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Abstract

In recent years, the field of gene and cell therapy has experienced major breakthroughs in the development of novel gene-based therapies and has been increasingly appreciated as versatile and innovative platform to cure hitherto untreatable diseases. Gene therapy resorts to biotechnological tools and approaches, such as recombinant (r)AAV vectors, RNAi and CRISPR/Cas9, that are employed to replace, silence or edit malfunctioning genes. The accomplishments of these technologies are highlighted in numerous clinical trials and culminated in the authorization of three rAAV-based therapies (Glybera, Luxturna and Zolgensma) and in one RNAi-based therapeutic (Onpattro). The presented doctoral thesis combines and further improves these technologies by drawing on several principles of synthetic biology and bioengineering and demonstrates their potential to tackle HBV infections and/or HBV/HDV co-infections as clinically relevant targets. The first part of this study was intended to validate an innovative generation of rAAV vectors previously established in our lab (by Florian Schmidt) and named TRISPR that enables the combination of the CRISPR/Cas9 and RNAi technologies. TRISPR facilitates the juxtaposition of three small RNA triggers (short-hairpin (sh)RNAs and single guide (sg)RNAs as required by the RNAi and CRISPR machineries, respectively) on a single rAAV vector and was experimentally reassessed in the presented study as superior strategy to knock-out HBV antigens via CRISPR. We further engineered TRISPR to co-express sgRNAs and shRNAs against HBV infections and HBV/HDV co-infections and showed that the combination of RNAi and CRISPR/Cas9 yielded a synergistic benefit in the elimination of both pathogens in cell culture experiments. Thus, this thesis paves the way for subsequent in vivo studies in HBV and HBV/HDV mouse models to test the effect of a combinatorial knock-down and knock-out strategy on the host immune response, which is typically blunted during HBV infection. Next, we evaluated the addition of 2.5% DMSO to the media as a new method to enhance rAAV transduction in cell culture experiments. A comprehensive screen via an automated microscopy assay with a fluorescence reporter revealed that the DMSO-mediated enhancement is dependent on selected rAAV variants and cell lines. Transduction rates increased up to 5-fold and 10-fold in Hepa16 and HepG2 cells, respectively, and culminated in N2A cells, in which transduction and expression rates of several rAAV variants that otherwise barely transduced these cells were boosted towards upper detection limits. Furthermore, the addition of DMSO also resulted in strongly enhanced rAAV uptake and improved expression of CRISPR components in N2A cells that yielded up to 20-fold increased targeted mutagenesis rates. Although the mechanism remains to be elucidated, DMSO represents an alternative method to improve rAAV transduction with less cytotoxic side effects compared to currently applied enhancers. We further harnessed the inherent power of double-stranded (ds)AAV vectors to express the CRISPR components more rapidly and more efficiently than conventional single-stranded (ss)AAV counterparts. Yet, expression from the superior dsAAV vector comes at the cost of a further reduction of the rAAV packaging capacity, from about 5 kb for ssAAVs, to only 2.4 kb for dsAAVs. Previously, others have circumvented the inherent size limit of ssAAV vectors by splitting the Streptococcus pyogenes (Sp)Cas9 endonuclease into two parts, which are reconstituted in the cell to the holo-enzyme by intein trans-splicing. We previously worked towards an intein-mediated splitCas9 system (MSc thesis, C. Schmelas) to split the smaller Cas9 ortholog from Staphylococcus aureus (SaCas9) in two halves, each of which was sufficiently small to allow packaging in dsAAV vectors. Here, we experimentally confirmed that the dsAAV/splitSaCas9 system can induce up to 5-fold higher knock-out efficiencies compared to the conventional ssAAV/full-lengthSaCas9, depending on cell type and incubation time. In addition, dsAAV/splitSaCas9 induced about 2-fold increased mutagenesis rates in a luciferase reporter in the liver of mice. In order to restrict the strong dsAAV/splitSaCas9 expression after successful gene editing, we further implemented a self-inactivating (SIN) approach developed by Julia Fakhiri from our lab that contributed to safeguard the rAAV/CRISPR technology. Lastly, we increased the safety profile of the CRISPR/Cas9 system from Staphylococcus aureus by collaborating with the Niopek and Correia labs that have engineered its first designer anti-CRISPR protein. To this end, they made use of AcrIIC1, a broad-spectrum inhibitor targeting various other Cas9 orthologs and re-designed its binding surface towards the SaCas9 HNH domain. Together, we then demonstrated on various genomic loci that the novel AcrX inhibitor efficiently prevents SaCas9 cleavage activity and also implemented a microRNA-based switch to restrict SaCas9 activity towards hepatocytes. In summary, this doctoral thesis demonstrated the compatibility and great potential of major tools in the field of gene therapy and presented several technological improvements that could be harnessed in future work either alone or in combination to enable a more efficient and safer approach to tackle HBV infections and HBV/HDV co-infections.