Prime Editing Enables Precise and Efficient Single Amino Acid Substitutions to Shield CD34+ Hematopoietic Stem Cells from Anti-CD117 Antibody-Based Conditioning

Hematopoietic stem cell transplantation (HSCT) requires conditioning to create space for the incoming graft. Conventional conditioning of chemotherapy or radiation is effective but causes multi-organ damage and risks treatment-associated morbidity and mortality. In previous nonclinical studies, antibody (Ab)-based conditioning has been shown to have reduced toxicity and may reduce cytopenia compared to conventional conditioning to improve HSCT outcomes.

CD117, the c-KIT receptor tyrosine kinase (encoded by KIT gene), is highly expressed on HSCs. The stem cell factor / CD117 signaling axis is required for HSC survival, proliferation, and differentiation. CD117 Ab depletes wild-type HSCs and could be used for conditioning. We have previously shown that installing a point mutation in KIT protects HSCs from CD117 Ab-mediated depletion while preserving c-KIT function (a ‘shielding edit’) (Marone et al., Blood (2022) 140 (Supplement 1): 4493–4494). In the context of gene corrected autologous HSCT, CD117 Ab treatment may deplete unedited cells and enrich shielded corrected cells. CRISPR-nuclease homology directed-repair (HDR)-mediated editing is not ideal for shielding edit installation due to low HDR efficiency and high unintended edits introduced through DNA double stranded breaks (DSBs). In contrast, Prime Editing can introduce precise edits without DSBs and may be tailored for potentially safer yet effective installation of shielding edits.

Prime Editors that introduce a shielding edit were designed and tested in human CD34+ cells (n=3 donors), which were evaluated for multipotency in colony forming cell (CFC) assays. Here, 78 ± 6 % of Prime Edited (PE) CD34+ cell CFCs contained the shielding edit, 50% of which contained 2 shielding KIT alleles/cell. No unintended edits were detected (0%). PE CD34+ cells were protected from anti-CD117 Ab CIM056 mediated depletion, forming erythroid, myeloid and mixed erythro-myeloid colonies, with 77 ± 6% of CD34+ cells shielded, similar to the % of cells with a shielding edit that were detected in the absence of CIM056. DNA sequencing of colonies derived from PE cells in presence of CIM056 confirmed depletion of the unedited cell fraction. Mock treated CFCs exposed to CD117 Ab lost multipotency with only small myeloid colonies detected and 32% of the number of colonies vs. control Ab. These findings show that shielded CD34+ cells maintain multipotency in the presence of CIM056. Optimization increased Prime Editing to 98% in CD34+ cells, 76% of which contained 2 shielding KIT alleles/cell. Use of shielding in lieu of toxic conditioning can support in vivo selection in context of a gene corrected autologous HSCT therapy. However, this would require two Prime Editors to install a shielding edit and create a Prime Edit at a therapeutically relevant gene target site to restore gene expression. This ‘multiplex’ approach was tested with Prime Editors targeting KIT and a proof-of-concept (PoC) gene. Ninety six percent (96%) of PE CD34+ cell CFCs had a shielding edit and within this population 93% of cells had the desired edit at the PoC site.

Toward development of a shielded PE CD34+ cell drug product, a clinical scale manufacturing cell process was developed. Prime Editing efficiencies at clinical scale and research scale were similar. In the clinical PE CD34+ cell batch produced with unoptimized Prime Editors, 73% of cells contained a shielding edit and ~50% of these cells had 2 shielding KIT alleles/cell. The shielded PE CD34+ cells produced at clinical scale were infused into immunodeficient mice that were previously engrafted with wild-type CD34+ cells and treated with anti-CD117 Ab before infusion of the PE CD34+ cells from the same donor to model Ab conditioning. Long-term engraftment, LT-HSC functionality, and in vivo selection after treatment with CD117 Ab are currently being evaluated. In summary, these results show that Prime Editing precisely installs a shielding mutation in the KIT gene in CD34+ cells with very low unintended edits, CD117 Ab depletes unedited CD34+ cells but does not deplete shielded PE CD34+ cells, and PE CD34+ cells exposed to CD117 Ab retain multipotency. Shielded PE CD34+ cells made at clinical scale achieved efficient Prime Editing with no impact on viability or multipotency. These findings indicate that Prime Editing can potentially be used to develop a shielded PE CD34+ product in combination with CD117 Ab conditioning and in vivo selection.