Infusion Of Hematopoietic Stem Cells Research Articles

Restoring T and B cell generation in X-linked severe combined immunodeficiency mice through hematopoietic stem cells adenine base editing

Base editing of hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematologic diseases. However, the feasibility of using adenine base-edited HSPCs for treating X-linked severe combined immunodeficiency (SCID-X1), the influence of dose–response relationships on immune cell generation, and the potential risks have not been demonstrated in vivo. Here, a humanized SCID-X1 mouse model was established and 86.67% ± 2.52% (n = 3) of mouse hematopoietic stem cell (HSC) pathogenic mutations were corrected, with no sgRNA-dependent off-target effects detected. Analysis of peripheral blood over 16 weeks post-transplantation in mice with different immunodeficiency backgrounds revealed efficient immune cell generation following transplantation of different amounts of modified HSCs. Therefore, a large-scale infusion of gene-corrected HSCs within a safe range can achieve rapid, stable, and durable immune cells regeneration. Tissue section staining further demonstrated the restoration of immune organ tissue structures, with no tumor formation in multiple organs. Collectively, these data suggest that base-edited HSCs are a potential therapeutic approach for SCID-X1 and that a threshold infusion dose of gene-corrected cells is required for immune cell regeneration. This study lays a theoretical foundation for the clinical application of base-edited HSCs in treating SCID-X1.

Gene editing improves endoplasmic reticulum-mitochondrial contacts and unfolded protein response in Friedreich's ataxia iPSC-derived neurons.

Friedreich ataxia (FRDA) is a multisystemic, autosomal recessive disorder caused by homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently, there is no effective treatment for FRDA. We have previously demonstrated that single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in prevention of neurologic and cardiac complications of FRDA in YG8R mice, and rescue was mediated by FXN transfer from tissue engrafted, HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation, we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients' CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. We generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. iPSC derived FRDA neurons displayed characteristic apoptotic and mitochondrial phenotype of the disease, such as non-homogenous microtubule staining in neurites, increased caspase-3 expression, mitochondrial superoxide levels, mitochondrial fragmentation, and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. Gene edited neurons demonstrated improved ER-calcium release, normalization of ER stress response gene, XBP-1, and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles, as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe ER structural damage in FRDA neurons, that was undetected in gene edited neurons. Taken together, these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage in FRDA, validate the efficacy profile of our FXN gene editing approach in a disease relevant model, and support our approach as an effective strategy for therapeutic intervention for Friedreich's ataxia.

Chapter 7 - The role of chemotherapy in hematopoietic stem cell transplantation for autoimmune disorders: From lymphoablative to myeloablative conditioning protocols

Hematopoietic stem cell transplantation (HSCT) is a medical procedure used mainly for the treatment of onco-hematologic disorders. Over the last two decades, autologous HSCT has been explored for the treatment of neurologic autoimmune diseases (ADs), being multiple sclerosis (MS) the most frequent indication in this setting. HSCT is characterized by the sequential administration of a conditioning regimen (CR) and the infusion of hematopoietic stem cells (HSCs), previously collected either by the individual himself in the autologous transplant (AHSCT), or by a healthy donor in allogeneic HSCT. CR consists of the administration of high-dose chemotherapy and/or total body irradiation (TBI), that in ADs is usually associated with an immunodepleting serotherapy, either by an animal-derived polyclonal serum or a monoclonal antibody (MoAb), to induce intense immunosuppression. CRs are classified according to the European Society for Blood and Marrow Transplantation (EBMT) guidelines for HSCT in ADs in three grades of intensity according to the degrees of depletion of the hemato-lymphopoietic system induced. In the present chapter, after a brief overview of mobilization and CR adopted in the neurologic autoimmune setting, the role of chemotherapy in HSCT will be discussed, providing a historical perspective on the use of different regimens and summarizing the available evidence on potential associations between CR and outcomes.

Chapter 9 - The HSCT procedure (II): Conditioning, hematopoietic stem cell infusion, supportive care, and monitoring

Autologous hematopoietic stem cell transplantation (aHSCT) is a complex process, designed to replace the blood and lymphoid systems of a patient with hematopoietic stem cells (HSCs) that have been previously collected and cryopreserved, derived from the same patient. The rationale of aHSCT in neurologic diseases is to eliminate self-reacting cell clones and induce self-tolerance through a profound renewal of the immune system. The steps analyzed in this chapter are conditioning, HSCs infusion, supportive care, and monitoring. Before transplantation, ablation of the hemato-lymphopoietic system is achieved with chemotherapy; this stage is known as the conditioning regimen. The EBMT guidelines support the use of "intermediate intensity" regimens, either cyclophosphamide 200mg/kg or BEAM (bis-chloroethyl-nitrosourea, etoposide, cytarabine, and melphalan), in combination with serotherapy that consists of rabbit antithymocyte globulin (ATG) in most protocols. The infusion of HSC is performed through a central intravenous line, after being thawed at 37°C using either a water bath or a heat bath; in this phase, the prevention and management of infusion-related adverse events are crucial. The supportive care consists mainly of infection prophylaxis and treatment, administration of blood product transfusions, and nutritional and electrolyte support. The monitoring phase is focused on hematologic recovery and monitoring for early and late complications of aHSCT.

CD117 Antibody-Drug Conjugate Conditioning Allows Efficient Engraftment of Gene-Modified CD34+ Cells with Fertility Preservation in a Rhesus Gene Therapy Model

Hematopoietic stem cell (HSC) gene therapy is curative for multiple genetic diseases; however, it is limited due to toxicities from chemotherapy-based conditioning. To target HSCs directly, we developed an anti-human CD117 ( c-KIT)-targeted antibody-drug conjugate (CD117-ADC) conjugated to a DNA-binding payload that cross-reacted with non-human primates. To investigate the activity of CD117-ADC in non-human primates, we administered a single injection of ADC without HSC infusion, resulting in a >99% depletion of CD34+CD90+ bone marrow cells in cynomolgus macaques (n=2 of 3 in 0.1 mg/kg and n=3 in 0.3 mg/kg); similar to the ablation observed with 4 doses of myeloablative busulfan (Bu) (n=3). With escalating ADC doses (0.2-0.6 mg/kg), the optimal timing of HSC infusion (6 days in 0.2 or 0.3 mg/kg and 10 days in 0.4mg/kg) was determined in rhesus macaques (n=1 each). The animal dosed 0.6 mg/kg required euthanasia on day 14 due to irreversible hematopoietic suppression and severe liver toxicity. In our preliminary transplantation study, dosing 0.2 mg/kg ADC to rhesus macaques (n=2) resulted in bone marrow depletion and modest-level engraftment of gene-modified cells (vector copy number per cell (VCN) 0.05±0.01) (ASH 2019). Therefore, we evaluated higher single doses of CD117-ADC in rhesus macaques (0.3 mg/kg for ZL13 and ZJ62, and 0.4 mg/kg for H635 and H96G, n=4) and directly compared to Bu conditioning (5.5 mg/kg x 4 days, n=2). Mobilized rhesus CD34+ cells (ADC 6.4±2.9e6/kg vs. Bu 4.1±0.1e6/kg, n.s.) were transduced with a lentiviral vector for stable fetal hemoglobin (HbF) induction (Sci Transl Med. 2021). These cells ( in vitro VCN 10.1±3.8 vs. 10.2±7.3, n.s.) were transplanted into autologous animals after ADC or Bu conditioning. The time course of blood cell count suppression and recovery was similar between ADC and Bu animals, except for a strong reduction of platelet counts observed only with ADC (ASH 2021). Two months post-transplant, substantial levels of vector-containing granulocytes were observed long-term in 1 of 2 animals receiving 0.3 mg/kg ADC (ZJ62, VCN 0.16) and in both animals receiving 0.4 mg/kg (H635, VCN 0.46 and H96G, VCN 0.21) at a similar range to the two Bu animals (0.44±0.17, n.s.). Robust HbF induction was detected in three ADC animals with substantial VCN as measured by both HbF-positive cells (F-cells 8.5±1.8% vs. 13.7±5.8%, n.s.) and HPLC measurement of total HbF (8.0±2.9% vs. 11.1±5.2%, n.s.). In ZL13 (0.3 mg/kg ADC), lower gene marking (VCN 0.02) was observed, along with lower HbF induction (F-cells 1.0% and HbF amounts 0.9%). Importantly, ADC resulted in minimal off-target toxicity, unlike those associated with Bu (such as infertility, veno-occlusive disease, pulmonary dysfunction, mucositis, or seizures). High diversity of gene-modified cells (Simpson's diversity index >0.99) without any large clone (maximal integration site 0.3-3.7%) was observed by integration site analysis 1-year post-transplant with both ADC (0.3-0.4 mg/kg) and Bu. Infertility is a major complication of conventional myeloablative conditioning that limits the uptake of curative therapies; therefore, we investigated fertility in CD117-ADC animals. When monitored daily for menstrual bleeding, the menstrual cycles of all 4 female macaques (ZL13, ZJ62, H635, and H96G) were maintained post-ADC conditioning (0.92±0.11 per month) in contrast to our previously transplanted animals following myeloablative Bu (0.23±0.11, n=4, p<0.01) and total body irradiation (0.33±0.3, n=6, p<0.05). To confirm fertility, all transplanted animals post-ADC including females (n=4) and males (n=2) were housed for mating at a rhesus breeding colony. Two ADC females (ZL13 and H635) gave birth, and an ADC male (12U032) was paired with a pregnant female. Importantly, an ADC female (H635) was paired with an ADC male (13U047). In summary, a single-injection CD117-ADC allows for efficient engraftment of gene-modified CD34+ HSCs in a rhesus gene therapy model, achieving similar levels as the current clinical standard of care, myeloablative Bu conditioning. Robust HbF induction was also confirmed at the protein level in our rhesus gene therapy model with ADC conditioning. The transplanted animals with ADC maintained their fertility. Overall, these data indicate that an ADC-based targeted approach offers safer conditioning and could improve the risk-benefit profile in HSC gene therapy.

ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION IN ELDERLY PATIENTS IN BRAZIL

The allogeneic hematopoietic stem cell transplant (allo-HCT) represents an important therapeutic strategy for acute leukemias, lymphomas and solid neoplasms, also used in benign diseases, such as aplastic anemia and inborn errors of immunity. This treatment requires myeloablative chemotherapy (conditioning regimen) followed by the infusion of donor-derived hematopoietic stem cells. However, this procedure carries some risks, such as infections, graft versus host disease (GVHD) and conditioning toxicity, which may result in transplant-related mortality. Over the decades, due to the increasing life expectancy and new advances in medicine, the cases of patients > 50 years with hematologic diseases that need allogeneic transplant have grown, requiring a comprehensive geriatric assessment as a mechanism for the best treatment option choice.
 Objective: To apply a clinical frailty score and Karnofsky score in allogeneic hematopoietic stem cell older than 50 years old for three years in Walter Cantídio University Hospital (Fortaleza/Ceará) and in Amaral Carvalho Hospital (Jaú/São Paulo), expecting to recognize the profile of this patients and to demonstrate the relation between the clinical frailty score and overall survived, besides to estimate the contribution of GVHD prophylaxis and relapse in overall survival. Methods: Multicentric, retrospective, descriptive, analytical and quantitative study, acquiring dates by means of exams and medical record from Walter Cantídio University Hospital in Fortaleza/Ceará and Amaral Carvalho Hospital in Jaú/São Paulo. Results: The study selected 252 patients, 147 males and 105 females, sort in gender, disease, HCTCI score, CFS and KPS. In three years, the overall survival in FIT score is 2,46 years, while in FRAILTY score is 1,82 years. About the prophylaxis, the combination of cyclosporine, mycophenolate mofetil, cyclophosphamide had worse results than others prophylaxis. As expected, in case of relapse, there is shorter survival. Conclusion: The elderly population require a geriatric score in order to evaluate the profile of this patients once the allogeneic transplant must happen, then FIT patients has longer survival than FRAILTY patients.

Pediatric Autologous Hematopoietic Stem Cell Transplantation: Safety, Efficacy, and Patient Outcomes. Literature Review.

Autologous stem cell transplantation (auto-HSCT) is a part of the therapeutic strategy for various oncohematological diseases. The auto-HSCT procedure enables hematological recovery after high-dose chemotherapy, otherwise not tolerable, by the infusion of autologous hematopoietic stem cells. Unlike allogeneic transplant (allo-HSCT), auto-HSCT has the advantage of lacking acute-graft-versus-host disease (GVHD) and prolonged immunosuppression, however, these advantages are counterbalanced by the absence of graft-versus-leukemia. Moreover, in hematological malignancies, the autologous hematopoietic stem cell source may be contaminated by neoplastic cells, leading to disease reappearance. In recent years, allogeneic transplant-related mortality (TRM) has progressively decreased, almost approaching auto-TRM, and many alternative donor sources are available for the majority of patients eligible for transplant procedures. In adults, the role of auto-HSCT compared to conventional chemotherapy (CT) in hematological malignancies has been well defined in many extended randomized trials; however, such trials are lacking in pediatric cohorts. Therefore, the role of auto-HSCT in pediatric oncohematology is limited, in both first- and second-line therapies and still remains to be defined. Nowadays, the accurate stratification in risk groups, according to the biological characteristics of the tumors and therapy response, and the introduction of new biological therapies, have to be taken into account in order to assign auto-HSCT a precise role in the therapeutic strategies, also considering that in the developmental age, auto-HSCT has a clear advantage over allo-HSCT, in terms of late sequelae, such as organ damage and second neoplasms. The purpose of this review is to report the results obtained with auto-HSCT in the different pediatric oncohematological diseases, focusing on the most significant literature data in the context of the various diseases and discussing this data in the light of the current therapeutic landscape.

Impact of oral microbiota on pathophysiology of GVHD.

Allogeneic transplantation of hematopoietic cells is the only curative therapy for several hematopoietic disease in which patients receive cytotoxic conditioning regimens followed by infusion of hematopoietic stem cells. Although the outcomes have improved over the past decades, graft-versus-host-disease (GVHD), the most common life-threatening complication, remains a major cause of non-relapse morbidity and mortality. Pathophysiology of acute GVHD characterized by host antigen-presenting cells after tissue damage and donor T-cells is well studied, and additionally the importance of recipient microbiota in the intestine is elucidated in the GVHD setting. Oral microbiota is the second most abundant bacterial flora in the body after the intestinal tract, and it is related to chronic inflammation and carcinogenesis. Recently, composition of the oral microbiome in GVHD related to transplantation has been characterized and several common patterns, dysbiosis and enrichment of the specific bacterial groups, have been reported. This review focuses on the role of the oral microbiota in the context of GVHD.

Chimeric Antigen Receptor T-Cell Therapy and Hematopoiesis.

Chimeric Antigen Receptor (CAR) T-cell therapy is a promising treatment option for patients suffering from B-cell- and plasma cell-derived hematologic malignancies and is being adapted for the treatment of solid cancers. However, CAR T is associated with frequently severe toxicities such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), macrophage activation syndrome (MAS), and prolonged cytopenias-a reduction in the number of mature blood cells of one or more lineage. Although we understand some drivers of these toxicities, their mechanisms remain under investigation. Since the CAR T regimen is a complex, multi-step process with frequent adverse events, ways to improve the benefit-to-risk ratio are needed. In this review, we discuss a variety of potential solutions being investigated to address the limitations of CAR T. First, we discuss the incidence and characteristics of CAR T-related cytopenias and their association with reduced CAR T-cell efficacy. We review approaches to managing or mitigating cytopenias during the CAR T regimen-including the use of growth factors, allogeneic rescue, autologous hematopoietic stem cell infusion, and alternative conditioning regimens. Finally, we introduce novel methods to improve CAR T-cell-infusion products and the implications of CAR T and clonal hematopoiesis.

Donor Hematopoietic Stem Cell/Lymphocyte Maintenance Treatment After CAR T-Cell Therapy in Patients With B-Cell Acute Lymphoblastic Leukemia Relapse Following Stem Cell Transplant

Maintaining the efficacy of anti-CD19 chimeric antigen receptor modified (CAR) T-cell therapy in patients with B-cell acute lymphoblastic leukemia (B-ALL) relapse after allogeneic hematopoietic stem cell transplant (allo-HSCT) is an urgent problem. In this study, we aimed to compare the efficacy of donor hematopoietic stem cell infusion (DSI) therapy and donor lymphocyte infusion (DLI) therapy as a maintenance therapy after R/R B-ALL patients achieved CR in anti-CD19-CAR T-cell therapy but relapsed after allo-HSCT. In total, 22 B-ALL patients who relapsed after allo-HSCT received anti-CD19-CAR T-cell therapy. Patients who responded to CAR T-cell therapy received DSI or DLI as maintenance therapy. We compared the clinical responses, acute graft versus host disease (aGVHD), expansion of CAR-T-cells, and adverse events between the two groups. In our study, 19 patients received DSI/DLI as maintenance therapy. After DSI/DLI therapy, progression-free survival and overall survival were higher in the DSI group than in the DLI group at 365 days. The grades I and II of aGVHD was observed in four patients (36.4%) in the DSI group. Only one patient developed grade II aGVHD in the DLI group. The peaks of CAR T-cells in the DSI group were higher than those in the DLI group. IL-6 and TNF-α levels increased again in nine of 11 patients after DSI but not in the DLI group. Our findings indicate that for B-ALL patients who relapse after allo-HSCT, DSI is a feasible maintenance therapy if CR is obtained with CAR-T-cell therapy.

Transplantation of Hematopoietic Stem and Progenitor Cells from Cadaveric Organ Donors Leads to Long-Term Multilineage Engraftment in NSG Mice

Background Infusion of Hematopoietic Stem and Progenitor Cells (HSPC) from cadaveric organ donors (CD) has been used anecdotally in the setting of allogeneic Hematopoietic Stem Cell transplants (HSCT) most commonly with the intention for induction of mixed hematopoietic chimerism and subsequent immunological tolerance. No systematic study has addressed the functional capacity of these cells for inducing long-term multi-lineage engraftment. Since the introduction of post-transplant Cyclophosphamide (PTCy) it has been possible to reliably overcome HLA barriers and achieve stable engraftment across extensively HLA-mismatched donor and recipient pairs. Banked HSPC from cadaveric donors may provide a new approach to expand the number of donors for HSCT. Usage of HSPC from bone marrow (BM) of banked cadaveric donors is viewed as no different than live donor aspirated marrow by the FDA but is not yet available on the registry. This emerging source could be of high importance to allow rapid on-demand access to transplants for patients who lack HSPC donors or induce stable mixed chimerism and immune tolerance in patients undergoing combined organ and BM transplantation. The objective of our studies was to investigate whether hCD34⁺HSC selected from cadaveric BM retain their capacity for long-term multi-lineage engraftment. Methods and Results We compared CD34⁺-selected HSPC obtained from CD and living donors (LD) regarding their in vitro and in vivo function and with particular focus on their ability to achieve long-term multi-lineage engraftment following transplantation into NSG mice. Bone marrow was recovered from vertebral bodies of CD and CD34⁺ selected. 5x10⁵ hCD34⁺ cells from 24 different CD were transplanted i.v. in to NSG or SGM3 mice (n=5 per donor) following 1Gy total body irradiation (TBI). Blood was drawn every two weeks, starting week 4 and human CD45⁺ chimerism was analyzed via flow cytometry. All donors engrafted successfully and robust long-term multi-lineage engraftment >16 weeks was seen in 19 out of 22 donors. To further investigate the long-term repopulating capacity, we performed secondary transplants. BM of 15 primary engrafted donors was harvested, hCD45⁺ were selected with immunobeads and transplanted into 1Gy irradiated NSG mice. Successful multi-lineage engraftment after secondary transfer was observed in 12 out of 15 donors in the peripheral blood (PB). Results of tissue engraftment are still outstanding for most experiments. So far they showed mean engraftment of human CD45⁺ cells of 6.16%+/-2.4% for donor 1 and 8.48%+/-0.74% for donor 2 in the BM. To compare CD and LD we set up two side-by-side transplantation experiments and intra-mouse competitive engraftment studies using HLA-mismatched donors. Using side-by-side experiments no significant difference in human CD45⁺ chimerism was detected in blood measured at weeks 6, 12 and 16 pos-transplant. The tissues showed a mean engraftment of human CD45⁺ cells of 87% +/- 9.6% vs 71% +/- 9.5% in spleen and 76% +/- 5.3% vs 78% +/- 4% (NS) in BM upon takedown 20 weeks post-transplant. For our competitive assays, we transplanted 2.5x10⁵ hCD34⁺ i.v. from LD and CD simultaneously into NSG mice (n=5 per donor pair) following 1Gy TBI. In total 8 CD and 3 LD were used, out of whom 4 CD reached higher engraftment than the LD. Conclusion Our data suggest that CD34+HSPC from CD retain their long-term multi-lineage engraftment capacity, reaching at least equipotent levels as HSPC from living donors. Based on our findings cadaveric bone marrow should be considered as HSPC source for HSCT.

Improved T-Cell Chimerism and Successful Withdrawal of Immunosuppression after Non-Myeloablative Stem Cell Transplantation for Sickle Cell Disease with Azathioprine/Hydroxyurea Preconditioning Added to Alemtuzumab/TBI Conditioning

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only established curative treatment option for sickle cell disease (SCD). In adult SCD patients, myeloablative conditioning is associated with significant toxicity, primarily because of cumulative organ damage. Recently, matched sibling donor (MSD) transplantation with non-myeloablative conditioning (alemtuzumab/3 Gy total body irradiation (TBI)) showed promising outcomes in adult SCD patients. This chemotherapy-free regimen, typically resulting in mixed chimerism, is associated with mild toxicity with no reports of graft-versus-host disease (GvHD). Despite mixed chimerism, the red blood cell (RBC) phenotype is completely donor-derived. However, in a large series, 13% of patients experienced graft failure. Another 8.2% of patients could not stop their immunosuppression (sirolimus) after one year, because of low donor T-cell chimerism (<50%). Reported mean donor T-cell chimerism at 12-months post-transplantation was 48% (Br J Haematol. 2021 Feb;192(4): 761-768). We hypothesized, that the addition of azathioprine and hydroxyurea as preconditioning to the alemtuzumab/TBI conditioning regimen will reduce the risk of graft failure and improve donor T-cell chimerism, enabling successful withdrawal of sirolimus in all patients. Aim: To investigate the effects of adding azathioprine/hydroxyurea preconditioning to alemtuzumab/TBI conditioning on donor chimerism levels and the occurrence of graft failure in adult SCD patients receiving non-myeloablative MSD-HSCT. Methods: Adult SCD patients with an available HLA-identical sibling donor were eligible for this treatment. All patients received three months of azathioprine 150mg qd and hydroxyurea 25mg/kg qd preconditioning. After exchange transfusion (target HbS <30%), conditioning with alemtuzumab 1mg/kg total dose and 3Gy TBI started on day -7, followed by unmanipulated peripheral hematopoietic stem cell infusion on day 0. Sirolimus as GvHD and rejection prophylaxis started on day -1. Results: Twenty SCD patients (median age 26 (range 19-49) years) were transplanted with a median follow-up of 24.5 months (range 1-52 months). Patient and transplant characteristics are shown in Table 1. Median donor myeloid and T-cell chimerism of engrafted patients was 100% (range 87-100%) and 68.5% (range 37-89%) at 12-months post-transplantation and 94% (range 86-100%) and 78.5% (range 68-88%) at 18-months post-transplantation, respectively (Figure 1). These donor T-cell chimerism percentages are higher than previously reported with alemtuzumab/TBI conditioning only. All engrafted patients had a corrected SCD phenotype with normalized hemoglobin levels. Importantly, all patients with a follow-up of ≥ 12 months (n=12) could successfully taper and stop sirolimus without decreases in donor chimerism. One year estimated disease-free survival and overall survival were 94.7%. and 93.3%, respectively. One patient (5%) experienced graft failure without autologous regeneration and subsequently died after a series of complications, including EBV reactivation, post-transplantation lymphoproliferative disease, and systemic aspergillosis. Acute gastrointestinal GvHD grade 2 occurred in one patient (5%) and resolved quickly after treatment with prednisolone. Summary/Conclusion: Azathioprine/hydroxyurea preconditioning prior to alemtuzumab/TBI resulted in improved donor T-cell chimerism, potentially reducing the risk of graft failure after non-myeloablatieve MSD transplantation in SCD patients. Importantly, all engrafted patients reached donor T-cell chimerism >50% and were able to stop immunosuppressives as scheduled. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immune tolerance induced by hematopoietic stem cell infusion after HLA identical sibling kidney transplantation.

After the first attempt to induce operational tolerance, it has taken decades to implement it in clinical practice. Recipients with Human leukocyte antigen (HLA) identical sibling donors were enrolled. Hematopoietic stem cells (HSCs) infusion was done after HLA identical sibling kidney transplantation (KTx). Three cases included were followed up for over 8 years. The perioperative conditioning protocol included anti-CD20, rabbit anti-thymocyte globulin (ATG), total lymphoid irradiation (TLI), and cyclophosphamide. Infusion of CD3+ cells and CD34+ cells was conducted. The withdrawal of immunosuppression was determined by mixed lymphocyte reaction (MLR) and graft biopsy. Case 1 and Case 2 showed persistent chimerism, while chimerism was not detected in Case 3. All three recipients showed a low-level response to donor-specific stimulation. Case 1 and Case 3 met the withdrawal rules at 16 and 32 months after transplantation, respectively. Graft function was stable, and no rejection signs were observed in routine biopsies until 94 and 61 months after transplantation. Case 2 was diagnosed with graft-versus-host disease (GVHD) 9 months after transplantation and recovered after an enhanced immunosuppression therapy. Steroids were withdrawn after 1 year, and 0.5 mg tacrolimus twice a day is currently the only immunosuppression at 8 years and 8 months. In conclusion, our clinical experience indicated the efficacy of non-myeloablative conditioning protocol for tolerance induction in HLA identical patients. Complete chimerism might be a risk factor for GVHD.

Genetic Disruption of KLF1 K74 SUMOylation in Hematopoietic System Promotes Healthy Longevity in Mice.

The quest for rejuvenation and prolonged lifespan through transfusion of young blood has been studied for decades with the hope of unlocking the mystery of the key substance(s) that exists in the circulating blood of juvenile organisms. However, a pivotal mediator has yet been identified. Here, atypical findings are presented that are observed in a knockin mouse model carrying a lysine to arginine substitution at residue 74 of Krüppel‐like factor 1 (KLF1/EKLF), the SUMOylation‐deficient Klf1 K74R/K74R mouse, that displayed significant improvement in geriatric disorders and lifespan extension. Klf1 K74R/K74R mice exhibit a marked delay in age‐related physical performance decline and disease progression as evidenced by physiological and pathological examinations. Furthermore, the KLF1(K74R) knockin affects a subset of lymphoid lineage cells; the abundance of tumor infiltrating effector CD8+ T cells and NKT cells is increased resulting in antitumor immune enhancement in response to tumor cell administration. Significantly, infusion of hematopoietic stem cells (HSCs) from Klf1 K74R/K74R mice extends the lifespan of the wild‐type mice. The Klf1 K74R/K74R mice appear to be an ideal animal model system for further understanding of the molecular/cellular basis of aging and development of new strategies for antiaging and prevention/treatment of age‐related diseases thus extending the healthspan as well as lifespan.

Cryopreserved versus non-cryopreserved stem cell autografts in multiple myeloma a restrospective cohort study.

The use of non-cryopreserved hematopoietic stem cells (HSC) can be an alternative to the traditional cryopreserved infusions of HSCs in autologous stem cell transplantation (aHSCT). After high-dose melphalan conditioning (HDM), we sought to compare time to engraftment, overall survival, and safety in multiple myeloma (MM) patients undergoing a first aHSCT after high-dose melphalan conditioning (HDM). We conducted a cohort study from March 2018 to December 2019. Of all autologous transplants performed during this period, 105 were for MM as the first consolidation. Fifty-one patients received a cryopreserved graft; the remaining 54 patients received a fresh infusion. General clinical characteristics were similar between these two groups. Cell viability was higher in non-cryopreserved grafts (95% vs. 86% p < 0.01). Four deaths occurred during hospitalization in the cryopreserved group, one in the non-cryopreserved group. The cumulative incidence of neutrophil and platelet engraftment on D + 25 was higher in the non-cryopreserved compared to the cryopreserved group (98% vs 90% p < 0.01 and 96.2% vs 72.54% p < 0.01 respectively). Additionally, the hospital length of stay was reduced by 4 days for patients for the non-cryopreserved cohort. In summary, the use of non-cryopreserved HSCs after HDM is safe and effective compared to patients who received a cryopreserved graft.

Gene Therapy for Sickle Cell Disease

Gene Therapy for Sickle Cell Disease

Pharmacologically Enhanced Regulatory Hematopoietic Stem Cells Revert Experimental Autoimmune Diabetes and Mitigate Other Autoimmune Disorders.

Type 1 diabetes (T1D) is characterized by the loss of immune self-tolerance, resulting in an aberrant immune responses against self-tissue. A few therapeutics have been partially successful in reverting or slowing down T1D progression in patients, and the infusion of autologous hematopoietic stem cells (HSCs) is emerging as an option to be explored. In this study, we proposed to pharmacologically enhance by ex vivo modulation with small molecules the immunoregulatory and trafficking properties of HSCs to provide a safer and more efficacious treatment option for patients with T1D and other autoimmune disorders. A high-throughput targeted RNA sequencing screening strategy was used to identify a combination of small molecules (16,16-dimethyl PGE2 and dexamethasone), which significantly upregulate key genes involved in trafficking (e.g., CXCR4) and immunoregulation (e.g., programmed death ligand 1). The pharmacologically enhanced, ex vivo-modulated HSCs (regulatory HSCs [HSC.Regs]) have strong trafficking properties to sites of inflammation in a mouse model of T1D, reverted autoimmune diabetes in NOD mice, and delayed experimental multiple sclerosis and rheumatoid arthritis in preclinical models. Mechanistically, HSC.Regs reduced lymphocytic infiltration of pancreatic β cells and inhibited the activity of autoreactive T cells. Moreover, when tested in clinically relevant in vitro autoimmune assays, HSC.Regs abrogated the autoimmune response. Ex vivo pharmacological modulation enhances the immunoregulatory and trafficking properties of HSCs, thus generating HSC.Regs, which mitigated autoimmune diabetes and other autoimmune disorders.

FBX-101, an intravenous AAV gene replacement therapy given after infusion of hematopoietic stem cells, extends efficacious dose ranging and corrects disease manifestations in Krabbe disease

FBX-101, an intravenous AAV gene replacement therapy given after infusion of hematopoietic stem cells, extends efficacious dose ranging and corrects disease manifestations in Krabbe disease

CRISPR Genome Editing: Into the Second Decade

CRISPR Genome Editing: Into the Second Decade

Engraftment Effects after Intra-Bone Marrow versus Intravenous Allogeneic Stem Cell Transplantation in a Reduced-Intensity Conditioning Dog Leukocyte Antigen-Identical Canine Model

Following conventional i.v. hematopoietic stem cell transplantation (IV-HSCT), most of the hematopoietic stem cells get trapped in peripheral organs and do not reach the bone marrow niche. A promising approach to overcome this cell loss during the homing process seems to be the infusion of hematopoietic stem cells directly into the bone marrow cavity (intra-bone marrow [IBM]-HSCT). This study aimed to investigate the engraftment efficiency of IBM-HSCT compared with IV-HSCT following reduced-intensity conditioning in a canine HSCT model. Furthermore, the impact of 2 different graft infusion rates during IBM-HSCT on the engraftment was evaluated. Dogs received 4.5 Gy total body irradiation for conditioning at day -1 and 15mg/kg cyclosporin A twice daily at days -1 to +35 as immunosuppression. The IV-HSCT group (n=7) received unmodified bone marrow. The IBM-HSCT cohorts received buffy coat-enriched bone marrow that was applied into the humerus and femur simultaneously with an infusion time of either 10 minutes (IBM10; n=8) or 60 minutes (IBM60; n=7). Statistical analyses were performed using the Kruskal-Wallis test followed by the Mann-Whitney U test with Bonferroni correction for multiple comparisons. Statistical significance was declared at Bonferroni-adjusted P < .017. All dogs initially engrafted. One dog of the IBM10 cohort died at day +15 from infection. All 21 evaluable dogs developed a durable mixed donor chimerism over the course of 112 days. Engraftment kinetics did not differ significantly across the 3 groups. Leukocyte and platelet nadirs, as well as the durations of leukopenia and thrombocytopenia, were comparable in the 3 groups. Signs of toxicity for ingestion, body temperature, activity, and defecation did not show statistically significant differences among the 3 groups; only weight loss was greater in the IBM60 group compared with the IV group. IBM-HSCT following reduced-intensity conditioning resulted in an engraftment efficiency and hematopoietic recovery comparable to that seen with conventional IV-HSCT. In addition, modification of the graft infusion rate had no impact on engraftment and hematopoietic recovery in the canine IBM-HSCT model.