Synthetic rewiring and boosting type I interferon responses for visualization and counteracting viral infections.

Mammalian first line of defense against viruses is accomplished by the interferon (IFN) system. Viruses have evolved numerous mechanisms to reduce the IFN action allowing them to invade the host and/or to establish latency. We generated an IFN responsive intracellular hub by integrating the synthetic transactivator tTA into the chromosomal Mx2 locus for IFN-based activation of tTA dependent expression modules. The additional implementation of a synthetic amplifier module with positive feedback even allowed for monitoring and reacting to infections of viruses that can antagonize the IFN system. Low and transient IFN amounts are sufficient to trigger these amplifier cells. This gives rise to higher and sustained-but optionally de-activatable-expression even when the initial stimulus has faded out. Amplification of the IFN response induced by IFN suppressing viruses is sufficient to protect cells from infection. Together, this interfaced sensor/actuator system provides a toolbox for robust sensing and counteracting viral infections.

Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.

Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.

Stable Expression by Lentiviral Transduction of Cells.

Lentiviral gene transfer represents a versatile and powerful method for genetic transduction of many cell lines and primary cells including "hard-to-transfect" cells. As a consequence of the integration of the recombinant lentiviral vector into the cellular genome, the transgene is stably maintained, and long-term producing cells are established. Here, we describe the current state of the art and give details for lab-scale production of lentiviral vectors as well as for infection and titration of the viral vectors.

A Ubiquitous Chromatin Opening Element and DNA Demethylation Facilitate Doxycycline-Controlled Expression during Differentiation and in Transgenic Mice.

Synthetic expression cassettes provide the ability to control transgene expression in experimental animal models through external triggers, enabling the study of gene function and the modulation of endogenous regulatory networks in vivo. The performance of synthetic expression cassettes in transgenic animals critically depends on the regulatory properties of the respective chromosomal integration sites, which are affected by the remodeling of the chromatin structure during development. The epigenetic status may affect the transcriptional activity of the synthetic cassettes and even lead to transcriptional silencing, depending on the chromosomal sites and the tissue. In this study, we investigated the influence of the ubiquitous chromosome opening element (UCOE) HNRPA2B1-CBX3 and its subfragments A2UCOE and CBX3 on doxycycline-controlled expression modules within the chromosomal Rosa26 locus. While HNRPA2B1-CBX3 and A2UCOE reduced the expression of the synthetic cassettes in mouse embryonic stem cells, CBX3 stabilized the expression and facilitated doxycycline-controlled expression after in vitro differentiation. In transgenic mice, the CBX3 element protected the cassettes from overt silencing although the expression was moderate and only partially controlled by doxycycline. We demonstrate that CBX3-flanked synthetic cassettes can be activated by decitabine-mediated blockade of DNA methylation or by specific recruitment of the catalytic demethylation domain of the ten-eleven translocation protein TET1 to the synthetic promoter. This suggests that CBX3 renders the synthetic cassettes permissive for subsequent epigenetic activation, thereby supporting doxycycline-controlled expression. Together, this study reveals a strategy for overcoming epigenetic constraints of synthetic expression cassettes, facilitating externally controlled transgene expression in mice.

Fibroblasts are a site of murine cytomegalovirus lytic replication and Stat1-dependent latent persistence in vivo.

To date, no herpesvirus has been shown to latently persist in fibroblastic cells. Here, we show that murine cytomegalovirus, a ß-herpesvirus, persists for the long term and across organs in PDGFRα-positive fibroblastic cells, with similar or higher genome loads than in the previously known sites of murine cytomegalovirus latency. Whereas murine cytomegalovirus gene transcription in PDGFRα-positive fibroblastic cells is almost completely silenced at 5 months post-infection, these cells give rise to reactivated virus ex vivo, arguing that they support latent murine cytomegalovirus infection. Notably, PDGFRα-positive fibroblastic cells also support productive virus replication during primary murine cytomegalovirus infection. Mechanistically, Stat1-deficiency promotes lytic infection but abolishes latent persistence of murine cytomegalovirus in PDGFRα-positive fibroblastic cells in vivo. In sum, fibroblastic cells have a dual role as a site of lytic murine cytomegalovirus replication and a reservoir of latent murine cytomegalovirus in vivo and STAT1 is required for murine cytomegalovirus latent persistence in vivo.

Evaluation of the Neutralizing Antibody STE90-C11 against SARS-CoV-2 Delta Infection and Its Recognition of Other Variants of Concerns.

As of now, the COVID-19 pandemic has spread to over 770 million confirmed cases and caused approximately 7 million deaths. While several vaccines and monoclonal antibodies (mAb) have been developed and deployed, natural selection against immune recognition of viral antigens by antibodies has fueled the evolution of new emerging variants and limited the immune protection by vaccines and mAb. To optimize the efficiency of mAb, it is imperative to understand how they neutralize the variants of concern (VoCs) and to investigate the mutations responsible for immune escape. In this study, we show the in vitro neutralizing effects of a previously described monoclonal antibody (STE90-C11) against the SARS-CoV-2 Delta variant (B.1.617.2) and its in vivo effects in therapeutic and prophylactic settings. We also show that the Omicron variant avoids recognition by this mAb. To define which mutations are responsible for the escape in the Omicron variant, we used a library of pseudovirus mutants carrying each of the mutations present in the Omicron VoC individually. We show that either 501Y or 417K point mutations were sufficient for the escape of Omicron recognition by STE90-C11. To test how escape mutations act against a combination of antibodies, we tested the same library against bispecific antibodies, recognizing two discrete regions of the spike antigen. While Omicron escaped the control by the bispecific antibodies, the same antibodies controlled all mutants with individual mutations.

Rational Design of Single Copy Expression Cassettes in Defined Chromosomal Sites Overcomes Intraclonal Cell-to-Cell Expression Heterogeneity and Ensures Robust Antibody Production.

The expression of endogenous genes as well as transgenes depends on regulatory elements within and surrounding genes as well as their epigenetic modifications. Members of a cloned cell population often show pronounced cell-to-cell heterogeneity with respect to the expression of a certain gene. To investigate the heterogeneity of recombinant protein expression we targeted cassettes into two preselected chromosomal hot-spots in Chinese hamster ovary (CHO) cells. Depending on the gene of interest and the design of the expression cassette, we found strong expression variability that could be reduced by epigenetic modifiers, but not by site-specific recruitment of the modulator dCas9-VPR. In particular, the implementation of ubiquitous chromatin opening elements (UCOEs) reduced cell-to-cell heterogeneity and concomitantly increased expression. The application of this method to recombinant antibody expression confirmed that rational design of cell lines for production of transgenes with predictable and high titers is a promising approach.

Stable Expression by Lentiviral Transduction of Cells.

Lentiviral gene transfer represents a versatile and powerful method for genetic transduction of many cell lines and primary cells including "hard-to-transfect" cells. As a consequence of the integration of the recombinant lentiviral vector into the cellular genome the transgene is stably maintained and long term producing cells are established. Here, we describe the current state of the art and give details for lab scale production of lentiviral vectors as well as for infection and titration of the viral vectors.

The CpG-sites of the CBX3 ubiquitous chromatin opening element are critical structural determinants for the anti-silencing function.

Suppression of therapeutic transgene expression from retroviral gene therapy vectors by epigenetic defence mechanisms represents a problem that is particularly encountered in pluripotent stem cells (PSCs) and their differentiated progeny. Transgene expression in these cells, however, can be stabilised by CpG-rich ubiquitous chromatin opening elements (UCOEs). In this context we recently demonstrated profound anti-silencing properties for the small (679 bp) CBX3-UCO element and we now confirmed this observation in the context of the defined murine chromosomal loci ROSA26 and TIGRE. Moreover, since the structural basis for the anti-silencing activity of UCOEs has remained poorly defined, we interrogated various CBX3 subfragments in the context of lentiviral vectors and murine PSCs. We demonstrated marked though distinct anti-silencing activity in the pluripotent state and during PSC-differentiation for several of the CBX3 subfragments. This activity was significantly correlated with CpG content as well as endogenous transcriptional activity. Interestingly, also a scrambled CBX3 version with preserved CpG-sites retained the anti-silencing activity despite the lack of endogenous promoter activity. Our data therefore highlight the importance of CpG-sites and transcriptional activity for UCOE functionality and suggest contributions from different mechanisms to the overall anti-silencing function of the CBX3 element.

Epigenetic modulations rendering cell-to-cell variability and phenotypic metastability.

Tumor cells display phenotypic plasticity and heterogeneity due to genetic and epigenetic variations which limit the predictability of therapeutic interventions. Chromatin modifications can arise stochastically but can also be a consequence of environmental influences such as the microenvironment of cancer cells. A better understanding of the impact and dynamics of epigenetic modulation at defined chromosomal sites is required to get access to the underlying mechanisms. We investigated the epigenetic modulations leading to cell-to-cell heterogeneity in a tumor cell line model. To this end, we analyzed expression variance in 80 genetically uniform cell populations having a single-copy reporter randomly integrated in the genome. Single-cell analysis showed high intraclonal heterogeneity. Epigenetic characterization revealed that expression heterogeneity was accompanied by differential histone marks whereas contribution of DNA methylation could be excluded. Strikingly, some clones revealed a highly dynamic, stochastically altered chromatin state of the transgene cassette which was accompanied with a metastable expression pattern. In contrast, other clones represented a robust chromatin state of the transgene cassette with a stable expression pattern. Together, these results elucidate locus-specific epigenetic modulation in gene expression that contributes to phenotypic heterogeneity of cells and might account for cellular plasticity.