Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells

Adenovirus-mediated gene transfer to muscle is a promising technology forgene therapy of Duchenne muscular dystrophy (DMD). However, currentlyavailable recombinant adenovirus vectors have several limitations,including a limited cloning capacity of approximately 8.5 kb, and theinduction of a host immune response that leads to transient gene expressionof 3-4 weeks in immunocompetent animals. Gene therapy for DMD could benefitfrom the development of adenoviral vectors with an increased cloningcapacity to accommodate a full-length (approximately 14 kb) dystrophincDNA. This increased capacity should also accommodate gene regulatoryelements to achieve expression of transduced genes in a tissue-specificmanner. Additional vector modifications that eliminate adenoviral genes,expression of which is associated with development of a host immuneresponse, might greatly increase long-term expression of virally deliveredgenes in vivo. We have constructed encapsidated adenovirus minichromosomestheoretically capable of delivering up to 35 kb of non-viral exogenous DNA.These minichromosomes are derived from bacterial plasmids containing twofused inverted adenovirus origins of replication embedded in a circulargenome, the adenovirus packaging signals, a beta-galactosidase reportergene and a full-length dystrophin cDNA regulated by a muscle-specificenhancer/promoter. The encapsidated minichromosomes are propagated in vitroby trans-complementation with a replication-defective (E1 + E3 deleted)helper virus. We show that the minichromosomes can be propagated to hightiter (> 10(8)/ml) and purified on CsCl gradients due to their buoyancydifference relative to helper virus. These vectors are able to transducemyogenic cell cultures and express dystrophin in myotubes. These resultssuggest that encapsidated adenovirus minichromosomes may be useful for genetransfer to muscle and other tissues.