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Vector & Gene Therapy

Vector & Gene Therapy

Vector & Gene Therapy

The Department Vector & Gene Therapy focuses on the RNA-based transfer of therapeutic genes for various therapeutic approaches. The Functional Unit Vectors is currently assigned to the department. The Vector & Gene Therapy Department is tackling the challenge of developing new therapeutic RNA that can better meet future requirements than established mRNA.

  • Dr. Tim Beißert

    Co-Director Vector & Gene Therapy

  • Dr. Mario Perkovic

    Co-Director Vector & Gene Therapy

Our vision

Tailored therapeutic RNA as an appropriate response to the different requirements of prophylactic vaccination and gene therapy.

Vectors

  • Dr. Tim Beißert

    Head of Functional Unit Vectors

  • Dr. Cristian Raileanu

    Deputy Head of Functional Unit Vectors

Our activities and competences

Our methods and technologies

Sequence design and cloning

Using both manual and AI-assisted literature searches, we specifically look for virus variants or artificially generated mutants that exhibit altered RNA replication behavior and thus have the potential to improve our self-replicating RNA. Using gene synthesis and conventional cloning, we bring these variants into the lab and test their effects on our vectors.

RNA production

We produce RNA using in vitro transcription, minimize contamination with double-stranded RNA, or incorporate various non-canonical nucleotides into the synthesis. Depending on the intended application, this allows us to obtain RNA with the desired immunogenicity.

Gene expression

When testing new RNA variants, key initial readouts are the expression levels and duration of reporter proteins in various cell types. We validate protein expression by monitoring RNA replication at the RNA level. To test replication in a cell lacking innate immunity, we use the BHK21 cell line. In these cells, we investigate the basic functionality of new RNA vectors and how their RNA replication behaves—without the inhibitory effect of the interferon response—compared to our established control vectors. Other important cell types include primary human fibroblasts, which respond to transfected and/or replicating RNA with pronounced IFN release, as well as dendritic cells or PBMCs, which are important later in the development of the immune response in human subjects.

Immunization

We insert model antigens into promising new RNA variants to study the adaptive immune response in immunization studies in mice. Influenza hemagglutinin (HA) or coronavirus spike protein are frequently used for this purpose, as are envelope proteins from alphaviruses. The selection of antigens can be expanded at any time.

T-cell reprogramming

To test the suitability of these vectors as vehicles for therapeutic genes beyond antigens, our primary focus is on the transfer of chimeric antigen receptors (CARs) or T-cell receptors into human T cells and their adoptive transfer. CAR T cells are also an established therapeutic approach that would benefit from cost-effective, long-lasting RNA expression. We are investigating CAR expression levels and duration, as well as the killing of target cells by T cells in vitro and in vivo.

Publications

Trans-amplifying RNA (taRNA) is a split-vector derivative of self-amplifying RNA (saRNA) and a promising vaccine platform. taRNA combines a non-replicating mRNA encoding an alphaviral replicase, and a transreplicon RNA (TR) coding for the antigen. Upon translation, the replicase amplifies the antigen-coding TR, thereby requiring minimal amounts of TR for immunization. TR-amplification by the replicase follows a complex mechanism orchestrated by genomic and subgenomic promoters (SGP), and generates genomic and subgenomic amplicons whereby only the latter are translated into therapeutic proteins. This complexity merits simplification to improve the platform. Here, we eliminated the SGP and redesigned the 5' untranslated region to shorten the TR (STR) thereby enabling translation of the remaining genomic amplicon. We then applied a directed evolution approach to select for faster replicating STRs. The resulting evolved STR (eSTR) had acquired A-rich 5'extensions, which improved taRNA expression thanks to accelerated replication. Consequently, we reduced the minimal required TR amount by more than 10-fold without losing taRNA expression in vitro. Accordingly, eSTR-immunized mice developed greater antibody titers to taRNA-encoded Influenza HA than TR-immunized mice. In summary, this work points the way for further optimization of taRNA by combining rational design and directed evolution.

Here we present a potent RNA vaccine approach based on a novel bipartite vector system using trans-amplifying RNA (taRNA). The vector cassette encoding the vaccine antigen originates from an alphaviral self-amplifying RNA (saRNA), from which the replicase was deleted to form a transreplicon. Replicase activity is provided in trans by a second molecule, either by a standard saRNA or an optimized non-replicating mRNA (nrRNA). The latter delivered 10-100 fold higher transreplicon expression than the former. Moreover, expression driven by the nrRNA-encoded replicase in the taRNA system was as efficient as in a conventional monopartite saRNA system. We show that the superiority of nrRNA- over saRNA-encoded replicase to drive expression of the transreplicon is most likely attributable to its higher translational efficiency and lack of interference with cellular translation. Testing the novel taRNA system in mice, we observed that doses of influenza hemagglutinin antigen-encoding RNA as low as 50 ng were sufficient to induce neutralizing antibodies and mount a protective immune response against live virus challenge. These findings, together with a favorable safety profile, a simpler production process and the universal applicability associated with this bipartite vector system, warrant further exploration of taRNA.

The arthritogenic alphavirus, chikungunya virus (CHIKV), is now present in almost 100 countries worldwide. Further spread is very likely, which raises public health concerns. CHIKV infections cause fever and arthralgia, which can be debilitating and last for years. Here, we describe a CHIKV vaccine candidate based on trans-amplifying RNA (taRNA). The vaccine candidate consists of two RNAs: a non-replicating mRNA encoding for the CHIKV nonstructural proteins, forming the replicase complex and a trans-replicon (TR) RNA encoding the CHIKV envelope proteins. The TR-RNA can be amplified by the replicase in trans, and small RNA amounts can induce a potent immune response. The TR-RNA was efficiently amplified by the CHIKV replicase in vitro, leading to high protein expression, comparable to that generated by a CHIKV infection. In addition, the taRNA system did not recombine to replication-competent CHIKV. Using a prime-boost schedule, the vaccine candidate induced potent CHIKV-specific humoral and cellular immune responses in vivo in a mouse model. Notably, mice were protected against a high-dose CHIKV challenge infection with two vaccine doses of only 1.5 μg RNA. Therefore, taRNAs are a promising safe and efficient vaccination strategy against CHIKV infections.

Replicating RNA, including self-amplifying RNA (saRNA) and trans-amplifying RNA (taRNA), holds great potential for advancing the next generation of RNA-based vaccines. Unlike in vitro transcribed mRNA found in most current RNA vaccines, saRNA or taRNA can be massively replicated within cells in the presence of RNA-amplifying enzymes known as replicases. We recently demonstrated that this property could enhance immune responses with minimal injected RNA amounts. In saRNA-based vaccines, replicase and antigens are encoded on the same mRNA molecule, resulting in very long RNA sequences, which poses significant challenges in production, delivery, and stability. In taRNA-based vaccines, these challenges can be overcome by splitting the replication system into two parts: one that encodes replicase and the other that encodes a short antigen-encoding RNA called transreplicon. Here, we review the identification and use of transreplicon RNA in alphavirus research, with a focus on the development of novel taRNA technology as a state-of-the art vaccine platform. Additionally, we discuss remaining challenges essential to the clinical application and highlight the potential benefits related to the unique properties of this future vaccine platform.

The co-delivery of microRNAs (miRNAs) and protein-coding RNA presents an opportunity for a combined approach to gene expression and gene regulation for therapeutic applications. Protein delivery is established using long mRNA, self-, and trans-amplifying RNA (taRNA), whereas miRNA delivery typically uses short synthetic oligonucleotides rather than incorporating it as a precursor into long RNA. Although miRNA delivery into the cell cytoplasm using long genomes of RNA viruses has been described, concerns have remained regarding low processing efficiency. However, miRNA precursors can be released from long cytoplasmic alphaviral RNA by a cytoplasmic fraction of Drosha. taRNA, a promising vector platform for infectious disease vaccination, uses a nonreplicating mRNA expressing an alphaviral replicase to amplify a protein-coding short transreplicon-RNA (STR) in trans. To investigate the possibility of simultaneously delivering protein expression and gene silencing, we tested whether a taRNA system can carry and release functional miRNA to target cells. Here, we show that mature miRNA is released from STRs and silences specific targets in a replication-dependent manner for several days without compromising the expression of STR-encoded proteins. Our findings suggest that incorporating miRNAs into the taRNA vector platform has the potential for gene regulation alongside the expression of therapeutic genes.