RESUMEN
The purpose was to determine the relative efficiency, toxicity and duration of expression following gene delivery by intramyocardial injection of naked DNA, naked DNA complexed to cationic liposomes, naked DNA complexed to cationic liposomes with integrin-targetting peptide, recombinant (E1-/E3-) adenovirus, recombinant adeno-associated virus and recombinant (ICP27-) herpes simplex virus. All vectors incorporated a LacZ reporter driven by a promoter containing the hCMV-IE promoter/enhancer. Efficiency was scored by counting positive cells in five standard microscopic sections harvested from the left ventricular apex. Rabbit hearts (n = 100) were examined from 2 to 56 days after injection. Uncomplexed and complexed naked DNA were very inefficient with less than one positive cell visible per heart. The viral vectors all resulted in robust gene expression with adenovirus being the most efficient by at least one order of magnitude before 21 days. However, despite disparate titres, the efficiency beyond 21 days of adenovirus and adeno-associated virus were comparable. In contrast to adeno-associated virus, both adenovirus and herpes-simplex virus were associated with a marked inflammatory response. Despite reporter gene activity appearing only after 21 days, adeno-associated virus shows comparative promise as a myocardial gene delivery vector.
Asunto(s)
Técnicas de Transferencia de Gen , Miocardio/metabolismo , Adenoviridae/genética , Adenoviridae/fisiología , Animales , Virus ADN/fisiología , ADN Recombinante/fisiología , Relación Dosis-Respuesta a Droga , Regulación Viral de la Expresión Génica/fisiología , Vectores Genéticos/fisiología , Inflamación/virología , Masculino , Modelos Animales , Conejos , Simplexvirus/genética , Simplexvirus/fisiología , Transducción GenéticaRESUMEN
Clinical reports suggest that intracoronary delivery of adenoviruses encoding angiogenic growth factors, or their transactivators, has a therapeutic benefit. However there has not been a systematic assessment of the transfection efficiency of this technique in vivo. In rabbits we investigated the efficiency of myocardial gene transfer following intracoronary infusion of 1 x 10(-10) -1 x 10(12) p.f.u. of adenovirus in combination with interventions to enhance transfection. In five standard short axis sections, we were barely able to detect reporter gene expression following unmodified intracoronary infusion. Efficiency was not enhanced by the exclusion of blood and the increase of intracoronary dwell time through occlusive engagement of the left coronary ostium enabled by oxygenated perfluorocarbon emulsion as viral diluent. Of the interventions and pretreatments designed to increase vascular permeability, VEGF, calcium-free viral diluent and adenosine, only the latter tended to increase efficiency. However an intervention designed to increase the myocardial transcapillary gradient, by increasing venular pressure with pulmonary artery occlusion and arteriolar pressure with occlusion of the aorta above the coronary ostia, increased transfection efficiency by two orders of magnitude. Unfortunately the clinical utility of this technique may be limited by accompanying cardiac dilation and marked elevations in intracardiac pressure.
Asunto(s)
Adenoviridae/genética , Vasos Coronarios , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Miocardio/enzimología , beta-Galactosidasa/genética , Adenosina/administración & dosificación , Animales , Permeabilidad Capilar/efectos de los fármacos , Factores de Crecimiento Endotelial/administración & dosificación , Expresión Génica , Terapia Genética , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Linfocinas/administración & dosificación , Masculino , Conejos , Proteínas Recombinantes/administración & dosificación , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Presión Ventricular/efectos de los fármacosRESUMEN
The myocardium is a potential target for the expression of exogenous genes to treat inherited and acquired diseases. Although adenovirus-mediated gene transfer has resulted in high-level gene transfer in vivo via direct intramyocardial injection and via a percutaneous intra-arterial route, the time-course of gene expression is limited by host immune responses. It was the aim of this study to test whether cationic liposome-mediated gene transfer, which does not suffer from the aforementioned problems, was feasible in the adult rabbit myocardium via a percutaneous transluminal approach. Doses of plasmid DNA encoding lacZ from 200-800 micrograms complexed to cationic liposomes resulted in X-gal conversion at day 3 with associated myocardial damage. We hypothesised that the damage was associated with macro-aggregates of cationic liposomes-DNA occluding the microcirculation. When such aggregates were excluded no X-gal conversion was seen in vivo. In order to show that X-gal conversion occurs in areas of infarction in the myocardium we caused closed chest infarction by deploying a platinum micro-embolisation coil in the circumflex coronary artery. At day 3 X-gal conversion was observed in the territory supplied by the occluded artery. Thus, microinfarction causes the false positive appearance of gene transfer when using a lacZ reporter gene.