Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice

<p>Abstract</p> <p>Background</p> <p>Adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. Viral vectors, based on the Adeno-Associated Virus (AAV) type 2, have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features.</p> <p>Results</p> <p>We designed recombinant adeno-associated virus (rAAV) vectors for the regulated expression of transgenes in different configurations. We integrated the macrolide-responsive E.REX systems (E_ONand E_OFF) into rAAV backbones and investigated the delivery and expression of intracellular as well as secreted transgenes for binary set-ups and for self- and auto-regulated one-vector configurations. Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications. We tested the performance of the different vectors in selected biotechnologically and therapeutically relevant cell types (CHO-K1, HT-1080, NHDF, MCF-7). Moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for <it>in vivo </it>studies. To validate the functionality of delivery and regulation we performed <it>in vivo </it>studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression.</p> <p>Conclusion</p> <p>Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as in mice.</p

The food additive vanillic acid controls transgene expression in mammalian cells and mice

Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapie

The food additive vanillic acid controls transgene expression in mammalian cells and mice

Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies

Impact of Cerebral Microbleeds in Stroke Patients with Atrial Fibrillation

OBJECTIVES: Cerebral microbleeds are associated with the risks of ischemic stroke and intracranial hemorrhage, causing clinical dilemmas for antithrombotic treatment decisions. We aimed to evaluate the risks of intracranial hemorrhage and ischemic stroke associated with microbleeds in patients with atrial fibrillation treated with Vitamin K antagonists, direct oral anticoagulants, antiplatelets, and combination therapy (i.e. concurrent oral anticoagulant and antiplatelet) METHODS: We included patients with documented atrial fibrillation from the pooled individual patient data analysis by the Microbleeds International Collaborative Network. Risks of subsequent intracranial hemorrhage and ischemic stroke were compared between patients with and without microbleeds, stratified by antithrombotic use. RESULTS: A total of 7,839 patients were included. The presence of microbleeds was associated with an increased relative risk of intracranial hemorrhage (aHR 2.74, 95% confidence interval 1.76 - 4.26) and ischemic stroke (aHR 1.29, 95% confidence interval 1.04 - 1.59). For the entire cohort, the absolute incidence of ischemic stroke was higher than intracranial hemorrhage regardless of microbleeds burden. However, for the subgroup of patients taking combination of anticoagulant and antiplatelet therapy, the absolute risk of intracranial hemorrhage exceeded that of ischemic stroke in those with 2-4 microbleeds (25 vs 12 per 1,000 patient-years) and ≥11 microbleeds (94 vs 48 per 1,000 patient-years). INTERPRETATION: Patients with atrial fibrillation and high burden of microbleeds receiving combination therapy have a tendency of higher rate of intracranial hemorrhage than ischemic stroke, with potential for net harm. Further studies are needed to help optimize stroke preventive strategies in this high-risk group. This article is protected by copyright. All rights reserved

Post-mortem assessment in vascular dementia: advances and aspirations.

BACKGROUND: Cerebrovascular lesions are a frequent finding in the elderly population. However, the impact of these lesions on cognitive performance, the prevalence of vascular dementia, and the pathophysiology behind characteristic in vivo imaging findings are subject to controversy. Moreover, there are no standardised criteria for the neuropathological assessment of cerebrovascular disease or its related lesions in human post-mortem brains, and conventional histological techniques may indeed be insufficient to fully reflect the consequences of cerebrovascular disease. DISCUSSION: Here, we review and discuss both the neuropathological and in vivo imaging characteristics of cerebrovascular disease, prevalence rates of vascular dementia, and clinico-pathological correlations. We also discuss the frequent comorbidity of cerebrovascular pathology and Alzheimer's disease pathology, as well as the difficult and controversial issue of clinically differentiating between Alzheimer's disease, vascular dementia and mixed Alzheimer's disease/vascular dementia. Finally, we consider additional novel approaches to complement and enhance current post-mortem assessment of cerebral human tissue. CONCLUSION: Elucidation of the pathophysiology of cerebrovascular disease, clarification of characteristic findings of in vivo imaging and knowledge about the impact of combined pathologies are needed to improve the diagnostic accuracy of clinical diagnoses

Queen mandibular pheromone: questions that remain to be resolved

The discovery of ‘queen substance’, and the subsequent identification and synthesis of keycomponents of queen mandibular pheromone, has been of significant importance to beekeepers and to thebeekeeping industry. Fifty years on, there is greater appreciation of the importance and complexity of queenpheromones, but many mysteries remain about the mechanisms through which pheromones operate. Thediscovery of sex pheromone communication in moths occurred within the same time period, but in this case,intense pressure to find better means of pest management resulted in a remarkable focusing of research activityon understanding pheromone detection mechanisms and the central processing of pheromone signals in themoth. We can benefit from this work and here, studies on moths are used to highlight some of the gaps in ourknowledge of pheromone communication in bees. A better understanding of pheromone communication inhoney bees promises improved strategies for the successful management of these extraordinary animals

Macrolide-triggered SEAP expression in mice injected with pDF143 (ITR-P-ET1-pA-P-SEAP-pA-ITR)-derived AAV type 2 particles

<p><b>Copyright information:</b></p><p>Taken from "Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice"</p><p>http://www.biomedcentral.com/1472-6750/7/75</p><p>BMC Biotechnology 2007;7():75-75.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2211474.</p><p></p> pDF143-derived AAV particles were administered intramuscularly and SEAP levels in the serum were measured at two different time points for one group in the absence of erythromycin, and for the other group with EM injections every 24 h. SEAP expression is shown in milliunits/liter (mU/l) as defined by Schlatter et al. [52]. Abbreviations: EM, erythromycin; SEAP, human placental secreted alkaline phosphatase

Reversibility and adjustability of macrolide-responsive SEAP expression in HT-1080 and MCF-7 transduced by transgenic AAV type 2-derived particles

<p><b>Copyright information:</b></p><p>Taken from "Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice"</p><p>http://www.biomedcentral.com/1472-6750/7/75</p><p>BMC Biotechnology 2007;7():75-75.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2211474.</p><p></p> (A) HT-1080 and (B) MCF-7 were transduced with pDF143-derived AAV particles (2000 genomic particles/cell) and six equal populations (1–6) were cultivated in media containing different antibiotic concentrations. Cells were (i) cultivated in the presence (+++) or absence (---) of EM over 9 days, (ii) cultivated in the presence (++-) or absence (--+) of EM over 6 days and then incubated in reversed EM conditions for the remaining three days or (iii) cells were cultivated in medium whose EM status was alternated every three days +EM to --EM to +EM (+-+) or from --EM to +EM to --EM (-+-) and SEAP expression was quantified. Adjustability of SEAP expression of pDF143 (1000 genomic particles/cell) (C) and pDF51/77 (500 genomic particles/cell) (D) in HT-1080 and MCF-7 cultivated for 48 h in the presence of increasing EM concentrations. SEAP expression is shown in units/liter (U/l) as defined by Schlatter et al. [52]. Abbreviations: EM, erythromycin; SEAP, human placental secreted alkaline phosphatase. Abbreviations: EM, erythromycin; SEAP, human placental secreted alkaline phosphatase

Self- and auto-regulated AAV type 2-based expression of fluorescent proteins

<p><b>Copyright information:</b></p><p>Taken from "Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice"</p><p>http://www.biomedcentral.com/1472-6750/7/75</p><p>BMC Biotechnology 2007;7():75-75.</p><p>Published online 6 Nov 2007</p><p>PMCID:PMC2211474.</p><p></p> (A) Schematic representation of an auto-regulated (pDF124), a bidirectional (pDF89) and a self-regulated (pDF141) AAV type 2-based expression unit. (B) Fluorescence micrographs of human fibrosarcoma cells (HT-1080) and a human breast cancer cell line (MCF-7) transduced with pDF124-, pDF89- and pDF141-derived AAV particles (2000 genomic particles/cell) cultivated in the presence (+) and absence (-) of EM. (C) FACS-mediated quantification of EYFP in HT-1080 transduced with equal amounts of viral particles (2000 genomic particles/cell) and cultivated in the presence (+EM) and absence (-EM) of erythromycin. Abbreviations: EM, erythromycin; ET1, erythromycin-dependent transactivator; ETR, ET1-specific operator; EYFP, enhanced yellow fluorescent protein; IRES, internal ribosome entry site; ITR, inverted terminal repeat; pA, synthetic polyadenylation signal; pA, simian virus 40 polyadenylation signal; P, erythromycin-responsive promoter; P, minimal version of the heat-shock protein 70 promoter; P, simian virus 40 promoter