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Biochemically modified messenger RNA encoding nucleases for in vivo gene correction of severe inherited lung diseases

Periodic Reporting for period 4 - BREATHE (Biochemically modified messenger RNA encoding nucleases for in vivo gene correction of severe inherited lung diseases)

Berichtszeitraum: 2019-10-01 bis 2020-03-31

In the last century gene therapy evolved as a new hope for a wide range of diseases, particularly monogenetic diseases. Unfortunately, early trials using vector-based approaches yielded a general low efficiency. On contrast the usage of integrating viral vectors led to drastic consequences due to the insertional mutagenesis and the development of cancers in patients. Nonetheless, the field of gene therapy progressed with the discovery of new tools to be more specific in changing the genome. In particular, the discovery of site-specific endonucleases opened the perspective of base-specific gene correction within the human genome.

This action aimed to investigate the possible usage of site-specific endonucleases for gene correction, more specifically the correction of specific mutations within two different genes: the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) gene associated with Cystic Fibrosis (CF) and the Surfactant Protein B (SFTPB) gene associated with Surfactant Protein B (SP-B) deficiency. Both diseases are rare monogenetic diseases, but have rather different characters. CF is the most common monogenetic disease in the Caucasian population and leads to reduced life expectancy due to a defect in a chloride channel in mucus-producing cells. This leads to organ degradation especially in the lungs, pancreas and small intestine with the lung defect being the most life-limiting. On the contrary SP-B deficiency is a very rare monogenetic disease leading to lethal respiratory failure within the first year of life. The lungs do not produce the important Surfactant which is required for O2/CO2 gas exchange. The only cure available for SP-B deficiency to date is lung transplantation, while the drug class of CFTR modulators improves the condition of CF without providing a long lasting cure.

In light of these limitations for the treatment of CF and SP-B deficiency, this action set the goal of advancing the method of gene correction in order to develop a possible therapy for monogenetic diseases like CF and SP-B deficiency. This included the evaluation of the available tools for gene correction, like transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas). Furthermore, the optimization of gene therapeutic approaches in vitro, ex vivo and in vivo with emphasis on the usage of chemically modified messenger RNA (cmRNA) and in vivo delivery to the lung were in focus of this action. For testing of gene therapy agents multiple murine disease models were created harboring mutations in CFTR and SFTPB gene.

We can conclude for this action that for gene therapy the CRISPR/Cas system displays a more promising approach due to the extended flexibility in targeting sequence-specific genomic regions solely by the use of distinctive single guide RNAs (sgRNA). We showed that targeting of the selected mutations in the CFTR and SFTPB genes is possible in in vitro models. The main task that was identified is reaching an adequate percentage of gene correction using CRISPR/Cas systems while maintaining acceptable levels of off-target effects to ensure the safety of the gene correction approach. In this matter, this action was able to provide significant advances for the usage of cmRNA for in vitro and in vivo delivery of genetic material. We believe that the combination of the advances in RNA-based delivery of gene correction tools and the new approaches for gene correction tools (e.g. prime editing system) will yield a long lasting cure for monogenetic diseases in the future.
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.
"1. By screening the different kinetic profiles of mRNA encoded endonucleases, we have a significantly higher efficacy protocol for in vivo gene editing as well as a significantly lower chance for off-target effects.
2. The combination with certain nanoparticles (in cooperation with HelmholtzZentrum Saarbrücken) we do now not only have the intratracheal spray application as primary application method, but also the i.v. route - which is a huge advantage to reach lung (stem) cells - as natural ""defense"" systems like cilia or disease-correlated symptomatic issues such as mucus can be circumvented.
3. In comprehensive analyses we are understanding mRNA immunogenicity and how to avoid or completely abrogate that. This will be mandatory for first in men studies."
Scheme of project research approach