Human Human Hematopoietic Stem And Progenitor Cells Research Articles

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.

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.

The Humanized Mouse Model: What Added Value Does It Offer for HIV Research?

In the early 2000s, novel humanized mouse models based on the transplantation of human hematopoietic stem and progenitor cells (HSPCs) into immunocompromised mice were introduced (hu mice). The human HSPCs gave rise to a lymphoid system of human origin. The HIV research community has greatly benefitted from these hu mice. Since human immunodeficiency virus (HIV) type 1 infection results in a high-titer disseminated HIV infection, hu mice have been of great value for all types of HIV research from pathogenesis to novel therapies. Since the first description of this new generation of hu mice, great efforts have been expended to improve humanization by creating other immunodeficient mouse models or supplementing mice with human transgenes to improve human engraftment. Many labs have their own customized hu mouse models, making comparisons quite difficult. Here, we discuss the different hu mouse models in the context of specific research questions in order to define which characteristics should be considered when determining which hu mouse model is appropriate for the question posed. We strongly believe that researchers must first define their research question and then determine whether a hu mouse model exists, allowing the research question to be studied.

Human gene-engineered calreticulin mutant stem cells recapitulate MPN hallmarks and identify targetable vulnerabilities

Calreticulin (CALR) mutations present the main oncogenic drivers in JAK2 wildtype (WT) myeloproliferative neoplasms (MPN), including essential thrombocythemia and myelofibrosis, where mutant (MUT) CALR is increasingly recognized as a suitable mutation-specific drug target. However, our current understanding of its mechanism-of-action is derived from mouse models or immortalized cell lines, where cross-species differences, ectopic over-expression and lack of disease penetrance are hampering translational research. Here, we describe the first human gene-engineered model of CALR MUT MPN using a CRISPR/Cas9 and adeno-associated viral vector-mediated knock-in strategy in primary human hematopoietic stem and progenitor cells (HSPCs) to establish a reproducible and trackable phenotype in vitro and in xenografted mice. Our humanized model recapitulates many disease hallmarks: thrombopoietin-independent megakaryopoiesis, myeloid-lineage skewing, splenomegaly, bone marrow fibrosis, and expansion of megakaryocyte-primed CD41+ progenitors. Strikingly, introduction of CALR mutations enforced early reprogramming of human HSPCs and the induction of an endoplasmic reticulum stress response. The observed compensatory upregulation of chaperones revealed novel mutation-specific vulnerabilities with preferential sensitivity of CALR mutant cells to inhibition of the BiP chaperone and the proteasome. Overall, our humanized model improves purely murine models and provides a readily usable basis for testing of novel therapeutic strategies in a human setting.

Application of a Spacer-nick Gene-targeting Approach to Repair Disease-causing Mutations with Increased Safety.

The CRISPR/Cas9 system is a powerful tool for gene repair that holds great potential for gene therapy to cure monogenic diseases. Despite intensive improvement, the safety of this system remains a major clinical concern. In contrast to Cas9 nuclease, Cas9 nickases with a pair of short-distance (38-68 bp) PAM-out single-guide RNAs (sgRNAs) preserve gene repair efficiency while strongly reducing off-target effects. However, this approach still leads to efficient unwanted on-target mutations that may cause tumorigenesis or abnormal hematopoiesis. We establish a precise and safe spacer-nick gene repair approach that combines Cas9D10A nickase with a pair of PAM-out sgRNAs at a distance of 200-350 bp. In combination with adeno-associated virus (AAV) serotype 6 donor templates, this approach leads to efficient gene repair with minimal unintended on- and off-target mutations in human hematopoietic stem and progenitor cells (HSPCs). Here, we provide detailed protocols to use the spacer-nick approach for gene repair and to assess the safety of this system in human HSPCs. The spacer-nick approach enables efficient gene correction for repair of disease-causing mutations with increased safety and suitability for gene therapy. Graphical overview.

Pre-Clinical Investigation of a Novel Small Molecule Inhibitor Targeting YBX1 in AML

Recently, our group identified the pleiotropic DNA and RNA binding protein YBX1 as a specific dependency and therapeutic target in acute myeloid leukemia (AML) that is essential for stabilizing oncogenic transcripts and thereby maintains oncogenic protein expression (Perner et al., 2021). Selective pharmacologic targeting of YBX1 in AML, however, has not been achieved so far. Here, we investigate the functional consequences of a novel small molecule inhibitor (SU056) of YBX1 in AML. We examined the viability of several human AML cell lines (MOLM13, HL60, EOL1, THP1, Kasumi1, ML2, Mv4;11, OCI-AML3, NOMO1) upon SU056 treatment and found significant reduction of cell growth in a concentration dependent manner. Inhibition of cell growth could be mainly attributed to impaired proliferative capacity at nanomolar concentrations. Pharmacologic inhibition of YBX1 resulted in impaired colony formation (mean colony number DMSO: 231.3±13.88 vs. SU056 500nM: 154.7±8.29; p<0.0001), cell cycle arrest (cells in S phase: DMSO: 37.3±2.4 vs. SU056 500nM: 27.2±1.67; p=0.0127) and induction of myeloid differentiation. Consistently, in a xenograft model of human AML, SU056 treatment resulted in prolonged disease latency (MOLM13 diluent: 62 days OS vs. SU056: not reached, p=0.0013; OCI-AML3 diluent: 79 days OS vs. not reached, p=0.0001) and significant reduction of disease penetrance (MOLM13 diluent: 100% vs. SU056: 24,9%; OCI-AML3 diluent: 100% vs. SU056: 9,1%). Consistently, percentage of SU056 treated human leukemic cells in the bone marrow of recipient mice was decreased for both, MOLM13 (mean diluent: 41,3% vs. SU056: 7,6%) and OCI-AML3 (mean diluent: 28,9% vs. SU056: 2,9%). Likewise, pharmacologic inhibition of YBX1 reduced colony formation and total cell numbers of primary human AML cells in a dose-dependent manner. To be clinically relevant, such treatment must have a suitable therapeutic index between human hematopoietic stem- and progenitor cells (HSPCs) and their malignant counterpart. This is particularly important, considering the role of YBX1 in mRNA splicing and processing. Of note, pharmacologic inhibition of YBX1 did not perturb colony formation of normal HSPCs derived from apheresis donors. These findings were confirmed by investigating primary human leukemic cells in a patient-derived xenograft (PDX) model of AML. We aimed to assess whether loss in leukemia cell competition induced by pharmacologic targeting of YBX1 is sufficient for normal hematopoietic cells to outcompete leukemic cells in vivo. 1x106 normal human HSPCs were transplanted along with 2x104 leukemic cells to assess for efficacy of SU056-treatment versus diluent control. In vivo treatment with SU056 (20mg/kg, i.p.) for 3 weeks resulted in a significant decrease in the AML blast number in the PB (mean blast %: diluent: 38.5%; SU056: 2.25%; p<0.0001). This resulted in increased survival of SU056 treated recipients (OS diluent: 83 days vs. SU056: not reached). To confirm the proposed mechanism by which SU056 impairs leukemia growth we performed global transcriptome and proteome analysis following treatment with SU056 (500nM, 96h). Proteome profiling revealed a total of 2641 deregulated proteins. Gene-ontology analysis indicated deregulation of YBX1 targets and proteins associated with cell cycle, DNA repair, mRNA splicing/processing, and transcriptional regulation. Of note, overlap between transcriptional regulation (as determined by mRNA-Seq) and changes in proteome abundance revealed less than 5% target genes/proteins, indicating that SU056 may in fact interfere with binding of oncogenic mRNAs to YBX1 and thereby may reduce translation of oncogenic transcripts. In order to define functional consequences of pharmacologic YBX1 inhibition and explore potential vulnerabilities of drug combinations, we performed a genome-wide CRISPR/Cas9 screen in human AML cells treated with SU056 versus diluent control. Validation experiments are currently ongoing. Taken together, our data confirm the therapeutic potential to target YBX1 using the novel small molecule SU056. Given its broad applicability across genetic subtypes of AML and lack of toxicity on normal HSPCs, SU056 as a single agent or in combination with established targeted therapies may efficiently help to target and outcompete leukemic cells. Experiments to assess for potential synergy with already established targeted therapies will be presented.

RNAi Screen Identifies MTA1 as an Epigenetic Modifier of Differentiation Commitment in Human HSPCs

The molecular mechanisms regulating key fate decisions of hematopoietic stem cells (HSCs) remain incompletely understood. Here, we targeted global shRNA libraries to primary human hematopoietic stem and progenitor cells (HSPCs) to screen for modifiers of self-renewal and differentiation, and identified metastasis-associated 1 (MTA1) as a negative regulator of human HSPC propagation in vitro. Knockdown of MTA1 by independent shRNAs in primary human cord blood (CB) HSPCs led to a cell expansion during culture and a relative accumulation of immature CD34+CD90+ cells with perturbed in vitro differentiation potential. Transplantation experiments in immunodeficient mice revealed a significant reduction in human chimerism in both blood and bone marrow from HSPCs with knockdown of MTA1, possibly caused by reduced maturation of blood cells. We further found that MTA1 associates with the nucleosome remodeling deacetylase (NuRD) complex in human HSPCs, and on knockdown of MTA1, we observed an increase in H3K27Ac marks coupled with a downregulation of genes linked to differentiation toward the erythroid lineage. Together, our findings identify MTA1 as a novel regulator of human HSPCs in vitro and in vivo with critical functions for differentiation commitment.

Increased expression of RUNX3 inhibits normal human myeloid development

RUNX3 is a transcription factor dysregulated in acute myeloid leukemia (AML). However, its role in normal myeloid development and leukemia is poorly understood. Here we investigate RUNX3 expression in both settings and the impact of its dysregulation on myelopoiesis. We found that RUNX3 mRNA expression was stable during hematopoiesis but decreased with granulocytic differentiation. In AML, RUNX3 mRNA was overexpressed in many disease subtypes, but downregulated in AML with core binding factor abnormalities, such as RUNX1::ETO. Overexpression of RUNX3 in human hematopoietic stem and progenitor cells (HSPC) inhibited myeloid differentiation, particularly of the granulocytic lineage. Proliferation and myeloid colony formation were also inhibited. Conversely, RUNX3 knockdown did not impact the myeloid growth and development of human HSPC. Overexpression of RUNX3 in the context of RUNX1::ETO did not rescue the RUNX1::ETO-mediated block in differentiation. RNA-sequencing showed that RUNX3 overexpression downregulates key developmental genes, such as KIT and RUNX1, while upregulating lymphoid genes, such as KLRB1 and TBX21. Overall, these data show that increased RUNX3 expression observed in AML could contribute to the developmental arrest characteristic of this disease, possibly by driving a competing transcriptional program favoring a lymphoid fate.

Editing TINF2 as a potential therapeutic approach to restore telomere length in dyskeratosis congenita

AbstractMutations in the TINF2 gene, encoding the shelterin protein TIN2, cause telomere shortening and the inherited bone marrow (BM) failure syndrome dyskeratosis congenita (DC). A lack of suitable model systems limits the mechanistic understanding of telomere shortening in the stem cells and thus hinders the development of treatment options for BM failure. Here, we endogenously introduced TIN2-DC mutations in human embryonic stem cells (hESCs) and human hematopoietic stem and progenitor cells (HSPCs) to dissect the disease mechanism and identify a gene-editing strategy that rescued the disease phenotypes. The hESCs with the T284R disease mutation exhibited the short telomere phenotype observed in DC patients. Yet, telomeres in mutant hESCs did not trigger DNA damage responses at telomeres or show exacerbated telomere shortening when differentiated into telomerase-negative cells. Disruption of the mutant TINF2 allele by introducing a frameshift mutation in exon 2 restored telomere length in stem cells and the replicative potential of differentiated cells. Similarly, we introduced TIN2-DC disease variants in human HSPCs to assess the changes in telomere length and proliferative capacity. Lastly, we showed that editing at exon 2 of TINF2 that restored telomere length in hESCs could be generated in TINF2-DC patient HSPCs. Our study demonstrates a simple genetic intervention that rescues the TIN2-DC disease phenotype in stem cells and provides a versatile platform to assess the efficacy of potential therapeutic approaches in vivo.

Ex Vivo Generation of CAR Macrophages from Hematopoietic Stem and Progenitor Cells for Use in Cancer Therapy.

Chimeric antigen receptor (CAR) T-cell therapies have shown impressive results in patients with hematological malignancies; however, little success has been achieved in the treatment of solid tumors. Recently, macrophages (MΦs) were identified as an additional candidate for the CAR approach, and initial proof of concept studies using peripheral blood-derived monocytes showed antigen-redirected activation of CAR MΦs. However, some patients may not be suitable for monocyte-apheresis, and prior cancer treatment regimens may negatively affect immune cell number and functionality. To address this problem, we here introduce primary human hematopoietic stem and progenitor cells (HSPCs) as a cell source to generate functional CAR MΦs ex vivo. Our data showed successful CAR expression in cord blood (CB)-derived HSPCs, with considerable cell expansion during differentiation to CAR MΦs. HSPC-derived MΦs showed typical MΦ morphology, phenotype, and basic anti-bacterial functionality. CAR MΦs targeting the carcinoembryonic antigen (CEA) and containing either a DAP12- or a CD3ζ-derived signaling domain showed antigen redirected activation as they secreted pro-inflammatory cytokines specifically upon contact with CEA+ target cells. In addition, CD3ζ-expressing CAR MΦs exhibited significantly enhanced phagocytosis of CEA+ HT1080 cells. Our data establish human HSPCs as a suitable cell source to generate functional CAR MΦs and further support the use of CAR MΦs in the context of solid tumor therapy.

Glucose Metabolism and Aging of Hematopoietic Stem and Progenitor Cells.

Comprehensive proteomics studies of human hematopoietic stem and progenitor cells (HSPC) have revealed that aging of the HSPC compartment is characterized by elevated glycolysis. This is in addition to deregulations found in murine transcriptomics studies, such as an increased differentiation bias towards the myeloid lineage, alterations in DNA repair, and a decrease in lymphoid development. The increase in glycolytic enzyme activity is caused by the expansion of a more glycolytic HSPC subset. We therefore developed a method to isolate HSPC into three distinct categories according to their glucose uptake (GU) levels, namely the GUhigh, GUinter and GUlow subsets. Single-cell transcriptomics studies showed that the GUhigh subset is highly enriched for HSPC with a differentiation bias towards myeloid lineages. Gene set enrichment analysis (GSEA) demonstrated that the gene sets for cell cycle arrest, senescence-associated secretory phenotype, and the anti-apoptosis and P53 pathways are significantly upregulated in the GUhigh population. With this series of studies, we have produced a comprehensive proteomics and single-cell transcriptomics atlas of molecular changes in human HSPC upon aging. Although many of the molecular deregulations are similar to those found in mice, there are significant differences. The most unique finding is the association of elevated central carbon metabolism with senescence. Due to the lack of specific markers, the isolation and collection of senescent cells have yet to be developed, especially for human HSPC. The GUhigh subset from the human HSPC compartment possesses all the transcriptome characteristics of senescence. This property may be exploited to accurately enrich, visualize, and trace senescence development in human bone marrow.

ZNF384 Fusion Oncoproteins Drive Lineage Aberrancy in Acute Leukemia.

Expression of ZNF384 FO early in hematopoiesis results in binding and deregulation of key hematopoietic regulators, skewing of hematopoiesis, and priming for leukemic transformation. These results reveal the interplay between cell of origin and expression of ZNF384 FO to mediate lineage ambiguity and leukemia development. This article is highlighted in the In This Issue feature, p. 171.

Assessing Stealth and Sensed Base Editing in Human Hematopoietic Stem/Progenitor Cells

Genome editing represents a promising tool to manipulate human hematopoietic stem and progenitor cells (HSPCs) opening the possibility to correct hematopoietic diseases avoiding the risk of insertional mutagenesis and uncontrolled expression of the transgene, issues that emerged with retroviral and lentiviral gene therapy. Engineered nucleases such as CRISPR/Cas9 have enable targeted genetic manipulation in human HSPCs for therapeutic purposes. Still, nuclease-induced DNA double-strand breaks (DSBs) trigger p53-dependent DNA damage response affecting HSPC properties and may lead to unintended chromosomal rearrangements. Base editing (BE) holds the promise for precise editing by the introduction of specific single-nucleotide variants (SNVs) while bypassing the requirement for DSBs. In particular, base editors are composed by: i) a deamination domain that directly modifies nucleotides comprised within a defined editing window in one of the two genomic strands, and ii) a nickase Cas9 that introduces a single-strand break (SSB) on the other strand to promote more efficient base editing. Depending on the type of modification introduced editors are classified in Cytosine (C-) BE (C-G transition to T-A) and Adenine (A-) BE (A-T transition to G-C). However, a comprehensive characterization of efficiency, tolerability and genotoxicity of CBE and ABE in human HSPCs is lacking and is required to instruct the rationale towards safe and effective clinical translation. Here, we developed an optimized mRNA-based protocol for BE in human HSPCs and compared CBE4max, ABE8.20-m and Cas9 nuclease by targeting the same locus (B2M) using the same sgRNA. Common outcome for all editors is disruption of targeted gene expression, which is measured by flow cytometry and Next Generation Sequencing. ABE8.20-m showed higher efficiency than CBE4max and Cas9 nuclease at the target locus (up to 90, 40 and 50%), which was consistent across HSPC subpopulations comprising the most primitive compartment endowed with long term repopulation potential and cell sources (such as cord blood- and mobilized peripheral blood-derived HSPCs). Importantly, Cas9, but not CBE4max and ABE8.20m, treated HSPCs showed lower in-vitro clonogenic capacity than mock electroporated cells. Transcriptional analyses uncovered that CBE4max, but not ABE8.20-m, triggered p53 pathway activation, albeit at lower extent as compared to Cas9 and presumably consequent to a fraction of single-strand nicks turning into DSB upon DNA replication. Additionally, BE, and particularly CBE4max, upregulated the expression of interferon-stimulated genes, which was not ascribed to mRNA delivery. Remarkably, despite edited HSPCs showed long-term multilineage capacity in xenotransplanted mice, CBE4max edited cells tended to decrease over time in the graft pointing to some detrimental response to the treatment of the long-term engrafting HSC subset. Overall, our results prompt further investigation on BE sensing in human HSPCs. On-going studies are aimed to investigate clonal dynamics and genome integrity of base-edited HSPCs with the final goal of building confidence for their perspective clinical translation. DisclosuresNaldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Modeling the Development of SRSF2 Mutated Myeloid Malignancies By CRISPR/Cas9 Mediated Genome Engineering of Primary Human Hematopoietic Stem and Progenitor Cells

Introduction: Acute Myeloid Leukemia (AML) is a malignant disease of the bone marrow that can arise from a premalignant condition called clonal hematopoiesis of indeterminate potential (CHIP). Mutations in Serine and Arginine-rich Splicing Factor 2 (SRSF2) are detected in CHIP and mediate a high risk for AML development. Here we used CRISPR/Cas9-mediated genome engineering to introduce a heterozygous SRSF2P95H mutation into primary human hematopoietic stem and progenitor cells (HSPCs) and investigated its functional consequences using both in vitro and in vivo assays.Methods: We used CRISPR/Cas9 technology to introduce a heterozygous mutant (mut) SRSF2P95H into the endogenous SRSF2 gene locus of healthy cord blood HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6) (Figure A). This allows for targeted in-frame integration of mut and/or wildtype (WT) SRSF2 cDNA under the control of the endogenous SRSF2 promoter. Notably, an integrated fluorescent reporter enables the isolation and tracking of heterozygously mutated HSPCs (Figure B). Methylcellulose colony and long-term competition assays of SRSF2 mut and WT HSPCs were performed in vitro. Cells were analyzed by flow cytometry and characterized cytomorphologically. In addition, bulk RNA-seq analyses were performed to characterize differential gene expression and abnormal splicing events. Xenotransplantation into NSG-SGM3 mice was performed in order to assess stem cell characteristics and the in vivo leukemogenic potential of SRSF2 mut HSPCs. Finally, we investigated the mutation-specific effect of the splicing inhibitor Indisulam to determine if SRSF2 mut cells are particularly vulnerable to splicing inhibition.Results: Colony assays (n=9) revealed impaired erythroid and increased monocytic differentiation of SRSF2 mut HSPCs. Quantification of colonies showed a lower frequency of erythroid BFU-E in SRSF2 mut compared to SRSF2 WT HSPCs (mean ± SD; 33.3 ± 12.5% vs. 17.4 ± 10.8%, p=0.00002). In contrast, the frequency of myeloid CFU-M colonies was higher in SRSF2 mut HSPCs compared to SRSF2 WT HSPCs (38.3 ± 7.3% vs. 22.6 ± 6.8%, p = 0.0003) (Figure C). Long-term in vitro competition assays revealed an outgrowth of SRSF2 mut over WT cells in 2 out of 7 donors. Strikingly, after three months of in vitro culture, in one donor, the SRSF2 mut cells developed a blast-like morphology with strong CD34 expression (Figure D). To assess stem cell characteristics and the leukemogenic potential in vivo, we transplanted SRSF2 mut HSPCs from 4 different donors into immunodeficient NSG-SGM3 mice (n=11). SRSF2 mut cells showed a myeloid-skewed engraftment. Cytomorphologic analysis of long-term engrafted SRSF2 mut myeloid cells revealed dysplastic changes such as nuclear abnormalities and extensive cytoplasmic vacuolization. In 4 out of 11 xenografts, human engraftment substantially increased over time with a parallel outgrowth of the SRSF2 mut clone and the appearance of blast-like cells resembling transformation into myeloid leukemia (Figure E). Comparative RNA-seq analysis identified 138 differentially spliced genes, with exon skipping being the dominant altered splicing type. Gene ontology (GO) analysis on differentially expressed genes revealed “Acute Myeloid Leukemia” among the most enriched terms (p-val = 8.2E-07, min FDR = 1.486E-04). When testing the SRSF2-mutation specific effect of the splicing inhibitor Indisulam, SRSF2 mut HSPCs show a significantly lower IC-50 than WT cells (977nM vs. 3574 nM). Strikingly, in competition- and CFU-assays, Indisulam preferentially eradicates SRSF2 mut hematopoietic cells, while sparing WT cells.Conclusion: Using our CRISPR/Cas9 approach, we can successfully introduce heterozygous SRSF2P95H mutants in primary human HSPCs. Mutant SRSF2P95H leads to increased monocytic differentiation, impaired erythroid differentiation, and phenocopy SRSF2P95H driven diseases in patients. Importantly, we show for the first time that the SRSF2 mutation alone is sufficient to induce dysplastic features and even transform healthy human HSPCs into AML-like blasts. Our model allows the identification and therapeutic investigation of specific cellular vulnerabilities caused by SRSF2 mutations and highlights Indisulam as a potential compound to specifically treat individuals carrying a SRSF2 mutation. [Display omitted] DisclosuresEdiriwickrema: Nanosive SAS: Patents & Royalties. Greinix: Novartis: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Therakos: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Celgene: Consultancy, Honoraria; AbbVie: Consultancy; Novartis: Consultancy. Majeti: BeyondSpring Inc.: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc.. Reinisch: Pfizer: Consultancy; Celgene: Research Funding.

Reticular Dysgenesis-Associated Adenylate Kinase 2 Deficiency Impairs Purine Metabolism and Ribosomal Biogenesis during Myelopoiesis

Reticular Dysgenesis (RD) is a particularly grave form of severe combined immunodeficiency (SCID), characterized by maturation arrest of both myeloid and lymphoid lineages paired with sensorineural hearing loss. RD is caused by biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2). AK2 catalyzes the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) in the mitochondrial intermembrane space. Using a CRISPR/Cas9 AK2 biallelic knock out model in human hematopoietic stem and progenitor cells (HSPCs), we have shown that AK2 -/- HSPCs mimic the neutrophil maturation defect in RD patients. Mitochondrial respiration is compromised in AK2 -/- HSPCs, which leads to a decreased NAD +/NADH ratio resulting in reductive stress. Metabolomics analysis by LC-MS/MS showed a significant accumulation of AMP, along with increased AMP/ADP and AMP/ATP ratios in AK2 -/- HSPCs, suggesting that purine metabolism is compromised by AK2 deficiency. Purine metabolism defects, such as deficiencies in adenosine deaminase (ADA) and purine nucleotide phosphorylase (PNP), have long been recognized as a cause of SCID. Furthermore, pharmacological interference with purine metabolism is a highly effective antiproliferative strategy in cancer therapy. In this study, we sought to investigate whether impaired purine metabolism contributes to the myelopoietic defect caused by AK2 deficiency.ResultsWe explored how purine metabolism affects myelopoiesis by differentiating HSPCs in media containing no nucleosides (nucleoside-), mixed nucleosides (nucleoside+) or adenosine only (adenosine+). We observed no difference in proliferation or neutrophil maturation between nucleosides- and nucleoside+ media for both control and AK2 -/- HSPCs, suggesting that AK2 -/- HSPCs do not rely on exogenous nucleosides. Interestingly, control HSPCs cultured in adenosine+ media showed severe proliferation and neutrophil maturation defects that mimic AK2 deficiency, suggesting that purine imbalance is detrimental to myelopoiesis.Previous metabolomics analysis showed a significant accumulation of inosine monophosphate (IMP) in AK2 -/- HSPCs. Since IMP can be produced through AMP deamination by AMPD, we asked whether the IMP accumulation in AK2 -/- HSPCs is caused by converting excess AMP to IMP. An LC-MS/MS analysis showed that AMPD inhibitor (AMPDi) treatment significantly lowered IMP levels and increased AMP levels in AK2 -/- HSPCs, indicating that AMP deamination is a major source of IMP accumulation in AK2 -/- HSPCs. Furthermore, AMPDi treatment did not improve, but rather slightly aggravated neutrophil differentiation in AK2 -/- HSPCs, suggesting that the AK2 -/- neutrophil maturation defect is not caused by IMP accumulation. This raises the possibility that AK2 -/- HSPCs employ AMP deamination as a mechanism to curtail the toxicity of excess AMP.Since purine is a building block of RNA, and ribosomal RNA (rRNA) constitutes >85% of cellular RNA content, we asked whether rRNA synthesis is compromised by AK2 deficiency. Pyronin Y staining showed a significant decrease in rRNA content in AK2 -/- HSPCs. Nascent peptide synthesis rate was also decreased in AK2 -/- HSPCs, as quantified by OP-puromycin uptake. These findings are corroborated by RNA-seq analysis of AK2 -/- and control HSPCs, which showed that ribosomal subunits, ribosomal biogenesis and ribonucleoprotein complex assembly are among the top down-regulated pathways. The data suggest that defective purine metabolism in AK2 -/- HSPCs impairs ribosomal biogenesis and protein synthesis.ConclusionOur studies showed that purine imbalance in HSPCs impairs myeloid proliferation and neutrophil maturation. AK2 depletion in HSPCs leads to AMP accumulation and defective ribosomal biogenesis. AK2 -/- HSPCs convert excess AMP to IMP, possibly as a means to mitigate AMP toxicity. However, AMP deamination activities alone are not sufficient to lower AMP levels to those of control HSPCs. We are currently testing whether boosting 5'-nucleotidase activities (cNIA, cN1B and cNII) in AK2 -/- HSPCs can decrease AMP levels and rescue the neutrophil maturation defect. As purine metabolic defects are associated with diverse immune and non-immune abnormalities, further understanding of how purine metabolism governs differentiation of human HSPCs will enable us to develop novel therapeutic strategies for RD and other purine disorders. DisclosuresPorteus: CRISPR Therapeutics: Current equity holder in publicly-traded company; Allogene Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Versant Ventures: Consultancy; Ziopharm: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Graphite Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Morrison: Garuda Therapeutics: Other: founder and SAB member ; Kojin Therapeutics: Other: SAB member ; Frequency Therapeutics: Other: SAB member ; Ona Terapeutics: Other: SAB member ; Protein Fluidics: Other: SAB member .

Introduction and Genetic Correction of Calreticulin Mutations in Human Hematopoietic Stem and Progenitor Cells Sheds Light on MPN Pathogenesis

Introduction:Recurrent mutations in calreticulin (CALR) are present in 70% to 80% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without a JAK2 or MPL mutation. Despite recent advances in understanding mutant CALR, the detailed mechanisms are not fully elucidated, and current knowledge is mainly based on transgenic mouse models or human cancer cell lines. Thus, to more faithfully model MPN pathogenesis, we first aimed to introduce heterozygous type-1 and type-2 CALR mutations into healthy human hematopoietic stem and progenitor cells (HSPCs) via targeted CRISPR/Cas9-mediated gene knock-in (KI) and investigate its impact on HSPC function in vitro and in vivo. Second, we aimed to correct CALR mutations in patient-derived HSPCs to study their dependence on the initial driver event to exert an MPN phenotype.Methods:We used CRISPR/Cas9 to introduce heterozygous CALR mutations into the endogenous gene locus of healthy cord blood-derived HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6). Briefly, Cas9-sgRNA ribonucleoprotein (RNP) was used to cut the DNA. Simultaneously AAV6, carrying either a mutation-bearing or a wildtype control cDNA, was co-delivered to allow for targeted in-frame integration. This way, mutant CALR remains under the control of the endogenous promoter. Concurrent integration of a fluorescent reporter downstream of the mutated exon, enabled purification and tracking of modified cells via flow cytometry. Purified CRISPR-modified HSPCs were used for in vitro collagen-based colony-forming assays, proliferation and differentiation assays in liquid culture, and intrafemoral transplantation into immunodeficient NSG mice to assess their pathogenic potential.Results:Our CRISPR/Cas9 KI strategy enabled us to efficiently generate and enrich for heterozygous CALR mutant human HSPCs. Modified cells harbor the mutation at the endogenous CALR locus with intact gene regulatory regions. Correct integration and transcript expression were confirmed on DNA and RNA level by sanger sequencing. Additionally, CALR mutant protein expression was confirmed via immunohistochemistry using a diagnostically approved mutant-specific antibody.Type-1 and type-2 CALR mutations led to TPO-independent growth of CD34 + HSPC-derived cells and a two-fold (p<0.01) increase of megakaryocyte colonies in collagen-based media compared to wildtype control KI. These findings were corroborated by significantly enhanced CD41 + CD42b + megakaryocyte formation of CALR mutant HSPCs upon liquid culture differentiation.When transplanted into sublethally irradiated immunodeficient NSG mice, CALR mutant HSPCs showed robust engraftment in the bone marrow with a myeloid lineage skewing, outcompetition of wildtype cells and increased formation of CALR mutant CD41 + megakaryocyte progenitors.To investigate, if removal of type-1 and type-2 CALR mutations can ameliorate MPNs, we utilized our KI strategy to correct both CALR mutations in MPN patient-derived HSPCs by replacing them with wildtype sequences. A successful correction was confirmed on DNA and RNA level and by the absence of mutant CALR protein. Opposite to the results from introducing CALR mutations, correcting the mutations led to a two-fold decrease in megakaryocyte colony formation. Interestingly this was only seen in ET and post-ET MF samples, whereas primary MF samples were unaffected, underscoring the importance of other secondary genetic driver events in the pathogenesis of primary MF.Conclusion:Our system allows us to investigate human MPN pathogenesis prospectively and shed light on the transforming mechanisms of mutant CALR in primary HSPCs. We could show that CALR mutations prime HSPCs toward the formation of platelet-producing megakaryocytes. Genetic correction of CALR mutations in MPN patient-derived HSPCs revealed a dependence on the oncogenic mutant CALR driver event in ET and post-ET MF patients, opening the possibility of an ex vivo gene correction approach to remove mutant CALR in patient-derived HSPCs . Lastly, since MPN patient-derived cells have notoriously low engraftment potential in mice, our CRISPR/Cas9-engineered CALR mutant model also provides a powerful new strategy to generate MPN xenotransplants with defined genotypes for the evaluation of novel therapies. DisclosuresGreinix: Celgene: Consultancy; Therakos: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Novartis: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Novartis: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

Longitudinal clonal tracking in humanized mice reveals sustained polyclonal repopulation of gene-modified human-HSPC despite vector integration bias

BackgroundCurrent understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment.MethodsHumanized bone marrow-liver-thymus (hu-BLT) mice were generated by transplanting lentiviral vector-transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25 µl of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias.ResultsA total of 897 HSPC clones were longitudinally tracked in hu-BLT mice—providing a first-ever demonstration of clonal dynamics and coordinated expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 (~ 60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within ± 1 kb of H3K36me3-enriched regions.ConclusionsHuman repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings.

Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells

The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to lentiviral transduction of Cas9 being extremely inefficient in these cells. Here, to overcome these hurdles, we developed a combinatorial system using stable lentiviral delivery of single guide RNA (sgRNA) followed by transient transfection of Cas9 mRNA by electroporation in human cord blood-derived CD34+ HSPCs. We further applied an optimized sgRNA structure, that significantly improved editing efficiency in this context, and we obtained knockout levels reaching 90% for the cell surface proteins CD45 and CD44 in sgRNA transduced HSPCs. Our combinatorial CRISPR/Cas9 delivery approach had no negative influence on CD34 expression or colony forming capacity in vitro compared to non-treated HSPCs. Furthermore, gene edited HSPCs showed intact in vivo reconstitution capacity following transplantation to immunodeficient mice. Taken together, we developed a paradigm for combinatorial CRISPR/Cas9 delivery that enables efficient and traceable gene editing in primary human HSPCs, and is compatible with high functionality both in vitro and in vivo.

SATB1 Regulates GATA1 Protein Expression in Early Hematopoiesis and Is Deregulated in Diamond Blackfan Anemia

The induction of the master erythroid transcription factor, GATA1 during early erythropoiesis is critical for efficient red blood cell production. However, GATA1 is expressed at low levels in hematopoietic stem cells (HSCs) and is moderately induced at both the common myeloid progenitor (CMP) and megakaryocyte/erythroid progenitor (MEP) stages prior to lineage commitment. Diamond Blackfan Anemia is a rare disease, usually associated with ribosomal gene mutations, leading to significant decrease in GATA1 expression and block in early committed erythroid differentiation. Mild defects in other myeloid lineages are also observed, with limited clinical relevance. The importance of GATA1 downregulation in disease pathogenesis is manifested by rare patients with DBA carrying GATA1 mutations. To understand signaling pathways that contribute to the pathogenesis DBA, we perform RNA-seq with mRNA from human CD34+ fetal liver cells and found that the chromatin organizer, Special AT-rich sequence binding protein 1 (SATB1) was prematurely downregulated. Our results further demonstrated that sustained SATB1 expression is critical to maintain required levels of GATA1 protein at both the CMP and MEP stages of differentiation, but not in committed erythroid progenitors. In mice, SATB1 is modestly expressed in HSCs and upregulated during lymphopoiesis. SATB1 is downregulated during myeloid and erythroid differentiation and antagonizes myeloid and erythroid expansion. However, in human hematopoietic stem and progenitor cells (HSPCs), SATB1 is required for efficient expansion of these lineages. SATB1 maintains 78% expression in human MEPs, but is undetectable in early committed erythroid progenitors. In RPS19-insufficient human HSPCs, SATB1 was downregulated to 22% in MEPs (p=0.02). Re-expression of SATB1 corrected a significant subset of deregulated mRNAs, including GATA1 regulators. In the absence of SATB1, one such GATA1 regulator, heat shock protein 70 (HSP70), failed to be induced in ribosome-competent human MEPs, reducing GATA1 protein expression by 35.7% (p= 0.026). Concurrently, MEP expansion was inhibited by 64.5% (p=0.023), reducing erythroid and megakaryocyte expansion by 18.2% (p=0.024) and 20.4% (p=0.183) respectively. SATB1 facilitated the formation of chromatin loops linking together an enhancer element with HSP70 promoters required for HSP70 induction in early differentiation. Although GATA1 is significantly upregulated in committed erythroid progenitors, RPS19-insufficient human CD235+ erythrocytes express GATA1 28.4% of controls (p= 0.011). SATB1 re-expression increased GATA1 expression to 31.4% (p=0.089). Similarly, SATB1 re-expression increased CD235+ expansion from 13.9% to 39.5% (p=0.02) compared to controls. Our data indicate that premature SATB1 downregulation contributes to erythroid failure in DBA by reducing MEP expansion, but aberrant GATA1 expression observed in more mature erythrocytes is predominantly SATB1-independent. However, SATB1-re-expression improved CD11b+ myeloid expansion from 81.2% to 90.4% (p=0.045) and CD41a+ megakaryocyte expansion from 76.7% to 214.7% (p=0.038) respectively. Our results demonstrate that SATB1 plays an important role in human hematopoiesis and is an important regulator of GATA1. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy.

Efficient and nontoxic biomolecule delivery to primary human hematopoietic stem cells using nanostraws

Introduction of exogenous genetic material into primary stem cells is essential for studying biological function and for clinical applications. Traditional delivery methods for nucleic acids, such as electroporation, have advanced the field, but have negative effects on stem cell function and viability. We introduce nanostraw-assisted transfection as an alternative method for RNA delivery to human hematopoietic stem and progenitor cells (HSPCs). Nanostraws are hollow alumina nanotubes that can be used to deliver biomolecules to living cells. We use nanostraws to target human primary HSPCs and show efficient delivery of mRNA, short interfering RNAs (siRNAs), DNA oligonucleotides, and dextrans of sizes ranging from 6 kDa to 2,000 kDa. Nanostraw-treated cells were fully functional and viable, with no impairment in their proliferative or colony-forming capacity, and showed similar long-term engraftment potential in vivo as untreated cells. Additionally, we found that gene expression of the cells was not perturbed by nanostraw treatment, while conventional electroporation changed the expression of more than 2,000 genes. Our results show that nanostraw-mediated transfection is a gentle alternative to established gene delivery methods, and uniquely suited for nonperturbative treatment of sensitive primary stem cells.