RESUMO
For gene therapy to improve lung function in cystic fibrosis (CF) subjects, repeated administration of the gene transfer agent over the lifetime of patients is likely to be necessary. This requirement limits the utility of adenoviral and adeno-associated viral vectors (both previously evaluated in CF gene therapy trials) because of induced adaptive immune responses that render repeated dosing ineffective. For CF gene therapy trials, non-viral vectors are currently the only viable option. We previously showed that the cationic lipid formulation GL67A is the most efficient of several non-viral vectors analysed for airway gene transfer. Here, we assessed the efficacy and safety of administering 12 inhaled doses of GL67A complexed with pGM169, a CpG-free plasmid encoding human CFTR complementary DNA, into mice. We show that repeated administration of pGM169/GL67A to murine lungs is feasible, safe and achieves reproducible, dose-related and persistent gene expression (>140 days after each dose) using an aerosol generated by a clinically relevant nebuliser. This study supports progression into the first non-viral multidose lung trial in CF patients.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Terapia Genética , Vetores Genéticos , Lipídeos/administração & dosagem , Lipídeos/toxicidade , Pulmão/efeitos dos fármacos , Plasmídeos , Administração por Inalação , Animais , Terapia Combinada , Fibrose Cística/patologia , Fibrose Cística/terapia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos TestesRESUMO
BACKGROUND: CF is traditionally assessed in clinic. It is unclear if home monitoring of young people with CF is feasible or acceptable. The COVID-19 pandemic has made home monitoring more of a necessity. We report the results of CLIMB-CF, exploring home monitoring's feasibility and potential obstacles. METHODS: We designed a mobile app and enrolled participants with CF aged 2-17 years and their parents for six months. They were asked to complete a variety of measures either daily or twice a week. During the study, participants and their parents completed questionnaires exploring depression, anxiety and quality of life. At the end of the study parents and participants completed acceptability questionnaires. RESULTS: 148 participants were recruited, 4 withdrew prior to starting the study. 82 participants were female with median (IQR) age 7.9 (5.2-12 years). Median data completeness was 40.1% (13.6-69.9%) for the whole cohort; when assessed by age participants aged ≥ 12 years contributed significantly less (15.6% [9.8-30%]). Data completeness decreased over time. There was no significant difference between parental depression and anxiety scores at the start and the end of the study nor in CFQ-R respiratory domain scores for participants ≥ 14 years. The majority of participants did not feel the introduction of home monitoring impacted their daily lives. CONCLUSIONS: Most participants felt home monitoring did not negatively impact their lives and it did not increase depression, anxiety or decrease quality of life. However, uptake was variable, and not well sustained. The teenage years pose a particular challenge and further work is required.
Assuntos
Fibrose Cística/terapia , Aplicativos Móveis , Monitorização Fisiológica/métodos , Monitorização Fisiológica/psicologia , Qualidade de Vida , Adolescente , Ansiedade , COVID-19/epidemiologia , Criança , Pré-Escolar , Depressão , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pandemias , SARS-CoV-2RESUMO
We have previously shown that recombinant Sendai virus (SeV) vector, derived from murine parainfluenza virus, is one of the most efficient vectors for airway gene transfer. We have also shown that SeV-mediated transfection on second administration, although reduced by 60% when compared with levels achieved after a single dose, is still high because of the efficient transfection achieved by SeV vector in murine airways. Here, we show that these levels further decrease on subsequent doses. In addition, we validated SeV vector repeat administration in a non-natural host model, the sheep. As part of these studies we first assessed viral stability in a Pari LC Plus nebuliser, a polyethylene catheter (PEC) and the Trudell AeroProbe. We also compared the distribution of gene expression after PEC and Trudell AeroProbe administration and quantified virus shedding after sheep transduction. In addition, we show that bronchial brushings and biopsies, collected in anaesthetized sheep, can be used to assess SeV-mediated gene expression over time. Similar to mice, gene expression in sheep was transient and had returned to baseline values by day 14. In conclusion, the SeV vector should be strongly considered for lung-related applications requiring a single administration of the vector even though it might not be suitable for diseases requiring repeat administration.
Assuntos
Expressão Gênica , Vetores Genéticos , Vírus Sendai/genética , Ovinos/genética , Transdução Genética , Animais , Catéteres , Feminino , Técnicas de Transferência de Genes/instrumentação , Pulmão , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , RetratamentoRESUMO
We use both large and small animal models in our pre-clinical evaluation of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy. Here, we report the use of a large animal model to assess three non-viral GTAs: 25 kDa-branched polyethyleneimine (PEI), the cationic liposome (GL67A) and compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. GTAs complexed with plasmids expressing human cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA were administered to the sheep lung (n=8 per group) by aerosol. All GTAs gave evidence of gene transfer and expression 1 day after treatment. Vector-derived mRNA was expressed in lung tissues, including epithelial cell-enriched bronchial brushing samples, with median group values reaching 1-10% of endogenous CFTR mRNA levels. GL67A gave the highest levels of expression. Human CFTR protein was detected in small airway epithelial cells in some animals treated with GL67A (two out of eight) and PEI (one out of eight). Bronchoalveolar lavage neutrophilia, lung histology and elevated serum haptoglobin levels indicated that gene delivery was associated with mild local and systemic inflammation. Our conclusion was that GL67A was the best non-viral GTA currently available for aerosol delivery to the sheep lung, led to the selection of GL67A as our lead GTA for clinical trials in CF patients.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Lipossomos/administração & dosagem , Nanopartículas/administração & dosagem , Polietilenoimina/administração & dosagem , Administração por Inalação , Animais , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , DNA Complementar/administração & dosagem , DNA Complementar/genética , Humanos , Polietilenoglicóis , RNA Mensageiro/metabolismo , OvinosRESUMO
Significant survival heterogeneity exists in cystic fibrosis. Our aim was to determine whether residual function of the cystic fibrosis transmembrane conductance regulator (CFTR) is present in long-term survivors with severe mutations. Nasal potential difference (PD) and sweat chloride were measured in 34 long-term survivors (aged ≥ 40 yrs) and compared with young patients (18-23 yrs) with severe (n = 30) and mild (n = 31) lung disease. Baseline PD was not significantly different across the three groups (long-term survivors, -42.8 (range -71.0- -20.5) mV; young/mild, -40.5 (-58.8- -19.5) mV; young/severe,-46.3 (-74.0- -20.0) mV). Response to amiloride (ΔAmil) was significantly different across the three groups (p = 0.01); long-term survivors had values (27.8 (range 8.5-46) mV) which were not different to either young group, but the young/severe group had significantly higher values (29.5 (11-47) mV) than those in the young/mild group (22.0 (7-39) mV; p<0.01). Baseline PD and ΔAmil were associated with forced expiratory volume in 1 s (FEV1) (co-efficient (95% CI) -0.13 (-0.23- -0.03); p = 0.009 and -0.12 (-0.20- -0.04); p = 0.003, respectively). Sweat chloride was lowest (p <0.05) in the young/severe group (93.5 (74-111) mmol·L⻹ versus 98.8 (76.5-116.0) mmol·L⻹; long-term survivors; and 99.5 (80.0-113.5) mmol·L⻹; young/mild). Δ Amil is associated with FEV1 but our findings indicate that long-term survival cannot be explained by residual CFTR function when measurements are taken in later life.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Fibrose Cística/mortalidade , Adolescente , Adulto , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Humanos , Masculino , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Mutação , Mucosa Nasal/fisiopatologia , Estudos Prospectivos , Testes de Função Respiratória , Índice de Gravidade de Doença , Sobreviventes , Suor/química , Suor/fisiologia , Adulto JovemRESUMO
BACKGROUND: Mannose-binding lectin (MBL) deficiency has been associated with infections of the respiratory tract and with increased disease severity in cystic fibrosis (CF). The mechanism is uncertain, and could relate either to systemic or local effects. The aim of this study was to determine, in a large cohort of children, whether MBL is present on the airway surface in health or disease. METHODS: Bronchoalveolar lavage (BAL) fluid from children with and without respiratory infection (some with underlying disease) was analysed for MBL and neutrophil elastase (NE). Levels were compared between groups, and correlations were examined with local and systemic inflammatory markers, infective organisms and load. RESULTS: 85 children were recruited to the study. MBL was absent in the lavage of all 7 children without lung infection but present in 62% (8/13) of those with acute pneumonia/pneumonitis, 23% (5/22) with recurrent respiratory tract infections, 17% (1/6) with primary ciliary dyskinesia and 8% (3/37) with CF (p<0.01). Children with acute pneumonia/pneumonitis had significantly higher levels than those in the other groups. There was no relationship with organisms cultured or systemic markers of inflammation, although in the group with detectable MBL in the BAL fluid, the levels correlated positively with levels of NE. CONCLUSIONS: MBL is undetectable in the non-infected airway but is present in a significant number of samples from children with lung infection. The levels found in the BAL fluid could be physiologically active and the protein may therefore be playing a role in host defence.
Assuntos
Brônquios/química , Broncopatias/metabolismo , Líquido da Lavagem Broncoalveolar/química , Lectina de Ligação a Manose/metabolismo , Infecções Respiratórias/metabolismo , Adolescente , Bactérias/isolamento & purificação , Broncopatias/microbiologia , Líquido da Lavagem Broncoalveolar/microbiologia , Criança , Pré-Escolar , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Haplótipos , Humanos , Lactente , Elastase de Leucócito/metabolismo , Masculino , Inibidores de Proteases/farmacologia , Recidiva , Infecções Respiratórias/microbiologia , Vírus/isolamento & purificaçãoRESUMO
Bacteria-mediated gene transfer ('bactofection') has emerged as an alternative approach for genetic vaccination and gene therapy. Here, we assessed bactofection of airway epithelial cells in vitro and in vivo using an attenuated Escherichia coli genetically engineered to invade non-phagocytic cells. Invasive E. coli expressing green fluorescent protein (GFP) under the control of a prokaryotic promoter was efficiently taken up into the cytoplasm of cystic fibrosis tracheal epithelial (CFTE29o-) cells and led to dose-related reporter gene expression. In vivo experiments showed that following nasal instillation the vast majority of GFP-positive bacteria pooled in the alveoli. Further, bactofection was assessed in vivo. Mice receiving 5 x 10(8) E. coli carrying pCIKLux, in which luciferase (lux) expression is under control of the eukaryotic cytomegalovirus (CMV) promoter, showed a significant increase (P<0.01) in lux activity in lung homogenates compared to untransfected mice. Surprisingly, similar level of lux activity was observed for the non-invasive control strain indicating that the eukaryotic CMV promoter might be active in E. coli. Insertion of prokaryotic transcription termination sequences into pCIKLux significantly reduced prokaryotic expression from the CMV promoter allowing bactofection to be detected in vitro and in vivo. However, bacteria-mediated gene transfer leads to a significantly lower lux expression than cationic lipid GL67-mediated gene transfer. In conclusion, although proof-of-principle for lung bactofection has been demonstrated, levels were low and further modification to the bacterial vector, vector administration and the plasmids will be required.
Assuntos
Células Epiteliais/microbiologia , Escherichia coli/fisiologia , Terapia Genética/métodos , Alvéolos Pulmonares/microbiologia , Animais , Linhagem Celular , Citomegalovirus/genética , Escherichia coli/genética , Infecções por Escherichia coli/transmissão , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Luciferases/genética , Pneumopatias/microbiologia , Camundongos , Camundongos Knockout , Viabilidade Microbiana , Organismos Geneticamente Modificados , Plasmídeos/administração & dosagem , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Lung clearance index (LCI) is a sensitive marker of early lung disease in children but has not been assessed in adults. Measurement is hindered by the complexity of the equipment required. The aims of this study were to assess performance of a novel gas analyser (Innocor) and to use it as a clinical tool for the measurement of LCI in cystic fibrosis (CF). METHODS: LCI was measured in 48 healthy adults, 12 healthy school-age children and 33 adults with CF by performing an inert gas washout from 0.2% sulfur hexafluoride (SF6). SF6 signal:noise ratio and 10-90% rise time of Innocor were compared with a mass spectrometer used in similar studies in children. RESULTS: Compared with the mass spectrometer, Innocor had a superior signal:noise ratio but a slower rise time (150 ms vs 60 ms) which may limit its use in very young children. Mean (SD) LCI in healthy adults was significantly different from that in patients with CF: 6.7 (0.4) vs 13.1 (3.8), p<0.001. Ten of the patients with CF had forced expiratory volume in 1 s > or = 80% predicted but only one had a normal LCI. LCI repeats were reproducible in all three groups of subjects (mean intra-visit coefficient of variation ranged from 3.6% to 5.4%). CONCLUSIONS: Innocor can be adapted to measure LCI and affords a simpler alternative to a mass spectrometer. LCI is raised in adults with CF with normal spirometry, and may prove to be a more sensitive marker of the effects of treatment in this group.
Assuntos
Fibrose Cística/complicações , Testes de Função Respiratória/métodos , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Fibrose Cística/fisiopatologia , Feminino , Volume Expiratório Forçado , Humanos , Pneumopatias/diagnóstico , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Testes de Função Respiratória/normas , Sensibilidade e EspecificidadeRESUMO
Magnetic nanoparticle-based gene transfection has been shown to be effective in combination with both viral vectors and with non-viral agents. In these systems, therapeutic or reporter genes are attached to magnetic nanoparticles which are then focused to the target site/cells via high-field/high-gradient magnets. The technique has been shown to be efficient and rapid for in vitro transfection and compares well with cationic lipid-based reagents, producing good overall transfection levels with lower doses and shorter transfection times. In spite of its potential advantages (particularly for in vivo targeting), the overall transfection levels do not generally exceed those of other non-viral agents. In order to improve the overall transfection levels while maintaining the advantages inherent in this technique, we have developed a novel, oscillating magnet array system which adds lateral motion to the particle/gene complex in order to promote transfection. Experimental results indicate that the system significantly enhances overall in vitro transfection levels in human airway epithelial cells compared to both static field techniques (p<0.005) and the cationic lipids (p<0.001) tested. In addition, it has the previously demonstrated advantages of magnetofection-rapid transfection times and requiring lower levels of DNA than cationic lipid-based transfection agents. This method shows potential for non-viral gene delivery both in vitro and in vivo.
RESUMO
Sendai virus (SeV) vector has been shown to efficiently transduce airway epithelial cells. As a precursor to the potential use of this vector for cystic fibrosis (CF) gene therapy, the correct maturation of the SeV vector-derived CF transmembrane conductance regulator (CFTR) protein was examined using biochemical and functional analyses. We constructed a recombinant SeV vector, based on the fusion (F) gene-deleted non-transmissible SeV vector, carrying the GFP-CFTR gene in which the N terminus of CFTR was fused to green fluorescence protein (GFP). This vector was recovered and propagated to high titers in the packaging cell line. Western blotting using an anti-GFP antibody detected both the fully glycosylated (mature) and the core-glycosylated (immature) proteins, indicating that SeV vector-derived GFP-CFTR was similar to endogenous CFTR. We also confirmed the functional channel activity of GFP-CFTR in an iodide efflux assay. The efficient expression of GFP-CFTR, and its apical surface localization, were observed in both MDCK cells in vitro, and in the nasal epithelium of mice in vivo. We concluded that recombinant SeV vector, a cytoplasmically maintained RNA vector, is able to direct production of a correctly localized, mature form of CFTR, suggesting the value of this vector for studies of CF gene therapy.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vírus Sendai/genética , Transdução Genética/métodos , Animais , Linhagem Celular , Fibrose Cística/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Cavidade Nasal , Perfusão , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/metabolismo , Mucosa Respiratória/metabolismoRESUMO
We have assessed if high-frequency ultrasound (US) can enhance nonviral gene transfer to the mouse lung. Cationic lipid GL67/pDNA, polyethylenimine (PEI)/pDNA and naked plasmid DNA (pDNA) were delivered via intranasal instillation, mixed with Optison microbubbles, and the animals were then exposed to 1 MHz US. Addition of Optison alone significantly reduced the transfection efficiency of all three gene transfer agents. US exposure did not increase GL67/pDNA or PEI/pDNA gene transfer compared to Optison-treated animals. However, it increased naked pDNA transfection efficiency by approximately 15-fold compared to Optison-treated animals, suggesting that despite ultrasound being attenuated by air in the lung, sufficient energy penetrates the tissue to increase gene transfer. US-induced lung haemorrhage, assessed histologically, increased with prolonged US exposure. The left lung was more affected than the right and this was mirrored by a lesser increase in naked pDNA gene transfer, in the left lung. The positive effect of US was dependent on Optison, as in its absence US did not increase naked pDNA transfection efficiency. We have thus established proof of principle that US can increase nonviral gene transfer, in the air-filled murine lung.
Assuntos
Albuminas , DNA/administração & dosagem , Fluorocarbonos , Terapia Genética/métodos , Pulmão/metabolismo , Transfecção/métodos , Ultrassom , Animais , DNA/genética , Expressão Gênica , Luciferases/genética , Pneumopatias/terapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , PolietilenoiminaRESUMO
The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr(G551D) and Cftr(tm1Unc)TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vírus Sendai/genética , Aerossóis , Animais , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Feminino , Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Iodetos/metabolismo , Canais Iônicos/metabolismo , Pulmão , Masculino , Camundongos , Camundongos Knockout , Mutação , Técnicas de Patch-Clamp , Transdução Genética/métodosRESUMO
Our first review on progress and prospects in cystic fibrosis (CF) gene therapy was published in this series in October 2002. We now summarize the progress made since then and comment on the prospects for CF gene therapy over the next couple of years. Three clinical trials have been carried out, further supporting the proof-of-principle that gene transfer to the airway epithelium is feasible. Developments in viral and non-viral vectors, as well as recent alternative strategies such as gene repair, trans-splicing and stem cell therapy will be reviewed.
Assuntos
Fibrose Cística/terapia , Terapia Genética/métodos , Adenoviridae/genética , Ensaios Clínicos como Assunto , Dependovirus/genética , Previsões , Técnicas de Transferência de Genes , Terapia Genética/tendências , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Mucosa Respiratória/metabolismo , Células-Tronco/metabolismo , Células-Tronco/virologiaRESUMO
Topical gene transfer to the airways of cystic fibrosis (CF) patients has been inefficient, partly due to extracellular barriers such as sputum. In an attempt to circumvent these, we assessed whether airway epithelial cells can be transfected by intravenous (i.v.) administration of liposome-complexed or "naked" oligonucleotides (ODNs). The conducting airways are the likely target for CF therapy and are supplied by the bronchial circulation. Consequently, we assessed ODN transfer in the mouse trachea and main bronchi as these are supplied by the bronchial circulation. Liposome-protamine-DNA (LPD) complexes were detected in the bronchial circulation but did not transfect conducting airway epithelial cells, even in the presence of microvascular leakage. In contrast, 'naked' ODNs were delivered to 17% (inter-quartile range (IQR) 10-34%) and 35% (IQR 24-59%) of epithelial cells when injected at 500 microg/animal, without and with microvascular leakage, respectively. Two types of nuclear signal were observed; punctate in cells throughout the airways (3%, IQR 2-6%, and 6%, IQR 4-7%, of cells when delivered without and with microvascular leakage, respectively) and diffuse in a small number of epithelial cells in the proximal trachea. ODNs may be relevant to CF in a variety of ways and these data suggest one way towards implementing their use.
Assuntos
Brônquios/irrigação sanguínea , Terapia Genética/métodos , Oligonucleotídeos/administração & dosagem , Mucosa Respiratória/metabolismo , Transfecção/métodos , Animais , Transporte Biológico , Bradicinina/farmacologia , Núcleo Celular/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/terapia , Citoplasma/metabolismo , Células Epiteliais/metabolismo , Extravasamento de Materiais Terapêuticos e Diagnósticos , Expressão Gênica , Histamina/farmacologia , Injeções Intravenosas , Lipossomos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microcirculação , Microscopia de Fluorescência , NF-kappa B/genética , Fator de Ativação de Plaquetas/farmacologiaRESUMO
We have assessed whether magnetic forces (magnetofection) can enhance non-viral gene transfer to the airways. TransMAG(PEI), a superparamagnetic particle was coupled to Lipofectamine 2000 or cationic lipid 67 (GL67)/plasmid DNA (pDNA) liposome complexes. In vitro transfection with these formulations resulted in approximately 300- and 30-fold increase in reporter gene expression, respectively, after exposure to a magnetic field, but only at suboptimal pDNA concentrations. Because GL67 has been formulated for in vivo use, we next assessed TransMAG(PEI) in the murine nasal epithelium in vivo, and compared this to naked pDNA. At the concentrations required for in vivo experiments, precipitation of magnetic complexes was seen. After extensive optimization, addition of non-precipitated magnetic particles resulted in approximately seven- and 90-fold decrease in gene expression for naked pDNA and GL67/pDNA liposome complexes, respectively, compared to non-magnetic particles. Thus, whereas exposure to a magnetic field improved in vitro transfection efficiency, translation to the in vivo setting remains difficult.
Assuntos
DNA/farmacologia , Terapia Genética/métodos , Magnetismo , Mucosa Respiratória/metabolismo , Transfecção/métodos , Animais , Cátions , Linhagem Celular Tumoral , Fibrose Cística/metabolismo , Fibrose Cística/terapia , Expressão Gênica , Engenharia Genética , Humanos , Lipídeos/farmacologia , Masculino , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Material ParticuladoRESUMO
Sendai virus (SeV) is able to transfect airway epithelial cells efficiently in vivo. However, as with other viral vectors, repeated administration leads to reduced gene expression. We have investigated the impact of inducing immunological tolerance to immunodominant T-cell epitopes on gene expression following repeated administration. Immunodominant CD4 and CD8 T-cell peptide epitopes of SeV were administered to C57BL/6 mice intranasally 10 days before the first virus administration with transmission-incompetent F-protein-deleted DeltaF/SeV-GFP. At 21 days after the first virus administration, mice were again transfected with DeltaF/SeV. To avoid interference of anti-GFP antibodies, the second transfection was carried out with DeltaF/SeV-lacZ. At 2 days after the final transfection lung beta-galactosidase expression, T-cell proliferation and antibody responses were measured. A state of 'split tolerance' was achieved with reduced T-cell proliferation, but no impact on antiviral antibody production. There was no enhancement of expression on repeat administration; instead, T-cell tolerance was, paradoxically, associated with a more profound extinction of viral expression. Multiple immune mechanisms operate to eradicate viruses from the lung, and these findings indicate that impeding the adaptive T-cell response to the immunodominant viral epitope is not sufficient to prevent the process.
Assuntos
Terapia Genética/métodos , Pulmão/imunologia , Vírus Sendai/genética , Linfócitos T/imunologia , Vacinas Virais/administração & dosagem , Animais , Antígenos CD4/imunologia , Antígenos CD8/imunologia , Proliferação de Células , Feminino , Expressão Gênica , Engenharia Genética , Tolerância Imunológica/genética , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Vírus Sendai/imunologia , Vacinas Virais/imunologiaRESUMO
Alveolar type II (ATII) cell proliferation and differentiation are important mechanisms in repair following injury to the alveolar epithelium. KGF is a potent ATII cell mitogen, which has been demonstrated to be protective in a number of animal models of lung injury. We have assessed the effect of recombinant human KGF (rhKGF) and liposome-mediated KGF gene delivery in vivo and evaluated the potential of KGF as a therapy for acute lung injury in mice. rhKGF was administered intratracheally in male BALB/c mice to assess dose response and time course of proliferation. SP-B immunohistochemistry demonstrated significant increases in ATII cell numbers at all rhKGF doses compared with control animals and peaked 2 days following administration of 10 mg/kg rhKGF. Protein therapy in general is very expensive, and gene therapy has been suggested as a cheaper alternative for many protein replacement therapies. We evaluated the effect of topical and systemic liposome-mediated KGF-gene delivery on ATII cell proliferation. SP-B immunohistochemistry showed only modest increases in ATII cell numbers following gene delivery, and these approaches were therefore not believed to be capable of reaching therapeutic levels. The effect of rhKGF was evaluated in a murine model of OA-induced lung injury. This model was found to be associated with significant alveolar damage leading to severe impairment of gas exchange and lung compliance. Pretreatment with rhKGF 2 days before intravenous OA challenge resulted in significant improvements in PO2, PCO2, and lung compliance. This study suggests the feasibility of KGF as a therapy for acute lung injury.
Assuntos
Fatores de Crescimento de Fibroblastos/uso terapêutico , Pneumopatias/induzido quimicamente , Pneumopatias/prevenção & controle , Lesão Pulmonar , Ácido Oleico/toxicidade , Alvéolos Pulmonares/efeitos dos fármacos , Doença Aguda , Animais , Diferenciação Celular , Proliferação de Células , Fator 7 de Crescimento de Fibroblastos , Humanos , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Alvéolos Pulmonares/citologia , Proteína B Associada a Surfactante Pulmonar/metabolismo , Proteínas Recombinantes/uso terapêuticoRESUMO
Gene therapy is currently being evaluated for a wide range of acute and chronic lung diseases. The requirement of gene transfer into the individual cell types of the complex lung structure will very much depend on the target disease. Over the last decade, the gene therapy community has recognized that there is not even one vector that is good for all applications, but that the gene transfer agent has to be carefully chosen. Gene therapy is particularly attractive for diseases that currently do not have satisfactory treatment options and probably easier for monogenic disorders than for complex diseases. Cystic fibrosis (CF) fulfills these criteria and is therefore a good candidate for gene therapy-based treatment. This review will focus on CF as an example for lung gene therapy and discuss the progress made in this field over the last couple of years.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Pulmão/metabolismo , Animais , Fibrose Cística/metabolismo , Humanos , Pulmão/virologia , Modelos Animais , Transdução Genética/métodosRESUMO
Repeat administration of gene therapy for cystic fibrosis is likely to be essential for long-term clinical efficacy. This may be minimized by the use of slow-release gene transfer preparations with more prolonged expression and longer dosing intervals for the patient. Poly(D-L-lactide-co-glycolide) (PLG) is a biodegradable and biocompatible polymer that has been used to encapsulate plasmid DNA. PLG-DNA microspheres were generated and characterized with respect to morphology, size (80% of particles <5.2 microm), and encapsulation efficiency (50.7+/-2.3%, n=6). Gel electrophoresis of DNA re-extracted from the microspheres confirmed that despite a decrease in the proportion of supercoiled conformation, it had not been degraded by the preparation process. Gene transfer efficiency was tested using microspheres encapsulating the reporter gene beta-galactosidase in vitro on Cos 7 cells and a CF airway epithelial line (CFTEo approximately ) and ex vivo in a sheep tracheal (s.t.) model. In both cases, transgene expression was significantly (P<0.01) lower at the first time point tested (24 h in vitro, 48 h ex vivo) compared to lipid-#67-mediated gene transfer. However, PLG-mediated expression in vitro was sustained at 48 h, while lipid #67-mediated expression levels had dropped significantly (P<0.05) to 50.3+/-13.7 and 38.2+/-2.7% (Cos 7 and CFTEo approximately cells, respectively) of the 24-h level. This pattern was also seen in the s.t. model where at 72 h, PLG-mediated expression was 125.4+/-7.2% of the 48-h level demonstrating significantly (P<0.05) better retention of transfection efficiency than lipid #67, where levels had fallen to approximately half the 48 h level. By 96 h, expression was still retained in the PLG-transfected group (87.3+/-12.5% of 48 h expression) but was undetectable in the lipid -#67-transfected s.t. Finally, PLG microspheres, encapsulating the reporter gene chloramphenicol transferase (CAT, 80 microg) were instilled intranasally into Balb/C mice. Compared to lipid-#67-mediated delivery, where whole lung CAT expression was highest at 48 h (13.7 x 10(3)+/-0.05 CAT U/microg protein, n=6) and then not detectable at further time points, CAT expression was not detectable in PLG-transfected mice at 48 h, but was detectable at 7, 14 and 21 days after transfection. These data demonstrate that PLG-mediated gene transfer can produce prolonged gene expression in airway epithelia. However, gene transfer efficiency still requires significant improvement.