Recombinant Adenovirus Deleted of All Viral Genes for Gene Therapy of Cystic Fibrosis

Abstract

Recombinant adenoviruses are being developed for gene therapy of inherited disorders such as cystic fibrosis because they efficiently transduce recombinant genes into nondividing cells in vivo. First generation recombinant adenoviruses, rendered defective by deletion of sequences spanning E1a and E1b, express low levels of early and late viral genes that activate destructive cellular immune responses. Current strategies for improving recombinant adenoviruses attempt to inactivate other essential genes through deletion and growth in new packaging cell lines or incorporation of temperature sensitive mutations which allow propagation of the virus in available packaging cell lines at permissive temperatures. We describe in this report a new type of recombinant adenovirus that is deleted of all viral open reading frames. This recombinant (called delta-rAd), which contains only the essential cis elements (i.e., ITRs and contiguous packaging sequence), is propagated in 293 cells in the presence of E1-deleted helper virus. Concatamers of the monomeric vector genome were passaged and capable of transduction. The delta-rAd genome is packaged into virions that sediment at a lower density than the helper virus in cesium gradients forming the basis for a purification protocol. A fully deleted recombinant adenovirus that expresses human cystic fibrosis transmembrane conductance regulator was produced and used to transduce human airway epithelial cells derived from a cystic fibrosis patient. Packaging and propagation of a fully deleted adenovirus is an important step toward the development of a safer vector. Improved production and purification strategies need to be developed before this new vector system can be evaluated in vivo.

In vitro and in vivo asessment of adenovirus 41 as a vector for gene delivery to the intestine

“…[46][47][48] This 19 kDa protein is responsible for the down-regulation of majorhistocompatibility complex class I expression on the surface of cells infected with the virus. 49,50 Much effort has been made to avoid the immune response with the use of immunosuppressive agents, 19,51,52 administration of vectors with specific parts of the viral genome known to induce immune response deleted, 48,[53][54][55] and initiation of gene therapy early in development before the immune system is fully formed. 56,57 We have demonstrated in vitro that even in the absence of the aforementioned factors, gene delivery with an adenovirus type 5 vector to differentiated enterocytes is extremely inefficient.…”

ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo

“…12 We showed prestrated. 4 Recently, synthetic vectors have been suggested as an viously that DOGS/DOPE was able to transfer marker genes into the mouse upper airways after intratracheal alternative to replication defective viruses. Their major advantage is the lack of inflammatory reaction or instillation.…”

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.…”