Periodic Reporting for period 4 - GENSURGE (Designer recombinases for efficient and safe genome surgery)
Période du rapport: 2022-07-01 au 2024-06-30
GENSURGE aimed at developing gene editing technologies based on designer-recombinases to offer cure strategies. GENSURGE in particular addressed important limitations of current genome editing technologies, which often rely on creating double-strand DNA breaks (DSBs) at a target locus as the first step for gene correction. These breaks are typically repaired by the cell's intrinsic DNA repair pathways, leading to the introduction of unwanted insertions and deletions (indels) compromising the precision and safety of genome editing. Especially for therapeutic applications this could lead to unwanted side effects. Nevertheless, most known genome-editing tools such as e.g. homing endonucleases (HEs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas system work by introducing targeted DSBs at specific sites in the genome. Site specific recombinases (SSRs) offer important advantages over nuclease-based genome editing technologies because they allow precise genome editing without triggering endogenous DNA repair pathways and possess the unique ability to fulfill both cleavage and immediate resealing of the processed DNA in vivo. The importance of GENSURGE for society lies in its potential to bring about significant advancements in biomedicine and biotechnology. By deepening our understanding of directed molecular evolution, the obtained project results could accelerate the generation of genome editing enzymes with therapeutic utility, which could lead to innovative treatments for diseases caused by mutations.
Aim1: We set out to leverage the information collected during the directed evolution of SSRs by deep sequencing. The PacBio-based long-read protocol was fully implemented and more than 80 evolved SSR libraries were sequenced at a depth of at least 20.000 individual clones. This data allowed us to e.g. identify when specific mutations emerged in the library. We also observed that 157 positions in the 324 amino acid long recombinase coding sequence had been selected to be altered during at least one evolved enzyme library. More interestingly, certain positions were found to change to different amino acids, depending on the selected target site the recombinases were evolved to recombine. In some extreme cases, eight different amino acids were found to be selected from the different projects. Overall, we have obtained excellent insight into the evolution dynamics of evolved recombinases. Using this data, we could further optimize the SLiDE procedure. The obtained data was implemented into the computer algorithms and analyses helped to improve the directed evolution procedure and to rationalize the evolution dynamics and structure-function relationships. The data also revealed that certain amino acids repeatedly co-evolved during directed evolution of SSRs. Furthermore, the sequencing data was helpful to uncover that nearest-neighbour amino acids play an important role in enhancing SSR activity on foreign target sites. The deep sequencing data from additional designer-recombinase libraries were generated and integrated into the model to further optimize the evolution conditions. Based on these results, we have determined the best inflection point to reduce the arabinose concentration during the evolution cycles. Together with other measures the generation of new designer-recombinase was significantly accelerated. The sequencing data was also implemented into a first generative deep-learning algorithm to predict designer-recombinases with predefined specificities. Aim 2: To expand the sequences that can be addressed by designer-SSRs, we first demonstrated that the generation of heterodimer recombinases is feasible. Employing SLiDE, we developed a fused heterodimeric recombinase system (RecF8) that is able to correct the int1h inversion frequently found in patients with a severe form of haemophilia A. Importantly, we achieved 30% inversion of the target sequence in human tissue culture cells, indicating that RecF8 can efficiently function in the target cells. Transient RecF8 treatment of endothelial cells, differentiated from patient derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, resulted in prominent correction of the inversion and restored Factor VIII mRNA expression. As the expression of 2 different designer-recombinases might increase potential off-target recombination, we developed an assay to screen for mutations that would render the monomers inactive, without compromising the dimer functionality. This screen identified single catalytic mutations in the individual monomers that fulfilled these criteria. We first explored the possibility of transplanting human liver organoids into TK-NOG mice in collaboration with Prof. Takanori Takebe (USA). However, due to the complexity and risks associated with these experiments, and after consultations with the German regulatory authorities, we decided to pursue an alternative approach. Aim 3: The principle of designer-recombinase induced gene replacement was demonstrated with loxLTR and loxBTR-specific recombinases by building a pSLIDE-4R system. Furthermore, we have generated a designer recombinase that catalyzes recombination of two genomic human sites to replace exon 8 of the F9 gene. Importantly, we could show that this recombinase can carry out the cassette exchange reaction in mammalian cells, when the cells were co-transfected with a donor and a receptor plasmid, plus a recombinase expression plasmid. Therefore, we achieved proof of concept for replacing a gene segment by DRiGR.
Genetic therapies are transforming the treatment of different diseases. Therapeutic human gene editing technologies have advanced significantly with CRISPR. However, despite CRISPR’s transformative impact on gene editing, concerns about its safety have arisen due to reports of potentially harmful side effects. In response, newer technologies have been developed. Nonetheless, these methods still rely on cellular DNA repair mechanisms, which can limit the size of edits and carry the risk of unintended side effects. The search for the "best-in-class" editing tools that can make precise, flexible changes at specific genomic sites without causing unwanted effects continues. GENSURGE had foreseen these limitations and has early on predicted that fully programmable recombinases would represent the ultimate therapeutic genome editing agent. That this was a wise decision can be seen in efforts of leaders in the field to shift their attention to the development of recombination-based genome editing. The progress made during GENSURGE has cemented the leading global position in evolving and engineering tyrosine-type designer-recombinases. Today, programmable recombinases are seen as the therapeutic editing enzymes of the future and our progress obtained as a result of GENSURGE puts us at the forefront of this development. A remarkable example is our recent discovery of conditioning recombinase enzyme activity through the internal fusion of a DNA-binding domain. In summary, GENSURGE has delivered impressive results to overcome road-blocks to allow accelerated generation of designer-recombinases for therapeutic applications.