Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 216

(Clontech, Mountain View, CA), AdEasy™ XL System (Stratagene, La Jolla, CA),
and ViraPowerì Adenoviral Expression System (Invitrogen, San Diego, CA) are
available for construction of first-generation Ad vectors. Construction of a full-length
adenovirus genome can be captured in a plasmid form that forms infectious virus
following transfection into permissive cell lines but the large size of these plasmids
(  40 kb) makes standard cloning procedures difficult [20] . For benefit of the natural
recombination pathways present in prokaryotic and eukaryotic cells, several methods
for viral construction have been developed. One of the earliest systems for creating
first-generation Ad employed the recombination pathway in mammalian cells follow-
ing the transfection of two DNAs [19] :
1. A plasmid containing the left-hand portion of the virus (including the packaging signal and
5 ITR) with an appropriate expression cassette replacing the E1 sequences.
2. The right-hand portion of the viral genome (either purified viral DNA digested with Xba
I/C1aI to remove the packaging signal or a plasmid containing a circularized genome).
Recombinant viral genomes are produced by recombination between homologous
Ad sequences on the two plasmids. Unfortunately, this method was not found to be
highly efficient. Recently, the system of using a site-specific recombinase (Cre) to
mediate recombination between the two plasmids, instead of the natural homologous
recombination pathways in mammalian cells, has been improved 100-fold [21] .
The gene of interest was cloned into a shuttle plasmid containing the Ad5 packaging
signal and both 5 and 3 ITRs. The shuttle plasmid was cotransfected with a genomic
plasmid that lacks a packaging signal and contains an expression cassette encoding the
Cre recombinase. Cre mediates recombination between lox P sites contained in both the
shuttle plasmid and genomic plasmid, facilitating the generation of viral genomes [21]
( Fig. 5.7 ). An alternative method has been developed, using homologous recombination
in bacteria, which is more efficient than the mammalian system [22] . In this method,
bacteria were transformed with two plasmids: (1) a shuttle plasmid similar to the one
used for rescue in mammalian cells and (2) a circularized Ad genome with a deletion
of the entire 5 region, including the 5 ITR, packaging signal, and E1 region.
The advantages of this design are as follows:
1. Recombinants are identified by a conversion of antibiotic resistance from ampicillin
(parental circularized Ad) to kanamycin (recombinant circularized Ad).
2. By screening recombinants in Escherichia coli rather than mammalian cells, the length of
time required to identify the appropriate virus is greatly reduced.
The plasmid can be isolated, linearized (Ad ITRs must be present at the termi-
nal of a linear molecule for efficient DNA replication), and transfected into an E1-
complementing cell line when the correct viral genome is identified, where it will
generate recombinant Ad. More recent improvement to this approach utilizes a cosmid-
based vector (cosmid is a type of hybrid plasmid often used as a cloning vector) that
contains cos sequences , DNA sequences originally from the Lambda phage [23] .
Rather than depending on homologous recombination, the shuttle plasmid is ligated
directly to the cosmid prior to packaging in  particles. The shuttle plasmid provides
both the left cosmid arm, necessary for packaging into phage particles, and the lacZ