TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells.

Background: In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells. Methods: We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models. Results: We show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells. Conclusion: These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.

Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency.

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1-/- mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

Metformin reduces the clonal fitness of Dnmt3aR878H hematopoietic stem and progenitor cells by reversing their aberrant metabolic and epigenetic state.

Clonal hematopoiesis (CH) arises when a hematopoietic stem cell (HSC) acquires a mutation that confers a competitive advantage over wild-type (WT) HSCs, resulting in its clonal expansion. Individuals with CH are at an increased risk of developing hematologic neoplasms and a range of age-related inflammatory illnesses1-3. Therapeutic interventions that suppress the expansion of mutant HSCs have the potential to prevent these CH-related illnesses; however, such interventions have not yet been identified. The most common CH driver mutations are in the DNA methyltransferase 3 alpha (DNMT3A) gene with arginine 882 (R882) being a mutation hotspot. Here we show that murine hematopoietic stem and progenitor cells (HSPCs) carrying the Dnmt3aR878H/+ mutation, which is equivalent to human DNMT3AR882H/+, have increased mitochondrial respiration compared with WT cells and are dependent on this metabolic reprogramming for their competitive advantage. Importantly, treatment with metformin, an oral anti-diabetic drug with inhibitory activity against complex I in the electron transport chain (ETC), reduced the fitness of Dnmt3aR878H/+ HSCs. Through a multi-omics approach, we discovered that metformin acts by enhancing the methylation potential in Dnmt3aR878H/+ HSPCs and reversing their aberrant DNA CpG methylation and histone H3K27 trimethylation (H3K27me3) profiles. Metformin also reduced the fitness of human DNMT3AR882H HSPCs generated by prime editing. Our findings provide preclinical rationale for investigating metformin as a preventive intervention against illnesses associated with DNMT3AR882 mutation-driven CH in humans.

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4+ T-cells for the treatment of Hyper IgM1.

Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4+ T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.

IL-1ß+ macrophages fuel pathogenic inflammation in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with high resistance to therapies1. Inflammatory and immunomodulatory signals co-exist in the pancreatic tumour microenvironment, leading to dysregulated repair and cytotoxic responses. Tumour-associated macrophages (TAMs) have key roles in PDAC2, but their diversity has prevented therapeutic exploitation. Here we combined single-cell and spatial genomics with functional experiments to unravel macrophage functions in pancreatic cancer. We uncovered an inflammatory loop between tumour cells and interleukin-1ß (IL-1ß)-expressing TAMs, a subset of macrophages elicited by a local synergy between prostaglandin E2 (PGE2) and tumour necrosis factor (TNF). Physical proximity with IL-1ß+ TAMs was associated with inflammatory reprogramming and acquisition of pathogenic properties by a subset of PDAC cells. This occurrence was an early event in pancreatic tumorigenesis and led to persistent transcriptional changes associated with disease progression and poor outcomes for patients. Blocking PGE2 or IL-1ß activity elicited TAM reprogramming and antagonized tumour cell-intrinsic and -extrinsic inflammation, leading to PDAC control in vivo. Targeting the PGE2-IL-1ß axis may enable preventive or therapeutic strategies for reprogramming of immune dynamics in pancreatic cancer.

In vivo macrophage engineering reshapes the tumor microenvironment leading to eradication of liver metastases.

Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma liver metastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8+ T cells. Conversely, increased IL-10 signaling, expansion of Eomes CD4+ T cells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory T cell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive T cells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need.

Genotoxic effects of base and prime editing in human hematopoietic stem cells.

Base and prime editors (BEs and PEs) may provide more precise genetic engineering than nuclease-based approaches because they bypass the dependence on DNA double-strand breaks. However, little is known about their cellular responses and genotoxicity. Here, we compared state-of-the-art BEs and PEs and Cas9 in human hematopoietic stem and progenitor cells with respect to editing efficiency, cytotoxicity, transcriptomic changes and on-target and genome-wide genotoxicity. BEs and PEs induced detrimental transcriptional responses that reduced editing efficiency and hematopoietic repopulation in xenotransplants and also generated DNA double-strand breaks and genotoxic byproducts, including deletions and translocations, at a lower frequency than Cas9. These effects were strongest for cytidine BEs due to suboptimal inhibition of base excision repair and were mitigated by tailoring delivery timing and editor expression through optimized mRNA design. However, BEs altered the mutational landscape of hematopoietic stem and progenitor cells across the genome by increasing the load and relative proportions of nucleotide variants. These findings raise concerns about the genotoxicity of BEs and PEs and warrant further investigation in view of their clinical application.

Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated in vitro and in vivo.

Hyper-IgM1 is a rare X-linked combined immunodeficiency caused by mutations in the CD40 ligand (CD40LG) gene with a median survival of 25 years, potentially treatable with in situ CD4+ T cell gene editing with Cas9 and a one-size-fits-most corrective donor template. Here, starting from our research-grade editing protocol, we pursued the development of a good manufacturing practice (GMP)-compliant, scalable process that allows for correction, selection and expansion of edited cells, using an integrase defective lentiviral vector as donor template. After systematic optimization of reagents and conditions we proved maintenance of stem and central memory phenotypes and expression and function of CD40LG in edited healthy donor and patient cells recapitulating the physiological CD40LG regulation. We then documented the preserved fitness of edited cells by xenotransplantation into immunodeficient mice. Finally, we transitioned to large-scale manufacturing, and developed a panel of quality control assays. Overall, our GMP-compliant process takes long-range gene editing one step closer to clinical application with a reassuring safety profile.

Mobilization-based engraftment of haematopoietic stem cells: a new perspective for chemotherapy-free gene therapy and transplantation.

INTRODUCTION: In haematopoietic stem cell transplantation (HSCT), haematopoietic stem cells (HSCs) from a healthy donor replace the patient's ones. Ex vivo HSC gene therapy (HSC-GT) is a form of HSCT in which HSCs, usually from an autologous source, are genetically modified before infusion, to generate a progeny of gene-modified cells. In HSCT and HSC-GT, chemotherapy is administered before infusion to free space in the bone marrow (BM) niche, which is required for the engraftment of infused cells. Here, we review alternative chemotherapy-free approaches to niche voidance that could replace conventional regimens and alleviate the morbidity of the procedure. SOURCES OF DATA: Literature was reviewed from PubMed-listed peer-reviewed articles. No new data are presented in this article. AREAS OF AGREEMENT: Chemotherapy exerts short and long-term toxicity to haematopoietic and non-haematopoietic organs. Whenever chemotherapy is solely used to allow engraftment of donor HSCs, rather than eliminating malignant cells, as in the case of HSC-GT for inborn genetic diseases, non-genotoxic approaches sparing off-target tissues are highly desirable. AREAS OF CONTROVERSY: In principle, HSCs can be temporarily moved from the BM niches using mobilizing drugs or selectively cleared with targeted antibodies or immunotoxins to make space for the infused cells. However, translation of these principles into clinically relevant settings is only at the beginning, and whether therapeutically meaningful levels of chimerism can be safely established with these approaches remains to be determined. GROWING POINTS: In pre-clinical models, mobilization of HSCs from the niche can be tailored to accommodate the exchange and engraftment of infused cells. Infused cells can be further endowed with a transient engraftment advantage. AREAS TIMELY FOR DEVELOPING RESEARCH: Inter-individual efficiency and kinetics of HSC mobilization need to be carefully assessed. Investigations in large animal models of emerging non-genotoxic approaches will further strengthen the rationale and encourage application to the treatment of selected diseases.

A step toward stem cell engineering in vivo.

mRNA-based delivery may change the paradigm of hematopoietic stem cell gene therapy.

Lipid nanoparticles allow efficient and harmless ex vivo gene editing of human hematopoietic cells.

Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses the delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo via electroporation, and when aiming for homology-driven correction of a DNA template, often provided by viral vectors together with a nuclease editor. Although HSPCs activate a robust p53-dependent DNA damage response upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multiomics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism, and inducing an inflammatory response. Nuclease RNA delivery using lipid nanoparticles (LNPs) nearly abolished cell death and ameliorated cell growth, improving tolerance to the procedure and yielding a higher number of edited cells compared with using electroporation. Transient transcriptomic changes upon LNP treatment were mostly caused by cellular loading with exogenous cholesterol, whose potentially detrimental impact could be overcome by limiting exposure. Notably, LNP-based HSPC editing dampened p53 pathway induction and supported higher clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared with electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for the treatment of human diseases.

Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy.

The growing clinical success of hematopoietic stem/progenitor cell (HSPC) gene therapy (GT) relies on the development of viral vectors as portable "Trojan horses" for safe and efficient gene transfer. The recent advent of novel technologies enabling site-specific gene editing is broadening the scope and means of GT, paving the way to more precise genetic engineering and expanding the spectrum of diseases amenable to HSPC-GT. Here, we provide an overview of state-of-the-art and prospective developments of the HSPC-GT field, highlighting how advances in biological characterization and manipulation of HSPCs will enable the design of the next generation of these transforming therapeutics.

Constitutive IL-1RA production by modified immune cells protects against IL-1-mediated inflammatory disorders.

Dysregulation of the interleukin-1 (IL-1) pathway leads to immune diseases that can result in chronic tissue and organ inflammation. Although IL-1 blockade has shown promise in ameliorating these symptoms and improving patients' quality of life, there is an urgent need for more effective, long-lasting treatments. We developed a lentivirus (LV)-mediated gene transfer strategy using transplanted autologous hematopoietic stem/progenitor cells (HSPCs) as a source of IL-1 receptor antagonist (IL-1RA) for systemic delivery to tissues and organs. Transplantation of mouse and human HSPCs transduced with an IL-1RA-encoding LV ensured stable IL-1RA production while maintaining the clonogenic and differentiation capacities of HSPCs in vivo. We examined the efficacy of cell-mediated IL-1RA delivery in three models of IL-1-dependent inflammation, for which treatment hindered neutrophil recruitment in an inducible model of gout, prevented systemic and multi-tissue inflammation in a genetic model of cryopyrin-associated periodic syndromes, and reduced disease severity in an experimental autoimmune encephalomyelitis model of multiple sclerosis. Our findings demonstrate HSPC-mediated IL-1RA delivery as a potential therapeutic modality that can be exploited to suppress tissue and organ inflammation in diverse immune-related diseases involving IL-1-driven inflammation.

Gene-based delivery of immune-activating cytokines for cancer treatment.

Tumors evolve together with the tumor microenvironment (TME) and reshape it towards immunosuppression. Immunostimulating cytokines can be used to revert this state leading to effective antitumor immune responses, but their exploitation as anticancer drugs has been hampered by severe toxicity associated with systemic administration. Local, TME-targeted delivery of immune activating cytokines can deploy their antitumoral function more effectively than systemic administration while, at the same time, avoiding exposure of healthy organs and limiting toxicity. Here, we review different gene and cell therapy platforms developed for tumor-directed cytokine delivery highlighting their potential for clinical translation.

Mesenchymal stromal cells improve the transplantation outcome of CRISPR-Cas9 gene-edited human HSPCs.

Mesenchymal stromal cells (MSCs) have been employed in vitro to support hematopoietic stem and progenitor cell (HSPC) expansion and in vivo to promote HSPC engraftment. Based on these studies, we developed an MSC-based co-culture system to optimize the transplantation outcome of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene-edited (GE) human HSPCs. We show that bone marrow (BM)-MSCs produce several hematopoietic supportive and anti-inflammatory factors capable of alleviating the proliferation arrest and mitigating the apoptotic and inflammatory programs activated in GE-HSPCs, improving their expansion and clonogenic potential in vitro. The use of BM-MSCs resulted in superior human engraftment and increased clonal output of GE-HSPCs contributing to the early phase of hematological reconstitution in the peripheral blood of transplanted mice. In conclusion, our work poses the biological bases for a novel clinical use of BM-MSCs to promote engraftment of GE-HSPCs and improve their transplantation outcome.

Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells.

Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long-term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.

Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress.

Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools.