Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 218

safety and increased transgene expression in some cases, but they cannot solve virus-
associated immunologic problems in vivo . Therefore, production of stable transgene
expression has not yet been established [36] . Construction and preparation of second-
generation vectors are relatively difficult compared with first-generation vectors.
Although second-generation Ads are easier to generate than fully deleted Ad (fdAd)
vectors, they have not gained widespread use because of their inconsistent perfor-
mance and marginal increase in cloning capacity compared with first-generation Ads.

5.2.5.4 Gutted Adenoviral Vectors
The gutted, or gutless, vector system has been developed in which all viral-coding
sequences are deleted, and this gutless vector system has reduced immunogenicity
and improved safety ( Fig. 5.7 ). Deletion of all Ad protein-coding sequences gives
rise to fdAd vectors. The only Ad sequences that must be retained in the fdAd are
approximately 500 bp of cis -acting DNA elements, including the viral ITRs located
at each end of the genome that are necessary for viral DNA replication, and the viral
packaging signal (  ). This is the only important element necessary for the packag-
ing of DNA into virions. Gutless vectors are also called helper-dependent Ad vectors
because the production of these gutless vectors depends on a helper virus to provide
viral gene products in trans to support the vector DNA replication and package.
The earliest production of gutless Ad vectors removed only a part of the viral-coding
sequences and had a mutated packaging region to decrease helper production [37] . But
with this system, high levels of helper virus contamination could not be avoided in the
final preparations [38] . An improved method for production of gutless Ad involved the
application of site-specific recombination of the bacterial phage P1 Cre recombinase
and loxP to remove the helper packaging region [39] . In this method, 293-derived cell
line 293Cre4 is used, because the Cre recombinase expressed in a 293-derived cell
line 293Cre4 cleaves the packaging signal flanked with loxP sites in a helper virus
(AdLC8cluc). The packaging signal in the helper virus DNA is excised through the
Cre/ loxP interaction by 293Cre4 infection so that the helper virus DNA, without its
packaging signal, cannot be packaged into viral particles but can still produce viral
proteins [40] . The viral proteins produced by the helper virus support the replication of
gutless Ad vectors, while the helper virus itself is restricted from packaging into viral
particles because its packaging signal has been deleted. Using this method, the helper
virus contamination decreased to 0.1% or lower levels in gutless Ad vector prepara-
tions, but this strategy cannot completely eliminate helper viruses in the final purified
gutless vector production [40] .
Morral et al. 1998 1998 [17] has reported that gutless Ad vectors are substantially

less toxic than other Ad vectors when high doses are administered intravascularly
in mice and that they have remarkably long-term expression of transgenes for 1 to 2
years after a single intravascular dose for delivery to the liver in mice and baboons
[41] . Using gutless vector with low-density lipoprotein (LDL) receptor gene therapy,
a long-term protection against atherosclerosis in a mouse model of familial hyper-
cholesterolemia has been produced [42] . Lifelong elimination of hyperbilirubinemia
in the Gunn rat with negligible chronic toxicity was achieved by gutless Ad vector