Molecular medicine has entered a high-tech age that provides curative treatments of complex genetic diseases through genetically engineered cellular medicinal products. Their clinical imple- mentation requires the ability to stably integrate genetic information through gene transfer vec- tors in a safe, effective and economically viable manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are prevalent in ongoing pre-clinical and translational research. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here we review several recent refinements of the system, including the development of optimized transposons and hyperactive SB variants, the vectorization of transposase and transposon as mRNA and DNA minicircles (MCs) to enhance performance and facilitate vector production, as well as a detailed understanding of SB’s genomic integration and biosafety features. This review also provides a perspective on the regulatory framework for clinical trials of gene delivery with SB, and illustrates the path to successful clinical implementation by using, as examples, gene therapy for age- related macular degeneration (AMD) and the engineering of chimeric antigen receptor (CAR)- modified T cells in cancer immunotherapy.
Thyroid cancer, even in advanced metastatic disease, can be effectively treated by radioiodine therapy, due to thyroidal expression of NIS (Dai et al., 1996; Smanik et al., 1996; Spitzweg and Morris, 2002a). NIS expressing thyroid cancer metastases can be detected and treated by administration of radioiodine, while avoiding adverse effects of ionising radiation to other organs, which do not express NIS and thus do not concentrate radioiodine. NIS therefore represents one of the oldest and most successful targets for molecular imaging and targeted radionuclide therapy. Cloning and characterization of the NIS gene has therefore allowed the development of the NIS gene therapy concept based on NIS gene transfer into nonthyroidal tumor cells, followed by diagnostic and therapeutic application of radioiodine (Dai et al., 1996; Smanik et al., 1996; Hingorani et al., 2010a). One of the major challenges on the way to efficient application of the NIS gene therapy concept in the clinical setting of metastatic cancer is optimal tumor targeting in the presence of low toxicity and sufficiently high transduction efficiency after systemic administration of gene delivery vectors. Only a limited number of studies have investigated systemic NIS gene delivery approaches with the aim of NIS-targeted radionuclide therapy of metastatic disease using an oncolytic measles virus or vesicular stomatitis virus encoding human NIS in multiple myeloma mouse models (Dingli et al., 2004; Goel et al., 2007; Liu et al., 2010). In a recent study we have utilized a promising non-viral gene delivery system for tumor- targeted NIS gene transfer in the syngeneic Neuro2A neuroblastoma mouse model. Branched polycations based on OEI-grafted polypropylenimine dendrimers (G2-HD-OEI) have recently been characterized as biodegradable synthetic gene delivery vectors with high
A lot of studies described apoptotic cell death as one event in PEI mediated toxicity [40, 41], but Fischer et al. (2003) postulated necrosis as alternative pathway of cell death for PEI treated cells . In our study, the cytokine profile did not match the structure-function relationship of cytotoxicity and lipid peroxidation. Mainly four acute-phase cytokines such TNF-α, IL-6, IL-1α and G-CSF were released after polymer treatment. PEI(25)-PEG(2)10 caused the highest proinflammatory response in both cell lines, e.g. up to 80-fold elevated levels of G-CSF (Fig. 3), whereas this polymer was one of the very promising polymers for in vivo application because of its low cytotoxic and oxidative stress potential. For all other polymers, the four acute-phase cytokine levels (TNF- α, IL-6, IL-1α and G-CSF) were up to 5-fold elevated in both cell lines without any structure-function relationship. The apoptotic cell death seemed to be more prominent because of the highly dose-dependent reduction of metabolic activity in the mitochondria and the alteration of the mitochondrial membrane potential and most probably the mitochondrial membrane itself. Due to very high cytoxicity of PEI 25 kDa and the lower PEGylated PEI-PEG copolymers it could be suggested that the cells were leaking of energy and therefore could not release any cytokine for their defence at 24h treatment. Nevertheless, these data highlighted the membrane interaction with PEI as one of the most prominent effect to understand the toxicity of this polymer. PEI (25)-PEG(2)10 and PEI(25)-PEG(0.55)30 seemed to be promising non-viral vector systems for pulmonary application of siRNA or pDNA in vivo regarding their cytotoxic profiles.
In viral transfections, also named transductions, most vector constructs are based on retroviruses (RNA virus), adenoviruses (dsDNA virus), adeno-associated virus (ssDNA virus) and herpes simplex virus (DNA virus) (reviewed in: Robbins and Ghivizzani, 1998). Retrovirus (RV) vectors have the advantage that they integrate into the genome and are thus stable over a long period. However, they require mitotic cells for integration and due to that for a transduction success. An exception is the lentivirus subgroup which does not need mitotic events for infections. The HI virus belongs to the lentivirus subgroup. Most RV vectors used in clinical approaches have been based on the Moloney murine leukaemia virus (MMLV). Adenovirus (AV) vectors can infect both dividing and non-dividing cells, but expression is not very stable using AV vectors. Cells infected by an adenoviral construct are easily recognized by the immune system, resulting in an immune response and loss of expression soon after infection, e.g in mouse (Yang et al, 1995). Additionally, a humoral immune response has also been observed when using AV constructs (Mack et al, 1997). Adeno-associated virus (AAV) vectors infect both dividing and non- dividing cells such as adenoviral vectors and AAV stabilly integrates into the genome in chromosome 19 in humans. However, the amount of DNA delivered by an AAV vector construct is strictly limited, and needs a helper virus to be successful (AV or herpes simplex virus (HSV)). Furthermore, the integration ability of the AAV vector has been lost in many constructs. HSV constructs can deliver large amounts of DNA, but this vector is highly cytotoxic, only resulting in short term expressions of a maximum of one week. Finally, rarely used viral vectors are plus-stranded RNA viridae as polio or hepatitis A derived vectors, or combinations of human papilloma virus (HPV) or sindbis virus derived vectors with additional helper virus constructs. The problem in most viral gene therapy studies has been that in several cases patients have developed cancer, even in an ex vivo application, most probably as a result of the viral gene therapy approach (Hacein-Bey-Abina et al, 2003). This leads to the pressing need to enhance the efficiency of non-viral gene therapeutic approaches for following clinical applications.
In accordance with the given literature, 5 human ES cell-specific miRs derived from the miR-302a-367 and miR-371-373 cluster were applied in non-viral reprogramming because they are potent regulators of pluripotency, namely miR-302a-d & miR-372 (FIG. 15). Importantly, human ES cell-specific miRs are regulated by reprogramming factors. Oct4, Nanog, and Sox2 predominantly bind to promoters of ES cell-associated miRs including the miR-302a-367 and miR-371-373 cluster (Barroso-delJesus et al., 2008; Card et al., 2008; Marson et al., 2008). Therefore, several approaches aim at the combination of miRs with other reprogramming techniques to improve efficiency and quality of reprogramming. Viral reprogramming of MEFs by retroviruses was more efficient when synthetic miRs from the miR-290-295 cluster were transfected (Judson et al., 2009). Further, viral mediated expression of the miR-302a-367 cluster and depletion of Hdac2 was sufficient to generate iPS cells from MEFs and human dermal fibroblasts (Anokye-Danso et al., 2011). Besides the miR-302a-367cluster, expression of miR-25 was useful for more efficient reprogramming when retroviral vectors were applied in mouse fibroblasts (Lu et al., 2012). MicroRNA-25 targets ubiquitin ligases, which are proposed to be regulators of Oct4 and c-Myc. Another approach reported that Nanog expression was indirectly up regulated by miR-214 in ovarian cancer cell lines (Xu et al., 2012a). Xu and co-authors suggested that Nanog induction was achieved by miR-214-mediated repression of the p53 signaling pathway. Reprogramming is still challenging, because the underlying mechanisms have yet to be fully elucidated (Sridharan and Plath, 2008; Okita and Yamanaka, 2011). It is important to highlight that efficient miR delivery and preservation of functional miRs (as mentioned before) as well as repression of unwanted targets is required for miR-mediated reprogramming (Guan et al., 2013). It is highly debated as to which step in the reprogramming process is the most crucial. However, ES cell-enriched miRs are immediately elevated during reprogramming and miRs therefore appear to be a powerful tool for non-viral reprogramming.
The primary prerequisites of delivery vehicles for nucleic acids that will finally make it from bench to bedside are biocompatibility and robust processes of assembly, conjugation and purification (7). Pre-formulation studies are commonly followed by the optimization of biophysicochemical parameters, and if successful, by scale-up for the manufacturing of therapeutic amounts. A broad variety of lipid based vectors, polymers, biopolymers, dendrimers, polypeptides, and inorganic nanoparticles have been investigated by groups all around the world (44). The most prominent polymeric vector is certainly poly (ethylene imine) (PEI), which is commercially available or can be polymerized as low or high molecular weight PEI (45). PEI was first introduced as non-viral gene delivery vector by Bousif et al. in 1995 (46) who described its outstanding property called the “proton-sponge- effect”. While liposomes escape the endo-/lysosomal compartment after endocytosis due to fusogenic properties, PEI is believed to attract an influx of chloride and subsequently an osmotic influx of water into the lysosome as it is protonated. This altogether leads to swelling and bursting of the lysosomes which release the polymer and nucleic acid into the cytosol.
Especially for tumor gene therapy, vectors are desired that show a high specificity for tumor cells while leaving normal cells unaffected. This would offer the possibility of applying high doses of gene constructs with minimal side effects. Several studies de- scribed specific ligand-mediated gene transfer using non-viral vectors, e.g. by transfer- rin (12, 13), folate (14, 15) saccharides (9, 16, 17) or by antibodies or antibody frag- ments such as anti-CD3 (13, 18), anti-PECAM (19), anti-OA3 (20), anti-GAD (21) or anti-PSMA antibodies (22). The coupling of antibodies is an advantageous strategy for targeted delivery due to their versatility, the highly specific binding to target cells and, if an appropriate target was chosen, subsequent uptake by receptor mediated endocytosis. Trastuzumab, a monoclonal antibody directed against the Her2 epitope, offers the possi- bility of specific targeting of various cancers, such as breast, lung, ovarian and prostate cancers (23-26). In normal tissues Her2 is only expressed in low levels and only in cer- tain epithelial cell types (27). Upon ligand binding it is fully internalized, which is mim- icked by Trastuzumab (28). Anti-Her2 antibodies were already successfully used for targeted drug delivery employing immunoliposomes (29) or immunoconjugates with different chemo- or radiotherapeutics (26, 30-32), in virus-like particles for gene deliv- ery (33), and most recently, as targeting moieties coupled to linear (34) and branched PEI (35).
Gene therapy is a controversial topic due to the variety of problems that come along with its application, However, it might be the future hope and cure of many diseases, ranging from cancer, to autoimmune diseases and genetic disorders. One challenge in the field is the development of safe and efficient administration vehicles for transgenes, so-called gene vectors. But after years of intense research scientists are still dealing with basic questions of this therapeutic approach like e.g. safety, administration and long- term stable and consistent transgene expression. Most currently used gene vectors have a viral background, mainly for the reasons of efficient administration into the target cell and stable transgene expression. Adenovirus-based vectors and vectors based on adeno- associated virus (AAV) that have a depleted integrative potential, make up roughly one quarter of applied vectors for gene therapeutic approaches 1 and offer the advantage of a non-integrating character. However, because adenovirus is a common human virus the application of adenovirus-based vectors can easily lead to immunological reactions of the patient. AAV-based vectors display low immunogenicity but this vector type has only a low transgene capacity up to a maximum of 4.7 kbp (Daya and Berns, 2008). Other vectors, like e.g. retro- and lentivirus-based vectors bring a big disadvantage along - integration into the host’s genome. The main problem of integration is
RNA interference has been regarded not only as an innovative approach to suppress the expression of a target gene, but also as a new therapeutic strategy to combat many diseases such as cancer, autoimmune diseases and viral infections (Iorns et al., 2007). SiRNAs and miRNAs can theoretically interfere with the expression of many genes through transcriptional or translational repression (de Fougerolles et al., 2007). Much progress has been made in clinical trials using siRNAs to treat many diseases such as age-related macular degeneration and respiratory syncytial virus infection (Melnikova, 2007). Moreover, the first evidence of targeted in vivo gene silencing for human cancer therapy through systemic delivery of siRNA using transferrin-tagged, cyclodextrin-based polymeric nanoparticles has been recently presented (Oh and Park, 2009).
Background: The dynamic interaction between HIV and its host governs the replication of the virus and the study of the virus-host interplay is key to understand the viral lifecycle. The host factor lens epithelium-derived growth factor (LEDGF/p75) tethers the HIV preintegration complex to the chromatin through a direct interaction with integrase (IN). Small molecules that bind the LEDGF/p75 binding pocket of the HIV IN dimer (LEDGINs) block HIV replication through a multimodal mechanism impacting early and late stage replication including HIV maturation. Furthermore, LEDGF/p75 has been identified as a Pol interaction partner. This raised the question whether LEDGF/p75 besides acting as a molecular tether in the target cell, also affects late steps of HIV replication.
Many RNA and DNA virus families have acquired one or more gene product(s) that reduce the induction of the latent kinase PKR or phosphorylation of its substrate eIF2 by very diverse mechanisms. Research in the past decade has revealed that the capacity to antagonize this cellular defence is an important aspect of the virulence and/or host specificity of these viral pathogens. There is recent evidence for rapid evolution of PKR genes in primates and positive selection at specific amino acid sites, supporting the view that this kinase evolves under the constant pressure of antagonistic viral gene functions [151,152]. Although a lot of information has accumulated on PKR, a number of questions remain concerning the biology of this conserved kinase. These include the precise sequence of events leading from the latent, monomeric, form to the fully phosphorylated dimer, whether allelic differences in PKR are associated with different susceptibility to certain viruses, and the precise roles of several of its cellular interaction partners. A particular technical challenge concerns the characterization of structures within natural viral nucleic acids, which trigger PKR activation inside cells. Clearly, research directed to close these gaps in our knowledge will provide valuable insights into the ongoing arms race between viral pathogens and their hosts.
tegie, die es ermöglicht Konsumenten über ein innovatives Produkt oder eine Marke zu informieren und zu werben, ohne dass der Werbecharakter erkannt und boykottiert wird. Daher achten Marketingverantwortliche vermehrt auf die Wirkung von alternativen Werbeformen, wie Mundpropaganda Marketing und viralem Marketing. 4 Dies geht aus einer Studie der GfK und der Münchener Agentur Robert&Horst hervor. Content soll sich vermehrt durch die Kommunikation von Kunden, Unternehmens- bzw. Markeninte- ressierten verbreiten. Vor allem, da Kundenempfehlungen ein großes Potenzial auf- grund ihrer geringen Kosten und großen Wirkung bieten. 5 Dabei bestehen keine Barrieren und geschehen offline, wie online. Gespräche finden online in sozialen Netz- werken, wie Facebook, Twitter, Xing, Google+ etc. statt. Allein Facebook hat im ersten Quartal im Jahr 2014 über eine Milliarde aktive Nutzer monatlich. 6 So kann unterhalt- samer oder einzigartiger Content mit einem Klick auf den "Teilen" Button auf Facebook oder durch die Benutzung von "Hashtags" auf Twitter, mit Freunden und Bekannten geteilt werden. Somit bieten diese Plattformen ein optimales Umfeld um mit Kunden und Markeninteressierten in Kontakt zu treten und Werbekampagnen viral zu verbrei- ten. Mit der "Like", "Share" oder auch "Comment" Funktion besitzt Facebook eine bestmögliche Grundlage für virale Kampagnen und die damit auszulösende Mundpro- paganda. Doch auch durch die Vielzahl an Usern und Digital Natives, deren hohen Grad an Kommunikations- und Informationsbedürfnis, sowie Markenaffinität ist Face- book hinsichtlich Viral Marketing die optimale Plattform für langfristige Erfolgschancen in Bezug auf Steigerung der Markenbekanntheit durch Zielgruppenerweiterung, Traffic, Umsatz und Neukundengewinnung.
Marine phytoplankton constituting of unicellular eukaryotic pico-algae, form the basis of most marine food webs. Picophytoplankton Micromonas pusilla is a wide-spread, non-bloom forming small flagellated unicellular algae. M. pusilla has been identified as a major component of the phytoplankton populations throughout the year (Not et al., 2004). The occurrence of M. pusilla has been documented in varied oceanic environments such as polar and temperate marine regions as well as in nutrient rich coastal environments (Kuylenstierna and Karlson, 1994; Not et al., 2005). A characteristic feature of M. pusilla cells is the presence of a sub-cellular strucutre called pyrenoid, which hosts carbon fixation enzymes and is surrounded by starch sheath (Salisbury and Floyd, 1978). Viruses infecting M. pusilla (MpVs), just like their host, have been found in many oceanic environments (Cottrell and Suttle, 1991, 1995). Previous studies conducted under natural systems have suggested that MpVs can have a profound impact on M. pusilla population dynamics (Zingone et al., 1999). However, the impact of M. pusilla viral infections structuring bacterial community is currently unknown.
sarcoma-associated herpesvirus (KSHV), co-infected by both KSHV and Epstein-Barr-Virus (EBV) or not infected. Thus, each cell line provides a distinct context for microRNA-mediated regulation. All datasets were re-analyzed using a new algorithm called PARma [Erhard et al., 2013a]. PARma considers the topology of the microRNA/target interaction and the position of UV-light induced cross-links in more detail than state-of-the-art methods and provides quality control scores for both, the identification of microRNA target site clusters and for the annotation of the interacting microRNA to these sites. For two of these four cell lines, we generated three additional data sets including RIP-Chip, 4sU-tagging-derived RNA half-lives and large-scale SILAC-based proteomics. This allowed us to comprehensively analyze the effect of context- dependent microRNA/target interactions on the recruitment of the target mRNAs to Argonaute-2 complexes, on target RNA stability and on target protein levels. By considering viral as well as host microRNAs, we investigated both microRNA/target interactions that coevolved within a species as well as interactions of an exogenous microRNA with endogenous target sites. The results provide compelling evidence that context-dependency of microRNA-mediated regulation is not restricted to a few examples but is a widespread and general feature of post-transcriptional regulation mediated by both cellular and viral microRNAs.
(Dörig et al, 1993; Galanis et al, 2010). CD46, a complement regulatory protein, is expressed by all human nucleated cells but most importantly it is frequently overexpressed in tumor cells (Riley-Vargas et al, 2004; Russell & Peng, 2009). Key to the measles virus attractiveness as oncolytic agent is that cytopathic effects are only observed when CD46 expression level reaches a certain threshold leaving non-malignant tissues unaffected by the virus and leading to preferential killing of tumor cells (Anderson et al, 2004). Nowadays, these favorable characteristics are combined with engineering strategies to monitor virus replication, enhance systemic delivery and tumor specificity and improve efficacy (Miest & Cattaneo, 2014). Promising preclinical results have already led to the initiation of several clinical phase trials. In July 2014, Russel et al. reported complete remission of one patient with advanced incurable myeloma after systemic administration of a high dose recombinant MV engineered to express the human thyroidal sodiumiodide symporter (NIS) allowing noninvasive tumor imaging (Russell et al, 2014; Bell, 2014). Remarkably, this was the first case of intravenous virotherapy to completely eliminate disseminated malignancies.
The value of PCT for differentiating viral and bacterial diseases has been evaluated by Schültzle H. et al 52 . They retrospectively analysed serum PCT levels of 327 children with acute respiratory tract infections presenting in the emergency department and in whom a nasopharyngeal swab reverse-transcription polymerase chain reaction (RT-PCR) assay for 13 respiratory viruses and 4 “atypical” bacterial has been performed. In 327 patients with serum PCT levels below 0.1ng/ml 86 had a positive virus testing and 90.2% had a CRP value below 40mg/l, indicative for non-bacterial infection. 40% of patients with PCT values >0.5ng/ml did not receive antibiotics without adverse effects. In these patients virus testing was positive in 54%, maybe due to viral-bacterial co-infections. The authors hypothesized that these PCT levels may also reflect the invasiveness and severity of microbial invasion. The benefits of this study are that a highly sensitive PCT assay was used and the RT-PCR, which detected 13 viral agents. However, it is a retrospective study and lacks evidence of bacterial agents as cause of respiratory infection in the majority of the patients, calling for a more comprehensive pathogen testing.
on treatment success for different drugs. Second, the detected level of resistance for a minor viral population may be inaccurate due to biological and technical factors. On the one hand, minority resistance mutations are not always biologically meaningful because they may be found on reads that belong to viral variants with low replicative capacity (due to low fitness) or without any replicative capacity (due to the action of APOBEC). On the other hand, applying prediction models that were developed for Sanger sequences to NGS data may incur inaccuracies. This is because currently available approaches consider only the amino acid with the greatest impact on drug resis- tance when multiple amino acids are observed at a single position. Selecting only the amino acids most strongly associated with drug resistance is particularly problematic for consensus sequences at low abundances (e.g. at 1%) as it is likely that constructed sequences contain resistance-associated mutations that do not occur on the same viral strand in vivo. This can lead to overestimated levels of drug resistance because all of these mutations are taken into account in unison. Models that do not rely on consensus generation would allow for more accurate estimates of biological reality.
The ability of CMV to replicate in endothelial cells strongly defines the extent of viral spread through the body and viral dissemination from the circulating blood to the organs . For human cytomegalovirus (HCMV), the viral tropism for endothelial cells greatly varies between different strains and it is dependent by the UL128-131 gene region. UL128, UL130, and UL131 along with gH and gL form the pentameric complex, which is exposed on the viral envelope and is essential for the viral entry into endothelial and epithelial cells. It has been shown that this region is under selective pressure and continuous passaging of HCMV in fibroblasts leads to mutations in this region and loss of endothelial cells tropism . Besides the UL128-131 gene region, HCMV-encoded proteins UL24, UL135, UL136, and US16 have been shown to contribute to viral endothelial tropism . While the HCMV-encoded proteins UL135 and UL136 are required for efficient formation of the viral assembly compartment (VAC) and maturation of virus particles , the US16 protein is critical for the entry and post-entry events in both endothelial and epithelial cells . For MCMV, it has been shown that deletion of the tegument protein M45 abrogates viral replication in endothelial SVEC4-10 cells, as M45 expression is needed to prevent necroptosis in these cells [161, 162]. m139-deficient MCMV mutants replicate less efficiently than MCMV WT in endothelial SVEC4-10 cells, indicating that m139 is a novel factor for MCMV endothelial cell tropism.
Transduction of the T cell line RF33.70 was performed as following. A 24-well plate was incubated with 400 µl (12.5 µg/ml) RetroNectin for 2 h at RT. RetroNectin is a recombinant human fibronectin fragment with three functional domains that are able to interact with integrin on target cells and the virus particles. After incubation, the plate was blocked with 2% BSA for 30 min at 37°C, before washed once with 2 ml 1x PBS 25 mM HEPES. Thereafter, 10 5 cells (in 1 ml) of RF33.70 cell line and 1 ml filtered viral supernatant of Plat-E cells (through 0.45 µm filter) together with 4 µg/ml protamine sulfate and 1 % HEPES were added to each well and the plate was centrifuged at 32°C for 1.5 hours prior to incubation overnight at 37°C, 5% CO2. One day after, cells were pelleted, replated with 3 ml medium on a 6-well plate and incubated for 3 days. On the 8 th day of experiment, transduction efficiency was analyzed via FACS.