Periodic Reporting for period 3 - DNA-DOCK (Precision Docking of Very Large DNA Cargos in Mammalian Genomes)
Reporting period: 2022-09-01 to 2024-02-29
The human genome was sequenced 15 years ago. Today, thousands of sequenced genomes provide a dizzying array of new information, constituting the list of parts that define organisms, and simultaneously the programming code that dictates the functions that drive life. Tools were developed that achieve precise cleavage of single, unique DNA sequences enable scientists to edit genes by creating double-stranded breaks at any user defined sequence in the massive genomes of eukaryotes, providing means to decipher, and alter, function. A game change occurred with the discovery of CRISPR/Cas9, which effectively democratized gene editing.
To unlock the full potential of this revolution, however, our capacities to disrupt or rewrite small local elements of code by gene editing must be complemented by capabilities to efficiently dock very large DNA cargos with a wide range of functions into genomic sites, by inserting large, custom designed, multifunctional DNAs at base pair precision. Large designer DNA cargoes would carry multicomponent DNA circuitry including programmable and fine-tuneable functionalities and represent the vital interface between gene editing which is the state-of-the-art at present, and genome engineering, which is the future. This challenge remains largely unresolved to date.
The research here proposed tackles precisely this fundamental bottleneck. By bringing to bear an array of sophisticated tools, integrating unique know-how with leading synthetic, biological, chemical and biomedical assets, we will establish new technology to enable highly efficient, easy-to-use docking of custom designed very large multifunctional designer DNA cargos into mammalian genomes with base pair precision, addressing significant unmet academic, biomedical and industrial needs.
To unlock the full potential of this revolution, however, our capacities to disrupt or rewrite small local elements of code by gene editing must be complemented by capabilities to efficiently dock very large DNA cargos with a wide range of functions into genomic sites, by inserting large, custom designed, multifunctional DNAs at base pair precision. Large designer DNA cargoes would carry multicomponent DNA circuitry including programmable and fine-tuneable functionalities and represent the vital interface between gene editing which is the state-of-the-art at present, and genome engineering, which is the future. This challenge remains largely unresolved to date.
The research here proposed tackles precisely this fundamental bottleneck. By bringing to bear an array of sophisticated tools, integrating unique know-how with leading synthetic, biological, chemical and biomedical assets, we will establish new technology to enable highly efficient, easy-to-use docking of custom designed very large multifunctional designer DNA cargos into mammalian genomes with base pair precision, addressing significant unmet academic, biomedical and industrial needs.
During the DNA-DOCK project to date, we had applied a genetic screen to identify regions of our vector genome that can be disposed of for functional SVNs. We validated 67 distinct genes earmarked for elimination in a subsequent screen assaying viral replication and heterologous expression properties. This allowed us to generate an array of functional SVN prototypes with reduced, compacted genomes and enabled us to add modalities to integrate synthetic multifunctional DNA circuitry in particularly well suited and stable spots in the SVN genome. We identified an SVN prototype with enhanced genomic stability and validated this prototype thoroughly in the laboratory. Importantly, during the lockdowns owed tot he COVID-19 crisis, we utilized our SVNs to create a highly efficient low-cost COVID-19 vaccine which efficiently immunizes and neutralizes SARS-CoV-2 in mice. This resulted in a collaboration with a leading vaccine manufacturer in Vietnam to develop a low-cost vaccine candidate that can be locally manufactured. The results of our SVN development are described in a PhD thesis from the DNA-DOCK team which has been successfully defended end of 2021, and peer-reviewed publications from the data in this thesis are being prepared.
Our major objective in DNA-DOCK is to make decisive progress towards ‘minimal-invasive genome surgery’ by using our SVNs, ultimately in a therapeutic context. Towards this end, we have implemented and thoroughly validated CRISPR/Cas HITI and axon replacement, and we integrated cutting edge Base- and PRIME editing modalities in our SVNs. Intriguingly, we now routinely reach very high genomic intervention efficacies (40%) with a single administration of our SVNs in cell culture, also with very large DNA cargoes that cannot be delivered with other currently used vector technology (e.g. AAV or lentivirus). These results resulted in a second PhD thesis that was succesfully defended end of 2021. A corresponding manuscript has been published on a preprint server (bioRXiv) and is currently in final peer review in a high-impact journal. A second publication is being prepared.
Moreover, we established an in vitro Darwinian selection/evolution method, Ribosome Display, in the lab, to select genetically encoded nanobody inhibitors to modulate cell functions with the aim to maximise genomic intervention efficiencies with our SVN approach. Our aim is to suppress transiently mechanisms in cells that counteract productive gene editing. This work is in progress and we aim to integrate such genetically encoded modalities in due course in our SVN approach.
We are preparing to take our SVN approach from bench to bedside. We have established collaborations with gene therapy experts in the health sector to banhmark our SVN approach against currently deployed vector technology (AAV) in patient derived cell lines and animal models, for validating our SVN approach towards the clinic. We have already made significant progress by developing SVNs capable of to repairing a genetic defect in kidney cells. This genetic defect gives rise to steroid-resistant nephrotic syndrome (SRNS), a debilitating hereditary disease affecting children and young adults, severely impacting quality of life, for which a cure remains elusive to date.
Our major objective in DNA-DOCK is to make decisive progress towards ‘minimal-invasive genome surgery’ by using our SVNs, ultimately in a therapeutic context. Towards this end, we have implemented and thoroughly validated CRISPR/Cas HITI and axon replacement, and we integrated cutting edge Base- and PRIME editing modalities in our SVNs. Intriguingly, we now routinely reach very high genomic intervention efficacies (40%) with a single administration of our SVNs in cell culture, also with very large DNA cargoes that cannot be delivered with other currently used vector technology (e.g. AAV or lentivirus). These results resulted in a second PhD thesis that was succesfully defended end of 2021. A corresponding manuscript has been published on a preprint server (bioRXiv) and is currently in final peer review in a high-impact journal. A second publication is being prepared.
Moreover, we established an in vitro Darwinian selection/evolution method, Ribosome Display, in the lab, to select genetically encoded nanobody inhibitors to modulate cell functions with the aim to maximise genomic intervention efficiencies with our SVN approach. Our aim is to suppress transiently mechanisms in cells that counteract productive gene editing. This work is in progress and we aim to integrate such genetically encoded modalities in due course in our SVN approach.
We are preparing to take our SVN approach from bench to bedside. We have established collaborations with gene therapy experts in the health sector to banhmark our SVN approach against currently deployed vector technology (AAV) in patient derived cell lines and animal models, for validating our SVN approach towards the clinic. We have already made significant progress by developing SVNs capable of to repairing a genetic defect in kidney cells. This genetic defect gives rise to steroid-resistant nephrotic syndrome (SRNS), a debilitating hereditary disease affecting children and young adults, severely impacting quality of life, for which a cure remains elusive to date.
In DNA-DOCK, we aim to develop our synthetic virus-derived nanosystems (SVNs) as a bona fide replacement technology for currently used vector technology (AAV, lenti-, adenvirus) to overcome their shortcomings (cost of goods, complicated manufacturing, low DNA payload capacity), to ultimately transform therapeutic genomic intervention (gene therapy 2.0). Towards this end, we have re-engineered and optimized a non-human virus, which does not replicate in humans and is therefore safe. We chose this approach owed to our large experience with this virus in other contexts, and the unprecedented DNA cargo capacity (>100kb) that can be loaded into the virus and faithfully delivered to target cells and organisms. We applied a range of synthetic biology techniques to create SVNs based on this virus, and outfitted our SVNs with cutting-edge CRISPR based functionalities. We thoroughly validated our SVNs and are now in possession of a powerful prototype SVN that can be readied for the clinic. We enlisted world-leading collaborators who have patient derived cells, organoids and animal models, to benchmark and validate our SVNs. We are in a very exciting phase of the DNA-DOCK project as we are approaching critical milestones towards realizing our ambition and unlock, with our SVNs, many hereditary diseases that can currently not be treated, to gene therapy.