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.
Public Health Relevance Statement: PROJECT NARRATIVE Gene therapies have the potential to treat the millions of people suffering with a genetic health condition by correcting the underlying genetic problem. Better, more modular and easily programmable molecular vehicles for carrying and delivering genetic repair machinery would help researchers develop such treatments faster and more affordably. In this project, we will develop and test a new class of such programmable carriers for gene therapy treatments and a nanoengineering platform for rapidly and reliably producing them.
Project Terms: Affect; Algorithms; Biological Sciences; Biologic Sciences; Bioscience; Life Sciences; Bone Marrow; Bone Marrow Reticuloendothelial System; Capital; capsule; Capsules; Carrying Capacities; Cell Line; CellLine; Strains Cell Lines; cultured cell line; Cells; Cell Body; Charge; Disease; Disorder; DNA; Deoxyribonucleic Acid; Elements; Engineering; Face; faces; facial; Flow Cytometry; Flow Cytofluorometries; Flow Cytofluorometry; Flow Microfluorimetry; Flow Microfluorometry; flow cytophotometry; Gene Expression; gene therapy; DNA Therapy; Gene Transfer Clinical; Genetic Intervention; gene repair therapy; gene-based therapy; genetic therapy; genomic therapy; Genes; Genome; Goals; Health; Marketing; Methods; Persons; Nucleic Acids; Patients; Peptides; Polymers; polymer; polymeric; Production; Endosomes; Receptosomes; Research; Research Personnel; Investigators; Researchers; Safety; Software Design; Designing computer software; Specificity; Medical Technology; Testing; Vendor; Virus; Insertional Mutagenesis; falls; Computer-Assisted Design; Computer-Aided Design; Label; improved; Site; Surface; Clinical; Encapsulated; repair; repaired; Phase; Medical; electroporative delivery; gene electrotransfer; Electroporation; Endothelial Cells; Confocal Microscopy; transgene; Transgenes; Immunological response; host response; immune system response; immunoresponse; Immune response; Genetic; Shapes; Inflammatory; Reporter; Machine Learning; machine based learning; Jurkat Cells; Complex; System; Nuclear; Services; particle; fluorophore; Rare Diseases; Orphan Disease; Rare Disorder; orphan disorder; Structure; novel; Human Cell Line; Reporting; Nanotechnology; nano tech; nano technology; nano-technological; nanotech; nanotechnological; Genomics; Effectiveness; virus-like nanoparticles; viruslike particle; Virus-like particle; Dose; Nanostructures; nano-sized structures; nano-structures; Therapeutic Studies; Therapy Research; in vivo; Gene Transfer; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Viral Vector; Molecular; Gene Delivery; vector; human stem cells; designing; design; nanofabricate; nanofabrication; nanovessel; nanocarrier; nano engineering; nanoengineering; Outcome; scale up; Heritability; nonviral gene delivery; non-viral gene delivery; nonviral gene therapy; non-viral gene therapy; commercial application; commercialization; FDA approved; operations; operation; CRISPR; CRISPR/Cas system; Clustered Regularly Interspaced Short Palindromic Repeats; genomic editing; genome editing; CRISPR approach; CRISPR based approach; CRISPR method; CRISPR methodology; CRISPR technique; CRISPR technology; CRISPR tools; CRISPR-CAS-9; CRISPR-based method; CRISPR-based technique; CRISPR-based technology; CRISPR-based tool; CRISPR/CAS approach; CRISPR/Cas method; CRISPR/Cas9; CRISPR/Cas9 technology; Cas nuclease technology; Clustered Regularly Interspaced Short Palindromic Repeats approach; Clustered Regularly Interspaced Short Palindromic Repeats method; Clustered Regularly Interspaced Short Palindromic Repeats methodology; Clustered Regularly Interspaced Short Palindromic Repeats technique; Clustered Regularly Interspaced Short Palindromic Repeats technology; CRISPR/Cas technology; knockin; Knock-in; clinically translatable; clinical translation; genetic condition; genetic disorder; Genetic Diseases; gene-editing therapy; genome editing based therapy; genome editing therapy; genome editing treatment; genome editing-based therapeutics; therapeutic editing; therapeutic genome editing; lipid based nanoparticle; lipid nanoparticle; rare genetic disease; rare genetic disorder; prime editing; base editing; delivery vehicle; delivery vector; manufacturing cost; fabrication cost
