Chromosomal integration of adenoviral vector DNA in vivo

Abstract

So far there has been no report of any clinical or preclinical evidence for chromosomal vector integration following adenovirus (Ad) vector-mediated gene transfer in vivo. We used liver gene transfer with high-capacity Ad vectors in the FAH(Deltaexon5) mouse model to analyze homologous and heterologous recombination events between vector and chromosomal DNA. Intravenous injection of Ad vectors either expressing a fumarylacetoacetate hydrolase (FAH) cDNA or carrying part of the FAH genomic locus resulted in liver nodules of FAH-expressing hepatocytes, demonstrating chromosomal vector integration. Analysis of junctions between vector and chromosomal DNA following heterologous recombination indicated integration of the vector genome through its termini. Heterologous recombination occurred with a median frequency of 6.72 x 10(-5) per transduced hepatocyte, while homologous recombination occurred more rarely with a median frequency of 3.88 x 10(-7). This study has established quantitative and qualitative data on recombination of adenoviral vector DNA with genomic DNA in vivo, contributing to a risk-benefit assessment of the biosafety of Ad vector-mediated gene transfer.

FIG. 1.

Analysis of random integration of HC-Ad vector DNA into chromosomal DNA in vivo. (A) Map of HC-AdSLS16 vector expressing the murine FAH cDNA from the RSV promoter. ITR, Ad5 inverted terminal repeat; Ψ, Ad5 packaging signal; stuffer DNA, 16-kb stuffer from human HPRT genomic DNA (nt 1799 to 17853 of human HPRT gene); RSV, RSV promoter; mFAH cDNA, murine FAH cDNA; stuffer DNA, 9-kb c346 stuffer (nt 12421 to 21484 of cosmid c346). The HC-AdSLS16 vector was used to analyze random integration events. (B and C) Representative photomicrographs of liver sections stained with hematoxylin-eosin. FAH expression is detected with a polyclonal rabbit antibody to rat FAH.

FIG. 2.

Analysis of homologous recombination of HC-Ad vector DNA with chromosomal DNA in vivo. (A) Schematic of the HC-AdSLS14 vector carrying a 12.3-kb genomic fragment of the murine DNA and the scheme of homologous recombination (X) whereby exon 5 in the FAH locus may be replaced by the targeting vector. ITR, Ad5 inverted terminal repeat; Ψ, Ad5 packaging signal; murine FAH fragment, 12.3-kb murine FAH genomic DNA fragment beginning at nt 91754097 of the murine FAH gene from introns 1 to 9; E2 to E9, wild-type exons 2 to 9, respectively; E5*, mutated exon 5 with NeoR insertion; Neo, NeoR gene; stuffer DNA, 16-kb stuffer from human HPRT genomic DNA (nt 1799 to 17853 of human HPRT gene). The positions of the primers used in the PCR to demonstrate the presence of an uninterrupted exon 5 in the livers are indicated as the horizontal arrows FAHA, FAHB, and FAHC. (B) The 2% agarose gel used for separation of the PCR products; Lanes: −, water controls; f+* and f+, C57/BL6 and 129 mouse controls; respectively; p, pSLS14 plasmid control; f−, FAH^−/− mouse negative control; 10, FAH^−/− mouse injected with the first-generation Ad vector negative control; 9, transplantation recipient of HC-AdSLS16 vector negative control; 4 to 8, transplantation recipients; 1 to 3, injected animals. (C and D) Representative photomicrographs of liver sections stained with hematoxylin-eosin. FAH expression is detected with a polyclonal rabbit antibody to rat FAH.