Development and Analysis of Recombinant Adenoviruses for Gene Therapy of Cystic Fibrosis

Abstract

A new adenovirus-based vector (Ad2/CFTR-1) has been constructed in which the cDNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, replaces the early region 1 coding sequences, E1a and E1b. The virus retains the E3 region. Ad2/CFTR-1 and a related construct encoding beta-galactosidase replicate in human 293 cells which provide E1 gene functions in trans. Replication of these recombinant viruses was not detected in a variety of other cells, although very limited viral DNA synthesis and transcription from the E4 and L5 regions could be measured. These E1-deletion vectors were also deficient in cellular transformation, shut-off of host cell protein synthesis, and production of cytopathic effects, even at high multiplicities of infection. Ad2/CFTR-1 produced CFTR protein in a variety of cells including airway epithelia from CF patients. Expression of functional CFTR protein in a CF airway epithelial monolayer was detected by correction of the Cl- transport defect characteristic of CF. Surprisingly low multiplicities of infection (0.1 moi) were sufficient to generate CFTR Cl- current across a CF epithelial monolayer in vitro. These data, together with the lack of obvious toxicity, suggest that Ad2/CFTR-1 should be suitable for CF gene therapy.

Adenovirus‐mediated gene transfer to murine retinal cells in vitro and in vivo

“…Adenoviral stocks were prepared, purified and titred as described previously [12]. AdUCMVlacZ-1 is a replication-deficient recombinant adenovirus deleted in the El region and retaining the E3 region [lo].…”

Human PBMC-derived dendritic cells transduced with an adenovirus vector induce cytotoxic T-lymphocyte responses against a vector-encoded antigen in vitro

“…19,[22][23][24][28][29][30] Moreover, murine DC genetically modified by adenovirus transduction have been shown to induce antigen-specific therapeutic antitumour immunity. 19,22,24 While murine studies using human-tropic adenovirus vectors are promising, these findings are not necessarily predictive of the outcome of experiments using human DC, because the adenovirus vectors used retained the capacity for viral gene expression [31][32][33][34] and the potential to have different effects on function in mouse and human cells. 35 Human DC have been successfully transduced using adenovirus vectors with efficiencies exceeding 90% at high MOI.…”

An Adenovirus Type 5 (Ad5) Amplicon-Based Packaging Cell Line for Production of High-Capacity Helper-Independent ΔE1-E2-E3-E4 Ad5 Vectors

“…Despite the great potential of Ad vectors for gene therapy and vaccination purposes, their possible applications have been hampered as a consequence of several problems limiting their overall in vivo efficacy (11,31,33,38,45). To date, several vector systems based on Ad5 have been proposed with the aim of improving the safety profile of FG vectors and increasing their cloning capacities.…”

“…Early region 1 (E1)-deleted first-generation (FG) Ad vectors are ideal recombinant viral vaccines due to their ability to elicit strong cell-mediated and humoral immunities (7,23,42,43,(45)(46)(47). In contrast, use of FG Ad for gene therapy has been hampered by its shortterm transgene expression as a result of leaky adenoviral structural-gene expression, leading to immune clearance of transduced cells (11,33,38,45). To improve longevity of expression, new generations of Ad type 5 (Ad5)-based vectors have been constructed by introducing additional deletions of viral genes, resulting in further attenuation of the adenoviral gene expression in vivo.…”