Biology Reference
In-Depth Information
Chapter 15
Production of High-Capacity Adenovirus Vectors
Florian Kreppel
Abstract
High-capacity adenoviral vectors (HC-Ad), also known as “helper-dependent” (HD-Ad), “gutless”, “gutted”,
or “third-generation” Ad vectors, are devoid of all viral coding sequences and have shown promising
potential for a wide variety of different applications—from classic gene therapy to genetic vaccination and
tumor treatment. However, compared to fi rst-generation adenoviral vectors their production is more com-
plex and requires specifi c in-depth knowledge. This chapter delivers a detailed protocol for the successful
production of HC-Ad vectors to high titers.
Key words
High-capacity adenovirus vectors, Helper-dependent adenovirus vectors, Gutless vectors
1
Introduction
High-capacity adenoviral vectors (HC-Ad), also known as “helper-
dependent” (HD-Ad), “gutless”, “gutted”, or “third-generation”
Ad vectors, are devoid of all viral coding sequences. While earlier
generations of adenoviral vectors (e.g.,
1.1 Features
of HC-Ad Vectors
E3 Ad vectors) are
replication-defi cient, they still harbor adenovirus genes which are
expressed at a low level after transduction of target cells. This
expression of adenovirus genes from the vectors induces very
potent CD8+ T cell responses against the transduced cells and,
therefore, fi rst- and second-generation adenoviral vectors typically
only mediate short-term transgene expression in vivo [
1
-
3
].
In contrast, the absence of adenovirus gene expression from
HC-Ad vectors allows for long-term transgene expression without
chronic toxicity in small and large animal models [
4
-
8
]. While
being suitable for long-term transgene expression in quiescent or
slowly proliferating tissues in vivo, HC-Ad vectors are also charac-
terized by a large cloning capacity. Up to 36 kB of transgenic
sequences can be incorporated into the vector genome. This allows
for example to clone several expression cassettes and/or large
cis-acting elements into one vector genome in order to regulate
Δ
E1/
Δ
