Abstract
A major hurdle to the successful application of autologous hematopoietic stem cell transplant (HSCT) gene therapy, a potential treatment for a variety of hemoglobinopathies including sickle cell disease (SCD), is risk of severe adverse effects associated with use of the chemotherapeutic drug busulfan for pre-transplant myeloablative conditioning. Aiming to overcome this hurdle, we developed a non-genotoxic investigational therapy for hemoglobinopathies that combines multi-plex base edited engineered HSCs (eHSCs) with antibody-based conditioning.
Our engineered stem cell antibody paired evasion (ESCAPE) strategy consists of a multiplex base-edited eHSC that includes, a therapeutic edit at the promoter region of HBG1/2, and a missense mutation in the extracellular domain of CD117 (cKIT), a receptor tyrosine kinase expressed by hematopoietic stem and progenitor cells (HSPCs) that regulates HSPC survival, proliferation, and differentiation. While the HBG1/2 base edit enables upregulation of fetal hemoglobin, the CD117 base edit leads to loss of recognition by a monoclonal antibody (mAb) identified to bind orthogonally to wild-type (WT) CD117, thus allowing for a myeloablative conditioning that specifically targets the diseased unedited bone marrow HSCs and retention of base edited eHSCs.
We achieved ~80% bi-allelic CD117 editing and near complete editing of the HBG1/2 locus in HSCs. Single clonal analysis of CD34+ HSPCs multiplex-edited by ex vivo electroporation of mRNA and sgRNAs revealed that each cell that contains a CD117 edit, also contains the HBG1/2 edit, confirming highly efficient multi-plex editing.
Additionally, we compared ligand binding, phosphorylation, and internalization of our modified CD117 to wild type CD117, in vitro. Results indicate that our CD117 variant protein binds normally to SCF, undergoes similar levels of phosphorylation (compared to WT protein), and is internalized normally after SCF binding. We compared edited and unedited primary CD34+ HSPCs in in vitro erythroid differentiation (IVED) and CFU assays and observed that editing CD117 in the HSPCs does not alter HSPCs ability to differentiate into myeloid or erythroid lineages. Xenotransplantation studies demonstrate that CD117-edited HSPCs are capable of long-term multi-lineage hematopoietic engraftment in immunocompromised mice.
Our lead anti-CD117 monoclonal antibody, mAb7, binds with high affinity to unedited HSPCs but not to CD117-edited cells. mAb7 binding to CD117 blocks SCF-CD117 interaction thereby selectively suppressing survival of unedited HSPCs. Treatment of HSPCs with mAb7 in vitro resulted in >85% reduction in viability of unedited HSPCs, while CD117-edited cells remained unaffected.
Since CD117 signaling has been linked with mast cell degranulation, we evaluated effects of native and Fc-engineered versions of mAb7 on in vitro culture-differentiated mast cells. Our results indicate that Fc-engineered versions of mAb7 do not lead to any level of mast cell degranulation in vitro.
Altogether, our ESCAPE strategy may enable less toxic pre-transplant conditioning for autologous HSC-based SCD therapies, while minimizing treatment-related toxicities that arise from current busulfan-based conditioning.
Disclosures
Mondal:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Harmon:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Budak:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhang:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wong:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rinaldi:Beam Theraputics: Current Employment, Current equity holder in publicly-traded company. Olins:Beam Theraputics: Current Employment, Current equity holder in publicly-traded company. Hoar:Beam Theraputics: Current Employment, Current equity holder in publicly-traded company. Menon:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. White:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Musenge:Beam Theraputics: Current Employment, Current equity holder in publicly-traded company. Camblin:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wolin:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Coisman:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bai:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Qiao:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Patel:Beam Theraputics: Current Employment, Current equity holder in publicly-traded company. Decker:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gantzer:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Leete:Beam Theraputics: Current Employment, Current equity holder in publicly-traded company. Yu:Beam Theraputics: Current Employment, Current equity holder in publicly-traded company. Bohnuud:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Smith:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonagh:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gaudelli:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chu:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hartigan:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ciaramella:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company.
Author notes
Asterisk with author names denotes non-ASH members.
