Erratum: Development of a gene edited next-generation hematopoietic cell transplant to enable acute myeloid leukemia treatment by solving off-tumor toxicity.

[This corrects the article DOI: 10.1016/j.omtm.2023.101135.].

Development of a gene edited next-generation hematopoietic cell transplant to enable acute myeloid leukemia treatment by solving off-tumor toxicity.

Immunotherapy of acute myeloid leukemia (AML) has been challenging because the lack of tumor-specific antigens results in "on-target, off-tumor" toxicity. To unlock the full potential of AML therapies, we used CRISPR-Cas9 to genetically ablate the myeloid protein CD33 from healthy donor hematopoietic stem and progenitor cells (HSPCs), creating tremtelectogene empogeditemcel (trem-cel). Trem-cel is a HSPC transplant product designed to provide a reconstituted hematopoietic compartment that is resistant to anti-CD33 drug cytotoxicity. Here, we describe preclinical studies and process development of clinical-scale manufacturing of trem-cel. Preclinical data showed proof-of-concept with loss of CD33 surface protein and no impact on myeloid cell differentiation or function. At clinical scale, trem-cel could be manufactured reproducibly, routinely achieving >70% CD33 editing with no effect on cell viability, differentiation, and function. Trem-cel pharmacology studies using mouse xenograft models showed long-term engraftment, multilineage differentiation, and persistence of gene editing. Toxicology assessment revealed no adverse findings, and no significant or reproducible off-target editing events. Importantly, CD33-knockout myeloid cells were resistant to the CD33-targeted agent gemtuzumab ozogamicin in vitro and in vivo. These studies supported the initiation of the first-in-human, multicenter clinical trial evaluating the safety and efficacy of trem-cel in patients with AML (NCT04849910).

Small-molecule screen reveals pathways that regulate C4 secretion in stem cell-derived astrocytes.

In the brain, the complement system plays a crucial role in the immune response and in synaptic elimination during normal development and disease. Here, we sought to identify pathways that modulate the production of complement component 4 (C4), recently associated with an increased risk of schizophrenia. To design a disease-relevant assay, we first developed a rapid and robust 3D protocol capable of producing large numbers of astrocytes from pluripotent cells. Transcriptional profiling of these astrocytes confirmed the homogeneity of this population of dorsal fetal-like astrocytes. Using a novel ELISA-based small-molecule screen, we identified epigenetic regulators, as well as inhibitors of intracellular signaling pathways, able to modulate C4 secretion from astrocytes. We then built a connectivity map to predict and validate additional key regulatory pathways, including one involving c-Jun-kinase. This work provides a foundation for developing therapies for CNS diseases involving the complement cascade.

Molecular convergence between Down syndrome and fragile X syndrome identified using human pluripotent stem cell models.

Down syndrome (DS), driven by an extra copy of chromosome 21 (HSA21), and fragile X syndrome (FXS), driven by loss of the RNA-binding protein FMRP, are two common genetic causes of intellectual disability and autism. Based upon the number of DS-implicated transcripts bound by FMRP, we hypothesize that DS and FXS may share underlying mechanisms. Comparing DS and FXS human pluripotent stem cell (hPSC) and glutamatergic neuron models, we identify increased protein expression of select targets and overlapping transcriptional perturbations. Moreover, acute upregulation of endogenous FMRP in DS patient cells using CRISPRa is sufficient to significantly reduce expression levels of candidate proteins and reverse 40% of global transcriptional perturbations. These results pinpoint specific molecular perturbations shared between DS and FXS that can be leveraged as a strategy for target prioritization; they also provide evidence for the functional relevance of previous associations between FMRP targets and disease-implicated genes.

A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells.

CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional gene studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design and generate new fluorescently labeled piggyBac (PB) vectors to deliver uniform and sustained expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target the TCF4 gene in hPSCs and assess expression levels of the dCas9 effectors, individual gRNAs and targeted gene. We also assess the performance of our PB system for single gRNA delivery, confirming its utility for library format applications. Collectively, our results provide proof-of-principle application of a stable, multiplexed PB gRNA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system can facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.