Hematopoietic Stem And Progenitor Cells Transplantation Research Articles

Prospective Phase I/II Study of Eltrombopag for the Treatment of Bone Marrow Failure in Fanconi Anemia

Fanconi anemia (FA) is an inherited disorder associated with loss of function mutations in gene members of the FA-BRCA pathway involved in interstrand cross-link DNA damage repair during cell division. Progressive bone marrow failure (BMF) is a primary cause of morbidity and mortality in patients with FA. Allogeneic hematopoietic stem and progenitor cell (HSPC) transplantation is the only curative treatment for FA-associated BMF. However, donor availability, graft failure, and FA-specific transplant toxicities remain significant hurdles. Attempts at genetic correction of FA are underway but collection of sufficient numbers of autologous HSPCs is challenging in subjects with advanced BMF. Androgens have been successfully used but side effects often prevent prolonged therapy. The orally bioavailable small molecule mimetic of thrombopoietin, eltrombopag (EPAG), was recently shown to stimulate multipotent long-term repopulating HSPCs in patients with acquired BMF, resulting in persistent trilineage hematopoiesis. EPAG promotes DNA repair (Guenther, Exp Hematol 2019) and maintains HSPCs under inflammatory conditions (Alvarado, Blood 2019), indicating potential relevance in FA. In this study, we investigated whether EPAG may offer a novel therapeutic modality for FA-associated BMF.We have enrolled 10 patients (FANCA, n=9 and FANCC, n=1) to date in a prospective phase I/II study of EPAG in Fanconi anemia (NCT03204188). All subjects received EPAG at an oral daily dose adjusted for age and ethnicity. The primary efficacy endpoint was the proportion of subjects with at least 2-fold increase in marrow cellularity or CD34+ HSPC frequency (marrow response), or clinically significant improvement in peripheral blood (PB) counts at 6 months (blood response). The primary safety endpoint was the global toxicity profile assessed at 6 months using CTCAE criteria. Responders were invited to continue on the extension phase of the study for an additional 3 years.Four of 10 patients have reached the 6-month assessment endpoint, and a pre-specified 3-month interim analysis is available on two additional subjects. A marrow response was observed in all 4 subjects at 6 months. Mean marrow cellularity increased by a factor of 4 (Fig. A/B), and CD34+ HSPC frequency improved 2.5-fold at 6 months relative to pre-treatment values (Fig. C/D). Marrow response criteria were also met in the other two subjects at the 3-month interim assessment. Primary PB response was observed in 2 of 4 patients in one (hemoglobin) or two lineages (hemoglobin and platelets) at 6 months of treatment. Responders continue to receive EPAG at 10 or 25 months with sustained improvements in blood counts and transfusion independence. The subject treated for 25 months recently met response criteria in a third lineage (neutrophils) (Fig. E). Both patients who had a marrow but no PB response are clinically well; one subject (FANCA) underwent an unrelated allogeneic HSPC transplantation one year ago, and the other subject (FANCC) recently entered the extension phase of this study. Unilineage PB response criteria were also met in one of two subjects at the 3-month interim assessment.No drug-related serious adverse events have occurred during EPAG treatment. All patients tolerated the maximum dose of EPAG and no interruption or dose reduction was required for adverse events attributable to EPAG. Patients without transfusional iron overload at study entry developed progressive depletion of iron stores attributable to the known potent iron chelating and mobilizing properties of EPAG. At a median of 3 months after initiating EPAG (range, 1 to 6 months), affected patients initiated daily oral iron supplementation with gradual amelioration of iron store levels. To date, there has been no occurrence of marrow fibrosis, solid malignancies or clonal evolution, defined as abnormalities on standard metaphase analysis of bone marrow or overt clinical transformation to MDS/AML.In sum, treatment with EPAG was associated with increased marrow HSPCs in all subjects and PB responses in a subset of patients with Fanconi anemia. The overall safety profile was favorable. Further follow-up and expansion of the ongoing patient cohort will confirm EPAG's potential as a safe and efficient long-term therapeutic modality for FA-associated BMF. Alternatively, EPAG could also be used safely to boost marrow CD34+ cell numbers prior to autologous gene therapy applications. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Administration of Granulocyte Colony-Stimulating Factor Post-Transplant Impedes Engraftment of CRISPR-Cas9 Edited Long-Term Repopulating Hematopoietic Stem and Progenitor Cells

Transplantation of genetically modified autologous hematopoietic stem and progenitor cells (HSPCs) holds a curative potential for subjects with inherited blood disorders. In recent years, transfer of a therapeutic gene to HSPCs has been successfully achieved using replication-incompetent integrating lentiviral vectors. More recently, advances have emerged to more precisely edit cellular genomes by specific correction of mutations or targeted gene addition at endogenous genomic loci. However, cellular processes triggered in HSPCs by the programmable nucleases utilized in these gene editing approaches may negatively impact their ability to reconstitute and maintain hematopoiesis long-term in recipient hosts. Granulocyte colony-stimulating factor (G-CSF) use after autologous HSPC transplantation is generally recommended to shorten the duration of severe neutropenia. However, little is known about the safety and efficacy of G-CSF use after transplantation of genetically modified autologous HSPCs. G-CSF is the principal cytokine regulating granulopoiesis, but also plays an important role in regulating hematopoietic stem cell (HSC) function (Schuttpelz, Leukemia 2014). Studies have suggested that G-CSF can exacerbate HSC damage caused by chemotherapeutic agents and irradiation by promoting differentiation at the expense of self-renewal and by inducing cellular senescence (van Os, Stem Cells 2000; Li, Cell Biosci 2015). Here, we asked whether G-CSF use after transplantation of gene edited HSPCs may negatively affect their long-term repopulating (LTR) and self-renewal capacities.To assess the effect of G-CSF use post-transplant on HSPC repopulating function after gene editing, mobilized human CD34+ cells were stimulated for 2 days, electroporated with AAVS1-specific sgRNA/Cas9 ribonucleoprotein complexes, and subsequently transplanted into NSG mice following busulfan conditioning. We subcutaneously injected G-CSF (125 mcg/kg/day) or PBS from post-transplant day 1 to 14 and compared hematopoietic reconstitution between both groups. The use of G-CSF initially increased human CD45+ cells in peripheral blood (PB) at 2 weeks post-transplant by enhancing CD13+ myeloid cell proliferation from committed progenitors (Fig. A). However, starting at 10 weeks post-transplant when hematopoiesis begins to emerge from the most primitive HSPCs, administration of G-CSF resulted in a 3 to 4-fold reduction in PB human cell engraftment compared to untreated mice (Fig. A). Similarly, G-CSF treated mice had significantly lower bone marrow (BM) and splenic engraftment at the endpoint (22 weeks) analysis, with comparable editing efficiency and lineage composition detected within human CD45+ cells (Fig. B, C). Importantly, percentages of immunophenotypic HSCs were 2-fold lower within the BM of G-CSF treated mice relative to the untreated group (Fig. D). To determine whether the negative effect of G-CSF post-transplant is specific to CRISPR-Cas9 gene editing, similar experiments were conducted using unmanipulated CD34+ cells or CD34+ cells transduced with a lentivirus vector expressing GFP. Interestingly, we found no differences in engraftment levels or immunophenotypic HSC frequencies between G-CSF treated and untreated mice. To assess the self-renewal capacity and quantify the frequency of gene edited LTR-HSCs, human CD45+ cells obtained from the BM of primary mice were serially transplanted into secondary recipient (NBSGW) mice at limiting dilution and BM engraftment was analyzed at 20 weeks post-transplant (total period of engraftment was 42 weeks). Notably, the secondary mice in the untreated group showed significantly superior human CD45+ cell engraftment compared with those in G-CSF treated group at the highest dose tested (Fig. E). The extreme limiting dilution analysis indicated that the frequency of LTR-HSCs was 5.1-fold higher (p = 0.011) in the untreated group compared with G-CSF treated group (Fig. F, G). Considering total engraftment in primary mice and the frequency of edited LTR-HSCs in secondary mice, we estimated the frequency of edited LTR-HSCs was reduced by 10-fold with G-CSF administration post-transplant.Collectively, our data suggest that G-CSF use post-transplant significantly reduces LTR and self-renewal capacities of CRISPR-Cas9 gene edited HSPCs. This understanding could have important clinical implications in HSPC gene therapy protocol. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Non-Human Primate CRISPR/Cas9 Model of Clonal Hematopoiesis of Indeterminate Potential Demonstrates Expansion of TET2-Disrupted Clones

Recent large-scale genetic screening of human blood cells have identified that somatic mutations associated with clonal expansions commonly arise with aging, even in the absence of cytopenias, myelodysplasia, or leukemia. Loss of function or dominant negative mutations in genes encoding epigenetic modifier enzymes such as DNMT3, TET2, and ASXL1 are most common in this “clonal hematopoiesis of indeterminate potential (CHIP)”. However, a causal relationship between these mutations and clonal expansions or progression to myelodysplasia or leukemia is unclear, as it has been difficult to extrapolate from murine models due to the differences in lifespan, aging phenotypes and hematopoietic cell transformation susceptibility. We utilized CRISPR/Cas9 gene editing of hematopoietic stem and progenitor cells (HSPC) in the rhesus macaque model to study the relationship between mutations in these genes, and clonal expansion and progression to abnormal hematopoiesis.We optimized CRISPR/Cas9 gene-editing strategies to introduce loss-of-function mutations in rhesus macaque CD34+ HSPC and applied this methodology to macaque autologous HSPC transplantation. We initially focused on the DNMT3A gene, which has been reported as most frequently mutated in CHIP. Macaque CD34+ HSPC were split into three equal fractions, transduced with one of three lentiviral vectors carrying either a gRNA for the control AAVS1 locus, DNMT3A exon 3, or DNMT3 exon 19 (the location of the “hotspot” putative dominant negative mutation in CHIP), and then electroporated with Cas9 mRNA. These edited cells were then transplanted into the autologous macaque following ablative total body irradiation. A second macaque also received autologous CD34+ cells electroporated with either AAVS1- or DNMT3A-targeting Cas9 ribonucleoprotein (RNP) complexes. Both animals engrafted promptly, with detection of low level (0-4%) indels at the on-target sites for AAVS1 and DNMT3A for up to 12 months post transplantation, but so far, we have not detected clonal expansion of DNMT3A-mutated HSPC in these two animals.We next delivered Cas9 and a gRNA pool targeting DNMT3A, TET2, and ASXL1 (the top three mutated and expanded genes in CHIP) (80%) or AAVS1 (20%) into CD34+ HSPC, followed by autologous transplantation (Fig 1A). After engraftment, the level of indels at each target site in granulocytes was 2% or less. However, during the 12 months post-transplant, there was a gradual and marked expansion of the granulocyte fraction containing indels predicted to knock out TET2 function (Fig 1B). Of the 17 different indel signatures identified at 12 months, only two were over the limit of detection in the infusion product or at 1 month post-transplant (Fig 1C), suggesting that the expanding TET2 mutant clones originated from long-term HSPC rather than short-term progenitors or that rare, initially mutated clones were sufficient to initiate clonal expansions. Finally, TET2 mutation frequency in bone marrow CD34+ cells was more than 2-fold higher (~20%) than in mature granulocytes (8%) at 12 months post-transplant (Fig 1C), suggesting a possible block in differentiation or a defect in release from the marrow for TET2 mutated cells.In conclusion, we have successfully created a non-human primate model of CHIP utilizing autologous transplantation of CRISPR/Cas9-edited HSPC, demonstrating gradual expansion of TET2-mutated clones. In contrast, simple indel formation at the DNMT3A locus was not sufficient to result in clonal expansion in the three animals studied to date, and a strategy utilizing HDR to specifically knock-in the hotspot base pair recurrently mutated in CHIP will be required to further investigate the relationship of DNMT3A to CHIP. Taken together, these approaches should improve our understanding of the effects of TET2 loss of function on the development of CHIP and its link to hematologic and cardiovascular disease, as well as provide a model for the testing of preventative interventions for clonal expansion and progression to frank malignancy. [Display omitted] DisclosuresDunbar:Novartis/GSK to institute: Research Funding. Winkler:Novartis/GSK to institute: Research Funding.

CRISPR/Cas9 Gene-Edited Hematopoietic Stem Cell Therapy for Sickle Cell Disease

Sickle cell disease (SCD) is a life-threatening hereditary disorder that affects thirty million individuals worldwide. The disease results from a single amino acid change in the β-globin gene, which causes polymerization of hemoglobin and deformation of red blood cells, leading to vaso-occlusion, severe pain crisis, multi-organ dysfunction, and ultimately reduction of lifespan in affected individuals. Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative therapy for SCD, yet its success is limited by donor availability and high risk of graft-versus-host disease. Increasing expression of fetal γ-globin in adults is an accepted therapeutic strategy for treating SCD. Supporting this approach are GWAS studies that have identified genetic loci, including within the BCL11A gene, that are associated with elevated fetal hemoglobin (HbF) and mitigated disease severity. Leveraging CRISPR/Cas9 genome editing technology, we aim to develop a safe and curative therapy by autologous, genome-modified HSCT leading to sustained generation of HbF-containing, functional red blood cells. Guide RNAs (gRNAs) targeting the erythroid-specific enhancer region of the BCL11A gene were assayed in CD34+ hematopoietic stem and progenitor cells (HSPCs), and one gRNA was selected for in vivo evaluation based on editing efficiency and functional potency. We achieved approximately 80% target site editing in human CD34+ HSPCs and 65% in CD34+CD38-CD90+CD45RA-CD49f+ defined LT-HSCs. Potential off-target editing sites were identified by two approaches: in silico prediction and unbiased oligo insertion method. These sites were then directly interrogated in HSPCs by targeted PCR amplification and NGS analysis, and to date our selected gRNA had zero validated off-target sites. In addition, HSPC composition was not altered following electroporation of ribonucleoprotein complex, and cells were capable of in vitro expansion and multi-lineage differentiation as measured by CFU assays. Upon in vitro differentiation into the erythroid lineage, edited cells demonstrated ~40% reduction in BCL11A mRNA with a corresponding two-fold increase in γ-globin transcript, translating to a 30-40% increase in HbF positive cells. Most importantly, when CD34+ cells derived from SCD patients were gene-edited and differentiated into erythroid cells, a similar editing efficiency and increase in HbF positive cells was observed. To evaluate whether gene editing alters the self-renewal, engraftment or lineage differentiation capacity of HSCs, gene-edited HSPCs were transplanted into NOD- scid IL2rγnull (NSG) mice to assess reconstitution of the hematopoietic system. Mice displayed 30-40% human CD45+ cell engraftment and multi-lineage reconstitution at 16 weeks following transplantation. When long-term engrafted bone marrow cells were harvested from the recipients and differentiated into erythroid cells in vitro, gene-edited bone marrow cells produced significantly higher numbers of HbF positive cells compared to mock-edited bone marrow cells. These data support the development of autologous genome-modified HSPC transplantation as a novel cell therapy for SCD. DisclosuresYu:Novartis Institutes for BioMedical Research: Employment. Liu:Novartis Institutes for BioMedical Research: Employment. Curran:Novartis Institutes for BioMedical Research: Employment. Zhang:Novartis: Employment. Snead:Novartis: Employment. Schmedt:Novartis: Employment. Yang:Novartis Institutes for BioMedical Research: Employment. Lin:Novartis: Employment. Tschantz:Novartis Institutes for BioMedical Research: Employment. Quinn:Novartis Institutes for BioMedical Research: Employment. Russ:Novartis: Employment. Clarkson:Novartis: Employment. Janiak:Novartis: Employment. Stewart:Intellia Therapeutics: Employment. Mulumba:Intellia Therapeutics: Employment. Lescarbeau:Intellia Therapeutics: Employment. Murray:Intellia Therapeutics: Employment. Seitzer:Intellia Therapeutics: Employment. Strapps:Intellia Therapeutics: Employment. Huang:Intellia Therapeutics: Employment. Sloan:Intellia Therapeutics: Employment. Mickanin:Novartis: Employment. Klickstein:Novartis: Employment. Stevenson:Novartis: Employment.

Manipulating Osteogenic/Adipogenic Balance in Mesenchymal Stromal Cells (MSCs) for Hematopoietic Applications

Aims: Bone marrow-derived mesenchymal stromal cells (BM-MSCs) have the potential to improve hematopoietic transplantation by supporting hematopoietic stem and progenitor cell (HSPC) expansion as well as by promoting HSPC engraftment to the BM. However, culture expanded MSCs becomes a mixture of progenitor cells committed to different cell lineages, each with different potencies for any one desired application. Clinically, the accumulation of adipogenic MSCs in the BM is believed to contribute towards age-related impairment of regeneration and hematopoiesis; this observation, together with our understanding of the inverse relationship between MSC osteogenic and adipogenic differentiation prompted a more in depth study of the ability of these different MSC lineages to support hematopoietic clinical applications. Clinically relevant methods for engineering a pure cell population from heterogeneous MSC culture will also be discussed.Methods: MSCs were derived from the healthy BM of patients aged 30-50 years. At various stages of osteogenic and adipogenic differentiation in vitro , cultures were evaluated for their ability to support cord blood (CB) CD34+ HSC expansion and stimulate regenerative processes such as angiogenesis and cell proliferation. qPCR, coupled with secretome analysis was performed to discover gene expression markers for MSCs with potent hematopoietic supportive capabilities. Finally, due to the lack of unique surface markers for identification of MSC subpopulations, we utilized multivariate biophysical analysis (microfluidic sorting, atomic force microscopy, etc.), correlating these quantitative measures with biomolecular markers as well as in vitro and in vivo functionality. Novel label-free cell separation strategies that take advantage of unique biophysical traits in a potent MSC subpopulation for HSPC transplantation was also developed to enable clinical translation.Results: Differentiated osteoblasts (OB) as well as pre-OB demonstrated a higher ability to support CB CD34+ HSC expansion compared to undifferentiated MSCs or MSCs in the adipogenic state. Over a period of 10 days, the total HSPC fold expansion was 44.4 ± 4.83, 57.8 ± 10.67, 40.2 ± 4.65, 34.2 ± 6.18 and 13.8 ± 5.50 for OB, pre-OB, MSCs, pre-AD and AD, respectively (n = 5). Absolute expanded CD34+ HSCs was highest with pre-OB cultures (~1.7 fold, n = 5) compared to undifferentiated MSCs. Adipogenic MSCs had much reduced capabilities for supporting HSPC expansion. In vitro angiogenic and proliferation assays showed the greatest degree of HUVEC sprouting and proliferation (~ 2.6 fold greater than undifferentiated MSCs) when exposed to a pre-OB secretome. Pre-OBs are distinct in their transcriptome (up-regulated osteopontin, runx2, angiopoietin-1, FGF1, etc) but no surface marker reliably distinguishes them from other MSCs. We subsequently found a minimal set of three biophysical markers: cell diameter, membrane stiffness and nuclear membrane fluctuations, which collectively are predictive of MSC subpopulations (multipotent vs committed progenitors). We hypothesized and experimentally verified that a biophysically derived pre-OB subpopulation, defined biophysically as MSCs with >20 μm diameter, >375Pa stiffness and <1.2 % nuclear fluctuation, exhibits significantly greater efficacy over other MSC subpopulations for promoting bone marrow repair (~2-3 fold improvements in medial survival following BM injury) and expansion of HSPCs (~1.5-2 fold increase in HSPC numbers). Following co-transplantation with HSPCs, biophysically derived pre-OB promoted faster engraftment of donor cells in the peripheral blood by week 4 (~ 9% vs < 5% in other MSC groups, P<0.05).Conclusion: These results demonstrate a correlation between MSCs with high osteogenic activity and the capabilities for BM repair as well as hematopoietic support, making such cells ideal for augmentation of HSC transplantations. In the absence of reliable surface markers for isolation of these pre-OBs, we developed a clinically relevant biophysical isolation strategy for derivation of pre-OBs that can markedly improve the efficacy of MSC-based approaches for augmentation of hematopoietic transplants. DisclosuresNo relevant conflicts of interest to declare.

Role of ex vivo Expanded Mesenchymal Stromal Cells in Determining Hematopoietic Stem Cell Transplantation Outcome

Overall, the human organism requires the production of ∼1 trillion new blood cells per day. Such goal is achieved via hematopoiesis occurring within the bone marrow (BM) under the tight regulation of hematopoietic stem and progenitor cell (HSPC) homeostasis made by the BM microenvironment. The BM niche is defined by the close interactions of HSPCs and non-hematopoietic cells of different origin, which control the maintenance of HSPCs and orchestrate hematopoiesis in response to the body’s requirements. The activity of the BM niche is regulated by specific signaling pathways in physiological conditions and in case of stress, including the one induced by the HSPC transplantation (HSCT) procedures. HSCT is the curative option for several hematological and non-hematological diseases, despite being associated with early and late complications, mainly due to a low level of HSPC engraftment, impaired hematopoietic recovery, immune-mediated graft rejection, and graft-versus-host disease (GvHD) in case of allogenic transplant. Mesenchymal stromal cells (MSCs) are key elements of the BM niche, regulating HSPC homeostasis by direct contact and secreting several paracrine factors. In this review, we will explore the several mechanisms through which MSCs impact on the supportive activity of the BM niche and regulate HSPC homeostasis. We will further discuss how the growing understanding of such mechanisms have impacted, under a clinical point of view, on the transplantation field. In more recent years, these results have instructed the design of clinical trials to ameliorate the outcome of HSCT, especially in the allogenic setting, and when low doses of HSPCs were available for transplantation.

The Strategies and Challenges of CCR5 Gene Editing in Hematopoietic Stem and Progenitor Cells for the Treatment of HIV.

HIV infection continues to be a serious health issue with an alarming global spread, owing to the fact that attempts at developing an effective vaccine or a permanent cure remains futile. So far, the only available treatment for the clinical management of HIV is the combined Anti-Retroviral Therapy (cART), but the long-term cART is associated with metabolic changes, organ damages, and development and transmission of drug resistant HIV strains. Thus, there is a need for the development of one-time curative treatment for HIV infection. The allogeneic transplantation with the Hematopoietic Stem and Progenitor cells (HSPCs) having 32bp deletion in Chemokine receptor 5 gene (CCR5 Δ32) demonstrated successful HIV remission in the Berlin and London patients, and highlighted that transplantation of CCR5 null HSPCs is a promising approach for a long- term HIV remission. The advent of gene editing technologies offers a new choice of generating ex vivo CCR5 ablated allogeneic or autologous HSPCs for stem cell transplantation into HIV patients. Many groups are attempting CCR5 disruption in HSPCs using various gene-editing strategies. At least two such studies, involving CCR5 gene editing in HSPCs have entered the clinical trials. This review aims to outline the strategies taken for CCR5 gene editing and discuss the challenges associated with the development of CCR5 manipulated HSPCs for the gene therapy of HIV infection.

In Vivo Tracking of Hematopoietic Stem and Progenitor Cell Ontogeny by Cellular Barcoding.

Cellular barcoding is a powerful technique that allows for high-throughput mapping of the fate of single cells, notably hematopoietic stem and progenitor cells (HSPCs) after transplantation. Unique artificial DNA fragments, termed barcodes, are stably inserted into HSPCs using lentiviral transduction, making sure that each individual cell receives a single unique barcode. Barcoded HSPCs are transplanted into sublethally irradiated mice where they reconstitute the hematopoietic system through proliferation and differentiation. During this process, the barcode of each HSPC is inherited by all of its daughter cells and their subsequent mature hematopoietic cell progeny. After sorting mature hematopoietic cell subsets, their barcodes can be retrieved from genomic DNA through nested PCR and sequencing. Analysis of barcode sequencing results allows for determination of clonal relationships between the mature cells, that is, which cell types were produced by a single barcoded HSPC, as well as the heterogeneity of the initial HSPC population. Here, we give a detailed protocol of a complete HSPC cellular barcoding experiment, starting with barcode lentivirus production, isolation, transduction, and transplantation of HSPCs, isolation of target cells followed by PCR amplification and sequencing of DNA barcodes. Finally, we describe the basic filtering and analysis steps of barcode sequencing data to ensure high-quality results.

In Vivo Gene Therapy for Canine SCID-X1 Using Cocal-Pseudotyped Lentiviral Vector.

Hematopoietic stem and progenitor cell (HSPC)-based ex vivo gene therapy has demonstrated clinical success for X-linked severe combined immunodeficiency (SCID-X1) patients who lack a suitable donor for HSPC transplantation. Nevertheless, this form of treatment is associated with an increased risk of infectious disease complications and genotoxicity mainly due to the conditioning regimen. In addition, ex vivo gene therapy approaches require sophisticated facilities to manufacture gene-modified cells and to care for the patients after chemotherapy. Considering these impediments, we have developed an in vivo gene therapy approach to treat canine SCID-X1 after HSPC mobilization and systemic delivery of the therapeutic vector. Here, we investigated the use of the cocal envelope to pseudotype a lentiviral (LV) vector expressing a functional gammaC gene. The cocal envelope is resistant to serum inactivation compared with the commonly used vesicular stomatitis virus envelope glycoprotein (VSV-G) envelope and thus well suited for systemic delivery. Two SCID-X1 neonatal canines treated with this approach achieved long-term therapeutic immune reconstitution with no prior conditioning. Therapeutic levels of gene-corrected CD3+ T cells were demonstrated for at least 16 months, and all other correlates of T cell functionality were within normal range. Retroviral integration-site analysis demonstrated polyclonal T cell reconstitution. Comparative analysis of integration profiles of foamy viral (FV) vector and cocal LV vector after in vivo gene therapy found distinct integration-site patterns. These data demonstrate that clinically relevant and durable correction of canine SCID-X1 can be achieved with in vivo delivery of cocal LV. Since manufacturing of cocal LV is similar to VSV-G LV, this approach is easily translatable to a clinical setting, thus providing for a highly portable and accessible gene therapy platform for SCID-X1.

CRISPR-Cas9-Mediated ELANE Mutation Correction in Hematopoietic Stem and Progenitor Cells to Treat Severe Congenital Neutropenia

Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation. In this study, we use CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)6 as a donor template delivery system to repair the ELANEL172P mutation in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs). We used a single guide RNA (sgRNA) specifically targeting the mutant allele, and an sgRNA targeting exon 4 of ELANE. Using the latter sgRNA, ∼34% of the known ELANE mutations can in principle be repaired. We achieved gene correction efficiencies of up to 40% (with sgELANE-ex4) and 56% (with sgELANE-L172P) in the SCN patient-derived HSPCs. Gene repair restored neutrophil differentiation in vitro and in vivo upon HSPC transplantation into humanized mice. Mature edited neutrophils expressed normal elastase levels and behaved normally in functional assays. Thus, we provide a proof of principle for using CRISPR-Cas9 to correct ELANE mutations in patient-derived HSPCs, which may translate into gene therapy for SCN.

3117 – GPRASP2 REGULATES CXCR4 STABILITY AND SIGNAL TRANSDUCTION IN HUMAN HEMATOPOIETIC CELLS WITH IMPLICATIONS FOR HSPC TRANSPLANTATION

Transplantation of hematopoietic stem and progenitor cells (HSPCs) is a life-saving therapy that is limited by donor availability and low cell numbers in sources such as cord blood. Advances in our understanding of the biochemical pathways that determine HSPC engraftment will allow for the optimization of transplantation strategies. We previously found that knockdown of GPRASP (G-protein Coupled Receptor Associated Sorting Protein) protein family members increases CXCR4 abundance and transplantation efficiency in murine HSPCs. Here, we demonstrate that GPRASP2 facilitates the acute agonist-induced degradation of CXCR4 in human hematopoietic cells and limits signaling downstream of CXCR4. Initial data also indicate that loss of GPRASP2 may increase the transplantation efficiency of human CD34+ cells. An antibody protection assay performed in HL60 cells demonstrated that knockdown of GPRASP2 significantly increases stability of CXCR4 following SDF-1 treatment. Phosphoflow experiments were performed to quantify the impact of GPRASP2 on signaling pathways downstream of CXCR4 including AKT, ERK1/2, p38, and JAK/STAT. SDF-1 treatment primarily resulted in phosphorylation of ERK1/2 and this was significantly increased by GPRASP2 knockdown. To test the role of GPRASP2 in human HSPC transplantation, CD34+ cells were edited by CRISPR/Cas9 with sgRNAs targeting GPRASP2 or the AAVS1 safe harbor site and then xenotransplanted into NBSGW mice. Initial results indicate that GPRASP2 KO results in higher human chimerism than is seen for AAVS1-targeted cells. In addition to assessing chimerism between transplants, changes in INDEL frequency within each transplant will be tracked for 16 weeks to determine the fitness of GPRASP2 knockout cells as compared to non-edited cells. This work reveals novel aspects of CXCR4 regulation by GPRASP2 and illuminates a potential point of intervention for the optimization of HSPC transplantation.

Tetraspanin Scaffold Proteins Function as Key Regulators of Hematopoietic Stem Cells.

Hematopoietic stem and progenitor cells (HSPCs) are responsible for the development, maintenance, and regeneration of all the blood forming cells in the body, and as such, are critical for a number of patient therapies. For successful HSPC transplantation, stem cells must traffic through the blood and home to the bone marrow (BM) microenvironment or “niche,” which is composed of soluble factors, matrix proteins, and supportive cells. HSPC adhesion to, and signaling with, cellular and extracellular components of the niche provide instructional cues to balance stem cell self-renewal and differentiation. In this review, we will explore the regulation of these stem cell properties with a focus on the tetraspanin family of membrane proteins. Tetraspanins are molecular scaffolds that uniquely function to distribute proteins into highly organized microdomains comprising adhesion, signaling, and adaptor proteins. As such, tetraspanins contribute to many aspects of cell physiology as mediators of cell adhesion, trafficking, and signaling. We will summarize the many reports that identify tetraspanins as markers of specific HSPC populations. Moreover, we will discuss the various studies establishing the functional importance of tetraspanins in the regulation of essential HSPC processes including quiescence, migration, and niche adhesion. When taken together, studies outlined in this review suggest that several tetraspanins may serve as potential targets to modulate HSPC interactions with the BM niche, ultimately impacting future HSPC therapies.

CRISPR-Cas9 Gene Editing of Hematopoietic Stem Cells from Patients with Friedreich’s Ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by expansion of GAA repeats in intron 1 of the frataxin (FXN) gene, leading to significant decreased expression of frataxin, a mitochondrial iron-binding protein. We previously reported that syngeneic hematopoietic stem and progenitor cell (HSPC) transplantation prevented neurodegeneration in the FRDA mouse model YG8R. We showed that the mechanism of rescue was mediated by the transfer of the functional frataxin from HSPC-derived microglia/macrophage cells to neurons/myocytes. In this study, we report the first step toward an autologous HSPC transplantation using the CRISPR-Cas9 system for FRDA. We first identified a pair of CRISPR RNAs (crRNAs) that efficiently removes the GAA expansions in human FRDA lymphoblasts, restoring the non-pathologic level of frataxin expression and normalizing mitochondrial activity. We also optimized the gene-editing approach in HSPCs isolated from healthy and FRDA patients’ peripheral blood and demonstrated normal hematopoiesis of gene-edited cells in vitro and in vivo. The procedure did not induce cellular toxic effect or major off-target events, but a p53-mediated cell proliferation delay was observed in the gene-edited cells. This study provides the foundation for the clinical translation of autologous transplantation of gene-corrected HSPCs for FRDA.

Anti-human CD117 CAR T-cells efficiently eliminate healthy and malignant CD117-expressing hematopoietic cells.

Acute myeloid leukemia (AML) initiating and sustaining cells maintain high cell-surface similarity with their cells-of-origin, i.e., hematopoietic stem and progenitor cells (HSPCs), and identification of truly distinguishing leukemia-private antigens has remained elusive to date. To nonetheless utilize surface antigen-directed immunotherapy in AML, we here propose targeting both, healthy and malignant human HSPC, by chimeric antigen receptor (CAR) T-cells with specificity against CD117, the cognate receptor for stem cell factor. This approach should spare most mature hematopoietic cells and would require CAR T termination followed by subsequent transplantation of healthy HSPCs to rescue hematopoiesis. We successfully generated anti-CD117 CAR T-cells from healthy donors and AML patients. Anti-CD117 CAR T-cells efficiently targeted healthy and leukemic CD117-positive cells in vitro. In mice xenografted with healthy human hematopoiesis, they eliminated CD117-expressing, but not CD117-negative human cells. Importantly, in mice xenografted with primary human CD117-positive AML, they eradicated disease in a therapeutic setting. Administration of ATG in combination with rituximab, which binds to the co-expressed CAR T-cell transduction/selection marker RQR8, led to CAR T-cell depletion. Thus, we here provide the first proof of concept for the generation and preclinical efficacy of CAR T-cells directed against CD117-expressing human hematopoietic cells.

CXCR4 expression in the bone marrow microenvironment is required for hematopoietic stem and progenitor cell maintenance and early hematopoietic regeneration after myeloablation.

The bone marrow (BM) microenvironment/niche plays a key role in regulating hematopoietic stem and progenitor cell (HSPC) activities; however, mechanisms regulating niche cell function are not well understood. In this study, we show that niche intrinsic expression of the CXCR4 chemokine receptor critically regulates HSPC maintenance during steady state, and promotes early hematopoietic regeneration after myeloablative irradiation. At steady state, chimeric mice with wild-type (WT) HSPC and marrow stroma that lack CXCR4 show decreased HSPC quiescence, and their repopulation capacity was markedly reduced. Mesenchymal stromal cells (MSC) were significantly reduced in the BM of CXCR4 deficient mice, which was accompanied by decreased levels of the HSPC supporting factors stromal cell-derived factor-1 (SDF-1) and stem cell factor (SCF). CXCR4 also plays a crucial role in survival and restoration of BM stromal cells after myeloablative irradiation, where the loss of BM stromal cells was more severe in CXCR4-deficient mice compared to WT mice. In addition, transplantation of WT donor HSPC into CXCR4-deficient recipient mice demonstrated reduced HSPC homing and early hematopoietic reconstitution. We found that CXCR4 signaling attenuates irradiation-induced BM stromal cell loss by upregulating the expression of the antiapoptotic protein Survivin via the PI3K pathway. Our study suggests that SDF-1-CXCR4 signaling in the stromal microenvironment cells plays a crucial role in maintenance of HSPCs during homeostasis, and promotes niche regeneration and early hematopoietic reconstitution after transplantation. Modulation of CXCR4 signaling in the HSPC microenvironment could be a means to enhance hematopoietic recovery after clinical hematopoietic cell transplantation.

Modeling NOTCH1 driven T-cell Acute Lymphoblastic Leukemia in Mice.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that arises from transformation of T-cell primed hematopoietic progenitors. Although T-ALL is a heterogenous and molecularly complex disease, more than 65% of T-ALL patients carry activating mutations in the NOTCH1 gene. The majority of T-ALL-associated NOTCH1 mutations either disrupt the negative regulatory region, allowing signal activation in the absence of ligand binding, or result in truncation of the C-terminal PEST domain involved in the termination of NOTCH1 signaling by proteasomal degradation. To date, retroviral transduction models have relied heavily on the overexpression of aggressively truncated variants of NOTCH1 (such as ICN1 or ΔE-NOTCH1), which result in supraphysiological levels of signaling activity and are rarely found in human T-ALL. The current protocol describes the method for mouse bone marrow isolation, hematopoietic stem and progenitor cell (HSC) enrichment, followed by retroviral transduction with an oncogenic mutant form of the NOTCH1 receptor (NOTCH1-L1601P-ΔP) that closely resembles the gain-of-function mutations most commonly found in patient samples. A hallmark of this forced expression of constitutively active NOTCH1 is a transient wave of extrathymic immature T-cell development, which precedes oncogenic transformation to T-ALL. Furthermore, this approach models leukemic transformation and progression in vivo by allowing for crosstalk between leukemia cells and the microenvironment, an aspect unaccounted for in cell-line based in vitro studies. Thus, the HSC transduction and transplantation model more faithfully recapitulates development of the human disease, providing a highly comprehensive and versatile tool for further in vivo and ex vivo functional studies.

R4 Rgs Subfamily Proteins Suppress Engraftment and Regulate SDF-1/CXCR4 Signaling in Human CD34+ Hematopoietic Stem/Progenitor Cells

Members of the Regulators of G-protein Signaling (RGS) are GTPase-accelerating proteins and have been implicated in SDF-1-directed trafficking of mature hematopoietic cells. However, their roles in hematopoietic stem and progenitor cells (HSPC) remain largely unknown. In this study, we investigated the expression, functions and mechanism of R4 RGS subfamily members on migration and engraftment of human HSPC. Our results demonstrated that cord blood (CB), bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells expressed specific RGS mRNAs, of which RGS1, RGS2, RGS13 and RGS16 were significantly upregulated by SDF-1 (1.6-1.9 fold, n=5, P&amp;lt;0.05). In the presence of AMD3100, a CXCR4 inhibitor, the stimulating effects of SDF-1 on RGS expression were completely abolished (n=6). SDF-1-directed functions (chemotaxis, trans-matrigel migration and calcium flux) and signaling (phosphorylation of Akt, ERK and Stat3) were significantly inhibited in RGS1, RGS13 and RGS16-overexpressing CD34+ cells (n=4-6, P&amp;lt;0.05) but not in RGS2-overexpressing cells, whereas actin polymerization, adhesion and colony formation were unaffected by these RGS members. In the NOD/SCID mouse xenotransplantation model, overexpression of RGS1, RGS13 or RGS16 in CD34+ cells did not impact their short-term homing but substantially compromised their long-term engraftment efficiency in bone marrow and spleens of recipient mice by 91.4%, 83.7% and 71.2%, respectively (n= 8-9; P&amp;lt;0.05). Genome-wide expression microarray and qPCR validation identified 32 common differentially expressed genes (1 upregulated and 31 downregulated) in RGS1, RGS13 or RGS16-overexpressing CD34+ cells. Network analysis revealed the potential mechanisms of RGS1, RGS13 and RGS16 downstream of SDF1/CXCR4 and Gαi protein, leading to compromised Akt, ERK and Stat3 phosphorylation and negative regulation of stem cell functions (CCNA1, SPP1, LPAR5, IL1RL1, HPSE), complement activation (C3AR1, C5AR2, C5AR1), proteolysis (TIMP3, MMP14) and cell migration (THBS1, F2RL2, PROS1, CCL1). Our results highlight the unprecedented functions of R4 RGS proteins in HSPC migration and engraftment, and provide the foundation of future design of RGS-targeting strategies to enhance the efficiency of clinical HSPC transplantation. Disclosures No relevant conflicts of interest to declare.

Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood.

Granulocyte colony‐stimulating factor (G‐CSF) is used in clinical practice to mobilize cells from the bone marrow to the blood; however, it is not always effective. We show that cobalt protoporphyrin IX (CoPP) increases plasma concentrations of G‐CSF, IL‐6, and MCP‐1 in mice, triggering the mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC). Compared with recombinant G‐CSF, CoPP mobilizes higher number of HSPC and mature granulocytes. In contrast to G‐CSF, CoPP does not increase the number of circulating T cells. Transplantation of CoPP‐mobilized peripheral blood mononuclear cells (PBMC) results in higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G‐CSF. Although CoPP is used to activate Nrf2/HO‐1 axis, the observed effects are Nrf2/HO‐1 independent. Concluding, CoPP increases expression of mobilization‐related cytokines and has superior mobilizing efficiency compared with recombinant G‐CSF. This observation could lead to the development of new strategies for the treatment of neutropenia and HSPC transplantation.

Macrophage polarization impacts tunneling nanotube formation and intercellular organelle trafficking

Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M1-like M(LPS/IFNγ) or anti-inflammatory M2-like M(IL-4/IL-10) affected TNT-like protrusion formation, intercellular transport and, ultimately, the efficacy of cystinosis prevention. We designed new automated image processing algorithms used to demonstrate that LPS/IFNγ polarization decreased bone marrow-derived macrophages (BMDMs) formation of protrusions, some of which displayed characteristics of TNTs, including cytoskeletal structure, 3D morphology and size. In contrast, co-culture of macrophages with cystinotic fibroblasts yielded more frequent and larger protrusions, as well as increased lysosomal and mitochondrial intercellular trafficking to the diseased fibroblasts. Unexpectedly, we observed normal protrusion formation and therapeutic efficacy following disruption of anti-inflammatory IL-4/IL-10 polarization in vivo by transplantation of HSPCs isolated from the Rac2−/− mouse model. Altogether, we developed unbiased image quantification systems that probe mechanistic aspects of TNT formation and function in vitro, while HSPC transplantation into cystinotic mice provides a complex in vivo disease model. While the differences between polarization cell culture and mouse models exemplify the oversimplicity of in vitro cytokine treatment, they simultaneously demonstrate the utility of our co-culture model which recapitulates the in vivo phenomenon of diseased cystinotic cells stimulating thicker TNT formation and intercellular trafficking from macrophages. Ultimately, we can use both approaches to expand the utility of TNT-like protrusions as a delivery system for regenerative medicine.

CRISPR-Edited Stem Cells in a Patient with HIV and Acute Lymphocytic Leukemia

SummaryThe safety of CRISPR (clustered regularly interspaced short palindromic repeats)–based genome editing in the context of human gene therapy is largely unknown. CCR5 is a reasonable but not absolutely protective target for a cure of human immunodeficiency virus type 1 (HIV-1) infection, because CCR5-null blood cells are largely resistant to HIV-1 entry. We transplanted CRISPR-edited CCR5-ablated hematopoietic stem and progenitor cells (HSPCs) into a patient with HIV-1 infection and acute lymphoblastic leukemia. The acute lymphoblastic leukemia was in complete remission with full donor chimerism, and donor cells carrying the ablated CCR5 persisted for more than 19 months without gene editing–related adverse events. The percentage of CD4+ cells with CCR5 ablation increased by a small degree during a period of antiretroviral-therapy interruption. Although we achieved successful transplantation and long-term engraftment of CRISPR-edited HSPCs, the percentage of CCR5 disruption in lymphocytes was only approximately 5%, which indicates the need for further research into this approach. (Funded by the Beijing Municipal Science and Technology Commission and others; ClinicalTrials.gov number, NCT03164135.)