Explore the vast range of titles available.

Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs. Limits of AAV-mediated gene therapy include targeting dominant mutations and inducing long-term transgene expression. Here, the authors show that AAV-HITI results in efficient allele-independent integration of a donor DNA in both retina and liver providing therapeutic benefit in mouse models of either a genetic form of blindness or a lysosomal storage disease, respectively.

Enzyme replacement therapy (ERT) is the standard of care for several lysosomal storage diseases (LSDs). ERT, however, requires multiple and costly administrations and has limited efficacy. We recently showed that a single high dose administration of adeno-associated viral vector serotype 8 (AAV2/8) is at least as effective as weekly ERT in a mouse model of mucopolysaccharidosis type VI (MPS VI). However, systemic administration of high doses of AAV might result in both cell-mediated immune responses and insertional mutagenesis. Here we evaluated whether the combination of low doses of AAV2/8 with a less frequent (monthly) than canonical (weekly) ERT schedule may be as effective as the single treatments at high doses or frequent regimen. A greater reduction of both urinary glycosaminoglycans, considered a sensitive biomarker of therapeutic efficacy, and storage in the myocardium and heart valves was observed in mice receiving the combined than the single therapies. Importantly, these levels of correction were similar to those we obtained in a previous study following either high doses of AAV2/8 or weekly ERT. Our data show that low-dose gene therapy can be used as a means to rarify ERT administration, thus reducing both the risks and costs associated with either therapies.

Background Leptin is a pleiotropic hormone whose mitogenic and angiogenic activity has been implicated in the development and progression of several malignancies, including brain tumors. In human brain cancer, especially in glioblastoma multiforme (GBM), leptin and its receptor (ObR) are overexpressed relative to normal tissue. Until present, the potential of intratumoral leptin to exert proangiogenic effects on endothelial cells has not been addressed. Using in vitro models, we investigated if GBM can express leptin, if leptin can affect angiogenic and mitogenic potential of endothelial cells, and if its action can be inhibited with specific ObR antagonists. Leptin effects were compared with that induced by the best-characterized angiogenic regulator, VEGF. Results We found that GBM cell lines LN18 and LN229 express leptin mRNA and LN18 cells secrete detectable amounts of leptin protein. Both lines also expressed and secreted VEGF. The conditioned medium (CM) of LN18 and LN 229 cultures as well as 200 ng/mL pure leptin or 50 ng/mL pure VEGF stimulated proliferation of human umbilical vein endothelial cells (HUVEC) at 24 h of treatment. Mitogenic effects of CM were ~2-fold greater than that of pure growth factors. Furthermore, CM treatment of HUVEC for 24 h increased tube formation by ~5.5-fold, while leptin increased tube formation by ~ 80% and VEGF by ~60% at 8 h. The mitogenic and angiogenic effects of both CM were blocked by Aca 1, a peptide ObR antagonist, and by SU1498, which inhibits the VEGF receptor. The best anti-angiogenic and cytostatic effects of Aca1 were obtained with 10 nM and 25 nM, respectively, while for SU1498, the best growth and angiogenic inhibition was observed at 5 μM. The combination of 5 μM SU1498 and Aca1 at 25 nM (growth inhibition) or at 10 nM (reduction of tube formation) produced superior effects compared with single agent treatments. Conclusions Our data provide the first evidence that LN18 and LN 229 human GBM cells express leptin mRNA and might produce biologically active leptin, which can stimulate tube formation and enhance proliferation of endothelial cells. Furthermore, we demonstrate for the first time that a peptide ObR antagonist inhibits proangiogenic and growth effects of leptin on endothelial cells, and that the pharmacological potential of this compound might be combined with drugs targeting the VEGF pathway.

In vivo gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. We recently demonstrated that AAV8-mediated liver gene transfer is effective in animal models of mucopolysaccharidosis type VI (MPS VI), a rare lysosomal storage disease that is caused by arylsulfatase B (ARSB) deficiency. In preparing for a first-in-human trial, we performed non-clinical studies to assess the safety of intravenous administrations of AAV2/8.TBG. produced under good manufacturing practice-like conditions. No toxicity was observed in AAV-treated mice, except for a transient increase in alanine aminotransferase in females and thyroid epithelial hypertrophy. AAV2/8.TBG. biodistribution and expression confirmed the liver as the main site of both infection and transduction. Shedding and breeding studies suggest that the risk of both horizontal and germline transmission is minimal. An AAV dose-response study in MPS VI mice was performed to define the range of doses to be used in the clinical study. Overall, these data support the non-clinical safety and efficacy of AAV2/8.TBG. and pave the way for a phase I/II clinical trial based on intravascular infusions of AAV8 in patients with MPS VI.

Metformin, a derivative of biguanide, is a first-line therapy for type 2 diabetes mellitus. Since the drug has been shown to significantly reduce the risk of various cancers and cancer mortality in diabetic patients, it is being considered as a potential anticancer therapeutic or preventive agent. In cellular models, metformin inhibits the growth of many types of cancer cells; however, its effects on glioblastoma multi-forme (GBM) are not well characterized. Here, we analyzed the effects of metformin on the growth and migration of LN18 and LN229 GBM cells cultured under basal conditions or exposed to leptin, a cytokine that has recently been implicated in GBM development. We found that 2-16 mM metformin reduced basal and leptin-stimulated growth of GBM cells in a dose-dependent manner. Furthermore, the drug significantly inhibited the migration of GBM cells. The action of metformin was mediated through the upregulation of its main signaling molecule, the adenosine monophosphate-activated protein kinase (AMPK), as well as through the downregulation of the signal transducer and activator of transcription 3 (STAT3) and the Akt/PKB serine/threonine protein kinase. In leptin-treated cells, the drug reversed the effects of the cytokine on the AMPK and STAT3 pathways, but modulated Akt activity in a cell-dependent manner. Our results suggest that metformin or similar AMPK-targeting agents with optimized blood-brain-barrier penetrability could be developed as potential treatments of GBM and could be used in conjunction with other target drugs such as leptin receptor antagonists.

 Phytophagous mites can cause economic losses in many crops, including grapevines. The changes in their population levels may be associated with changes in the predator-prey relationship. Knowledge of the distribution of mites in plants is important for planning sampling strategies and facilitating control decisions. The aims of this study were to (i) evaluate the abundances of Tetranychus urticae (Tetranychidae) and Neoseiulus californicus (Phytoseiidae), and the correlations between them and environmental factors; (ii) determine their distribution on the top, middle, or base strata of the evaluated grapevines (Vitis vinifera, Chardonnay cultivar) and, additionally, report the first occurrence of damage caused by T. urticae in grapevine leaves in the state of Rio Grande do Sul, Southern Brazil. Sixteen samplings were conducted, divided between the 2018 and 2019 seasons. In each sampling, three leaves from the three strata of the plant (top, middle, and base) were collected from 20 plants, totaling 60 leaves per sampling. The predator-prey relationship and their association with environmental variables were evaluated with multivariate correlation, whereas the number of mites per plant leaf strata was compared using a generalized linear mixed model in R software. It was possible to observe the symptoms of damage caused by T. urticae attacks on grapevines in Southern Brazil, characterized by the presence of yellow spots and general yellowing of the vineyard. Our findings indicate that T. urticae and N. californicus individuals are strongly associated with each other regardless of the environmental variables, and such relationship occurs mainly on the lower strata (middle and base leaves) of grapevines. Thus, by taking into account the damage on the leaves of grapevines and the occurrence of T. urticae and N. californicus majorities on specific strata of these vineyards, we suggest that the lower strata of grapevines should be the priority targets for management strategies to control such mites.

The aim of this study was to investigate the biology and life table parameters of the predatory mite Agistemus floridanus Gonzalez (Stigmaeidae) feeding on Panonychus ulmi (Koch) (Tetranychidae). The experiment was initiated with 34 eggs of A. floridanus isolated in experimental units and maintained in a controlled environment at 25 ± 1 °C, 12 L:12D, and 80 ± 5% relative humidity. The development time of females was 11.7 ± 1.0 days and the immature phase of males 11.2 ± 1.0 days. Adult longevity of females was 17.1 ± 5.3 days, with mean production of 29.2 ± 17.4 eggs/female. Life table parameters obtained were: net reproduction rate (Ro) 19.8; mean generation time (T) 19.1 days; intrinsic rate of increase (rm) 0.2 female per female per day; finite rate of increase (λ) 1.2 female/female, and population doubling time (DT) 4.4 days. Agistemus floridanus demonstrated survival and reproduction capacity when feeding exclusively on P. ulmi.