Active non-viral nuclear delivery of DNA templates independent of mitosis

PROJECT SUMMARY Progress in genomic medicine is hampered by safety, toxicity, immunogenicity and cargo capacity/size concerns inherent to the use of lentiviral and retroviral methods. Non-viral methods of gene delivery are highly attractive to meet the demand for effective, scalable gene therapies and largely address insertional mutagenesis; however, nuclear delivery after endosomal escape is a large bottleneck that needs to be overcome. Lipid nanoparticles are clinically approved for delivery of nucleic acids, but payloads are largely limited to the cytosol, with very modest nuclear entry. To address the nuclear delivery bottleneck, Kano Therapeutics’ proposed solution utilizes gene repair templates with DNA targeting sequences, designed by our team of experts using our proprietary in silico platform to enable nuclear entry, achieve efficient integration, and minimize potential immunogenicity. Kano Therapeutics’ fully custom, circular single strand DNA (cssDNA) is an optimal payload for gene editing, with high integration efficiency and minimal off-target effects. Our pipeline for fully custom cssDNA production enables production and proven integration of kb-size templates, which can revolutionize the field of genomic medicine and lead to permanent cures of monogenetic disorders. Our nucleic acid engineering know-how, together with a highly scalable and low-cost patented manufacturing process, makes us hopeful that we can drive progress in the gene editing field for a variety of indications. In this Fast-Track application we aim to thoroughly investigate active nuclear delivery independent of mitosis and demonstrate proof-of-concept of our platform for the treatment of X-linked severe combined immunodeficiency (X-SCID), a rare monogenic disorder caused by mutations in the IL2RG gene. X-SCID affects 1 in 58,000 births and is rapidly fatal if untreated; current treatments like HSCT offer only a 70% success rate for long-term immune reconstitution, and even then, significant complications can arise. Despite advances in clinical methodologies, effective, safe treatments are still lacking for X-SCID and many other diseases with genetic origins. In Phase I, we will (1) identify and validate DNA targeting sequence motifs for incorporation to cssDNA that will enable efficient nuclear targeting, and (2) demonstrate and optimize nuclear delivery of custom cssDNA payloads into established human cell lines. In Phase II we will turn our focus to X- SCID and (1) validate nuclear delivery of cssDNA-LNPs into clinically relevant CD34+ hematopoietic stem and progenitor cells (HSPCs), and (2) successfully insert full-length IL2RG gene using a genomic nuclease system and confirm on-target integration. Lastly (3), we will demonstrate the safety and efficacy of this therapeutic approach in an in vivo murine X-SCID model. A successful outcome of this project will lead to the establishment of a proof-of-concept for our gene editing platform both in vitro and in vivo, and lay the foundation for an improved gene therapy for X-SCID, as well as additional monogenic diseases.