Search

Impaired calcium uptake resulting from reduced expression and activity of the cardiac sarco-endoplasmic reticulum Ca2+ ATPase (SERCA2a) is a hallmark of heart failure (HF). Recently, new mechanisms of SERCA2a regulation, including post-translational modifications (PTMs), have emerged. Our latest analysis of SERCA2a PTMs has identified lysine acetylation as another PTM which might play a significant role in regulating SERCA2a activity. SERCA2a is acetylated, and that acetylation is more prominent in failing human hearts. In this study, we confirmed that p300 interacts with and acetylates SERCA2a in cardiac tissues. Several lysine residues in SERCA2a modulated by p300 were identified using in vitro acetylation assay. Analysis of in vitro acetylated SERCA2a revealed several lysine residues in SERCA2a susceptible to acetylation by p300. Among them, SERCA2a Lys514 (K514) was confirmed to be essential for SERCA2a activity and stability using an acetylated mimicking mutant. Finally, the reintroduction of an acetyl-mimicking mutant of SERCA2a (K514Q) into SERCA2 knockout cardiomyocytes resulted in deteriorated cardiomyocyte function. Taken together, our data demonstrated that p300-mediated acetylation of SERCA2a is a critical PTM that decreases the pump's function and contributes to cardiac impairment in HF. SERCA2a acetylation can be targeted for therapeutic aims for the treatment of HF. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:24 issue:4 ispartof: location:Switzerland status: published

Biolistic intracellular delivery of functional macromolecules makes use of dense microparticles which are ballistically fired onto cells with a pressurized gun. While it has been used to transfect plant cells, its application to mammalian cells has met with limited success mainly due to high toxicity. Here we present a more refined nanotechnological approach to biolistic delivery with light-triggered self-assembled nanobombs (NBs) that consist of a photothermal core particle surrounded by smaller nanoprojectiles. Upon irradiation with pulsed laser light, fast heating of the core particle results in vapor bubble formation, which propels the nanoprojectiles through the cell membrane of nearby cells. We show successful transfection of both adherent and non-adherent cells with mRNA and pDNA, outperforming electroporation as the most used physical transfection technology by a factor of 5.5–7.6 in transfection yield. With a throughput of 104-105 cells per second, biolistic delivery with NBs offers scalable and highly efficient transfections of mammalian cells.

Cardiovascular diseases are a major health problem, and long-term survival for people diagnosed with heart failure is, still, unrealistic. Pathological cardiac hypertrophy largely contributes to morbidity and mortality, as effective therapeutic approaches are lacking. Non-coding RNAs (ncRNAs) arise as active regulators of the signaling pathways and mechanisms that govern this pathology, and their therapeutic potential has received great attention in the last decades. Preclinical studies in large animal models have been successful in ameliorating cardiac hypertrophy, and an antisense drug for the treatment of heart failure has, already, entered clinical trials. In this review, we provide an overview of the molecular mechanisms underlying cardiac hypertrophy, the involvement of ncRNAs, and the current therapeutic landscape of oligonucleotides targeting these regulators. Strategies to improve the delivery of such therapeutics and overcome the actual challenges are, also, defined and discussed. With the fast advance in the improvement of oligonucleotide drug delivery, the inclusion of ncRNAs-targeting therapies for cardiac hypertrophy seems, increasingly, a closer reality.

Cardiovascular diseases are a major health problem, and long-term survival for people diagnosed with heart failure is, still, unrealistic. Pathological cardiac hypertrophy largely contributes to morbidity and mortality, as effective therapeutic approaches are lacking. Non-coding RNAs (ncRNAs) arise as active regulators of the signaling pathways and mechanisms that govern this pathology, and their therapeutic potential has received great attention in the last decades. Preclinical studies in large animal models have been successful in ameliorating cardiac hypertrophy, and an antisense drug for the treatment of heart failure has, already, entered clinical trials. In this review, we provide an overview of the molecular mechanisms underlying cardiac hypertrophy, the involvement of ncRNAs, and the current therapeutic landscape of oligonucleotides targeting these regulators. Strategies to improve the delivery of such therapeutics and overcome the actual challenges are, also, defined and discussed. With the fast advance in the improvement of oligonucleotide drug delivery, the inclusion of ncRNAs-targeting therapies for cardiac hypertrophy seems, increasingly, a closer reality.

Midbrain dopamine neurons deteriorate in Parkinson's disease (PD) that is a progressive neurodegenerative movement disorder. No cure is available that would stop the dopaminergic decline or restore function of injured neurons in PD. Neurotrophic factors (NTFs), e.g., glial cell line-derived neurotrophic factor (GDNF) are small, secreted proteins that promote neuron survival during mammalian development and regulate adult neuronal plasticity, and they are studied as potential therapeutic agents for the treatment of neurodegenerative diseases. However, results from clinical trials of GDNF and related NTF neurturin (NRTN) in PD have been modest so far. In this review, we focus on cerebral dopamine neurotrophic factor (CDNF), an unconventional neurotrophic protein. CDNF delivered to the brain parenchyma protects and restores dopamine neurons in animal models of PD. In a recent Phase I-II clinical trial CDNF was found safe and well tolerated. CDNF deletion in mice led to age-dependent functional changes in the brain dopaminergic system and loss of enteric neurons resulting in slower gastrointestinal motility. These defects in Cdnf(-/-) mice intriguingly resemble deficiencies observed in early stage PD. Different from classical NTFs, CDNF can function both as an extracellular trophic factor and as an intracellular, endoplasmic reticulum (ER) luminal protein that protects neurons and other cell types against ER stress. Similarly to the homologous mesencephalic astrocyte-derived neurotrophic factor (MANF), CDNF is able to regulate ER stress-induced unfolded protein response (UPR) signaling and promote protein homeostasis in the ER. Since ER stress is thought to be one of the pathophysiological mechanisms contributing to the dopaminergic degeneration in PD, CDNF, and its small-molecule derivatives that are under development may provide useful tools for experimental medicine and future therapies for the treatment of PD and other neurodegenerative protein-misfolding diseases.

Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy., Adeno-associated virus is the basis of many gene therapies and gene transfer vectors. Here the authors report a pipeline to enable side-by-side comparison of pre-selected capsids in a high throughput manner.

Oncolytic adenoviruses are promising cancer therapeutic agents. Clinical data have shown adenoviruses’ ability to transduce tumors after systemic delivery in human cancer patients, despite antibodies. In the present work, we have focused on the interaction of a chimeric adenovirus Ad5/3 with human lymphocytes and human erythrocytes. Ad5/3 binding with human lymphocytes and erythrocytes was observed to occur in a reversible manner, which allowed viral transduction of tumors, and oncolytic potency of Ad5/3 in vitro and in vivo, with or without neutralizing antibodies. Immunodeficient mice bearing xenograft tumors showed enhanced tumor transduction following systemic administration, when Ad5/3 virus was bound to lymphocytes or erythrocytes (P

Adeno-associated virus (AAV) vector serotypes vary in their ability to transduce hepatocytes from different species. Chimeric mouse models harboring human hepatocytes have shown translational promise for liver-directed gene therapies. However, many variables that influence human hepatocyte transduction and transgene expression in such models remain poorly defined. Here, we aimed to test whether three experimental conditions influence AAV transgene expression in immunodeficient, fumaryl-acetoactetate-hydrolase-deficient (Fah−/−) chimeric mice repopulated with primary human hepatocytes. We examined the effects of the murine liver injury cycle, human donor variability, and vector doses on hepatocyte transduction with various AAV serotypes expressing a green fluorescent protein (GFP). We determined that the timing of AAV vector challenge in the liver injury cycle resulted in up to 7-fold differences in the percentage of GFP expressing human hepatocytes. The GFP+ hepatocyte frequency varied 7-fold between human donors without, however, changing the relative transduction efficiency between serotypes for an individual donor. There was also a clear relationship between AAV vector doses and human hepatocyte transduction and transgene expression. We conclude that several experimental variables substantially affect human hepatocyte transduction in the Fah−/− chimera model, attention to which may improve reproducibility between findings from different laboratories., Graphical Abstract, Liver chimeric mice are widely used to model human hepatocyte susceptibility to adeno-associated virus (AAV) gene vector transduction with inconsistent finding between laboratories. Zou et al. quantify to what extent three experimental conditions—namely, mouse liver injury, human donor variability, and AAV doses—affect human hepatocyte transduction in chimeric mice.

Therapeutic interventions with proven efficacy in heart failure with reduced ejection fraction (HFrEF) have been unsuccessful in heart failure with preserved ejection fraction (HFpEF). The modifiable risk factor with the greatest impact on the development of HFpEF is hypertension. The objectives of this study were to establish a murine model of HFpEF associated with hypertension and to evaluate the effect of apo A-IMilano nanoparticles (MDCO-216) on established HFpEF in this model. Subcutaneous infusion of angiotensin II in combination with 1% NaCl in the drinking water was started at the age of 12 weeks in male C57BL/6 N mice and continued for the entire duration of the experiment. Treatment with MDCO-216 partially reversed established cardiac hypertrophy, cardiomyocyte hypertrophy, capillary rarefaction, and perivascular fibrosis in this model. Pressure-volume loop analysis was consistent with HFpEF in hypertension mice as evidenced by the preserved ejection fraction and a significant reduction of cardiac output (7.78 ± 0.56 ml/min versus 10.5 ± 0.7 ml/min; p Milano nanoparticles constitute an effective treatment for established hypertension-associated HFpEF.

The development of eukaryote-derived antimicrobial peptides as systemically administered drugs has proven a challenging task. Here, we report the first human oral actinomyces-sourced defensin-actinomycesin-that shows promise for systemic therapy. Actinomycesin and its homologs are only present in actinobacteria and myxobacteria, and share similarity with a group of ancient invertebrate-type defensins reported in fungi and invertebrates. Signatures of natural selection were detected in defensins from the actinomyces colonized in human oral cavity and ruminant rumen and dental plaque, highlighting their role in adaptation to complex multispecies bacterial communities. Consistently, actinomycesin exhibited potent antibacterial activity against oral bacteria and clinical isolates of Staphylococcus and synergized with two classes of human salivary antibacterial factors. Actinomycesin specifically inhibited bacterial peptidoglycan synthesis and displayed weak immunomodulatory activity and low toxicity on human and mammalian cells and ion channels in the heart and central nervous system. Actinomycesin was highly efficient in mice infected with Streptococcus pneumoniae and mice with MRSA-induced experimental peritoneal infection. This work identifies human oral bacteria as a new source of systemic anti-infective drugs. ispartof: EMBO MOLECULAR MEDICINE vol:14 issue:2 ispartof: location:England status: published

Virus-based cancer vaccines are nowadays considered an interesting approach in the field of cancer immunotherapy, despite the observation that the majority of the immune responses they elicit are against the virus and not against the tumor. In contrast, targeting tumor associated antigens is effective, however the identification of these antigens remains challenging. Here, we describe ExtraCRAd, a multi-vaccination strategy focused on an oncolytic virus artificially wrapped with tumor cancer membranes carrying tumor antigens. We demonstrate that ExtraCRAd displays increased infectivity and oncolytic effect in vitro and in vivo. We show that this nanoparticle platform controls the growth of aggressive melanoma and lung tumors in vivo both in preventive and therapeutic setting, creating a highly specific anti-cancer immune response. In conclusion, ExtraCRAd might serve as the next generation of personalized cancer vaccines with enhanced features over standard vaccination regimens, representing an alternative way to target cancer., Cancer therapy using oncolytic virus has shown pre-clinical and clinical efficacy. Here, the authors report ExtraCRAd, an oncolytic virus cloaked with tumour cell membrane and report its therapeutic effects in vitro and in vivo in multiple mouse tumour models.

Due to morphological reduction and absence of amplifiable plastid genes, the identification of photosynthetic relatives of heterotrophic plants is problematic. Although nuclear and mitochondrial gene sequences may offer a welcome alternative source of phylogenetic markers, the presence of rate heterogeneity in these genes may introduce bias/systematic error in phylogenetic analyses. We examine the phylogenetic position of Thismiaceae based on nuclear 18S rDNA and mitochondrial atpA DNA sequence data, as well as using parsimony, likelihood and Bayesian inference methods. Significant differences in evolutionary rates of these genes between closely related taxa lead to conflicting results: while parsimony analyses of 18S rDNA and combined data strongly support the monophyly of Thismiaceae, Bayesian inference, with and without a relaxed molecular clock, as well as the Swofford-Olsen-Waddell-Hillis (SOWH) test confidently reject this hypothesis. We show that rate heterogeneity in our data leads to long-branch attraction artifacts in parsimony analysis. However, using model-based inference methods the question of whether Thismiaceae are monophyletic remains elusive. On the one hand maximum likelihood nonparametric bootstrapping and parametric hypothesis tests fail to support a paraphyletic Thismiaceae, on the other hand Bayesian inference methods (both without and with a relaxed clock) significantly reject a monophyletic Thismiaceae. These results show that an adequate sampling, the use of rate homogeneous data, and the application of different inference methods are important factors for developing phylogenetic hypotheses of myco-heterotrophic plants. (C) The Willi Hennig Society 2009. ispartof: Cladistics vol:25 issue:1 pages:64-77 ispartof: location:United States status: published

Aims Takotsubo syndrome (TTS) is an acute heart failure, typically triggered by high adrenaline during physical or emotional stress. It is distinguished from myocardial infarction (MI) by a characteristic pattern of ventricular basal hypercontractility with hypokinesis of apical segments, and absence of coronary occlusion. We aimed to understand whether recently discovered circulating biomarkers miR-16 and miR-26a, which differentiate TTS from MI at presentation, were mechanistically involved in the pathophysiology of TTS. Methods and results miR-16 and miR-26a were co-overexpressed in rats with AAV and TTS induced with an adrenaline bolus. Untreated isolated rat cardiomyocytes were transfected with pre-/anti-miRs and functionally assessed. Ventricular basal hypercontraction and apical depression were accentuated in miR-transfected animals after induction of TTS. In vitro miR-16 and/or miR-26a overexpression in isolated apical (but not basal) cardiomyocytes produced strong depression of contraction, with loss of adrenaline sensitivity. They also enhanced the initial positive inotropic effect of adrenaline in basal cells. Decreased contractility after TTS-miRs was reproduced in non-failing human apical cardiomyocytes. Bioinformatic profiling of miR targets, followed by expression assays and functional experiments, identified reductions of CACNB1 (L-type calcium channel Cavβ subunit), RGS4 (regulator of G-protein signalling 4) and G-protein subunit Gβ (GNB1) as underlying these effects. Conclusion miR-16 and miR-26a sensitise the heart to TTS-like changes produced by adrenaline. Since these miRs have been associated with anxiety and depression, they could provide a mechanism whereby priming of the heart by previous stress causes an increased likelihood of TTS in the future. Translational perspective TTS-associated miRs have the potential to be active players predisposing to TTS. Feasibly, their measurement in recovered TTS patients during subsequent periods of stress could be used to predict likelihood of recurrence, a significant risk in this population, and allow preventative action. Since they have been reported as raised in anxiety and depression, they could be part of a priming mechanism where chronic stress predisposes to an acute episode. Understanding the mechanistic basis for the sensitisation may also allow design of other prophylactic pharmacological therapies, including the pre/anti-miR constructs which are now starting to reach the clinic.

Many functions of eukaryotic cells are compartmentalized within membrane-bound organelles. One or more cis-encoded signals within a polypeptide sequence typically govern protein targeting to and within destination organelles. Perhaps unexpectedly, organelle targeting does not occur with high specificity, but instead is characterized by considerable degeneracy and inefficiency. Indeed, the same peptide signals can target proteins to more than one location, randomized sequences can easily direct proteins to organelles, and many enzymes appear to traverse different subcellular settings across eukaryotic phylogeny. We discuss the potential benefits provided by flexibility in organelle targeting, with a special emphasis on horizontally transferred and de novo proteins. Moreover, we consider how these new organelle residents can be protected and maintained before they contribute to the needs of the cell and promote fitness.

Horizontal gene transfer is an essential component of bacterial evolution. Quantitative information on transfer rates is particularly useful to better understand and possibly predict the spread of antimicrobial resistance. A variety of methods has been proposed to estimate the rates of plasmid-mediated gene transfer all of which require substantial labor input or financial resources. A cheap but reliable method with high-throughput capabilities is yet to be developed in order to better capture the variability of plasmid transfer rates, e.g. among strains or in response to environmental cues. We explored a new approach to the culture-based estimation of plasmid transfer rates in liquid media allowing for a large number of parallel experiments. It deviates from established approaches in the fact that it exploits data on the absence/presence of transconjugant cells in the wells of a well plate observed over time. Specifically, the binary observations are compared to the probability of transconjugant detection as predicted by a dynamic model. The bulk transfer rate is found as the best-fit value of a designated model parameter. The feasibility of the approach is demonstrated on mating experiments where the RP4 plasmid is transfered from Serratia marcescens to several Escherichia coil recipients. The methods uncertainty is explored via split sampling and virtual experiments.

Immunotherapy with tumor-infiltrating lymphocytes (TIL) or oncolytic adenoviruses, have shown promising results in cancer treatment, when used as separate therapies. When used in combination, the antitumor effect is synergistically potentiated due oncolytic adenovirus infection and its immune stimulating effects on T cells. Indeed, studies in hamsters have shown a 100% complete response rate when animals were treated with oncolytic adenovirus coding for TNFa and IL-2 (Ad5/3-E2F-D24-hTNFa-IRES-hIL2, TILT-123) and TIL therapy. In humans, one caveat with oncolytic virus therapy is that intratumoral injection has been traditionally preferred over systemic administration, for achieving sufficient virus concentrations in tumors, especially when neutralizing antibodies emerge. We have previously shown that 5/3 chimeric oncolytic adenovirus can bind to human lymphocytes for avoidance of neutralization. In this study, we hypothesized that incubation of oncolytic adenovirus (TILT-123) with TILs prior to systemic injection would allow delivery of virus to tumors. This approach would deliver both components in one self-amplifying product. TILs would help deliver TILT-123, whose replication will recruit more TILs and increase their cytotoxicity. In vitro, TILT-123 was seen binding efficiently to lymphocytes, supporting the idea of dual administration. We show in vivo in different models that virus could be delivered to tumors with TILs as carriers.

Deposits of different abnormal forms of tau in neurons and astrocytes represent key anatomo-pathological features of tauopathies. Although tau protein is highly enriched in neurons and poorly expressed by astrocytes, the origin of astrocytic tau is still elusive. Here, we used innovative gene transfer tools to model tauopathies in adult mouse brains and to investigate the origin of astrocytic tau. We showed in our adeno-associated virus (AAV)-based models and in Thy-Tau22 transgenic mice that astrocytic tau pathology can emerge secondarily to neuronal pathology. By designing an in vivo reporter system, we further demonstrated bidirectional exchanges of tau species between neurons and astrocytes. We then determined the consequences of tau accumulation in astrocytes on their survival in models displaying various status of tau aggregation. Using stereological counting of astrocytes, we report that, as for neurons, soluble tau species are highly toxic to some subpopulations of astrocytes in the hippocampus, whereas the accumulation of tau aggregates does not affect their survival. Thus, astrocytes are not mere bystanders of neuronal pathology. Our results strongly suggest that tau pathology in astrocytes may significantly contribute to clinical symptoms.

Dendritic cell (DC)-based vaccines have shown some degree of success for the treatment of prostate cancer (PC). However, the highly immunosuppressive tumor microenvironment leads to DC dysfunction, which has limited the effectiveness of these vaccines. We hypothesized that use of a fully serotype 3 oncolytic adenovirus (Ad3-hTERT-CMV-hCD40L; TILT-234) could stimulate DCs in the prostate tumor microenvironment by expressing CD40L. Activated DCs would then activate cytotoxic T cells against the tumor, resulting in therapeutic immune responses. Oncolytic cell killing due to cancer cell-specific virus replication adds to antitumor effects but also enhances the immunological effect by releasing tumor epitopes for sampling by DC, in the presence of danger signals. In this study, we evaluated the companion effect of Ad3-hTERT-CMV-hCD40L and DC-therapy in a humanized mouse model and PC histocultures. Treatment with Ad3-hTERT-CMV-hCD40L and DC resulted in enhanced antitumor responses in vivo. Treatment of established histocultures with Ad3-hTERT-CMV-hCD40L induced DC maturation and notable increase in proinflammatory cytokines. In conclusion, Ad3-hTERT-CMV-hCD40L is able to modulate an immunosuppressive prostate tumor microenvironment and improve the effectiveness of DC vaccination in PC models and patient histocultures, setting the stage for clinical translation.

Introduction Dendritic cells (DCs) and macrophages, two important antigen presenting cells (APCs) of the innate immune system, are being explored for the use in cell-based cancer immunotherapy. For this application, the therapeutic potential of patient-derived APCs is increased by delivering different types of functional macromolecules, such as mRNA and pDNA, into their cytosol. Compared to the use of viral and non-viral delivery vectors, physical intracellular delivery techniques are known to be more straightforward, more controllable, faster and generate high delivery efficiencies. Areas covered This review starts with electroporation as the most traditional physical transfection method, before continuing with the more recent technologies such as sonoporation, nanowires and microfluidic cell squeezing. A description is provided of each of those intracellular delivery technologies with their strengths and weaknesses, especially paying attention to delivery efficiency and safety profile. Expert opinion Given the common use of electroporation for the production of therapeutic APCs, it is recommended that more detailed studies are performed on the effect of electroporation on APC fitness, even down to the genetic level. Newer intracellular delivery technologies seem to have less impact on APC functionality but further work is needed to fully uncover their suitability to transfect APCs with different types of macromolecules.

In this review, we discuss the use of oncolytic viruses in cancer immunotherapy treatments in general, with a particular focus on adenoviruses. These serve as a model to elucidate how versatile viruses are, and how they can be used to complement other cancer therapies to gain optimal patient benefits. Historical reports from over a hundred years suggest treatment efficacy and safety with adenovirus and other oncolytic viruses. This is confirmed in more contemporary patient series and multiple clinical trials. Yet, while the first viruses have already been granted approval from several regulatory authorities, room for improvement remains. As good safety and tolerability have been seen, the oncolytic virus field has now moved on to increase efficacy in a wide array of approaches. Adding different immunomodulatory transgenes to the viruses is one strategy gaining momentum. Immunostimulatory molecules can thus be produced at the tumor with reduced systemic side effects. On the other hand, preclinical work suggests additive or synergistic effects with conventional treatments such as radiotherapy and chemotherapy. In addition, the newly introduced checkpoint inhibitors and other immunomodulatory drugs could make perfect companions to oncolytic viruses. Especially tumors that seem not to be recognized by the immune system can be made immunogenic by oncolytic viruses. Logically, the combination with checkpoint inhibitors is being evaluated in ongoing trials. Another promising avenue is modulating the tumor microenvironment with oncolytic viruses to allow T cell therapies to work in solid tumors. Oncolytic viruses could be the next remarkable wave in cancer immunotherapy.

In vivo reprogramming of somatic cells into induced pluripotent stem cells (iPSC) holds vast potential for basic research and regenerative medicine. However, it remains hampered by a need for vectors to express reprogramming factors (Oct-3/4, Klf4, Sox2, c-Myc; OKSM) in selected organs. Here, we report OKSM delivery vectors based on pseudotyped Adeno-associated virus (AAV). Using the AAV-DJ capsid, we could robustly reprogram mouse embryonic fibroblasts with low vector doses. Swapping to AAV8 permitted to efficiently reprogram somatic cells in adult mice by intravenous vector delivery, evidenced by hepatic or extra-hepatic teratomas and iPSC in the blood. Notably, we accomplished full in vivo reprogramming without c-Myc. Most iPSC generated in vitro or in vivo showed transcriptionally silent, intronic or intergenic vector integration, likely reflecting the increased host genome accessibility during reprogramming. Our approach crucially advances in vivo reprogramming technology, and concurrently facilitates investigations into the mechanisms and consequences of AAV persistence., In vivo reprogramming of somatic cells is hampered by the need for vectors to express the OKSM factors in selected organs. Here the authors report new AAV-based vectors capable of in vivo reprogramming at low doses.

Genetic engineering by viral infection of single cells is useful to study complex systems such as the brain. However, available methods for infecting single cells have drawbacks that limit their applications. Here we describe 'virus stamping', in which viruses are reversibly bound to a delivery vehicle - a functionalized glass pipette tip or magnetic nanoparticles in a pipette - that is brought into physical contact with the target cell on a surface or in tissue, using mechanical or magnetic forces. Different single cells in the same tissue can be infected with different viruses and an individual cell can be simultaneously infected with different viruses. We use rabies, lenti, herpes simplex, and adeno-associated viruses to drive expression of fluorescent markers or a calcium indicator in target cells in cell culture, mouse retina, human brain organoid, and the brains of live mice. Virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types, both in vitro and in vivo.

Lentiviral vectors are gene delivery vehicles that integrate into the host genome of dividing and non-dividing mammalian cells facilitating long-term transgene expression. Lentiviral vector versatility is greatly increased by incorporating heterologous viral envelope proteins onto the vector particles instead of the native envelope, conferring on these pseudotyped vectors a modified tropism and host range specificity. We investigated the pseudotyping efficiency of HIV-1 based lentiviral vectors with alphaviral envelope proteins from the Chikungunya Virus (CHIKV-G) and Sindbis Virus (SINV-G). Following vector production optimisation, titres for the CHIKV-G pseudotype were comparable to the VSV-G pseudotype but those for the SINV-G pseudotype were significantly lower. High titre CHIKV-G pseudotyped vector efficiently transduced various human and mouse neural cell lines and normal human astrocytes (NHA) in vitro. Although transduction was broad, tropism for NHAs was observed. In vivo stereotaxic delivery in striatum, thalamus and hippocampus respectively in the adult rat brain revealed localised transduction restricted to striatal astrocytes and hippocampal dentate granule neurons. Transduction of different subtypes of granule neurons from precursor to post-mitotic stages of differentiation was evident in the sub-granular zone and dentate granule cell layer. No significant inflammatory response was observed, but comparable to that of VSV-G pseudotyped lentiviral vectors. Robust long-term expression followed for three months post-transduction along with absence of neuroinflammation, coupled to the selective and unique neuron/glial tropism indicates that these vectors could be useful for modelling and gene therapy studies in the CNS.

Objective/Background: Neointimal hyperplasia (NIH) remains one of the leading causes of graft failure after vascular anastomoses. Cytotoxic drugs, such as rapamycin and tacrolimus, have been shown to inhibit the development of NIH. In this study, the aim was to test the impact of a sustained releasing tacrolimus-chitosan-eluting suture on the development of NIH in a rat model. Methods: After tacrolimus-chitosan coating of a 7/0 polyvinylidene difluoride (PVDF) Trofilen (R) suture, the tacrolimus concentration on the coated suture and in vitro release trials were performed spectrophotometrically. Twelve Wistar rats were included. After midline laparotomy, a 7-8 mm longitudinal aortotomy in the infrarenal aorta was made and then closed by a bare 7/0 PVDF (group C, n = 6) and a 7/0 tacrolimus-chitosan coated PVDF suture (0.65 mu g/cm tacrolimus [0.9 wt%] + 1.82 mu/cm chitosan [2.28 wt%]) (group T, n = 6). After 1 month, rats were sacrificed and aortotomy sites were examined histologically by ratio of intimal area (including neointima) and immunohistochemically by alpha-smooth muscle actin (ASMA) and proliferating cell nuclear antigen (PCNA) immunostaining. The PCNA positive cells were indexed to total cell number and expressed as percentage. Results: In vitro tacrolimus release tests for a 7/0 tacrolimus-chitosan coated PVDF suture were confirmed for 1 month without an initial burst release. Endothelialisation over the aortotomy line occurred in both groups. The area of neointima was significantly reduced in group T compared with group C (ratio 0.22 +/- 0.12 vs. 0.42 +/- 0.11; p =.017) 1 month post-operatively. Likewise, the percentage of PCNA immunostaining significantly decreased in group C compared with group T (3.83 +/- 2.85% vs. 11.17 +/- 7.78%; p =.026). The cells constituting NIH were positive for ASMA immunostaining. Conclusions: Tacrolimus-chitosan-eluting suture is shown to be an effective way to reduce NIH without interfering with normal endothelialisation. (C) 2016 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal blood vessels growing into the subretinal space, leading to retinal pigment epithelial (RPE) cell degeneration and vision loss. Vessel growth results from an imbalance of pro-angiogenic (e.g., vascular endothelial growth factor [VEGF]) and anti-angiogenic factors (e.g., pigment epithelium-derived factor [PEDF]). Current treatment using intravitreal injections of anti-VEGF antibodies improves vision in about 30% of patients but may be accompanied by side effects and non-compliance. To avoid the difficulties posed by frequent intravitreal injections, we have proposed the transplantation of pigment epithelial cells modified to overexpress human PEDF. Stable transgene integration and expression is ensured by the hyperactive Sleeping Beauty transposon system delivered by pFAR4 miniplasmids, which have a backbone free of antibiotic resistance markers. We demonstrated efficient expression of the PEDF gene and an optimized PEDF cDNA sequence in as few as 5 × 103 primary cells. At 3 weeks post-transfection, PEDF secretion was significantly elevated and long-term follow-up indicated a more stable secretion by cells transfected with the optimized PEDF transgene. Analysis of transgene insertion sites in human RPE cells showed an almost random genomic distribution. The results represent an important contribution toward a clinical trial aiming at a non-viral gene therapy of nvAMD. Keywords: neovascular age-related macular degeneration, non-viral ex vivo gene therapy, sleeping beauty transposon system, pFAR4 miniplasmids, primary pigment epithelial cells, pigment epithelium-derived factor

Epidemiological studies support a strong association between non-high-density lipoprotein cholesterol levels and heart failure incidence. The objective of the current study was to evaluate the effect of selective cholesterol lowering adeno-associated viral serotype 8 (AAV8)-mediated low-density lipoprotein receptor (LDLr) gene transfer on cardiac remodelling and myocardial oxidative stress following transverse aortic constriction (TAC) in female C57BL/6 LDLr-/- mice with mild hypercholesterolemia. Cholesterol lowering gene transfer resulted in a 65.9% (p

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells. Keywords: adeno-associated virus, AAV8, AAV9, suspension HEK293 cells, transient transfection, orbitally shaken bioreactors, immune affinity chromatography, CNS transduction

Domestication refers to artificial selection and breeding of wild species to obtain cultivated variants that thrive in man-made niches and meet human or industrial requirements. Several genotypic and phenotypic signatures of domestication have been described in crops, livestock and pets. However, domestication is not unique to plants and animals. Microbial diversity has also been shaped by the emergence of novel and highly specific man-made environments, like food and beverage fermentations. This allowed rapid adaptation and diversification of various microbes, such as certain Lactococcus, Lactobacillus, Oenococcus, Saccharomyces and Aspergillus species. During the domestication process, microbes gained the capacity to efficiently consume particular nutrients, cope with a multitude of industry-specific stress factors and produce desirable compounds, often at the cost of a reduction in fitness in their original, natural environments. Moreover, different lineages of the same species adapted to highly diverse niches, resulting in genetically and phenotypically distinct strains. In this Review, we discuss the basic principles of microbial domestication and describe how recent research is uncovering its genetic underpinnings. ispartof: CURRENT BIOLOGY vol:29 issue:10 pages:R381-R393 ispartof: location:England status: published

Ca2+ activated non-selective (CAN) cation channels have been described in cardiomyocytes since the advent of the patch clamp technique. It has been hypothesized that this type of ion channel contributes to the triggering of cardiac arrhythmias. TRPM4 is to date the only molecular candidate for a CAN cation channel in cardiomyocytes. Its significance for arrhythmogenesis in living animals remains, however, unclear. In this study, we have tested whether increased expression of wild-type (WT) TRPM4 augments the risk of arrhythmias in living mice. Overexpression of WT TRPM4 was achieved via tail vein injection of adeno-associated viral vector serotype 9 (AAV9) particles, which have been described to be relatively cardiac specific in mice. Subsequently, we performed ECG-measurements in freely moving mice to determine their in vivo cardiac phenotype. Though cardiac muscle was transduced with TRPM4 viral particles, the majority of viral particles accumulated in the liver. We did not observe any difference in arrhythmic incidents during baseline conditions. Instead, WT mice that overexpress TRPM4 were more vulnerable to develop premature ventricular ectopic beats during exercise-induced β-adrenergic stress. Conduction abnormalities were rare and not more frequent in transduced mice compare to WT mice. Taken together, we provide evidence that overexpression of TRPM4 increases the susceptibility of living mice to stress-induced arrhythmias. ispartof: FRONTIERS IN PHYSIOLOGY vol:10 ispartof: location:Switzerland status: published

The risk of heart failure (HF) is prominently increased in patients with type 2 diabetes mellitus. The objectives of this study were to establish a murine model of diabetic cardiomyopathy induced by feeding a high-sugar/high-fat (HSHF) diet and to evaluate the effect of reconstituted HDLMilano administration on established HF in this model. The HSHF diet was initiated at the age of 12 weeks and continued for 16 weeks. To investigate the effect of reconstituted HDLMilano on HF, eight intraperitoneal administrations of MDCO-216 (100 mg/kg protein concentration) or of an identical volume of control buffer were executed with a 48-h interval starting at the age of 28 weeks. The HSHF diet-induced obesity, hyperinsulinemia, and type 2 diabetes mellitus. Diabetic cardiomyopathy was present in HSHF diet mice as evidenced by cardiac hypertrophy, increased interstitial and perivascular fibrosis, and decreased myocardial capillary density. Pressure-volume loop analysis indicated the presence of both systolic and diastolic dysfunction and of decreased cardiac output in HSHF diet mice. Treatment with MDCO-216 reversed pathological remodelling and cardiac dysfunction and normalized wet lung weight, indicating effective treatment of HF. No effect of control buffer injection was observed. In conclusion, reconstituted HDLMilano reverses HF in type 2 diabetic mice. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:20 issue:6 ispartof: location:Switzerland status: published

Extracellular and intracellular degradation of nucleic acids remains an issue in non-viral gene therapy. Understanding biodegradation is critical for the rational design of gene therapeutics in order to maintain stability and functionality at the target site. However, there are only limited methods available that allow determining the stability of genetic materials in biological environments. In this context, the decay kinetics of fluorescently labeled plasmid DNA (pDNA) and messenger RNA (mRNA) in undiluted biological samples (i.e., human serum, human ascites, bovine vitreous) and cell extracts is studied using fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT). It is demonstrated that FCS is suitable to follow mRNA degradation, while SPT is better suited to investigate pDNA integrity. The half-life of mRNA and pDNA is approximate to 1-2 min and 1-4 h in biological samples, respectively. The resistance against biodegradation drastically improves by complexation with lipid-based carriers. Taken together, FCS and SPT are able to quantify the integrity of mRNA and pDNA, respectively, as a function of time, both in the extracellular biological fluids and cell extracts. This in turn allows to focus on the important but less understood issue of nucleic acids degradation in more detail and to rationally optimize gene delivery system as therapeutics.

Glial cell line-derived neurotrophic factor (GDNF) is one of the most studied neurotrophic factors. GDNF has two splice isoforms, full-length pre-alpha-pro-GDNF (u-GDNF) and pre-beta-pro-GDNF (beta-GDNF), which has a 26 amino acid deletion in the pro-region. Thus far, studies have focused solely on the u-GDNF isoform, and nothing is known about the in vivo effects of the shorter beta-GDNF variant. Here we compare for the first time the effects of overexpressed cx-GDNF and beta-GDNF in non-lesioned rat striatum and the partial 6-hydroxydopamine lesion model of Parkinson's disease. GDNF isoforms were overexpressed with their native pre-pro-sequences in the striatum using an adeno-associated virus (AAV) vector, and the effects on motor performance and dopaminergic phenotype of the nigrostriatal pathway were assessed. In the non-lesioned striatum, both isoforms increased the density of dopamine transporter-positive fibers at 3 weeks after viral vector delivery. Although both isoforms increased the activity of the animals in cylinder assay, only u-GDNF enhanced the use of contralateral paw. Four weeks later, the striatal tyrosine hydroxylase (TH)-immunoreactivity was decreased in both u-GDNF and 1-GDNF treated animals. In the neuroprotection assay, both GDNF splice isoforms increased the number of TH-immunoreactive cells in the substantia nigra but did not promote behavioral recovery based on amphetamine-induced rotation or cylinder assays. Thus, the shorter GDNF isoform, beta-GDNF, and the full-length alpha-isoform have comparable neuroprotective efficacy on dopamine neurons of the nigrostriatal circuitry.

Aims Glucokinase (GK) serves as a glucose sensor in several tissues including glucose‐sensitive neurons of the arcuate nucleus within the hypothalamus. We have previously demonstrated a role for arcuate GK in the regulation of food and glucose intake. However, its role in the regulation of glucose homeostasis is less clear. We therefore sought to investigate the role of arcuate GK in the regulation of glucose homeostasis. Materials and Methods Recombinant adeno‐associated virus expressing either GK or an antisense GK construct was used to alter GK activity specifically in the hypothalamic arcuate nucleus. GK activity in this nucleus was also increased by stereotactic injection of the GK activator, compound A. The effect of altered arcuate nucleus GK activity on glucose homeostasis was subsequently investigated using glucose and insulin tolerance tests. Results Increased GK activity specifically within the arcuate nucleus increased insulin secretion and improved glucose tolerance in rats during oral glucose tolerance tests. Decreased GK activity in this nucleus reduced insulin secretion and increased glucose levels during the same tests. Insulin sensitivity was not affected in either case. The effect of arcuate nucleus glucokinase was maintained in a model of type 2 diabetes. Conclusions These results demonstrate a role for arcuate nucleus GK in systemic glucose homeostasis.

To accurately recapitulate the heterogeneity of human diseases, animal models require to recreate multiple complex genetic alterations. Here, we combine the RCAS-TVA system with the CRISPR-Cas9 genome editing tools for precise modeling of human tumors. We show that somatic deletion in neural stem cells of a variety of known tumor suppressor genes (Trp53, Cdkn2a, and Pten) leads to high-grade glioma formation. Moreover, by simultaneous delivery of pairs of guide RNAs we generate different gene fusions with oncogenic potential, either by chromosomal deletion (Bcan-Ntrk1) or by chromosomal translocation (Myb-Qk). Lastly, using homology-directed-repair, we also produce tumors carrying the homologous mutation to human BRAF V600E, frequently identified in a variety of tumors, including different types of gliomas. In summary, we have developed an extremely versatile mouse model for in vivo somatic genome editing, that will elicit the generation of more accurate cancer models particularly appropriate for pre-clinical testing., Accurate recapitulation of human disease in animal models requires generation of complex and heterogeneous genetic variation. Here the authors combine RCAS-TVA with CRISPR-Cas9 to generate mouse models of cancer.

The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a 'function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes. ispartof: MOLECULAR OMICS vol:14 issue:3 pages:181-196 ispartof: location:England status: published