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X-linked chronic granulomatous disease (CGD) is associated with defective phagocytosis, life-threatening infections, and inflammatory complications. We performed a clinical trial of lentivirus-based gene therapy in four patients (NCT02757911). Two patients show stable engraftment and clinical benefits, whereas the other two have progressively lost gene-corrected cells. Single-cell transcriptomic analysis reveals a significantly lower frequency of hematopoietic stem cells (HSCs) in CGD patients, especially in the two patients with defective engraftment. These two present a profound change in HSC status, a high interferon score, and elevated myeloid progenitor frequency. We use elastic-net logistic regression to identify a set of 51 interferon genes and transcription factors that predict the failure of HSC engraftment. In one patient, an aberrant HSC state with elevated CEBPß expression drives HSC exhaustion, as demonstrated by low repopulation in a xenotransplantation model. Targeted treatments to protect HSCs, coupled to targeted gene expression screening, might improve clinical outcomes in CGD.
Assuntos
Doença Granulomatosa Crônica , Transplante de Células-Tronco Hematopoéticas , Humanos , Terapia Genética/efeitos adversos , Doença Granulomatosa Crônica/diagnóstico , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/terapia , Células-Tronco Hematopoéticas/metabolismo , Inflamação/metabolismo , Interferons/metabolismoRESUMO
BACKGROUND: Fanconi anemia (FA) is an inherited disorder characterized clinically by congenital abnormalities, progressive bone marrow failure (BMF), and a predisposition to malignancy. Gene therapy (GT) of FA, via the infusion of gene-corrected peripheral blood (PB) autologous hematopoietic stem cells (HSCs), may constitute a cure for BMF. GT bypasses the donor restrictions and adverse events associated with allogenic HSC transplantation. However, adequate harvesting of PB-HSCs is a crucial determinant of successful engraftment in gene therapy. Harvesting the low numbers of HSCs in patients with FA is particularly challenging. STUDY DESIGN AND METHODS: This open-label phase I/II trial evaluates the feasibility and safety of co-administration of G-CSF and plerixafor in patients with FA for the mobilization and harvesting of peripheral HSCs, intending to use them in a gene therapy trial. Patients with mutations in the FANCA gene received two subcutaneous injections of G-CSF (6 µg/kg × 2/d from D1 to D8. Plerixafor (0.24 mg/kg/d) was administered 2 h before apheresis (from D5 onward). RESULTS: CD34+ cells were mobilized for four patients quickly but transiently after the plerixafor injection. One patient had a CD34+ cell count of over 100/µl; the mobilization peaked 2 h after the injection and lasted for more than 9 h. There were no short-term adverse events associated with the mobilization or harvesting procedures. CONCLUSION: Our data in patients with FA show that the mobilization of HSCs with G-CSF and plerixafor is safe and more efficient in younger individuals without BMF.
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Anemia de Fanconi , Transplante de Células-Tronco Hematopoéticas , Compostos Heterocíclicos , Antígenos CD34/metabolismo , Anemia de Fanconi/induzido quimicamente , Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Terapia Genética/métodos , Fator Estimulador de Colônias de Granulócitos , Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , HumanosRESUMO
BACKGROUND: The immunogenicity of a two-dose mRNA COVID-19 vaccine regimen is low in kidney transplant (KT) recipients. Here, we provide a thorough assessment of the immunogenicity of a three-dose COVID-19 vaccine regimen in this population. METHODS: We performed a prospective longitudinal study in sixty-one KT recipients given three doses of the BNT162b2 COVID-19 vaccine. We performed semi-structured pharmacovigilance interviews and monitored donor-specific antibodies and kidney function. We compared levels of anti-spike IgG, pseudo-neutralization activity against vaccine homologous and heterologous variants, frequency of spike-specific interferon (IFN)-γ-secreting cells, and antigen-induced cytokine production 28 days after the second and third doses. FINDINGS: Reactions to vaccine were mild. One patient developed donor-specific anti-HLA antibodies after the second dose which could be explained by non-adherence to immunosuppressive therapy. Spike-specific IgG seroconversion raised from 44·3% (n=27) after the second dose to 62·3% (n=38) after the third dose (p<0·05). The mean level of spike-specific IgG increased from 1620 (SD, 3460) to 8772 (SD, 16733) AU/ml (p<0·0001). Serum neutralizing activity increased after the third dose for all variants of concern tested including the Delta variant (p<0·0001). The frequency of spike-specific IFN-γ-secreting cells increased from 19·9 (SD, 56·0) to 64·0 (SD, 76·8) cells/million PBMCs after the third dose (p<0·0001). A significant increase in IFN-γ responses was also observed in patients who remained seronegative after three doses (p<0·0001). INTERPRETATION: A third dose of the BNT162b2 vaccine increases both cross-variant neutralizing antibody and cellular responses in KT recipients with an acceptable tolerability profile. FUNDING: Nice University Hospital, University Cote d'Azur.
Assuntos
Anticorpos Neutralizantes/imunologia , Vacina BNT162/imunologia , COVID-19/imunologia , Transplante de Rim , Idoso , Anticorpos Neutralizantes/sangue , Autoanticorpos/sangue , Vacina BNT162/administração & dosagem , Vacina BNT162/efeitos adversos , COVID-19/prevenção & controle , COVID-19/virologia , Feminino , Rejeição de Enxerto/prevenção & controle , Antígenos HLA/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunossupressores/uso terapêutico , Interferon gama/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fatores de Risco , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Severe combined immunodeficiencies (SCIDs) correspond to the most severe form of primary immunodeficiency. Allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy are curative treatments, depending on the donor's availability and molecular diagnostics. A partially human leukocyte antigen (HLA)-compatible donor used has been developed for this specific HSCT indication in the absence of a matched donor. However, the CD34+ selected process induces prolonged post-transplant T-cell immunodeficiency. The aim here was to investigate a modeling approach to predict the time course and the extent of CD4+ T-cell immune reconstitution after CD34+ selected transplantation. We performed a Bayesian approach based on the age-related changes in thymic output and the cell proliferation/loss model. For that purpose, we defined specific individual covariates from the data collected from 10 years of clinical practice and then evaluated the model's predicted performances and accuracy. We have shown that this Bayesian modeling approach predicted the time course and extent of CD4+ T-cell immune reconstitution after SCID transplantation.
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BACKGROUND: Autologous and allogeneic hematopoietic stem cell transplantation of cytokine-mobilized peripheral blood stem cells (PBSCs) is increasingly used to treat patients with hematologic disorders. Different types of vascular access have been exploited for the apheresis procedure, including peripheral veins (PV) and central venous catheter (CVC). In some cases, PV access is unavailable. There are few published data on the efficiency and quality of harvesting with different types of vascular access. This study brings out complications and morbidity of this procedure linked to these different access. METHODS: We performed a comparative, retrospective, single-center study of hematopoietic stem cell collection using these two types of vascular access. We compared the efficiency and complication rate for 617 adults apheresis sessions in 401 patients and healthy donors, for PBSC collection via PV or CVC between 2010 and 2016. The quality of the HSC product was evaluated in terms of the total CD34 + count and neutrophil contamination. RESULTS: The PV and CVC groups did not differ significantly in terms of the quality of the apheresis product, mean ± SD CD34 + cells collected in PV group was 383.1 ± 402.7 × 10e6 and 298.8 ± 372.7 × 10e6 and the level of neutrophil contamination was 21.0 ± 17.8% in the PV group and 20.6 ± 18.4% in the CVC group. The complication rate did not differ between the two groups. CONCLUSION: The type of vascular access for apheresis hematopoietic stem cell harvesting must be determined by trained staff. Successful harvesting can be performed via PV then CVC is not needed or not available.
Assuntos
Remoção de Componentes Sanguíneos/métodos , Cateterismo Venoso Central/métodos , Cateterismo Periférico/métodos , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Transplante de Células-Tronco de Sangue Periférico/métodos , Adolescente , Adulto , Idoso , Cateterismo Venoso Central/efeitos adversos , Cateterismo Periférico/efeitos adversos , Feminino , Mobilização de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
Although studies of mixed chimerism following hematopoietic stem cell transplantation in patients with sickle cell disease (SCD) may provide insights into the engraftment needed to correct the disease and into immunological reconstitution, an extensive multilineage analysis is lacking. We analyzed chimerism simultaneously in peripheral erythroid and granulomonocytic precursors/progenitors, highly purified B and T lymphocytes, monocytes, granulocytes and red blood cells (RBC). Thirty-four patients with mixed chimerism and ≥12 months of follow-up were included. A selective advantage of donor RBC and their progenitors/precursors led to full chimerism in mature RBC (despite partial engraftment of other lineages), and resulted in the clinical control of the disease. Six patients with donor chimerism <50% had hemolysis (reticulocytosis) and higher HbS than their donor. Four of them had donor chimerism <30%, including a patient with AA donor (hemoglobin >10 g/dL) and three with AS donors (hemoglobin <10 g/dL). However, only one vaso-occlusive crisis occurred with 68.7% HbS. Except in the patients with the lowest chimerism, the donor engraftment was lower for T cells than for the other lineages. In a context of mixed chimerism after hematopoietic stem cell transplantation for SCD, myeloid (rather than T cell) engraftment was the key efficacy criterion. Results show that myeloid chimerism as low as 30% was sufficient to prevent a vaso-occlusive crisis in transplants from an AA donor but not constantly from an AS donor. However, the correction of hemolysis requires higher donor chimerism levels (i.e ≥50%) in both AA and AS recipients. In the future, this group of patients may need a different therapeutic approach.
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Anemia Falciforme , Transplante de Células-Tronco Hematopoéticas , Anemia Falciforme/terapia , Quimerismo , Terapia Genética , Hematopoese , Humanos , Quimeras de Transplante , Transplante HomólogoRESUMO
Prolonged T-cell immunodeficiency following HLA- incompatible hematopoietic stem cell transplantation (HSCT) represents a major obstacle hampering the more widespread use of this approach. Strategies to fasten T-cell reconstitution in this setting are highly warranted as opportunistic infections and an increased risk of relapse account for high rates of morbidity and mortality especially during early month following this type of HSCT. We have implemented a feeder free cell system based on the use of the notch ligand DL4 and cytokines allowing for the in vitro differentiation of human T-Lymphoid Progenitor cells (HTLPs) from various sources of CD34+ hematopoietic stem and precursor cells (HSPCs). Co- transplantion of human T-lymphoid progenitors (HTLPs) and non- manipulated HSPCs into immunodeficient mice successfully accelerated the reconstitution of a polyclonal T-cell repertoire. This review summarizes preclinical data on the use of T-cell progenitors for treatment of post- transplantation immunodeficiency and gives insights into the development of GMP based protocols for potential clinical applications including gene therapy approaches. Future clinical trials implementing this protocol will aim at the acceleration of immune reconstitution in different clinical settings such as SCID and leukemia patients undergoing allogeneic transplantation. Apart from pure cell-therapy approaches, the combination of DL-4 culture with gene transduction protocols will open new perspectives in terms of gene therapy applications for primary immunodeficiencies.
Assuntos
Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Reconstituição Imune/imunologia , Linfócitos T/imunologia , Condicionamento Pré-Transplante/métodos , Animais , Humanos , CamundongosRESUMO
Although the risk of developing lymphoma has decreased in the highly active antiretroviral therapy era, this cancer remains the major cause of mortality in HIV-infected patients. Autologous hematopoietic stem cell transplantation (ASCT) outcome does not differ for HIV-infected versus HIV-uninfected patients. We propose to develop a new treatment for HIV-associated high-risk lymphoma based on autologous transplantation of two genetically modified products: CD4+ T lymphocytes and CD34+ hematopoietic stem cells (HSPCs). The cells will be transduced ex vivo with the Cal-1 lentiviral vector encoding for both a short hairpin RNA (shRNA) against CCR5 (sh5) and the HIV-1 fusion inhibitor C46. The transduced cells will be resistant to HIV infection by two complementary mechanisms: impaired binding of the virus to the cellular CCR5 co-receptor and decreased fusion of the virus as C46 interacts with gp41 and inhibits HIV infection. This phase I/II pilot study, also entitled GENHIV, will involve two French participating centers: Saint Louis Hospital and Necker Hospital in Paris. We plan to enroll five HIV-1-infected patients presenting with high-risk lymphoma and require a treatment with ASCT. The primary objective of this study is to evaluate the safety, feasibility, and success of engraftment of Cal-1 gene-transduced CD4+ T lymphocytes and CD34+ HSPCs.
RESUMO
Sickle cell disease is characterized by chronic anemia and vaso-occlusive crises, which eventually lead to multi-organ damage and premature death. Hematopoietic stem cell transplantation is the only curative treatment but it is limited by toxicity and poor availability of HLA-compatible donors. A gene therapy approach based on the autologous transplantation of lentiviral-corrected hematopoietic stem and progenitor cells was shown to be efficacious in one patient. However, alterations of the bone marrow environment and properties of the red blood cells hamper the harvesting and immunoselection of patients' stem cells from bone marrow. The use of Filgrastim to mobilize large numbers of hematopoietic stem and progenitor cells into the circulation has been associated with severe adverse events in sickle cell patients. Thus, broader application of the gene therapy approach requires the development of alternative mobilization methods. We set up a phase I/II clinical trial whose primary objective was to assess the safety of a single injection of Plerixafor in sickle cell patients undergoing red blood cell exchange to decrease the hemoglobin S level to below 30%. The secondary objective was to measure the efficiency of mobilization and isolation of hematopoietic stem and progenitor cells. No adverse events were observed. Large numbers of CD34+ cells were mobilized extremely quickly. Importantly, the mobilized cells contained high numbers of hematopoietic stem cells, expressed high levels of stemness genes, and engrafted very efficiently in immunodeficient mice. Thus, Plerixafor can be safely used to mobilize hematopoietic stem cells in sickle cell patients; this finding opens up new avenues for treatment approaches based on gene addition and genome editing. Clinicaltrials.gov identifier: NCT02212535.
Assuntos
Anemia Falciforme/terapia , Transfusão de Sangue , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Compostos Heterocíclicos/administração & dosagem , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Fármacos Anti-HIV/administração & dosagem , Antígenos CD34/metabolismo , Antidrepanocíticos/administração & dosagem , Benzilaminas , Estudos de Casos e Controles , Células Cultivadas , Estudos de Coortes , Ciclamos , Células-Tronco Hematopoéticas/citologia , Humanos , Hidroxiureia/administração & dosagemRESUMO
After 30 years of hematopoietic stem cell use for various indications, umbilical cord blood is considered as an established source of cells with marrow and postmobilization peripheral blood. The limited number of cells still remains a problematic element restricting their use, especially in adults who require to be grafted with a higher cell number. Improving the quality of harvested cord blood, at least in terms of volume and amount of cells, is essential to decrease the number of discarded units. In this review, we examine several variables related to parturient, pregnancy, labor, delivery, collection, the newborn, umbilical cord, and placenta. We aim to understand the biologic mechanisms that can impact cord blood quality. This knowledge will ultimately allow targeting donors, which could provide a rich graft and improve the efficiency of the collection.