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Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues.

Clonal hematopoiesis (CH) is an age-related clonal expansion of hematopoietic stem cells (HSCs). Factors that shape the progression of CH are only beginning to be characterized. HSCs are a heterogeneous population of cells with distinct clonal outputs over time, which are hypothesized to be largely scripted in epigenetic and transcriptional cell state. Molecular properties of HSC states that mediate clonal behaviors of two major driver mutations in CH - DNMT3A and TET2 - represent potential therapeutical targets. Recent studies have identified the landscape of human CH mutant cells compared to non-mutated ones. However, the notion of a specific molecular target identified in CH cells associated with selective advantage or predisposition for subsequent transformation requires a formal validation by longitudinal studies. To uncover molecular underpinnings of CH-driven clonal phenotypes in human HSCs in vivo, we combined clonal analysis using heritable expressed DNA barcodes with single-cell transcriptomics using transplant in immunodeficient mice. We developed a statistical approach to associate transcriptional differences of HSCs with the clone phenotypes, such as size and HSC density, across more than a hundred of detected clones. It revealed robust transcriptional signatures underlying clone size and HSC density, which are largely complementary to transcriptional programs activated in TET2 and DNMT3A mutant HSCs. Of note, the clone size and density of HSCs in the clone tend to be uncoupled and associated with distinct transcriptional processes. Citation Format: Ruslan Soldatov, Giulia Schiroli, Peter Kharchenko, David Scadden. Transcriptional signatures of stem cell phenotypes in normal and clonal hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2456.

A sleepless night may feel awful in its aftermath, but sleep’s revitalizing powers are substantial, perpetuating the idea that convalescent sleep is a consequence-free physiological reset. Although recent studies have shown that catch-up sleep insufficiently neutralizes the negative effects of sleep debt, the mechanisms that control prolonged effects of sleep disruption are not understood. Here, we show that sleep interruption restructures the epigenome of hematopoietic stem and progenitor cells (HSPCs) and increases their proliferation, thus reducing hematopoietic clonal diversity through accelerated genetic drift. Sleep fragmentation exerts a lasting influence on the HSPC epigenome, skewing commitment toward a myeloid fate and priming cells for exaggerated inflammatory bursts. Combining hematopoietic clonal tracking with mathematical modeling, we infer that sleep preserves clonal diversity by limiting neutral drift. In humans, sleep restriction alters the HSPC epigenome and activates hematopoiesis. These findings show that sleep slows decay of the hematopoietic system by calibrating the hematopoietic epigenome, constraining inflammatory output, and maintaining clonal diversity.

Myeloid cells are the most evolutionarily ancient aspect of a specialized immune system and the cornerstone of innate immunity in vertebrates. Innate immunity serves at the front line of host defense playing essential roles in directly clearing infection while also activating adaptive immune cell populations. Even with the importance in its roles, the myeloid system has often been regarded as the 'dumb brute' side of cell-based immunity with limited specificity and variability of responses. Heterogeneity in innate immune cells is increasingly recognized but still modest compared to other cell types and constrained by limited investigative tools. Monocytes have traditionally been categorized into two subsets: classical and non-classical. Classical monocytes are thought to readily convert to non-classical monocytes with exogenous cues. Studies on bulk monocyte populations have shown that monocytes acquire functional features dependent on physiological needs supporting a prevailing model that monocytes are highly plastic. We adapted a system for inducible clonal expansion of mouse granulocyte-monocyte progenitor (GMP) capable of differentiating into mature myeloid cells to address at a clonal level the issues of monocyte heterogeneity, plasticity, and whether changes in monocyte functional groups are due to induction or selection, Using inducible HoxB8-ER, large numbers of primary self-renewing GMP can undergo progressive maturation to fully functional granulocytes or monocytes upon removal of estrogen. The resulting GMP clones could then be characterized molecularly and immunophenotypically in correlation with the specialized functions of their descendent monocytes (Figure 1A). Four GMP differentiation trajectories that yield functionally distinct monocytes were defined (Figures 1B and 1C). Notably the functional monocyte groups were evident at the GMP level based on unsupervised clustering of chromatin configuration data (Figure 1D). GMP bore epigenetic scripting of the potential of their descendent monocytes and this fate had little flexibility once differentiation had begun. Testing cells under in vitro and in vivo homeostasis and stress conditions revealed that the cells maintain their differentiation path and do not transition from one state to another (Figures 1E and 1F). These data imply a model whereby monocyte stimulus-specific selection may occur and may contribute to an innate immune memory that resembles adaptive immune memory. Figure 1 Figure 1. Disclosures Sykes: Keros Therapeutics: Consultancy; SAFI Biosolutions: Consultancy, Current equity holder in publicly-traded company; Clear Creek Bio: Current equity holder in publicly-traded company. Scadden: Magenta Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Editas Medicines: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Current holder of individual stocks in a privately-held company; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Dainippon Sumitomo Pharma: Other: Sponsored research; FOG Pharma:: Consultancy; Garuda Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; VCanBio: Consultancy; Inzen Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Innate immune factors may restrict hematopoietic stem cell (HSC) genetic engineering and contribute to broad individual variability in gene therapy outcomes. Here, we show that HSCs harbor an early, constitutively active innate immune block to lentiviral transduction that can be efficiently overcome by cyclosporine H (CsH). CsH potently enhances gene transfer and editing in human long-term repopulating HSCs by inhibiting interferon-induced transmembrane protein 3 (IFITM3), which potently restricts VSV glycoprotein-mediated vector entry. Importantly, individual variability in endogenous IFITM3 levels correlated with permissiveness of HSCs to lentiviral transduction, suggesting that CsH treatment will be useful for improving ex vivo gene therapy and standardizing HSC transduction across patients. Overall, our work unravels the involvement of innate pathogen recognition molecules in immune blocks to gene correction in primary human HSCs and highlights how these roadblocks can be overcome to develop innovative cell and gene therapies.

Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases.

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. Precise gene editing has the potential to treat immune and hematological diseases. Genovese, Naldini, Di Micco, and colleagues now show that gene-editing procedures are well tolerated by hematopoietic stem cells and provide molecular evidence of the feasibility of seamless gene editing, strengthening translation of such approaches to humans.

Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all IL2RG (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the IL2RG-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the AAVS1 "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34+ cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34+ cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells. The authors would like to thank Aurora de la Cal for her assistance with thecoordination in the delivery of the samples from the patients. RebecaSánchez-Domínguez for her expertise in flow cytometry and Centro de Trans-fusiones de la Comunidad de Madrid for providing cord blood samples. Theauthors are also indebted to the FA patients, families, and clinicians from theFA spanish network. This work was supported by grants from the“7th Frame-work Program European Commission (HEALTH-F5-2012-305421; EUROFAN-COLEN)”,“Ministerio de Sanidad, Servicios Sociales e Igualdad”(EC11/060andEC11/550),“Ministerio de Economía, Comercio y Competitividad y FondoEuropeo de Desarrollo Regional (FEDER)”(SAF2015-68073-R), and“Fondo deInvestigaciones Sanitarias, Instituto de Salud Carlos III”(RD12/0019/0023). Theauthors also thank the Fundación Marcelino Botín for promoting translationalresearch at the Hematopoietic Innovative Therapies Division of the CIEMAT.CIBERER is an initiative of the Instituto de Salud Carlos III, Spain. Sí

Targeted genome editing by artificial nucleases has brought the goal of gene correction within the reach of gene therapy. A candidate disease for HSPC gene correction is SCID-X1, because gene therapy with early generation integrating vectors showed robust clinical efficacy even from few corrected cells but also the occurrence of adverse events due to insertional mutagenesis and unregulated transgene expression. We recently reported a strategy that enabled targeted integration of a corrective cDNA into the IL2RG gene in 6% of human HSPC with high specificity. Gene corrected HSPC generated polyclonal lymphoid cells that express the IL2RG protein and have a selective growth advantage over those carrying disruptive IL2RG mutations (Genovese, Nature, 2014). Here, to model SCID-X1 disease correction, we developed a mouse model carrying the IL2RG human gene including a common disease-causing mutation in place of the murine Il2rg gene, allowing to use the same reagents utilized for gene correction of human cells. These mice have impairment in lymphoid development which phenocopies that reported for Il2rg-/- mice. To assess the minimal level of corrected HSPC required to achieve immune reconstitution we first performed competitive transplants with wild-type (WT) and Il2rg-/- HSPC and found that 1% of WT cells are sufficient to reconstitute at least in part the T and B cell compartments. We then developed a protocol to obtain gene correction in murine Lin- HSPC based on the delivery of donor DNA template by IDLVs followed by transfection of ZFN mRNAs. This protocol was associated with high on-target nuclease activity (40%) and a mean of 6% transgene integration by HDR, but also with high levels of acute cytotoxicity (65% cell loss). The surviving cells remained capable of expansion in culture and preserved their clonogenic potential. Importantly, upon transplant into lethally irradiated mice, only the gene corrected cells were able to generate lymphoid lineages (B and T cells), showing a clear selective advantage over un-corrected cells. These data indicate functional correction of the IL2RG gene by our strategy. Yet, measuring percentage of correction within myeloid cells at long-term we found that it was almost undetectable. Despite the lack of HSC marking, gene corrected lymphoid cells stably persisted in the mice up to 7 months post transplantation within all the hematopoietic organs. Furthermore, upon challenging the transplanted mice with a murine pathogen (LCMV Arm.) we observed viral-specific γIFN production by CD8+ gene corrected cells at a similar extent as for WT mice, proving in vivo the functionality of corrected T cells. These results suggest that our protocol achieves biologically relevant levels of gene correction in progenitors that sustain long-term lymphopoiesis but is limited in multipotent HSC. Ongoing studies aim to improve murine HSC gene targeting and to compare safety and efficacy of gene correction vs gene replacement in our disease model.

Gene targeting is rapidly progressing thanks to the development of improved nucleases and donor constructs, and represents a new gene therapy-based strategy for the treatment of inherited diseases, such as Fanconi anemia (FA). Because FA can be generated by multiple mutations in up to 19 different genes, we focused our interests in the insertion of therapeutic FA genes in safe harbor loci, since this approach could serve as a platform for treating all FA subtypes and pathogenic mutations. In previous studies we demonstrated the feasibility of correcting the phenotype of hematopoietic progenitors through the genome editing and reprogramming of fibroblasts from FA patients. Because of the limited repopulating properties so far reported for iPSC-derived hematopoietic precursor cells, here we aimed to investigate the possibility of conducting a similar gene targeting approach using human primary CD34+ cells. In a first set of experiments we investigated the efficiency of targeting the AAVS1 locus in cord blood HSPCs. Pre-stimulated CD34+ cells were transduced with integrase-defective lentiviral vectors (IDLV) encoding an EGFP reporter gene under the control of the PGK promoter, flanked by sequences homologous to the AAVS1 target site. The cells were then nucleofected with AAVS1 -specific ZFN mRNAs. The average targeting efficiencies ranged from 10-20% both in in vitro cultured cells and NSG mice-repopulating cells. In subsequent experiments, the EGFP reporter gene was replaced by the hFANCA therapeutic gene. Gene targeting experiments conducted in lymphoblast cell lines and primary CD34+ cells from FA-A patients reverted the characteristic hypersensitivity of FA cells to mitomycin C (MMC), and also restored the formation of FANCD2 foci after DNA damage, evidencing the phenotypic correction of these cells. Our data confirm the efficacy of AAVS1-gene targeting in hHSPCs and demonstrate the feasibility of conducting these approaches in primary CD34+ cells from FA patients.