This action enabled us to perform new research in the field of gene therapy and monogenetic diseases. Our work included the establishment, identification and optimization of different gene correction tools. For the specific approach of gene correction of multiple target sites in different genes the clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) system exerted a higher flexibility was therefore much easier to handle than the other tested tools transcription activator-like effector nucleases (TALENs) and Zinc finger nucleases (ZFNs). In particular, the CRISPR/Cas system including single guide RNAs (sgRNA) and single strand oligo deoxynucleotides (ssODNs) as repair template was successfully designed to target two mutations in the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) and Surfactant Protein B (SFTPB) genes in the human genome, responsible for Cystic Fibrosis (CF) and Surfactant Protein B (SP-B) deficiency, respectively. Furthermore, we could show that gene correction using a CRISPR/Cas system is possible by enlisting an ex vivo model of hematopoietic stem cells (HSC, CD34+) and a CRISPR/Cas system targeting the human beta-globin (HBB) gene (Antony et al. 2018).
Besides the work with gene correction systems, our lab designed and created several mouse models that serve as disease models for the in vivo gene correction approach. The work with these mouse models is still ongoing at the time of this report.
In accordance with the goal to use optimized CRISPR/Cas systems in vivo, this action also focused on the optimization of RNA-based delivery in a murine system. This included the investigation of different RNA modifications, especially the use of chemical modifications of bases in in vitro transcribed RNA. Additionally, this action studied the impact of sequence optimization on expression and established a simple method of evaluating immunogenicity of RNA with RNA ImmunoGenic assay (Haque et al. 2020). These extensive studies yielded several improvements of the RNA system, like the inclusion of chemical modifications like 2-Thiouridine or 5-Methylcytidine and the reduction of overall Uridine content in the sequence (Vaidyanathan et al. 2018). These optimizations showed an overall enhanced function of RNA after in vivo delivery and even an improvement in lung function in CF mouse model after treatment with chemically modified mRNA encoding for human CFTR (cmRNAhCFTR, Haque et al. 2018).
Detailed results can be found in the peer-reviewed scientific publications. Additionally, results of this action were presented to the scientific community at various international conferences and a European patent for “Chemically modified mRNA for use in the treatment of a disease associated with the CFTR gene” (5402P571EP) was granted. Finally, we believe that RNA-based gene correction approaches have the power to provide a therapy for monogenetic diseases and that this action provided a big step forward in the advancement of these tools towards the patient.
