Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 213

to loop. Adenovirus is a successful method of gene delivery because the adenoviral
genome is easily manipulated; it can be propagated to high titers and can infect both
dividing and nondividing cells.
5.2.5 Design and Construction of Adenoviral Vectors
Two components are used for the production of recombinant adenoviral vectors:
(1) viral DNA vector and (2) packaging cell line. The adenoviral DNA vector is a
plasmid DNA that contains a portion of the viral genome having the E1A region
deleted, and desired genes are cloned into a multicloning site that has been inserted
in place of the E1A region of the genome. The large size of the adenoviral genome
prevents the use of a single plasmid-based system in vector production. Instead of
the above method, the adenoviral vector is produced using either in vitro ligation or
homologous recombination [5] .
In the in vitro ligation method, wild-type adenoviral DNA is isolated and cleaved
with the restriction endonuclease ClaI, and the digested viral DNA is then ligated
onto the adenoviral DNA vector of interest that has previously been digested with
ClaI. Finally, the ligated DNA species is transfected into the packaging cell line.
In the homologous recombination system, we can use either ClaI digested viral
DNA or a plasmid containing all the adenoviral sequence downstream from the ClaI
site. Adenoviral DNA vector and the viral DNA component are both cotransfected
into the packaging cell line, and these DNA species are then left to undergo homolo-
gous recombination within the cells, resulting in vector production. The different cell
lines used for the production of adenoviral vectors are shown in Table 5.2 .
Over the years, many methods for manipulating the viral genome have been
developed. Currently, there are three classes of adenovirus vectors being developed
for gene therapy purposes ( Fig. 5.6 ) [6,7] .
1. First-generation vectors
2. Second-generation vectors
3. Gutted vectors
5.2.5.1 First-Generation Vectors
The early versions of adenoviral vectors (Ad) had modest mutations. First-generation
Ad vectors are those having deletions of the E1a and E1b genes. Expression cassettes
of therapeutic or reporter genes are inserted in the deleted E1 region [8] . Removal of
the E1 region hampers the transcription of E2 and other viral genes and consequently
blocks viral DNA replication. E1 products are most important for virus replication;
vectors cannot be replicated in infected cells with deletion of E1. The defective E1
viruses could only be produced in E1-complementing cells, such as 293 cells [9,10] .
With deletion of the E1 regions, the vector can carry a transgene of 3- to 4-kb DNA.
For improvement of the carrying capacity in first-generation vectors, the E3 region
is also removed in some of the first-generation vectors, because E3 products are not
essential for adenovirus replication in cell cultures. Deletions of both E1 and E3 allow
the introduction of 5 to 6 kb foreign DNA into first-generation vectors. A further