Thalassemia or sickle cell patients with hereditary persistence of fetal hemoglobin (HbF) have an ameliorated clinical phenotype and, in some cases, can achieve transfusion independence. Inactivation via genome editing of γ-globin developmental suppressors, such as BCL11A or LRF/ZBTB7A, or of their binding sites, have been shown to significantly increase expression of endogenous HbF. To broaden the therapeutic window beyond a single-editing approach, we have explored combinations of cis- and trans-editing targets to enhance HbF reactivation. Multiplex mutagenesis in adult CD34+ cells was well tolerated and did not lead to any detectable defect in the cells’ proliferation and differentiation, either in vitro or in vivo. The combination of 1 trans and 1 cis mutation resulted in high editing retention in vivo, coupled with almost pancellular HbF expression in NBSGW mice. The greater in vivo performance of this combination was also recapitulated using a novel helper-dependent adenoviral-CRISPR vector (HD-Ad-dualCRISPR) in CD34+ cells from β-thalassemia patients transplanted to NBSGW mice. A pronounced increase in HbF expression was observed in human red blood cells in mice with established predominant β0/β0-thalassemic hemopoiesis after in vivo injection of the HD-Ad-dualCRISPR vector. Collectively, our data suggest that the combination of cis and trans fetal globin reactivation mutations has the potential to significantly increase HbF both totally and on a per cell basis over single editing and could thus provide significant clinical benefit to patients with severe β-globin phenotype.

T-cell-based, personalized immunotherapy can nowadays be considered the mainstream treatment for certain blood cancers, with a high potential for expanding indications. Chimeric antigen receptor T cells (CAR-Ts), an ex vivo genetically modified T-cell therapy product redirected to target an antigen of interest, have achieved unforeseen successes in patients with B-cell hematologic malignancies. Frequently, however, CAR-T cell therapies fail to provide durable responses while they have met with only limited success in treating solid cancers because unique, unaddressed challenges, including poor persistence, impaired trafficking to the tumor, and site penetration through a hostile microenvironment, impede their efficacy. Increasing evidence suggests that CAR-Ts’ in vivo performance is associated with T-cell intrinsic features that may be epigenetically altered or dysregulated. In this review, we focus on the impact of epigenetic regulation on T-cell differentiation, exhaustion, and tumor infiltration and discuss how epigenetic reprogramming may enhance CAR-Ts’ memory phenotype, trafficking, and fitness, contributing to the development of a new generation of potent CAR-T immunotherapies.

Beta-hemoglobinopathies are the most common genetic disorders worldwide, caused by a wide spectrum of mutations in the β-globin locus, and associated with morbidity and early mortality in case of patient non-adherence to supportive treatment. Allogeneic transplantation of hematopoietic stem cells (allo-HSCT) used to be the only curative option, although the indispensable need for an HLA-matched donor markedly restricted its universal application. The evolution of gene therapy approaches made possible the ex vivo delivery of a therapeutic β- or γ- globin gene into patient-derived hematopoietic stem cells followed by the transplantation of corrected cells into myeloablated patients, having led to high rates of transfusion independence (thalassemia) or complete resolution of painful crises (sickle cell disease-SCD). Hereditary persistence of fetal hemoglobin (HPFH), a syndrome characterized by increased γ-globin levels, when co-inherited with β-thalassemia or SCD, converts hemoglobinopathies to a benign condition with mild clinical phenotype. The rapid development of precise genome editing tools (ZFN, TALENs, CRISPR/Cas9) over the last decade has allowed the targeted introduction of mutations, resulting in disease-modifying outcomes. In this context, genome editing tools have successfully been used for the introduction of HPFH-like mutations both in HBG1/HBG2 promoters or/and in the erythroid enhancer of BCL11A to increase HbF expression as an alternative curative approach for β-hemoglobinopathies. The current investigation of new HbF modulators, such as ZBTB7A, KLF-1, SOX6, and ZNF410, further expands the range of possible genome editing targets. Importantly, genome editing approaches have recently reached clinical translation in trials investigating HbF reactivation in both SCD and thalassemic patients. Showing promising outcomes, these approaches are yet to be confirmed in long-term follow-up studies.

Lineage commitment and differentiation is driven by the concerted action of master transcriptional regulators at their target chromatin sites. Multiple efforts have characterized the key transcription factors (TFs) that determine the various hematopoietic lineages. However, the temporal interactions between individual TFs and their chromatin targets during differentiation and how these interactions dictate lineage commitment remains poorly understood. Here we perform dense, daily, temporal profiling of chromatin accessibility (DNase I-seq) and gene expression changes (total RNA-seq) along ex vivo human erythropoiesis to comprehensively define developmentally regulated DNase I hypersensitive sites (DHSs) and transcripts. We link both distal DHSs to their target gene promoters and individual TFs to their target DHSs, revealing that the regulatory landscape is organized in distinct sequential regulatory modules that regulate lineage restriction and maturation. Finally, direct comparison of transcriptional dynamics (bulk and single-cell) and lineage potential between erythropoiesis and megakaryopoiesis uncovers differential fate commitment dynamics between the two lineages as they exit the stem and progenitor stage. Collectively, these data provide insights into the temporally regulated synergy of thecis- and thetrans-regulatory components underlying hematopoietic lineage commitment and differentiation.

Regulatory T cells (Tregs) are fundamental to maintaining immune homeostasis by inhibiting immune responses to self-antigens and preventing the excessive activation of the immune system. Their functions extend beyond immune surveillance and subpopulations of tissue-resident Treg cells can also facilitate tissue repair and homeostasis. The unique ability to regulate aberrant immune responses has generated the concept of harnessing Tregs as a new cellular immunotherapy approach for reshaping undesired immune reactions in autoimmune diseases and allo-responses in transplantation to ultimately re-establish tolerance. However, a number of issues limit the broad clinical applicability of Treg adoptive immunotherapy, including the lack of antigen specificity, heterogeneity within the Treg population, poor persistence, functional Treg impairment in disease states, and in vivo plasticity that results in the loss of suppressive function. Although the early-phase clinical trials of Treg cell therapy have shown the feasibility and tolerability of the approach in several conditions, its efficacy has remained questionable. Leveraging the smart tools and platforms that have been successfully developed for primary T cell engineering in cancer, the field has now shifted towards “next-generation” adoptive Treg immunotherapy, where genetically modified Treg products with improved characteristics are being generated, as regards antigen specificity, function, persistence, and immunogenicity. Here, we review the state of the art on Treg adoptive immunotherapy and progress beyond it, while critically evaluating the hurdles and opportunities towards the materialization of Tregs as a living drug therapy for various inflammation states and the broad clinical translation of Treg therapeutics.

High numbers of genetically modified hematopoietic stem cells (HSCs) equipped with enhanced engrafting potential are required for successful stem cell gene therapy. By using thalassemia as a model, we investigated the functional properties of hematopoietic stem and progenitor cells (HSPCs) from Hbb(th3)/45.2(+) mice after mobilization with G-CSF, plerixafor, or G-CSF+plerixafor and the engraftment kinetics of primed cells after competitive primary and noncompetitive secondary transplantation. G-CSF+plerixafor yielded the highest numbers of HSPCs, while G-CSF+plerixafor-mobilized Hbb(th3)/45.2(+) cells, either unmanipulated or transduced with a reporter vector, achieved faster hematologic reconstitution and higher levels of donor chimerism over all other types of mobilized cells, after competitive transplantation to B6.BoyJ/45.1(+) recipients. The engraftment benefit observed in the G-CSF+plerixafor group was attributed to the more primitive stem cell phenotype of G-CSF+plerixafor-LSK cells, characterized by higher CD150(+)/CD48 expression. Moreover, secondary G-CSF+plerixafor recipients displayed stable or even higher chimerism levels as compared with primary engrafted mice, thus maintaining or further improving engraftment levels over G-CSF- or plerixafor-secondary recipients. Plerixafor-primed cells displayed the lowest competiveness over all other mobilized cells after primary or secondary transplantation, probably because of the higher frequency of more actively proliferating LK cells. Overall, the higher HSC yields, the faster hematological recovery, and the superiority in long-term engraftment indicate G-CSF+plerixafor-mobilized blood as an optimal graft source, not only for thalassemia gene therapy, but also for stem cell gene therapy applications in general.