In Vivo Expression of Full-Length Human Dystrophin from Adenoviral Vectors Deleted of All Viral Genes

Abstract

Adenoviral vectors have been shown to effect efficient somatic gene transfer in skeletal muscle and thus offer potential for the development of therapy for Duchenne muscular dystrophy (DMD). Efficient transfer of recombinant genes has been demonstrated in skeletal muscle using recombinant adenoviruses deleted of E1. Application of this vector system to the treatment of DMD is limited by the vector immunogenicity, as well as by size constraints for insertion of recombinant genes, precluding the incorporation of a full-length dystrophin minigene construct. We describe in this study the use of helper adenovirus to generate a recombinant vector deleted of all viral open reading frames and containing a full-length dystrophin minigene. We show that this deleted vector (Δ vector) is capable of efficiently transducing dystrophin in mdx mice, in myotubes in vitro and muscle fibers in vivo. Our modification of adenoviral vector technology may be useful for the development of gene therapies for DMD and other diseases. A new type of adenoviral vector, fully deleted of adenoviral genes, has been developed for gene therapy of Duchenne muscular dystrophy (DMD). A minimal vector contains the inverted terminal repeats and contiguous packaging sequence, as well as a minigene harboring a full-length dystrophin cDNA. This deleted vector (Δ vector) was packaged into virions through the use of a helper adenovirus. The resulting preparation, which contains a mixture of Δ vector and helper virus, was propagated to high titers. The majority of helper virus was removed by sedimentation through cesium gradients. Preparations of the dystrophin Δ vectors efficiently transduced muscle fibers when injected directly into skeletal muscle of the murine model for DMD. The advantage of this system is that it can accommodate larger recombinant genes such as that containing full-length dystrophin and has potential for minimizing destructive cellular immune responses.