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.

Depression-like episodes in mice harboring mtDNA deletions in paraventricular thalamus.

Depression is a common debilitating human disease whose etiology has defied decades of research. A critical bottleneck is the difficulty in modeling depressive episodes in animals. Here, we show that a transgenic mouse with chronic forebrain expression of a dominant negative mutant of Polg1, a mitochondrial DNA (mtDNA) polymerase, exhibits lethargic behavioral changes, which are associated with emotional, vegetative and psychomotor disturbances, and response to antidepression drug treatment. The results suggested a symptomatic similarity between the lethargic behavioral change that was recurrently and spontaneously experienced by the mutant mice and major depressive episode as defined by DSM-5. A comprehensive screen of mutant brain revealed a hotspot for mtDNA deletions and mitochondrial dysfunction in the paraventricular thalamic nucleus (PVT) with similar defects observed in postmortem brains of patients with mitochondrial disease with mood symptoms. Remarkably, the genetic inhibition of PVT synaptic output by Cre-loxP-dependent expression of tetanus toxin triggered de novo depression-like episodes. These findings identify a novel preclinical mouse model and brain area for major depressive episodes with mitochondrial dysfunction as its cellular mechanism.

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.

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.

Can malignant transformation in mature cystic teratoma be preoperatively predicted?

PURPOSE OF INVESTIGATION: The study aimed to determine whether malignant transformation of mature cystic teratoma (MCT) can be preoperatively predicted by presenting two cases of MCT with malignant transformation and comparing their clinical factors with those of benign MCT encountered at around the same time. MATERIALS AND METHODS: Age, maximum tumor diameter, tumor marker levels (serum squamous cell carcinoma (SCC) and carbohydrate antigen (CA) 19-9, the presence of solid tumor masses, and the presence or absence of contrast enhancement in pelvic magnetic resonance imaging (MRI) were investigated in two cases of MCT with malignant transformation and 76 cases of benign MCT in which surgery was performed and a pathological diagnosis given by the department from 2004 to 2010. RESULTS: The mean ages of the two cases with malignant transformation and the cases of benign MCT were 42.5 years and 34.2 years, respectively. The mean maximum diameter of the two tumors with malignant transformation and the cases of benign MCT were 130 mm and 73.6 mm, respectively. The mean serum levels of SCC in the two cases with malignant transformation and the cases of benign MCT were 31.5 ng/ml and 0.92 ng/ml, respectively. Contrast enhancement and the presence of solid masses in images of MCT with malignant transformation were apparent. CONCLUSION: In order to accurately detect malignant transformation of MCT, the authors found it to be important to determine whether tumors larger than 100 mm in diameter were present and to check for the presence of solid masses enhanced in pelvic MRI examination, as well as to measure at least serum SCC and CA19-9 even in relatively young patients.

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.

Ganglioside GM3 promotes cell migration by regulating MAPK and c-Fos/AP-1.

Gangliosides have been proposed as modulators of transmembrane signaling. Recently, GM3, a glycosphingolipid containing monosaialic acids, is thought to be one of the key molecules of signal transduction in mammalian cells. In this study, we used mouse embryonic fibroblast cell lines (MEFs) established from sialyltransferase-I knockout mice (GM3 synthase KO mice) to evaluate the regulation of mitogenic signals by gangliosides. Cell proliferation assay revealed a higher growth potential of GM3 KO MEFs. Immunoblots showed upregulation of Ras/Raf/MEK/ERK pathway in GM3 KO MEFs, and these signals resulted in enhanced translocation of ERK into the nuclei. Further, both exogenous and endogenous add-back of GM3 decreased the activities of MAPK in GM3 KO MEFs. In addition, GM3 KO MEFs formed foci in high-density culture condition, and analyses of cell cycle modulators revealed the resistance of GM3 KO MEFs for entering cell cycle arrest. Finally, sustained expressions of c-Fos in GM3 KO MEFs were shown to correlate with DNA-binding activity between c-Fos and AP-1. These results demonstrate that the deletion of sialyltransferase-I changes the character of MEFs to a highly activated state of the MAPK pathway, indicating the critical role of GM3 as a regulator of membrane-transmitted signals.

Effects of Hydroxychloroquine in Patients With Cutaneous Lupus Erythematosus: A Multicenter, Double-Blind, Randomized, Parallel-Group Trial.

OBJECTIVE: To assess the efficacy and tolerability of hydroxychloroquine (HCQ) in patients with cutaneous lupus erythematosus (CLE), in a phase III clinical trial conducted in Japan. METHODS: We conducted a double-blind, randomized, parallel-group clinical trial. This was a baseline-controlled study, and the group differences were evaluated in an exploratory analysis. A total of 103 patients with active CLE (according to a Cutaneous Lupus Erythematosus Disease Area and Severity Index [CLASI] activity score of ≥4) were included. Patients were randomized 3:1 to receive HCQ or placebo during the 16-week double-blind period, and all patients were given HCQ during the following 36-week single-blind period. The primary efficacy end point was a reduction in the CLASI activity score at week 16. The secondary end points included the central photo evaluation (5-point scale), patient's global assessment (7-point scale), the Skindex-29 score, and investigator's global assessment (7-point scale, based on the other 3 secondary end points). In patients with systemic lupus erythematosus, fatigue and musculoskeletal pain were assessed. Safety was assessed up to week 55. RESULTS: The mean CLASI score at week 16 was significantly improved from baseline in both the HCQ group and the placebo group: mean change -4.6 (95% confidence interval [95% CI] -6.1, -3.1) (P < 0.0001), and mean change -3.2 (95% CI -5.1, -1.3) (P = 0.002), respectively, without between-group difference (P = 0.197). The investigator's global assessment demonstrated a greater proportion of "improved" and "remarkably improved" patients in the HCQ group (51.4% versus 8.7% in the placebo group [P = 0.0002 between groups]). The other secondary end points supported the efficacy of HCQ. Cellulitis, drug eruption, hepatic dysfunction, and Stevens-Johnson syndrome were shown to be serious adverse events related to HCQ use. CONCLUSION: The results of this randomized clinical trial support the efficacy and tolerability of HCQ in patients with CLE.

Increased expression and activation of matrix metalloproteinase-2 (MMP-2) in O-1N: hamster oral squamous cell carcinoma with high potential lymph node metastasis.

Oral squamous cell carcinoma (OSCC) is one of the most common head and neck cancers with poor clinical outcome. MMP-2 was implied to contribute to the invasiveness and metastatization of various malignancies because of the degradation of type IV collagen. In this experiment, ELISA, Western blot and Q-RT-PCR was performed to investigate the expression and activation of MMP-2 in serum and tumor from hamster oral cancer model with high lymph node metastasis based on the various stages of OSCC development. Active MMP-2 in the serum was found elevated during oral cancer progression. In the metastatic stage, total MMP-2 level was 46% higher than it in the expansive stage, while active MMP-2 level increased 6-fold than it in the expansive stage. MMP-2 serum activation ratio was significantly enhanced in the metastatic stage than before tumor transplantation and that in the expansive stage. MMP-2 protein and MMP-2 mRNA was found to be increased during oral cancer development in hamster models. The increase of MMP-2 expression and activation starts prior to the metastasis occurrence, indicating the important role of MMP-2 in the early phase of oral cancer progression. Active MMP-2 level in serum may be a useful indicator for monitoring oral cancer progression.

ESR correlation times of 2,2,6,6-tetramethyl piperidone-N-oxyl (Tempone) in solutions of hemoglobin A and hemoglobin S.

Correlation times for the tumbling motion of the spin probe 2,2,6,6,-tetramethyl piperidone-N-oxyl (Tempone) were obtained in the presence of different concentrations of oxyhemoglobin A, oxyhemoglobin S, and deoxyhemoglobin S and compared to the viscosity of non-gelling hemoglobin solutions. Reorientational motion (or tumbling) of Tempone in gelled solutions of deoxyhemoglobin S is as great as that in non-gelled hemoglobins of the same total concentration. It is concluded that the gel does not exclusively partition Tempone into an aqueous phase of lower solute concentration after gel formation. The gel at room temperature is a highly mobile and dynamic structure on the microscopic level.

N-terminal spin label studies of hemoglobin, Ligand and pH dependence.

Human hemoglobin was spin labeled with 4-isothiocanato-2,2,6,6-tetramethyl-piperdinooxyl, which is known to bind specifically to the N-terminal alpha-amino groups of proteins and slightly to the reactive sulfhydryl groups. Electron spin resonance (ESR) analysis indicated a partially resolved five-line spectrum, suggesting that the label was attached to at least two different binding sites. Using specific blocking reagents prior to spin labeling, the two binding sites were attributed to the sulfhydryl group of beta-93 (immobile) and the alpha-amino group of the N-terminal valines (mobile). The relative motion of the spin at one set of binding sites was restricted regardless of the state of ligation and pH, while the motion at the other site showed dependence on those parameters, e.g. the spin-labeled N-terminal ends of deoxyhemoglobin have restricted motion at all pH ranges studied, while those of oxyhemoglobin are relatively free to move at the basic pH range, but become more restricted in the acidic pH range.

Double blockade of cell cycle progression by coptisine in vascular smooth muscle cells.

Coptisine, an isoquinoline alkaloid isolated from rhizome of Coptis japonica, inhibits proliferation of vascular smooth muscle cells (VSMCs). The aim of this study was to evaluate the action of coptisine, along with berberine (a structurally similar isoquinoline alkaloid), on progression of the cell cycle in VSMCs. Coptisine displayed antiproliferative action against VSMCs by blocking the cell cycle at G(1) and G(2)/M phases. The G(1) block was shown by inhibition of [(3)H]thymidine incorporation into VSMCs at coptisine concentrations higher than 15 microM. The mechanism underlying the G(1) arrest involved a decrease in cyclin D1 protein, although cyclin E, A, and B were not affected by coptisine treatment. The selective reduction in cyclin D1 protein was mainly attributable to accelerated proteolysis via proteasome-dependent pathway, since it was inhibited by a proteasome inhibitor, N-carbobenzoxy-L-leucinyl-L-leucinyl-L-norleucinal (MG132) and further the mRNA level of cyclin D1, protein synthesis, and mitogen-activated protein kinase (MAPK) activity remained unaltered. The mechanism underlying the G(2)/M arrest involved partial inhibition of tubulin polymerization, which was apparent at coptisine concentration of 3 microM. Berberine arrested the cell cycle at G(1) phase via a mechanism identical with coptisine, but did not cause block at G(2)/M phase. The results demonstrate that a small difference in the structure between isoquinoline alkaloids produces a big difference in activity, and that coptisine has a unique double action in arresting the cell cycle of VSMCs.

Retinoic acid acts to neutralize the inhibitory effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on alkaline phosphatase activity of neutrophils that is induced by granulocyte colony-stimulating factor (G-CSF).

Granulocyte colony-stimulating factor (G-CSF) is known to act on the neutrophilic granulocytes from chronic myelogenous leukemia (CML) patients to induce neutrophil alkaline phosphatase (NAP) activity. Gamma-interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been reported to suppress NAP induction with G-CSF. We confirmed that this inhibitory effect of GM-CSF is accompanied by the decrease of the NAP mRNA level. Moreover, we found that the simultaneous addition of retinoic acid completely neutralized this inhibitory effect of GM-CSF. Recovery of the NAP activity brought about by the retinoic acid was also accompanied by the increase of NAP mRNA level. These results indicate that retinoic acid neutralizes the inhibitory effect of GM-CSF on the induction of NAP activity through the change of the NAP mRNA level.

New approaches to gene and cell therapy for hemophilia.

Hemophilia is considered suitable for gene therapy because it is caused by a single gene abnormality, and therapeutic coagulation factor levels may vary across a broad range. Recent success of hemophilia B gene therapy with an adeno-associated virus (AAV) vector in a clinical trial showed the real prospect that, through gene therapy, a cure for hemophilia may become a reality. However, AAV-mediated gene therapy is not applicable to patients with hemophilia A at present, and neutralizing antibodies against AAV reduce the efficacy of AAV-mediated strategies. Because patients that benefit from AAV treatment (hemophilia B without neutralizing antibodies) are estimated to represent only 15% of total patients with hemophilia, the development of basic technologies for hemophilia A and those that result in higher therapeutic effects are critical. In this review, we present an outline of gene therapy methods for hemophilia, including the transition of technical developments thus far and our novel techniques.

A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus.

Recombinant adeno-associated virus (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector). A single injection of the AAV-HIV vector induced strong production of HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV-HIV vector was coadministered with AAV-IL2 vector, the HIV-specific cell-mediated immunity (CMI) was significantly enhanced. Boosting with AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin (HA) gene showed protective immunity against homologous influenza virus challenge. These results demonstrate that AAV-HIV vector immunization may provide a novel and promising HIV vaccination strategy.

Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson's disease.

Parkinson's disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.

A metal-linked gapped zipper model is proposed for the hsp90-glucocorticoid receptor interaction.

In the presence of certain metals, regions of the hormone binding domain of the glucocorticoid receptor (GR) are capable of binding the 90 kDa heat shock protein (hsp90). Using secondary structure prediction methods in correlation with the experimental data, we propose a model which predicts the presence of two widely spaced leucine zipper-like heptads on either side of a central subdomain. The heptads could interact hydrophobically with similar regions on the hsp90 homodimer, bringing putative metal binding residues on each protein close enough to establish a shared metal bridge. The central subdomain between heptads is suggested to contain regions involved in metal binding, steroid binding, and conformational mobility. The hypothetical model that we are proposing therefore addresses the nature of the structural link between hsp90 binding, hormone binding, and conformational changes in the receptor.

Gamma-rays enhance rAAV-mediated transgene expression and cytocidal effect of AAV-HSVtk/ganciclovir on cancer cells.

Adeno-associated virus (AAV) vector has several unique properties suited for gene therapy applications. However, relatively low efficiency of transgene expression, which is mainly due to a limited second-strand synthesis from the single-stranded AAV genome, can be a problem in some applications that require potent gene expression such as antitumor applications. Recently, gamma-ray irradiation has been reported to enhance the second-strand synthesis of the AAV genome, and consequently transgene expression. We demonstrate here that an AAV vector harboring the herpes simplex virus type-1 thymidine kinase (HSVtk) is able to kill cancer cells more efficiently when used in combination with gamma-ray irradiation. A human maxillary sinus cancer cell line, NKO-1, was efficiently killed in combination with HSVtk transduction and ganciclovir (GCV), as expected. More importantly, gamma-ray irradiation of practical dosages augmented the cytocidal effect of the HSVtk/GCV system. Southern analysis indicated that gamma-rays enhanced the double-strand synthesis of the rAAV genome in NKO-1 cells. These findings suggest that the combination of rAAVtk/GCV suicide gene therapy with radiotherapy has synergistic effects in the treatment of cancers and may lead to a reduction of the potential toxicity of both rAAVtk/GCV and gamma-ray irradiation.

Specific detection of human coagulation factor IX in cynomolgus macaques.

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.