High level of transgene expression in cell cultures and in the mouse by replication-incompetent adenoviruses harboring modified VAI genes.

Abstract

Replication-incompetent adenoviruses are currently used in gene therapy trials. Most of the work designed to increase the expression from these vectors concerns the modification of cis sequences of the foreign transcription unit, so as to improve the transcription level or the stability of the mRNA. In this report, we show that an alternative strategy based on the coexpression of modified VAI genes can efficiently increase gene expression both in cell cultures and in animals. The VAI RNA is synthesized mainly during the late phase of the adenovirus cycle and increases the translation of late adenovirus gene products by counteracting the effect of an interferon-induced kinase, the PKR. We have constructed several modified VAI genes in which the central domain was deleted or substituted by exogenous sequences. These modified VAI genes, or the native VAI gene, were cloned into the left part of adenovirus type 5 genomes harboring their own endogenous VAI gene. One of the resulting viruses (Ad-VAr) increased 12.5- to 502-fold the expression level of reporter genes, either expressed as a constitutive cell line from an extrachromosomal DNA or introduced into cells by coinfection with another adenovirus vector. This effect was independent of the promoter, the coding sequence, and the 5' untranslated mRNA sequence and was obvious in the two non-E1-complementing cell lines tested (HeLa and Vero). Coinfection of Ad-VAr with adenoviruses expressing the luciferase gene from the major late promoter or Rous sarcoma virus (RSV) promoter by the intravenous route in mice increased by more than 33 (MLP)- to 128 (RSV)- and 4,700 (MLP)- to 30,000 (RSV)-fold the expression level of the reporter gene in the lungs and liver, respectively. The intramuscular coinoculation of Ad-VAr and Ad-MLP-gD (a recombinant adenovirus vaccine expressing gD from the pseudorabies herpes virus) led to a 10-fold decrease in the protective dose of Ad-gD in mice. Ad-VAfull, a similar adenovirus in which the native VAI gene was cloned at the left part of the genome, showed no evidence of efficacy in cell culture and in mice. These results suggest that the use of modified VAI genes expressed at the early phase of the cycle can be helpful in the design of potent adenovirus vectors.