Optimization of adeno-associated virus vector-mediated gene transfer to the respiratory tract.

An efficient adeno-associated virus (AAV) vector was constructed for the treatment of respiratory diseases. AAV serotypes, promoters and routes of administration potentially influencing the efficiency of gene transfer to airway cells were examined in the present study. Among the nine AAV serotypes (AAV1-9) screened in vitro and four serotypes (AAV1, 2, 6, 9) evaluated in vivo, AAV6 showed the strongest transgene expression. As for promoters, the cytomegalovirus (CMV) early enhancer/chicken ß-actin (CAG) promoter resulted in more robust transduction than the CMV promoter. Regarding delivery routes, intratracheal administration resulted in strong transgene expression in the lung, whereas the intravenous and intranasal administration routes yielded negligible expression. The combination of the AAV6 capsid and CAG promoter resulted in sustained expression, and the intratracheally administered AAV6-CAG vector transduced bronchial cells and pericytes in the lung. These results suggest that AAV6-CAG vectors are more promising than the previously preferred AAV2 vectors for airway transduction, particularly when administered into the trachea. The present study offers an optimized strategy for AAV-mediated gene therapy for lung diseases, such as cystic fibrosis and pulmonary fibrosis.

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

Serum pyridoxal concentrations and depressive symptoms among Japanese adults: results from a prospective study.

BACKGROUND/OBJECTIVES: Vitamin B6 is suggested to have a protective role against depression. However, the association between vitamin B6 intake and depression remains inconclusive, and few studies have examined the relationship between circulating vitamin B6 concentrations and depressive symptoms. Here, we investigated the cross-sectional and prospective associations between serum pyridoxal concentrations and depressive symptoms among Japanese workers. SUBJECTS/METHODS: Participants were 422 municipal employees (aged 21-67 years) who participated in a baseline survey in 2006 for cross-sectional analysis, and 210 subjects without depressive symptoms at baseline (2006) who completed both baseline and follow-up (2009) surveys for prospective analysis. Depressive symptoms were assessed using the Center for Epidemiologic Studies Depression (CES-D) scale. Logistic regression analysis was used to estimate the odds ratio of depressive symptoms (CES-D scale of ≥ 19) according to tertile of serum pyridoxal with adjustment for potential confounding variables. RESULTS: In the cross-sectional analysis, serum pyridoxal concentrations were significantly associated with a decreased prevalence of depressive symptoms (P for trend=0.03); the multivariable-adjusted odds ratio of depressive symptoms for the highest tertile of pyridoxal was 0.54 (95% confidence interval 0.30-0.96) compared with the lowest tertile. In longitudinal analyses, higher serum pyridoxal concentrations at baseline were associated with a trend toward reduced depressive symptoms after 3 years; the multivariable-adjusted odds ratio of depressive symptoms for the highest versus the lowest tertile of pyridoxal concentration was 0.55 (95% confidence interval 0.13-2.32). CONCLUSIONS: A higher vitamin B6 status may be associated with a decreased risk of depressive symptoms in Japanese.

Improvement in skin color achieved by smoking cessation.

It has been well known that habitual smoking accelerates premature skin ageing recognized as 'smoker's face'. However, the effect of smoking cessation on the appearance of skin has not been elucidated. The aim of this study was to evaluate objectively the effect of smoking cessation on the skin's appearance. The stratum corneum carbonyl protein level and skin colour of the cheek and the hand were measured. The change before and during the smoking cessation treatment (0, 2, 4, 8 and 12 weeks), and the success or failure in smoking cessation, was compared and examined. Eighty-four cases who had smoking cessation treatment were examined. The level of the stratum corneum carbonyl protein did not show any difference comparing before and after treatment for the smoking cessation success group and the failure group. The lightness of skin colour showed an upward tendency 4-12 weeks after starting the treatment in the success group and increased significantly compared with the failure group. The redness showed a significant decrease in comparison with before the treatment, and it also showed a significant decrease compared with the failure group. The yellowness did not show any clear tendency. Also, the haemoglobin showed a decreased tendency. Furthermore, multivariate statistical analysis showed a possibility that the lightness and haemoglobin could be changed by smoking cessation treatment. In conclusion, our study showed that an upward tendency of skin lightness was seen to correspond with a haemoglobin decrease accompanied by smoking cessation. If we can easily measure skin improvement as an effect of smoking cessation, it is thought to be a useful aid for smoking cessation support.

Interleukin-10 expression induced by adeno-associated virus vector suppresses proteinuria in Zucker obese rats.

Varying degrees of metabolic abnormalities mediated by chronic inflammation are implicated in the chronic glomerular injuries associated with obesity. Interleukin (IL)-10, a pleiotropic cytokine, exerts anti-inflammatory effects in numerous biological settings. In the present study, we explored the biological benefits of adeno-associated virus (AAV) vector-mediated sustained IL-10 expression against the pathological renal characteristics observed in Zucker fatty rats (ZFRs). We injected an AAV vector, encoding rat IL-10 or enhanced green fluorescent protein (GFP) into male ZFRs at 5 weeks of age. Subsequently, the renal pathophysiological changes were analyzed. Persistent IL-10 expression significantly reduced the urinary protein excretion of ZFRs compared with GFP expression (47.1±11.6 mg per mg·creatinine versus 88.8±30.0 mg per mg·creatinine, P<0.01). The serum levels of IL-10 negatively correlated with the urinary protein in AAV-treated rats (r=-0.78, P<0.01). Renal hypertrophy, increased widths in the glomerular basement membrane, and the lack of uniformity and regularity of the foot process of the visceral glomerular epithelial cells of ZFRs were significantly blunted by IL-10 expression. IL-10 also abrogated the downregulation of glomerular nephrin observed in ZFRs treated with the GFP vector. Our findings provide insights into the potential benefit of the anti-inflammatory effects of IL-10 on the overall management of glomerulopathy induced by the metabolic disorders associated with obesity.

Reciprocal upregulation of Notch signaling molecules in hematopoietic progenitor and mesenchymal stromal cells.

Although mesenchymal stem cells (MSCs) play pivotal supportive roles in hematopoiesis, how they interact with hematopoietic stem cells (HSCs) is not well understood. We investigated the interaction between HSCs and surrogate MSCs (C3H10T1/2 stromal cells), focusing on the molecular events induced by cell contact of these bipartite populations. C3H10T1/2 is a mesenchymal stromal cell line that can be induced to differentiate into preadipocytes (A54) and myoblasts (M1601). The stromal cell derivatives were cocultured with murine HSCs (Lineage(-)Sca1(+)), and gene expression profiles in stromal cells and HSCs were compared before and after the coculture. HSCs gave rise to cobblestone areas only on A54 cells, with ninefold more progenitors than on M1601 or undifferentiated C3H10T1/2 cells. Microarray-based screening and a quantitative reverse transcriptase directed-polymerase chain reaction showed that the levels of Notch ligands (Jagged1 and Delta-like 3) were increased in A54 cells upon interaction with HSCs. On the other hand, the expression of Notch1 and Hes1 was upregulated in the HSCs cocultured with A54 cells. A transwell assay revealed that the reciprocal upregulation was dependent on cell-to-cell contact. The result suggested that in the hematopoietic niche, HSCs help MSCs to produce Notch ligands, and in turn, MSCs help HSCs to express Notch receptor. Such a reciprocal upregulation would reinforce the downstream signaling to determine the fate of hematopoietic cell lineage. Clarification of the initiating events on cell contact should lead to the identification of specific molecular targets to facilitate HSC engraftment in transplantation therapy.

Systemic delivery of IL-10 by an AAV vector prevents vascular remodeling and end-organ damage in stroke-prone spontaneously hypertensive rat.

Interleukin-10 (IL-10) ameliorates various T-helper type 1 cell-mediated chronic inflammatory diseases. Although the therapeutic benefits of IL-10 include antiatherosclerotic effects, pathophysiological effects of IL-10 on vascular remodeling in hypertension have not yet been elucidated. These studies were designed to determine whether sustained IL-10 expression, mediated by an adeno-associated virus (AAV) vector, prevents vascular remodeling and target-organ damage in the stroke-prone spontaneously hypertensive rat (SHR-SP)-an animal model of malignant hypertension. A single intramuscular injection of an AAV1 vector encoding rat IL-10 introduced long-term IL-10 expression. These IL-10-transduced rats had decreased stroke episodes and proteinuria, resulting in improved survival. Histological examination revealed a reduced level of deleterious vascular remodeling of resistance vessels in the brain and kidney of these rats. Immunohistochemical analysis indicated that IL-10 inhibited the enhanced renal transforming growth factor-beta expression and perivascular infiltration of monocytes/macrophages and nuclear factor-kappaB-positive cells normally observed in the SHR-SP. Four weeks after IL-10 vector injection, systolic blood pressure significantly decreased and this effect persisted for several months. Overall, AAV vector-mediated systemic IL-10 expression prevented vascular remodeling and inflammatory lesions of target organs in the SHR-SP. This approach provides significant insights into the prevention strategy of disease onset with unknown genetic predisposition or intractable polygenic disorders.

[C-reactive protein, white blood cell and body temperature following cardiovascular surgery, as predicting factors of postoperative infection].

The aim of this study is to clarify the relationship between CRP and postoperative infection after cardiovascular surgery. We had 5 cases of surgical site infection, and 3 cases of infective endocarditis (IE) among 57 patients selected for this study out of 405 patients who had undergone cardiovascular surgery from May 1995 to March 2005. CRP, WBC and body temperature (BT) were evaluated during 1 week after the operation. Our results showed not only that the mean value of CRP level in the 49 non-infection patients attained the peak on the 2nd or 3rd day after the operation (18.2 +/- 4.7 and 17.7 +/- 5.7 mg/dl), but also that each patient in this group showed the same pattern of CRP sequence. CRP in the 5 cases of postoperative infection showed different patterns from that in the non-infection group. CRP in 3 cases of valve replacement for IE showed significantly higher level than that in 16 cases of valve replacement without IE through 1 week after the surgery. WBC level in the non-infection group reached the peak just after the operation (11.3 +/- 4.4 x 10(3)/microl) and then decreased gradually during 1 week after the operation. WBC in the 3 cases of valve replacement for IE, did not show different sequence pattern from that in the 16 cases of valve replacement without IE. WBC in a case of postoperative mediastinal infection showed a similar pattern of sequence to that in the non-infection group although it showed a remarkably high level of CRP sequence through 1 week after the surgery. BT in the non-infection group became the lowest just after the operation and reached the peak 8 hours after the operation. It then decreased gradually during 1 week after the operation. Our study demonstrates that CRP sequence after the surgery might be useful to detect postoperative infection after cardiovascular surgery.

[Coronary artery bypass grafting to left anterior descending coronary artery diagnosed by multidetector-row computed tomography].

A 64-year-old male received coronary angiography because of chest pain. Although coronary angiography showed total occlusion of right coronary artery (RCA) # 2 and left anterior descending branch (LAD) #6, and a significant stenosis of left circumflex (LCx) #11, it could not visualize LAD distal to LAD # 6. Since coronary multidetector-row computed tomography (MD CT) could visualize the distal LAD, coronary artery bypass grafting (CABG) was indicated for this patient. Left internal thoracic artery (LITA) was anastomosed to LAD and saphenous vein graft (SVG) was used for distal anastomoses to obtuse marginal branch (OM) and 4-posterior descending branch (# 4 PD). Postoperative course was uneventful. LITA anastomosed to LAD and SVG to OM and # 4 PD were visualized by postoperative coronary angiography. MD CT in addition to coronary angiography was demonstrated useful to assess precise lesions of the coronary artery disease in this case.

Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits atherosclerosis in apolipoprotein E-deficient mice.

Inflammation is a major contributor to atherosclerosis by its effects on arterial wall biology and lipoprotein metabolism. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that may modulate the atherosclerotic disease process. We investigated the effects of adeno-associated virus (AAV) vector-mediated gene transfer of IL-10 on atherogenesis in apolipoprotein E (ApoE)-deficient mice. A murine myoblast cell line, C2C12, transduced with AAV encoding murine IL-10 (AAV2-mIL10) secreted substantial amounts of IL-10 into conditioned medium. The production of monocyte chemoattractant protein-1 (MCP-1) by the murine macrophage cell line, J774, was significantly inhibited by conditioned medium from AAV2-mIL10-transduced C2C12 cells. ApoE-deficient mice were injected with AAV5-mIL10 into their anterior tibial muscle at 8 weeks of age. The expression of MCP-1 in the vascular wall of the ascending aorta and serum MCP-1 concentration were decreased in AAV5-mIL10-transduced mice compared with AAV5-LacZ-transduced mice. Oil red-O staining of the ascending aorta revealed that IL-10 gene transfer resulted in a 31% reduction in plaque surface area. Serum cholesterol concentrations were also significantly reduced in AAV5-mIL10-transduced mice. To understand the cholesterol-lowering mechanism of IL-10, we measured the cellular cholesterol level in HepG2 cells, resulting in its significant decrease by the addition of IL-10 in a dose-dependent manner. Furthermore, IL-10 suppressed HMG-CoA reductase expression in the HepG2 cells. These observations suggest that intramuscular injection of AAV5-mIL10 into ApoE-deficient mice inhibits atherogenesis through anti-inflammatory and cholesterol-lowering effects.

Expansion of genetically corrected neutrophils in chronic granulomatous disease mice by cotransferring a therapeutic gene and a selective amplifier gene.

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 gp91phox subunit. Bone marrow cells from X-CGD mice were transduced with a bicistronic retrovirus encoding GcRER and gp91phox, and transplanted to lethally irradiated X-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.

Long-term correction of hyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylketonuria mice.

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 hyperphenylalaninemia 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 PAH(enu2) 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.

Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice.

The application of adeno-associated virus (AAV) vectors to cancers is limited by their low transduction efficiency. Previously, we reported that gamma-ray enhanced the second-strand synthesis, leading to the improvement of the transgene expression, and cytocidal effect of the herpes simplex virus type-1 thymidine kinase (HSVtk) and ganciclovir (GCV) system. In this study, we extended this in vitro findings to in vivo. First, the laryngeal cancer cell line (HEp-2) and HeLa were treated with AAVtk/GCV, the number of surviving cells was reduced as the concentration of GCV increased. Furthermore, the 4 Gy irradiation enhanced the killing effects of AAVtk/GCV by four-fold on HeLa cells and 15-fold on HEp-2 cells. Following the in vitro experiments, we evaluated the transgene expression and the antitumor activity of the AAV vectors in combination with gamma-ray in nude mice inoculated with HEp-2 subcutaneously. The LacZ expression was observed in the xenografted tumors and significantly increased by gamma-ray. The AAVtk/GCV system suppressed the tumors growth, and gamma-ray augmented the antitumor activity by five-fold. These findings suggest that the combination of AAVtk/GCV system with radiotherapy is significantly effective in the treatment of cancers and may lead to reduction of the potential toxicity of both AAVtk/GCV and gamma-ray.

Selective growth advantage of wild-type lymphocytes in X-linked SCID recipients.

The cytokine receptor common gamma chain (gamma c) plays a pivotal role in multiple interleukin signaling, and gamma c gene mutations cause an X-linked form of SCID (X-SCID). Recently, gamma c gene transfer into the autologous X-SCID BM achieved appreciable lymphocyte reconstitution, contrasting with the limited success in previous gene therapy trials targeting hematopoietic stem cells. To understand the mechanisms underlying this success, we examined the repopulating potential of the wild-type (WT) BM cells using an X-SCID mouse model. Limited numbers of WT cells were infused into non-ablated WT and X-SCID hosts. Whereas no appreciable engraftment was observed in WT recipients, donor-derived lymphocytes repopulated well in X-SCID, reaching 37% (10(6)cells given) and 53% (10(7) cells given) of the normal control value 5 months post BMT. A lineage analysis showed a predominance of the donor-derived lymphocytes (CD4(+) T, CD8(+) T, B and NK cells) in X-SCID while the donor-derived granulocytes and monocytes engrafted poorly. These results showed a selective advantage of WT cells in X-SCID, and that the advantage was restricted to lymphocytes. In human gene therapy for X-SCID, an analogous growth advantage would greatly enhance the repopulation of lymphocytes derived from a very small number of gamma c gene-supplemented precursors.

In vivo selective expansion of gene-modified hematopoietic cells in a nonhuman primate model.

A major problem limiting hematopoietic stem cell (HSC) gene therapy is the low efficiency of gene transfer into human HSCs using retroviral vectors. Strategies, which would allow in vivo expansion of gene-modified hematopoietic cells, could circumvent the problem. To this end, we developed a selective amplifier gene (SAG) consisting of a chimeric gene composed of the granulocyte colony-stimulating factor (G-CSF) receptor gene and the estrogen receptor gene hormone-binding domain. We have previously demonstrated that primary bone marrow progenitor cells transduced with the SAG could be expanded in response to estrogen in vitro. In the present study, we evaluated the efficacy of the SAG in the setting of a clinically applicable cynomolgus monkey transplantation protocol. Cynomolgus bone marrow CD34(+) cells were transduced with retroviral vectors encoding the SAG and reinfused into each myeloablated monkey. Three of the six monkeys that received SAG transduced HSCs showed an increase in the levels of circulating progeny containing the provirus in vivo following administration of estrogen or tamoxifen without any serious adverse effects. In one monkey examined in detail, transduced hematopoietic progenitor cells were increased by several-fold (from 5% to 30%). Retroviral integration site analysis revealed that this observed increase was polyclonal and no outgrowth of a dominant single clonal population was observed. These results demonstrate that the inclusion of our SAG in the retroviral construct allows selective in vivo expansion of genetically modified cells by a non-toxic hormone treatment.

Variation in CO2 assimilation rate induced by simulated dew waters with different sources of hydroxyl radical (*OH) on the needle surfaces of Japanese red pine (Pinus densifora Sieb. et Zucc.).

The hydroxyl radical (*OH) is generated in polluted dew on the needle surfaces of Japanese red pine (Pinus densiflora Sieb. et Zucc.). This free radical, which is a potent oxidant, is assumed to be a cause of ecophysiological disorders of declining trees on the urban-facing side of Mt. Gokurakuji, western Japan. Mists of *OH-generating N(III) (HNO2 and NO2-) and HOOH + Fe + oxalate solutions (50 and 100 microM, pH 5.1-5.4) simulating the dew water were applied to the foliage of pine seedlings grown in open-top chambers in the early morning. Needles treated with 100 microM N(III) tended to have a greater maximum CO2 assimilation rate (Amax), a greater stomatal conductance (g(s)) and a greater needle nitrogen content (Nneedle), suggesting that N(III) mist acts as a fertilizer rather than as a phytotoxin. On the other hand, needles treated with 100 microM HOOH + Fe + oxalate solution showed the smallest Amax, g(s), and Nneedle, suggesting that the combination of HOOH + Fe + oxalate caused a decrease in needle productivity. The effects of HOOH + Fe + oxalate mist on pine needles were very similar to the symptoms of declining trees at Mt. Gokurakuji.

Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AAV) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AAV vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or beta-galactosidase (AAV-LacZ) into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AAV-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4-20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AAV vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.

Expression of HGF/NK4 in ovarian cancer cells suppresses intraperitoneal dissemination and extends host survival.

Peritoneal dissemination is the most frequent progression pathway of ovarian cancer and is therefore a key step to improve the prognosis. NK4, a large part of the alpha-chain of hepatocyte growth factor, is known to inhibit cancer cell migration. To characterize the function of NK4 and investigate its potential role in gene therapy of ovarian cancer, we introduced NK4 cDNA to an ovarian cancer cell line HRA and investigated its effects both in vitro and in vivo. HRA cells were transfected with either NK4 or luciferase-expression plasmids. After selection, NK4-expressing HRA cells (HRA/NK4) and the control cells (HRA/LUC) were obtained. NK4 was detected in the culture supernatant of HRA/NK4 by Western analysis. Migration capabilities of the cells were evaluated in vitro by scratch wound healing assay. The number of migrated cells was significantly smaller in the HRA/NK4 cultures than that in the control cultures (HRA or HRA/LUC). Also, the culture supernatant of HRA/NK4 significantly suppressed migration of control cells. This suppressive effect was observed when NK4-expressing cells were mixed with control cells at the ratio of 25% or more. In the in vivo experiments, HRA transfectants were injected intraperitoneally. The number of intraperitoneal tumors of HRA/NK4 was much smaller than that of control. In mice injected with HRA/NK4, ascites formation was suppressed and the survival was significantly prolonged. These findings suggest that NK4-mediated gene therapy can improve the prognosis of ovarian cancer by suppressing peritoneal dissemination.

Development and characterization of an antisense-mediated prepackaging cell line for adeno-associated virus vector production.

One of the limitations of recombinant adeno-associated virus (rAAV) vector systems for gene therapy applications has been the difficulty in producing the vector in sufficient quantity for adequate evaluation. Since the AAV Rep proteins are cytotoxic, it is not easy to establish stable cell lines that express them constitutively. We describe a novel 293-derived prepackaging cell line which constitutively expresses the antisense rep/cap driven by a loxP-flanked CMV promoter. This cell line was converted into a packaging cell line expressing Rep/Cap for rAAV vector production through adenovirus-mediated introduction of a Cre recombinase gene. Without the introduction of the Cre recombinase gene, the cell line was shown to produce neither Rep nor Cap. rAAV vector was produced (1 x 10(9) genome copies/3.5-cm dish) 4 days after the transduction with Cre-expression adenovirus vector together with transfection of AAV vector plasmid. We further showed that the addition of Cap-expression adenovirus vector caused a 10-fold increase in the yield of rAAV vector. This system is also capable of producing rAAV as a transfection-free system by using a small amount of rAAV instead of vector plasmid.

Adeno-associated virus-mediated transfer of endothelial nitric oxide synthase gene inhibits protein synthesis of rat ventricular cardiomyocytes.

We investigated whether nitric oxide (NO) synthase gene transfer could attenuate growth of cultured cardiac myocytes. First, we investigated the effects of exogenous NO and cGMP analog on protein synthesis of cultured neonatal rat cardiac myocytes. The NO donor 3-morpholino-sydnonimine-hydrochloride (SIN-1) and 8-bromo-cGMP caused concentration-dependent decreases in phenylephrine-stimulated incorporation of 3H-leucine into cardiac myocytes. We then transferred endothelial constitutive NO synthase (ecNOS) gene into cultured neonatal rat cardiac myocytes using adeno-associated virus (AAV) vectors. ecNOS gene transfer into cardiac myocytes induced 140 kD ecNOS protein expression and significantly increased cGMP contents of myocytes compared with control cells. ecNOS gene transfer inhibited 3H-leucine incorporation into cardiac myocytes in response to phenylephrine, which was significantly recovered in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine acetate. These results indicate that endogenously generated NO by ecNOS gene transfer using AAV vectors inhibits the alpha-adrenergic agonist-induced cardiac protein synthesis at least partially via cGMP production.