Construction

Although the pathology associated with wild-type adenovirus infections is generally mild, there is a potential risk of using E1+ Ad for gene transfer in that the inflammatory host responses to Ad infection may alter organ function (9). There is also the possibility of overwhelming infection if Ad replication is allowed to progress when there are deficiencies in the host defense system. Because the E1A products are essential for expression of other early and late genes and for DNA replication, the most direct approach to eliminating replication is to delete the E1A genes. To produce E1" Ad vectors, the classic approach is to transfect the recombinant E1 ~ Ad vector genome into the human embryonic kidney cell line 293, a cell line originally established by transforming primary cells with Ad5 (27,28). The 293 cells contain approximately 11 map units of the Ad5 genome, originating at the left-hand end (29).

One example of a so-called ''first-generation'' adenovirus vector expresses the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA under control of the constitutively highly active cytomegalovirus (CMV) immediate/early promoter (Fig. 4) (30). A polyadenylation site is located following the cDNA, and the whole expression cassette in a left to right orientation replaces the E1A and part of the E1B genes. Because the expression cassette is 5601 bp in length and the E1 deletion is 3062 bp, it is necessary to delete part of the E3 region to construct the vector. Because the E3 region is nonessential in vitro, this deletion does not affect propagation of the replication-deficient virus in 293 cells. However, for some therapeutic genes, if the extra space is not necessary, the E3 region can be retained.

The genomes of first-generation (E1~, E3~) Ad are now typically constructed and amplified as Escherichia coli plasmids (Fig. 4), followed by transfection into 293 cells for the production of vector. In one widely used system (31), the Ad genome is created by homologous recombination between a shuttle plasmid and a backbone plasmid. The shuttle plasmid consists of the extreme right- and left-hand ends (map units

1-16 and 97-100) of the Ad genome with the expression cassette for the transgene in the deleted E1 region (map units

2-10 deleted). The backbone plasmid contains most the Ad genome (map units 10-100), except for the extreme left-hand end and a deletion of E3. Recombinants between the shuttle and backbone plasmids containing the whole vector genome are selected by appropriate antibiotic resistance markers.

Once made, a new vector is plaque purified repeatedly in 293 cells (to remove any contaminating wild-type virus) and is then propagated to produce the required amounts of the vector. Under standard laboratory conditions, it is possible to produce up to 2 X 1013 viral particles from fifty 150-mm cell culture plates (about 109 293 cells). The recombinant Ad is easily purified from cell lysates on equilibrium cesium chloride density gradients. After purification, the vector is assayed for infectivity by plaquing efficiency on 293 cells, the presence of contaminating replication-competent Ad (RCA) by the plaquing efficiency on A549 cells (an E1" cell line) (32), and the activity of the transgene (using whatever assay is relevant). Titer on 293 cells gives the titer in plaque-forming units (pfu) per milliliter. This has historically been the activity unit used to standardize doses for experimental animals and patients. However, it has become evident that the pfu is an arbitrary, poorly reproducible measurement, and thus most laboratories now use particle units (pu) as the dosing unit, based on the premise that highly purified viruses made by a standard protocol represent a uniform population of potentially infec tious units. The particle count is calculated from the absor-bance at 260 nm using the formula 1A260 = 1.25 X 1012 particles/mL, and is typically 10 to 100 times the titer in pfu (33).

0 0

Post a comment