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Gene Manipulation

Gene Manipulation

Cutting-edge plasmid DNA cloning and gene transfer technologies for next-generation RNA therapeutics.

Gene Manipulation

The Department of Gene Manipulation consists of the Cloning and Gene Transfer Units.

The Cloning Unit provides cutting-edge plasmid DNA cloning services that enable deeper insights into cellular processes at the molecular level and help lay the foundation for the development of new therapeutics. In the Gene Transfer Unit, we apply different approaches to manipulate or transfer genes into host cells. Here, we use retroviral particles to deliver genes for permanent expression, CRISPR-based approaches for gene manipulation and trans-amplifying RNA for therapeutic RNA expression at low doses.

  • Maximilian Mustermann

    Department Director

  • Max Mustermann

    Deputy Director

Our vision

Deepening our understanding of cellular processes and advancing next-generation RNA therapeutics through molecular cloning expertise, genome manipulation and mechanistic research.

Cloning Unit

  • Maximilian Mustermann

    Director Functional Unit

  • Maximilian Mustermann

    Deputy Director Functional Unit

Our activities and competences

Our methods and platforms

Polymerase chain and cloning reactions

Polymerase chain reactions (PCRs) are used to amplify target sequences for downstream cloning applications, including ligation, Cold Fusion, Gateway cloning, Gibson and Golden Gate assembly

Cultivation of E. coli and plasmid DNA isolation

Bacterial transformation with plasmid DNA, liquid culture cultivation, and manual or semi-automated plasmid DNA preparation at mini, midi, and maxi scale

DNA quality assessment

Assessment of DNA quantity and quality using NanoDrop and Qubit, restriction digestion followed by agarose gel electrophoresis, and evaluation of Sanger and Oxford Nanopore Technologies (ONT) sequencing results

Plasmid verification

In-house NGS library preparation and plasmid DNA analysis using an Illumina MiSeq sequencer and a custom-built analysis pipeline

Linearization service

Preparation of plasmid DNA for downstream RNA synthesis via in vitro transcription (IVT), including linearization and purification of DNA templates using the KingFisher Duo Prime Purification System

Publications

200 days ago, TRON took a decisive step toward advancing clinical translation by introducing the new role of Medical Director and appointing Univ.-Prof. Dr. Matthias Gaida. This move reflects our clear commitment to aligning research with clinical reality and patient care.

Why does this matter?
As Medical Director, Matthias ensures that TRON’s scientific strategy addresses real-world medical challenges. By integrating clinical perspectives into decision-making, research questions become more relevant, and promising discoveries can move faster into application.

What drives this approach?
A strong belief that science should serve patients. The goal is to accelerate translational research and strengthen collaborations—locally, nationally, and internationally. This includes building structured partnerships, developing early-stage study concepts, and ensuring access to clinical data and samples.

What’s next?
Under Matthias’ leadership, TRON is creating flexible pipelines for pilot projects and fostering research that responds directly to patient needs. This strategy will position TRON as a leading center for personalized immuno-oncology—while exploring new areas such as cardiology, where scientific discovery and patient care go hand in hand.

Cancer therapy is an emergentapplication for mRNA therapeutics. While in tumor immunotherapy, mRNA encodingfor tumor-associated antigens is delivered to antigen-presenting cells inspleen and lymph nodes, other therapeutic options benefit from immediate deliveryof mRNA nanomedicines directly to the tumor. However, tumor targeting of mRNAtherapeutics is still a challenge, since, in addition to delivery of the cargoto the tumor, specifics of the targeted cell type as well as its interplay withthe tumor microenvironment are crucial for successful intervention. This studyinvestigated lipoplex nanoparticle-mediated mRNA delivery to spheroid cellculture models of melanoma. Insights into cell-type specific targeting,non-cell-autonomous effects, and penetration capacity in tumor and stroma cellsof the mRNA lipoplex nanoparticles were obtained. It was shown that bothcoculture of different cell types as well as three-dimensional cell growthcharacteristics can modulate distribution and transfection efficiency of mRNAlipoplex formulations. The results demonstrate that three-dimensional coculturespheroids can provide a valuable surplus of information in comparison toadherent cells. Thus, they may represent in vitro models with enhancedpredictivity for the in vivo activity of cancer nanotherapeutics.

Alphaviruses such as the human pathogenic chikungunya virus (CHIKV) and Ross River virus (RRV) can cause explosive outbreaks raising public health concerns. However, no vaccine or specific antiviral treatment is yet available. We recently established a CHIKV vaccine candidate based on trans-amplifying RNA (taRNA). This novel system consists of a replicase-encoding mRNA and a trans-replicon (TR) RNA encoding the antigen. The TR-RNA is amplified by the replicase in situ. We were interested in determining whether multiple TR-RNAs can be amplified in parallel and if, thus, a multivalent vaccine candidate can be generated. In vitro, we observed an efficient amplification of two TR-RNAs, encoding for the CHIKV and the RRV envelope proteins, by the replicase, which resulted in a high antigen expression. Vaccination of BALB/c mice with the two TR-RNAs induced CHIKV- and RRV-specific humoral and cellular immune responses. However, antibody titers and neutralization capacity were higher after immunization with a single TR-RNA. In contrast, alphavirus-specific T cell responses were equally potent after the bivalent vaccination. These data show the proof-of-principle that the taRNA system can be used to generate multivalent vaccines; however, further optimizations will be needed for clinical application.

Enteroviruses (EV) are implicated in an extensive range of clinical manifestations, such as pancreatic failure, cardiovascular disease, hepatitis, and meningoencephalitis. We recently reported on the biochemical properties of the highly conserved cysteine residue at position 38 (C38) of enteroviral protein 3A and demonstrated a C38-mediated homodimerization of the Coxsackievirus B3 protein 3A (CVB3-3A) that resulted in its profound stabilization. Here, we show that residue C38 of protein 3A supports the replication of CVB3, a clinically relevant member of the enterovirus genus. The infection of HeLa cells with protein 3A cysteine 38 to alanine mutants (C38A) attenuates virus replication, resulting in comparably lower virus particle formation. Consistently, in a mouse infection model, the enhanced virus propagation of CVB3-3A wt in comparison to the CVB3-3A[C38A] mutant was confirmed and found to promote severe liver tissue damage. In contrast, infection with the CVB3-3A[C38A] mutant mitigated hepatic tissue injury and ameliorated the signs of systemic inflammatory responses, such as hypoglycemia and hypothermia. Based on these data and our previous report on the C38-mediated stabilization of the CVB3-3A protein, we conclude that the highly conserved amino acid C38 in protein 3A enhances the virulence of CVB3.

Gene Transfer Unit

  • Maximilian Mustermann

    Director Functional Unit

  • Maximilian Mustermann

    Deputy Director Functional Unit

Our activities and competences

Our methods and platforms

Trans-amplifying RNA

Together with the Vectors Unit we developed trans-amplifying RNA, our proprietary variant of self-amplifying RNA. Suitable to reduce the amount of antigen coding RNA needed for vaccination. Detailed description of scientific background and methods:

Production of retroviral particles and generation of cell lines

A technology that utilize modified retroviruses to deliver genes into cells for permanent expression. Gene transfer is mediated via viral particles. The vector system allows efficient but random insertion of genetic material with the size up to ~10 kb in vast majority of cells.

CRISPR/Cas9 knockout

CRISPR-Cas9 is a transformative gene-editing technology that revolutionizes genomic modifications because it allows precise genome editing directly within cells. The system consists of a short, non-coding guide RNA (gRNA) and the Cas9 nuclease, which together act as a genetic-engineering tool capable of disabling, repairing, or introducing new genetic material.

CRISPRa/i (activation/inhibition)

CRISPRa/i is based on Cas9 mutant which is not able to cut the DNA, so called dead Cas9 (dCas9). Fusing one or more transcription factors to dCas9 in combination with gRNAs targeting promotor regions turns the system into a tool suitable to specifically up- or downregulate expression of desired genes.

Publications

Epithelial-to-mesenchymal transition (EMT) renders epithelial cells migratory properties. While epigenetic and splicing changes have been implicated in EMT, the mechanisms governing their crosstalk remain poorly understood. Here we discovered that a C2H2 zinc finger protein, ZNF827, is strongly induced during various contexts of EMT, including in brain development and breast cancer metastasis, and is required for the molecular and phenotypic changes underlying EMT in these processes. Mechanistically, ZNF827 mediated these responses by orchestrating a large-scale remodelling of the splicing landscape by recruiting HDAC1 for epigenetic modulation of distinct genomic loci, thereby slowing RNA polymerase II progression and altering the splicing of genes encoding key EMT regulators in cis. Our findings reveal an unprecedented complexity of crosstalk between epigenetic landscape and splicing programme in governing EMT and identify ZNF827 as a master regulator coupling these processes during EMT in brain development and breast cancer metastasis.

Chimeric antigen receptor (CAR) T cells are efficacious in patients with B-cell malignancies, while their activity is limited in patients with solid tumors. We developed a novel heterodimeric TCR-like CAR (TCAR) designed to achieve optimal chain pairing and integration into the T-cell CD3 signaling complex. The TCAR mediated high antigen sensitivity and potent antigen-specific T-cell effector functions in short-term in vitro assays. Both persistence and functionality of TCAR T cells were augmented by provision of costimulatory signals, which improved proliferation in vitro and in vivo. Combination with a nanoparticulate RNA vaccine, developed for in vivo expansion of CAR T cells, promoted tightly controlled expansion, survival, and antitumor efficacy of TCAR T cells in vivo.

Glioblastomas (GBM) are the most aggressive tumors affecting the central nervous system in adults, causing death within, on average, 15 months after diagnosis. Immunocompetent in-vivo models that closely mirror human GBM are urgently needed for deciphering glioma biology and for the development of effective treatment options. The murine GBM cell lines currently available for engraftment in immunocompetent mice are not only exiguous but also inadequate in representing prominent characteristics of human GBM such as infiltrative behavior, necrotic areas, and pronounced tumor heterogeneity. Therefore, we generated a set of glioblastoma cell lines by repeated in vivo passaging of cells isolated from a neural stem cell-specific Pten/p53 double-knockout genetic mouse brain tumor model. Transcriptome and genome analyses of the cell lines revealed molecular heterogeneity comparable to that observed in human glioblastoma. Upon orthotopic transplantation into syngeneic hosts, they formed high-grade gliomas that faithfully recapitulated the histopathological features, invasiveness and immune cell infiltration characteristic of human glioblastoma. These features make our cell lines unique and useful tools to study multiple aspects of glioblastoma pathomechanism and to test novel treatments in an intact immune microenvironment.