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.

Nascent-polypeptide-associated complex and coactivator alpha (␣NAC) is a protein shuttling between the nucleus and the cytoplasm. Upon phosphorylation at residue serine 43 by integrin-linked kinase, ␣NAC is translocated to the nuclei of osteoblasts, where it acts as an AP-1 coactivator to increase osteocalcin gene transcription. To determine the physiological role of nuclear ␣NAC, we engineered a knock-in mouse model with a serine-to-alanine mutation at position 43 (S43A). The S43A mutation resulted in a decrease in the amount of nuclear ␣NAC with reduced osteocalcin gene promoter occupancy, leading to a significant decrease in osteocalcin gene transcription. The S43A mutant bones also expressed decreased levels of ␣ 1 (I) collagen mRNA and as a consequence had significantly less osteoid tissue. Transient transfection assays and chromatin immunoprecipitation confirmed the ␣ 1 (I) collagen gene as a novel ␣NAC target. The reduced quantity of bone matrix in S43A mutant bones was mineralized faster, as demonstrated by the significantly reduced mineralization lag time, producing a lower volume of immature, woven-type bone characterized by poor lamellation and an increase in the number of osteocytes. Accordingly, the expression of the osteocyte differentiation marker genes DMP-1 (dentin matrix protein 1), E11, and SOST (sclerostin) was increased. The accelerated mineralization phenotype was cell autonomous, as osteoblasts isolated from the calvaria of S43A mutant mice mineralized their matrix faster than did wild-type cells. Thus, inhibition of ␣NAC nuclear translocation results in an osteopenic phenotype caused by reduced expression of osteocalcin and type I collagen, accelerated mineralization, and immature woven-bone formation.

Abnormalities in folate and/or homocysteine metabolism may adversely influence embryonic development, leading to the birth of infants with a variety of congenital malformations, including neural tube defects (NTDs) and craniofacial abnormalities. Based upon suggestive evidence that periconceptional folic acid supplementation is effective in preventing a significant proportion of the aforementioned birth defects, genetic variation in the folate biosynthetic pathways may influence the infant's susceptibility to these birth defects. The goal of our study was to investigate sequence variations in the betaine-homocysteine methyltransferase (BHMT) and betaine-homocysteine methyltransferase (BHMT2) genes as modifiers of risk of spina bifida, cleft palate, and cleft lip and palate. The results of this study indicated that individuals homozygous for the single nucleotide polymorphism R239Q in BHMT did not have elevated risks for spina bifida. Genotype frequencies for the BHMT2 rs626105 polymorphism also did not reveal any elevated risks for spina bifida, and only a modest, imprecise elevation of risk for orofacial clefts. The results of these experiments suggest that variants of the BHMT/BHMT2 genes in infants do not substantially contribute to the risk of spina bifida or orofacial clefts in our study population.