Efficient long-term multilineage engraftment of CD33-edited hematopoietic stem/progenitor cells in nonhuman primates

Abstract

Current immunotherapeutic targets are often shared between neoplastic and normal hematopoietic stem and progenitor cells (HSPCs), leading to unwanted on-target, off-tumor toxicities. Deletion or modification of such targets to protect normal HSPCs is, therefore, of great interest. Although HSPC modifications commonly aim to mimic naturally occurring phenotypes, the long-term persistence and safety of gene-edited cells need to be evaluated. Here, we deleted the V-set domain of CD33, the immune-dominant domain targeted by most anti-CD33 antibodies used to treat CD33-positive malignancies, including acute myeloid leukemia, in the HSPCs of two rhesus macaques, performed autologous transplantation after myeloablative conditioning, and followed the animals for up to 3 years. CD33-edited HSPCs engrafted without any delay in recovery of neutrophils, the primary cell type expressing CD33. No impact on the blood composition, reconstitution of the bone marrow stem cell compartment, or myeloid differentiation potential was observed. Up to 20% long-term gene editing in HSPCs and blood cell lineages was seen with robust loss of CD33 detection on myeloid lineages. In conclusion, deletion of the V-set domain of CD33 on HSPCs, progenitors, and myeloid lineages did not show any adverse effects on their homing and engraftment potential or the differentiation and functionality of myeloid progenitors and lineages.

Keywords: CD33; CRISPR; HSCs; NHP; autologous transplantation; gene therapy and editing; hematopoietic stem cells; non-human primate.

Graphical abstract

Figure 1

Efficient deletion of CD33 in NHP CD34⁺ cells without impacting the in vitro differentiation potential (A) Experimental workflow for the CRISPR-Cas9-mediated deletion of CD33. (B and C) Loss of CD33 signal on CD34⁺ cells 7 days post-editing measured by flow cytometry. (D) Colony-forming potential of gene-modified CD34⁺ cells and FACS-purified CD34-subsets enriched for HSCs (CD34⁺CD90⁺), EMPs (CD34⁺CD90^–), and LMPs (CD34⁺CD45RA⁺). BFU, burst-forming unit; E, erythrocyte; G, granulocytes; GM, granulocyte-macrophage/monocyte; M, macrophages/monocytes; MIX, erythro-myeloid colony.

Figure 2

Hematopoietic recovery of the PB after transplant (A–D) Neutrophil, platelet, white blood cell (WBC), and lymphocyte counts measured by complete blood cell counts. Horizontal dotted lines indicate range of normal as determined by historical data as well as published values. (E) Phenotypic composition of the PB measured by flow cytometry.

Figure 3

Long-term persisting multi-lineage gene-editing in the PB (A) Loss of CD33 signal on PB granulocytes measured with flow cytometry. (B) Genomic deletion of CD33 measured by ddPCR. (C) CD33 deletion efficiency in FACS-purified PB lineages measured by ddPCR at 1 year post transplant.

Figure 4

Long-term engraftment of CD33-edited HSPCs in the BM (A) Flow-cytometric assessment of the recovery of the BM stem cell compartment and loss of CD33 on the surface of CD34⁺ cells 3 years post transplant. (B) Quantification of CD33 editing by ddPCR 1 year and 3 years post transplant.

Figure 5

CFC potential of CD33^ΔE2 HSPCs in the BM stem cell compartment (A) (top) Total CFC potential and (bottom) erythro-myeloid differentiation potential of HSPCs 1 year and 3 years post-transplant. BFU-E, burst-forming unit erythrocyte; G, granulocytes; GM, granulocytes-macrophage/monocytes; GEMM, granulocytes-erythrocytes-monocytes-megakaryocytes; M, macrophage/monocytes. (B) Representative PCR-based assessment of CD33 deletion in individual colonies. ΔE2, CD33^ΔE2; WT, wildtype. (C) Quantification of colonies with WT CD33 or mono-/biallelic editing of CD33^ΔE2.