Efficiency Of Genomic Integration In Vitro By A Hybrid Adenoretroviral Vector

Previously, we reported that a replication-deficient recombinant adenoviral vector carrying the 5' and 3' long terminal repeats (LTRs), and additional sequences, of the Moloney murine leukemia virus (MoMLV), can randomly integrate into the genome of host cells both in vitro and in vivo (Zheng et al., Nat Biotechnol, 2000). Objectives: To easily visualize transgene expression and to readily determine vector integration efficiency. Methods: Two recombinant vectors, AdLTR-Redf and AdCMV-Redf were constructed. Both vectors had a type 5, E1- adenoviral backbone and contained a transgene cassette encoding the red fluorescence (Redf) protein as a reporter gene. AdLTR-Redf was constructed with MoMLV elements as in Zheng et al (2000), while AdCMV-Redf was a conventional vector using the cytomegalovirus promoter/enhancer. A5 rat salivary epithelial cells were infected with either vector at low doses (5 plaque forming units (pfu)/cell or 1pfu/cell), and allowed to form individual clones by a single limiting dilution procedure. Transgene expression was assessed microscopically two weeks after infection. Results: Out of 450 (5 pfu/cell) and 675 (1 pfu/cell) clones established with AdLTR-Redf, 36 and 9 clones, respectively, expressed the Redf gene corresponding to an integration efficiency of ~ 8 % and 1.3%. Conversely, none of the 449 (5 pfu/cell) and 669 (1pfu/cell) clones obtained from AdCMV-Redf infected cells exhibited Redf gene expression. Among the AdLTR-Redf infected positive clones, there was considerable variability in both the proportion of cells appearing red (1-50%), as well as the strength of transgene expression within positive cells. Conclusion: While further studies are needed to more accurately quantify the true integration efficiency, and understand the heterogeneity of expression, these results are consistent with AdLTR-Redf infection in vitro resulting in considerable genomic integration; much greater than the extremely low levels (10e-3; Harui et al., J Virol, 1999) seen with conventional adenoviral vectors.