Autologous Hematopoietic Stem Cell Research Articles

Evaluation of the effectiveness of hematopoietic blood stem cell mobilization using empegfilgrastim (Extimia®, BIOCAD) in patients with lymphoproliferative diseases: the experience of several centers in the Russian Federation. A retrospective study

Aim. To evaluate the effectiveness of autologous hematopoietic stem cell (HSC) mobilization with empegfilgrastim in patients with lymphoproliferative diseases (LPDs) and to identify prognostic factors affecting the effectiveness of mobilization. Materials and methods. The paper analyzes the effectiveness of HSC mobilization with Extimia® by JSC "BIOCAD" (INN: empegfilgrastim) in patients with LPDs. The study included 89 patients with LPDs (Hodgkin's lymphoma, B- and T-cell lymphomas, plasma cell tumors) who were treated in 5 research centers of the Russian Federation (in Moscow and St. Petersburg). The median age of patients was 39 (18–67) years. In 26 (29%) patients, HSC mobilization was performed "in a stable state of hematopoiesis" using only pegylated granulocyte colony-stimulating factor (PEG-G-CSF), and in 63 (71%) patients after chemotherapy (CT). Empegfilgrastim was administered as a single dose of 7.5 mg subcutaneously in two regimens: 24 hours after the end of CT or at a random time. Results. Two groups of patients were identified, analyzed, and compared: those with ineffective (n=23, 26%) and effective (n=66, 74%) HSC mobilization. Univariate and multivariate statistical analyses (binary logistic regression and multiple bidirectional binary logistic regression, respectively) were sequentially performed to determine the factors associated with ineffective mobilization. In a one-factor analysis, we studied the sex, age, diagnosis, bone/bone marrow involvement, the number of previous CT lines, the status of the disease (remission/no remission), the history of radiation therapy, the time interval from the administration of empegfilgrastim to the decision to perform leukapheresis, the mode of granulocytopoiesis stimulation ("in a stable state of hematopoiesis" or after CT), the leukocyte and platelet counts. The subsequent multifactorial analysis of the two selected parameters (diagnosis and time interval from drug administration to the decision to perform leukapheresis) showed the following statistically significant risk factors for ineffective mobilization: T-cell lymphoma; T-cell lymphoma versus other (combination) diagnoses (B-cell lymphoma, Hodgkin's lymphoma and plasma cell tumor): p=0.046, Wald test, odds ratio 4.18 (95% confidence interval 0.96–19.4). The time interval from the administration of empegfilgrastim to the first leukapheresis in the effective mobilization group depended on the diagnosis. The longest period was observed in patients with plasma cell tumors and the shortest in those with T-cell lymphomas: 10 (4–10) vs 5.5 (4–6) [p=0.01, Kruskal–Wallis test]. Empegfilgrastim proved equally effective for HSC mobilization both "on stable hematopoiesis" and after CT. Conclusion. The results of the first multicenter experience with Extimia® by JSC "BIOCAD" (INN: empegfilgrastim) to mobilize autologous HSCs in LPD patients demonstrate its high efficacy with a favorable safety and tolerability profile: 74% (66/89) successful mobilizations after a single administration of a fixed dose of the drug.

Preclinical ex vivo IL2RG gene therapy using autologous hematopoietic stem cells as an effective and safe treatment for X-linked severe combined immunodeficiency disease

X-linked severe combined immunodeficiency disease (X-SCID) is a rare inherited disease caused by mutations in the interleukin 2 receptor subunit gamma gene (IL2RG), which encodes the common γ chain protein, a subunit of the receptor for lymphocytes. X-SCID is characterized by profound defects in T-cell, B-cell, and natural killer cell function. Here, we report a Chinese cohort of nine X-SCID patients with six novel IL2RG mutations. Among those, the two adolescent patients carrying missense mutation with an atypical immunotype were confirmed by further analyzing IL-2-JAK-STAT5 signaling, T cell proliferation, and T cell receptor excision circles (Trecs). Interestingly, Bacillus Calmette-Guérin (BCG) disease occurred commonly in this cohort. Although allogeneic hematopoietic stem-cell transplantation is curative for the disease, it is not available to all patients due to the lack of suitable matched donors. Autologous gene therapy using a self-inactivating lentiviral vector (SIN-LV) technology, developed in recent decades has provided an alternative therapy for treating such mono-genetic diseases. Here, we performed the pre-clinical studies to assess our SIN-LV carrying IL2RG on human ED7R cells deficient in IL2RG and CD34+ stem cells derived from the bone marrow of a healthy donor and a patient with X-SCID. This work is done complied with the established "Good Manufacturing Practice" (GMP) used in the clinical trials. In addition, a safety study is performed using the transduced CD34+ cells implanted into the axilla of nude mice in vivo. Overall, our studies have demonstrated the efficiency and safety of SIN-IL2RG-LV, which paves the way for conducting X-SCID gene therapy clinical trials in China in the near future.

Comparative assessment of the effectiveness of hemapoyesis restoration in patients with multiple myeloma at autologous bone marrow transplantation using frozen and native stem cells

Purpose of the study. To compare the effectiveness and safety of using autologous hematopoietic stem cells without freezing in multiple myeloma.Materials and methods. The study was carried out in the transplantation department of the Republican Scientific and Practical Medical Center of the Ministry of Health of the Republic of Uzbekistan. The study included 82 patients with multiple myeloma who underwent autologous bone marrow transplantation between 2014 and 2020 and was retrospective in nature based on medical records. The patients were divided into 2 groups – I without freezing (40 patients), II – with freezing of stem cells (42 patients). The average age was 47±4.5 years, the male to female ratio was 32 (39%) and 50 (61%), respectively. The timing of hematopoietic restoration, length of hospital stay, viability of hematopoietic cells and 5-year overall and relapse-free survival in both groups were analyzed.Results and discussion. In the study groups, immediately after apheresis of peripheral hematopoietic stem cells, viability was 96-98%. In group I, at the time of administration, the number of living cells in the graft was the same 96-98%, and in group II, at the time of transplantation, viability was at the level of 79-93%, which was associated with cell loss during freezing, thawing and exposure to Dimethylsulfoxide. Also, the use of an unfrozen transplant increased the recovery time of hematopoiesis after transplantation to 11 days, while the frozen transplant was restored by the 15th day. Complications associated with preservative toxicity occurred in group II in 100% of patients. The study did not reveal any difference in 5-year overall and disease-free survival. In group I, relapse-free survival was 63%, in group II -60% (p = 0.2). When assessing overall 5-year survival between groups I and II, there was also no significant difference: 78% and 74% (p = 0.1).Conclusions. The data obtained in the study allow us to conclude that transplantation with non-frozen peripheral hematopoietic stem cells is an effective and safe method and does not have a significant difference when comparing long-term results in cryofrozen peripheral hematopoietic stem cells.

One size does NOT fit all – factoring weight into current stem cell transplant guidelines in multiple myeloma patients

Abstract Autologous hematopoietic stem cell (HPSC) transplantation serves as the standard of care treatment for multiple myeloma patients. The standard ASCT procedures for multiple myeloma (MM) patients often includes induction therapy, stem cell collection via apheresis and autologous hematopoietic cell transplantation (HCT) for eligible patients. The current guideline for HPSC collection for multiple myeloma established that 3x106 cells/kg of actual body weight (ABW) by the 1997 study of stem cell transplants in breast cancer. Recent research indicates that ideal body weight (IBW) utilization is a stronger predictor of post-autotransplantation neutrophil recovery than calculations based on ABW. Reducing the cost of HPSC collection and cryopreservation is critical to mitigate the cost incurred by the transplant center and the patient. This study evaluates whether using a patient’s IBW or Adjusted Ideal Body Weight (AIBW) would be satisfactory in determining the amount of stem cells required for transfusion when compared to ABW. After institutional review board approval, a retrospective chart review study analyzed a cohort of 224 deceased MM patients, who received HCT at the UAB Medicine Hospital within the fiscal years of 2017 to 2019. The ABW for each patient was used to calculate their IBW and AIBW using the formulas shown below. The target CD34+ cell amount for patients was calculated using each body weight parameter. The platelet count and absolute neutrophil count (ANC) defined a successful platelet and neutrophil engraftment. The engraftment date was determined as the day the platelet value exceeded 20 and ANC exceeded 0.5 for three consecutive days (without any transfusion within a seven day window) and a statistical analysis was utilized to determine whether the IBW and AIBW parameters were adequate to achieve successful engraftment and HCT. This study finds that the cohort of multiple myeloma patients are significantly more overweight than the prior study that established the current guidelines for autologous hematopoietic cell transplantation. The results for CD34+/kg cell targets for ideal and adjusted ideal body weight suggests that there are minimal differences in platelet and neutrophil engraftment and number of platelet transfusions when compared to CD34+/kg targets using actual body weight. Some methods to reduce excessive stem cell collection include decreasing target cell dosage from previous standards of 6.0 x 106 cells/kg or calculating total cell dosage with IBW or AIBW instead of ABW. Future studies should evaluate the efficacy of cell dosage decrease to lowered targets and total cell calculations using IBW or AIBW instead of ABW. Equation 1. ABW to IBW Conversion (H = height over 5 ft. in inches) IBW(males) = 50 kg + (2.3 kg x H) IBW(females) = 45.5 kg + (2.3 kg x H) Equation 2. ABW to AIBW Conversion AIBW = IBW + 0.5(ABW-IBW)

Semaglutide treatment for PRevention Of Toxicity in high-dosE Chemotherapy with autologous haematopoietic stem-cell Transplantation (PROTECT): study protocol for a randomised, double-blind, placebo-controlled, investigator-initiated study

IntroductionCancer treatment with high-dose chemotherapy damages the mucosal barrier of the gastrointestinal (GI) tract and is associated with severe toxicity involving mucositis, severe inflammation and organ dysfunction. Currently, there is...

The role of the haematopoietic stem cell niche in development and ageing.

Blood production depends on rare haematopoietic stem cells (HSCs) and haematopoietic stem and progenitor cells (HSPCs) that ultimately take up residence in the bone marrow during development. HSPCs and HSCs are subject to extrinsic regulation by the bone marrow microenvironment, or niche. Studying the interactions between HSCs and their niche is critical for improving ex vivo culturing conditions and genetic manipulation of HSCs, which is pivotal for improving autologous HSC therapies and transplantations. Additionally, understanding how the complex molecular network in the bone marrow is altered during ageing is paramount for developing novel therapeutics for ageing-related haematopoietic disorders. HSCs are unique amongst stem and progenitor cell pools in that they engage with multiple physically distinct niches during their ontogeny. HSCs are specified from haemogenic endothelium in the aorta, migrate to the fetal liver and, ultimately, colonize their final niche in the bone marrow. Recent studies employing single-cell transcriptomics and microscopy have identified novel cellular interactions thatgovern HSC specification and engagement with their niches throughout ontogeny. New lineage-tracing models and microscopy tools have raised questions about the numbers of HSCs specified, as well as the functional consequences of HSCs interacting with each developmental niche. Advances have also been made in understanding how these niches are modified and perturbed during ageing, and the role of these altered interactions in haematopoietic diseases. In this Review, we discuss these new findings and highlight the questions that remain to be explored.

Impact of age on safety of Busulfan-Melphalan followed by autologous hematopoietic stem-cell transplantation versus standard chemotherapy in the patients of the EURO-E.W.I.N.G. 99 and Ewing 2008 clinical trials

IntroductionEwing sarcoma (ES), is a rare cancer affecting children, adolescents and adults. After VIDE (vincristine-ifosfamide-doxorobucin-etoposide) induction chemotherapy, Busulfan-Melphalan (BuMel) high-dose chemotherapy followed by autologous hematopoietic stem cells transplantation improved outcomes in unfavourable localized ES, but with more toxicities than conventional chemotherapy (VAI: Vincristine-dactinomycin-Ifosfamide). We evaluated whether the risk of acute toxicity associated with BuMel compared to VAI varied according to age in patients recruited in the R2Loc and R2Pulm randomised trials of the Euro-E.W.I.N.G.99 and Ewing-2008 trials. MethodsWe included patients with a localized high-risk disease, or pulmonary or pleural metastasis. We analysed the risk of severe toxicity according to randomised treatment group (VAI versus BuMel) and age group (<12 years, 12–17 years, 18–24 years, ≥25 years). We evaluated the heterogeneity of treatment effects by age group using interaction terms in logistic multivariable models. ResultsThe analysis included 243 patients treated with VAI and 205 with BuMel. Overall, BuMel was associated with a higher risk of severe acute toxicity than VAI particularly haematological, gastrointestinal, liver, sinusoidal occlusive syndrome, and infections. Severe haematological toxicity and lower general condition were significantly more frequent in younger patients, whatever treatment. We did not observe any significant heterogeneity in terms of the excess risk of severe toxicities associated with BuMel compared to VAI according to age group. ConclusionThe excess of acute toxicity associated with BuMel compared to VAI does not vary significantly with age, suggesting the feasibility of BuMel across all age groups.

In-Vitro Proliferation and Enrichment of CD34+ Autologous Haematopoietic Stem Cells, Obtained from Patients with End-Stage Left Ventricular Failure

With a rising global prevalence of end-stage heart failure and a limited availability of cardiac transplantation programmes or left ventricular assist devices in many parts of the world, there is a renewed interest in potential alternative treatment options.Along with other groups, we have previously demonstrated the beneficial effect of intracoronary autologous bone marrow derived peripheral haemopoietic stem cell transplantation, to improve myocardial contractility in end-stage heart failure.We now assess the ability of these cells to expand in numbers in-vitro, to achieve enrichment of stem cell counts, using standard cell culture methods and flow- cytometric analysis.We demonstrate that with in-vitro culturing (within 3 passages post-harvest), the CD34+ stem cell fraction increases in standard culture media, across multiple samples. However, optimal culture conditions to achieve near pure stem cell populations with rapid cell proliferation, still needs to be defined.

Predictive model of the efficiency of hematopoietic stem cell collection in patients with multiple myeloma and lymphoma based on multiple peripheral blood markers.

Autologous hematopoietic stem cell transplantation (ASCT) has gained extensive application in the treatment of lymphoma and multiple myeloma (MM). Plenty of studies demonstrate that peripheral blood indicators could be considered potential predictive biomarkers for hematopoietic stem cells (HSCs) collection efficiency, including white blood cell count (WBC), monocyte count (Mono), platelet count (PLT), hematocrit, and hemoglobin levels. Currently, clinically practical predictive models based on these peripheral detection indicators to quickly, conveniently, and accurately predict collection efficiency are lacking. In total, 139 patients with MM and lymphoma undergoing mobilization and collection of ASCT were retrospectively studied. The study endpoint was successful collection of autologous HSCs. We analyzed the effects of clinical characteristics and peripheral blood markers on collection success, and screened variables to establish a prediction model. We determined the optimal cutoff value of peripheral blood markers for predicting successful stem cell collection and the clinical value of a multi-marker prediction approach. We also established a prediction model for collection efficacy. Univariate and multivariate logistic regression analyses showed that the mobilization regimen, Mono, PLT, mononuclear cell count (MNC), and peripheral blood CD34+ cell count (PB CD34+ counts) were significant predictors of successful collection of peripheral blood stem cells (PBSC). Two predictive models were constructed based on the results of multivariate logistic analyses. Model 1 included the mobilization regimen, Mono, PLT, and MNC, whereas Model 2 included the mobilization regimen, Mono, PLT, MNC, and PB CD34+ counts. Receiver operating characteristic (ROC) curve analysis showed that the PB CD34+ counts, Model 1, and Model 2 could predict successful HSCs collection, with cutoff values of 26.92 × 106/L, 0.548, and 0.355, respectively. Model 1 could predict successful HSCs collection with a sensitivity of 84.62%, specificity of 75.73%, and area under the curve (AUC) of 0.863. Model 2 could predict successful HSCs collection with a sensitivity of 83.52%, specificity of 94.17%, and AUC of 0.946; thus, it was superior to the PB CD34+ counts alone. Our findings suggest that the combination of the mobilization regimen, Mono, PLT, MNC, and PB CD34+ counts before collection has predictive value for the efficacy of autologous HSCs collection in patients with MM and lymphoma. Using models based on these predictive markers may help to avoid over-collection and improve patient outcomes.

The first genome-edited cell product receives FDA approval

The promise of genome editing technologies (GETs) for gene therapy is one of the key drivers for development of this field to treat monogenic hereditary diseases. However, the general consensus of research community and many physicians is that in some cases the balance of risk and benefit of GET-based drugs and CRISPR/Cas9, in particular, has not been fully studied to date. However, on December 03, 2023, the US Food and Drug Administration (FDA) approved marketing of two products for sickle cell disease. One of them (“Casgevy”) is a cellular product consisting of CD34+ autologous hematopoietic stem cells, in which, using the CRISPR/Cas9 system, it was possible to increase the production of fetal hemoglobin, which leads to compensation of patient’s condition. Thus, for the first time in world practice, national regulator has approved not just a genetically modified cell product, but a product obtained using GET. This short message is dedicated to this historical event and its importance, as well as possible consequences.

Anti-CD45 PBD-based antibody-drug conjugates are effective targeted conditioning agents for gene therapy and stem cell transplant

Stem cell gene therapy and hematopoietic stem cell transplantation (SCT) require conditioning to ablate the recipient's hematopoietic stem cells (HSCs) and create a niche for gene-corrected/donor HSCs. Conventional conditioning agents are non-specific, leading to off-target toxicities and resulting in significant morbidity and mortality. We developed tissue-specific anti-human CD45 antibody-drug conjugates (ADCs), using rat IgG2b anti-human CD45 antibody clones YTH24.5 and YTH54.12, conjugated to cytotoxic pyrrolobenzodiazepine (PBD) dimer payloads with cleavable (SG3249) or non-cleavable (SG3376) linkers. Invitro, these ADCs internalized to lysosomes for drug release, resulting in potent and specific killing of human CD45+ cells. In humanized NSG mice, the ADCs completely ablated human HSCs without toxicity to non-hematopoietic tissues, enabling successful engraftment of gene-modified autologous and allogeneic human HSCs. The ADCs also delayed leukemia onset and improved survival in CD45+ tumor models. These data provide proof of concept that conditioning with anti-human CD45-PBD ADCs allows engraftment of donor/gene-corrected HSCs with minimal toxicity to non-hematopoietic tissues. Our anti-CD45-PBDs or similar agents could potentially shift the paradigm in transplantation medicine that intensive chemo/radiotherapy is required for HSC engraftment after gene therapy and allogeneic SCT. Targeted conditioning both improve the safety and minimize late effects of these procedures, which would greatly increase their applicability.

Current state of gene therapy in sickle cell disease.

Sickle cell disease (SCD) is a type of hemoglobinopathy due to an autosomal recessive genetic defect, causing significant red cell sickling, multi-organ damage and long-term severe morbidities. Due to its complicated care and the impact on quality of life, a curative treatment for SCD is highly desirable. In recent years, gene therapy is emerging as a curative option for SCD, where autologous haematopoietic stem cells are collected from SCD patients and genetically modified ex vivo to reduce its sickling tendency before reinfusion. Although still largely investigational, a limited number of gene therapy options have been recently granted approval for SCD patients. Published data are still currently limited, but early studies have so far demonstrated the intended outcomes of less vaso-occlusive crisis and haemolysis. Nonetheless, despite its curative potential, larger clinical trials and longer follow-up period are still necessary to evaluate the safety of this treatment option, especially the risk of unintended genetic modifications. Furthermore, SCD patients frequently have limited access to specialty care; hence, the issues of affordability and accessibility to SCD gene therapy must also be addressed for it to benefit the appropriate patient population.

Comparative Efficacy of Autologous Hematopoietic Stem Cell Mobilization Regimens in Multiple Myeloma Patients

Aim. To compare the efficacy of mobilization regimens (MR) differing in their composition and intensity, with the purpose of defining the criteria to personalize the choice of MR based on clinical and hematological characteristics of multiple myeloma (MM) patients.&#x0D; Materials &amp; Methods. A retrospective analysis of the autologous hematopoietic stem cell (HSC) mobilization and autograft harvesting results was performed in 177 patients with newly diagnosed MM. The patients were divided into 4 groups. Group 1 included 62 patients with the median age of 53 years who were treated with single injection of cyclophosphamide (CF) dose 3 g/m2 as MR. Group 2 consisted of 71 patients with the median age of 58 years who received vinorelbine 35 mg/m2. Granulocyte colony-stimulating factor (G-CSF) as a monoregimen was administered to group 3 consisting of 33 patients with the median age of 55 years. Group 4 included 11 patients with the median age of 57 years who received G-CSF enhanced by plerixafor administration. G-CSF 10 µg/kg was used as MR. In all chemomobilization cases, daily G-CSF 10 µg/kg started on Day 4 from the administration of the chemotherapy drug prescribed as MR.&#x0D; Results. In the analyzed groups, the median time from MR start to the first leukocyte apheresis session was 11, 8, 5, and 5 days, respectively. On the first leukocyte apheresis day, the median CD34+ cell collection in group 3 was significantly lower than in groups 1, 2, and 4: 2.2 × 106/kg vs. 3.79 × 106/kg, 7.22 × 106/kg, and 3.9 × 106/kg, respectively. The total CD34+ cell collection after two leukocyte apheresis sessions was also the lowest in group 3 compared with groups 1, 2, and 4: 3.22 × 106/kg vs. 5.2 × 106/kg, 4.95 × 106/kg, and 7.5 × 106/kg, respectively. In the analyzed groups, the rate of mobilization with CD34+ cell collection &lt; 2.0 × 106/kg was 6.5 %, 5.6 %, 18.2 %, and 9.1 %. The evaluation of the results in all patients showed a direct correlation of CD34+ cell collection with lenalidomide administered before autologous HSC mobilization. A significant difference in CD34+ cell collection in lenalidomide recipients vs. non-recipients was reported when vinorelbine as MR and G-CSF as monoregimen (р = 0.001 and р = 0.022, respectively) were used. No significant differences were observed either with CF or G-CSF combined with plerixafor treatment.&#x0D; Conclusion. Based on the findings, age of a MM patient, comorbidities, and prior lenalidomide administration can be regarded as key criteria for choosing one of 4 MRs.

OP29 Haematopoietic stem cell gene therapy as a treatment for severe Crohn’s Disease associated with pathogenic NOD2 genetic variants

Abstract Background Pathogenic variants of the NOD2 (nucleotide-binding oligomerization domain containing protein 2) gene demonstrate the strongest genetic association to Crohn’s inflammatory bowel disease (CD), with mounting evidence linking NOD2 deficiency with poor clinical outcome. CD patients with NOD2 variants, particularly carriers of more than one risk allele, frequently present an aggressive, fistulizing and fibrostenotic disease, requiring multiple surgical resections. NOD2-deficiency is implicated to drive CD pathogenesis through failure of gut innate immunity to resolve bacterial infections and by loss of tissue homeostasis within the intestinal microenvironment. Here we present preclinical data on OTL-104, an autologous haematopoietic stemcell gene therapy (HSC-GT) which aims to stably restore NOD2 expression in gut resident macrophages, to correct immune dysfunction linked to NOD2-deficient CD pathogenesis. Methods We used in vitro and in vivo models of NOD2 deficiency to demonstrate the mechanism of action and the efficacy of OTL-104 autologous HSC-GT. Results NOD2KO human myeloid cells differentiated in vitro from CRISPR-generated NOD2KO CD34+ HSCs are unable to mount a proinflammatory cytokine response to MDP stimulation. Similarly, myeloid cells differentiated from CD34+ cells obtained from peripheral blood of genetically characterized NOD2-deficient CD patients, are also refractory to MDP stimulation and unable to generate a normal cytokine response profile. In both NOD2 deficient CD34+ derived monocyte models, transduction with a lentiviral vector (LVV) expressing NOD2 fully restores NOD2-dependent cytokine and chemokine responses, restoring an immune profile that is comparable to monocytes derived from CD34+ cells from NOD2 wild-type healthy donors (Figure 1). Transplantation of lineage negative (Lin-) haematopoietic stem/progenitor cells (HSPCs) transduced with the OTL-104 LVV in NOD2KO mice was used as an in vivo model of gene therapy for CD. Compared to WT mice, NOD2KO mice fail to release systemic inflammatory mediators and recruit myeloid cells in response to MDP administration. Transplantation of transduced Lin- HSPCs restores MDP-induced systemic release of IL-6 and CXCL1 as well as innate mobilization of monocyte/macrophage cells and gut associated gene expression. Key to our therapeutic approach, histopathological analysis of intestinal lamina propria from transplanted mice shows a normal biodistribution and physiological NOD2 gene expression in tissue resident cells. Conclusion These results confirm the impact of NOD2 deficiency in primary immune activation and demonstrates the therapeutic potential of OTL-104 HSC-GT for long-term correction of NOD2-deficient CD.

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.

Early Autologous Hematopoietic Stem Cells (AHSC) Boost (HCB) to Overcome Immune Effector Cell-Associated Hematotoxicity (ICAHT) after CAR T-Cell Therapy for Lymphoma: A Single Center Experience

Sustained cytopenias designed as Immune Effector Cell-Associated Hematotoxicity (ICAHT) represent one of the most frequent side effect of CAR-T-cell therapy (30 to 90 % patients in recent real life studies) resulting in significant morbidity, mortality and resources consumption. We report single center experience of early reinjection of previously cryopreserved AHSC (HCB) in that setting. From July 2021 until January 2023, 27 patients received CAR T-cell therapy (25 Axi-Cel, 2 Brexu-Cel) after Fludarabine and Cyclophosphamide lymphodepletion for relapsed/refractory large B-cell lymphomas (24), mantle lymphoma (2), or follicular lymphoma (1). Median age was 62 years (19-75). CAR-HEMATOTOX score was ≥ 2 for 46% of patients. All patients received Filgrastim from D0. Hematopoietic recovery was longitudinally evaluated until D90 or censoring for relapse and/or subsequent cytotoxic treatment: severe (CTAE grade ≥ 3) neutropenia, anemia or thrombopenia occurred respectively for 88, 44 and 76% of patients until D28, and 60, 24 and 60% until D90. Median duration of severe neutropenia was 7 days (0-39) and 28 days for severe thrombocytopenia (0-118). Prolonged neutropenia occurred in 18/25 evaluable patients, mostly with a biphasic course (intermittent recovery and aplastic phenotype for 14 and 4 patients respectively). CAR-HEMATOTOX score was significantly correlated with sustained thrombopenia (p = 0,005) but only marginally with ICAHT (p = 0,054) and not with severe neutropenia (p = 0,44) . We observed significant correlation of severe ICAHT with progressive disease at CAR T-cell infusion (p = 0,015), platelets nadir &amp;lt; 30 giga/l before D28 (p &amp;lt; 0,005) and CAR T-cell expansion (&amp;gt; 200/µl) at D7 (p = 0,023). 7 patients received HCB (no conditioning) at a median D24 (15-41) post CAR T-cell infusion: after D14 for patients with aplastic phenotype, or for cytopenic recurrence for patients with biphasic course. Filgrastim was pursued or reintroduced until neutrophil recovery (&amp;gt; 1 giga/l) which occurred after a median of 10 days (4-16); platelet recovery (&amp;gt; 20 giga/l and &amp;gt; 50 giga/l) occurred after a median of respectively 12 (8-14) and 17 (11-22) days. If we consider D0 as CAR T-cell infusion, neutrophil recovery occurred at median D37 (22-49) and platelet recovery (&amp;gt; 50 giga/l) at median D37 (26-63). Median complete hematopoietic reconstitution (defined as PMNC &amp;gt; 1 giga/l, and Hb &amp;gt; 100 g/l, and PLT &amp;gt; 100 giga/l) was significantly shorter (median 23 days, 14-30) for patients receiving HCB compared with patients with prolonged cytopenia (&amp;gt; 28 days) who do not receive HCB (60 days, p = 0,018). Transfusion and hematopoietic growth factors (HGF) consumption was significantly reduced after HCB but we observe no difference between 2 groups considering hospitalization, EFS or OS. 2 patients receiving HCB experienced late recurrence of cytopenia (after D90) requiring intermittent HGF support but no transfusion or hospitalization. Discussion - Perspectives Recent case series highlighted clinical feasibility and safety and of this approach across a broad population of pediatric and adult patients: we validate this strategy in a more homogeneous and systematic practice. As study population is small, we failed to demonstrate that earlier application of an HCB is associated with superior survival outcomes as shown by others. We agree with recent updated recommendation of experts panel considering application of HCB without prior conditioning chemotherapy for grade ≥ 3 ICAHT beyond D14 (so considered early) if AHSC graft is readily available. Prophylactic AHSC harvest in high-risk candidates has been proposed but collection process may add to already high logistic burden and costs of CAR T-cell therapy in the lymphoma setting. Conversely, cryopreserved ASHC is mainly available for patients with myeloma and this approach would be readily proposed and useful for around 20% patients as recently described. Gagelmann N et al. Hematopoietic stem cell boost for persistent neutropenia after CAR T-cell therapy: a GLA/DRST study. Blood Adv. 2023;7(4):555 Rejeski K et al, Immune Effector Cell-Associated Hematotoxicity (ICAHT): EHA/EBMT Consensus Grading and Best Practice Recommendations. Blood. 2023 doi: 10.1182/blood.2023020578 Davis JA et al. Efficacy and safety of CD34+ Stem Cell Boost for Delayed hematopoietic Recovery After BCMA Directed CAR T-cell Therapy. Transplant Cell Ther. 2023 doi: 10.1016/j.jtct.2023.05.012

Autologous MPO Knock-out Hematopoietic Stem and Progenitor Cells for Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is a progressive condition for which there is no cure. Even with substantial pharmacologic advances in the modern treatment era, existing treatments provide only symptomatic relief and survival remains unacceptably poor. Patients who fail to respond to medications usually have the worst prognosis and patients with persistently elevated pulmonary pressure and right heart failure usually die within 5 years. PAH is a severe complication of Systemic sclerosis (SSc) affecting approximately 8%-12% of patients. SSc affects 13.5-44.3 per 100,000 individuals and is complex and diverse, with limited treatment options. SSc-associated PAH is the second largest group of patients with PAH. Once diagnosed, it becomes the leading cause of mortality among this patient population, with a 50% mortality rate within 3 years of PAH diagnosis. Inflammation and endothelial injury being the main components of the syndrome, investigation regarding the underlying mechanisms of this immune response may lead to the development of a new efficient therapy. There is extensive evidence for a pathogenic role for neutrophil myeloperoxidase (MPO) in PAH disease. PAH patients with higher levels of MPO have a poorer outcome. Studies in murine models of PAH demonstrate significant disease amelioration by elimination of neutrophil MPO by hematopoietic stem cell (HSC) transplantation with HSC lacking MPO from germline MPO gene knock-out. We developed a gene therapy approach to PAH in which autologous HSCs will undergo gene editing using CRISPR/Cas9 to achieve bi-allelic disruption of the MPO gene. These MPO-knock-out HSCs will then be autologous transplanted. That autologous transplantation of HSPC in which the MPO gene is disrupted is expected to prevent progression of PAH due to the essential role of neutrophil MPO activity on disease development. We identified a sgRNA that leads to high frequency disruption of MPO in primary human CD34+ hematopoietic stem and progenitor cells (HSPC), leading to efficient elimination of MPO protein in the resultant neutrophils. That sequence was assessed for genome-wide off-target and showed no detectable locus, validating it as a lead candidate for safe gene editing strategy. We validated in an in vitro neutrophil differentiation model the functional efficiency of the knock-out. MPO KO differentiated cells exhibit reduced neutrophil extracellular traps formation (NETosis) and absence of MPO protein. An optimized gene editing protocol for HSC was optimized and showed consistent allelic disruption over 80%. The edited cells retain hematopoietic potential and showed no skewing in the generated lineages in an in vitro colony forming assay. We developed a surrogate gene editing protocol for murine cells and optimized electroporation to achieve high allelic disruption of the murine MPO gene in lineage negative murine hematopoietic cells. The protocol will be tested in a hypoxia-induced PAH murine model. The presented work can represent a major improvement for PAH patient. The limited therapeutic options and severity of the disease urges to develop new approaches. The gene therapy protocol developed showed promising result in vitro and rely on clinical evidences for the implication of MPO in the disease. Results will be tested in a pre-clinical murine model

A Single Center Real-World Data Comparing Salvage Autologous Hematopoietic Stem-Cell Transplantation, Selinexor Combo and Elranatamab Alone Among Triple Class Refractory Myeloma Patients: Better Efficacy with Elranatamab

Introduction: A vast majority of multiple myeloma (MM) patients may experience relapse and require multiple lines of therapy. The choice of the next treatment depends on multiple factors including the response, tolerance to prior therapies, as well as access to novel treatments. Among triple-class refractory patients, in real-world settings where CAR-T therapies are not available, options are limited. Here we report a single-center experience comparing salvage autologous hematopoietic stem-cell transplantation (sAHCT), selinexor combinations, and elranatamab in this highly unmet needed group. Patients &amp; Methods: Between January 2015 and June 2023, 50 patients at our center underwent sAHCT for treatment of refractoriness to all available drugs. Salvage AHCT is defined as being transplanted for treatment of MM after evidence of disease progression not responding to second-generation proteasome inhibitors (PI), immunomodulating agents (IMID), or antiCD38 monoclonal antibody. We compared the outcomes of these patients with RRMM patients who had exhausted their treatment options and were progressing on their last line of therapy to receive single agent Elranatamab at a step-up dosing starting with escalating to 72 mg once a week for six months and on alternate weeks thereafter. Selinexor was administered with either Bortezomib(n:8) or Pomalidomide(n:9) at a dose of 60-100 mg/week every 5-6 weeks. Response and disease progression were assessed according to the IMWG criteria. Results: Patients' characteristics and the outcomes are shown in Table. The median age of patients was similar in both groups. Patients were heavily treated with a median of 4 prior lines of therapy (1-17) and 7 out of 13 carried high-risk cytogenetics in elranatamab group. All patients were triple-class refractory. Five patients responded to DCEP bridging therapy prior to sAHCT. Selinexor (n: 11, 64.7%) and elranatamab (n:5 patients,38.5%) were administered after sAHCT. One patient underwent sAHCT due to progression under Selinexor treatment. In the sAHCT group, 68% of patients relapsed within a median of 7.4 months. Median DFS was significantly prolonged with single agent elranatamab (NR) compared to sAHCT (7.3 mos) and Selinexor-combos (7.3 mos) (p=0.4) (Figure). Adverse events on selinexor regimens included fatigue, nausea, and thrombocytopenia which were managed with Selinexor dose adjustment and supportive care. All patients received the recommended step-up priming dose elranatamab regimen, 69.2% of our patients experienced a Grade 1 cytokine release syndrome (CRS) during the first cycle. None of these patients had to stop treatment due to toxicity. Early sAHSCT mortality was not observed either. At the time of the analysis, all patients on Selinexor have progressed after a median follow-up of 14.7 months. Elranatamab patients are currently at 5 months follow-up and 7/ 13 are still on treatment. Conclusion: Upfront AHCT is a cost-effective treatment option with a decreasing efficacy in late relapses. In this RWE study, we have been able to confirm the inferiority of AHCT at late stages. As seen in our series cytogenetic profile worsens with an increase in the prior number of lines of treatment which are both highest among patients who have received elranatamab. Despite these unfavorable features, single-agent elranatamab has been able to attain the highest overall response rates, unfortunately not reflecting on PFS yet, due to short follow-up. Furthermore, two of these had progressed while on a BCMA immunotoxin Rx and have responded to elranatamab. In conclusion, even with short follow-up single-agent elranatamab is a tolerable and highly effective regimen

Expression of a large coding sequence: Gene therapy vectors for Ataxia Telangiectasia

Ataxia telangiectasia is a monogenetic disorder caused by mutations in the ATM gene. Its encoded protein kinase ATM plays a fundamental role in DNA repair of double strand breaks (DSBs). Impaired function of this kinase leads to a multisystemic disorder including immunodeficiency, progressive cerebellar degeneration, radiation sensitivity, dilated blood vessels, premature aging and a predisposition to cancer. Since allogenic hematopoietic stem cell (HSC) transplantation improved disease outcome, gene therapy based on autologous HSCs is an alternative promising concept. However, due to the large cDNA of ATM (9.2 kb), efficient packaging of retroviral particles and sufficient transduction of HSCs remains challenging.We generated lentiviral, gammaretroviral and foamy viral vectors with a GFP.F2A.Atm fusion or a GFP transgene and systematically compared transduction efficiencies. Vector titers dropped with increasing transgene size, but despite their described limited packaging capacity, we were able to produce lentiviral and gammaretroviral particles. The reduction in titers could not be explained by impaired packaging of the viral genomes, but the main differences occurred after transduction. Finally, after transduction of Atm-deficient (ATM-KO) murine fibroblasts with the lentiviral vector expressing Atm, we could show the expression of ATM protein which phosphorylated its downstream substrates (pKap1 and p-p53).

Gene Therapy of Transfusion-Dependent β-Thalassemia Patients with Quick Engraftment of Reinfused Hematopoietic Stem Cells: An Investigator-Initiated Trial of KL003

Introduction In recent years, autologous hematopoietic stem cell (HSC)-based gene therapy has emerged as one of the most promising curative therapies for the treatment of severe hereditary anemia, including β-thalassemia major. Although several clinical trials of gene therapy for β-thalassemia major patients have been completed in developed countries, clinical data of gene therapy remain scarce for patients in developing countries. These patients are often in poor clinical condition because of severe iron overload and insufficient blood transfusion due to limited blood supply. They account for the majority of the β-thalassemia major patients in developing countries. Unlike in developed countries, most β-thalassemia major patients in developing countries cannot survive to adulthood as a consequence of irregular treatments due to the limitation of medical facilities and/or personal financial conditions. Methods We conducted a non-randomized, single-dose, open-label investigator-initiated clinical trial (ChiCTR2200055565) in representative patients in southern China, including those with ferritin above 5000 µg/L. Among the 11 β-thalassemia major patients (aged 6-22) we enrolled, 6 of the patients were in poor clinical conditions (e.g., medium (3000-5000 µg/L ferritin) to severe (&amp;gt;5000 µg/L ferritin) iron overload) due to inconsistent blood transfusions and iron chelation therapy before enrollment (Table). We treated patients using their own HSCs after transduced ex vivo with KL003, a lentiviral vector expressing a functional β-globin gene. We then monitored patients closely for safety and evaluated efficacy of KL003, including stem cell engraftment, hematopoietic reconstitution/recovery, and blood transfusion requirement. Results Blood transfusion was no longer needed for all 11 patients from median day 14 (range: 10-30 days) after treatment. The longest duration of no blood transfusion has been 18 months. The median engraftment time was 14 days for neutrophils (range: 8-15 days) and 14 days (range: 11-33 days) for platelets. This is a significant improvement compared with much longer engraftment time from previous autologous HSC-based gene therapies in the literature, which were around 4 weeks for neutrophils and 6 weeks for platelets. It is worth noting that the platelet engraftment time (33 days) for patient No. 07, who had a severe iron overload, was much longer than others (Table). This may represent a common challenge for treating patients with severe iron overload. The safety profile was consistent with that typical of busulfan-based myeloablation conditioning. There were no severe adverse events associated with KL003. Conclusion Our clinical trial results demonstrate potentially curative results in β-thalassemia major patients, including those in poor clinical condition. In addition, the optimized transduction process of HSCs may have contributed to maintaining stemness of HSCs, which is essential for quick engraftment and often gets lost during ex vivo manipulation. This has led to significantly faster engraftment and better efficacy. Thus, KL003 could be a potential functional cure for β-thalassemia major patients.