In Vivo Correction of a Genetically Humanized Fanconi Anemia Mouse Bone Marrow Failure Model Using Digital Editing Technologies

Fanconi anemia (FA) is a rare genetic disease caused by mutations in certain DNA repair genes (i.e. FA genes), whose gene products make up the FA pathway for interstrand crosslink (ICL) repair. FA patients are predisposed to bone marrow failure (BMF) and cancer development. The current standard-of-care for >90% of FA patients that develop BMF or hematopoietic malignancy is allogeneic hematopoietic stem cell transplant (HSCT). However, conditioning toxicities and development of graft-vs-host disease after transplant are associated with increased risks of cancer after age 20 years, particularly in the aero-esophageal tract. Therefore, there is significant interest in the development of an effective and safe gene therapy approach for FA. While the development of CRISPR/Cas9-mediated gene editing has made it possible to induce site-specific double-strand breaks (DSBs) and correct gene mutations through homology-directed repair (HDR), this approach is impeded in FA cells because of their underlying defect in HDR. Furthermore, the number of FA hematopoietic stem and progenitor cells (HSPC) available for ex vivo manipulation is limiting. To overcome these barriers, we will optimize the efficiency of in vivo HSPC correction using newly developed ‘digital’ base editors (BEs) and prime editors (PEs), which do not require DSB repair. For this work, we developed a humanized murine model that harbors the human FANCA Spanish founder mutation c.295 C>T. We have successfully shown that BEs efficiently correct FANCA Spanish founder mutation in patient-derived cells in vitro (PMID: 35955545). To show that in vivo correction of the FANCA c.295 C>T mutation can be accomplished with BEs or PEs and prevent bone marrow failure typical in FA patients, we are modifying the FA mouse model in the 129S4 background that not only harbors the FANCA Spanish founder mutation at Fanca exon 4, but also a mutated alcohol dehydrogenase 5 (Aldh5) which predisposes cells to genotoxic ICLs and subsequent marrow failure. Using these mice, we will measure correction of the FANCA Spanish founder mutation in vivo by adenosine base editor (ABE) or PE delivered by recombinant adeno-associated viruses or lipid nanoparticles. Furthermore, we will assess FA phenotypes after FANCA correction (i.e. bone marrow HSPC population size, function, sensitivity to ICLs, and repopulation capacity). Completion of our studies will generate preclinical data demonstrating proof-of-concept for the use and superiority of in vivo digital gene editing of bone marrow HSPCs for treatment of FA regardless of FA genotype and potentially other congenital marrow failure syndromes.

Moriarity:Luminary Therapeutics: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Webber:Luminary Therapeutics: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Wagner:Magenta Therapeutics: Current equity holder in private company; Vertex Pharmaceuticals: Consultancy; ASC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Garuda Therapeutics: Membership on an entity's Board of Directors or advisory committees.