Optimization of the helper-dependent adenovirus system for production and potency invivo

Optimization of the helper-dependent adenovirus system for production and potency invivo

Departments of ^*Virus and Cell Biology, ^‡Human Genetics, and ^‖Bioprocess and Bioanalytical Research, Merck Research Laboratories, West Point, PA 19486; and ^¶Department of Genetics, I.R.B.M. P. Angeletti, 00040 Pomezia, Italy

Contributed by C. Thomas Caskey

Abstract

Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences provide for safe and highly efficient gene transfer with long-lasting transgene expression. High titer stocks of HD vectors can be generated by using the cre-recombinase system. However, we have encountered difficulties with this system, including rearranged HD vectors and variable efficiency of HD vector rescue. These problems represent a major hindrance, particularly with regard to large-scale production. To overcome these limitations, we have modified the system in two ways: We constructed a new helper virus with a modified packaging signal and enhanced growth characteristics. We also redesigned the vector backbones by including noncoding adenovirus sequences adjacent to the right inverted terminal repeat and by incorporated a number of different segments of noncoding DNA of human origin as “stuffer.” Comparison of these vectors showed that the nature of the stuffer sequence affects replication of the HD vector. Optimization of the system resulted in a more robust and consistent production of HD vectors with low helper contamination and high in vivo potency.

Footnotes

↵ † To whom reprint requests should be addressed at: Department of Virus and Cell Biology, Merck Research Laboratories, WP26A-3000, West Point, PA, 19486. E-mail: volker_sandig{at}merck.com.
↵ § Present address: Banyu Tsukuba Research Institute, Tsukuba 300-2611, Japan.
Abbreviations:

HD,

helper-dependent;

FG,

first generation;

CMV,

cytomegalovirus;

ITR,

inverted terminal repeat;

MAR,

matrix attachment region