Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.
Adenoviral vectors are widely used as highly efficient gene transfer vehicles in a variety of biological research strategies including human gene therapy. One of the limitations of the currently available adenoviral vector system is the presence of the majority of the viral genome in the vector, resulting in leaky expression of viral genes particularly at high multiplicity of infection and limited cloning capacity of exogenous sequences. As a first step to overcome this problem, we attempted to rescue a defective human adenovirus serotype 5 DNA, which had an essential region of the viral genome (L1, L2, VAI + II, pTP) deleted and replaced with an indicator gene. In the presence of wild-type adenovirus as a helper, this DNA was packaged and propagated as transducing viral particles. After several rounds of amplification, the titer of the recombinant virus reached at least 4 x 10(6) transducing particles per ml. The recombinant virus could be partially purified from the helper virus by CsCl equilibrium density-gradient centrifugation. The structure of the recombinant virus around the marker gene remained intact after serial propagation, while the pBR sequence inserted in the E1 region was deleted from the recombinant virus. Our results suggest that it should be possible to develop a helper-dependent adenoviral vector, which does not encode any viral proteins, as an alternative to the currently available adenoviral vector systems.
Mitani
K
K
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Graham
F L
FL
Caskey
C T
CT
Kochanek
S
S
D000260:Adenoviruses, Human / Q000302:isolation & purification / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002522:Chlorocebus aethiops; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D003673:Defective Viruses / Q000502:physiology; D005822:Genetic Vectors; D006378:Helper Viruses / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D007668:Kidney; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D015183:Restriction Mapping; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000096:biosynthesis
D004279:DNA, Viral; D011994:Recombinant Proteins; D001616:beta-Galactosidase