Mobilization-based chemotherapy-free engraftment of gene-edited human hematopoietic stem cells.

Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) is proving successful to treat several genetic diseases. HSPCs are mobilized, harvested, genetically corrected ex vivo, and infused, after the administration of toxic myeloablative conditioning to deplete the bone marrow (BM) for the modified cells. We show that mobilizers create an opportunity for seamless engraftment of exogenous cells, which effectively outcompete those mobilized, to repopulate the depleted BM. The competitive advantage results from the rescue during ex vivo culture of a detrimental impact of mobilization on HSPCs and can be further enhanced by the transient overexpression of engraftment effectors exploiting optimized mRNA-based delivery. We show the therapeutic efficacy in a mouse model of hyper IgM syndrome and further developed it in human hematochimeric mice, showing its applicability and versatility when coupled with gene transfer and editing strategies. Overall, our findings provide a potentially valuable strategy paving the way to broader and safer use of HSPC-GT.

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.

Correcting inborn errors of immunity: From viral mediated gene addition to gene editing.

Allogeneic hematopoietic stem cell transplantation is an effective treatment to cure inborn errors of immunity. Remarkable progress has been achieved thanks to the development and optimization of effective combination of advanced conditioning regimens and use of immunoablative/suppressive agents preventing rejection as well as graft versus host disease. Despite these tremendous advances, autologous hematopoietic stem/progenitor cell therapy based on ex vivo gene addition exploiting integrating γ-retro- or lenti-viral vectors, has demonstrated to be an innovative and safe therapeutic strategy providing proof of correction without the complications of the allogeneic approach. The recent advent of targeted gene editing able to precisely correct genomic variants in an intended locus of the genome, by introducing deletions, insertions, nucleotide substitutions or introducing a corrective cassette, is emerging in the clinical setting, further extending the therapeutic armamentarium and offering a cure to inherited immune defects not approachable by conventional gene addition. In this review, we will analyze the current state-of-the art of conventional gene therapy and innovative protocols of genome editing in various primary immunodeficiencies, describing preclinical models and clinical data obtained from different trials, highlighting potential advantages and limits of gene correction.

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.

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.

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.

Diaphragm ultrasound evaluation during weaning from mechanical ventilation in COVID-19 patients: a pragmatic, cross-section, multicenter study.

BACKGROUND: Diaphragmatic dysfunction is a major factor responsible for weaning failure in patients that underwent prolonged invasive mechanical ventilation for acute severe respiratory failure from COVID-19. This study hypothesizes that ultrasound measured diaphragmatic thickening fraction (DTF) could provide corroborating information for weaning COVID-19 patients from mechanical ventilation. METHODS: This was an observational, pragmatic, cross-section, multicenter study in 6 Italian intensive care units. DTF was assessed in COVID-19 patients undergoing weaning from mechanical ventilation from 1st March 2020 to 30th June 2021. Primary aim was to evaluate whether DTF is a predictive factor for weaning failure. RESULTS: Fifty-seven patients were enrolled, 25 patients failed spontaneous breathing trial (44%). Median length of invasive ventilation was 14 days (IQR 7-22). Median DTF within 24 h since the start of weaning was 28% (IQR 22-39%), RASS score (- 2 vs - 2; p = 0.031); Kelly-Matthay score (2 vs 1; p = 0.002); inspiratory oxygen fraction (0.45 vs 0.40; p = 0.033). PaO2/FiO2 ratio was lower (176 vs 241; p = 0.032) and length of intensive care stay was longer (27 vs 16.5 days; p = 0.025) in patients who failed weaning. The generalized linear regression model did not select any variables that could predict weaning failure. DTF was correlated with pH (RR 1.56 × 1027; p = 0.002); Kelly-Matthay score (RR 353; p < 0.001); RASS (RR 2.11; p = 0.003); PaO2/FiO2 ratio (RR 1.03; p = 0.05); SAPS2 (RR 0.71; p = 0.005); hospital and ICU length of stay (RR 1.22 and 0.79, respectively; p < 0.001 and p = 0.004). CONCLUSIONS: DTF in COVID-19 patients was not predictive of weaning failure from mechanical ventilation, and larger studies are needed to evaluate it in clinical practice further. Registered: ClinicalTrial.gov (NCT05019313, 24 August 2021).

Transcriptomic analysis of BM-MSCs identified EGR1 as a transcription factor to fully exploit their therapeutic potential.

Bone marrow-mesenchymal stromal cells (BM-MSCs) are key components of the BM niche, where they regulate hematopoietic stem progenitor cell (HSPC) homeostasis by direct contact and secreting soluble factors. BM-MSCs also protect the BM niche from excessive inflammation by releasing anti-inflammatory factors and modulating immune cell activity. Thanks to these properties, BM-MSCs were successfully employed in pre-clinical HSPC transplantation models, increasing the rate of HSPC engraftment, accelerating the hematological reconstitution, and reducing the risk of graft failure. However, their clinical use requires extensive in vitro expansion, potentially altering their biological and functional properties. In this work, we analyzed the transcriptomic profile of human BM-MSCs sorted as CD45-, CD105+, CD73+, and CD90+ cells from the BM aspirates of heathy-donors and corresponding ex-vivo expanded BM-MSCs. We found the expression of immune and inflammatory genes downregulated upon cell culture and selected the transcription factor EGR1 to restore the MSC properties. We overexpressed EGR1 in BM-MSCs and performed in vitro tests to study the functional properties of EGR1-overexpressing BM-MSCs. We concluded that EGR1 increased the MSC response to inflammatory stimuli and immune cell control and potentiated the MSC hematopoietic supportive activity in co-culture assay, suggesting that the EGR1-based reprogramming may improve the BM-MSC clinical use.

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.

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.

A step toward stem cell engineering in vivo.

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

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.

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.

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.

BAR-Seq clonal tracking of gene-edited cells.

Gene editing by engineered nucleases has revolutionized the field of gene therapy by enabling targeted and precise modification of the genome. However, the limited availability of methods for clonal tracking of edited cells has resulted in a paucity of information on the diversity, abundance and behavior of engineered clones. Here we detail the wet laboratory and bioinformatic BAR-Seq pipeline, a strategy for clonal tracking of cells harboring homology-directed targeted integration of a barcoding cassette. We present the BAR-Seq web application, an online, freely available and easy-to-use software that allows performing clonal tracking analyses on raw sequencing data without any computational resources or advanced bioinformatic skills. BAR-Seq can be applied to most editing strategies, and we describe its use to investigate the clonal dynamics of human edited hematopoietic stem/progenitor cells in xenotransplanted hosts. Notably, BAR-Seq may be applied in both basic and translational research contexts to investigate the biology of edited cells and stringently compare editing protocols at a clonal level. Our BAR-Seq pipeline allows library preparation and validation in a few days and clonal analyses of edited cell populations in 1 week.

Gene Editing of Hematopoietic Stem Cells: Hopes and Hurdles Toward Clinical Translation.

In the field of hematology, gene therapies based on integrating vectors have reached outstanding results for a number of human diseases. With the advent of novel programmable nucleases, such as CRISPR/Cas9, it has been possible to expand the applications of gene therapy beyond semi-random gene addition to site-specific modification of the genome, holding the promise for safer genetic manipulation. Here we review the state of the art of ex vivo gene editing with programmable nucleases in human hematopoietic stem and progenitor cells (HSPCs). We highlight the potential advantages and the current challenges toward safe and effective clinical translation of gene editing for the treatment of hematological diseases.

Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking.

Targeted gene editing in hematopoietic stem cells (HSCs) is a promising treatment for several diseases. However, the limited efficiency of homology-directed repair (HDR) in HSCs and the unknown impact of the procedure on clonal composition and dynamics of transplantation have hampered clinical translation. Here, we apply a barcoding strategy to clonal tracking of edited cells (BAR-Seq) and show that editing activates p53, which substantially shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserve multilineage and self-renewing capacity. Transient p53 inhibition restored polyclonal graft composition. We increased HDR efficiency by forcing cell-cycle progression and upregulating components of the HDR machinery through transient expression of the adenovirus 5 E4orf6/7 protein, which recruits the cell-cycle controller E2F on its target genes. Combined E4orf6/7 expression and p53 inhibition resulted in HDR editing efficiencies of up to 50% in the long-term human graft, without perturbing repopulation and self-renewal of edited HSCs. This enhanced protocol should broaden applicability of HSC gene editing and pave its way to clinical translation.

Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response.

Precise gene editing in hematopoietic stem and progenitor cells (HSPCs) holds promise for treating genetic diseases. However, responses triggered by programmable nucleases in HSPCs are poorly characterized and may negatively impact HSPC engraftment and long-term repopulation capacity. Here, we induced either one or several DNA double-stranded breaks (DSBs) with optimized zinc-finger and CRISPR/Cas9 nucleases and monitored DNA damage response (DDR) foci induction, cell-cycle progression, and transcriptional responses in HSPC subpopulations, with up to single-cell resolution. p53-mediated DDR pathway activation was the predominant response to even single-nuclease-induced DSBs across all HSPC subtypes analyzed. Excess DSB load and/or adeno-associated virus (AAV)-mediated delivery of DNA repair templates induced cumulative p53 pathway activation, constraining proliferation, yield, and engraftment of edited HSPCs. However, functional impairment was reversible when DDR burden was low and could be overcome by transient p53 inhibition. These findings provide molecular and functional evidence for feasible and seamless gene editing in HSPCs.