 vector expressing IL-10, followed by monocrotaline injection at 7 weeks old. IL-10 transduction significantly improved survival rates of the PAH rats 8 weeks after monocrotaline administration compared with control gene transduction (75% versus 0%, P < 0.01). IL-10 also significantly reduced mean PA pressure (22.8 +/- 1.5 versus 29.7 +/- 2.8 mm Hg, P < 0.05), a weight ratio of right ventricle to left ventricle plus septum (0.35 +/- 0.04 versus 0.42 +/- 0.05, P < 0.05), and percent medial thickness of the PA (12.9 +/- 0.3% versus 21.4 +/- 0.4%, P < 0.01) compared with controls. IL-10 significantly reduced macrophage infiltration and vascular cell proliferation in the remodeled PA in vivo. It also significantly decreased the lung levels of transforming growth factor-beta(1) and IL-6, which are indicative of PA remodeling. In addition, IL-10 increased the lung level of heme oxygenase-1, which strongly prevents PA remodeling. In vitro analysis revealed that IL-10 significantly inhibited excessive proliferation of cultured human PA smooth muscle cells treated with transforming growth factor-beta(1) or the heme oxygenase inhibitor tin protoporphyrin IX. Thus, IL-10 prevented the development of monocrotaline-induced PAH, and these results provide new insights into the molecular mechanisms of human PAH.

Prostacyclin synthase (PGIS) is the final committed enzyme in the metabolic pathway of prostacyclin production. The therapeutic option of intravenous prostacyclin infusion in patients with pulmonary arterial hypertension is limited by the short half-life of the drug and life-threatening catheter-related complications. To develop a better delivery system for prostacyclin, we examined the feasibility of intramuscular injection of an adenoassociated virus (AAV) vector expressing PGIS for preventing monocrotaline-induced pulmonary arterial hypertension in rats. We developed an AAV serotype 1-based vector carrying a human PGIS gene (AAV-PGIS). AAV-PGIS or the control AAV vector expressing enhanced green fluorescent protein was injected into the anterior tibial muscles of 3-week-old male Wistar rats; this was followed by the monocrotaline administration at 7 weeks. Eight weeks after injecting the vector, the plasma levels of 6-keto-prostaglandin F-1 alpha increased in a vector dose-dependent manner. At this time point, the PGIS transduction (1x10(10) genome copies per body) significantly decreased mean pulmonary arterial pressure (33.9 +/- 2.4 versus 46.1 +/- 3.0 mm Hg; P < 0.05), pulmonary vascular resistance (0.26 +/- 0.03 versus 0.41 +/- 0.03 mm Hg . mL(-1) . min(-1) . kg(-1); P < 0.05), and medial thickness of the peripheral pulmonary artery (14.6 +/- 1.5% versus 23.5 +/- 0.5%; P < 0.01) as compared with the controls. Furthermore, the PGIS-transduced rats demonstrated significantly improved survival rates as compared with the controls (100% versus 50%; P < 0.05) at 8 weeks postmonocrotaline administration. An intramuscular injection of AAV-PGIS prevents monocrotaline-pulmonary arterial hypertension in rats and provides a new therapeutic alternative for preventing pulmonary arterial hypertension in humans.

Pulmonary arterial hypertension (PAH) is a fatal disease associated with inflammation and pathological remodeling of the pulmonary artery (PA). Interleukin (IL)-10 is a pleiotropic antiinflammatory cytokine with vasculoprotective properties. Here, we report the preventive effects of IL-10 on monocrotaline-induced PAH. Three-week-old Wistar rats were intramuscularly injected with an adeno-associated virus serotype 1 vector expressing IL-10, followed by monocrotaline injection at 7 weeks old. IL-10 transduction significantly improved survival rates of the PAH rats 8 weeks after monocrotaline administration compared with control gene transduction (75% versus 0%, P < 0.01). IL-10 also significantly reduced mean PA pressure (22.8 +/- 1.5 versus 29.7 +/- 2.8 mm Hg, P < 0.05), a weight ratio of right ventricle to left ventricle plus septum (0.35 +/- 0.04 versus 0.42 +/- 0.05, P < 0.05), and percent medial thickness of the PA (12.9 +/- 0.3% versus 21.4 +/- 0.4%, P < 0.01) compared with controls. IL-10 significantly reduced macrophage infiltration and vascular cell proliferation in the remodeled PA in vivo. It also significantly decreased the lung levels of transforming growth factor-beta(1) and IL-6, which are indicative of PA remodeling. In addition, IL-10 increased the lung level of heme oxygenase-1, which strongly prevents PA remodeling. In vitro analysis revealed that IL-10 significantly inhibited excessive proliferation of cultured human PA smooth muscle cells treated with transforming growth factor-beta(1) or the heme oxygenase inhibitor tin protoporphyrin IX. Thus, IL-10 prevented the development of monocrotaline-induced PAH, and these results provide new insights into the molecular mechanisms of human PAH.

Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis in a variety of tumors. A soluble form of Flt-1 (sFlt-1), a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidences suggest the applicability of sFlt-1 in tumor suppression by means of anti-angiogenesis. We previously demonstrated the efficacy of sflt-1 gene expression in situ to suppress tumor growth and ascites in ovarian cancer. Here, we demonstrate the therapeutic applicability of muscle-mediated expression of sFlt-1 in tumor-bearing mice. Initially, tumor suppressive action was confirmed by inoculating sFlt-1-expressing ovarian cancer (SHIN-3) cells into mice, both subcutaneously and intraperitoneally. To validate the therapeutic efficacy in a more clinically relevant model, adeno-associated virus vectors encoding sflt-1 were introduced into mouse skeletal muscles and were subsequently inoculated with tumor cells. As a result, high serum sFlt-1 levels were constantly observed, and the growth of both subcutaneously- and intraperitoneally-inoculated tumors was significantly suppressed. No delay in wound healing or adverse events of neuromuscular damage were noted, body weight did not change, and laboratory data, such as those representing liver and renal functions, were not affected. These results indicate that sFlt-1 suppresses growth and peritoneal dissemination of ovarian cancer by the inhibition of angiogenesis, and thus suggest the usefulness of gene therapy for ovarian cancer. (c) 2006 Wiley-Liss, Inc.

Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis in a variety of tumors. A soluble form of Flt-1 (sFlt-1), a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidences suggest the applicability of sFlt-1 in tumor suppression by means of anti-angiogenesis. We previously demonstrated the efficacy of sflt-1 gene expression in situ to suppress tumor growth and ascites in ovarian cancer. Here, we demonstrate the therapeutic applicability of muscle-mediated expression of sFlt-1 in tumor-bearing mice. Initially, tumor suppressive action was confirmed by inoculating sFlt-1-expressing ovarian cancer (SHIN-3) cells into mice, both subcutaneously and intraperitoneally. To validate the therapeutic efficacy in a more clinically relevant model, adeno-associated virus vectors encoding sflt-1 were introduced into mouse skeletal muscles and were subsequently inoculated with tumor cells. As a result, high serum sFlt-1 levels were constantly observed, and the growth of both subcutaneously- and intraperitoneally-inoculated tumors was significantly suppressed. No delay in wound healing or adverse events of neuromuscular damage were noted, body weight did not change, and laboratory data, such as those representing liver and renal functions, were not affected. These results indicate that sFlt-1 suppresses growth and peritoneal dissemination of ovarian cancer by the inhibition of angiogenesis, and thus suggest the usefulness of gene therapy for ovarian cancer. (c) 2006 Wiley-Liss, Inc.

The number of patients suffering from diabetes mellitus is constantly rising worldwide, and diabetic retinopathy (DR) has become the most frequent cause of postnatal blindness. Vascular endothelial growth factor (VEGF) is known to play a central role during DR development. Thus, inhibiting the effects of VEGF may hamper the disease progression, and gene transfer of the soluble VEGF receptor sflt-1 is an attractive approach for this purpose. However, the lack of suitable animal models hindered the evaluation of this strategy. Recently, the spontaneously diabetic non-obese Torii (SDT) rat was established and is considered as one of the ideal models for human DR. In this study, we evaluated the efficacy of gene therapy in SDT rats by using adeno-associated viral vectors (AAV-sflt-1) injected into the subretinal space. Thirty weeks later, the progression of DR was assessed by fluorescein angiography using three parameters; the presence of an avascular area, extensive hyperfluorescein and arterial narrowing. These changes were significantly less evident in the 'treated' eyes than in the control. No adverse effects were observed throughout the study. These results indicate that local sflt-1 gene transfer inhibits DR progression in SDT rats and offers powerful therapeutic potential for the management of human DR.

Background: Transfer of exogenous growth factor genes to injured tendons offers a promising method for strengthening tendon repairs. Adeno-associated virus vectors have advantages of being both nonpathogenic and nontoxic. The authors explored the efficiency of transduction of intrasynovial tenocytes with different serotypes of adeno-associated virus (AAV) and the persistency of its expression of a growth factor transgene. Methods: Tenocytes were obtained from cultures of rat intrasynovial tendons and distributed to 82 wells in eight culture plates and to 30 culture dishes. The tenocytes in the wells were treated with AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, and AAV8 vectors containing the lacZ gene, and plasmid vectors (pCMV beta-lacZ). The tenocytes were stained with in situ P-galactosidase 5 days later. The basic fibroblast growth factor (bFGF) gene was cloned to the AAV2 vector to construct the AAV2-bFGF vector, which transduced tenocytes in culture dishes. Expression of the transgene was measured over 3 weeks and analyzed statistically. Results: AAV2 effectively delivered exogenous genes to proliferating intrasynovial tenocytes. In contrast, other tested adeno-associated viruses transduced tenocytes minimally or not at all. The efficiency of gene transfer by AAV2, indicated by the percentage of cells with positive beta-galactosidase staining, was significantly greater than that by a plasmid vector (p = 0.001). Expression of the bFGF gene in tenocytes transduced with the AAV2-bFGF was significantly higher than that in the control over the 3-week period (p < 0.01). Conclusions: Gene transfer to tenocytes by AAV2 is more efficient than that by a plasmid vector. However, other adeno-associated virus serotypes cannot effectively transduce tenocytes. The bFGF gene can be delivered to intrasynovial tenocytes by the AAV2 vector effectively, and the gene transfer significantly increases expression of bFGF gene over 3 weeks.

Site-specific integration of the therapeutic transgene is favorable for gene therapy applications since it minimizes the risk of insertional mutagenesis and thereby prevents target cells from developing tumors. Adeno-associated virus (AAV), a member of parvovirus, is unique in that it integrates its genome into a specific site termed the AAVS1 locus (19q13.4) in the human genome. A non-structural replication initiator protein of AAV, Rep78 or Rep68, binds the inverted terminal repeat (ITR) sequence at either end of the AAV genome via tandem repeats of the GAGC/GCTC motif. A homologous sequence exists at theAAVS1 site. The Rep protein recognizes it and drives the integration of theAAVgenome intoAAVS1. The ITR is a cis element sufficient for AAVS1-specific integration. The incorporation of the ITR sequence into plasmidDNAis thus discussed in terms of Rep-mediated sitespecific integration and of AAVS1 as a hazard-free target for transgene integration. Therefore, the use of the AAV integration machinery should allow us to develop a safer gene delivery system.

The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy.

Traditionally, skeletal muscle and liver are the preferred target organs for gene transfer to supply a transgene product into the systemic circulation. In this respect, adipose tissue presents a number of attractive features. However, adipose tissue transduction in vivo has not been feasible by conventional methods. To solve this issue, we tested the utility of excipients in adeno-associated virus (AAV) vector-mediated gene transfer and found that Pluronics are suitable for this purpose. In a histological analysis of adipose tissue in db/db mice, Pluronic F88 showed the greatest augmentative effect on beta-galactosidase expression in combination with the AAV1 vector. When the vector encoding mouse erythropoietin (Epo) was used in the same manner, increased plasma Epo concentrations were observed (230 +/- 80 versus 58 +/- 14 mU/ml). Moreover, the plasma Epo concentration returned to the normal level after the surgical removal of transduced adipose tissue. No damage was observed in the transduced tissue. Our results indicate that the proposed method is safe and efficient for gene transfer into adipose tissues, thus providing an alternative for supplemental gene therapy.

Background Gene transfer into a fetus or neonate can be a fundamental approach for treating genetic diseases, particularly disorders that have irreversible manifestations in adulthood. Although the potential utility of this technique has been suggested, the advantages of neonatal gene transfer, have not been widely investigated. Here, we tested the usefulness of neonatal gene transfer using adeno-associated virus (AAV) vectors by comparing the, administration routes and vector doses. Methods To determine the optimal administration route neonates were, subjected to intravenous (iv) or intraperitoneal (ip) injections of AAV5-based vectors encoding the human coagulation factor IX (hfIX) gene, and the dose response was examined. To determine the distribution of transgene expression, vectors encoding lacZ or luciferase (luc) genes were used and assessed by X-gal staining and in vivo imaging, respectively. After the observation period, the vector distribution across tissues was quantified. Results The factor IX concentration was higher in ip-injected mice than in iv-injected mice. All transgenes administered by ip injection were more efficiently expressed in neonates than in adults. The expression was confined to the peritoneal tissue. Interestingly, a sex-related difference was observed in transgene expression in adults, whereas this difference was not apparent in neonates. Conclusions AAV vector administration to neonates using the ip route was clearly advantageous in obtaining robust transgene expression. Vector genomes and transgene expression were observed mainly in the peritoneal tissue. These findings indicate the advantages of neonatal gene therapy and would help in designing strategies for gene therapy using AAV vectors. Copyright (c) 2006 John Wiley & Sons, Ltd.

The transduction of cancer cells using recombinant adeno-associated virus (rAAV) occurs with low efficiency, which limits its utility in cancer gene therapy. We have previously sought to enhance rAAV-mediated transduction of cancer cells by applying DNA-damaging stresses. In this study, we examined the effects of the histone deacetylase inhibitor FR901228 on tumor transduction mediated by rAAV types 2 and 5. FR901228 treatment significantly improved the expression of the transgene in four cancer cell lines. The cell surface levels of alpha v integrin, FGF-R1, and PDGF-R were modestly enhanced by the presence of FR901228. These results suggest that the superior transduction induced by the HDAC inhibitor was due to an enhancement of transgene expression rather than increased viral entry. Furthermore, we characterized the association of the acetylated histone H3 in the episomal AAV vector genome by using the chromatin immunoprecipitation assay. The results suggest that the superior transduction may be related to the proposed histone-associated chromatin form of the rAAV concatemer in transduced cells. In the analysis with subcutaneous tumor models, strong enhancement of the transgene expression as well as therapeutic effect was confirmed in vivo. The use of this HDAC inhibitor may enhance the utility of rAAV-mediated transduction strategies for cancer gene therapy.

Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), virus protein shells without the vector genome. The contaminating VLPs would interfere with transduction by competing for cell-surface receptors and, when administered in vivo, contribute to antigen load, which may elicit a stronger immune response. Density-gradient ultracentrifugation provides a means to separate VLPs from rAAV particles, but is not feasible for large-scale preparations of vectors. Since the compositions of the VLP and vector differ by the single-stranded DNA genome, we hypothesized that the isoelectric point of the vector may differ from that of the VLP. In an attempt to separate type I rAAV particles from VLPs by ionexchange chromatography, we tested a number of buffer systems and found that trimethylammonium sulfate, or [(CH3)(4)N](2)SO4, effectively separated rAAV1 particles from VLPs. The rAAV1-GFP chromatographically separated from VLPs induced stronger GFP expression in HEK293 cells than rAAV1-GFP contaminated with VLPs. The transduction of mouse muscles with rAAV1-SEAP (secreted form of alkaline phosphatase) isolated from VLPs also showed higher serum SEAP levels than rAAV1 SEAP with VLPs. These results suggest that chromatographic separation of rAAV1 from empty capsids increased the efficacy of rAAV1.

We established a method for production of recombinant adeno-associated virus type 5 (rAAV5) in insect cells by use of baculovirus expression vectors. One baculovirus harbors a transgene between the inverted terminal repeat sequences of type 5, and the second expresses Rep78 and Rep52. Interestingly, the replacement of type 5 Rep52 with type 1 Rep52 generated four times more rAAV5 particles. We replaced the N-terminal portion of type 5 VP1 with the equivalent portion of type 2 to generate infectious AAV5 particles. The rAAV5 with the modified VP1 required alpha 2-3 sialic acid for transduction, as revealed by a competition experiment with an analog of alpha 2-3 sialic acid. rAAV5-GFP/Neo with a 4.4-kb vector genome produced in HEK293 cells or Sf9 cells transduced COS cells with similar efficiencies. Surprisingly, Sf9-produced humanized Renilla green fluorescent protein (hGFP) vector with a 2.4-kb vector genome induced stronger GFP expression than the 293-produced one. Transduction of murine skeletal muscles with Sf9-generated rAAV5 with a 3.4-kb vector genome carrying a human secreted alkaline phosphatase (SEAP) expression cassette induced levels of SEAP more than 30 times higher than those for 293-produced vector 1 week after injection. Analysis of virion DNA revealed that in addition to a 2.4- or 3.4-kb single-stranded vector genome, Sf9-rAAV5 had more-abundant forms of approximately 4.7 kb, which appeared to correspond to the monomer duplex form of hGFP vector or truncated monomer duplex SEAP vector DNA. These results indicated that rAAV5 can be generated in insect cells, although the difference in incorporated virion DNA may induce different expression patterns of the transgene.

Adeno-associated virus (AAV) vectors carrying the B domain-deleted canine FVIII (BDD cFVIII) gene utilizing the beta-actin minimum promoter (167b) pseudotyped with serotype 1 (AAV1-beta-actin-cFVIII) and serotype 8 (AAV8-beta-actin-cFVIII) were developed to express cFVIII in hemophilia A mice. FVIII clotting activities measured by the APTT method increased in hemophilia A mice with intramuscular injection of AAV1-beta-actin-cFVIII in a dose-dependent manner. Therapeutic FVIII levels (2.9 +/- 1.0%) in hemophilia A mice with the AAV1-beta-actin-cFVIII dose of 1x10(12) gc/body were achieved, suggesting partial correction of the phenotype with AAV1-beta-actin-cFVIII vectors. FVIII clotting activity levels in hemophilia A mice with intravenous injection of AAV8-beta-actin-cFVIII also were increased dose-dependently, achieving therapeutic FVIII levels (5-90%) in hemophilia A mice with the AAV8-beta-actin-cFVIII doses of 1-3 x 10(11) gc/body and supernormal FVIII levels (180-670%) in hemophilia A mice with the AAV8-beta-actin-cFVIII dose of 1 x 10(12) gc/body. Transduction of the liver with AAV8-beta-actin-cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding the FVIII production and antibody formation. These data suggested that both AM and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy. (c) 2005 Elsevier Ltd. All rights reserved.

The objective of this study was to establish the potency of adeno-associated virus (AAV) as a viral vector to transport the basic fibroblast growth factor (bFGF) gene into synovial tissue, and to evaluate the consequent repair of articular cartilage defects. In the in vitro study, LacZ- and bFGF-encoding genes were transduced into rabbit synoviocytes by recombinant adeno-associated virus (AAV) vector, and the cells were cultured for 2 weeks. The percentage of successfully transduced LacZ-positive cells was assessed by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining, and the concentration of bFGF in the culture supernatant was confirmed by bFGF-specific enzyme-linked immunosorbent assay. In the in vivo study, 12- to 14-week-old Japanese white rabbits (all female) were used. AAV-bFGF was administered into an artificially created full-thickness defect (5 mm in diameter and 3 nun deep) in the patellar groove of the distal femur. Cartilage repair was subsequently monitored at 4, 8, and 12 weeks, by macroscopic and histological examination, and results were graded on the basis of semiquantitative scores. lacZ gene expression in synoviocytes reached more than 93% within the first 2 weeks, and the mean bFGF concentration in the culture supernatant of the bFGF gene-transduced group was significantly increased (p < 0.01). Semiquantitative macroscopic and histological assessment indicated that the average score was significantly better in the bFGF-transduced group throughout the observation period, suggesting better cartilage repair. These results demonstrate that gene transfer into synoviocytes, using the AAV vector, was a potent method of gene transduction. Moreover, after intraarticular administration of AAV-bFGF, constant expression of bFGF in the knee joints resulted in substantial cartilage regeneration that, with further long-term study, could possibly merit consideration for clinical application.

Recombinant adeno-associated virus (AAV) vectors are of interest for cochlear gene therapy because of their ability to mediate the efficient transfer and long-term stable expression of therapeutic genes in a wide variety of postmitotic tissues with minimal vector-related cytotoxicity. In the present study, seven AAV serotypes (AAV1-5, 7, 8) were used to construct vectors. The expression of EGFP by the chicken P-actin promoter associated with the cytomegalovirus immediate-early enhancer in cochlear cells showed that each of these serotypes successfully targets distinct cochlear cell types. In contrast to the other serotypes, the AAV3 vector specifically transduced cochlear inner hair cells with high efficiency in vivo, while the AAV1, 2, 5, 7, and 8 vectors also transduced these and other cell types, including spiral ganglion and spiral ligament cells. There was no loss of cochlear function with respect to evoked auditory brain-stem responses over the range of frequencies tested after the injection of AAV vectors. These findings are of value for further molecular studies of cochlear inner hair cells and for gene replacement strategies to correct recessive genetic hearing loss due to monogenic mutations in these cells.

Adenovirus and adeno-associated virus (AAV) vectors are increasingly used for gene transduction experiments. However, to produce a sufficient amount of these vectors for in vivo experiments requires large-capacity tissue culture facilities, which may not be practical in limited laboratory space. We describe here a large-scale method to produce adenovirus and AAV vectors with an active gassing system that uses large culture vessels to process labor-and cost-effective infection or transfection in a closed system. Development of this system was based on the infection or transfection of 293 cells on a large scale, using a large culture vessel with a surface area of 6320 cm(2). A minipump was connected to the gas inlet of the large vessel, which was placed inside the incubator, so that the incubator atmosphere was circulated through the vessel. When active gassing was employed, the productivity of the adenovirus and AAV vectors significantly increased. This vector production system was achieved by improved CO2 and air exchange and maintenance of pH in the culture medium. Viral production with active gassing is particularly promising, as it can be used with existing incubators and the large culture vessel can readily be converted for use with the active gassing system.

Amyloid β peptide (Aβ) has been implicated in Alzheimer's disease (AD) as an initiator of the pathological cascades. Several lines of compelling evidence have supported major roles of Aβ-degrading enzyme neprilysin in the pathogenesis of sporadic AD. Here, we have shown a substantial reduction of cerebrospinal fluid (CSF) neprilysin activity (CSF-NEP) in patients with AD-converted mild cognitive impairment and early AD as compared with age-matched control subjects. The altered CSF-NEP likely reflects changes in neuronal neprilysin, since transfer of neprilysin from brain tissue into CSF was demonstrated by injecting neprilysin-carrying viral vector into the brains of neprilysin-deficient mice. Interestingly, CSF-NEP showed an elevation with the progression of AD. Along with a close association of CSF-NEP with CSF tau proteins, this finding suggests that presynaptically located neprilysin can be released into CSF as a consequence of synaptic disruption. The impact of neuronal damages on CSF-NEP was further demonstrated by a prominent increase of CSF-NEP in rats exhibiting kainate-induced neurodegeneration. Our results unequivocally indicate significance of CSF-NEP as a biochemical indicator to pursue a pathological process that involves decreased neprilysin activity and Aβ-induced synaptic toxicity, and the support the potential benefits of neprilysin up-regulation in ameliorating neuropathology in prodromal and early AD.

A Kunitz-type protease inhibitor, bikunin, is known to suppress the invasion and metastasis of cancer cells. HI8, a carboxyl-terminal domain of bikunin, is an active site of this glycoprotein. To increase its affinity for cancer cells, we constructed a chimeric gene, ATF-HI8, and investigated the anti-invasive and anti-migratory activity of ATF-HI8 on ovarian cancer cells. ATF-HI8-expressing plasmid and ATF-expressing plasmid were introduced into the highly invasive and metastatic ovarian cancer cell line HRA. The properties of the established cell line (HRA/ATF-HI8) were compared to those of the HRA/ATF and the HRA/luciferase (HRA/LUC, control) cell lines in terms of cell proliferation, invasion and migration. As a result, (i) there were no differences in cell proliferation between HRA/ATF-HI8 and HRA/LUC; (ii) the invasion and migration of HRA/ATF-HI8 cells were significantly inhibited compared to those of HRA/LUC cells; (iii) the migration, but not the invasion, of HRA/ATF cells was significantly inhibited compared to that of HRA/LUC. These results indicate that the overexpression of ATF-HI8 inhibits the invasion and migration of ovarian cancer cells without affecting cell proliferation and suggest that HI8 is involved in the anti-invasive and the anti-migratory activities, and the addition of ATF brought about the increase in the anti-migratory activity of HI8. The above findings suggest the applicability of therapeutic strategies targeting the inhibition of peritoneal invasion and dissemination of ovarian cancer by the use of the chimeric gene ATF-HI8. (C) 2004 Wiley-Liss, Inc.

A Kunitz-type protease inhibitor, bikunin, is known to suppress the invasion and metastasis of cancer cells. HI8, a carboxyl-terminal domain of bikunin, is an active site of this glycoprotein. To increase its affinity for cancer cells, we constructed a chimeric gene, ATF-HI8, and investigated the anti-invasive and anti-migratory activity of ATF-HI8 on ovarian cancer cells. ATF-HI8-expressing plasmid and ATF-expressing plasmid were introduced into the highly invasive and metastatic ovarian cancer cell line HRA. The properties of the established cell line (HRA/ATF-HI8) were compared to those of the HRA/ATF and the HRA/luciferase (HRA/LUC, control) cell lines in terms of cell proliferation, invasion and migration. As a result, (i) there were no differences in cell proliferation between HRA/ATF-HI8 and HRA/LUC; (ii) the invasion and migration of HRA/ATF-HI8 cells were significantly inhibited compared to those of HRA/LUC cells; (iii) the migration, but not the invasion, of HRA/ATF cells was significantly inhibited compared to that of HRA/LUC. These results indicate that the overexpression of ATF-HI8 inhibits the invasion and migration of ovarian cancer cells without affecting cell proliferation and suggest that HI8 is involved in the anti-invasive and the anti-migratory activities, and the addition of ATF brought about the increase in the anti-migratory activity of HI8. The above findings suggest the applicability of therapeutic strategies targeting the inhibition of peritoneal invasion and dissemination of ovarian cancer by the use of the chimeric gene ATF-HI8. (C) 2004 Wiley-Liss, Inc.

Objective. To examine the effects of basic fibroblast growth factor (bFGF) gene-transduced chondrocytes on the repair of articular cartilage defects. Methods. LacZ gene or bFGF gene was transduced into primary isolated rabbit chondrocytes with the use of a recombinant adeno-associated virus (AAV) vector. These gene-transduced chondrocytes were embedded in collagen gel and transplanted into a full-thickness defect in the articular cartilage of the patellar groove of a rabbit. The efficiency of gene transduction was assessed according to the percentage of LacZ-positive cells among the total number of living cells. The concentration of bFGF in the culture supernatant was measured by enzyme-linked immunosorbent assay to confirm the production by bFGF gene-transduced chondrocytes. At 4, 8, and 12 weeks after transplantation, cartilage repair was evaluated histologically and graded semiquantitatively using a histologic scoring system ranging from 0 (complete regeneration) to 14 (no regeneration) points. Results. LacZ gene expression by chondrocytes was maintained until 8 weeks in >85% of the in vitro population. LacZ-positive cells were found at the transplant sites for at least 4 weeks after surgery. The mean concentration of bFGF was significantly increased in bFGF gene-transduced cells compared with control cells (P < 0.01). Semiquantitative histologic scoring indicated that the total score was significantly lower in the bFGF-transduced group than in the control group throughout the observation period. Conclusion. These results demonstrated that gene transfer to chondrocytes by an ex vivo method was established with the AAV vector, and transplantation of bFGF gene-transduced chondrocytes had a clear beneficial effect on the repair of rabbit articular cartilage defects.

Background Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34(+) cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII). Methods CD34(+) cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34(+) cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34(+) cells and expression of transgenes were studied. Results We could efficiently transduce CD34(+) cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 +/- 0.1%) at MOI of 5 x 10(3) vector genome/cell. After transducing CD34(+) cells with SIVhFVIII, hFVIII was produced (274.3 +/- 20.1 ng) from 106 CD34(+) cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34(+) cells (5-10 x 10(5)) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34(+) cells and production of hFVIII (minimum 1.2 +/- 0.9 ng/mL, maximum 3.6 +/- 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells. Conclusions Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients. Copyright (C) 2004 John Wiley Sons, Ltd.

Adeno-associated virus (AAV) is a non-pathogenic virus with a single-strand DNA genome. AAV vectors have several unique properties suited for gene therapy applications. However, an obstacle to their application is a low efficiency of transgene expression, mainly due to a limited second-strand synthesis. Previously, we reported that gamma-rays enhanced the transduction efficiency and cytocidal effect of AAV vector harboring the herpes simplex virus-thymidine kinase (AAVtk) and ganciclovir (GCV) system. In the present study, we investigated whether topoisomerase inhibitors (etoposide and camptothecin) enhance the AAV vector-mediated transgene expression and the killing effect by AAVtk/GCV system. The enhancement of transgene expression was observed in a concentration -dependent manner on human laryngeal carcinoma cells (HEp-2 cells) and HeLa cells. Southern analysis confirmed that etoposide enhanced the double-strand synthesis of the AAV vector genome in HEp-2 cells and HeLa cells. The cells were efficiently killed by AAVtk/GCV system, as expected. More importantly, both etoposide and camptothecin augmented the cytocidal effect of the AAVtk/GCV system. These findings suggest that the combination of AAV-mediated suicide gene therapy and treatment with topoisomerase inhibitors may have synergistic therapeutic effects in the treatment of cancers.

Hematopoietic stem cell gene therapy has not provided clinical success in disorders such as chronic granulomatous disease (CGD), where genetically corrected cells do not show a selective advantage in vivo. To facilitate selective expansion of transduced cells, we have developed a fusion receptor system that confers drug-induced proliferation. Here, a 'selective amplifier gene (SAG)' encodes a chimeric receptor (GcRER) that generates a mitotic signal in response to estrogen. We evaluated the in vivo efficacy of SAG-mediated cell expansion in a mouse disease model of X-linked CGD (X-CGD) that is deficient in the NADPH oxidase gp91(phox) subunit. Bone marrow cells from X-CGD mice were transduced with a bicistronic retrovirus encoding GcRER and gp91(phox), and transplanted to lethally irradiated CGD recipients. Estrogen was administered to a cohort of the transplants, and neutrophil superoxide production was monitored. A significant increase in oxidase-positive cells was observed in the estrogen-treated mice, and repeated estrogen administration maintained the elevation of transduced cells for 20 weeks. In addition, oxidase-positive neutrophils were increased in the X-CGD transplants given the first estrogen even at 9 months post-transplantation. These results showed that the SAG system would enhance the therapeutic effects by boosting genetically modified, functionally corrected cells in vivo.

Although the adenoviral Ell, E2A, E4 and VA RNA regions are required for efficient adeno-associated virus (AAV) vector production, the role that the individual Ell genes (E1A, E1B19K, E1B55K and protein IX) play in AAV vector production has not been clearly determined. Ell mutants were analysed for their ability to mediate AAV vector production in HeLa or KB cells, when cotransfected with plasmids encoding all other packaging functions. Disruption of E1A and E1B19K genes resulted in vector yield reduction by up to 10- and 1 00-fold, respectively, relative to the wild-type Ell. Interruption of the E1B55K and protein IX genes had a modest effect on vector production. Interestingly, expression of anti-apoptotic E1B19K cellular homologues such as BcI-2 or Bcl-X-L fully complemented E1B19K mutants for AAV vector production. These findings may be valuable for the future development of packaging cell lines for AAV vector production.

Classical phenylketonuria (PKU) is a metabolic disorder caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). If untreated, accumulation of phenylalanine will damage the developing brain of affected individuals, leading to severe mental retardation. Here, we show that a liver-directed PAH gene transfer brought about long-term correction of hyperphenylalaninmia and behavioral improvement in a mouse model of PKU. A recombinant adeno-associated virus (AAV) vector carrying the murine PAH cDNA was constructed and administered to PAH-deficient mice ( strain PAH(enu2)) via the portal vein. Within 2 weeks of treatment, the hyperphenylalaninemic phenotype improved and completely normalized in the animals treated with higher vector doses. The therapeutic effect persisted for 40 weeks in male mice, while serum phenylalanine concentrations in female animals gradually returned to pretreatment levels. Notably, this long-term correction of hyperphenylalaninemia was associated with a reversal of hypoactivity observed in PAH(enu2) mice. While locomotory activity over 24 h and exploratory behavior were significantly decreased in untreated PAHenu2 mice compared with the age-matched controls, these indices were completely normalized in 12-month- old male PKU mice with lowered serum phenylalanine. These results demonstrate that AAV-mediated liver transduction ameliorated the PKU phenotype, including central nervous system dysfunctions.

We report a 67-year-old man with highly symptomatic polycystic liver disease. Fenestration was selected to treat symptoms because the cysts were scattered diffusely and the normal liver volume was limited. Although this patient was relieved from symptoms of liver cysts consequently, several severe postoperative complications including disseminated intravascular coagulation, respiratory failure, liver failure, and biliary leakage occurred resulting in a 6-month postoperative hospital stay. Although various treatments for symptomatic adult polycystic liver disease have been advocated, a definitive treatment remains controversial, especially in diffuse adult polycystic liver disease. Fenestration is one of the alternative treatments for the patients whose cysts are difficult to resect. However high morbidity rate should be carefully assessed, if extensive fenestration is needed to treat diffuse adult polycystic liver disease. Further consideration of appropriate treatments for diffuse adult polycystic liver disease is needed.

Adeno-associated virus (AAV) vectors are a practical choice for gene transfer, and demand for them is increasing. To cope with the necessity in the near future, we have developed a number of approaches to establish packaging cell lines for the production of AAV vectors. In our previous study, a-highly regulated expression of large Rep proteins was obtained by using,the Cke-loxP. switching system. Therefore, in the present study, to regulate Cap expression as well, we developed an inducible expression system for both Rep and Cap proteins by using an additional set of mutant loxP sequences. The mutants possess two base alterations in the spacer region of loxP and recombine specifically with the same counterpart in the presence of Cre. By using two separate plasmids, one with mutant and the other with wild-type loxP sequences, the expression of two different proteins can be induced simultaneously by Cre recombinase. When the LacZ-encoding plasmid vector was used as a packaging model, a significant packaging titer of 2.1 x 10(10) genome copies per 10-cm dish was obtained. These results indicate the importance of controlling Cap expression, in addition to Rep, to achieve an optimum production rate for AAV vectors.

After screening for species-specific antihuman factor (F)IX monoclonal antibodies, we found that antibody 3A6 did not bind to cynomolgus FIX. The 3A6 epitope was found to include Ala262 of human FIX. The 3A6 antibody was used as a catching antibody in an enzyme immunoassay (EIA) for specific detection of human FIX in cynomolgus macaque plasma. No significant increase of substrate hydrolysis was observed when EIA buffer containing cynomolgus macaque plasma was subjected to the 3A6-based EIA. Addition of up to 30% cynomolgus macaque plasma or canine plasma to the assay did not alter detection of human FIX. Three cynomolgus macaques were injected with human FIX (10 U kg(-1); i.v.) and the circulating human FIX was quantified in the macaque plasma. The FIX level in the circulation increased to 470 +/- 37.6 ng mL(-1) at 1 h after the injection and gradually decreased to 1.79 +/- 1.1 ng mL(-1) by day 5, which is approximately 0.06% of the normal human plasma FIX concentration. These data suggest that the cynomolgus macaque can be used as a primate model for studying hemophilia B gene therapy by transduction of macaque organs with vectors to express human FIX in vivo and detection of human FIX using the 3A6 monoclonal antibody.

A local increase in amyloid-beta peptide (Abeta) is closely associated with synaptic dysfunction in the brain in Alzheimer's disease. Here, we report on the catabolic mechanism of Abeta at the presynaptic sites. Neprilysin, an Abeta-degrading enzyme, expressed by recombinant adeno-associated viral vector-mediated gene transfer, was axonally transported to presynaptic sites through afferent projections of neuronal circuits. This gene transfer abolished the increase in Abeta levels in the hippocampal formations of neprilysin-deficient mice and also reduced the increase in young mutant amyloid precursor protein transgenic mice. In the latter case, Abeta levels in the hippocampal formation contralateral to the vector-injected side were also significantly reduced as a result of transport of neprilysin from the ipsilateral side, and in both sides soluble Abeta was degraded more efficiently than insoluble Abeta. Furthermore, amyloid deposition in aged mutant amyloid precursor protein transgenic mice was remarkably decelerated. Thus, presynaptic neprilysin has been demonstrated to degrade Abeta efficiently and to retard development of amyloid pathology.

Background/Aim: Gene transfer into the kidney has great potential as a novel therapeutic approach. However, an efficient method of gene transfer into the kidney has not been established. We explored the transduction efficiency of renal cells in vitro and in vivo using adeno-associated virus (AAV) serotype 1-5 vectors encoding the β-galactosidase gene. Methods: In the in vitro study, rat kidney epithelial cell line NRK52E cells were transfected with AAV serotype derived vectors. In the in vivo study, AAV serotype derived vectors were selectively injected into the kidney using a catheter-based gene delivery system in rats and mice mimicking the clinical procedure. The efficiency of gene expression was histologically evaluated on the basis of the β-galactosidase expression. Results: AAV serotype 1, 2, and 5 vectors transduced in rat kidney epithelial cell line NRK52E cells in vitro, whereas AAV serotype 3 or 4 vectors showed no transduction. In addition, the kidney-specific injection of AAV serotype 2 vectors successfully transduced in tubular epithelial cells, but not in glomerular, blood vessel, or interstitial cells in vivo, whereas the rest of the serotypes showed no transduction. Conclusion: Since kidney-specific gene delivery via the renal artery by catheterization is highly feasible in humans, these findings provide useful information for promising strategies in renal gene therapy. Copyright © 2004 S. Karger AG, Basel.