Periodic Reporting for period 2 - GENESHUTTLE (What does it take to build an artificial virus?)
Reporting period: 2023-11-01 to 2025-04-30
DNA-Based Nanocarriers for Advanced Gene Delivery and Therapeutic Applications
The project aims at creating synthetic virus-like assemblies capable of accomplishing cell-invading and gene expression functionalities known so far only from natural viruses.The primary objectives of this project are to develop DNA-based shells—both non-enveloped and enveloped—for advanced gene delivery, membrane fusion, and programmed cargo release. These shells integrate functional elements such as antibodies, ligands, and pH-responsive motifs to enable efficient cellular uptake and endosomal escape. To achieve these objectives, we set out to recreate and experimentally test mechanisms believed to be used by viruses, including receptor-mediated endocytosis, stimulus-dependent lipid membrane penetration, membrane fusion, active cytosolic transport, and nuclear import. The project promises to yield a gene delivery system with capabilities beyond current synthetic gene delivery vectors, which struggle to overcome the many cellular barriers to deliver and express genetic cargo. For safety reasons, the gene shuttle will by design be unable to assemble in the context of a cell to prevent uncontrolled autonomous replication.
The project aims at creating synthetic virus-like assemblies capable of accomplishing cell-invading and gene expression functionalities known so far only from natural viruses.The primary objectives of this project are to develop DNA-based shells—both non-enveloped and enveloped—for advanced gene delivery, membrane fusion, and programmed cargo release. These shells integrate functional elements such as antibodies, ligands, and pH-responsive motifs to enable efficient cellular uptake and endosomal escape. To achieve these objectives, we set out to recreate and experimentally test mechanisms believed to be used by viruses, including receptor-mediated endocytosis, stimulus-dependent lipid membrane penetration, membrane fusion, active cytosolic transport, and nuclear import. The project promises to yield a gene delivery system with capabilities beyond current synthetic gene delivery vectors, which struggle to overcome the many cellular barriers to deliver and express genetic cargo. For safety reasons, the gene shuttle will by design be unable to assemble in the context of a cell to prevent uncontrolled autonomous replication.
This project has indeed successfully developed prototypes of DNA-based delivery systems inspired by viral mechanisms to enable efficient gene delivery, controlled membrane fusion, and programmed cargo release. The team created both non-enveloped and lipid-enveloped nanoscale shells constructed from rationally designed DNA components, incorporating functional elements such as antibodies, ligands, and pH-responsive segments that facilitate targeted cellular uptake and endosomal escape. Early results demonstrated that DNA origami structures can deliver and express genes in mammalian cells, thereby confirming the fundamental premise of this work. Moreover, newly developed DNA constructs containing nuclear targeting sequences have enabled active import into the nucleus—even in non-dividing cells—broadening the scope for successful transfection.
Another significant outcome is the design of cholesterol-modified DNA triangles that self-assemble into polyhedral shells on lipid vesicles. This capability induces membrane budding in a process reminiscent of natural viral pathways and suggests a promising route for constructing membrane-enveloped nanocarriers. Concurrently, researchers established triple-stranded DNA (triplex) as a reliable, pH-sensitive motif to drive controlled conformational changes, thereby allowing cargo release in acidic environments such as endosomes. A newly introduced computational protein design pipeline also provides a foundation for creating hybrid nanostructures with enhanced functionality, stability, and specificity.
By integrating DNA nanotechnology, membrane engineering, and protein design, the project is advancing the state of the art in multifunctional gene delivery. These DNA-based carriers demonstrate a potential for targeted drug and gene delivery with programmable cargo release in specific intracellular compartments, and biomimetic approaches to membrane manipulation with wide-ranging applications in synthetic biology. Overall, this effort not only points toward new opportunities in therapeutic development but also offers insights into cellular processes and the future of gene delivery technologies.
Another significant outcome is the design of cholesterol-modified DNA triangles that self-assemble into polyhedral shells on lipid vesicles. This capability induces membrane budding in a process reminiscent of natural viral pathways and suggests a promising route for constructing membrane-enveloped nanocarriers. Concurrently, researchers established triple-stranded DNA (triplex) as a reliable, pH-sensitive motif to drive controlled conformational changes, thereby allowing cargo release in acidic environments such as endosomes. A newly introduced computational protein design pipeline also provides a foundation for creating hybrid nanostructures with enhanced functionality, stability, and specificity.
By integrating DNA nanotechnology, membrane engineering, and protein design, the project is advancing the state of the art in multifunctional gene delivery. These DNA-based carriers demonstrate a potential for targeted drug and gene delivery with programmable cargo release in specific intracellular compartments, and biomimetic approaches to membrane manipulation with wide-ranging applications in synthetic biology. Overall, this effort not only points toward new opportunities in therapeutic development but also offers insights into cellular processes and the future of gene delivery technologies.
Over the course of this project, we have developed and implemented a new AI-supported de novo protein design pipeline (Frank et al., Science 2024), which makes it possible to construct unpredecently large custom proteins with high design accuracy. We expect this approach to enable combining de novo protein design with DNA nanotechnology to leverage the distinct strengths of both molecular frameworks. DNA origami contributes a rigid, programmable scaffold, while designed proteins introduce highly specific binding sites, enzymatic functions, or increased thermal stability. By precisely positioning these proteins on DNA scaffolds, we may achieve multivalent interactions with cells and can selectively incorporate catalytic modules. This synergy has the potential to enable co-delivery of DNA and protein therapeutics in a single carrier, outclassing conventional single-platform systems in robustness and versatility.
Prior work predominantly focused on either DNA- or protein-based carriers, but the project’s integration of pH-responsive DNA motifs with sophisticated membrane fusion strategies and fully customized protein scaffolds offers an unprecedented degree of control over target recognition, cargo release, and intracellular trafficking. The high efficiency of nuclear import observed in arrested cells (Liedl et al., JACS 2023) was an unanticipated outcome, indicating new opportunities for transfection in otherwise challenging cell populations. Another noteworthy surprise involved the rapid kinetics of membrane budding enabled by cholesterol-modified origami shells (Pinner et al., under review). Experimental data show that geometry-optimized DNA assemblies can efficiently drive membrane remodeling. Until the end of the project we expect to make significant strides toward improving bottleneck steps such as endosomal escape and trafficking to the nucleus.
Overall, these findings extend well beyond previous achievements in gene delivery, membrane engineering, and synthetic biology. Going forward, the project’s hybrid design paradigm, uniting protein functionalities with carefully assembled DNA architectures, will provide a powerful new toolbox for targeted delivery and triggered release but also sheds light on fundamental processes like membrane remodeling and nuclear transport. In doing so, it opens up new possibilities for precisely engineered therapeutic vehicles that surpass the limitations of earlier generation platforms, setting a precedent for advanced cellular applications.
Prior work predominantly focused on either DNA- or protein-based carriers, but the project’s integration of pH-responsive DNA motifs with sophisticated membrane fusion strategies and fully customized protein scaffolds offers an unprecedented degree of control over target recognition, cargo release, and intracellular trafficking. The high efficiency of nuclear import observed in arrested cells (Liedl et al., JACS 2023) was an unanticipated outcome, indicating new opportunities for transfection in otherwise challenging cell populations. Another noteworthy surprise involved the rapid kinetics of membrane budding enabled by cholesterol-modified origami shells (Pinner et al., under review). Experimental data show that geometry-optimized DNA assemblies can efficiently drive membrane remodeling. Until the end of the project we expect to make significant strides toward improving bottleneck steps such as endosomal escape and trafficking to the nucleus.
Overall, these findings extend well beyond previous achievements in gene delivery, membrane engineering, and synthetic biology. Going forward, the project’s hybrid design paradigm, uniting protein functionalities with carefully assembled DNA architectures, will provide a powerful new toolbox for targeted delivery and triggered release but also sheds light on fundamental processes like membrane remodeling and nuclear transport. In doing so, it opens up new possibilities for precisely engineered therapeutic vehicles that surpass the limitations of earlier generation platforms, setting a precedent for advanced cellular applications.