A Cre-expressing cell line and an E1/E2a double-deleted virus for preparation of helper-dependent adenovirus vector

Adenoviral vectors are attractive for the delivery of transgenes into mamma lian cells because of their efficient transduction, high titer, and stabili ty. The major concerns with using El-deleted adenoviral vectors in gene the rapy are the pathogenic potential of the virus backbone and the leaky viral protein synthesis that leads to host immune responses and a short duration of transgene expression. Helper-dependent (HD) adenoviral vectors that are devoid of all viral protein-coding sequences have significantly increased the safety and reduced the immunogenicity of these vectors. Currently avail able HD vectors depend on an El-deleted adenovirus as a helper to provide v iral proteins in trans. As a consequence, contamination with helper virus c annot be avoided in the HD vector preparation though it can be decreased to 0.01% using a Cre/IoxP mechanism. Since the presence of El-deleted helper virus may have substantial unwanted effects, we have developed a new Cre-ex pressing cell line based on an E1- and E2a-complementing cell. This new cel l line can efficiently cleave the packaging region in the helper virus geno me. We have also developed an El and E2a double-deleted helper virus. By us ing the CreE cell with the helper virus deleted in both the El and the E2a genes it may be possible to further improve the safety of the vectors.