RESUMEN
BACKGROUND: Efficient adenovirus (AdV)-mediated gene transfer is possible only in immature muscle or regenerating muscle, suggesting that a developmentally regulated event plays a major role in limiting AdV uptake in mature skeletal muscle. Previously, we showed that the expression of the primary coxsackie and adenovirus receptor (CAR) is severely down-regulated during muscle maturation and that, in muscle-specific CAR transgenic mice, there is significant enhancement of AdV-mediated gene transfer to mature skeletal muscle. METHODS: To evaluate whether increasing CAR expression can also augment gene transfer to dystrophic muscle that has many regenerating fibers, we crossed CAR transgenics with dystrophin-deficient mice (mdx/CAR). We also tested a two-step protocol in which CAR levels were increased in the target muscle, prior to administration of AdV, through the use of recombinant adeno-associated virus (AAV2) expressing CAR. Lastly, we assessed the effect of histone deacetylase inhibitors on CAR and AdV transduction efficiency in myoblasts and mdx muscle. RESULTS: Although somewhat higher rates of transduction can be achieved in adult mdx mice than in normal mice as a result of ongoing muscle regeneration in these animals, CAR expression in the mdx background (mdx/CAR transgenics) still markedly improved the susceptibility of mature muscle to AdV-mediated gene transfer of dystrophin. Prior administration of AAV2-CAR to normal muscle led to significantly increased transduction by subsequent injection of AdV. The histone deacetylase inhibitor valproate increased CAR transcript and protein levels in myoblasts and mdx muscle, and also increased AdV-mediated gene transfer. CONCLUSIONS: We have developed a method of increasing CAR levels in both normal and regenerating muscle.
Asunto(s)
Fibras Musculares Esqueléticas/metabolismo , Distrofias Musculares/genética , Receptores Virales/genética , Transducción Genética/métodos , Adenoviridae , Animales , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Distrofina/genética , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Fibras Musculares Esqueléticas/efectos de los fármacos , Regeneración , Transcripción Genética/efectos de los fármacos , Ácido Valproico/farmacologíaRESUMEN
Adenoviral vectors that use the coxsackievirus and adenovirus receptor do not transduce mature muscle efficiently. Group B adenoviruses use CD46 as their cell attachment receptor. To evaluate the utility of vectors based on group B adenoviruses for gene transfer to human skeletal muscle, we assessed the expression of CD46 in biopsied normal skeletal muscle samples and in muscles from patients with Duchenne muscular dystrophy. Transcript levels of CD46 were extremely low in mature muscle and CD46 immunoreactivity was detected only on blood vessels in the muscle sections. Although myoblasts cultured from biopsied samples had robust cell surface CD46 expression by flow cytometry, CD46 transcript levels were barely detectable after differentiation of the myoblasts into myotubes. The myotubes were also much less susceptible to infection with an adenoviral vector carrying the fiber of serotype 35 adenovirus (AdF35). These results suggest that for skeletal muscle, vectors derived from group B adenoviruses may not be a suitable alternative to the commonly used Ad5 vectors.
Asunto(s)
Adenoviridae/metabolismo , Diferenciación Celular , Regulación hacia Abajo/genética , Proteína Cofactora de Membrana/genética , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Transducción Genética/métodos , Biopsia , Permeabilidad de la Membrana Celular , Células Cultivadas , Citometría de Flujo , Humanos , Fibras Musculares Esqueléticas , Distrofia Muscular de Duchenne , Mioblastos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , beta-GalactosidasaRESUMEN
Helper-dependent adenovirus vector (AdV)-mediated full-length dystrophin expression leads to significant mitigation of the dystrophic phenotype of the mdx mouse. However, dystrophin, as a neoantigen, elicits antibody formation. As an alternative approach, we evaluated gene transfer of full-length murine utrophin, a functional homologue of dystrophin that is normally present only at the neuromuscular junction. A single injection in the tibialis anterior (TA) muscle of the helper-dependent adenovirus vector encoding utrophin provided very good transduction, with 58% of fibers demonstrating sarcolemmal utrophin expression in the neonates, and 35% utrophin-positive (Utr(+)) fibers in adults. The presence of utrophin prevented extensive necrosis in the neonates, halted further necrosis in the adults, and led to restoration of sarcolemmal expression of dystrophin-associated proteins up to 1 year after injection. Marked physiological improvement was observed in both neonates and adults. Neither increased humoral responses nor cellular immune responses were evident. However, there was a time-related decline of the initial high utrophin expression. Although viral DNA persisted in animals that were injected in the neonatal stage, viral DNA levels decreased in muscles of adult mice. These results demonstrate that although utrophin gene transfer leads to amelioration of the dystrophic phenotype, the effects are not sustained upon loss of utrophin expression.