Autologous Hematopoietic Stem Cell Research Articles

High-level correction of the sickle mutation is amplified in vivo during erythroid differentiation

SummaryBackgroundA point mutation in sickle cell disease (SCD) alters one amino acid in the β-globin subunit of hemoglobin, with resultant anemia and multiorgan damage that typically shortens lifespan by decades. Because SCD is caused by a single mutation, and hematopoietic stem cells (HSCs) can be harvested, manipulated, and returned to an individual, it is an attractive target for gene correction.ResultsAn optimized Cas9 ribonucleoprotein (RNP) with an ssDNA oligonucleotide donor together generated correction of at least one β-globin allele in more than 30% of long-term engrafting human HSCs. After adopting a high-fidelity Cas9 variant, efficient correction with minimal off-target events also was observed. In vivo erythroid differentiation markedly enriches for corrected β-globin alleles, indicating that erythroblasts carrying one or more corrected alleles have a survival advantage.SignificanceThese findings indicate that the sickle mutation can be corrected in autologous HSCs with an optimized protocol suitable for clinical translation.

Gene Therapy for Sickle Cell Disease

Gene Therapy for Sickle Cell Disease

Immunological barriers to haematopoietic stem cell gene therapy.

Cell and gene therapies using haematopoietic stem cells (HSCs) epitomize the transformative potential of regenerative medicine. Recent clinical successes for gene therapies involving autologous HSC transplantation (HSCT) demonstrate the potential of genetic engineering in this stem cell type for curing disease. With recent advances in CRISPR gene-editing technologies, methodologies for the ex vivo expansion of HSCs and non-genotoxic conditioning protocols, the range of clinical indications for HSC-based gene therapies is expected to significantly expand. However, substantial immunological challenges need to be overcome. These include pre-existing immunity to gene-therapy reagents, immune responses to neoantigens introduced into HSCs by genetic engineering, and unique challenges associated with next-generation and off-the-shelf HSC products. By synthesizing these factors in this Review, we hope to encourage more research to address the immunological issues associated with current and next-generation HSC-based gene therapies to help realize the full potential of this field.

Hematopoietic Cells from Pluripotent Stem Cells: Hope and Promise for the Treatment of Inherited Blood Disorders

Inherited blood disorders comprise a large spectrum of diseases due to germline mutations in genes with key function in the hematopoietic system; they include immunodeficiencies, anemia or metabolic diseases. For most of them the only curative treatment is bone marrow transplantation, a procedure associated to severe complications; other therapies include red blood cell and platelet transfusions, which are dependent on donor availability. An alternative option is gene therapy, in which the wild-type form of the mutated gene is delivered into autologous hematopoietic stem cells using viral vectors. A more recent therapeutic perspective is gene correction through CRISPR/Cas9-mediated gene editing, that overcomes safety concerns due to insertional mutagenesis and allows correction of base substitutions in large size genes difficult to incorporate into vectors. However, applying this technique to genomic disorders caused by large gene deletions is challenging. Chromosomal transplantation has been proposed as a solution, using a universal source of wild-type chromosomes as donor, and induced pluripotent stem cells (iPSCs) as acceptor. One of the obstacles to be addressed for translating PSC research into clinical practice is the still unsatisfactory differentiation into transplantable hematopoietic stem or mature cells. We provide an overview of the recent progresses in this field and discuss challenges and potential of iPSC-based therapies for the treatment of inherited blood disorders.

Результаты трансплантации аутологичных периферических гемопоэтических стволовых клеток при множественной миеломе в Кыргызстане

The article is devoted to performing a series of autologous transplantations of peripheral hematopoietic stem cells in multiple myeloma (MM). For the first time in Central Asia, in the Kyrgyz Scientific Center of Hematology of the Ministry of Health of the Kyrgyz Republic, autologous hematopoietic stem cells were used in the treatment of 26 patients with MM. Comprehensive laboratory and functional analyses were carried out to diagnose MM. Autologous stem cells were collected using the blood cell separator. Conditioning mode and an algorithm of therapeutic approaches of patients before autotransplantation and post-transplantation monitoring were used.

Skin Disinfection with 2% Chlorine Gluconate-Adine in Autologous Peripheral Hematopoietic Stem Cell Transplantation Patients

Skin Disinfection with 2% Chlorine Gluconate-Adine in Autologous Peripheral Hematopoietic Stem Cell Transplantation Patients

Autologous or allogeneic hematopoietic stem cells transplantation combined with high-dose chemotherapy for refractory neuroblastoma: A protocol for systematic review and meta-analysis.

Background:Neuroblastoma is a common solid malignant tumor in children. Despite the development of new treatment options, the prognosis of high-risk neuroblastoma patients is still poor. High-dose chemotherapy and hematopoietic stem cell (HSC) transplantation might improve survival of patients with refractory neuroblastoma. In this study, we aimed to summarize the efficacy of autologous or allogeneic HSC transplantation combined with high-dose chemotherapy for patients with refractory neuroblastoma through the meta-analysis.Methods and analysis:Relevant clinical trials of autologous or allogeneic HSC transplantation for the treatment refractory neuroblastoma patients will be searched in Web of Science, Cochrane Library, PubMed, Google Scholar, Embase, Medline, China National Knowledge Infrastructure, China Scientific Journal Database, Chinese Biomedical Literature Database and Wanfang Database from their inception to December 2020. Two researchers will perform data extraction and risk of bias assessment independently. The clinical outcomes including tumor response, overall survival, event-free survival (EFS), quality of life (QoL) and adverse events, were systematically evaluated by using Review Manager 5.3 and Stata 14.0 statistical software.Results:The results of this study will provide high-quality evidence for the effect of autologous or allogeneic HSC transplantation combined with high-dose chemotherapy on tumor response, survival, and QoL in patients with refractory neuroblastoma.Conclusions:The conclusions of this meta-analysis will be published in a peer-reviewed journal, and provide more evidence-based guidance in clinical practice.

The role of nutritional support in autologous hematopoietic stem cell (autohsct) transplantation in patients with hodgkin's lymphoma. (interim results)

Rationale: Correction of metabolic disorders is one of the important point’s supportive care during high-dose chemotherapy with autologous stem cell transplantation (autoHSCT) in patients with Hodgkin's lymphoma (HL). The impact of nutritional support on recovery during autoHSCT for this group of patients is not clearly understood. To assess the effect of combined nutritional support on the intensity of body weight loss, sarcopenia developing, the duration of neutropenic fever (NL), the frequency of blood transfusions during high-dose chemotherapy with autoHSCT in patients with HL.

Hematopoietic Stem- and Progenitor-Cell Gene Therapy for Hurler Syndrome

BackgroundAllogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant [MPSIH]). However, this treatment is only partially curative and is associated with complications.MethodsWe are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an α-L-iduronidase (IDUA)–encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years.ResultsWe now report interim results. The children’s mean (±SD) age at the time of HSPC gene therapy was 1.9±0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced joint stiffness, and normal growth in line with World Health Organization growth charts.ConclusionsThe delivery of HSPC gene therapy in patients with MPSIH resulted in extensive metabolic correction in peripheral tissues and the central nervous system. (Funded by Fondazione Telethon and others; ClinicalTrials.gov number, NCT03488394; EudraCT number, 2017-002430-23.)

Efficient Nanoparticle-Mediated Delivery of Gene Editing Reagents into Human Hematopoietic Stem and Progenitor Cells

Autologous hematopoietic stem cell (HSC) gene therapy has the potential to cure millions of patients suffering from hematological diseases and disorders. Recent HSCs gene therapy trials using CRISPR/Cas9 nucleases to treat sickle cell disease (SCD) have shown promising results paving the way for gene editing approaches for other diseases. However, current applications depend on expensive and rare GMP facilities for the manipulation of HSCs ex vivo. Consequently, this promising treatment option remains inaccessible to many patients especially in low- and middle-income settings. HSC-targeted in vivo delivery of gene therapy reagents could overcome this bottleneck and thereby enhance the portability and availability of gene therapy.Various kinds of nanoparticles (lipid, gold, polymer, etc.) are currently used to develop targeted ex vivo as well as in vivo gene therapy approaches. We have previously shown that poly (β-amino ester) (PBAE)-based nanoparticle (NP) formulations can be used to efficiently deliver mRNA into human T cells and umbilical cord blood-derived CD34 + hematopoietic stem and progenitor cells (HSPCs) (Moffet et al. 2017, Nature Communications). Here, we optimized our NP formulation to deliver mRNA into GCSF-mobilized adult human CD34 + HSPCs, a more clinically relevant and frequently used cell source for ex vivo and the primary target for in vivo gene therapy. Furthermore, we specifically focused on the evaluation of NP-mediated delivery of CRISPR/Cas9 gene editing reagents. The efficiency of our NP-mediated delivery of gene editing reagents was comprehensively tested in comparison to electroporation, the current experimental, pre-clinical as well as clinical standard for gene editing. Most important for the clinical translation of this technology, we defined quality control parameters for NPs, identified standards that can predict the editing efficiency, and established protocols to lyophilize and store formulated NPs for enhanced portability and future in vivo applications.Nanoformulations were loaded with Cas9 ribonucleoprotein (RNP) complexes to knock out CD33, an established strategy in our lab to protect HSCs from anti-CD33 targeted acute myeloid leukemia (AML) immunotherapy (Humbert et al. 2019, Leukemia). RNP-loaded NPs were evaluated for size and charge to correlate physiochemical properties with the outcome as well as establish quality control standards. NPs passing the QC were incubated with human GCSF-mobilized CD34 + hematopoietic stem and progenitor cells (HSPCs). In parallel, RNPs were delivered into CD34 + cells using our established EP protocol. NP- and EP-edited CD34 + cells were evaluated phenotypically by flow cytometry and functionally in colony-forming cell (CFC) assays as well as in NSG xenograft model.The optimal characteristics for RNP-loaded NPs were determined at 150-250 nm and 25-35 mV. Physiochemical assessment of RNP-loaded NP formations provided an upfront quality control of RNP components reliably detecting degraded components. Most importantly, NP charge directly correlated with the editing efficiency (Figure A). NPs achieved more than 85% CD33 knockout using 3-fold lower dose of CRISPR nucleases compared to EP. No impact on the erythromyeloid differentiation potential of gene-edited cells in CFC assays was observed. Finally, NP-modified CD34 + cells showed efficient and sustained gene editing in vivo with improved long-term multilineage engraftment potential in the peripheral blood (PB) and bone marrow stem cell compartment of NSG mice in comparison to EP-edited cells (Figure B).Here we show that PBAE-NPs enable efficient CRISPR/Cas9 gene editing of human GCSF-mobilized CD34 + cells without compromising the viability and long-term multilineage engraftment of human HSPCs in vivo. Most importantly, we defined physiochemical properties of PBAE-NPs that enable us to not only determine the integrity of our gene-editing agents but also predict the efficiency of editing in HSPCs. The requirement of 3-fold less reagents compared to EP, the ability to lyophilize quality-controlled and ready to administer gene therapy reagents, and the opportunity to engineer the surface of PBAE-NPs with HSC-targeting molecules (e.g. antibodies) could make this also a highly attractive and portable editing platform for in vivo HSC gene therapy. [Display omitted] DisclosuresKiem: VOR Biopharma: Consultancy; Homology Medicines: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company. Radtke: Ensoma Inc.: Consultancy; 47 Inc.: Consultancy.

Inhibition of Fibroblast Growth Factor-23 (FGF-23) Rescues Bone and Hematopoietic Stem Cell Niche Defects in Beta-Thalassemia, Uncovering the Missing Link between Hematopoiesis and Bone

The bone marrow (BM) niche regulation and interactions with hematopoietic stem cells (HSC) have been extensively studied in steady state conditions and malignancies, but are still underexplored in hematological inherited disorders. We provided the first demonstration of impaired HSC function caused by an altered BM niche in a non-malignant disease, beta-thalassemia (BT) (Aprile et al., Blood 2020). BT is a globally widespread congenital hemoglobin disorder, resulting in severe anemia, ineffective erythropoiesis and multi-organ secondary complications, including bone alterations. Correction of the genetic defect is achieved by transplantation of HSC from healthy donors or autologous HSC from patients upon gene therapy. Since the quality and the engraftment of HSC depend on the BM microenvironment, niche-HSC crosstalk plays a crucial role for transplantation outcome. During the analysis of different components of the niche, we found reduced bone density in BT th3 mice, along with a defective HSC supporting activity by the BM stromal niche. Interestingly, osteoporosis is a constant hallmark in BT patients.We investigated the mechanisms underlying bone and HSC niche defects focusing on the role of fibroblast growth factor-23 (FGF-23), a hormone mainly secreted by osteocytes, but also by erythroid cells, which negatively modulates bone metabolism. Since FGF-23 is stimulated by the anemia-related factor erythropoietin (EPO), we hypothesized that the high EPO levels in BT might contribute to increase FGF-23, potentially affecting bone and BM niche homeostasis.We found high levels of circulating FGF-23 in th3 mice (wt vs. th3: 290.5±27.3 vs. 1823±136.1 pg/ml, p<0.0001) and in BT patients (HD vs. THAL: 94.7±1.8 vs. 117.2±5.3 RU/ml, p<0.01), associated to its increased expression by bone and BM erythroid cells. In vivo neutralization of EPO axis was sufficient to normalize FGF-23 levels (th3 vs. th3+anti-EPO: 1591±162.2 vs. 496.1±33.3 pg/ml, p<0.001), thus demonstrating the causative role of EPO. EPO stimulation and signaling inhibition strategies highlighted the involvement of Erk1/2 and Stat5 pathways in activating Fgf-23 transcription in bone and BM erythroid cells, respectively.To provide proof of concept data on the contribution of FGF-23 to BT bone and stromal niche alterations, we inhibited FGF-23 signaling. In vivo administration of FGF-23 blocking peptide rescued the trabecular bone density in th3 mice (th3 vs. th3+FGF23inh: 138.2±4.9 vs. 166.9±5.2 mg/cm 3, p<0.01). Short-term inhibition treatment (38 hours) was sufficient to enhance bone mineralization by acting on Alkaline Phosphatase and on the expression of the main regulators of mineralization Dmp1 and Mepe by osteocyte, whereas long-term administration (12 days) restored also osteoblast number and bone deposition. Importantly, FGF-23 inhibition normalized the expression of key niche molecules, such as Jagged-1 and osteopontin, involved in the functional crosstalk between HSC and the stromal niche. Consistently, the treatment restored the frequency of th3 HSC by expanding the pool of quiescent cells (th3 vs. th3+FGF23inh: 0.026 vs. 0.045% on Lin neg BM cells, p<0.01). FGF-23 inhibition had also a positive anti-apoptotic effect on the expanded BM erythroid compartment (th3 vs. th3+FGF23inh: 61.6±1.3 vs. 51.1±2.1% of BM Ter119 + cells, p<0.001), promoting the maturation of early erythroid precursors (th3 vs. th3+FGF23inh: 8.5±1 vs. 16.4±1.1% of BM Pro-Erythroblasts, p<0.0001), as already shown in models of secondary anemias.Evidence in BT patients showed negative correlations between FGF-23 levels and markers of bone homeostasis (e.g. osteocalcin R 2=0.88, p<0.05) and positive correlations with makers of ineffective erythropoiesis (e.g. circulating reticulocytes R 2=0.83, p<0.05), thus positioning FGF-23 as the molecule at the crossroads of erythropoiesis and bone metabolism in BT.Our findings uncover an underexplored role of FGF-23 in bone and BM niche defects in a primary anemia, as a condition of chronic EPO stimulation, and propose FGF-23 as the missing link between hematopoiesis and bone regulation. The inhibition of FGF-23 signaling might provide a novel strategy to ameliorate bone compartment and restore HSC-BM niche interactions in BT by a ‘two birds with one stone’ approach, with a potential translational relevance in improving HSC transplantation and gene therapy. DisclosuresCappellini: Celgene: Consultancy, Research Funding; Vifor: Consultancy; La Jolla: Research Funding; Protagonist Therapeutics: Research Funding; IONIS Pharmaceuticals: Consultancy; CRISPR Therapeutics: Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Cedar Trial in Progress: A First in Human, Phase 1/2 Study of the Correction of a Single Nucleotide Mutation in Autologous HSCs (GPH101) to Convert HbS to HbA for Treating Severe SCD

Cedar Trial in Progress: A First in Human, Phase 1/2 Study of the Correction of a Single Nucleotide Mutation in Autologous HSCs (GPH101) to Convert HbS to HbA for Treating Severe SCD

P53 Is a Crucial Regulator of Hemogenic Endothelial Cell Differentiation from Human Induced Pluripotent Stem Cells

De novo generation of hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (hiPSCs) could provide a virtually unlimited supply of autologous HSCs for clinical transplantation, and offer various approaches that enable gene therapy, drug discovery, disease modeling, and in vitro modeling of human hematopoietic development. However, the derivation of long-term self-renewing HSCs from hiPSCs in culture remains elusive. The tumor suppressor protein p53 plays important roles in normal and malignant hematopoiesis, and Trp53-deficient mice exhibit increased number of HSCs. Although activation of p53 is known to promote differentiation of hPSCs and hPSCs recurrently acquire TP53 dominant negative mutations, its role in hematopoietic differentiation of hiPSCs has not been explored.To differentiate hiPSCs into hematopoietic stem and progenitor cells (HSPCs), we used embryoid body (EB) formation method to first differentiate hiPSCs into hemogenic endothelial (HE) cells that express the CD34 highCD144 +CD73 -CD184 -CD43 -CD235a - cell-surface markers. HE cells were then transferred onto a Matrigel-coated plate to undergo endothelial-to-hematopoietic transition (EHT) to generate HSPCs that express the CD34 midCD45 mid cell-surface markers. Developed HSPCs were functionally evaluated by colony forming assay.We observed that the expression of CDKN1A, a p53 target gene, was upregulated in hiPSC-derived EBs and HSPCs over the course of differentiation. To investigate the role of p53 in the generation of HSPCs from hiPSCs, we genetically deleted TP53 in hiPSCs followed by hematopoietic differentiation. While TP53 deletion increased the growth of EBs, it resulted in severe impairment of differentiation into HE cells and overall production of HSPCs that can form colonies. During HE differentiation from hiPSCs, TP53-deficient EBs showed significant reduction of endothelial-lineage gene expression, such as ETV2, CDH5, and PECAM1, as well as expression of RUNX1, a master transcription factor required for HE specification. These results indicate the indispensable role of p53 in HE differentiation from hiPSCs.We then examined the effect of p53 activation on HE differentiation from hiPSCs by pharmacological activation of p53 in hiPSC-derived cells. Transient activation of p53 by Nutlin-3, a small molecule that inhibits the p53-HDM2 interaction and protects p53 from proteasomal degradation, only during HE differentiation but not during EHT significantly promoted HSPC generation as compared to the vehicle treated control.Our findings shed light on the importance of selecting hiPSC lines that retain normal p53 activity for HE differentiation, and provide an approach to promote hematopoietic differentiation of hiPSCs by transiently activating p53 during HE differentiation. DisclosuresKanaujiya: Synthego: Other: Scientific Advisory; eGenesis: Other: Scientific Advisory.

Multiple sclerosis in 2020

Recent developments in the management of multiple sclerosis (MS) are ushering in a new era in MS treatment. The novel therapeutic disease-modifying agents that have been recently approved for the treatment of MS are B-cell therapies and various sphingosine-1-phosphate inhibitors. The Bruton's tyrosine kinase inhibitor therapies provide a novel therapeutic approach in the treatment of MS. Numerous studies have demonstrated a high degree of efficacy with autologous hematopoietic stem cells. There are upcoming trials focusing on remyelination strategies in MS. In this yearly review, we will focus on all these latest developments which are leading to a promising change in the diagnosis, treatment, and prognosis of this disease.

Development of a Treg Lineage Specific Lentiviral Vector for the Treatment of IPEX Syndrome

▪Autologous hematopoietic stem cell (HSC) transplant with gene therapy has recently demonstrated remarkable clinical success in the correction of genetic blood cell disorders. One excellent candidate disease for HSC gene therapy is Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome. IPEX is a devastating autoimmune disease caused by mutations in FOXP3, a transcription factor required for the proper development and function of regulatory T (Treg) cells. Tregs are critical for providing dominant suppression of autoreactive conventional T (Tconv) cells, and thus their absence in IPEX patients causes severe and often fatal multi-organ autoimmunity. Allogeneic HSC transplant has been shown to be curative for IPEX patients, but is limited due to a lack of suitable donors and immunologic transplant complications. In order to explore an autologous HSC gene therapy strategy for IPEX, we sought to design an endogenously regulated lentiviral vector (LV) which confers targeted and developmentally appropriate FoxP3 expression. Here, we utilized three intronic regulatory regions of the FoxP3 gene which have verified enhancer/regulatory function: conserved noncoding sequence (CNS) 1, CNS2, and CNS3. These sequences were added upstream from the FoxP3 promoter and cloned into the CCL vector backbone to generate two LV expressing either mStrawberry (FoxP3 reporter vector), or a human FoxP3 cDNA and mStrawberry (FoxP3 cDNA vector). A primary screen of the FoxP3 reporter vector in human cell lines from different hematopoietic lineages (K562 [erythromyeloid, FoxP3-], Jurkat [Tconv, FoxP3-] and MT-2 [Treg-like, FoxP3+]) revealed mStrawberry expression restricted to the FoxP3+ cell line with low inherent “leakiness” of expression in FoxP3- cell types. In order to evaluate in vivo lineage specificity of the FoxP3 reporter vector, we utilized both murine congenic transplants and humanized NSG xenografts to quantify LV expression in transduced HSPC and their progeny throughout hematopoietic development. In both models, we observed high levels of mStrawberry expression in the Treg lineage with low expression in CD4 Tconv cells and CD8 T cells, and negligible expression in HSPC, B cells, myeloid cells, suggesting that the regulatory elements included in this vector confer developmentally appropriate FoxP3 expression. We next evaluated the capacity of this vector to restore Treg development when introduced into FoxP3-deficient HSC from the scurfy mouse. Scurfy HSC were transduced with the FoxP3 cDNA vector and transplanted into WT CD45.1 congenic recipients to generate corrected scurfy Tregs (cSf-Tregs). Thymic cSf-Tregs phenotypically resembled WT-Treg showing equally high expression of the Treg surface markers CD25, GITR, and CTLA-4. Furthermore, FACS-sorted cSf-Tregs were equivalent to WT-Tregs in their ability to suppress Tconv cell proliferation in vitro. We next evaluated the in vivo functionality of cSf-Tregs by assessing their ability to reverse clinical disease signs in neonatal scurfy mice. Donor CD45.2+CD4+ cells (containing either WT-Tregs, cSf-Tregs, or uncorrected Sf T cells) were purified from the spleens of WT CD45.1 transplant recipients and administered to scurfy neonates by i.p. injection. Treated scurfy mice were evaluated for disease progression at 21 days post-injection. In comparison to WT littermate controls, untreated scurfy mice showed typical signs of disease progression including scaly skin, malformed ears, splenomegaly, and a high percentage of activated (CD62L-CD44+) CD4 T cells. Administration of WT-Tregs or cSf-Tregs resulted in complete correction of all measures of the scurfy phenotype, such that these mice were indistinguishable from WT littermate controls. In contrast, scurfy mice receiving uncorrected Sf T cells showed no signs of disease amelioration, confirming that disease correction was not an artifact of the transplant/CD4 isolation procedure. These results demonstrate that correction of scurfy HSC with a lineage-specific FoxP3 cDNA LV produces CD4+ Tregs capable of controlling autoimmune T cell responses in vivo. These findings pave the way for the treatment of IPEX patients by autologous HSC transplant and may further provide valuable insights into new treatments for patients with autoimmune disorders of different origin. DisclosuresKohn:Orchard Therapeutics Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioMarin Pharmaceutical: Research Funding; Biogen IDEC: Research Funding.

Clinical Outcomes up to 3 Years Following Lentiglobin Gene Therapy for Transfusion-Dependent β-Thalassemia in the Northstar Hgb-204 Study

BACKGROUNDAutologous hematopoietic stem cell (HSC) gene therapy could be an effective treatment for transfusion-dependent β-thalassemia (TDT) without some of the limitations and risks associated with allogeneic HSC transplantation. LentiGlobin Drug Product (DP) contains autologous HSC (CD34+ cells) transduced ex vivo with the betibeglogene darolentivec (BB305) lentiviral vector encoding a human β-globin gene with the anti-sickling T87Q mutation (HbAT87Q). The Northstar Study(HGB-204; NCT01745120) is an international, multi-center phase 1/2 clinical trial examining the safety and efficacy of LentiGlobin investigational gene therapy in patients with TDT. As of the most recent data cut (June 2, 2017), all patients have 1 to 3 years of follow-up post DP infusion. METHODSPatients (12-35 years of age) with TDT were enrolled at participating sites in the U.S., Australia, and Thailand. Autologous CD34+ cells were collected by mobilization and apheresis and transduced with the BB305 vector in a centralized facility. Patients underwent myeloablative conditioning with intravenous busulfan prior to infusion of transduced cells. Patients were monitored for hematologic engraftment, vector copy number (VCN), levels of HbAT87Q, and red blood cell (RBC) transfusion requirements post-infusion. Safety data, including adverse events (AEs), vector integration site analysis, and surveillance for replication competent lentivirus (RCL), were evaluated post-infusion. The primary analysis period for the study is 24 months following infusion; patients then transition into a long-term study for an additional 13 years of follow-up. RESULTSEighteen patients with TDT (8 with β0/β0 and 10 with non-β0/β0 genotypes; aged 12-35 years) received LentiGlobin DP. The median DP VCN was 0.7 (range: 0.3-1.5) copies/diploid genome, the median cell dose was 8.1 x 106 (range: 5.2-18.1 x 106) CD34+ cells/kg, and the proportion of transduced CD34+ cells was 17-58%. The toxicity profile observed to date has been typical of myeloablative conditioning. During a follow-up period of up to 38.3 months post-infusion (median 25.5 months; range: 14.9-38.3 months), there have been no ≥ grade 3 DP-related AEs and no evidence of clonal dominance or RCL. Serious AEs occurring after DP infusion have been reported in 6/18 (33%) patients: venoocclusive liver disease (n=2) and 1 case each of cellulitis, diarrhea, gastroenteritis, Klebisella infection, cardiac ventricular thrombosis, device-related thrombosis, and hyperglycemia. No serious AEs were considered related to DP.Of the 10 patients with non-β0/β0 genotypes, 8 have been free of transfusions for a median of 27.1 (range 12.5-35.2) months. At their latest study visit (n=8: Months 15-36), their total Hb level ranged from 9.3-13.7 g/dL, and their HbAT87Q level was 3.6-9.6 g/dL. The peripheral VCN ranged from 0.1−1.0. Notably, of these 8 patients, 6 have achieved “transfusion independence” (TI), defined as ≥12 months without transfusions with a weighted average Hb ≥9 g/dL. The 2 patients with non-β0/β0 genotypes who still require intermittent transfusions had annual transfusion volumes reduced by 30% and 94%; both received DP with a VCN in the lower range (DP VCNs: 0.3 and 0.4).Two patients with β0/β0 genotypes have not received a transfusion in more than a year. At the patients' last study visit (Month 24/Month 12), total Hb levels were 9.0 and 10.2 g/dL, HbAT87Q levels were 8.4 and 6.8 g/dL, and peripheral VCNs were 0.9 and 0.6, respectively. Six patients with β0/β0 genotypes have continued transfusions. However, their annual transfusion volumes have decreased by a median of 63% (range: 19% to 81%), from an annualized volume of 124.4-261.3 ml/kg/year at baseline to an annualized volume of 31.4-212.6 ml/kg/year (from 6 months post-infusion to data cut). CONCLUSIONSWith up to 3 years of follow-up, the Northstar Study is the largest international gene therapy trial in TDT to date. All patients, 9 of whom have at least 2 years of follow-up, have demonstrated ongoing clinical benefit with a manageable safety profile. A total of 10 patients (8/10 with non-β0/β0, 2/10 with β0/β0 genotypes) have been able to discontinue RBC transfusions and remain clinically well. The 8/18 patients still receiving transfusions have significantly reduced annualized transfusion volumes (up to 81% and 94% in patients with β0/β0 and non-β0/β0 genotypes, respectively). DisclosuresKwiatkowski:Bluebird Bio: Research Funding; Apopharma: Research Funding; Novartis: Research Funding; Agios: Consultancy, Honoraria; Ionis: Consultancy, Honoraria. Thompson:Novartis: Consultancy, Research Funding; Baxalta: Research Funding; Celgene: Consultancy, Research Funding; bluebird bio: Consultancy, Research Funding. Rasko:Genea: Equity Ownership; IMAGO Biosciences: Consultancy, Equity Ownership; Rarecyte: Consultancy, Equity Ownership; Novartis: Other: Clinical trials, Speakers Bureau; Spark: Equity Ownership, Other: clinical trials, Speakers Bureau; President-Elect International Society for Cellular Therapy: Membership on an entity's Board of Directors or advisory committees. Schiller:bluebird bio: Research Funding; mateon therapeutics: Research Funding. von Kalle:bluebird bio: Consultancy; GeneWerk: Equity Ownership. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Deary:bluebird bio: Employment, Equity Ownership. Asmal:bluebird bio: Employment, Equity Ownership. Walters:bluebird bio: Research Funding; AllCells, Inc: Other: Medical Director; Sangamo Therapeutics: Consultancy; ViaCord Processing Lab: Other: Medical Director.

Enhanced Expression of CXCR4 Facilitates the Trafficking of Anti-c-Kit Chimeric Antigen Receptor (CAR) T Cells to Bone Marrow and Achieves Effective Donor Stem Cell Engraftment

Allogeneic hematopoietic stem cell (HSC) transplantation is one of the curative therapeutic options for patients with a variety of inherited disorders of blood cells, including immune deficiencies and hemoglobinopathies. In addition, considerable progress has been made toward achieving clinical benefit from autologous HSC ex vivo gene therapy for some of these disorders. Generally, some level of conditioning using chemotherapy and/or radiation is needed to achieve the required engraftment of allogeneic HSC or gene corrected autologous HSC. There is considerable interest in finding less toxic and more focused approaches to achieve the conditioning. Promising results were observed using antibody-based approaches including anti-c-kit (CD117) or anti-CD45 which directly target HSCs, and these results can be enhanced by linking the antibody to a toxin such as saponin. Here, we explored a related approach in mouse models in which chimeric antigen receptor (CAR)-T cells targeting c-kit (c-kit CAR-T cells) were employed as conditioning to achieve significant engraftment of congenic HSCs. Our initial attempts at efficient conditioning with c-kit CAR-T cells failed because there was insufficient trafficking of CAR-T cells to bone marrow (BM) and insufficient CAR-T cell expansion in vivo and because we did not eliminate the CAR-T cells before transplanting congenic HSCs.To address these barriers, before injecting CAR-T cells (derived from C57BL/6 Thy1.1/CD45.2 mouse spleen on Day1 and transduced with CAR retrovirus on Day2) we pre-treated the recipient mice (Thy1.2/CD45.2) with low-dose cyclophosphamide (CY, 125 mg/kg; Day2) and co-transduced the c-kit CAR-T cells with a CXCR4 (CD184) expression vector (Day2), followed by injection of 5×106 CAR-T cells on Day3. We later removed these CAR-T cells by treating the mice with anti-Thy1.1 antibody (Ab) on Day11 prior to HSC transplantation from congenic Thy1.2/CD45.1 mice (Day12; 3×106 cells/mouse).After single CAR retrovirus transduction, >90% of T cells expressed CAR. When used in vitro, CAR-T cells efficiently depleted BM c-kit+ population (10.5% reduced to 1.6%) and completely suppressed colony formation in nitrocellulose medium. In initial in vivo studies, we used a luciferase marker in the CAR-T cells to allow whole body imaging in addition to flow cytometry (FACS) of BM to assess trafficking. With no pre-treatment of mice, both in vivo imaging and FACS demonstrated poor trafficking of CAR-T cells to BM (< 1% of cells), and there was no decrease of the BM c-kit+ population. In contrast, pre-treatment with low-dose CY and overexpression of CXCR4 on the CAR-T cell surface improved the CAR-T cell expansion in vivo and the migration into BM (mean Thy1.1+ cells, 11.9%; n= 5), and this was associated with significant in vivo depletion of BM c-kit+ population (9.0% reduced to 0.0%). Severe pancytopenia was confirmed in peripheral blood (Hgb 2.2 g/dL compared to the normal, 13.0 g/dL) along with aplastic BM (Figure A), and these mice died within 4 weeks without BM transplant. Subsequent injection of anti-Thy1.1 Ab completely depleted CAR-T cells in viv o. When congenic BM cells were transplanted into mice conditioned in this manner (CY+CAR-T+Thy1.1 Ab), these mice achieved complete recovery of hematopoiesis with significant engraftment of donor BM in all lineages in peripheral blood (Figure B) and long-term HSCs in BM (> 20 % donor chimerism).Our findings provide proof-of-concept that c-kit CAR-T cells can be used to achieve sufficient conditioning to allow donor chimerism after congenic transplantation without use of high-dose chemotherapy or radiation. Low-dose CY was required to achieve the reduction of host T cells and stimulate in vivo expansion of CAR-T cells. A key element to achieve BM HSC depletion was the co-transduction of CXCR4 that greatly increased trafficking of the CAR-T cells to BM. This finding may be one of the most important aspects of this study, with broader implications for the CAR-T cell field in suggesting that use of trafficking receptors or ligands might be generalizable as a method to enhance targeting to designated organs or solid tumors. In summary, our studies add to the list of tools that should be further explored in the effort to seek less toxic conditioning regimens for BM transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cognitive Impairment and Quality of Life of Patients Subjected to Hematopoietic Stem Cell Transplantation

Objective: To assess and correlate overall quality of life and the cognitive function of adult patients with hematologic cancer subjected to autologous and allogeneic hematopoietic stem cell transplantations up to three years after treatment. Materials and method: A longitudinal, observational, and analytical study was conducted with 55 patients in a reference hospital in Latin America, from September 2013 to February 2019, with the Quality of Life Questionnaire-Core 30, analyzed with the Spearman’s correlation coefficient and Generalized Linear Mixed Model tests. Results: Overall quality of life in autologous and allogeneic transplantations presented a decline in the pancytopenia phase (59.3 and 55.3, respectively). There was impairment of the cognitive function in the autologous group in post-transplantation after two years (61.90) and, in the allogeneic group (74), in pancytopenia. In the autologous group, a positive (0.76) and significant (p &lt; 0.04) correlation is observed between the cognitive domain and quality of life in post-transplantation after two years. In the allogeneic group, there was a positive (0.55) and significant (p &lt; 0.00) correlation from 180 days after transplantation. Conclusions: Quality of life and the cognitive function present impairment and there is a correlation after the hematopoietic stem cell transplantation for both groups: autologous and allogeneic.

Identification of T Cell Receptors Specific to Antigens Presented By HLA-B5401 on IPS Cell-Derived Hematopoietic Stem Cells in a Patient with Acquired Aplastic Anemia Carrying B5401-Lacking Leukocytes

[Background] Cytotoxic T lymphocytes (CTLs) specific to hematopoietic stem cells (HSCs) are thought to play an important role in the development of acquired aplastic anemia (AA). The presence of HLA class I allele-lacking leukocytes as a result of 6pLOH or allelic mutations in about 25% of AA patients strongly suggests that CTLs specific to antigens presented by the specific HLA alleles are involved in the destruction of normal HSCs as well as in the escape of HLA-lacking HSCs. Establishing CTLs from these patients by culturing the patients' CD8+ cells with targeted HSCs may be useful for identifying autoantigens of AA. However, it is generally impossible to obtain sufficient numbers of HSCs from the patients for functional assays and in vitro stimulation of patient's T cells with HSCs may induce clonal expansion of CTLs irrelevant to pathogenesis of AA. To solve these problems, we generated iPS cells (iPSCs) from an untreated AA patient who possessed HLA-B5401-lacking leukocytes for inducing HSCs and attempted to identify cDNAs of T-cell receptor (TCR) α and β chains of predominant T cells in the patient's bone marrow (BM) using a single T-cell analysis, which may specifically recognize autologous HSCs. [Methods] iPSCs were generated from the peripheral blood mononuclear cells of an AA patient (56-year-old man), whose moderate AA later responded to cyclosporine therapy, using Yamanaka's method. The patient's monocytes accounted for 20% wild-type, 20% 6pLOH(+) and 60% B5401-lacking cells at the time of sampling. CD34+ cells were induced from these iPSCs and injected into the BM of C57BL/6.Rag2 mice harboring NOD-Sirpa (BRGS). CD8+ T cells in the BM of the patient were subjected to a single cell sorting followed by cDNA amplification of TCRVβ chains. TCRα chains of five T cells that showed the five most recurrent TCRVβs were determined, and the TCRβ cDNAs were retrovirally transfected to Jurkat cells that lacked TCRβ chain and were transfected with IFN-γ cDNA. Specific responses of each TCR transfectant to iPSC-derived CD34+ cells were determined using ELISA for detecting IFN-γ. [Results] Five iPSC clones, including wild type (n=2), 6pLOH(+) (n=1), and B5401-lacking (B5401[-]) (n=2) clones, that had different mutations in B*54:01 were generated. Three-week culture of the iPSCs in the presence of various growth factors produced hematopoietic cells consisting of 50% CD34+ cells of each phenotype, which were able to reconstitute of BRGS mice with 3.5% to 8.9% human CD45+ cells in the peripheral blood at 9-12 weeks after injection. When the four TCR transfectants were tested for their response to wild-type and B5401-lacking CD34+ cells, all four transfectants secreted higher amounts of IFN-γ in response to wild-type cells than to B5401(-) CD34+ cells. [Conclusions] HSCs with original genotypes capable of reconstituting hematopoiesis in immunodeficient mice were successfully induced from iPSCs that had been generated from an AA patient's monocytes. The TCR transfectants showing a specific response to B5401(+) wild-type CD34+ cells will be useful for identifying the ligand peptides presented by B5401. DisclosuresNo relevant conflicts of interest to declare.

Multiple myeloma and autologous haematopoietic stem-cell transplantation without cryopreservation: experiences of the Clinical Hematology Department of Casablanca, Morocco

La chimiothérapie à haute dose suivie d´autogreffe des cellules souches hématopoïétiques (ASCT) reste le traitement de choix du myélome multiple chez les sujets âgés ≤ 65 ans. Dans les pays en développement, l´ASCT sans cryoconservation, permet de réduire les coûts d´hospitalisation et des installations nécessaires. Pour évaluer cette procédure, une étude prospective, longitudinale et ouverte a été conduite au Service d´Hématologie Clinique de Casablanca au Maroc. Sur 24 mois, 64 patients ont été colligés. Après le traitement d´induction, la réponse globale (Rémission complète + Très bonne réponse partielle) était à 67,2% (43 patients). La richesse moyenne du greffon autologue était de 12.97x106 CD34+/Kg [2.4- 53x106 CD34+/Kg] et la durée médiane d´hospitalisation était de 20,5 jours [14-60 jours]. La réponse globale en post autogreffe était de 84% (54 patients). A 24 mois, l´estimation de la survie globale (OS) était à 83,5%, la médiane d´OS n´était pas atteinte et la survie sans progression (PFS) à 65,9% avec une médiane de la PFS à 24,1 mois avec un intervalle de confiance à 95% [21,7-26,5mois]. L´ASCT sans cryoconservation reste une excellente alternative dans notre contexte, ce qui permet de réduire les délais d´attente, et les coûts de congélation.