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

Abstract

Adenovirus-mediated gene transfer to muscle is a promising technology for gene therapy of Duchenne muscular dystrophy (DMD). However, currently available recombinant adenovirus vectors have several limitations, including a limited cloning capacity of approximately 8.5 kb, and the induction of a host immune response that leads to transient gene expression of 3-4 weeks in immunocompetent animals. Gene therapy for DMD could benefit from the development of adenoviral vectors with an increased cloning capacity to accommodate a full-length (approximately 14 kb) dystrophin cDNA. This increased capacity should also accommodate gene regulatory elements to achieve expression of transduced genes in a tissue-specific manner. Additional vector modifications that eliminate adenoviral genes, expression of which is associated with development of a host immune response, might greatly increase long-term expression of virally delivered genes in vivo. We have constructed encapsidated adenovirus minichromosomes theoretically capable of delivering up to 35 kb of non-viral exogenous DNA. These minichromosomes are derived from bacterial plasmids containing two fused inverted adenovirus origins of replication embedded in a circular genome, the adenovirus packaging signals, a beta-galactosidase reporter gene and a full-length dystrophin cDNA regulated by a muscle-specific enhancer/promoter. The encapsidated minichromosomes are propagated in vitro by trans-complementation with a replication-defective (E1 + E3 deleted) helper virus. We show that the minichromosomes can be propagated to high titer (> 10(8)/ml) and purified on CsCl gradients due to their buoyancy difference relative to helper virus. These vectors are able to transduce myogenic cell cultures and express dystrophin in myotubes. These results suggest that encapsidated adenovirus minichromosomes may be useful for gene transfer to muscle and other tissues.

Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool

“…One goal of cancer gene therapy is the development of gene delivery tools with lowered immunogenicity. While the construction of some viral vectors with reduced immunogenicity have been reported (Fisher et al, 1996;Haecker et al, 1996;Kochanek et al, 1996;Kumar-Singh and Chamberlain, 1996;Morral et al, 1999), preparation of these vectors is difficult because the virus is composed of several kinds of large molecules.…”

Helper-dependent adenovirus vectors: their use as a gene delivery system to neurons

“…30,[39][40][41][42][43] To minimize difficulties associated with contaminating helper virus, a Cre/loxP helper-dependent system was developed. 30 These newly developed vectors have been tested in vivo in liver tissue and virally mediated expression of the ␣1-antitrypsin gene was maintained for up to 1 year.…”

Development and application of adenoviral vectors for gene therapy of cancer

“…213 Another study used the E1-deleted Ad with the human placental alkaline phosphatase expression cassette as a helper Ad. 214 Both designs did not control the propagation or encapsidation of the helper virus along with that of the rAd. The contamination and dominant suppression of helper virus in the preparation of the rAd vectors were the crucial problems that remained to be solved.…”

“…213 Other vectors known as encapsidated Ad minichromosomes have the left end (0 -810 bp) and right end (994 bp) of Ad5 cis element flanking the dystrophin gene (for muscular dystrophy) and ␤-gal gene expression cassettes. 214 The mini-Ad vector AdDYS␤-gal is different from the two vectors described above, and uses the same left terminus of Ad5 (1-358 bp) to hold a nonviral recombinant genome containing both the dystrophin gene and the ␤-gal gene expression cassette. 215 Whether this design can enhance mini-Ad packaging needs to be further tested.…”

“…The plasmids can be cotransfected with a helper Ad genome into helper cells or transfected and subsequently rescued by helper Ad infection. 213,214,216,218 One approach used in vitro ligation of the Ad left end to the endless mini-Ad genome before cotransfection with the helper Ad genome into 293 cells. 215 Propagation of the mini-Ad is different when using the helper virus containing a packaging signal partial dele-tion as opposed to the helper virus that is used in the LoxP/Cre helper system.…”