PACKAGING CAPACITY AND STABILITY OF HUMAN ADENOVIRUS TYPE-5 VECTORS

Adenovirus vectors are extensively used for high-level expression of p roteins in mammalian cells and are receiving increasing attention for their potential use as live recombinant vaccines and as transducing vi ruses for use in gene therapy. Although it is commonly argued that one of the chief advantages of adenovirus vectors is their relative stabi lity, this has not been thoroughly investigated. To examine the geneti c stability of adenovirus type 5 vectors and in particular to examine the relationship between genetic stability and genome size, adenovirus vectors were constructed with inserts of 4.88 (herpes simplex virus t ype 1 gB), 4.10 (herpes simplex virus type 1 gB), or 3.82 (LacZ) kb co mbined with a 1.88-kb E3 deletion or with a newly generated 2.69-kb E3 deletion. The net excess of DNA over the wild-type (wt) genome size r anged from 1.13 to 3.00 kb or 3.1 to 8.3%. Analysis of these vectors d uring serial passage in tissue culture revealed that when the size exc eeded 105% of the wt genome length by approximately 1.2 kb (4.88-kb in sert combined with a 1.88-kb deletion), the resulting vector grew very poorly and underwent rapid rearrangement, resulting in loss of the in sert after only a few passages. In contrast, vectors with inserts resu lting in viral DNA close to or less than a net genome size of 105% of that of the wt grew well and were relatively stable. In general, virus es with genomes only slightly above 105% of that of the wt were unstab le and the rapidity with which rearrangement occurred correlated with the size of the insert. These findings suggest that there is a relativ ely tight constraint on the amount of DNA which can be packaged into v irions and that exceeding the limit results in a sharply decreased rat e of virus growth. The resultant strong selection for variants which h ave undergone rearrangement, generating smaller genomes, is manifested as genetic instability of the virus population.