Research

Nucleic acids are fantastic molecules. Due to their unique base pairing properties, hybridization between nucleic acids is highly predictable and thus rationally programmable. We combine oligonucleotide chemistry with bioengineering to create RNA-guided tools for the study and control of fundamental biochemical processes surrounding the function and processing of RNA inside living cells and organisms. In addition, we pioneered the field of RNA base editing (recently highlighted in Nature) and are working on the clinical development of RNA drugs that apply the RNA base editing mechanism. The lab works, in collaboration with various labs, on the whole pipeline from chemistry - to cell biology - to organoids - to in vivo proof of concept. The lab is founding member of the Gene and RNA Therapy Center Tübingen (GRTC).

Engineering Tools for RNA Base Editing

RNA-guided machineries perfectly satisfy the demand for rationally programmable tools that enable to precisely interfere with cellular gene expression. Since the last years it is becaming increasingly clear that there is an additional layer of information encoded into the RNA transcript referred to as epiranscriptomics. These included many chemical modification of transcriped message. To study and to harness such epitroanscirptomic events, we develop tools for the site-specific manipulation of epitranscriptomics marks, like inosine, for instance. With respect to the latter, we engineered artifical A-to-I editases based on the human protein ADAR. Since inosine is biochemically read as guanosine, A-to-I editing allows for reprogramming genetic information directly on the RNA-level. The natural editing process allows the cell to conditionally include point mutations into proteins by reprogramming single amino acid triplets on the mRNA. Thus protein isoforms with distinct properties are formed without the need for additional genomic copies. Furthermore, RNA processing signals including splice sites, miRNA binding sites, Start and Stop signals are targets of RNA editing. Another sort of RNA Base editing is the reprogramming of cytidine to uridine (C-to-U) RNA base editing, which decently complements A-to-I editing. Again, tools based on the SNAP-Tag platform are highly effective.

Key publications

Review: Precision RNA base editing with engineered and endogenous effectors

L. S. Pfeiffer and T. Stafforst*, Nature Biotechnology 2023, 41, 1526-42.

Precise and efficient C-to-U RNA Base Editing with SNAP-CDAR-S

N. Latifi, A. M. Mack, I. Tellioglu, S. Di Giorgio, T. Stafforst*, Nucl. Acids Res. 2023, gkad598.

Efficient and Precise Editing of Endogenous Transcripts with SNAP-tagged ADARs

P. Vogel, M. Moschref, Q. Li, T. Merkle, K. D. Selvasaravanan, J. B. Li, T. Stafforst*. Nature Methods 2018, 15, 535-38.

An RNA-Deaminase Conjugate Selectively Repairs Point Mutations

T. Stafforst^*, M. F. Schneider, Angew. Chem. Int. Ed. 2012, 51, 11166-9.

Therapeutic RNA Base Editing

The lab is currently working on the clinical implementation of RNA Base editing. We consider the harnessing of the endogenous ADAR enzyme as particularly promising in this context, as engineered base editing tools suffer from global off-target editing and are more complex to deliver. We are persuing programs using virus-driven, fully encodable guide RNAs, e.g. CLUSTER guide RNAs, and we are developing chemically modified oligonucleotides for this purpose, e.g. the RESTORE approach. Our work includes basic design and engineering, optimization in cell culture, and proof of concept studies in organoids and animal models of human disease.

This work let to the foundation of the US/German Biotech Start-Up AIRNA with sites in Cambridge, Massachusetts and Tübingen, together with Jin Billy Li (Stanford University), and Paul and Tobias, two former members of the lab. The commercial perspectives have recently been highlighted by Nature Reviews Drug Discovery.

Key publications

Precise in-vivo RNA base editing with a wobble-enhanced circular CLUSTER guide RNA

P. Reautschnig*, ... , E. Y. Levanon, G. Mandel, T. Stafforst*, Nature Biotechnology, accepted

CLUSTER guide RNAs enable precise and efficient RNA editing in cell culture and in vivo

P. Reautschnig, N. Wahn, J. Wettengel, A. E. Schulz, N. Latifi, P. Vogel, T.-W. Kang, L. S. Pfeiffer, C. Zarges, U. Naumann, L. Zender, J. B. Li and T. Stafforst*, Nature Biotechnology 2022, 40, 759–768.

Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides

T. Merkle, S. Merz, P. Reautschnig, A. Blaha, Q. Li, P. Vogel, J. Wettengel, J. B. Li, T. Stafforst*, Nature Biotechnology 2019, 37, 133-138.

press release en/dt; highlighted by Nature Methods; highlighted by Biopro BW; highlighted by Laborjournal;highlighted by C&EN

Harnessing human ADAR2 for RNA repair – Recoding a PINK1 mutation rescues mitophagy

J. Wettengel, P. Reautschnig, S. Geisler, P. J. Kahle, T. Stafforst*, Nucl. Acids Res. 2017, 45, 2797-2808.

Photocontrol of biochemical processes

Light is an attractive external trigger for various processes as it enables a precise control in time, space and dosage. We are interested in controlling various biochemical processes by light. These include nucleic acid hybridization with psoralen crosslinks, ribonucleoprotein assembly via SNAP-tag technology, and photorelease of nitrogen oxide among others.

Key publications

Npom-protected NONOate enables light-triggered NO/cGMP signalling in primary vascular smooth muscle cells

A. S. Stroppel, M. Paolillo, T. Ziegler, R. Feil, T. Stafforst*. ChemBioChem 2018, 19, 1312-18.

Site-directed RNA editing in vivo can be triggered by the light-driven assembly of an artificial riboprotein

A. Hanswillemenke, T. Kuzdere, P. Vogel, G. Jekely, T. Stafforst*, J. Am. Chem. Soc. 2015, 137, 15 875-81.

Photoactivation of a Psoralen-blocked Luciferase Gene by Blue Light

T. Stafforst^*, J. M. Stadler, Angew. Chem. Int. Ed. 2013, 52, 12448-51.