Frequency and stability of chromosomal integration of adenovirus vectors

Abstract

One of the limitations of adenovirus vectors is the lack of machinery necessary for their integration into host chromosomes, resulting in short-term gene expression in dividing cells. We analyzed frequencies of integration and persistence of gene expression from integrated adenovirus vectors. Both E1-substituted and helper-dependent adenovirus vectors achieved similar integration efficiencies of approximately 10(-3) to 10(-5) per cell, with the helper-dependent vector showing slightly higher efficiencies. In stable cell pools, gene expression of the integrated vector persisted for at least 50 cell divisions without selection. However, some stable cell clones showed changes in gene expression, which were accompanied by structural changes in the integrated vector DNA.

FIG. 1

Structure of adenovirus vectors. H, HindIII sites used for Southern hybridization analysis of the structure of integrated vectors.

FIG. 2

β-Gal expression from the integrated vectors over passages of the cells infected with ΔE1 vector (A and C) and ΔAd vector (B and D) without G418 selection. The stable cell pools (A and B) and HeLa clones (C and D) were maintained without G418 selection and stained by X-Gal every five passages; the percentages of X-Gal-positive cells were determined by microscopic observation. Two independent pools were analyzed for each cell line.

FIG. 3

Southern analysis of integrated vector DNA. DNA from HeLa cell clones infected with the ΔE1 (A, C, and E) or ΔAd (B, D, and E) vector was digested with HindIII and subjected to Southern hybridization with the right-end (A and B), neo (C and D), and full-length Ad5 DNA (E) probes. (A and C) Lanes 1 to 3, ΔE1 clones 1 to 3, respectively; lane 4, clone 4 before the passages without selection; lane 5, clone 4 after the passages; lane 6, clone 5 before the passages; lane 7, clone 5 after the passages; lane 8, AdSRαβ-geo DNA. (B and D) Lanes 1 to 4, ΔAd clones 1 to 4, respectively; lane 5, clone 5 before the passages without selection; lane 6, clone 5 after the passages. (E) Lane 1, ΔE1 clone 5; lane 2, ΔE1 clone 3; lane 3, ΔAd clone 3; lane 4, ΔAd clone 4, lane 5, ΔE1 clone 1; lane 6, ΔE1 clone 2; lane 7, uninfected HeLa; lane 8, AdSRαβ-geo DNA.