In vivo hematopoietic stem cell gene therapy in a novel genetically humanized murine model of SCID-A

Project Summary/Abstract A founder mutation in the ARTEMIS gene resulting in Severe Combined Immunodeficiency (SCID-A) is common in the Athabascan-speaking Navajo and Apache populations (a minority group) in the United States. One in 2,000 live births in this community have this devastating genetic disorder, resulting in a complete lack of mature T and B lymphocytes. The current standard of care for these patients is a bone marrow transplant, which often does not result in B cell reconstitution, carries risk of graft versus host disease, and necessitates harsh preconditioning regimens. Clinical trials are ongoing for ex vivo gene therapy using a lentiviral vector to insert additional copies of ARTEMIS in patient hematopoietic stem cells. We are pursuing a more targeted treatment using Adenine Base Editor (ABE) to convert two bases of ARTEMIS without causing double-stranded breaks in the genome. ABE can edit this mutation with a conservative amino acid substitution, replacing the premature stop codon. The effect of this edit on hematopoietic stem cells' immune reconstitution ability has not yet been examined. This proposed work aims to validate ABE conservative substitution as a viable therapy for SCID-A and establish in vivo delivery methods to hematopoietic stem/progenitor cells (HSPCs) for immune reconstitution using a novel, genetically humanized SCID-A mouse model that we recently developed. Aim 1 will characterize the efficacy and safety of the conservative substitution strategy with multiple in vitro Artemis assays in both a K562 SCID-A cell line and SCID-A murine hematopoietic stem cells. Aim 2 explores in vivo delivery of therapeutic guide RNA and base editor mRNA to HSPCs in our SCID-A mouse model. Novel cationic polymer micelles from the Reineke group will be tested in vivo along with recombinant adeno-associated virus (rAAV) serotype 6 to establish the feasibility of immune reconstitution by edited HSPCs in vivo. This work will lay the groundwork for potential in vivo base editor therapy clinical trials to treat SCID-A patients. This project describes the work that Ella will perform throughout the remainder of graduate studies. During the fellowship period, Ella will also undertake a career development plan to prepare for a future career as an independent researcher at an academic institution. This includes support from numerous collaborators to accomplish the research objectives, and training in new areas of biological research, such as polymer complex delivery methods, rAAV production, and rigorous in vivo studies to assess the efficacy of SCID-A treatment using ABE. These experiences, along with programming from the UMN’s Office of Graduate and Postdoctoral Studies will provide Ella with the skills and knowledge necessary to achieve her career goals and desire to perform impactful translational research.