RESUMEN
BACKGROUND AND AIMS: Smooth muscle cells (SMCs), interstitial cells of Cajal (ICCs), and platelet-derived growth factor receptor alpha (PDGFRα+) cells (PαCs) form a functional syncytium in the bowel known as the "SIP syncytium." The SIP syncytium works in concert with the enteric nervous system (ENS) to coordinate bowel motility. However, our understanding of individual cell types that form this syncytium and how they interact with each other remains limited, with no prior single-cell RNAseq analyses focused on human SIP syncytium cells. METHODS: We analyzed single-nucleus RNA sequencing data from 10,749 human colon SIP syncytium cells (5572 SMC, 372 ICC, and 4805 PαC nuclei) derived from 15 individuals. RESULTS: Consistent with critical contractile and pacemaker functions and with known enteric nervous system interactions, SIP syncytium cell types express many ion channels, including mechanosensitive channels in ICCs and PαCs. PαCs also prominently express extracellular matrix-associated genes and the inhibitory neurotransmitter receptor for vasoactive intestinal peptide (VIPR2), a novel finding. We identified 2 PαC clusters that differ in the expression of many ion channels and transcriptional regulators. Interestingly, SIP syncytium cells co-express 6 transcription factors (FOS, MEIS1, MEIS2, PBX1, SCMH1, and ZBTB16) that may be part of a combinatorial signature that specifies these cells. Bowel region-specific differences in SIP syncytium gene expression may correlate with regional differences in function, with right (ascending) colon SMCs and PαCs expressing more transcriptional regulators and ion channels than SMCs and PαCs in left (sigmoid) colon. CONCLUSION: These studies provide new insights into SIP syncytium biology that may be valuable for understanding bowel motility disorders and lead to future investigation of highlighted genes and pathways.
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Transcriptional enhancers have been extensively characterized, but cis-regulatory elements involved in acute gene repression have received less attention. Transcription factor GATA1 promotes erythroid differentiation by activating and repressing distinct gene sets. Here, we study the mechanism by which GATA1 silences the proliferative gene Kit during murine erythroid cell maturation and define stages from initial loss of activation to heterochromatinization. We find that GATA1 inactivates a potent upstream enhancer but concomitantly creates a discrete intronic regulatory region marked by H3K27ac, short noncoding RNAs, and de novo chromatin looping. This enhancer-like element forms transiently and serves to delay Kit silencing. The element is ultimately erased via the FOG1/NuRD deacetylase complex, as revealed by the study of a disease-associated GATA1 variant. Hence, regulatory sites can be self-limiting by dynamic co-factor usage. Genome-wide analyses across cell types and species uncover transiently active elements at numerous genes during repression, suggesting that modulation of silencing kinetics is widespread.
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Estudio de Asociación del Genoma Completo , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Ratones , Intrones , Diferenciación Celular , Silenciador del Gen , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2RESUMEN
The fetal-to-adult hemoglobin transition is clinically relevant because reactivation of fetal hemoglobin (HbF) significantly reduces morbidity and mortality associated with sickle cell disease (SCD) and ß-thalassemia. Most studies on the developmental regulation of the globin genes, including genome-wide genetics screens, have focused on DNA binding proteins, including BCL11A and ZBTB7A/LRF and their cofactors. Our understanding of RNA binding proteins (RBPs) in this process is much more limited. Two RBPs, LIN28B and IGF2BP1, are known posttranscriptional regulators of HbF production, but a global view of RBPs is still lacking. Here, we carried out a CRISPR/Cas9-based screen targeting RBPs harboring RNA methyltransferase and/or RNA recognition motif (RRM) domains and identified RNA binding motif 12 (RBM12) as a novel HbF suppressor. Depletion of RBM12 induced HbF expression and attenuated cell sickling in erythroid cells derived from patients with SCD with minimal detrimental effects on cell maturation. Transcriptome and proteome profiling revealed that RBM12 functions independently of major known HbF regulators. Enhanced cross-linking and immunoprecipitation followed by high-throughput sequencing revealed strong preferential binding of RBM12 to 5' untranslated regions of transcripts, narrowing down the mechanism of RBM12 action. Notably, we pinpointed the first of 5 RRM domains as essential, and, in conjunction with a linker domain, sufficient for RBM12-mediated HbF regulation. Our characterization of RBM12 as a negative regulator of HbF points to an additional regulatory layer of the fetal-to-adult hemoglobin switch and broadens the pool of potential therapeutic targets for SCD and ß-thalassemia.
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Anemia de Células Falciformes , Talasemia beta , Adulto , Humanos , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Talasemia beta/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Proteínas de Unión al ARN/genéticaRESUMEN
Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.
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Terapia Genética/métodos , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas , Lentivirus/genética , Síndrome de Wiskott-Aldrich/terapia , Adolescente , Adulto , Niño , Preescolar , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Humanos , Lactante , Resultado del Tratamiento , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/inmunología , Adulto JovenRESUMEN
Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.
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Edición Génica , Inmunodeficiencia Combinada Grave/genética , Animales , Proteína Quinasa Activada por ADN/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones SCID , Proteínas Nucleares/genéticaRESUMEN
Primary immunodeficiencies have played a major role in the development of gene therapy for monogenic diseases of the bone marrow. The last decade has seen convincing evidence of long-term disease correction as a result of ex vivo viral vector-mediated gene transfer into autologous haematopoietic stem cells. The success of these early studies has been balanced by the development of vector-related insertional mutagenic events. More recently the use of alternative vector designs with self-inactivating designs, which have an improved safety profile has led to the initiation of a wave of new studies that are showing early signs of efficacy. The ongoing development of safer vector platforms and gene-correction technologies together with improvements in cell-transduction techniques and optimised conditioning regimes is likely to make gene therapy amenable for a greater number of PIDs. If long-term efficacy and safety are shown, gene therapy will become a standard treatment option for specific forms of PID.
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Terapia Genética , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas , Síndromes de Inmunodeficiencia/terapia , Inmunodeficiencia Combinada Grave/terapia , Animales , Ensayos Clínicos como Asunto , Humanos , Lentivirus/genética , Transducción Genética , Trasplante AutólogoRESUMEN
Patients with X-linked severe combined immunodeficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). However, 5 of 20 patients developed leukemia from activation of cellular proto-oncogenes by viral enhancers in the long-terminal repeats (LTR) of the integrated vector. These events prompted the design of a gRV vector with self-inactivating (SIN) LTRs to enhance vector safety. Herein we report on the production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. Successful clinical production required careful selection of culture medium without pre-added glutamine, reduced exposure of packaging cells to cell-dissociation enzyme, and presence of cations in wash buffer. The clinical vector was high titer; transduced 68-70% normal human CD34(+) cells, as determined by colony-forming unit assays and by xenotransplantation in immunodeficient NOD.CB17-Prkdc(scid)/J (nonobese diabetic/severe combined immunodeficiency (NOD/SCID)) and NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NOD/SCID gamma (NSG))) mice; and resulted in the production of T cells in vitro from human SCID-X1 CD34(+) cells. The vector was certified and released for the treatment of SCID-X1 in a multi-center international phase I/II trial.
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Vectores Genéticos , Subunidad gamma Común de Receptores de Interleucina/genética , Retroviridae/genética , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/terapia , Animales , Técnicas de Transferencia de Gen , Humanos , Virus de la Leucemia del Gibón/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Secuencias Repetidas Terminales , Transducción GenéticaRESUMEN
BACKGROUND: Immunotherapy using autologous dendritic cell (DC) vaccination has not been systematically evaluated in osteosarcoma. We therefore conducted a phase I trial to assess feasibility, safety and tumour-specific immune responses in patients with relapsed disease. PATIENTS AND METHODS: Of 13 recruited patients with relapsed osteosarcoma, 12 received 3 weekly vaccines of autologous DCs matured with autologous tumour lysate and keyhole limpet haemocyanin (KLH), to a maximum of 6 vaccinations. An additional 3 paediatric patients afflicted with other tumour types and with relapsed disease received vaccines generated with identical methodology. Immune responses were assessed using an ELISpot assay for the detection of interferon gamma, whilst interleukin-2 and granzyme B were additionally assessed in cases where interferon-γ responses were induced. RESULTS: In total 61 vaccines, of homogeneous maturation phenotype and viability, were administered with no significant toxicity. Only in 2 out of 12 treated osteosarcoma cases was there an induction of specific T-cell immune response to the tumour, whilst a strong but non-specific immune response was induced in 1 further osteosarcoma patient. Immune response against KLH was induced in only 3 out of 12 osteosarcoma patients. In contrast, three additional non-osteosarcoma patients showed significant T-cell responses to vaccine. CONCLUSION: We have shown the strategy of DC vaccination in relapsed osteosarcoma is safe and feasible. However, significant anti-tumour responses were induced in only 2 out of 12 vaccinated patients with no evidence of clinical benefit. Comparison of results with identically treated control patients suggests that osteosarcoma patients might be relatively insensitive to DC-based vaccine treatments.
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Vacunas contra el Cáncer , Osteosarcoma/inmunología , Osteosarcoma/terapia , Linfocitos T/citología , Adolescente , Adulto , Células Dendríticas/citología , Estudios de Factibilidad , Femenino , Granzimas/biosíntesis , Hemocianinas/química , Humanos , Inmunoterapia/métodos , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Masculino , Osteosarcoma/metabolismo , Fenotipo , Recurrencia , Resultado del TratamientoRESUMEN
The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10-14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 10(7) infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.
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Reactores Biológicos , Gammaretrovirus/genética , Vectores Genéticos/aislamiento & purificación , Vectores Genéticos/normas , Transfección/métodos , Animales , Técnicas de Cultivo Celular por Lotes/métodos , Técnicas de Cultivo Celular por Lotes/normas , Biotecnología , Línea Celular , Gammaretrovirus/aislamiento & purificación , Humanos , Ratones , Control de CalidadRESUMEN
Severe combined immunodeficiency (SCID) patients with an inactivating mutation in recombination activation gene 1 (RAG1) lack B and T cells due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment option for RAG-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy has proven challenging. As a preclinical model for RAG-SCID, we used Rag1-/- mice and lentiviral self-inactivating (SIN) vectors harboring different internal elements to deliver native or codon-optimized human RAG1 sequences. Treatment resulted in the appearance of B and T cells in peripheral blood and developing B and T cells were detected in central lymphoid organs. Serum Ig levels and Ig and TCR Vß gene segment usage was comparable to wild-type (WT) controls, indicating that RAG-mediated rearrangement took place. Remarkably, relatively low frequencies of B cells produced WT levels of serum immunoglobulins. Upon stimulation of the TCR, corrected spleen cells proliferated and produced cytokines. In vivo challenge resulted in production of antigen-specific antibodies. No leukemia development as consequence of insertional mutagenesis was observed. The functional reconstitution of the B- as well as the T-cell compartment provides proof-of-principle for therapeutic RAG1 gene transfer in Rag1-/- mice using lentiviral SIN vectors.
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Terapia Genética , Vectores Genéticos/administración & dosificación , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Lentivirus/genética , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapia , Animales , Linfocitos B/fisiología , Western Blotting , Médula Ósea/metabolismo , Médula Ósea/patología , Trasplante de Médula Ósea , Proliferación Celular , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Reordenamiento Génico , Técnicas de Transferencia de Gen , Humanos , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos T/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Linfocitos T/fisiología , Transgenes/fisiologíaRESUMEN
The efficacy of gene therapy for the treatment of inherited immunodeficiency has been highlighted in recent clinical trials, although in some cases complicated by insertional mutagenesis and silencing of vector genomes through methylation. To minimize these effects, we have evaluated the use of regulatory elements that confer reliability of gene expression, but also lack potent indiscriminate enhancer activity. The Vav1 proximal promoter is particularly attractive in this regard and may be useful in situations where high-level or complex regulation of gene expression is not necessary. X-linked severe combined immunodeficiency (SCID-X1) is a good candidate for such an approach, particularly as there may be additional disease-related intrinsic risks of leukemogenesis, and where safety is therefore a paramount concern. We have tested whether lentiviral vectors expressing the common cytokine receptor gamma chain under the control of the proximal Vav1 gene promoter are effective for correction of signaling defects and the disease phenotype. Despite low-level gene expression, we observed near-complete restoration of cytokine-mediated STAT5 phosphorylation in a model cell line. Furthermore, at low vector copy number, highly effective T- and B-lymphocyte reconstitution was achieved in vivo in a murine model of SCID-X1, in both primary and secondary graft recipients. This vector configuration deserves further evaluation and consideration for future clinical trials.
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Terapia Genética , Subunidad gamma Común de Receptores de Interleucina/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-vav/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Orden Génico , Vectores Genéticos/genética , Células HEK293 , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Interleucina-2/metabolismo , Lentivirus/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Transducción de Señal , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/genética , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/terapiaRESUMEN
BACKGROUND: Genetic polymorphisms that influence neutrophil function were proposed as possible risk factors for aggressive periodontitis (AgP). The aim of this study is to test whether a clinical diagnosis and specific genetic variants are associated with neutrophil activity in subjects with AgP and healthy subjects. METHODS: This study describes the results of neutrophil analyses performed in 40 subjects (20 subjects with AgP and 20 healthy control subjects). Neutrophils were extracted from peripheral blood and analyzed in a masked manner for phagocytosis and oxidative burst by flow cytometry in response to Escherichia coli, Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), and Porphyromonas gingivalis. Genomic DNA was extracted and genotyped for the nicotinamide adenine dinucleotide phosphate p22 phagocytic oxidase subunit (p22(phox)) cytochrome b alpha gene (CYBA) C242T, crystallizable gamma fragment (Fcgamma)IIa H/R, and FcgammaIIIb neutrophil antigen (NA)1/NA2 polymorphisms. RESULTS: Analyses in pairs of white subjects (n = 24) revealed that patients with AgP exhibited a higher oxidative burst in response to E. coli (P = 0.002) and P. gingivalis (P = 0.002) compared to healthy control subjects. The p22(phox) CYBA 242 T allele was associated with an oxidative burst in response to the challenge by two strains of A. actinomycetemcomitans (P = 0.018 and P = 0.046). The FcgammaIIa polymorphism was associated with the phagocytic index of E. coli (P = 0.024). CONCLUSION: This study confirms previous reports of a higher oxidative burst associated with AgP and presented preliminary evidence that genetic factors may influence neutrophil function in patients with AgP and healthy individuals.
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Periodontitis Agresiva/genética , Periodontitis Agresiva/inmunología , Neutrófilos/inmunología , Estallido Respiratorio/genética , Adulto , Aggregatibacter actinomycetemcomitans/fisiología , Periodontitis Agresiva/sangre , Periodontitis Agresiva/microbiología , Alelos , Análisis de Varianza , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Escherichia coli/fisiología , Femenino , Proteínas Ligadas a GPI , Predisposición Genética a la Enfermedad , Humanos , Isoantígenos/genética , Masculino , NADPH Oxidasas/genética , Neutrófilos/fisiología , Fagocitosis/genética , Polimorfismo de Nucleótido Simple , Porphyromonas gingivalis/fisiología , Receptores de IgG/genéticaRESUMEN
The clinical application of self-inactivating (SIN) retroviral vectors has been hampered by the lack of reliable and efficient vector production technologies. To enable production of SIN gamma-retroviral vectors from stable producer clones, a new PG13-based packaging cell, known as PG368, was developed. Viral vector expression constructs can be reliably inserted at a predefined genomic locus of PG368 packaging cells by an Flp-recombinase-mediated targeted cassette exchange (RMCE) reaction. A new, carefully designed vector-targeting construct, pEMTAR-1, eliminated the co-packaging of the selectable marker gene used for the identification of successful recombination at the predefined genomic locus and thus, improved the safety of the production system. Selected clones produced vector supernatants at consistent titers. The targeted insertion of therapeutically relevant SIN vectors for chronic granulomatous disease and X-linked severe combined immunodeficiency into PG368 cells results in stable titers within the range necessary for clinical application. The production of retroviral SIN vectors from stable clinical-grade producer cells is feasible and will contribute to the safe production and application of SIN gamma-retroviral vectors for clinical trials.
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ADN Nucleotidiltransferasas , Técnicas de Transferencia de Gen , Vectores Genéticos , Retroviridae/genética , Línea Celular , Estudios de Factibilidad , Marcación de Gen , Terapia Genética/métodos , Enfermedad Granulomatosa Crónica/terapia , Humanos , Inmunodeficiencia Combinada Grave/terapiaRESUMEN
Haematopoietic stem cell transplantation (HSCT) is now widely used to treat primary immunodeficiencies (PID). For patients with specific disorders (severe combined immunodeficiency (SCID)-X1, adenosine deaminase deficiency (ADA)-SCID, X-chronic granulomatous disease (CGD) and Wiskott-Aldrich Syndrome (WAS)) who lack a suitable human leukocyte antigen (HLA)-matched donor, gene therapy has offered an important alternative treatment option. The success of gene therapy can be attributed, in part, to the selective advantage offered to gene-corrected cells, the avoidance of graft-versus-host disease and to the use of pre-conditioning in patients with chemotherapy to facilitate engraftment of corrected cells. Adverse events have been encountered and this has led to detailed characterization of retroviral vector integration profiles. A new generation of self-inactivating retroviral and lentiviral vectors have been designed to address these safety concerns, and are at an advanced stage of preparation for the next phase of clinical testing.
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Terapia Genética/métodos , Síndromes de Inmunodeficiencia/terapia , Animales , Silenciador del Gen , Terapia Genética/tendencias , Humanos , Masculino , Mutagénesis Insercional , Inmunodeficiencia Combinada Grave/terapia , Síndrome de Wiskott-Aldrich/terapiaRESUMEN
The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is widely used in retroviral gene transfer vectors. However, this element contains an open-reading frame (ORF) encoding a truncated peptide of the woodchuck hepatitis virus X protein (WHX). Because we are developing a lentiviral vector for the gene therapy of Wiskott-Aldrich syndrome (WAS), we evaluated whether the WPRE was needed in the gene transfer cassette and tested the possibility of replacing it with a mutated derivative. The transcriptional activity of the WPRE was undetectable in the context of the lentiviral vector but the element was capable of translating a polypeptide. This capability was abrogated by mutating the WHX ORF translation start. The WPRE was required to express high levels of the transgene and for that, the native form or mutated derivatives functioned equivalently. The vector using a WAS gene promoter and the mut6 WPRE induced long-term expression of the WAS transgene in vivo, correcting cytoskeletal defects, thymocyte and B-cell numbers and improved the colitis of WAS-null mice. By providing additional evidence of efficacy of this WAS lentiviral vector with improved safety features, our results validate a mutated WPRE, which should be useful in future gene therapy applications.
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Terapia Genética/métodos , Vectores Genéticos/genética , Virus de la Hepatitis B de la Marmota/genética , Transactivadores/genética , Síndrome de Wiskott-Aldrich/terapia , Animales , Línea Celular , Colitis/patología , Colitis/terapia , Regulación de la Expresión Génica , Humanos , Lentivirus/genética , Ratones , Mutación , Sistemas de Lectura Abierta/genética , Plásmidos/genética , Transactivadores/biosíntesis , Transducción Genética , Transgenes , Síndrome de Wiskott-Aldrich/patologíaRESUMEN
Non-integrating lentiviral vectors show considerable promise for gene therapy applications as they persist as long-term episomes in non-dividing cells and diminish risks of insertional mutagenesis. In this study, non-integrating lentiviral vectors were evaluated for their use in the adult and fetal central nervous system of rodents. Vectors differentially pseudotyped with vesicular stomatitis virus, rabies and baculoviral envelope proteins allowed targeting of varied cell populations. Efficient gene delivery to discrete areas of the brain and spinal cord was observed following stereotactic administration. Furthermore, after direct in utero administration (E14), sustained and strong expression was observed 4 months into adulthood. Quantification of transduction and viral copy number was comparable when using non-integrating lentivirus and conventional integrating vector. These data support the use of non-integrating lentiviral vectors as an effective alternative to their integrating counterparts in gene therapy applications, and highlight their potential for treatment of inherited and acquired neurological disorders.
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Encéfalo/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Lentivirus/genética , Animales , Cuerpo Estriado/metabolismo , Terapias Fetales/métodos , Terapia Genética/métodos , Lentivirus/fisiología , Ratones , Ratas , Médula Espinal/metabolismo , Técnicas Estereotáxicas , Transducción Genética , Integración ViralRESUMEN
Gene therapy has proven to be of potential value for the correction of inherited hematopoietic disorders. However, the occurrence of severe side effects in some of the clinical trials has questioned the safety of this approach and has hampered the use of long terminal repeat-driven vectors for the treatment of a large number of patients. The development of self-inactivating (SIN) vectors with reduced genotoxicity provides an alternative to the currently used vectors. Our initial attempts to use SIN vectors for the correction of a myeloid disorder, chronic granulomatous disease, failed due to low vector titers and poor transgene expression. The optimization of the transgene cDNA (gp91(phox)) resulted in substantially increased titers and transgene expression. Most notably, transgene optimization significantly improved expression of a second cistron located downstream of gp91(phox). Thus, optimization of the transgene sequence results in higher expression levels and increased therapeutic index allowing the use of low vector copy numbers per transduced cell and weaker internal promoters.
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Terapia Genética/métodos , Enfermedad Granulomatosa Crónica/terapia , Células Madre Hematopoyéticas/metabolismo , Glicoproteínas de Membrana/genética , NADPH Oxidasas/genética , Animales , Línea Celular Tumoral , Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Enfermedad Granulomatosa Crónica/metabolismo , Células Madre Hematopoyéticas/virología , Humanos , Separación Inmunomagnética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Superóxidos/análisis , Transducción Genética/métodos , Transgenes , Inactivación de VirusRESUMEN
Podosomes are specialized adhesion sites found in rapidly migrating and invasive cells, most notably in cells from the myeloid lineage that participate in immune surveillance and phagocyte defence mechanisms. In this review, we describe the nature of leukocyte podosomes and the regulation of their turnover during migration by the key regulatory molecules Wiskott-Aldrich syndrome protein and WASP-interacting protein.
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Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucocitos/fisiología , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Leucocitos/diagnóstico por imagen , Modelos Biológicos , UltrasonografíaRESUMEN
Gene transfer for cystic fibrosis (CF) airway disease has been hampered by the lung's innate refractivity to pathogen infection. We hypothesized that early intervention with an integrating gene transfer vector capable of transducing the lung via the lumen may be a successful therapeutic approach. An HIV-based lentiviral vector pseudotyped with the baculovirus gp64 envelope was applied to the fetal, neonatal or adult airways. Fetal intra-amniotic administration resulted in transduction of approximately 14% of airway epithelial cells, including both ciliated and non-ciliated epithelia of the upper, mid and lower airways; there was negligible alveolar or nasal transduction. Following neonatal intra-nasal administration we observed significant transduction of the airway epithelium (approximately 11%), although mainly in the distal lung, and substantial alveolar transduction. This expression was still detectable at 1 year after application. In the adult, the majority of transduction was restricted to the alveoli. In contrast, vesicular stomatitis virus glycoprotein pseudotyped virus transduced only alveoli after adult and neonatal application and no transduction was observed after fetal administration. Repeat administration did not increase transduction levels of the conducting airway epithelia. These data demonstrate that application at early developmental stages in conjunction with an appropriately pseudotyped virus provides efficient, high-level transgene expression in the murine lung. This may provide a modality for treatment for lung disease in CF.