Biomedical subjects

Henry Fechner

Publications and source records attributed to Henry Fechner.

9 recordsLinked to original sources

PubMed2013

Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.

Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206-mediated skeletal muscle-specific silencing of miR-206TS-bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1-mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3' portion of the miR-206, even enhanced the miR-206- mediated transgene repression. In vivo expression of m206TS-3G- and miR-122TS-containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs.

Base Pairing↗

PubMed2006

A bidirectional Tet-dependent promotor construct regulating the expression of E1A for tight control of oncolytic adenovirus replication.

Tight regulation of oncolytic adenoviruses (oAdV) represents an important requirement for their safe application. Here we describe a new doxycycline (Dox)-dependent oAdV with a bidirectional expression cassette, which drives the expression of the reverse tetracycline-controlled transactivator (rtTA(s)-M2) from a lung tumor-specific promoter and, in the opposite direction, the expression of the adenoviral E1A gene from a second generation TetO(7) sequence linked to an isolated TATA box. In H441 lung cancer cells, this oAdV showed a strictly Dox-dependent E1A expression, adenoviral replication, cell killing activity and a 450-fold induction of progeny virus production. The virus could be shut off again by withdrawal of Dox and, in contrast to a control oAdV expressing E1A directly from the SP-B promoter, did not replicate in non-target cells. However, the absolute values of virus production and the cell killing activity in the presence of the inducer were still reduced as compared to the control oAdV. The results demonstrate, for the first time, Dox-dependent oAdV replication from a single adenoviral vector genome. Future improvement of the Dox-dependent E1A regulation cassette should lead to the generation of an oAdV well suited to meet the demands for a highly regulated and efficient oncolytic virus for in vivo applications.

Adenoviridae↗

PubMed2006

Gene therapy for gastric cancer: is it promising?

Gastric cancer is one of the most common tumors worldwide. The therapeutic outcome of conventional therapies is inefficient. Thus, new therapeutic strategies are urgently needed. Gene therapy is a promising molecular alternative in the treatment of gastric cancer, including the replacement of defective tumor suppressor genes, the inactivation of oncogenes, the introduction of suicide genes, genetic immunotherapy, anti-angiogenetic gene therapy, and virotherapy. Improved molecular biological techniques and a better understanding of gastric carcinogenesis have allowed us to validate a variety of genes as molecular targets for gene therapy. This review provides an update of the new developments in cancer gene therapy, new principles, techniques, strategies and vector systems, and shows how they may be applied in the treatment of gastric cancer.

Angiogenesis Inhibitors↗

PubMed2005

An improved Tet-On regulatable FasL-adenovirus vector system for lung cancer therapy.

Gene therapy is a new therapeutic approach for the treatment of human cancers. Gene expression systems that can be regulated by drugs have been developed to improve the safety and efficacy of therapeutic transgene delivery. One of the most promising systems is the tetracycline (Tet)-responsive system in the Tet-On configuration. A major problem of the Tet-On system if used in viral vectors is the high basal activity of the Tet response element (TRE) promoter leading to leaky expression of transgenes under uninduced conditions. We therefore evaluated novel TRE promoters for controlling gene expression in an adenovirus vector (AdV) Tet-On system and further investigated them for expression of the pro-apoptotic CD95/Fas ligand (FasL) in human epithelial carcinoma cell line (HeLa) and lung cancer cells. Plasmid-based reporter gene assays showed that modifications within the tetO (7) and minimal immediate early cytomegalovirus promoter (CMV)(min) sequence of the TRE promoter reduced its leakiness and led to a markedly improved regulatability by doxycycline. Among several TRE promoters tested, a new construct (TRE-Tight1) containing modifications of both the tetO (7) sequence and the CMV(min) showed 11-fold reduced leakiness and 1.5-fold increased absolute transgene expression levels after induction, as compared to the original TRE. Under induced conditions, a TRE-Tight1 promoter-dependent AdV expressing the pro-apoptotic CD95L/FasL induced apoptosis and cell lysis in HeLa cells as efficiently as an AdV containing the original TRE promoter. In contrast to the latter, however, the vector with the modified TRE promoter left cells totally unaffected in the absence of the inducer. Stringently regulated induction of apoptosis and cell death by TRE-Tight1-AdV was also demonstrated in three human lung cancer cell lines. These data show that the novel TRE-Tight1 promoter has a high potential for closely controlled and efficient expression of cytotoxic genes in AdV-based anti-cancer approaches.

Adenoviridae↗

PubMed2005

Viral and nonviral factors causing nonspecific replication of tumor- and tissue-specific promoter-dependent oncolytic adenoviruses.

Restricted replication-competent adenoviruses (RRCAs) using tumor- and tissue-specific promoters (ttsP's) are new tools for cancer gene therapy. In this study we investigated viral and nonviral factors affecting "leakiness" of several ttsP's and their relevance for nonspecific ttsP-dependent RRCA (ttsP-RRCA) replication. The leakiness of the ttsP's in nontarget cells was per se highly variable and correlated with levels of nonspecific ttsP-RRCA replication. Transcriptional regulator elements fused to ttsP's showed variable effects: a hypoxic response element reduced leakiness of an alpha-fetoprotein promoter. In contrast, a mouse tyrosinase enhancer increased leakiness of a tyrosinase promoter, although it was not affected by a human tyrosinase enhancer. Furthermore, leakiness of ttsP's was enhanced by 5'-terminal adenoviral E1A enhancers, and adenoviral E1A-13S was found to be a strong transactivator of ttsP's, leading to "autoactivation" of leaky ttsP-RRCAs. In a proof-of-principle study, ttsP-RRCA replication was shown to be inhibited by a tetracycline-controlled transcriptional silencer via direct ttsP silencing. This opens up the prospect of pharmacological regulation of ttsP-RRCAs. Together, these data indicate that leakiness of ttsP's induced by several factors is a major cause of nonspecific ttsP-RRCA replication. Consideration of these factors may help optimize ttsP-dependent RRCA vectors and may thereby improve their safety.

Adenoviridae↗

PubMed2005

Selective induction of apoptosis in melanoma cells by tyrosinase promoter-controlled CD95 ligand overexpression.

Induction of apoptosis has been demonstrated previously by overexpression of CD95 ligand (CD95L) in cultured human melanoma cells. For in vivo approaches based on CD95L, however, targeted expression is a prerequisite and tyrosinase promoters have been considered for selection. Luciferase reporter gene assays performed for a representative panel of melanoma cell lines characterized by strong (SK-Mel-19), moderate (SK-Mel-13, MeWo), weak (A-375), and missing expression (M-5) of endogenous tyrosinase revealed high tyrosinase promoter activities in SK-Mel-19, SK-Mel-13, and MeWo, but only weak activities in A-375 and M-5 as well as in non-melanoma cell lines. After transfection of a CMV promoter CD95L expression construct, melanoma cells were found highly sensitive, as compared with non-melanoma cells. By applying a tyrosinase promoter CD95L construct, apoptosis was selectively induced in SK-Mel-19, SK-Mel-13, MeWo as well as in A-375, which was characterized by high CD95 surface expression and high sensitivity to agonistic CD95 activation. M5 and non-melanoma cell lines remained uninfluenced. Also, resistance to agonistic CD95 activation seen in MeWo characterized by weak CD95 surface expression was overcome by overexpression of CD95L. Our investigations provide evidence that tyrosinase promoter CD95L constructs may be of value for selective induction of apoptosis in therapeutic strategies for melanoma.

Apoptosis↗

PubMed2003

Induction of coxsackievirus-adenovirus-receptor expression during myocardial tissue formation and remodeling: identification of a cell-to-cell contact-dependent regulatory mechanism.

BACKGROUND: The coxsackievirus-adenovirus receptor (CAR) was cloned as a receptor for both viruses, but its primary biological functions and regulatory mechanisms are unknown. CAR was low in healthy adult myocardium, whereas strong CAR reexpression was observed in human dilated cardiomyopathy. The molecular mechanisms of CAR induction in cardiomyocytes are unknown. METHODS AND RESULTS: We report on CAR regulation during development, CAR induction after myocardial infarction, and cell-to-cell contact-dependent CAR regulation in the rat. The high CAR expression during development in various organs decreased up to 190-fold after birth. After infarction resulting in severe cardiac dysfunction (dP/dt(max), -53%; dP/dt(min), -58%; left ventricular pressure, -45%), CAR was induced locally in cardiomyocytes of the infarct zone, where it was also expressed by capillary-like CD31+ structures and CD18+ interstitial cells, whereas it remained confined to subendothelial layers of arterioles and venules. In cultured cardiomyocytes, endothelin-1, cardiotrophin-1, leukemia-inhibiting factor, and cyclic stretch had no effect on CAR, whereas at high versus low cell density, CAR was suppressed up to 10-fold (P=0.006). Conditioned media from low- or high-density cardiomyocytes or cardiofibroblasts had no effect. CONCLUSIONS: The locally confined CAR upregulation after infarction makes induction by various humoral factors unlikely, because cardiac dysfunction results in high activities of sympathetic and renin-angiotensin systems and cytokines. The cell culture experiments identify a cell-to-cell contact-dependent mechanism of CAR regulation. Further characterization of the signals linking cell-to-cell interactions to CAR gene expression may provide insight into mechanisms and functional consequences of the generalized CAR induction in dilated cardiomyopathy, and of its local induction after myocardial infarction.

Alternative Splicing↗

PubMed2003

Regulation of human factor IX expression using doxycycline-inducible gene expression system.

Following substitution therapy with human factor IX (hFIX) concentrate, therapy of haemophilia B by viral gene transfer has become an attractive alternative therapy in recent years. However, high doses of expressed hFIX, which can already be achieved in animal studies, may cause thrombosis in humans (van Hylckama Vlieg et al., 2000). Thus, it should be possible to maintain transgene expression within the therapeutic range. Therefore, we inserted elements of the tetracycline (Tet)-dependent Tet-On gene regulatory system into replication deficient adenovectors. The new system consists of two adenovectors: a response vector expressing hFIX (Ad5.TRE.hFIX), and a regulator vector expressing a second generation reverse tetracycline transactivator controlled by a CMV- (Ad5.CMV.rtTA) or human alpha1-antitrypsin-promoter (Ad5.hAAT.rtTA). Expression studies in four human cell lines showed high expression of hFIX from Ad5.TRE.hFIX in all cell lines in combination with Ad5.CMV.rtTA regulator vector, but only high specific expression in HepG2-cells in combination with Ad5.hAAT.rtTA regulator vector. Additionally, up- and down-regulation of hFIX expression could be demonstrated in vitro with the Ad5.TRE.hFIX/Ad5.CMV.rtTA combination and modulating doxycycline concentrations. When SCID-mice were infected with the Ad5.TRE.hFIX/Ad5.CMV.rtTA combination, up- and down-regulation of hFIX expression was achieved by oral doses of doxycycline for a period of at least two months. Replacement of the Ad5.CMV.rtTA vector by the Ad5.hAAT.rtTA vector showed minimal expression of hFIX in vivo. Although hFIX expression showed a slow and gradual decrease over time in vivo with the Ad5.CMV.rtTA vector, it remained within the therapeutic range. To date, regulation of hFIX has not been described in this way.

Animals↗

PubMedDate not supplied

Molecular genetic and expression analysis of alpha-tocopherol transfer protein mRNA in German shepherd dogs with degenerative myelopathy.

Degenerative Myelopathy (DM) is a progressive neurological disorder of the spinal cord preferentially occurring in German shepherd dogs. The pathogenesis of the disease is unknown. However, there are indications that vitamin E deficiency may be involved in the pathogenesis of DM. Therefore, we analyzed the expression and the nucleotide sequence of the canine alpha-tocopherol transfer protein (alpha Ttp) of German shepherd dogs with DM in order to determine whether a deficiency or a defect of the alpha Ttp could be a primary factor in the pathogenesis of DM, as found in human patients with Ataxia with vitamin E deficiency (AVED). The cDNA of the coding region of the canine alpha Ttp-mRNA was generated from total liver RNA using RT-PCR and 5' RACE technique. We determined the sequence of 707 out of 834 base pairs or 84.8% of the canine alpha Ttp coding region. Sequence comparison of canine alpha Ttp between affected and control dogs revealed no differences in either nucleotide or predicted amino acid sequence. Using Northern blot analysis alpha Ttp-mRNA expression was solely found in the liver of the dogs, rats and humans, while various other organs showed no alpha Ttp-mRNA expression. No significant differences in expression levels of canine alpha Ttp mRNA were found between DM and control dogs. Our data suggest that the canine alpha Ttp gene is unlikely to be involved in the pathogenesis of DM in German shepherd dogs.

Amino Acid Sequence↗