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1.
Gene Ther ; 8(18): 1380-6, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11571577

RESUMO

Nonviral vectors have been shown to be a safe and valid alternative to recombinant viruses for gene therapy of cystic fibrosis (CF). Nevertheless, gene transfer efficiency needs to be increased before clinical efficacy is likely in man. One barrier to increased efficacy is normal airway mucus. Using an ex vivo model of sheep tracheal epithelium, we show that this barrier can, in part, be overcome by treatment with the mucolytic agents, Nacystelyn or N-acetylcysteine using either a cationic lipid or a cationic polymer as the gene transfer agent. Further, in vivo application of either Nacystelyn or the anticholinergic glycopyrrolate, both clinically used agents, resulted in increased reporter gene expression in the mouse lung, but no significant correction of the bioelectric defect in CF null mice. These results, whilst unlikely to be sufficient in themselves to achieve clinically relevant gene therapy, may be a further useful step in the attainment of this goal.


Assuntos
Acetilcisteína/farmacologia , Cloranfenicol O-Acetiltransferase/genética , Fibrose Cística/terapia , Expectorantes/farmacologia , Terapia Genética/métodos , Lisina/farmacologia , Traqueia/metabolismo , Acetilcisteína/análogos & derivados , Animais , Antagonistas Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/farmacologia , Glicopirrolato/administração & dosagem , Injeções Intramusculares , Lisina/análogos & derivados , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CFTR , Modelos Animais , Mucosa Nasal/metabolismo , Ovinos , Traqueia/efeitos dos fármacos
2.
Expert Opin Investig Drugs ; 9(7): 1523-35, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11060757

RESUMO

The gene for cystic fibrosis was identified in 1989 and this together with the emerging technology of gene therapy heralded a new dawn for the treatment of genetic disease. The initial optimism however gave way to the realisation that gene therapy for cystic fibrosis was unlikely to be straightforward. The lung was considered an ideal organ to target due to ease of access, but subsequent research has shown that the airway surface provides an efficient barrier to topically applied gene transfer agents. A number of Phase I clinical safety trials were carried out through the 1990s and provided proof of concept evidence that delivery of DNA by either viral or non-viral means was safe though not clinically efficacious. Current research is now focusing more on the barriers faced by delivery agents, with the aim that more efficient gene delivery will lead to a gene therapeutic for cystic fibrosis.


Assuntos
Fibrose Cística/terapia , Terapia Genética , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Terapia Genética/efeitos adversos , Vetores Genéticos , Humanos , Retroviridae/genética
3.
Nat Biotechnol ; 18(9): 970-3, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10973218

RESUMO

Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer.


Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Pulmão/metabolismo , Mucosa Nasal/metabolismo , Respirovirus/genética , Traqueia/metabolismo , Adenoviridae/genética , Animais , Brônquios/metabolismo , Células COS , Linhagem Celular , Células Cultivadas , Fibrose Cística/terapia , Cães , Relação Dose-Resposta a Droga , Epitélio/metabolismo , Feminino , Furões , Humanos , Lipossomos , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/metabolismo , Receptores de Superfície Celular/metabolismo , Ovinos , Fatores de Tempo , Transfecção
4.
Gene Ther ; 6(4): 534-46, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10476213

RESUMO

Gene transfer to the respiratory epithelium is currently suboptimal and may be helped by the identification of limiting biological barriers. We have, therefore, developed an ex vivo model which retains many of the characteristics of in vivo native airways including mucociliary clearance, mucus coverage and an intact cellular structure. Using this model we have demonstrated several barriers to gene transfer. Liposome-mediated gene transfer was inhibited by normal mucus, with removal of this layer increasing expression approximately 25-fold. In addition both liposome and adenovirus were inhibited by CF sputum. The apical membrane represented a significant barrier to both agents. Adenovirus-mediated expression could be significantly augmented by increasing contact time or by pre-treatment of tissues with a nominally calcium-free medium. The presence of these extracellular and plasma membrane barriers appeared to be the key parameters responsible for the approximately three log difference in gene expression found in vitro compared with our ex vivo model. Cytoskeletal elements and the cell cycle also influenced in vitro gene transfer, and represent further barriers which need to be overcome.


Assuntos
Fibrose Cística/terapia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Traqueia/metabolismo , Adenoviridae/genética , Animais , Células COS , Membrana Celular/metabolismo , Fibrose Cística/patologia , Epitélio/metabolismo , Citometria de Fluxo , Expressão Gênica , Vetores Genéticos/administração & dosagem , Lipossomos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Modelos Biológicos , Muco/metabolismo , Ovinos , Escarro/metabolismo , Traqueia/ultraestrutura , Falha de Tratamento , beta-Galactosidase/genética
5.
Int J Trauma Nurs ; 5(4): 128-31, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10661142

RESUMO

Trauma centers find it a challenge to provide appropriate, cost-effective outreach education. A needs assessment of rural health care providers was conducted to determine "what," "how," and "where" was preferred. The results showed that multidisciplinary rural providers were consistent with their preferences, that they considered case reviews the most effective method, that weekday evenings were the most convenient time, and that their lack of experience was their greatest barrier to learning.


Assuntos
Educação Continuada em Enfermagem/normas , Hospitais Rurais/normas , Avaliação das Necessidades , Recursos Humanos de Enfermagem Hospitalar/educação , Desenvolvimento de Pessoal/normas , Humanos
6.
Biochemistry ; 34(18): 6208-17, 1995 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-7742326

RESUMO

The kinetics of cholesterol and cholesterol sulfate (CS) movement between vesicles have been investigated. CS is widely distributed in cell membranes, plasma and skin and is similar in structure to cholesterol, but possesses an ionizable sulfate moiety at the 3 beta-position which imparts a negative charge at physiological pHs. Donor vesicles of various sizes ranging from 40 to 250 nm, composed of egg phosphatidylcholine (EPC)/sterol/N-palmitoyldihydrolactosylcerebroside (75:10:15 mole ratio) containing trace amounts of [3H]sterol, were used to monitor sterol transfer into a 10-fold excess of large unilamellar vesicles (LUV) composed of EPC with a diameter of 100 nm. The two populations of vesicles were separated by centrifugation following the addition of a lectin which caused the aggregation of donor vesicles. Both cholesterol and CS exhibited biphasic kinetics of exchange. The rate constants for efflux and transbilayer diffusion for both sterol molecules were determined after fitting kinetic data, using numerical integration, to a three-compartment model, which includes the inner and outer monolayers of donor vesicles and the acceptor bilayer. The rate of intermembrane exchange for CS was approximately 10-fold faster than for cholesterol in all liposomes tested. Using the kinetic model, a rate of transbilayer movement for cholesterol and CS was estimated. In both cases, it was found to be slower than the rate of efflux from the surface of vesicles. For vesicles containing CS, the surface charge was monitored to demonstrate that the slowly exchanging pool was located in the inner monolayer, and the rapidly exchanging pool in the outer half of the bilayer. For cholesterol, it was not possible to distinguish between this model and one where lateral domains of cholesterol within the plane of the bilayer may influence the kinetics of exchange.


Assuntos
Ésteres do Colesterol/metabolismo , Colesterol/metabolismo , Bicamadas Lipídicas/metabolismo , Transporte Biológico , Cinética , Lipossomos/metabolismo
7.
Dermatol Clin ; 11(1): 143-54, 1993 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8435909

RESUMO

We believe that it is unlikely that intact liposomes can cross the stratum corneum and act as carriers of drugs through the skin. However, it is probable that liposomes have the potential to modify the cutaneous biodistribution of applied chemicals (e.g., drugs, toxins, humectants) and thus modify their activity. Liposomal lipids can also penetrate the skin barrier. The effect of this is likely to be varied and lipid dependent, but this phenomenon could hold the most potential for the application of liposomes to topical therapy.


Assuntos
Fármacos Dermatológicos/administração & dosagem , Lipossomos , Fármacos Dermatológicos/metabolismo , Fármacos Dermatológicos/farmacocinética , Dermatologia , Portadores de Fármacos , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Absorção Cutânea
9.
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