Human Hematopoietic Progenitor Cells Research Articles

Generation of allogeneic CAR-NKT cells from hematopoietic stem and progenitor cells using a clinically guided culture method.

Cancer immunotherapy with autologous chimeric antigen receptor (CAR) T cells faces challenges in manufacturing and patient selection that could be avoided by using 'off-the-shelf' products, such as allogeneic CAR natural killer T (AlloCAR-NKT) cells. Previously, we reported a system for differentiating human hematopoietic stem and progenitor cells into AlloCAR-NKT cells, but the use of three-dimensional culture and xenogeneic feeders precluded its clinical application. Here we describe a clinically guided method to differentiate and expand IL-15-enhanced AlloCAR-NKT cells with high yield and purity. We generated AlloCAR-NKT cells targeting seven cancers and, in a multiple myeloma model, demonstrated their antitumor efficacy, expansion and persistence. The cells also selectively depleted immunosuppressive cells in the tumor microenviroment and antagonized tumor immune evasion via triple targeting of CAR, TCR and NK receptors. They exhibited a stable hypoimmunogenic phenotype associated with epigenetic and signaling regulation and did not induce detectable graft versus host disease or cytokine release syndrome. These properties of AlloCAR-NKT cells support their potential for clinical translation.

PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human hematopoietic cells

The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are "open" prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin "pioneering factor" GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of PML::RARA, Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA. These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.

Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair

Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing, synergize with a DNAPK inhibitor molecule, and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes.

Abstract P29: Zotatifin, the eukaryotic translation initiation factor 4A (eIF4A) inhibitor, synergizes with Venetoclax to inhibit acute myeloid leukemia cell growth

Abstract New targeted therapies such as the selective and potent BCL-2 inhibitor Venetoclax have revolutionized the treatment of acute myeloid leukemia (AML). However, due to the cellular heterogeneity of the disease, drug-resistant cell types often emerge and drive relapse. We found that the eukaryotic translation initiation factor 4A (EIF4A) is highly expressed in multiple primitive AML cell types compared to their healthy counterparts, suggesting its potential as a therapeutic target. By unwinding complex structures in the 5’ UTR of transcripts, eIF4A promotes the translation of oncogene transcripts. A first-in-class drug, Zotatifin, inhibits the helicase activity of eIF4A and curtails tumor growth in receptor tyrosine kinase-driven tumors. Zotatifin is currently being evaluated in phase 1/2 trials across multiple solid tumors (NCT04092673). However, its effectiveness in blood cancers such as AML remains unexplored, and it is unknown whether Zotatifin may work synergistically with Venetoclax. Here, we treated AML cell lines in vitro and primary healthy or AML bone marrow ex vivo with Zotatifin or Venetoclax alone and in combination, and demonstrate synergistic killing of AML cells by the combination therapy (ZIP synergy score = 57.0 in MOLM-13, 19.3 in MV4-11, P < 0.0001). Mechanistically, we show that Zotatifin decreases eIF4A and AKT expression, and its combination with Venetoclax drives increased BAK and cleaved BAK expression, as well as increased cleavage of MCL-1 and BCL-2, consistent with drug synergy by convergence on the apoptotic pathway. In pilot colony-forming unit (CFU) assays, we found the combination to be tolerable in healthy CD34+ human hematopoietic stem and progenitor cells (HSPCs). To test drug efficacy in vivo, we generated a patient-derived xenograft (PDX) AML model from a patient who experienced relapse with complex cytogenetics, FLT3 and NPM1 mutations. Only the combination of Zotatifin and Venetoclax but not monotherapies effectively suppressed tumor burden as assessed by human CD45 engraftment in the peripheral blood. Further, the combination of Zotatifin and Venetoclax significantly prolonged survival of xenografted mice (median survival after treatment: vehicle 11 days, Venetoclax 13 days, Zotatifin 15 days, combination 42 days, P = 0.0002). Together, our data indicates that Zotatifin is synergistic with Venetoclax and may represent an effective treatment option for patients who are either primary refractory or have developed resistance to existing Venetoclax-containing regimens. Citation Format: Yoke Seng Lee, Jonathan Good, Noha Awais, Benjamin Eschle, Jenny Noel, Jean Acosta, Erica DePasquale, David Cucchi, Prafulla Gokhale, Fadi Najm, Peter van Galen. Zotatifin, the eukaryotic translation initiation factor 4A (eIF4A) inhibitor, synergizes with Venetoclax to inhibit acute myeloid leukemia cell growth [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P29.

Ex vivo culture resting time impacts transplantation outcomes of genome-edited human hematopoietic stem and progenitor cells in xenograft mouse models

Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease.

Exagamglogene Autotemcel: First Approval.

Exagamglogene autotemcel (Casgevy™) is a genetically modified autologous CD34+ cell enriched population. It contains human haematopoietic stem and progenitor cells edited ex vivo by CRISPR/Cas9 (a DNA double strand break-inducing nuclease system) to differentiate into erythroid cells that produce high levels of foetal hemoglobin. Developed by Vertex Pharmaceuticals and CRISPR Therapeutics, exagamglogene autotemcel received its first approval on 16 November 2023 in the UK for the treatment of transfusion-dependent β-thalassemia (TDT) in patients aged ≥ 12years for whom haematopoietic stem cell (HSC) transplantation is appropriate and a human leukocyte antigen matched related HSC donor is not available. On the same day, it was also approved in the UK for the treatment of sickle cell disease (SCD) in patients aged ≥ 12years with recurrent vasoocclusive crises (VOCs) who have the βS/βS, βS/β+ or βS/β0 genotype for whom HSC transplantation is appropriate and a human leukocyte antigen matched related HSC donor is not available. Subsequently, exagamglogene autotemcel was approved in the USA on 8 December 2023 for the treatment of SCD in patients aged ≥ 12years with recurrent VOCs and received a positive opinion in the EU on 14 December 2023 for the treatment of TDT and SCD. A regulatory assessment of exagamglogene autotemcel is currently underway for the treatment of TDT in the USA. This article summarizes the milestones in the development of exagamglogene autotemcel leading to these first approvals.

Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells

Genome editing by homology directed repair (HDR) is leveraged to precisely modify the genome of therapeutically relevant hematopoietic stem and progenitor cells (HSPCs). Here, we present a new approach to increasing the frequency of HDR in human HSPCs by the delivery of an inhibitor of 53BP1 (named “i53”) as a recombinant peptide. We show that the use of i53 peptide effectively increases the frequency of HDR-mediated genome editing at a variety of therapeutically relevant loci in HSPCs as well as other primary human cell types. We show that incorporating the use of i53 recombinant protein allows high frequencies of HDR while lowering the amounts of AAV6 needed by 8-fold. HDR edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in NSG mice, suggesting that i53 recombinant protein might be safely integrated into the standard CRISPR/AAV6-mediated genome editing protocol to gain greater numbers of edited cells for transplantation of clinically meaningful cell populations.

Intrafemoral Injection of Human Hematopoietic Stem and Progenitor Cells into Immunocompromised Mice.

Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell populations containing HSCs into syngeneic or immunocompromised mice. The size and multilineage composition of the graft is then measured over time, usually by flow cytometry. Classically, a population containing HSCs is injected into the circulation of the animal, after which the HSCs home to the bone marrow, where they lodge and begin blood production. Alternatively, HSCs and/or progenitor cells (HSPCs) can be placed directly in the bone marrow cavity. This paper describes a protocol for intrafemoral injection of human HSPCs into immunodeficient mice. In short, preconditioned mice are anesthetized, and a small hole is drilled through the knee into the femur using a needle. Using a smaller insulin needle, cells are then injected directly into the same conduit created by the first needle. This method of transplantation can be applied in varied experimental designs, using either mouse or human cells as donor cells. It has been most widely used for xenotransplantation, because in this context, it is thought to provide improved engraftment over intravenous injections, therefore improving statistical power and reducing the number of mice to be used.

Scalable generation of functional human iPSC-derived CAR-macrophages that efficiently eradicate CD19-positive leukemia

BackgroundMacrophages have recently become attractive therapeutics in cancer immunotherapy. The potential of macrophages to infiltrate and influence solid malignancies makes them promising targets for the chimeric antigen receptor (CAR) technology...

Targeted, Safe, and Efficient Gene Delivery to Human Hematopoietic Stem and Progenitor Cells In Vivo Using Engineered Avid Adenovirus Vector Platform

Precise editing of human genome using CRISPR-Cas nucleases, base editors, prime editors, or other technologies represents a promising approach for ameliorating or correcting many human genetic diseases. Although numerous pre-clinical and clinical proof-of-concept studies demonstrated feasibility of gene correction in the desired regions of the human genome ex vivo, targeted delivery to and cell-type-specific expression of gene editing proteins in various cell types in vivo represent major challenges for all viral and non-viral delivery platforms developed to date. Here, we describe the development and analysis of AVIDs, a novel adenovirus-based gene delivery platform, that allows for a highly targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells (HSPCs) in vivo after intravenous vector administration. The AVID vectors attach to human HSPCs via engineered fibers that bind to either CD46 or DSG2 receptors, which are highly expressed on human HSPCs. The efficient entry of AVIDs into these cells is mediated via a mutated penton that was engineered to interact with CD49f, α6 integrin class, whose expression is restricted to cells with long-term repopulating and stem cell capacity. To improve virus safety after intravenous administration, as well as to increase the efficacy of virus production for a cost-effective high-yield vector manufacturing, AVIDs further comprise mutations in hypervariable loops of the major capsid protein hexon. While in vitro infection with AVIDs leads to about 30% transduction of a pool of human CD34 + cells, the efficacy of transduction of CD34 +CD38 -CD45RA -CD90 + subsets, highly enriched for hematopoietic stem cells (HSCs), ranged from 80% to 100% for individual donors. Single intravenous administration of AVIDs to humanized mice, grafted with human CD34 + cells, after mobilization led to up to 20% transduction of CD34 +CD38 -CD45RA- HSPC subsets in the bone marrow. Importantly, targeted in vivo transduction of CD34 +CD38 -CD45RA -CD90 -CD49f + subsets, highly enriched for human HSCs, reached up to 19%, which represented a 1900-fold selectivity in gene delivery to HSC-enriched over lineage committed CD34-negative cell populations in the bone marrow. Because the AVID platform allows for regulated, cell type-specific expression of gene editing technologies as well as for expression of immunomodulatory proteins to ensure persistence of corrected HSCs in vivo, the HSC-targeted AVID platform may enable development of novel curative therapies through safe and effective in vivo correction of disease-causing mutations in human HSCs after a single intravenous administration.

BCOR Loss Confers Increased Stemness and Partially Rescues RUNX1-Deficient Phenotypes in Human Hematopoietic Stem and Progenitor Cells

Introduction: Loss of function mutations in BCL-6 corepressor (BCOR) commonly occur in de novo acute myeloid leukemia (AML) and germline hematopoietic malignancies, most notably in those arising from RUNX1 familial platelet disorder (RUNX1-FPD). Second only to loss of heterozygosity of RUNX1, mutations in BCOR are the most commonly acquired mutations upon progression to myeloid malignancy in RUNX1-FPD. Additionally, RUNX1 and BCOR mutations commonly co-occur in de novo AML, and both are classified as “adverse risk” by the European LeukemiaNet if found at AML diagnosis. However, the role of BCOR in leukemogenesis is poorly understood, and there are limited studies investigating co-loss of BCOR and RUNX1 in hematopoietic stem and progenitor cells (HSPCs). Methods: We developed a model of BCOR loss in primary human CD34+ HSPCs using CRISPR/Cas9 and AAV6-mediated homology directed repair to knock-in a fluorescent reporter expression cassette into the BCOR locus. We used this system to investigate how BCOR knockout (KO) perturbs normal HSPC function. We combined this approach with a previously developed analogous system to disrupt the RUNX1 locus to investigatehow BCOR loss in the setting of RUNX1 KO modulates the effects of RUNX1 loss on HSPC function. Results: Here, we found that BCOR loss increased capacity for serial replating and conferred a competitive advantage over control HSPCs. In serial replating assays, BCOR-deficient HSPCs had a 5-fold increase in colony formation after one passage with a striking majority of colonies being erythroid. In liquid differentiation assays, BCOR-deficient HSPCs consistently had a 2-fold increase in CD34+ cells over a 21 day time course. When co-cultured with control HSPCs in a 1:1 ratio in stem expansion media (base media + SCF, FLT3-L, TPO, IL-6, UM-171), BCOR-deficient HSPCs expanded to comprise 70% of the culture at 14 days. This competitive advantage was recapitulated in vivo. At the 12-week timepoint of a competitive transplantation assay in NSG mice using HSPCs from 3 different donors, BCOR KO cells (marked by BFP) on average made up 75% of the engrafted human CD45+ population, while control cells (expressing GFP from the AAVS1 safe harbor locus) made up only 21%. Additionally, BCOR loss partially rescued RUNX1-induced stem cell dysfunction in vitro. Compared to RUNX1 deficiency alone, HSPCs deficient in both RUNX1 and BCOR had a 4-fold increase in colony formation after two passages in serial replating assays. Our lab has previously shown that RUNX1 loss ablates erythroid colony formation and skews towards monocytic colonies. Despite BCOR loss alone having a strong skew towards erythroid colonies, BCOR loss in RUNX1-deficient HSPCs did not induce erythroid colony formation. This suggests that in double mutant HSPCs, RUNX1 loss drives lineage decisions while BCOR loss drives the increase in serial replating potential. Ongoing efforts are aimed at characterizing combinatorial effects of BCOR and RUNX1 loss on hematopoiesis and stem cell function through in vitro liquid differentiation assays and in vivo transplantations, as well as investigating the molecular underpinnings of these phenotypes through RNA-seq. Conclusion: In summary, we have established a model of BCOR loss in primary human HSPCs to evaluate not only the role of BCOR in hematopoiesis, but also to characterize how BCOR is involved in leukemic transformation when combined with RUNX1. We show that BCOR deficiency enhances several attributes associated with stem cell function, including increased potential for serial replating, increased CD34 positive population, and increased engraftment compared to control HSPCs. When combined with RUNX1 loss, BCOR loss increases the serial replating potential of RUNX1-deficient cells, suggesting some rescue of RUNX1 KO-induced stem cell dysfunction. Ultimately, this model will enable us to elucidate the molecular mechanisms underlying increased stemness and competitive advantage in BCOR-deficient HSPCs, interrogate RUNX1-FPD disease progression associated with BCOR mutation, and validate potential therapeutics.

Human Hematopoietic Stem Cells Have Altered Mitochondrial Activity after Stem Cell Transplantation

Hematopoietic stem cells (HSCs) give rise to the entire blood system. With their high regenerative potential, they are sometimes the only curative option for many malignancies. The success of HSCT depends on both the quantity of HSCs and on their ability to produce life-long blood. However, their regenerative potential is not unlimited. Transplanted HSCs sustain injury that alters their subsequent ability to generate blood cells. This leads to ineffective hematopoiesis and can ultimately lead to graft failure. Injury to HSCs is also a risk factor for secondary neoplasms, including leukemia. Our goal is to elucidate the mechanisms behind post-transplant HSC decline. Mitochondria are critically important for HSC function (Filippi Exp Hematol 2021). During regeneration of the bone marrow, HSCs become activated and undergo mitochondrial reprogramming, including reorganization of the mitochondrial network and enhanced mitochondrial activity (Filippi et al Blood 2019, Ito et al Nat Rev Mol Cell Biol 2014). However, after transplantation, HSCs accumulate mitochondria that are abnormal in organization and activity. These abnormal mitochondria lead to decline of HSC function and ineffective hematopoiesis (Hinge et al Cell Stem Cell 2020). While these findings have been replicated in murine models, it has not been studied within the human system. Our hypothesis is that after transplantation, human HSCs have altered mitochondria leading to ineffective hematopoiesis. We examined changes in HSC function using post-transplant samples of patient's bone marrow and a xenotransplant system (human CD34+ peripheral blood stem cells transplanted into sublethally irradiated immunodeficient NOD/SCID/IL-2 receptor-γ null mice). We first measured mitochondrial membrane potential using tetramethyl rhodamine ethyl ester (TMRE) dye and analyzed via flow cytometry. Three to six months after transplant in the xenotransplant model, we found that the mitochondrial membrane potential of huCD34+CD38-CD90+ HSCs was similar to the non-transplanted HSCs. However, huCD34+CD38+ progenitors exhibited a drastic 3-fold increase in mitochondrial membrane potential in comparison to the non-transplanted, huCD34+CD38+ progenitor cells. The mitochondrial membrane potential of CD34+CD38+ progenitors from transplanted bone marrow patient samples was also higher than non-transplanted CD34+CD38+bone marrow control cells. To understand if alterations in mitochondrial activity affect HSC differentiation, we placed xenotransplanted HSCs into in vitro culture. Post-transplanted huCD34+ cells failed to expand within 6 days of in vitro culture (serum-free culture with self-renewing cytokines, SCF+TPO+FLT3) compared to huCD34+ control. In subsequent differentiation assays, post-transplanted huCD34+ cultures gave rise to myeloid cells only. This contrasts with control huCD34+ cultures that produced myeloid, erythroid, and megakaryocytic cells. Interestingly, if the post-transplanted HSCs were given serum and cytokines for differentiation after only 3 days in self-renewing conditions, the cultured cells expanded similarly to non-transplanted HSCs and generated myeloid, erythroid, and megakaryocytic cells. This suggests that transplanted HSCs have decreased proliferation and differentiation ability in comparison to non-transplanted HSCs and respond differently to cytokines in vitro. This could account for ineffective hematopoiesis observed in some post-transplant patients. In conclusion, this study indicates that both mitochondrial activity and in-vitro growth change in HSCs after transplantation, despite re-establishing quiescence. This indicates that human hematopoietic stem and progenitor cells incur injury after transplantation. These findings are clinically important as the changes in mitochondrial activity in HSCs may play a role in post-transplant hematopoietic stem cell decline. Future single cell RNA sequencing analyses will provide mechanistic information regarding post-transplant changes in metabolism and potential targets for improving HSC function in transplant patients.

Loss of Metabolic Control Beyond the Promyelocyte Stage Resolves Myeloid Maturation Arrest in Reticular Dysgenesis

Reticular Dysgenesis (RD) is a particularly grave syndromic form of severe combined immunodeficiency. The hematopoietic phenotype manifests in defective myeloid and lymphoid development with profound neutropenia and lymphopenia. RD is caused by biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2), which catalyzes the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) in the mitochondrial intermembrane space. ADP is then phosphorylated to adenosine triphosphate (ATP) during the OXPHOS process. While there are abundant other sources of APD, AK2 constitutes the only mitochondrial enzyme that contributes to AMP “recycling” by phosphorylation. Accordingly, AK2-deficient myeloid cells have been shown to exhibit increased AMP levels 1. Beyond this observation, the cellular basis for the combined hematopoietic defect in RD remains elusive. Using a CRISPR/Cas9 AK2 biallelic knock-out model in human hematopoietic stem and progenitor cells (HSPCs), we have previously shown that AK2 -/- HSPCs recapitulate RD myelopoiesis defects in vitro. AK2 -/- HSPCs exhibit a severe proliferative defect that becomes apparent at the myelocyte (MC) and neutrophil (NP) stages, while the promyelocyte (PM) stage appears relatively spared, consistent with observations in RD patients. Our prior metabolomic analysis showed that AK2 -/- MCs not only have increased levels of AMP but up of 20-fold increased levels of IMP derived by converting excess AMP to IMP conversion via AMP deaminases (AMPDs). In addition, we have shown that beginning at the MC stage, AK2 -/- cells exhibit an increase in NADH:NAD+ ratio, consistent with reductive stress and decreased aspartate levels. Our new data revealed that expressing the aspartate transporter GLAST and/or the water-forming NADH oxidase LbNOX in AK2 -/- HSPCs partially rescues differentiation but not the proliferation defect. It raises the possibility that reductive stress and aspartate deficiency compromise protein synthesis necessary for maturation, but are separate from the proliferative defect related to high IMP and AMP levels. There were no metabolomic abnormalities noted at the PM stage. PMs have a higher proliferative index than MCs and NPs. This raises the question why the most metabolically active stage is phenotypically spared by AK2 deficiency. To better understand why the metabolic abnormalities associated with RD selectively affect differentiation beyond the promyelocyte stage, we investigated key regulators of cellular metabolism and proliferation, i.e., AMPK, ACC1 and S6, a target of mTOR signaling, at PM, MC and NP stages. We found activation of AMPK, ACC1 and S6 only at the PM stage, while AMPK, ACC1 and S6 protein expression at MC and NP stages was virtually undetectable. Interestingly, AK2 -/- PMs exhibited notably lower ATP synthesis rates compared to controls, while ATP synthesis in AK2 -/- MCs and NPs was significantly higher than in controls of corresponding stages, paralleling the accumulation of AMP and IMP noted earlier. These findings raise the possibility that tight metabolic control at the promyelocyte stage prevents AMP levels from rising by curtailing OXPHOS metabolism in AK2 -/- PMs. In contrast, lack of metabolic control beyond the PM stage permits unopposed OXPHOS activity, thereby allowing AMP and IMP levels to rise to toxic levels that interfere with proliferation. In our ongoing work, we are testing whether pharmacologically redirecting metabolism from OXPHOS to glycolysis with UK5099, could rescue AK2 -/- myelopoiesis defects. UK5099 is a potent inhibitor of the mitochondrial pyruvate carrier, which facilitates pyruvate transport across the mitochondrial inner membrane to fuel the TCA cycle. Additionally, we developed a hematopoietic-specific and inducible Cre-mediated Ak2 knock-out mouse model, which recapitulates the profound neutropenia and lymphopenia observed in RD patients. This mouse model will allow us to corroborate our in vitro observations and to investigate if the same mechanistic underpinnings also apply to the lymphoid lineage. Rissone A, Weinacht KG, la Marca G, et al. Reticular dysgenesis-associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress. J Exp Med. Jul 27 2015;212(8):1185-202. doi:10.1084/jem.20141286

Chronic Alcohol Exposure Causes Myeloid-Biased Hematopoiesis Via Double-Strand RNA Sensors and Type 1 Interferon Response

Excessive alcohol use is well-known to cause cytopenias and bone marrow (BM) suppression. However, the mechanisms by which alcohol suppresses BM are poorly understood. Moreover, even though the prevalence of moderate drinking is high worldwide, the effects of chronic moderate alcohol consumption on the human hematopoietic system remain unknown. To fill this knowledge gap, we sought to investigate the effects of chronic moderate alcohol consumption on human hematopoiesis using cell-based and animal models. To examine the effects of alcohol on hematopoietic cells in vitro, we tested varying doses of alcohol (0-100 mM) on four leukemia cell lines (Jurkat, MOLM13, MV-4-11, and THP1) and measured population doubling over 6 days. Population doubling levels significantly decreased in a dose-dependent manner in all cell lines tested. We also measured mitochondrial (mt) ROS by MitoSOX-Red flow cytometry in Jurkat, which showed a dose-dependent increase of mtROS (trends only at 10, 25 mM; significantly increased at 50, 100 mM alcohol) compared to untreated. These findings confirm that alcohol negatively affects cell growth and increases mtROS in a physiologic range of doses. To determine the effects of alcohol on human hematopoietic stem progenitor cells (HSPCs) in vitro, we treated human BM CD34+ cells with 0 vs. 50 mM alcohol for 48 hours and performed bulk RNA-sequencing in triplicate. Gene Set Enrichment Analysis (GSEA) revealed that multiple pathways related to platelet activation and coagulation were enriched in alcohol-treated HSPCs, suggesting the induction of inflammation and/or platelet-biased differentiation by alcohol. To assess the effects of chronic moderate alcohol consumption on HSPCs in vivo, we employed a xenotransplant model. In brief, human BM CD34+ cells were transplanted to 5-to-6-week-old female NBSGW mice. We confirmed their engraftment by bone marrow aspiration at six weeks post-transplant, and then they were randomly assigned to the alcohol vs. control groups. After 2 weeks of adjustment, mice were fed a liquid diet (their only source of water and food) containing alcohol 5% vol/vol vs. iso-caloric control diet for 8 weeks. At 4 and 8 weeks, the alcohol-fed group showed significant myeloid bias, reaching greater than 3 times higher myeloid-to-lymphoid ratio than the control group at 8 weeks, while overall engraftment levels were not different. We will report the results of the secondary transplantation of this experiment at the annual meeting. After 8-week alcohol feeding, sorted human CD45+CD34+ cells from mouse BM were subject to 10x Genomics Chromium X 3' HT single-cell RNA-sequencing (scRNA-seq). Complementing the flow data, our transcriptomic data portrayed stark differences between the two groups. Over 50% of cells in the alcohol group comprised granulocyte progenitors, with B cell progenitors representing less than 25%. Inversely, control group data revealed approximately 30% granulocyte progenitors and 55% B cell progenitors, confirming the myeloid bias of HSPC caused by alcohol. Examining differentially expressed genes and GSEA highlighted a significant upregulation of genes associated with the response to virus and type 1 interferon (IFN-1) signature across all HSPC types. A closer look into the gene set showed increased expression of genes tied to cytosolic double-strand (ds) RNA sensing pathways and subsequent interferon type 1 response. Specifically, dsRNA sensors like MDA5, RIG1, PKR, and OAS1-3, as well as interferon regulatory or responsive genes, such as IRF7, ISG15, IFI6, IFI44L, MX1, and STAT1, were notably increased in HSCs and most progenitor populations. Unexpectedly, alcohol-exposed HSCs tended to be in the G0/G1 phase rather than the S or M phase compared to their counterpart upon cell cycle analysis using scRNA-seq data. This may be due to the activation of the PKR pathway, a known cell cycle inhibitor in mouse HSCs. In summary, our findings demonstrate that chronic moderate alcohol consumption can significantly reshape the hematopoietic system through myeloid-biased differentiation and potentiate inflammation by activating the cytosolic dsRNA sensing pathways and IFN-1 response.

IPSC-Derived Mesenchymal Stem Cells Supports the Ex Vivo expansion of Human Hematopoietic Stem and Progenitor Cells Via Sterile Inflammation Pathway

Ex-vivo expansion of human hematopoietic stem/progenitor cells (HSPCs) represents a promising technology for investigating HSPCs' function and overcoming donor shortage for transplantation. HSPCs appropriately regulate self-renewal and differentiation, relying on hematopoietic supporting cells within the bone marrow microenvironment. Notably, various strategies employing primary mesenchymal stem cells (MSCs) as hematopoietic supporting cells have demonstrated successful HSPC expansion (De Lima, et al, N. Engl. J. Med. 2012). However, the preparation of primary MSCs includes harmful procedures, hindering the stable supply of high-quality hematopoietic supporting cells, thereby limited expansion capability and enhancement of clone variation. To overcome these issues, we have established the high-quality type of an immortalized MSCs (imMSCs) cell line derived from human induced pluripotent cells (iPSCs) with knockdown of p53 gene. The imMSCs exhibited exponential proliferation over 2 months, while retaining their MSC phenotype and functionality. To examine the supporting capability of imMSCs on HSPC expansion, we performed coculture experiments with cord blood-derived CD34 + cells in the presence or absence of combination of small compounds StemRegenin-1 and UM171 (SU). We compared four distinct culture conditions, i.e., i) the basic culture with cytokines (SCF, THPO, and FLT3L), as a control; ii) cocultured with imMSCs (imMSC condition); iii) SU alone (SU condition); and iv) cocultured with imMSC and SU (imMSC+SU condition). After a 7-day culture period, we observed a 2 to 3-fold expansion of the CD34 +CD45RA -CD90 + (phenotypic HSC) population in the imMSC condition compared to control. Moreover, the addition of SU (imMSC+SU condition) demonstrated a synergistical effect in promoting HSC expansion (up to 9-fold). Meanwhile, to evaluate the functional potential of expanded HSPCs in vivo, we conducted a xenotransplantation assay using NOG mice as recipients. The expanded HSPCs obtained from the initial 1,000 CD34 + cells were transplanted into the femoral bone of sub-lethally irradiated mice (2Gy). After 12 weeks, mice were sacrificed, and the proportion of human CD45 + cells in the murine bone marrow were analyzed with flow cytometer. All experimental conditions (ii, iii, and iv in the above) enabled to increase chimerism levels compared to the control (i). Notably, the imMSC+SU condition showed a significantly higher chimerism level compared to that of the freshly isolated CD34 + cells, highlighting the efficacy of our expanded HSPCs. Furthermore, a limiting dilution assay using immunodeficient mice revealed that the imMSC, SU, and imMSC+SU conditions showed 3.5-fold, 7.4-fold, and 20-fold expansion of functional HSCs, respectively, in comparison to the control. To elucidate the underlying mechanism how intervention promote HSPC expansion, we employed RNA sequencing analysis. We found that interferon signaling genes (ISGs) were significantly elevated in HSPCs from either imMSC or imMSC+SU compared to other conditions. This finding of elevated ISGs was recapitulated by freshly isolated CB-derived HSCs (uncultured), indicating that ISGs may contribute to the self-renewal ability of both uncultured HSC and those cocultured with imMSCs. While multiple roles of inflammatory signaling, including ISGs, have been identified in hematopoiesis, its role in the fate decision of cord blood-derived HSCs remains poorly understood. To gain further insights, we examined the role of the IFN pathway through shRNA-mediated knockdown of IRF3/7 in CD34 + HSPCs. Our results revealed that the knockdown of IRF3/7 in HSPCs led to a significant reduction in the phenotypic HSC population in all conditions, suggesting the critical role of interferon signaling in HSC expansion. In this context, we attempted to recapitulate the effects of imMSCs by administering recombinant IFN-2α with various doses. However, IFN-2α administration at any doses failed to promote HSC expansion, suggesting transcript of ISGs-dependent but not IFN protein-dependent mechanism might be crucial for the HSC self-renewal. Taken together, our new co-culture system using imMSCs should contribute to a better understanding of the regulatory networks governing HSPC biology as well as the future clinical application of HSPC expansion.

VLA-4 Agonist Enhances the Capacity of HSC Engraftment and Immune Reconstitution

Poor donor cell implantation and delayed hematopoietic reconstitution are important factors affecting the prognosis of patients after allogeneic hematopoietic stem cell transplantation (Allo-HSCT). However, our understanding of the critical endogenous signals that reconstitute hematopoiesis in transplant patients remains limited，which is essential for achieving targeted interventions and preventing donor-recipient chimeric states after transplantation. Here, we revealed the reconstitution dynamics of human allogeneic hematopoietic stem and progenitor cells (HSPCs) and immune cell subpopulations in the post-transplantation mixed donor chimerism (MDC) to fully donor chimerism (FDC) state at single-cell resolution. The hematopoietic stem cell (HSC) implantation process involves extensive interactions with immune cells mainly through surface Kcnq5, Itga4 (encoding a subunit of VLA-4), Tfrc, CD44 receptor, and ligands for Rps19, B2m, etc. Further identification of VLA-4(integrin α4β1) as a potential regulatory target for HSC implantation by single-cell sequencing of a stable allogeneic bone marrow transplantation (Allo-BMT) MDC-FDC mouse model. Grafts Itgα4 and Itgβ1 deletion impair donor implantation and further diminish the efficacy of donor lymphocyte infusion (DLI) following Allo-BMT, rather than directly affecting DLI. Critically, we identified a VLA-4 agonist that promotes donor implantation in the mixed chimeric mouse model of Allo-BMT in a time-dependent manner, with the most pronounced promotion of early T cell implantation in particular. Moreover, it was found to enhance the self-renewal capacity of HSC and lymphoid reconstitution in competitive transplantation and human umbilical cord blood transplantation xenograft experiments. Fortunately, VLA-4 agonist does not exacerbate the acute graft-versus-host disease(aGVHD) process while maintaining the graft-versus-leukemia (GVL) effect. VLA-4 agonist not only upregulates of both Itgα4 and Itgβ1 expression, but also promotes the ERK/2 phosphorylation pathway, which affects HSC biological functions, and this process can be inhibited by ERK kinase inhibitors. These results provide insights into the regenerative process of transplanted HSPCs in human patients，and demonstrate a previously unknown positive regulation of HSC engraftment and immune reconstitution by VLA-4, and allow for a new and simple translational strategy to enhance HSC transplantation.

Efficient and Specific In Vivo Genetic Engineering of Human Hematopoietic Stem Progenitor Cells without Selective Conditioning

Achieving in vivo genetic engineering of hematopoietic stem progenitor cells (HSPC) has the potential to transform treatment for hematological disorders. An ideal in vivo delivery platform should provide access to resting HSPC at low doses, in different in vivo compartments and with high specificity. We developed a multi-model strategy to assess efficiency and specificity of access to human HSPC, without reliance on conditioning to selectively enrich for gene-modified cells, and we achieved efficient genetic engineering of Lin-CD34+ cells and individual HSPC subtypes using highly potent lentiviral vectors (LV) and gene editing virus-like particles (VLP). We first conducted a high-resolution characterization of access to resting HSPC and individual FACS-sorted HSPC subtypes in vitro with different LV envelopes, including a detailed analysis of insertion site distribution in resting vs cytokine activated CD34+ cells. With a BaEVTR LV we achieved transduction of ~100% resting HSPC at MOI 66 (vs ~40% with a VSV-G LV at MOI 50,750) and ~20% of FACS-sorted hematopoietic stem cells (HSC) at MOI 32. This translated into 2-4% transduction of bone marrow (BM) huCD45+ cells when the same BaEVTR LV was dosed in NBSGW long-term humanized mice. However, such models carry a highly lymphoid-skewed human hematopoiesis and are not well suited for measuring access to all HSPC subtypes in all compartments. We therefore established 2 additional in vivo experimental scenarios. To measure LV access to HSPC in the peripheral blood (PB), we followed long-term NBSGW mice where BaEVTR LV was dosed intravenously (IV) immediately after HSPC infusion. In this model we achieved targeting of 23% early engrafting BM HSPC which was mirrored by a ~20% transduction in PB myeloid cells during the first 6 weeks, later stabilizing at 5-7% from week 8 up to week 16. To then measure access to HSPC in the BM niche, we characterized early human HSPC dynamics in the BM of NBSGW mice along 5 timepoints over the first 19 days after CD34+ cell infusion, with or without AMD3100/G-CSF mobilization. We then dosed BaEVTR LV at D7 post-humanization, a timepoint when we established that engrafted human cells have a more physiological lineage distribution, are few and remained confined in the BM even upon mobilization. In this model we demonstrated that a single BaEVTR LV dose injected IV can reach 5-6.4% of BM-resident HSPC. Based on these data, we developed a BaEVTR VLP carrying a CRISPR/Cas9 editor targeting the B2M locus. This VLP displayed 93% editing efficiency in vitro in resting HSPC and 38 to 61% in FACS-sorted HSC at MOI 16 and MOI 83 respectively. When dosed in vivo in the D7 post-humanization model, our VLP achieved B2M KO in 16% and 31% BM-resident Lin-CD34+ cells with 1 and 3 consecutive IV injections respectively, including 14% of phenotypic HSC. Importantly, a comparable level of in vivo editing of BM Lin-CD34+ cells (12%) was achieved upon equivalent pilot single VLP dosing in long-term humanized mice despite them carrying substantially higher numbers of human HSPC in the BM and a complete and stable PB human blood cell engraftment. Secondary transplantation experiments to confirm editing of long-term HSC are ongoing. We proved delivery of genetic payloads to resting HSPC in vitro and access to HSPC in different compartments in vivo with high efficiency. Adding a specific tropism for HSPC has the potential to enhance on-target bioavailability impacting effectiveness and safety of our delivery systems. To this aim, we are now testing LV and VLP carrying HSPC-targeted envelopes engineered with viral fusion proteins (retargetable fusogens). We will present comprehensivedata showing that a LV pseudotyped with a CD133-targeted fusogen combines in vitro and in vivo potency on par with BaEVTR LV with high specificity for CD133+ cells, as seen upon titrations on 8 cell lines and 10 primary human cell types as well as in spike-in experiments aimed at measuring CD133+ HSPC transduction when diluted in a mixture of CD133- human blood cells in vitro and in NBSGW mice. Furthermore, we will show that a CD117-targeted LV can transduce, in vivo, a cell population exclusively located in the BM and comprising as little as 0.3% of total huCD45+ cells with a 175-fold increased specificity as compared to envelopes with broad specificity. These results set the basis for generating a potent candidate for in vivo delivery of genetic payloads to HSPC with high efficiency and specificity.

M6A Mediated Ribostasis of RNA Stress Granule Assembly Governs Blood Development and Regeneration

Hematopoietic stem cell (HSC) form diverse cell types and requires transcription and translation regulation. Using CD34+ human hematopoietic stem and progenitor cells (hCD34+) combined with N6-methyladenosine (m6A) sequencing, we identified a novel regulatory mechanism via stress granule regulation during erythropoiesis. Our single-nucleotide resolution m6A analysis of the whole transcriptome identified ~4800 transcripts to be m6A modified and showed demethylation (>2-14 fold) during erythropoiesis. Genome-wide CHIP-seq analysis of hCD34+ cell erythropoiesis with anti-Gata1 showed an enhancer enrichment at the ALKBH5 RNA demethylase gene. Using ALKBH5 CRISPR Cas9 KO strategy combined with hCD34+ cell in-vitro differentiation protocol we observed a block in hCD34+ cell erythropoiesis thus validating Alkbh5 role in erythropoiesis. hCD34+ ALKBH5 KO cells showed a failure of RNA m6A demethylation and discovered stress granule (SGs) transcripts m6A levels to be upregulated (>2-4 fold, p=0.01). Stress granules are membrane-less organelles composed of various RNA binding proteins. SGs form a hub in the cytoplasm that governs RNA metabolism and global proteome by translational regulation. Our analysis showed ATXN2, G3BP1/2, Tia-1,and PABPC1 SGs transcripts are m6a modified at 3'UTR. K562 cell ALKBH5 KO erythropoiesis model led to massive m6A upregulation on ATXN2 3'UTR (>14 fold, p=0.001) and other SGs transcripts. Mass spectrometry analysis of hCD34+ ALKBH5 KO cell proteome showed downregulation of ATXN2(>2 fold, -log P value2), confirming 3' UTR m6A mediated protein synthesis regulation. ALKBH5 KO hCD34+ cells showed aggregation of SGs protein in the cytoplasm due to altered SG protein stoichiometry(>4-10 fold, p=0.001). Zebrafish alkbh5 KO showed defective blood development and anemia. STED microscopy of the whole kidney marrow of alkbh5 KO fish showed the presence of G3BP and ATXN2 SGs with m6A labeled RNAs. alkbh5 KO zebrafish blood and kidney marrow analysis also showed increased myeloid and lymphoid lineages indicating early lineage bias. We used the alkbh5 KO zebrafish model and subjected it to phenyl hydrazine-induced anemia to study its role in stress-induced regeneration. Using FACS analysis of whole kidney marrow, we show red cell recovery failure in alkbh5 KO zebrafish models. Temporal analysis of alkbh5 KO marrow cells during PHZ-induced acute anemia and regeneration showed a systemic failure to resolve SGs compared to controls. We confirmed these results in the mouse ALKBH5 KO model establishing the role of ALKBH5 in stress erythropoiesis. Furthermore, hCD34+ ATXN2 KO combined with Methocult-colony formation assay showed defects in erythroid and myeloid lineages. Super-resolution microscopy of hCD34+ ATXN2 KO cells phenocopied the presence of disease-associated ALS (amyotrophic lateral sclerosis) stress granules with TDP-43, G3BP1/2, PABPC1, and Tia-1 enriched with m6A labeled RNAs. Using the K562 ALKBH5 KO erythropoiesis model and ATXN2 lentiviral overexpression, we effectively resolved stress granules and blocks in erythropoiesis. In summary, m6A marks on ATXN2 mRNA regulate SG biology, affecting HSC differentiation and impacting blood development. SG-mediated regulation of HSC fate may highlight new therapeutic strategies for blood disorders.

A novel FOXP3 knockout-humanized mouse model for pre-clinical safety and efficacy evaluation of Treg-like cell products

Forkhead box P3 (FOXP3) is an essential transcription factor for regulatory T cell (Treg) function. Defects in Tregs mediate many immune diseases including the monogenic autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), which is caused by FOXP3 mutations. Treg-cell products are a promising modality to induce allograft tolerance or reduce the use of immunosuppressive drugs to prevent rejection, as well as in the treatment of acquired autoimmune diseases. We have recently opened a Phase I clinical trial for IPEX patients using autologous engineered Treg-like cells, CD4LVFOXP3. To facilitate the preclinical studies, a novel humanized-mouse (hu-mouse) model was developed whereby immune-deficient mice were transplanted with human hematopoietic stem progenitor cells (HSPCs) in which the FOXP3 gene was knocked out (FOXP3KO) using CRISPR/Cas9. Mice transplanted with FOXP3KO HSPCs had impaired survival, developed lymphoproliferation 10-12 weeks post-transplant and T-cell infiltration of the gut, resembling human IPEX. Strikingly, injection of CD4LVFOXP3 into the FOXP3KO hu-mice restored in vivo regulatory functions including control of lymphoproliferation and inhibition of T cell infiltration in the colon. This humanized-mouse disease model can be reproducibly established and constitutes an ideal model to assess preclinical efficacy of human Treg cell investigational products.

Targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells in vivo using the engineered AVID adenovirus vector platform

Targeted delivery and cell-type-specific expression of gene-editing proteins in various cell types invivo represent major challenges for all viral and non-viral delivery platforms developed to date. Here, we describe the development and analysis of artificial vectors for intravascular delivery (AVIDs), an engineered adenovirus-based gene delivery platform that allows for highly targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells (HSPCs) invivo after intravenous vector administration. Due to a set of refined structural modifications, intravenous administration of AVIDs did not trigger cytokine storm, hepatotoxicity, or thrombocytopenia. Single intravenous administration of AVIDs to humanized mice, grafted with human CD34+ cells, led to up to 20% transduction of CD34+CD38-CD45RA- HSPC subsets in the bone marrow. Importantly, targeted invivo transduction of CD34+CD38-CD45RA-CD90-CD49f+ subsets, highly enriched for human hematopoietic stem cells (HSCs), reached up to 19%, which represented a 1,900-fold selectivity in gene delivery to HSC-enriched over lineage-committed CD34-negative cell populations. Because the AVID platform allows for regulated, cell-type-specific expression of gene-editing technologies as well as expression of immunomodulatory proteins to ensure persistence of corrected HSCs invivo, the HSC-targeted AVID platform may enable development of curative therapies through invivo gene correction in human HSCs after a single intravenous administration.