Hematopoietic Stem Cells In Patients Research Articles

Genome Editing Therapy for the Blood: Ex Vivo Success and In Vivo Prospects.

Hematopoietic stem cells (HSCs) provide the body with a continuous supply of healthy, functional blood cells. In patients with hematopoietic malignancies, immunodeficiencies, lysosomal storage disorders, and hemoglobinopathies, therapeutic genome editing offers hope for corrective intervention, with even modest editing efficiencies likely to provide clinical benefit. Engineered white blood cells, such as T cells, can be applied therapeutically to address monogenic disorders of the immune system, HIV infection, or cancer. The versatility of CRISPR-based tools allows countless new medical interventions for diseases of the blood, and rapid ex vivo success has been demonstrated in hemoglobinopathies via transplantation of the patient's HSCs following genome editing in a laboratory setting. Here we review recent advances in therapeutic genome editing of HSCs and T cells, focusing on the progress in ex vivo contexts, the promise of improved access via invivo delivery, as well as the ongoing preclinical efforts that may enable the transition from ex vivo to invivo administration. We discuss the challenges, limitations, and future prospects of this rapidly developing field, which may one day establish CRISPR as the standard of care for some diseases affecting the blood.

G-CSF + plerixafor versus G-CSF alone mobilized hematopoietic stem cells in patients with multiple myeloma and lymphoma: a systematic review and meta-analysis

Aim The combination of granulocyte-colony stimulating factor (G-CSF) and plerixafor is one of the approaches for hematopoietic stem cell mobilization in patients with multiple myeloma (MM), non-Hodgkin’s lymphoma (NHL), and Hodgkin’s lymphoma (HL). This systematic review and meta-analysis aimed to determine the ability of G-CSF + plerixafor to mobilize peripheral blood (PB) CD34+ cells and examine its safety profile. Methods We performed a database search using the terms ‘granulocyte colony stimulating factor’, ‘G-CSF’, ‘AMD3100’, and ‘plerixafor’, published up to May 1, 2023. The methodology is described in further detail in the PROSPERO database (CRD42023425760). Results Twenty-three studies were included in this systematic review and meta-analysis. G-CSF + plerixafor resulted in more patients achieving the predetermined apheresis yield of CD34+ cells than G-CSF alone (OR, 5.33; 95%, 4.34–6.55). It was further discovered that G-CSF + plerixafor could mobilize more CD34+ cells into PB, which was beneficial for the next transplantation in both randomized controlled (MD, 18.30; 95%, 8.74–27.85) and single-arm (MD, 20.67; 95%, 14.34–27.00) trials. Furthermore, G-CSF + plerixafor did not cause more treatment emergent adverse events than G-CSF alone (OR, 1.25; 95%, 0.87–1.80). Conclusions This study suggests that the combination of G-CSF and plerixafor, resulted in more patients with MM, NHL, and HL, achieving the predetermined apheresis yield of CD34+ cells, which is related to the more effective mobilization of CD34+ cells into PB.

Development of hematopoietic stem cell-targeted gene therapy

Hematopoietic stem cell (HSC)-targeted gene therapy is curative for various genetic blood diseases, and its efficacy has been demonstrated in recent clinical trials. HSCs have self-renewal and hematopoietic multipotency; therefore, repairing pathological mutations or defects in HSCs allows for a lifelong cure with a single treatment. Autologous HSC gene therapy has been developed by lentiviral gene addition or gene editing, and is an option for most patients because it does not require a compatible donor. Current HSC gene therapy is based on ex vivo methods, in which patient HSCs are harvested, genetically modified ex vivo, and autologously transplanted into patients. However, the complexity of this process and the high cost of treatment are hindering the spread of gene therapy. Therefore, in vivo HSC gene therapy is being developed to deliver gene therapy tools directly into bone marrow HSCs by administration without ex vivo culture.

Increased clonal hematopoiesis mutational burden and accelerated dynamic evolution of circulating clones in patients with ischemic heart failure

Abstract Background Clonal hematopoiesis (CH) - defined as the age-related acquisition of mutations in hematopoietic stem cells giving rise to mutated circulating leukocytes - is associated with increased risk of death in patients with heart failure with reduced ejection fraction (HFrEF). While the mutational landscape of CH in peripheral blood (PB) of patients with HFrEF is well characterized, the mutational burden in bone marrow (BM) samples of patients with HFrEF as well as its dynamics over time have not been investigated. Methods/Results We analyzed matched BM and PB cells from 68 patients with ischemic HFrEF by deep error-corrected targeted sequencing of 56 CH-driver genes with a detection threshold of VAF ≥0.5%. In PB, 56/68 patients carried at least one CH mutation, whereas 63/68 patients had mutations in BM. The absolute numbers of detectable mutations were significantly higher in BM compared to PB (215 mutations vs 120 mutations; p<0.0001) (Fig. 1A). Likewise, the cumulative VAF was significantly higher in BM compared to PB (3.83 +/- 0.5% vs 3.0% +/-0.7%; mean +/- SEM, p = 0.0025) (Fig. 1B). However, there was a strong correlation between mutation load in PB and BM (r = 0.54, p<0.0001). Among the two most common mutations, DNMT3A mutations were either present in BM only or both compartments (PB only/BM only/ both compartments 2/12/10), whereas TET2 mutations were strikingly enriched in BM cells (PB only/BM only/both compartments 0/16/2). To study the dynamic evolution of CH clones in patients with HFrEF, we serially analyzed PB samples from 19 patients. At baseline, 16/19 patients carried at least one mutation in a CH driver gene and a total of 31 mutations. After a median follow-up of 12 months, 19/19 patients had at least one detectable mutation and a total of 43 mutations were identified (mean number of mutations per patient 1.63 +/- 0.033 vs 2,26 +/- 0.27; mean +/- SEM, p = 0,0410), indicating an increased mutation load over time (Fig. 2A). Moreover, cumulative VAF increased from 1.59 +/-0.36 % to 2.01 +/- 0.38 %). Conclusion Patients with ischemic HFrEF display a high burden of CH mutations compared to values reported for healthy individuals, which is further amplified in the bone marrow compartment. Clones with new mutations arise rapidly in longitudinal samples of patients with HFrEF and clonal expansion occurs with high momentum. The overrepresentation of mutations in the BM is indicative for augmented DNA damage in the hematopoietic stem cells of patients with HFrEF, while the rapid expansion of mutated circulating clones over time suggests that heart failure itself may expedite the expansion of hematopoietic cells harboring CH-driver mutations.

Features of the leukocyte pool of umbilical cell blood cell concentrate prepared for long-term cryostorage

Allogeneic hematopoietic cell transplantation is the standard of care for patients with highrisk acute leukemia. Cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells for patients who need transplantation but do not have a related donor. The purpose of our study was to evaluate the characteristics of leukocyte pool (cellular composition, immunophenotype) of umbilical cord blood (UCB) cell concentrates, which were harvested for public long-term cryostorage for the needs of transplantology. A study of 1096 samples of umbilical cord blood of full-term newborns before and after processing [isolation of hematopoietic stem cells (HSC) concentrate] was carried out. The number of cells in the pooled concentrate of HSC at a volume of 25 mL, after long-term cryostorage was as follows: leukocytes, 17.41±0.36 × 108; HSCs with CD34+ immunophenotype, 5.25±3.6 × 106; natural killer cells (CD3-CD16+CD56+), 1.65+0.7 × 108. Analysis of the NKT cell population revealed 6.22±2.1% of cells with CD3+CD16/CD56+ immunophenotype. The contents of CD3+CD56+ cells was 2.69±2.4%, the relative amount of CD3+CD16+ was 2.53±1.2%. In the pooled NKT cell preparations, 5.15±2.1% of CD56+CD16 cells were detected, and two minor subpopulations were also identified, which differ in CD56 and CD16 antigen expression: the level of CD56-CD16+ was 2.91±1.2%, and the ratio of CD56+CD16+ cells was 0.69±0.3%. The obtained data on relative amounts and characteristics of cellular immunophenotype as well as the heterogeneity of the NK and NKT cell population in the HSC UCB concentrate, subjected to long-term cryostorage must be taken into account when selecting the HSC UCB concentrates for the purpose of transplantation in oncohematological diseases.

Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox

Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole-genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole-genome sequencing (WGS) but still generate hundreds of artifacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolbox (PTATO), to accurately detect single base substitutions, insertions-deletions (indels), and structural variants in PTA-based WGS data. PTATO includes a machine learning approach and filtering based on recurrence to distinguish PTA artifacts from true mutations with high sensitivity (up to 90%), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that hematopoietic stem cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS, have normal somatic single base substitution burdens but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy.

Efficacy and Safety of Plerixafor Combined with G-CSF for Autologous Peripheral Blood Hematopoietic Stem Cell Mobilization in Lymphoma Patients

To investigate the efficacy and safety of plerixafor combined with granulocyte colony-stimulating factor (G-CSF) in mobilizing peripheral blood hematopoietic stem cells in patients with lymphoma. The clinical data of lymphoma patients who received autologous hematopoietic stem cell mobilization using plerixafor combined with G-CSF from January 2019 to December 2021 were retrospectively analyzed. The patients received 3 kinds of mobilization regimens: front-line steady-state mobilization, preemptive intervention, and recuse mobilization. The acquisition success rate, excellent rate of collection, and incidence of treatment-related adverse reaction were counted. The influence of sex, age, disease remission status, bone marrow involvement at diagnosis, chemotherapy lines, number of chemotherapy, platelet count and number of CD34+ cells on the day before acquisition in peripheral blood on the collection results were analyzed to identify the risk factors associated with poor stem cell collection. A total of 43 patients with lymphoma were enrolled, including 7 cases who received front-line steady-state mobilization, 19 cases who received preemptive intervention, and 17 cases who received recuse mobilization. The overall acquisition success rate was 58.1% (25/43) after use of plerixafor combined with G-CSF, and acquisition success rate of front-line steady-state mobilization, preemptive intervention, and recuse mobilization was 100%, 57.9%(11/19), and 41.2%(7/17), respectively. The excellent rate of collection was 18.6%(8/43). A total of 15 patients experienced mild to moderate treatment-related adverse reactions. The number of CD34+ cells < 5 cells/μl in peripheral blood on the day before collection was an independent risk factor affecting stem cell collection. Plerixafor combined with G-CSF is a safe and effective mobilization regimen for patients with lymphoma. The number of CD34+ cells in peripheral blood on the day before collection is an predictable index for the evaluation of stem cell collection.

The CSN5/HSF/SPI1/PU.1 Axis Regulates Cell Proliferation in Hypocellular Myelodysplastic Syndrome Patients.

This study explored the relationship between the activation of the jak/stat3 signaling pathway and the CSN5 gene transcript and protein expression levels in the hematopoietic stem cells of patients with myelodysplastic syndromes (MDSs). This study also aimed to investigate the correlation between the expression level of CSN5 and the deubiquitination of HSF1, as well as the transcript level of the spi1/pu.1 genes to explore the pathogenesis of MDS. We isolated cells from normal individuals and MDS patients, and the mRNA and protein expression levels of spi1/pu.1 in cd34+ cells (hematopoietic stem cells) were measured by PCR and western blotting, respectively. A ChIP assay was used to detect the binding of HSF1 to the spi1/pu.1 promoter in cd34+ cells. The ubiquitination of HSF1 in cd34+ cells was detected by CO-IP. The binding of HSF1 and Fbxw7α was detected in in cd34+ cells by CO-IP. The binding of HSF1 and CSN5 was evaluated. A luciferase reporter assay was used to detect the effect of STAT3 on CSN5 promoter activation in cd34+ cells. Western blotting was used to detect the phosphorylation of STAT3 in cd34+ cells of MDS patients. The binding of STAT3 and C/EBP beta in cd34+ cells was detected by CO-IP. Inhibition of SPI1/PU.1 expression was observed in MDS samples with low proliferation ability. Further experiments proved that phosphorylation of STAT3 affected CSN5 function and mediated the ubiquitination of HSF, the upstream regulator of SPI1/PU.1 transcription, which led to the inhibition of SPI1/PU.1 expression. Restoration of CSN5 rescued the inhibition of HSF1 ubiquitination, causing SPI1/PU.1 transcription to resume and increasing SPI1/PU.1 expression, promoting the recovery of cell proliferation in hypocellular MDS. Our research revealed the regulatory role of the CSN5/HSF/SPI1/PU.1 axis in hypocellular MDS, providing a probable target for clinical intervention.

Killing two birds with one stone: CRISPR/Cas9 CCR5 knockout hematopoietic stem cells transplantation to treat patients with HIV infection and hematological malignancies concurrently.

Human immunodeficiency virus (HIV) is known to cause hematological malignancy. Hematopoietic stem cell transplantation (HPSCT) is an advanced treatment for that. Currently, there are three successful HIV-eliminated cases, and two received HPSCT from CCR5-absent donors. It is well established that the CCR5 protein on the cell surface assists human immunodeficiency virus entry. Preliminary studies have revealed that knocking out CCR5 and/or CXCR4 may inhibit the viral entry of HIV, which may prove promising in the further development of HIV treatment options. Herein, we suggest performing autologous or allogeneic HSCT with CCR5 KO hematopoietic stem cells in patients who suffer from complicated HIV conditions, particularly drug-resistant HIV or a concurrent diagnosis of HIV with lymphoma/leukemia, to achieve complete HIV remission. Nevertheless, at the clinical forefront of CRISPR-HIV technology, more efforts should be directed to advance nonhuman primate (NHP) models for studies of HIV pathogenesis and off-target assessments within this system. CRISPR-Cas9 knock out ofhost HSCT-expressing CCR5 or CXCR4 may confer HIV-resistance, which when applied to bedside therapeutics in an allogeneic or autologous manner can warrant a permanent and effective treatment outcome.

Study of Hematopoietic Stem Cells in Patients with Extensive Burns

Introduction. Assessment of tissue regeneration, including that at the cellular and subcellular levels, appears to be one of the important trends in the complex treatment of patients with extensive burns. It is known that hematopoietic stem cells (HSCs) contribute to tissue restoration and regeneration through paracrine effects or direct cell differentiation, being a central component of post-burn anemia recovery and capable of forming not only blood cells, but also other types of cells. Notably, the role of these cells in burn injury has not been studied yet.The aim of the study was to investigate in dynamics the content of hematopoietic stem cells of different phenotypes in patients with extensive burns in the process of complex treatment. Methods. Hematopoietic stem cells and their subpopulations in peripheral blood samples were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, USA) using the CellQuest program and CD45/CD34/CD38 and CD45/CD34/CD133 monoclonal antibody panels (BD, USA). The results obtained were statistically processed using the GraphPad Prism 7.0 program (USA), results were considered statistically significant at p0.05.Results. Hematopoietic stem cells and their subpopulations were studied at different stages of the complex treatment in 25 patients with a large burn area, ​​more than 30% of the body surface. The comparison group consisted of 15 healthy volunteers. Upon admission to the Burn Center, a group of severely burned patients revealed a significant deep deficiency of both total HSCs CD45+34+ (p=0.0002) and their subpopulations CD45dim34+38+ (p=0.019), with predominantly early precursors of hematopoiesis CD45dim34+38- (p=0.0001) and CD45dim34+133+ (p=0.0002). In the course of the complex treatment, including surgical necrectomy and autodermoplasty of burn wounds, there was observed normalization of total HSCs CD45+34+ (0.050.012%, p=0.031) and a subpopulation of early HSCs CD45dim34+38- (0.0390.009%, p=0.016) in 20 days of treatment in the group of burn patients. There was a significant increase of mature CD45dim34+133- HSCs (p=0.0380) as a result of treatment, while the deficit of the more differentiated population of CD45dim34+38+ HSCs did not fully recover (p=0.272).Conclusion. The firstly detected modulations in the content of hematopoietic stem cells of different phenotypes in patients with extensive burns may reflect the state of compensatory-adaptive reactions of hematopoiesis in the course of the complex treatment. The data obtained may support the predictive use of HSC subcellular markers to assess the regenerative potential in burn wounds healing, including that during staged surgical treatment and autodermoplasty, and to predict the development of local and general complications of burn disease.

A Phase 2, Open-Label Study to Evaluate the Efficacy and Safety of Mgta-145 in Combination with Plerixafor for the Mobilization of Hematopoietic Stem Cells in Patients with Sickle Cell Anemia

Background: In sickle cell anemia (SCA), a point mutation in the β-globin gene results in abnormal hemoglobin prone to polymerization under hypoxic conditions. Erythrocytes become sickle-shaped and rigid, leading to painful vaso-occlusion, ischemic tissue damage, and endothelial dysfunction. Despite significant, life-threatening risks, allogeneic hematopoietic cell transplant (allo-HCT) is the only cure for SCA. Results of autologous HCT, using the addition of a non-sickling, post-transcriptional gene silencing or gene editing to increase fetal hemoglobin, are promising and suggest an alternative option for those who lack a suitable donor. Procuring hematopoietic stem and progenitor cells (HSPCs) is challenging in patients with SCA, as granulocyte colony-stimulating factor is contraindicated due to severe adverse effects. Peripheral blood mobilization with Plerixafor (Mozobil®, Genzyme) has recently been shown to be safe and effective in SCA. While mobilization with plerixafor allows collection of an HSPC product, many SCA patients require ≥2 cycles of multiday plerixafor doses and apheresis to collect sufficient quantities of HSCs for genetic manipulation. Reports suggest 30% of patients experience vaso-occlusive events (VOEs) of grade ≥3 during and after apheresis; therefore, a considerable unmet need exists for agents that augment stem cell (SC) yields and reduce apheresis time. MGTA-145 is an agonist of cell-surface chemokine receptor 2 (CXCR2) under development for the mobilization of CD34+ cells in combination with plerixafor. MGTA-145 binds to CXCR2, resulting in HSC release, which when given with plerixafor, increases mobilization of HSCs. Preclinical studies in animal models and clinical trials in healthy subjects and patients with multiple myeloma demonstrated that MGTA-145 with plerixafor results in more rapid and robust mobilization of CD34+ cells, including primitive HSC populations, compared to plerixafor alone. Objective: To evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of 1 or 2 doses of MGTA-145 combined with plerixafor for the mobilization of HSCs in individuals with SCA. Study Design and Methods: This phase 2, open-label, 2-part study (NCT05445128) will enroll approximately 10-14 adults ages 18-35 years with SCA, at 3 sites in the US. Participants must have a documented diagnosis of SCA, have a hydroxyurea washout period, and have adequate white blood cell counts and cardiopulmonary, renal, and liver function. Key exclusion criteria include history of VOE requiring a visit to a healthcare facility within 30 days of screening, poorly controlled healthcare conditions, including but not limited to cardiopulmonary or hepatorenal events ≤6 months prior to screening, and cerebrovascular accident or retinal infarct ≤2 years prior to screening. In the 1st cohort of Part A, subjects will receive a single 0.24 mg/kg subcutaneous dose of plerixafor followed by a single 0.03 mg/kg IV dose of MGTA-145, with apheresis occurring within approximately 30-45 minutes of MGTA-145 dosing. After a 1-week follow-up visit and an Independent Data Monitoring Committee (DMC) review, the 2nd cohort with additional subjects may be enrolled and given plerixafor and a 0.03 or 0.015 mg/kg or lower dose of MGTA-145 (FIGURE). Following a 1-week follow-up visit and a 2nd DMC review, patients will be enrolled in Part B and will receive, on 2 consecutive days, plerixafor and the same dose of MGTA-145 as the 2nd Part A cohort, followed by apheresis on each dosing day. The primary endpoint is total yield of CD34+ cells (CD34+ cells/kg). Safety outcomes include incidence of treatment-emergent adverse events (TEAEs), drug-related TEAEs, Grade ≥3 TEAEs, treatment-emergent serious adverse events, and TEAEs leading to study drug discontinuation. Core exploratory endpoints include characterization of the phenotype and function of cells collected by apheresis and assessment of the gene-modifying potential of mobilized CD34+ cells. The results of this trial will establish if MGTA-145 with plerixafor will rapidly and safely mobilize robust numbers of high-quality SCs for HSCT in SCA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparison of Post Transplant Outcomes between Refrigerated and Cryopreserved Autologous Hematopoietic Stem Cells in Patients with Multiple Myeloma

Introduction: Approximately 10% of individuals with hematological malignancies are diagnosed with Multiple Myeloma (MM); in addition, the incidence and prevalence of the disease have progressively increased in recent decades. Despite the recent remarkable advances in treatment, autologous hematopoietic stem cell transplantation (ASCT) is still a fundamental part of the therapeutic management of this population. Particularly in developing countries, ASCT is an underused tool. Social and economic issues play an essential role in accessing the procedure. Refrigerated hematopoietic stem cells (R-HSC) can be an attractive alternative to the traditional use of cryopreserved hematopoietic stem cells (C-HSC) since cryopreservation demands highly trained human resources, high-cost supplies, and sophisticated equipment. Aim: In order to compare the applicability, safety, and possible clinical and economic benefits of ASCT with different types of cell storage before infusion, we analyzed the medical records of patients diagnosed with MM who were transplanted using R-HSC or C-HSC. Methods: The study was approved by the local Research Ethics Committee. We analyzed charts of patients diagnosed with MM and submitted to ASCT at Erasto Gaertner Hospital between January 2017 and July 2021. R-HSC: Cells were stored immediately after leukapheresis, without any handling procedure, kept in a standard refrigerator at a temperature between 2-6oC, and infused within 48 hours after collection. C-HSC: Cells were collected by leukapheresis and submitted to cryopreservation within 12 hours after the procedure. As a cryoprotectant, a solution with dimethyl sulfoxide (DMSO) at a final concentration of 5.5% was used. The product's temperature was reduced under a controlled cooling rate and stored in a freezer at -80oC. All patients received a single intravenous dose of melphalan (200-100mg/m2) 24 hours before receiving the cells as conditioning regimen. In the group of patients who received R-HSC, the cells were removed from the refrigerator and immediately infused at a rate of 10 ml/minute. However, in patients who received C-HSC, the cells were removed from the freezer, thawed in a water bath at 37oC, and infused intravenously at a rate of 10 ml/minute. All patients received G-CSF from D+5 until neutrophilic engraftment. Results: We reviewed records of 170 patients. One hundred eight subjects received R-HSC and 62 C-HSC. Demographic characteristics, CD34+ cell dose received, melphalan dose, febrile neutropenia, and early mortality related to the procedure were similar between the two groups. There was a significant statistical difference in favor of the group utilizing R-HSC related to days until neutrophil engraftment (10 vs. 14 days p<0.0001), days until platelet engraftment (12 vs. 15 days p<0.0001), days of hospitalization (13 vs. 16 days p<0.0001) and infusion-related adverse reactions (10.1% vs. 33.8% p=0.003). There were no cases of graft failure in both groups. A reduction of 32.4% in the total cost per transplant was noticed in the R-HSC group. Conclusions: We demonstrated in this analysis that the use of stem cells stored in a standard refrigerator, with a temperature between 2-6oC for up to 48 hours after the collection is a safe and efficient approach. It was capable of simplifying the ASCT, preventing infusion-related adverse reactions, assuring an early neutrophil and platelet engraftment, and reducing hospitalization days. This strategy should be considered especially in resource constrained settings to avoid unnecessary expenses, increase the number of transplant centers, and expand access to the procedure.

Comparison of etoposide combined with G-CSF and cyclophosphamide combined with G-CSF in mobilization of autologous peripheral hematopoietic stem cells in patients with newly diagnosed multiple myeloma

Comparison of etoposide combined with G-CSF and cyclophosphamide combined with G-CSF in mobilization of autologous peripheral hematopoietic stem cells in patients with newly diagnosed multiple myeloma

МЕДИКО-ГЕНЕТИЧЕСКИЙ КОНТРОЛЬ ВРОЖДЕННЫХ ДЕФЕКТОВ ИММУНИТЕТА – БУДУЩЕЕ НАПРАВЛЕНИЕ НАУЧНОЙ И КЛИНИЧЕСКОЙ ПЕДИАТРИИ

The new International Classification of Diseases, 11th Revision (ICD-11) has started in Russia and worldwide in January, 2022. The section Diseases of the blood and blood-forming organs and certain disorders involving the immune mechanism (D50-D89) now includes more than 400 genetic disorders leading to disruption of cell cycle regulation, systemic inflammation and primary immunodeficiency (PID) with clinical manifestations as severe infections, autoinflammatory, autoimmune, allergic and oncological diseases. The PID verification tools include records on the epidemiology of inborn defects of immunity based on the national PID register, on the development and practical implementation of original domestic tests on T and B lymphocyte abnormalities and the diagnostic genetic panel for differential diagnostics of PID, as well as on conducting multicenter studies on the effectiveness of immunoglobulin drugs and transplantation of hematopoietic stem cells in patients with PID and the results of pilot studies of neonatal screening of PID in Russia. The article presents the computational indicators of children at risk and working algorithms for differential clinical and genetic diagnostics of PID and clinical decision-making designed to provide dynamic follow-up and treatment of this group of patients and to reduce infant and child mortality.

Clinical features of methods for collecting hematopoietic stem cells in patients with malignant neoplasms weighing up to 15 kg and early age

Relevance. Autologous hematopoietic stem cell transplantation (auto-HSCT) is an integral part of the treatment of patients with high-risk malignancies. However, carrying out the mobilization and collection of hematopoietic stem cells (HSC) for patients weighing up to 15 kg is a complex task that requires a special approach and multidisciplinary interaction.The purpose of the study is to present the experience of collecting HSC in young children weighing up to 15 kg at the Research Institute of Pediatric Oncology and Hematology of the Federal State Budgetary Institution N.N. Blokhin National Medical Research Centre of Oncology, Ministry of Health of Russia.Materials and methods. The study included 30 patients weighing up to 15 kg who received treatment from January 2020 to May 2021 at the Research Institute of Pediatric Oncology and Hematology of the Federal State Budgetary Institution N.N. Blokhin National Medical Research Centre of Oncology, Ministry of Health of Russia. Median age was 30.6 (12–48) months, median body weight was 12.2 (7.8–15) kg. Apheresis in children weighing up to 15 kg was performed in the resuscitation and intensive care unit with the condition of pre-filling the cell separator circuit with a donor irradiated erythrocyte suspension to prevent hypovolemic complications. Thirty two apheresis was performed on a cell separator of the “Spectra Optia” type. To mobilize HSC, preparations of granulocyte colony-stimulating factor with the main active ingredient filgrastim were used.Results. Twenty eight HSC apheresis procedures were successful on the first attempt, 2 patients underwent repeated apheresis (a total of 32 procedures were performed). The median number of CD34+ cells obtained was 13.9 × 106/kg (0.04–92.0 × 106/kg), and the median apheresis duration was 251 (160–415) min. In 2 children during the procedure, an immediate organization of repeated venous access was required.Conclusions. Performing HSC apheresis in children weighing up to 15 kg and young children is a safe and effective technique with the participation of a multidisciplinary team. The apheresis algorithm proposed by us makes it possible to carry out a high-quality collection of CD34+ cells, sufficient for auto-HSCT.

Lentiviral vector containing beta-globin gene for beta thalassemia gene therapy

Background and aimBeta thalassemia is a common monogenic disorder caused by partial or complete reduction of beta globin chains synthesis. In recent years allogeneic bone marrow transplantation (BMT) has been considered to be the successful cure for patients with thalassemia major, however this is restricted due to limited number of HLA-matched donors. Therefore, molecular approaches including gene therapy for direct normal beta globin gene transmission seem quite promising to cure thalassemia. The goal of this study was to evaluate the beta globin gene expression in K562 cell line, as a model of hematopoietic cell, transduced with lentiviral vector carrying normal beta globin gene. MethodsFor our purpose, we designed the DEST Lentiviral vector (3rd generation) carrying normal beta globin gene and its promoter and packaged in LentiX-293T cell line. The K562 cell line was transduced by packaged lentivirus containing β-globin cassette (pLenti-HBB). Bulk cells suspension form K562 cells were diluted and seeded into 96-well micro plate by manual pipetting. After single cell expansion, β-globin protein expression level in single cell clones was determined by flowcytometry and Western blotting. ResultsOur results showed that we have successfully packaged and generated lentivirus in LentiX-293T cell line. Flowcytometry analysis showed that beta globin protein expression achieved 20.3% in bulk population of K562 cells in MOI = 20 and 74.4% in F2 clone. ConclusionThese data indicated that vector used in this study is efficient in vitro and could be applied in gene therapy in patient's hematopoietic stem cell. The final goal of this study is to examine designed vector in hematopoietic stem cells promising therapeutic strategy for beta thalassemia and sickle cell disease.

Severe aplastic anemia after COVID-19 mRNA vaccination: Causality or coincidence?

The development of various autoimmune diseases has been reported after COVID-19 infections or vaccinations. However, no method for assessing the relationships between vaccines and the development of autoimmune diseases has been established. Aplastic anemia (AA) is an immune-mediated bone marrow failure syndrome. We report a case of severe AA that arose after the administration of a COVID-19 vaccine (the Pfizer-BioNTech mRNA vaccine), which was treated with allogeneic hematopoietic stem cell transplantation (HSCT). In this patient, antibodies against the SARS-CoV-2 spike protein were detected both before and after the HSCT. After the patient's hematopoietic stem cells were replaced through HSCT, his AA improved despite the presence of anti-SARS-CoV-2 antibodies. In this case, antibodies derived from the COVID-19 vaccine may not have been directly involved in the development of AA. This case suggests that the measurement of vaccine antibody titers before and after allogeneic HSCT may provide clues to the pathogenesis of vaccine-related autoimmune diseases. Although causality was not proven in this case, further evaluations are warranted to assess the associations between vaccines and AA.

Efficacy and safety of hematopoietic stem cell transplantation for hematologic malignancies

Abstract Introduction: Hematopoietic stem cell transplantation is an essential and often the sole treatment strategy for relapsed and refractory hematologic malignancies (HMs). More and more systematic reviews and meta-analysis of clinical trials have investigated the effects of hematopoietic stem cell transplantation in patients with HMs. In order to systematically appraise and synthesize these results, we will conduct an overview of systematic review and meta-analysis. Methods: This is a protocol for an overview of systematic reviews and meta-analysis. We will search eight databases: PubMed, Embase, Cochrane Library, Web of Science Core Collection, China Biology Medicine disc, Chinese National Knowledge Infrastructure, Chinese Scientific Journals Database and Wan Fang Data. The time is limited from the construction of the library to May 2021.Systematic reviews and meta-analysis of clinical trials evaluating the efficacy and safety of hematopoietic stem cells in patients with HMs will be included. Hematopoietic stem cells included bone marrow, peripheral blood stem cells and cord blood stem cells. Patients with HMs including leukemia, malignant lymphoma, myelodysplastic syndrome, etc. Two independent authors will screen titles and abstracts retrieved in the literature search and select studies meeting the eligibility criteria for full text review. The methodological quality of the included reviews will be assessed using A Measurement Tool to Assess Systematic Reviews-2. The efficacy and safety of different stem cell types in the treatment of the same hematological disease and the same stem cell type in the treatment of different HMs will be analyzed. Additionally, the quality of evidence will use the Grading of Recommendations Assessment, Development and Evaluation system. Our reviewers will conduct systematic reviews, qualification evaluation, data extraction, methodological quality and evidence quality screening in pairs. Results: The results will be published in a peer-reviewed journal. Conclusion: This overview will provide comprehensive evidence for hematopoietic stem cell transplantation in patients with HMs. Protocol Registration: INPLASY202150064.

The Donor – Recipient Weight Ratio is a Reliable Marker for Cell Yield in Hematopoietic Stem Cell Donations

Bone marrow transplants remain an import source of hematopoietic stem cells for patients suffering from specific diseases like aplastic anemia, for pediatric patients with malignant and non-malignant blood cell disorders, and for situations in which graft-versus-host disease (GvHD) is a concern. Identifying the optimal donor to achieve a 3-5 x 108/kg of recipient weight TNC yield may be challenging. In an analysis of 687 consecutive donors, donor and procedure characteristics were related to TNC/kg of recipient weight using Spearman correlation coefficients as well as linear and multiple regression analysis. We found correlations between donor WBC (r = 0.17), donor platelet counts (r = 0.15), donor BMI (r = 0.10), and the percentage of donor estimated blood volume accessed for harvesting (r = -0.57) with TNC/kg of recipient weight. The strongest correlation existed between the donor-recipient weight ratio and the TNC/kg (r = 84). In a multivariate regression analysis, the donor-recipient weight ratio influenced the TNC/kg of recipient weight more significantly (adjusted R2 = 0.84) than all other related variables put together. The minimal donor-recipient weight ratio associated with a TNC/kg of at least 3x108/kg of recipient weight was 0.8 (mean 3.425; 95% CI 2.01, 5.8). Using this donor-recipient ratio provides national bone marrow donor registries with a practical and simple measure to assure optimal cell yield outcomes in hematopoietic stem cell donations.

Hospitalization and Healthcare Resource Use of Omidubicel Vs Cord Blood Transplantation for Hematological Malignancies in a Global Randomized Phase III Clinical Trial

IntroductionUmbilical cord blood (UCB) remains an important source of hematopoietic stem cells for patients, where no matched donor is available and for racial minorities. However, cord blood transplants have been associated with delayed hematopoietic recovery, prolonged hospitalization, and higher costs of transplant compared with other donor sources. Omidubicel is an advanced cell therapy that preserves stem cell function to optimize cell homing, engraftment, differentiation, and self-renewal and is manufactured from an appropriately HLA-matched single UCB unit for each patient. A recent phase III clinical trial (NCT02730299) reported that patients who received omidubicel experienced faster time to neutrophil engraftment, faster platelet recovery, reduced rates of infections and shorter hospitalization time than patients who received standard single (33%) or double (67%) UCB transplantation [Horwitz et al, Blood, 2021]. We report on an analysis of resource utilization including hospital length of stay, hospital care setting, visits by provider type, rates of transfusions and readmissions from the Phase III trial.MethodsThe Phase III clinical trial included patients aged 12-65 with hematological malignancies and eligible for an allogeneic transplant. The primary endpoint was time to neutrophil engraftment, with secondary endpoints of time to platelet engraftment, first grade 2/3 bacterial or fungal infections and days alive and out of hospital in the first 100 days. 125 patients were randomized in the study. We analyzed resource utilization data for patients treated with omidubicel (n=52) or UCB (n=56) (“as-treated population”) focusing on resource utilization within the first 100 days of transplantation. Summary statistics were compared between treatment arms, means and medians used to draw comparison and significance testing was performed.ResultsIn the Phase III clinical trial, omidubicel met its primary endpoint and secondary endpoints with statistical significance. The demographics and patient characteristics were overall well-balanced with slightly more males (52% vs 63%) and a lower median age (40 vs 36) in the UCB arm. The study population was diverse with 17.6% Black, 14.8% Asian, 14% Hispanic or Latino and 12% other/unknown. Most patients had AML (45%) or ALL (34.3%), with MDS, CML and lymphomas making up the rest of the study population. The disease risk index was high/very high in 35% of the patients and 24% of the patients had a Karnofsky/Lansky performance score of <90. The rapid hematopoietic recovery was supported by a reduced rate of infections. The rates of acute and chronic GVHD in the two arms were comparable.Within the first 100 days after transplant, omidubicel patients experienced shorter average total length of hospital stay than UCB recipients (mean 41.2 vs 50.8 days; p = 0.027) and more days alive and out of the hospital (mean 55.8 vs 43.7 days; p=0.023). 12% of patients on omidubicel arm died vs 16% on UCB arm before day 100. During the primary hospitalization (transplant to discharge), fewer omidubicel patients required intensive care unit (ICU) stay (9.6% vs 23.2%) and spent fewer days in the ICU (mean 0.4 vs 4.7 days; p =0.028) and the transplant unit (mean 25.3 vs 32.9 days; p =0.022) compared to UCB recipients. Omidubicel patients required fewer outpatient consultant visits and fewer outpatient non-consultant visits (X-rays, scans, biopsies etc.) and required fewer platelet or other transfusions (RBC, albumin, plasma, and factor product) within 100 days from transplant (Table 1).ConclusionsThis analysis shows that more rapid hematopoietic recovery in patients transplanted with omidubicel was associated with significantly shorter hospital length of stay and reduced healthcare resource use compared to UCB in the clinical trial. Although economic data were not collected as part of the clinical trial, the costs of providing transplantation care during the first 100 days are likely lower with omidubicel compared to UCB in the real-world setting, as hospital stay, outpatient visits, and blood product transfusions are among the major drivers of costs during this time period. [Display omitted] DisclosuresMajhail: Incyte Corporation: Consultancy; Anthem, Inc: Consultancy. Manghani: Gamida Cell, Inc.: Current Employment; Tricida, Inc.: Ended employment in the past 24 months. Sivaraman: Incyte Corporation: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Gamida Cell, Inc.: Current Employment. Galamidi-Cohen: Gamida Cell, Ltd: Current Employment. Maziarz: Bristol-Myers, Squibb/Celgene, Intellia, Kite: Honoraria; CRISPR Therapeutics: Consultancy; Artiva Therapeutics: Consultancy; Intellia: Honoraria; Omeros: Research Funding; Athersys: Other: Data and Safety Monitoring Board, Patents & Royalties; Incyte Corporation: Consultancy, Honoraria; Novartis: Consultancy, Other: Data and Safety Monitoring board, Research Funding; Allovir: Consultancy, Research Funding; Vor Pharma: Other: Data and Safety Monitoring Board.