NOTCH1 signaling during CD4+ T-cell activation alters transcription factor networks and enhances antigen responsiveness.

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR-T) effectively treats refractory hematologic malignancies in a subset of patients but can be limited by poor T-cell expansion and persistence in vivo. Less differentiated T-cell states correlate with the capacity of CAR-T to proliferate and mediate antitumor responses, and interventions that limit tumor-specific T-cell differentiation during ex vivo manufacturing enhance efficacy. NOTCH signaling is involved in fate decisions across diverse cell lineages and in memory CD8+ T cells was reported to upregulate the transcription factor FOXM1, attenuate differentiation, and enhance proliferation and antitumor efficacy in vivo. Here, we used a cell-free culture system to provide an agonistic NOTCH1 signal during naïve CD4+ T-cell activation and CAR-T production and studied the effects on differentiation, transcription factor expression, cytokine production, and responses to tumor. NOTCH1 agonism efficiently induced a stem cell memory phenotype in CAR-T derived from naïve but not memory CD4+ T cells and upregulated expression of AhR and c-MAF, driving heightened production of interleukin-22, interleukin-10, and granzyme B. NOTCH1-agonized CD4+ CAR-T demonstrated enhanced antigen responsiveness and proliferated to strikingly higher frequencies in mice bearing human lymphoma xenografts. NOTCH1-agonized CD4+ CAR-T also provided superior help to cotransferred CD8+ CAR-T, driving improved expansion and curative antitumor responses in vivo at low CAR-T doses. Our data expand the mechanisms by which NOTCH can shape CD4+ T-cell behavior and demonstrate that activating NOTCH1 signaling during genetic modification ex vivo is a potential strategy for enhancing the function of T cells engineered with tumor-targeting receptors.

CRISPR/Cas9 ß-globin gene targeting in human haematopoietic stem cells.

The ß-haemoglobinopathies, such as sickle cell disease and ß-thalassaemia, are caused by mutations in the ß-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure ß-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult ß-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for ß-haemoglobinopathies.

TIM-3+ CD8 T cells with a terminally exhausted phenotype retain functional capacity in hematological malignancies.

Chronic antigen stimulation is thought to generate dysfunctional CD8 T cells. Here, we identify a CD8 T cell subset in the bone marrow tumor microenvironment that, despite an apparent terminally exhausted phenotype (TPHEX), expressed granzymes, perforin, and IFN-γ. Concurrent gene expression and DNA accessibility revealed that genes encoding these functional proteins correlated with BATF expression and motif accessibility. IFN-γ+ TPHEX effectively killed myeloma with comparable efficacy to transitory effectors, and disease progression correlated with numerical deficits in IFN-γ+ TPHEX. We also observed IFN-γ+ TPHEX within CD19-targeted chimeric antigen receptor T cells, which killed CD19+ leukemia cells. An IFN-γ+ TPHEX gene signature was recapitulated in TEX cells from human cancers, including myeloma and lymphoma. Here, we characterize a TEX subset in hematological malignancies that paradoxically retains function and is distinct from dysfunctional TEX found in chronic viral infections. Thus, IFN-γ+ TPHEX represent a potential target for immunotherapy of blood cancers.

Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.

CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.