Hematopoietic Stem And Progenitor Cells Gene Research Articles

Preclinical lentiviral vector-mediated hematopoietic stem and progenitor cell gene therapy corrects Pompe disease-related muscle and neurological manifestations

Pompe disease, a rare genetic neuromuscular disorder, is caused by a deficiency of acid alpha-glucosidase (GAA), leading to an accumulation of glycogen in lysosomes, and resulting in the progressive development of muscle weakness. The current standard treatment, enzyme replacement therapy (ERT), is not curative and has limitations such as poor penetration into skeletal muscle and both the central and peripheral nervous systems, a risk of immune responses against the recombinant enzyme, and the requirement for high doses and frequent infusions. To overcome these limitations, lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy has been proposed as a next-generation approach for treating Pompe disease. This study demonstrates the potential of lentiviral HSPC gene therapy to reverse the pathological effects of Pompe disease in a preclinical mouse model. It includes a comprehensive safety assessment via integration site analysis, along with single-cell RNA sequencing analysis of central nervous tissue samples to gain insights into the underlying mechanisms of phenotype correction.

In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis

Hematopoiesis is a continuous process of blood cell production driven by hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Proliferation and differentiation of HSPCs are regulated by complex transcriptional networks. In order to identify transcription factors with key roles in HSPC-mediated hematopoietic reconstitution, we developed an efficient and robust CRISPR/Cas9-based in vivo genetic screen. Using this experimental system, we identified the TFDP1 transcription factor to be essential for HSPC proliferation and post-transplant hematopoiesis. We further discovered that E2F4, an E2F transcription factor, serves as a binding partner of TFDP1 and is required for HSPC proliferation. Deletion of TFDP1 caused downregulation of genes associated with the cell cycle, with around 50% of these genes being identified as direct targets of TFDP1 and E2F4. Thus, our study expands the transcriptional network governing hematopoietic development through an in vivo CRISPR/Cas9-based genetic screen and identifies TFDP1/E2F4 as positive regulators of cell cycle genes in HSPCs.

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

Gene therapy in hematopoietic stem and progenitor cells (HSPCs) shows great potential for the treatment of inborn metabolic diseases. Typical HSPC gene therapy approaches rely on constitutive promoters to express a therapeutic transgene, which is associated with multiple disadvantages. Here, we propose a novel promoter-less intronic gene editing approach that triggers transgene expression only after cellular differentiation into the myeloid lineage. We integrated a splicing-competent eGFP cassette into the first intron of CD11b and observed expression of eGFP in the myeloid lineage but minimal to no expression in HSPCs or differentiated non-myeloid lineages. In vivo, edited HSPCs successfully engrafted in immunodeficient mice and displayed transgene expression in the myeloid compartment of multiple tissues. Using the same approach, we expressed alpha-L-iduronidase (IDUA), the defective enzyme in Mucopolysaccharidosis type I, and observed a 10-fold supraendogenous IDUA expression exclusively after myeloid differentiation. Edited cells efficiently populated bone marrow, blood, and spleen of immunodeficient mice, and retained the capacity to secrete IDUA ex vivo. Importantly, cells edited with the eGFP and IDUA transgenes were also found in the brain. This approach may unlock new therapeutic strategies for inborn metabolic and neurological diseases that require the delivery of therapeutics in brain.

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

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

Influenza Training of Granulopoiesis Promotes Long-Term Anti-Leukemia Activity

Abstract Influenza viral infections have the ability to reprogram the immune system of the host and alter its functions to fight infections. However, the exact mechanisms and functions of virus-induced innate immunity in the context of leukemia are not yet fully understood. Our previous research has shown that a patient with refractory acute myeloid leukemia (AML) achieved complete remission (CR) after being infected with the Influenza A virus (IAV, H1N1 subtype), and this was functionally validated in two different animal disease relevant models. Our recent study demonstrates that mice pre-trained with the IAV promotes a long-term anti-leukemia effect. The anti-leukemia activity of IAV-induced trained immunity is due to the transcriptomic rewiring of hematopoietic stem and progenitor cells (HSPCs), resulting in a sustained enhancement of myeloid cells reprogramming toward an antitumor phenotype. This mechanism involves the upregulation of type I interferons (IFNs) and subsequent downregulation of inflammatory proteins. Our findings shed light on the dynamic changes in innate immune cells following viral infections and indicate a novel anti-leukemia facet of appropriate rewiring of granulopoiesis via upregulation of type I IFNs. Statement of significance The study profiles the dynamic changes in immune cells during exposure to the Influenza virus and sheds light on the role of Influenza trained innate immunity in anti-leukemia effect. The study also explores a novel anti-leukemia facet of appropriate rewiring of granulopoiesis via upregulation of the type I IFN pathway. These findings suggest that innate immune training could be a promising adjuvant therapy for cancer and warrant further investigation. Legend for Figure 5 . H1N1 reprogrammed granulopoiesis via up-regulation of type I interferon pathway. (A-C) FCM analyses and absolute counts of BM progenitor cells on day 7 after training with H1N1 or PBS. FCM, flow cytometry; BM, bone marrow; LK, Lin-c-kit+Sac1-; LSK, Lin-c-kit+Sac1+; GMP, granulocyte-monocyte progenitors (Lin-c-kit+Sac1-CD16/32 hiCD34+); CMP, common myeloid progenitors (Lin-c-kit+Sac1-CD16/32 intCD34+); MEP, megakaryocyte-erythroid progenitor cell (Lin-c-kit+Sac1-CD16/32-CD34-). (D) T-SNE plot of top 7 progenitor cells population changes. t-SNE, t-distributed stochastic neighbor embedding. (E) Heatmap visualization of the expression of different genes in HSPCs. The upregulated genes related to the type I IFNs pathway are listed on the right. HSPCs and hematopoietic stem/progenitor cells. (F) The top enriched GO terms of biological processes for upregulated genes in H1N1 trained group. GO, gene ontology. (G-H) Heatmap and t-SNE plot of type I IFN-associated upregulation genes in progenitor cells of HSPCs, GMP, CMP, and MEP. t-SNE, t-distributed stochastic neighbor embedding.

Maternal Obesity Causes Sex Specific Long-Term Hematopoietic and Metabolic Defects

Maternal obesity is increasingly common and can negatively impact offspring health. Children born to obese mothers are at higher risk of developing diseases involved with metabolic health (e.g., type 2 diabetes). Many of these diseases are associated with abnormalities within the hematopoietic system such as atypical immune profiles or hematopoiesis. Interestingly, disease incidence and/or severity is often dependent on the offspring's gender. This suggests that there may be sex-specific reprogramming of hematopoietic stem and progenitor cells (HSPCs) during immune cell development. This study aims to test the hypothesis that offspring of obese mice exhibit sex-differences in HSPC function that affect offspring's metabolic health. To test this, C57BL/6 (CD45.2+) dams were fed standard chow (2018SX, Envigo, IN) or western-style diet (TD.88137, Envigo, IN) for four weeks prior to mating, through pregnancy, and the lactating period. At postnatal day 21, HSPCs (Lineage negative, Sca1 positive, cKit positive cells) were flow-sorted from bone marrow (BM) of offspring from dams fed chow (Con) or western-style diet (MatOb) and used for competitive transplant (Boy/J BM competitor cells; CD45.1+) into same-sex lethally irradiated C57BL/6 x Boy/J F1 (CD45.1+CD45.2+) recipient mice (8-10 weeks of age) or for RNA-sequencing analysis. Secondary transplants were performed to look at long-term engraftment potential of HSPCs. Body weight, body adiposity, fasting insulin, glucose tolerance test (GTT), and pancreatic β and α-cell area were assessed on male and female recipients of both primary and secondary transplants. Compared to mice that received male Con HSPCs, recipient mice that received male MatOb HSPCs had significantly lower donor chimerism in the blood and BM post-secondary transplantation. This was associated with a significant reduction in donor hematopoietic stem cell (HSC) numbers in the BM 16 weeks post-secondary transplant, indicating male MatOb HSPCs had decreased long-term engraftment capability due to an exhaustion of the HSC pool. This reduction in HSPC engraftment seen between male MatOb versus Con HSPCs was not seen when using HSPCs from female offspring. Next, metabolic parameters of our recipient mice was examined. Sixteen weeks after primary transplant, there were no differences detected in body weight and adiposity in the Con and MatOb HSPC recipient mice. Compared to sex-matched controls, we found that recipients transplanted with male MatOb HSPCs developed glucose intolerance, increased fasting serum insulin concentrations, and decreased β-cell area, supporting the presence of impaired glucose metabolism due to a combination of insulin resistance and lower β-cell mass. There was no difference in recipients of HSPCs from female Con and female MatOb pups. RNA-seq of BM HSPCs at time of transplant (p21) from Con or MatOb revealed 100 differentially expressed genes (DEG) in male MatOb HSPCs and 1788 DEG in female MatOb HSPCs. There were more and higher magnitude of differential gene expression in female mice than male, despite the long-term phenotypic differences being evident in males but not females, and the majority of genes changed in males were also altered in females. This suggests that the gene programs responsible for the observed phenotypes may be altered by maternal obesity in both male and female offspring, but that female offspring have a transcriptomic profile that confers protection on the HSPC population. One of the most striking findings from the transcriptomic analysis was a marked enrichment in gene programs associated with immune response, interferon signaling, and cytokine production in female MatOb offspring compared to sex matched controls that is not seen in male MatOb offspring. Pathway analysis identified upregulation of programs involving both IFNγ and IL-10 in the female MatOb HSPCs when compared to sex-mated Con HSPCs which correlated with an increase in IL-10 concentrations in the serum of p21 female MatOb mice. These pathways were downregulated or unaltered in males with no change in systemic IL-10 concentration.Our results demonstrate that maternal obesity results in sex-specific changes in offspring HSPC function and a novel role HSPCs may play in regulating glucose metabolism in the offspring of obese dams.

CD90-Targeted Cocal-Pseudotyped Lentivirus As a Robust Platform for Human HSC Gene Therapy

Ex vivo gene delivery and editing in hematopoietic stem and progenitor cells (HSPCs) provide a promising approach for the treatment of many chronic conditions such as hemoglobinopathies, lysosomal storage diseases, and HIV. However, such approaches require highly specialized biomedical infrastructure limiting its accessibility to most patients who could benefit. The development of a targeted in vivo approach to HSPC gene delivery/editing is therefore necessary for the broadened use of gene therapies. The CD34 +CD90 + subset of HSPCs represents a highly enriched population for both short-term and long-term hematopoietic lineage repopulation capacity as seen in the non-human primate (NHP) transplant model. Furthermore, ex vivo gene editing of solely CD34 +CD90 + Human and NHP hematopoietic stem cells (HSCs) was sufficient to produce therapeutic thresholds for sickle cell phenotypic amelioration after transplantation. Thus, the development of novel gene therapy vectors targeting the CD34 +CD90 + HSC population is crucial for more accessible and robust gene therapy platforms. Here we report the development of a novel CD90-targeted Cocal pseudotyped lentivirus system capable of targeted transduction of HSPCs in vitro and in vivo. Additionally, we compare transduction efficiency and specificity of this new technology with the more routinely used VSVG targeting methodology. To produce targeted lentivirus, native targeting of VSVG or Cocal envelope to the LDL-Receptor (LDLR) was knocked out by amino acid substitution. An anti-CD90 single-chain variable fragment (scFv) was introduced onto the mutated capsids using CD8 hinge and ICAM transmembrane domains. Mutagenesis of Cocal envelope LDLR binding motif was performed by Gibson cloning and confirmed via nanopore sequencing. Successful mutated viral production was assessed using hydrodynamic titration methods followed by functional analysis of transduction on CD90 + cell lines. In vitro transduction specificity and efficiency assays were performed on Human GCSF-mobilized CD34 +enriched apheresis samples and analyzed by flow cytometry. Humanized NBSGW mice were treated with targeted-lentiviral particles via intravenous (IV) and intraosseous (IO) injection. Mice were necropsied 4-8 weeks post injection. Transgene expression was analyzed by flow cytometry or luminescence imaging and confirmed by PCR on human-CD34 + FACS-sorted cells from necropsied mice. CD90-Targeted Cocal-pseudotyped lentivirus (Cocal-CD90) transduced CD34 +CD90 + HSCs 2.12-fold (p=.017) more robustly than CD90-targeted VSVG-pseudotyped lentivirus (VSVG-CD90) in vitro. Cocal-CD90 virus also showed significantly greater on-target transduction specificity of CD34 +CD90 + HSCs (p=.0008) compared to untargeted wild-type Cocal-pseudotyped lentivirus (Cocal-WT); but similar specificity to VSVG-CD90 virus (p=.118). Unlike VSVG-CD90 virus, we did not observe decreased transduction efficiency of CD34 +CD90 + HSCs using Cocal-CD90 virus in comparison to Cocal-WT virus. IV and IO injection of both CD90-targeted viruses in humanized mice resulted in transduction of immobilized human HSPCs (CD34 +CD38 -CD133 +) in the bone marrow. Transduced cells maintained multilineage differentiation capacity as seen by transgene expression of T-cell, B-cell, and myeloid compartments in the bone marrow, spleen, and peripheral blood at necropsy. As observed in vitro, Cocal-CD90 treated mice consistently displayed more robust transgene expression than VSVG-CD90 treated mice. Importantly, transduction of off-target organs (e.g., liver and lungs) post-IV injection with targeted virus was not detected by luminescence imaging during 8 weeks of follow-up or at necropsy. We provide the first evidence of targeted engineering of a knockout Cocal envelope using the attenuation of native LDLR tropism coupled with a CD90-scFv fusogen protein. Cocal-CD90 virus has increased specificity for CD90 + transduction compared to Cocal-WT lentivirus, without lowering transduction efficiency of on-target cells. Furthermore, in comparison to targeted VSVG engineering, Cocal engineered virus does not decrease transduction efficiency of HSCs in vitro or in vivo. Coupled with Cocal lentiviruses' resistance to human serum inactivation and low immunogenicity, targeted Cocal viral vectors are more suitable for future in vivo gene delivery platforms than their VSVG counterparts.

Reduced Polycomb Repressor Complex 2 (PRC2) Activity and Increased TP53 Activity Mediate Hematopoietic Stem Cell Dysfunction in RPS19-Mutated Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia caused by heterozygous loss-of-function mutations in one of 23 ribosomal protein (RP) genes, with RPS19 being mutated in approximately 25% of patients. While DBA typically manifests in infancy as isolated anemia with reticulocytopenia, older patients can develop multilineage cytopenias and bone marrow (BM) hypocellularity suggesting impaired hematopoietic stem cell (HSC) function. Defects in HSCs have been observed in Rps19-depleted mice but studies on human DBA HSCs have been inconclusive, largely because patient BM cells are difficult to obtain for research. To address this gap, we developed a new, robust, and scalable cellular model using CRISPR/Cas9 to create heterozygous RPS19 loss-of-function mutations in healthy donor CD34 + hematopoietic stem and progenitor cells (HSPCs). Transplantation of RPS19+/- HSPCs into NBSGW immunodeficient mice revealed a defect in BM repopulation that was partially rescued by co-disruption of the TP53 gene (Bhoopalan et al, JCI Insight, 2023). Here we provide further mechanistic insight into this HSC defect by showing that RP imbalance caused by RPS19 deficiency leads to the activation of TP53 and inhibition of Polycomb Repressor Complex 2 (PRC2), which has been previously shown to be required for HSC maintenance. Healthy donor CD34 + HSPC were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 safe harbor locus as a negative control. RPS19 disruption was verified by next-generation sequencing of the targeted region and by Western blot analysis showing reduction of RPS19 protein. RNA-Seq at 3 days after electroporation showed enrichment of TP53 pathway genes in RPS19-edited HSPCs compared to control cells, confirming our previous observations. Additionally, we noted an enrichment of HSPC genes that were previously shown to harbor the repressive histone mark H3K27me3 in normal HSPCs. Methylation of H3K27 is catalyzed by EZH2, the catalytic component of PRC2, which maintains HSCs by inhibiting transcription of genes that promote differentiation. Gene set enrichment analysis (GSEA) revealed significant induction of PRC2 target genes in RPS19-disrupted HSPCs and in induced pluripotent stem cells (iPSCs) generated from two different DBA patients with heterozygous RPS19 mutations. The “Cleavage under targets and release using nuclease (CUT&RUN)” assay indicated globally reduced EZH2 binding to chromatin in RPS19-disrupted HSPCs. These findings suggest that PRC2 activity is inhibited by RPS19 haploinsufficiency. Previous studies have shown that RPS19 haploinsufficiency can cause accumulation of large RP subunits, RPL5 and RPL11, which bind 5S ribosomal RNA to form 5S RNP. This complex can sequester the ubiquitin ligase MDM2 to inhibit its function. MDM2 ubiquitinates TP53, targeting it for proteasomal degradation. Additionally, MDM2 binds PRC2 to augment its activity. Thus, we theorized that RPS19 haploinsufficiency leads to the accumulation of 5S RNP complex which sequesters MDM2, causing increased TP53 activity and suppression of PRC2 activity, both of which can inhibit HSC maintenance. To test this, we inhibited formation of 5S RNP complex by co-disrupting RPL5 or RPL11 along with RPS19 in CD34 + HSPCs, followed by RNA-Seq analysis or transplantation into NBSGW mice. Remarkably, haploinsufficiency of either large RP gene rescued the BM repopulation defect of RPS19 +/- HSPC (p<0.05) ( Fig. 1A), reduced TP53 activity and improved PRC2 activity (p<0.001 and FDR q<0.01) ( Fig. 1B). In summary, our data support the model that RPS19 haploinsufficiency leads to increased TP53 activity and reduced PRC2 activity through 5S RNP-mediated sequestration of MDM2, causing impairment of HSC function. These studies provide insights into the molecular mechanisms of DBA and could potentially inform new therapies.

Current approaches and potential challenges in the delivery of gene editing cargos into hematopoietic stem and progenitor cells.

Advancements in gene delivery and editing have expanded the applications of autologous hematopoietic stem and progenitor cells (HSPCs) for the treatment of monogenic and acquired diseases. The gene editing toolbox is growing, and the ability to achieve gene editing with mRNA or protein delivered intracellularly by vehicles, such as electroporation and nanoparticles, has highlighted the potential of gene editing in HSPCs. Ongoing phase I/II clinical trials with gene-edited HSPCs for β-hemoglobinopathies provide hope for treating monogenic diseases. The development of safe and efficient gene editing reagents and their delivery into hard-to-transfect HSPCs have been critical drivers in the rapid translation of HSPC gene editing into clinical studies. This review article summarizes the available payloads and delivery vehicles for gene editing HSPCs and their potential impact on therapeutic applications.

Depletion of tet2 results in age-dependent changes in DNA methylation and gene expression in a zebrafish model of myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis, cytopenias, and dysplasia. The gene encoding ten-eleven translocation 2 (tet2), a dioxygenase enzyme that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, is a recurrently mutated tumor suppressor gene in MDS and other myeloid malignancies. Previously, we reported a stable zebrafish line with a loss-of-function mutation in the tet2 gene. The tet2m/m-mutant zebrafish developed a pre-MDS state with kidney marrow dysplasia, but normal circulating blood counts by 11 months of age and accompanying anemia, signifying the onset of MDS, by 24 months of age. In the current study, we collected progenitor cells from the kidney marrows of the adult tet2m/m and tet2wt/wt fish at 4 and 15 months of age and conducted enhanced reduced representation of bisulfite sequencing (ERRBS) and bulk RNA-seq to measure changes in DNA methylation and gene expression of hematopoietic stem and progenitor cells (HSPCs). A global increase in DNA methylation of gene promoter regions and CpG islands was observed in tet2m/m HSPCs at 4 months of age when compared with the wild type. Furthermore, hypermethylated genes were significantly enriched for targets of SUZ12 and the metal-response-element-binding transcription factor 2 (MTF2)-involved in the polycomb repressive complex 2 (PRC2). However, between 4 and 15 months of age, we observed a paradoxical global decrease in DNA methylation in tet2m/m HSPCs. Gene expression analyses identified upregulation of genes associated with mTORC1 signaling and interferon gamma and alpha responses in tet2m/m HSPCs at 4 months of age when compared with the wild type. Downregulated genes in HSPCs of tet2-mutant fish at 4 months of age were enriched for cell cycle regulation, heme metabolism, and interleukin 2 (IL2)/signal transducer and activator of transcription 5 (STAT5) signaling, possibly related to increased self-renewal and clonal advantage in HSPCs with tet2 loss of function. Finally, there was an overall inverse correlation between overall increased promoter methylation and gene expression.

Efficient manufacturing and engraftment of CCR5 gene-edited HSPCs following busulfan conditioning in nonhuman primates

Hematopoietic stem cell gene therapy has been successfully used for a number of genetic diseases and is also being explored for HIV. However, toxicity of the conditioning regimens has been a major concern. Here we compared current conditioning approaches in a clinically relevant nonhuman primate model. We first customized various aspects of the therapeutic approach, including mobilization and cell collection protocols, conditioning regimens that support engraftment with minimal collateral damage, and cell manufacturing and infusing schema that reflect and build on current clinical approaches. Through a series of iterative invivo experiments in two macaque species, we show that busulfan conditioning significantly spares lymphocytes and maintains a superior immune response to mucosal challenge with simian/human immunodeficiency virus, compared to total body irradiation and melphalan regimens. Comparative mobilization experiments demonstrate higher cell yield relative to our historical standard, primed bone marrow and engraftment of CRISPR-edited hematopoietic stem and progenitor cells (HSPCs) after busulfan conditioning. Our findings establish a detailed workflow for preclinical HSPC gene therapy studies in the nonhuman primate model, which in turn will support testing of novel conditioning regimens and more advanced HSPC gene editing techniques tailored to any disease of interest.

Abstract PR01: Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells

Abstract Most patients with acute myeloid leukemia (AML) initially achieve clinical remission after chemotherapy but eventually die from disease relapse as they succumb to chemotherapy-resistant AML cells, including the rare fraction of leukemia stem cells (LSC) that persist and often reinitiate a more aggressive disease. While eliminating LSCs has become a desirable strategy to achieve durable AML remission, much remains unknown about LSC vulnerabilities and the specific cellular programs that enable their disease activity. Spurred by increasing evidence that disruptions in proteome homeostasis or “proteostasis” impair stem cell integrity, we investigated how acute myeloid leukemogenesis is impacted by the inositol-requiring enzyme-1 alpha (IRE1a), the most conserved enzyme of the endoplasmic reticulum unfolded protein response that resolves proteotoxic stress in eukaryotic cells. Activated IRE1a catalyzes an unconventional splicing of the XBP1U mRNA to generate an XBP1S transcript that encodes the transcription factor XBP1 (X-box binding protein-1), the main effector of IRE1a-transduced signals. Using transcriptomics and genomics to profile the activation and function of IRE1a in primary human AML samples and murine AML models, we identified the IRE1a-XBP1 axis as a cell-intrinsic brake against pro-myeloid leukemogenic programs in hematopoietic stem and progenitor cells (HSPC). Notably, compared to blast cells from the same AML patient, LSCs expressed significantly reduced levels of the genes encoding IRE1a (ERN1) and its spliced product XBP1S suggesting that downregulation of IRE1a-XBP1 activity promotes LSC activity and acute myeloid leukemogenesis. Consistent with this hypothesis, genetic induction of XBP1S-encoded XBP1 in patient-derived AML cells suppressed their LSC transcriptional program, impaired their clonogenic capacity, and limited their ability to cause AML in immunodeficient animals. By contrast, genetically engineered IRE1a or XBP1 insufficiency enriched the LSC signature in HSPCs and cooperated with the myeloproliferative oncogene FLT3-ITD to initiate a lethal AML disease in mice. Integrated RNA-Seq and ChIP-Seq of XBP1 target genes in HSPCs revealed a mechanism in which IRE1a-induced XBP1 functions as a potent transcriptional repressor of signature pro-LSC gene programs, exemplified by the Wnt/beta-catenin pathway. Accordingly, we defined an independently prognostic “18-gene” signature of XBP1-repressed beta-catenin target genes that when highly expressed correlated with poor survival outcomes in multiple cohorts of AML patients. Taken together, our findings uncover a novel anti-leukemia function for IRE1a-XBP1 signaling that restrains acute myeloid leukemogenesis in HSPCs and suggest that this pathway is likely subverted in LSCs to facilitate AML initiation and progression. Our results further suggest that IRE1a activation is a vulnerability in AML stem cells that can be harnessed therapeutically to achieve durable disease remission. Citation Format: Brendan M Barton, Francheska Son, Akanksha Verma, Laurie Glimcher, Stanley Adoro. Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr PR01.

Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of β-Hemoglobinopathies.

Cellular therapies hold enormous potential for the cure of severe hematological and oncological disorders. The forefront of innovative gene therapy approaches including therapeutic gene editing and hematopoietic stem cell transplantation needs to be processed by good manufacturing practice to ensure safe application in patients. In the present study, an effective transfection protocol for automated clinical-scale production of genetically modified hematopoietic stem and progenitor cells (HSPCs) using the CliniMACS Prodigy® system including the CliniMACS Electroporator (Miltenyi Biotec) was established. As a proof-of-concept, the enhancer of the BCL11A gene, clustered regularly interspaced short palindromic repeat (CRISPR) target in ongoing clinical trials for β-thalassemia and sickle-cell disease treatment, was disrupted by the CRISPR-Cas9 system simulating a large-scale clinical scenario, yielding 100 million HSPCs with high editing efficiency. In vitro erythroid differentiation and high-performance liquid chromatography analyses corroborated fetal hemoglobin resurgence in edited samples, supporting the feasibility of running the complete process of HSPC gene editing in an automated closed system.

The NLRC4 Inflammasome Drives Myelodysplastic Syndrome By Linking Epigenetic Reprogramming and Innate Immune Signaling

Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell (HSC) malignancies characterized by dysplastic and ineffective hematopoiesis. Despite emerging evidence that chronic inflammation contributes to the development of MDS, the mechanisms underlying the dysregulation of innate immune and inflammatory signaling pathways in hematopoietic stem and progenitor cells (HSPCs) remain poorly characterized. We have used multiple MDS mouse models to identify a tumor suppressive role for the EP300 epigenetic histone acetyltransferase enzyme in the progression of MDS to AML. Based on the gene expression changes we observed, we have used one specific mouse model, that lacks Tet2 expression, to define the role that chronic inflammation plays in promoting MDS development and the mechanisms involved. Methods We have conducted a series of in vitro, in vivo, and in silico experiments using Tet2-deficient mice, and chronic, low-dose LPS treatment to model chronic inflammation. Cytokine array immunoassays were performed to evaluate the inflammatory response of WT or Tet2-deficient mice to LPS treatment. Chimeric mice transplanted with bone marrow (BM) cells isolated from LPS or vehicle (PBS) treated WT or Tet2-deficient mice were monitored for the development of MDS and survival. Serial competitive bone marrow transplantation assays and flow cytometry profiling were used to determine the competitive advantage of LPS or PBS-treated BM cells, and their differentiation. Bioinformatic analyses of RNA sequencing (RNA-seq) results from our mouse model were performed to characterize the transcriptional profiling and identify novel innate immune pathways. Colony-forming unit (CFU) and qPCR assays were used to evaluate HSPC proliferative capacity, self-renewal, and gene expression changes. Results We find that mice lacking Tet2 develop a hyper-inflammatory status, marked by the expression of a number of pro-inflammatory cytokines, such as Il-1b and Il-6, which are further fueled by LPS-induced chronic inflammation. Chimeric mice transplanted with BM cells isolated from LPS-treated Tet2-deficient mice exhibit a significantly shorter life span and early onset of MDS phenotypes, including dysplastic hematopoiesis and thrombocytopenia, compared to age-matched chimeric mice transplanted with BM cells isolated from PBS-treated Tet2-deficient mice or LPS-treated WT mice. Chronic inflammation promotes the self-renewal and myeloid differentiation of Tet2-deficient HSPCs in the short term; however, it accelerates HSC exhaustion in the long run. Transcription profiling and subsequent bioinformatic analyses identify functional clusters in LPS-treated Tet2-deficient HSPCs, including genes involved in the regulation of epigenetics, immunity, metabolism, and cell cycle. Detailed inspection of the most highly differentially expressed genes reveals that many genes in the Nlrc4 inflammasome pathway are upregulated in LPS-treated Tet2-deficient HSPCs. We validate changes in the expression of these genes by qPCR, and then show that deleting Nlrc4 suppresses the colony formation of Tet2-deficient HSPCs in vitro. Ongoing work focuses on evaluating the effects of deleting Nlrc4 or blocking Nlrc4 function using a small molecule inhibitor on MDS development in vivo. Conclusions The combinatory role of intrinsic and extrinsic factors drives the development of MDS in this Tet2-deficient MDS mouse model. It is likely that different genetic mutations generate unique sensitivity to certain signaling pathways, and we demonstrate the epigenetic rewiring and activation of the Nlrc4 inflammasome pathway as a mechanistic basis for the inflammatory component of MDS [or clonal hematopoiesis of indeterminate potential (CHIP)] pathogenesis in Tet2-deficient cells. Our work has the potential to provide novel targeting opportunities in MDS management and confirm the importance of different components of inflammasome signaling in MDS.

Comparing the Impact of NHEJ-CRISPR/Cas9 and Base Gene Editing on Rhesus Macaque Hematopoietic Stem Cell Clonal Behavior and Integrity

Despite recent transformative advancements in gene editing tools and hematopoietic stem and progenitor cell (HSPC) gene therapies, there are still unanswered questions about the impact of CRISPR/Cas9 and base editing technologies on HSPC clonal behavior, function, and integrity in vivo. Better understanding of the impact of available gene therapy modalities on HSCs in a relevant animal model should help safely move these therapies forward for clinical use and provide information regarding their relative advantages and risks. CRISPR/Cas9 editing generates double strand DNA breaks (DSBs) at targeted genomic locations and potentially at off-target sites, resulting in potential HSC genotoxicity and loss. To counter DSB-related consequences, base editors (BEs) have been developed with the ability to produce single-nucleotide conversion without DSBs, avoiding potentially deleterious DNA damage repair pathways. CD33 is expressed on all myeloid cells downstream of the common myeloid progenitor and is used as both a diagnostic marker and a therapeutic target for acute myeloid leukemia. Our past work demonstrated successful Cas9/NHEJ-mediated CD33 KO in rhesus macaque (RM) HSPCs with no functional impact (Kim et al, Cell, 2018). In the current study, we first genetically barcoded RM HSPCs, and then knocked out the CD33 locus with the intent of comparing the clonal impact of CRISPR/Cas9 editing via non-homologous end joining (NHEJ) to base editing in RM HSPCs in vitro by myeloid differentiation assays and in vivo following autologous HSPC transplantation. Highly diverse DNA barcodes were introduced into RM HSPCs via transduction with a lentiviral vector also expressing GFP. 24 hours later, the cells were electroporated with either Cas9/NHEJ or BE complexes targeted to disrupt CD33. Cells were further cultured in myeloid differentiation media for 8 days in vitro for differentiation assays (IVD). Cell viability and proliferation were analyzed over time during IVD. DNA was collected on day 8 for editing efficiency and barcode clonal analysis. In addition, GFP and CD33 expression were analyzed by flow cytometry to assess CD33 KO efficiency and examine whether the addition of gene editors impacted the clonality of the HSPCs as assessed by barcode retrieval, and whether the initial lentiviral transduction impacted editing efficiency. HSPC colony-forming unit (CFU) assays were carried out to assess differentiation potential of the transduced and/or edited HSPCs and check for biallelic versus monoallelic editing status at a clonal level. Throughout the 8 days of IVD, cell numbers increased overtime and maintained high cell viability (80-95%) in all test groups, which indicates barcoded lentiviral transduction and gene editing procedures had minimal impact on cell proliferation and viability. Base editing resulted in 80-85% CD33 KO efficiency, regardless of barcode lentivirus transduction prior to gene editing. However, Cas9/NHEJ was significantly less efficient, with CD33 KO efficiency of 10-20%, requiring additional optimization (Figure 1A). Variant allele frequency analysis confirmed the relative levels of CD33 locus editing by both editing methods. CFU assays indicated the lentiviral transduction and gene editing methods do not alter the HSPC differentiation potential as assessed in vitro. Barcode clonal analysis revealed both NHEJ and BE test groups demonstrated retention of high clonal diversity as assessed by Shannon Diversity Index, all > 8 regardless of gene editing methods (Figure 1B). In summary, lentiviral barcode transduction and editing procedures had no negative impact on cell viability and proliferation. Base editing allowed very high gene editing efficiency in HSPCs and can serve as a powerful editing tool. Importantly, both editing methods resulted highly diverse polyclonal clonal patterns in vitro long term, with no significant differences compared to the non-edited group. Combining genetic barcode technology and the BE or Cas9/NHEJ editing in an in vivo RM autologous HSPC transplantation model to track the hematopoietic clonal reconstitution of the edited HSPCs over time and compare the impact of BE or Cas9/NHEJ editing on engrafting HSPCs clonal behavior is ongoing. Therefore, these clinically relevant findings can determine which gene editing method is more suitable for developing human HSPC gene therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Screening chimeric GAA variants in preclinical study results in hematopoietic stem cell gene therapy candidate vectors for Pompe disease

Pompe disease is a rare genetic neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy, the current standard of care, penetrates poorly into the skeletal muscles and the peripheral and central nervous system (CNS), risks recombinant enzyme immunogenicity, and requires high doses and frequent infusions. Lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy was investigated in a Pompe mouse model using a clinically relevant promoter driving nine engineered GAA coding sequences incorporating distinct peptide tags and codon optimizations. Vectors solely including glycosylation-independent lysosomal targeting tags enhanced secretion and improved reduction of glycogen, myofiber, and CNS vacuolation in key tissues, although GAA enzyme activity and protein was consistently lower compared with native GAA. Genetically modified microglial cells in brains were detected at low levels but provided robust phenotypic correction. Furthermore, an amino acid substitution introduced in the tag reduced insulin receptor-mediated signaling with no evidence of an effect on blood glucose levels in Pompe mice. This study demonstrated the therapeutic potential of lentiviral HSPC gene therapy exploiting optimized GAA tagged coding sequences to reverse Pompe disease pathology in a preclinical mouse model, providing promising vector candidates for further investigation.

Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing.

Most genome editing analyses to date are based on quantifying small insertions and deletions. Here, we show that CRISPR-Cas9 genome editing can induce large gene modifications, such as deletions, insertions, and complex local rearrangements in different primary cells and cell lines. We analyzed large deletion events in hematopoietic stem and progenitor cells (HSPCs) using different methods, including clonal genotyping, droplet digital polymerase chain reaction, single-molecule real-time sequencing with unique molecular identifier, and long-amplicon sequencing assay. Our results show that large deletions of up to several thousand bases occur with high frequencies at the Cas9 on-target cut sites on the HBB (11.7 to 35.4%), HBG (14.3%), and BCL11A (13.2%) genes in HSPCs and the PD-1 (15.2%) gene in T cells. Our findings have important implications to advancing genome editing technologies for treating human diseases, because unintended large gene modifications may persist, thus altering the biological functions and reducing the available therapeutic alleles.

S116: CELLULAR AND MOLECULAR MECHANISMS OF EVI1-EXPRESSING MLL-REARRANGED ACUTE MYELOID LEUKEMIA

Background: Expression of a doxycycline (DOX)-inducible acute myeloid leukaemia (AML)-associated iMLL-AF9 fusion transgene in long-term haematopoietic stem cells (LT-HSC) can lead to an invasive and chemo-resistant disease expressing the transcription factor EVI1. High EVI1 expression has been suggested as marker of poor outcome in AML patients even without rearrangements of the EVI1 locus at 3q26. Aims: We addressed the association between EVI1 expression, the cellular origin and poor disease outcome in AML driven by the iMLL-AF9 fusion gene. Methods: The role of EVI1 expression was studied in iMLL-AF9 transgenic mice carrying an Evi1-IRES GFP reporter in vitro using flow cytometry, colony formation and RT-qPCR assays, ex vivo with high-resolution bone marrow (BM) imaging, and in vivo, by transplantation of enriched naïve Evi1+ iMLL-AF9 hematopoietic stem and progenitor cells (HSPC) into irradiated recipients on DOX. Haematopoiesis of symptomatic mice was analysed by flow cytometry and histology. For mechanistic studies, single cell and bulk RNA sequencing was performed on enriched HSPC or BM samples from diseased mice. Results: Analysis of BM cells from Evi1-IRES-GFP reporter mice revealed that not only the mostly quiescent LT-HSC but also fractions of the more proliferating multipotent progenitors (MPP1-3) express abundant Evi1 (“Evi1high”). Induction of the iMLL-AF9 fusion did not result in significant changes in numbers of Evi1+ cells nor levels of Evi1 mRNA expression in the LT-HSC and MPP1 compartments. However, in colony assays, Evi1high iMLL-AF9 cells retained a more immature phenotype and produced more colonies with an invasive morphology than Evi1low cells (n=11, p<0.05). While Evi1 expression did not influence disease induction upon transplantation of LT-HSC, recipients of Evi1+ MPP1 cells developed AML earlier than Evi1- MPP1 (n=11, 79 vs. 269d, p<0.05). Disease induced by Evi1+ cells presented with more extensive leukemic organ infiltration than Evi1- AML. Evi1 expression also correlated with in vitro Ara-C resistance. We also examined whether some exogenous factors may increase AML susceptibility by expanding the Evi1+ HSPC. Although a single injection of recombinant mouse thrombopoietin (TPO) only increased the number of LT-HSC and not of MPP1, the Evi1high cell fraction was enlarged in both compartments (LT-HSC: 23 vs. 50%; MPP1: 22 vs. 47%; n=29, p<0.0001) supported by high-resolution imaging. Interestingly, increased TPO induced HSPC cycling was confined to the Evi1high cell population (n=3, p<0.05). Transplantation of TPO-treated iMLL-AF9 LT-HSC or MPP1 resulted in a significantly faster induction of Evi1+ AML than controls (n=19, MPP1: 35 vs. 79d, p<0.001; LT-HSC: 41 vs. 90d, p<0.001). To better understand mechanisms of aggravated AML after TPO mediated expansion of Evi1+ HSCP we performed multiplexed single cell RNA sequencing of highly enriched HSPC cells in iMLL-AF9 and control mice. While we observed no changes of cellular cluster organisation 2 days after TPO injection, we found some differentially expressed genes in TPO-stimulated cycling iMLL-AF9 HSPC, including potential stemness regulators. Summary/Conclusion: Our results suggest that expansion of Evi1-expressing HSPC by exogenous factors can result in a more aggressive MLL-AF9-driven AML. Ongoing data exploration and validation may characterize aberrantly expressed genes in TPO-stimulated Evi1+ iMLL-AF9-expressing HSPC as potential therapeutic targets to impair stemness of AML cells.

Preferential Expansion of Human CD34+CD133+CD90+ Hematopoietic Stem Cells Enhances Gene-Modified Cell Frequency for Gene Therapy.

CD34+CD133+CD90+ hematopoietic stem cells (HSCs) are responsible for long-term multilineage hematopoiesis, and the high frequency of gene-modified HSCs is crucial for the success of hematopoietic stem and progenitor cell (HSPC) gene therapy. However, the ex vivo culture and gene manipulation steps of HSPC graft preparation significantly reduce the frequency of HSCs, thus necessitating large doses of HSPCs and reagents for the manipulation. In this study, we identified a combination of small molecules, Resveratrol, UM729, and SR1 that preferentially expands CD34+CD133+CD90+ HSCs over other subpopulations of adult HSPCs in ex vivo culture. The preferential expansion enriches the HSCs in ex vivo culture, enhances the adhesion, and results in a sixfold increase in the long-term engraftment in NSG mice. Further, the culture-enriched HSCs are more responsive to gene modification by lentiviral transduction and gene editing, increasing the frequency of gene-modified HSCs up to 10-fold in vivo. The yield of gene-modified HSCs obtained by the culture enrichment is similar to the sort-purification of HSCs and superior to Cyclosporin-H treatment. Our study addresses a critical challenge of low frequency of gene modified HSCs in HSPC graft by developing and demonstrating a facile HSPC culture condition that increases the frequency of gene-modified cells in vivo. This strategy will improve the outcome of HSPC gene therapy and also simplify the gene manipulation process.

Hematopoietic Stem Cell Gene Therapy for Cystinosis: From Bench-to-Bedside.

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. The gene involved is the CTNS gene that encodes cystinosin, a seven-transmembrane domain lysosomal protein, which is a proton-driven cystine transporter. Cystinosis is characterized by the lysosomal accumulation of cystine, a dimer of cysteine, in all the cells of the body leading to multi-organ failure, including the failure of the kidney, eye, thyroid, muscle, and pancreas, and eventually causing premature death in early adulthood. The current treatment is the drug cysteamine, which is onerous and expensive, and only delays the progression of the disease. Employing the mouse model of cystinosis, using Ctns−/− mice, we first showed that the transplantation of syngeneic wild-type murine hematopoietic stem and progenitor cells (HSPCs) led to abundant tissue integration of bone marrow-derived cells, a significant decrease in tissue cystine accumulation, and long-term kidney, eye and thyroid preservation. To translate this result to a potential human therapeutic treatment, given the risks of mortality and morbidity associated with allogeneic HSPC transplantation, we developed an autologous transplantation approach of HSPCs modified ex vivo using a self-inactivated lentiviral vector to introduce a functional version of the CTNS cDNA, pCCL-CTNS, and showed its efficacy in Ctns−/− mice. Based on these promising results, we held a pre-IND meeting with the Food and Drug Administration (FDA) to carry out the FDA agreed-upon pharmacological and toxicological studies for our therapeutic candidate, manufacturing development, production of the GMP lentiviral vector, design Phase 1/2 of the clinical trial, and filing of an IND application. Our IND was cleared by the FDA on 19 December 2018, to proceed to the clinical trial using CD34+ HSPCs from the G-CSF/plerixafor-mobilized peripheral blood stem cells of patients with cystinosis, modified by ex vivo transduction using the pCCL-CTNS vector (investigational product name: CTNS-RD-04). The clinical trial evaluated the safety and efficacy of CTNS-RD-04 and takes place at the University of California, San Diego (UCSD) and will include up to six patients affected with cystinosis. Following leukapheresis and cell manufacturing, the subjects undergo myeloablation before HSPC infusion. Patients also undergo comprehensive assessments before and after treatment to evaluate the impact of CTNS-RD-04 on the clinical outcomes and cystine and cystine crystal levels in the blood and tissues for 2 years. If successful, this treatment could be a one-time therapy that may eliminate or reduce renal deterioration as well as the long-term complications associated with cystinosis. In this review, we will describe the long path from bench-to-bedside for autologous HSPC gene therapy used to treat cystinosis.