The development of a novel S/MAR DNA vector platform for the stable, persistent and safe genetic engineering of dividing cells

The development of episomally maintained DNA vectors that are capable of providing safe, persistent and stable modification of cells whilst avoiding the risk of integration-mediated genotoxicity would provide a valuable tool for genetic research. DNA vectors harboring a Scaffold/Matrix Attachment Region (S/MAR) can provide persistent and robust transgene expression in human cancer cell lines which can be used in in vitro, in vivo and ex vivo studies. A prototype S/MAR DNA vector with which we initiated this study replicates episomally, remains unsilenced and unmethylated following the genetic modification of cells. Although, it showed great promise it does have significant limitations which restricts its application. The establishment rate of the original DNA vector is an inefficient passive process and the selection procedure is lengthy and often produces drug-resistant but non-expressing colonies generating a mosaic of cells with differential transgene expression. Thus, these vectors represent a reasonable tool for simple studies such as cell labelling with reporter genes but are not suitable for more sophisticated work such as gene-rescue experiments or for the genetic engineering of primary human cells. In this project, we have refined and enhanced the S/MAR DNA vector system. The range of next-generation DNA vectors that we have produced provide several advances over the original vectors. We have demonstrated that this new S/MAR DNA vector platform is more efficient and stable with improved efficiency in establishing stable cell-lines. We have also demonstrated that the persistence of transgene expression and the molecular integrity of the vector has been improved in a range of cancer cell lines as well as in primary human cells. We have used this next-generation of DNA vector to generate labelled cells suitable for in vitro and in vivo drug screening. We have also generated isogenic tumor models which provide insights into the mechanism of pancreatic cancer development by restoring crucial tumor suppressor genes to the cells without altering the molecular or biochemical integrity of the cells. Additionally, we have utilized the vector system to persistently modify a range of dividing cell types including primary mouse embryonic stem cells and embryonic fibroblasts and primary human fibroblasts. As an ultimate demonstration of the efficacy of this DNA vector we have used it to genetically modify human T-cells for immunotherapy and have demonstrated it to be capable of expressing transgenes in these cells for over 1 month with minimal toxicity. We have demonstrated that this novel class of DNA vector can be used to persistently modify every cell-type tested providing sustained and high levels of transgene expression whilst avoiding the risk of insertional mutagenesis induced by the random integration of genetic material with minimal impact to the cell

Delivery of non-viral naked DNA vectors to liver in small weaned pigs by hydrodynamic retrograde intrabiliary injection

Hepatic gene therapy by delivering non-integrating therapeutic vectors in newborns remains challenging due to the risk of dilution and loss of efficacy in the growing liver. Previously we reported on hepatocyte transfection in piglets by intraportal injection of naked DNA vectors. Here, we established delivery of naked DNA vectors to target periportal hepatocytes in weaned pigs by hydrodynamic retrograde intrabiliary injection (HRII). The surgical procedure involved laparotomy and transient isolation of the liver. For vector delivery, a catheter was placed within the common bile duct by enterotomy. Under optimal conditions, no histological abnormalities were observed in liver tissue upon pressurized injections. The transfection of hepatocytes in all tested liver samples was observed with vectors expressing luciferase from a liver-specific promoter. However, vector copy number and luciferase expression were low compared to hydrodynamic intraportal injection. A 10-fold higher number of vector genomes and luciferase expression was observed in pigs using a non-integrating naked DNA vector with the potential for replication. In summary, the HRII application was less efficient (i.e., lower luciferase activity and vector copy numbers) than the intraportal delivery method but was significantly less distressful for the piglets and has the potential for injection (or re-injection) of vector DNA by endoscopic retrograde cholangiopancreatography

An episomal DNA vector platform for the persistent genetic modification of pluripotent stem cells and their differentiated progeny

The genetic modification of stem cells (SCs) is typically achieved using integrating vectors, whose potential integrative genotoxicity and propensity for epigenetic silencing during differentiation limit their application. The genetic modification of cells should provide sustainable levels of transgene expression, without compromising the viability of a cell or its progeny. We developed nonviral, nonintegrating, and autonomously replicating minimally sized DNA nanovectors to persistently genetically modify SCs and their differentiated progeny without causing any molecular or genetic damage. These DNA vectors are capable of efficiently modifying murine and human pluripotent SCs with minimal impact and without differentiation-mediated transgene silencing or vector loss. We demonstrate that these vectors remain episomal and provide robust and sustained transgene expression during self-renewal and targeted differentiation of SCs both in vitro and in vivo through embryogenesis and differentiation into adult tissues, without damaging their phenotypic characteristics

Identification of a Putative Crf Splice Variant and Generation of Recombinant Antibodies for the Specific Detection of Aspergillus fumigatus

BACKGROUND: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. RESULTS: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. CONCLUSION: Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus

Architecture and performance of the KM3NeT front-end firmware

The KM3NeT infrastructure consists of two deep-sea neutrino telescopes being deployed in the Mediterranean Sea. The telescopes will detect extraterrestrial and atmospheric neutrinos by means of the incident photons induced by the passage of relativistic charged particles through the seawater as a consequence of a neutrino interaction. The telescopes are configured in a three-dimensional grid of digital optical modules, each hosting 31 photomultipliers. The photomultiplier signals produced by the incident Cherenkov photons are converted into digital information consisting of the integrated pulse duration and the time at which it surpasses a chosen threshold. The digitization is done by means of time to digital converters (TDCs) embedded in the field programmable gate array of the central logic board. Subsequently, a state machine formats the acquired data for its transmission to shore. We present the architecture and performance of the front-end firmware consisting of the TDCs and the state machine

Event reconstruction for KM3NeT/ORCA using convolutional neural networks

The KM3NeT research infrastructure is currently under construction at two locations in the Mediterranean Sea. The KM3NeT/ORCA water-Cherenkov neutrino detector off the French coast will instrument several megatons of seawater with photosensors. Its main objective is the determination of the neutrino mass ordering. This work aims at demonstrating the general applicability of deep convolutional neural networks to neutrino telescopes, using simulated datasets for the KM3NeT/ORCA detector as an example. To this end, the networks are employed to achieve reconstruction and classification tasks that constitute an alternative to the analysis pipeline presented for KM3NeT/ORCA in the KM3NeT Letter of Intent. They are used to infer event reconstruction estimates for the energy, the direction, and the interaction point of incident neutrinos. The spatial distribution of Cherenkov light generated by charged particles induced in neutrino interactions is classified as shower- or track-like, and the main background processes associated with the detection of atmospheric neutrinos are recognized. Performance comparisons to machine-learning classification and maximum-likelihood reconstruction algorithms previously developed for KM3NeT/ORCA are provided. It is shown that this application of deep convolutional neural networks to simulated datasets for a large-volume neutrino telescope yields competitive reconstruction results and performance improvements with respect to classical approaches

Event reconstruction for KM3NeT/ORCA using convolutional neural networks

The KM3NeT research infrastructure is currently under construction at two locations in the Mediterranean Sea. The KM3NeT/ORCA water-Cherenkov neutrino de tector off the French coast will instrument several megatons of seawater with photosensors. Its main objective is the determination of the neutrino mass ordering. This work aims at demonstrating the general applicability of deep convolutional neural networks to neutrino telescopes, using simulated datasets for the KM3NeT/ORCA detector as an example. To this end, the networks are employed to achieve reconstruction and classification tasks that constitute an alternative to the analysis pipeline presented for KM3NeT/ORCA in the KM3NeT Letter of Intent. They are used to infer event reconstruction estimates for the energy, the direction, and the interaction point of incident neutrinos. The spatial distribution of Cherenkov light generated by charged particles induced in neutrino interactions is classified as shower-or track-like, and the main background processes associated with the detection of atmospheric neutrinos are recognized. Performance comparisons to machine-learning classification and maximum-likelihood reconstruction algorithms previously developed for KM3NeT/ORCA are provided. It is shown that this application of deep convolutional neural networks to simulated datasets for a large-volume neutrino telescope yields competitive reconstruction results and performance improvements with respect to classical approaches

Beta HPV38 oncoproteins act with a hit-and-run mechanism in ultraviolet radiation-induced skin carcinogenesis in mice.

Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation