Gene-edited stem cells enable CD33-directed immune therapy for myeloid malignancies.

Borot F, Wang H, Ma Y, Jafarov T, Raza A, Ali AM, Mukherjee S.
Source: Proc Natl Acad Sci USA
Publication Date: (2019)
Issue: 116(24): 11978-11987
Research Area:
Stem Cells
Gene Expression
Regenerative medicine
Cells used in publication:
CD34+ cell, human
Species: human
Tissue Origin: blood
4D-Nucleofector™ X-Unit

CRISPR/Cas9-Mediated CD33 Genomic Targeting. The TrueCut Cas9 protein V2 was purchased from Invitrogen. The chemically modified sgRNA targeting CD33 were designed using Synthego CRISPR Gene KO design tool and purchased from Synthego. Three micrograms of TrueCut Cas9 protein and 1.5 µg sgRNA for 200,000 CD34+ cells were mixed in P3 buffer (Lonza, Amaxa P3
Primary Cell 4D-Nucleofector Kit) and incubated 10 min at 37 °C. The cells were then washed with PBS, resuspended in P3 buffer, mixed with the Cas9/ sgRNA RNP complex, and then electroporated with the 4D-Nucleofector (program DZ100). After electroporation, cells were cultured at 37 °C until analysis or maintained 48–72 h in vitro and then intravenously injected (5 × 105–1 × 106 per mouse) into sublethally irradiated NSGS mice (The Jackson Laboratory).


Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-Ts) or antibody-drug conjugates (ADCs), are associated with severe toxicities due to the lack of unique targetable antigens that can distinguish leukemic cells from normal myeloid cells or myeloid progenitors. Here, we present an approach to treat AML by targeting the lineage-specific myeloid antigen CD33. Our approach combines CD33-targeted CAR-T cells, or the ADC Gemtuzumab Ozogamicin with the transplantation of hematopoietic stem cells that have been engineered to ablate CD33 expression using genomic engineering methods. We show highly efficient genetic ablation of CD33 antigen using CRISPR/Cas9 technology in human stem/progenitor cells (HSPC) and provide evidence that the deletion of CD33 in HSPC doesn't impair their ability to engraft and to repopulate a functional multilineage hematopoietic system in vivo. Whole-genome sequencing and RNA sequencing analysis revealed no detectable off-target mutagenesis and no loss of functional p53 pathways. Using a human AML cell line (HL-60), we modeled a postremission marrow with minimal residual disease and showed that the transplantation of CD33-ablated HSPCs with CD33-targeted immunotherapy leads to leukemia clearance, without myelosuppression, as demonstrated by the engraftment and recovery of multilineage descendants of CD33-ablated HSPCs. Our study thus contributes to the advancement of targeted immunotherapy and could be replicated in other malignancies.