A nanoengineering platform for programmable gene editing therapies against rare diseases

PROJECT SUMMARY More than 300 million people worldwide are affected by a genetic health condition. Over 4,400 genetic diseases have been identified; nearly all of which are considered rare, which limits the amount of research each receives. Gene therapy is an attractive approach for treatment of genetic disease because of its versatility and broad applicability. Genome editing systems such as CRISPR-Cas9, base editing and prime editing have revolutionized gene therapy research and other fields of life science, however, few gene editing treatments have reached the market and clinical translation still faces important challenges. Among them is the need for safe and effective gene therapy delivery vehicles and platforms for their creation. In this project, we will design and test a new class of programmable, non-viral gene therapy carriers and cargo – virus-inspired DNA origami (VIDO) vectors and repair templates – and Essemblix GT, a nanoengineering platform tailored for their production. In contrast to other gene therapy delivery vehicles, VIDO products are modular and easily modified for different diseases. Moreover, they are structurally well-defined with little intermolecular variability, facilitating regulatory approval and clinical translation. To our knowledge, this will be the first project to investigate the use of DNA origami for encapsulation and delivery of gene editing agents. In Aim 1, we will demonstrate that CRISPR-Cas9 knock-in efficiency is improved by folding and compacting homology-directed repair (HDR) templates with DNA origami methods. VIDO-folded reporter templates will be compared against unstructured controls when delivered via electroporation to HEK293T and Jurkat human cell lines at two different genome insertion sites. Nuclear entry will be determined by confocal microscopy of fluorophore-labeled template and knock-in efficiency will be assessed by flow cytometry. In Aim 2, using the same cell lines and genomic targets, we will demonstrate VIDO vectors can encapsulate and co-deliver CRISPR-Cas9 editing agents and VIDO templates, are readily taken up by cells and induce knock-in efficiency that is competitive with delivery of the same agents via virus-like particles (VLP). Endosomal escape and gene expression will be tracked via confocal microscopy and flow cytometry. In both aims, correct genomic integration will be confirmed via Illumina sequencing. Successful completion of these aims will establish VIDO vectors and templates as new, programmable gene therapy products with key advantages over existing alternatives. By making it practical to rapidly design and create such VIDO products, the Essemblix GT nanoengineering platform could shift gene therapy research toward a paradigm of gene therapy engineering, thus enabling researchers to deliver more treatments for rare diseases to more patients more quickly.