Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
1.
Haematologica ; 106(1): 74-86, 2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-31949009

RESUMEN

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Osteopetrosis , ATPasas de Translocación de Protón Vacuolares , Antígenos CD34 , Terapia Genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Osteoclastos/metabolismo , Osteopetrosis/genética , Osteopetrosis/terapia , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo
2.
Biochim Biophys Acta Mol Cell Res ; 1865(1): 117-131, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28986220

RESUMEN

The relevance of mitochondrial DNA (mtDNA) mutations in cancer process is still unknown. Since the mutagenesis of mitochondrial genome in mammals is not possible yet, we have exploited budding yeast S. cerevisiae as a model to study the effects of tumor-associated mutations in the mitochondrial MTATP6 gene, encoding subunit 6 of ATP synthase, on the energy metabolism. We previously reported that four mutations in this gene have a limited impact on the production of cellular energy. Here we show that two mutations, Atp6-P163S and Atp6-K90E (human MTATP6-P136S and MTATP6-K64E, found in prostate and thyroid cancer samples, respectively), increase sensitivity of yeast cells both to compounds inducing oxidative stress and to high concentrations of calcium ions in the medium, when Om45p, the component of porin complex in outer mitochondrial membrane (OM), was fused to GFP. In OM45-GFP background, these mutations affect the activation of yeast permeability transition pore (yPTP, also called YMUC, yeast mitochondrial unspecific channel) upon calcium induction. Moreover, we show that calcium addition to isolated mitochondria heavily induced the formation of ATP synthase dimers and oligomers, recently proposed to form the core of PTP, which was slower in the mutants. We show the genetic evidence for involvement of mitochondrial ATP synthase in calcium homeostasis and permeability transition in yeast. This paper is a first to show, although in yeast model organism, that mitochondrial ATP synthase mutations, which accumulate during carcinogenesis process, may be significant for cancer cell escape from apoptosis.


Asunto(s)
Calcio/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/genética , Neoplasias/genética , Especies Reactivas de Oxígeno/metabolismo , Sustitución de Aminoácidos , Apoptosis/genética , Homeostasis/genética , Humanos , Mutación Missense , Neoplasias/metabolismo , Neoplasias/patología , Organismos Modificados Genéticamente , Permeabilidad , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
3.
J Allergy Clin Immunol ; 142(3): 928-941.e8, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29241731

RESUMEN

BACKGROUND: Omenn syndrome (OS) is a rare severe combined immunodeficiency associated with autoimmunity and caused by defects in lymphoid-specific V(D)J recombination. Most patients carry hypomorphic mutations in recombination-activating gene (RAG) 1 or 2. Hematopoietic stem cell transplantation is the standard treatment; however, gene therapy (GT) might represent a valid alternative, especially for patients lacking a matched donor. OBJECTIVE: We sought to determine the efficacy of lentiviral vector (LV)-mediated GT in the murine model of OS (Rag2R229Q/R229Q) in correcting immunodeficiency and autoimmunity. METHODS: Lineage-negative cells from mice with OS were transduced with an LV encoding the human RAG2 gene and injected into irradiated recipients with OS. Control mice underwent transplantation with wild-type or OS-untransduced lineage-negative cells. Immunophenotyping, T-dependent and T-independent antigen challenge, immune spectratyping, autoantibody detection, and detailed tissue immunohistochemical analyses were performed. RESULTS: LV-mediated GT allowed immunologic reconstitution, although it was suboptimal compared with that seen in wild-type bone marrow (BM)-transplanted OS mice in peripheral blood and hematopoietic organs, such as the BM, thymus, and spleen. We observed in vivo variability in the efficacy of GT correlating with the levels of transduction achieved. Immunoglobulin levels and T-cell repertoire normalized, and gene-corrected mice responded properly to challenges in vivo. Autoimmune manifestations, such as skin infiltration and autoantibodies, dramatically improved in GT mice with a vector copy number/genome higher than 1 in the BM and 2 in the thymus. CONCLUSIONS: Our data show that LV-mediated GT for patients with OS significantly ameliorates the immunodeficiency, even in an inflammatory environment.


Asunto(s)
Proteínas de Unión al ADN/genética , Terapia Genética , Lentivirus/genética , Inmunodeficiencia Combinada Grave/terapia , Animales , Autoinmunidad , Linfocitos B/inmunología , Modelos Animales de Enfermedad , Femenino , Inflamación/inmunología , Inflamación/terapia , Recuento de Linfocitos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Inmunodeficiencia Combinada Grave/inmunología , Linfocitos T/inmunología
4.
Front Endocrinol (Lausanne) ; 15: 1450349, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39314524

RESUMEN

Introduction: Autosomal recessive osteopetrosis (ARO) is a rare genetic disease, characterized by increased bone density due to defective osteoclast function. Most of the cases are due to TCIRG1 gene mutation, leading to severe bone phenotype and death in the first years of life. The standard therapy is the hematopoietic stem cell transplantation (HSCT), but its success is limited by several constraints. Conversely, gene therapy (GT) could minimize the immune-mediated complications of allogeneic HSCT and offer a prompt treatment to these patients. Methods: The Tcirg1-defective oc/oc mouse model displays a short lifespan and high bone density, closely mirroring the human condition. In this work, we exploited the oc/oc neonate mice to optimize the critical steps for a successful therapy. Results: First, we showed that lentiviral vector GT can revert the osteopetrotic bone phenotype, allowing long-term survival and reducing extramedullary haematopoiesis. Then, we demonstrated that plerixafor-induced mobilization can further increase the high number of HSPCs circulating in peripheral blood, facilitating the collection of adequate numbers of cells for therapeutic purposes. Finally, pre-transplant non-genotoxic conditioning allowed the stable engraftment of HSPCs, albeit at lower level than conventional total body irradiation, and led to long-term survival and correction of bone phenotype, in the absence of acute toxicity. Conclusion: These results will pave the way to the implementation of an effective GT protocol, reducing the transplant-related complication risks in the very young and severely affected ARO patients.

5.
Front Immunol ; 14: 1268620, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38022635

RESUMEN

Introduction: Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. Methods: In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter. Results and discussion: Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients.


Asunto(s)
Síndromes de Inmunodeficiencia , Inmunodeficiencia Combinada Grave , Humanos , Ratones , Animales , Proteínas de Homeodominio/genética , Síndromes de Inmunodeficiencia/terapia , Linfocitos B , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapia , Terapia Genética , Inmunoproteínas , Mutación
6.
Dis Model Mech ; 14(5)2021 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-33970241

RESUMEN

Autosomal recessive osteopetrosis (ARO) is a severe inherited bone disease characterized by defective osteoclast resorption or differentiation. Clinical manifestations include dense and brittle bones, anemia and progressive nerve compression, which hamper the quality of patients' lives and cause death in the first 10 years of age. This Review describes the pathogenesis of ARO and highlights the strengths and weaknesses of the current standard of care, namely hematopoietic stem cell transplantation (HSCT). Despite an improvement in the overall survival and outcomes of HSCT, transplant-related morbidity and the pre-existence of neurological symptoms significantly limit the success of HSCT, while the availability of human leukocyte antigen (HLA)-matched donors still remains an open issue. Novel therapeutic approaches are needed for ARO patients, especially for those that cannot benefit from HSCT. Here, we review preclinical and proof-of-concept studies, such as gene therapy, systematic administration of deficient protein, in utero HSCT and gene editing.


Asunto(s)
Osteopetrosis/genética , Osteopetrosis/terapia , Consenso , Regulación de la Expresión Génica , Genes Recesivos , Humanos , Osteoclastos/metabolismo , Osteoclastos/patología , Osteopetrosis/patología , Guías de Práctica Clínica como Asunto
7.
Artículo en Inglés | MEDLINE | ID: mdl-30837952

RESUMEN

Osteopetrosis is a condition characterized by increased bone mass due to defects in osteoclast function or formation. In the last decades, the molecular dissection of osteopetrosis has unveiled a plethora of molecular players responsible for different forms of the disease, some of which present also primary neurodegeneration that severely limits the therapy. Hematopoietic stem cell transplantation can cure the majority of them when performed in the first months of life, highlighting the relevance of an early molecular diagnosis. However, clinical management of these patients is constrained by the severity of the disease and lack of a bone marrow niche that may delay immune reconstitution. Based on osteopetrosis genetic heterogeneity and disease severity, personalized therapies are required for patients that are not candidate to bone marrow transplantation. This review briefly describes the genetics of osteopetrosis, its clinical heterogeneity, current therapy and innovative approaches undergoing preclinical evaluation.

SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA