Biomedical Engineering Reference
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differences reflect the nonphysiological regulation of rep and cap expression that
may occur in complementation systems, as was alluded to above. However, a pack-
aging plasmid that has the Rep78/69 expression is downregulated by changing the
initiation codon AUG to ACG. This modification is reported to give higher cap
expression tTA and higher yields of vector particles [112] .
Infectious adenovirus (Ad) can be replaced as the helper with a plasmid contain-
ing only the adenoviral E2A , E4 , and VA genes. All these genes together with the
E1A genes supplied by 293 cells provide a complete helper function in the absence
of adenoviral production [113] . Further simplification in the three plasmid transfec-
tion systems involved combining all three adenoviral genes ( E1A , E4 , VA ) and the
rep-cap genes into a single plasmid [114] . In general, all of these types of modifica-
tions increased vector productivity compared to earlier systems such as pAAV/Ad
[115] (104 particles per cell have been reported). In order to avoid DNA transfection,
the cells must still be infected by a helper adenovirus, but this can be removed read-
ily as a result of advances in downstream purification processes.
Release of the vector from a producer line having the vector stably integrated was
demonstrated by transfecting a rep - cap helper plasmid and infecting it with adenovi-
rus [116] . Yang et al. have constructed stable cell lines containing a rep gene capable
of generating functional rep protein by replacing the P5 promoter with a heterologous
transcription promoter [50] . Clark et al. [117,118] generated cell lines containing the
rep and cap gene cassettes having deleted for AAV ITRs. Infection of these cells with
adenovirus activates rep and cap gene expression. Moreover, the vector can be stably
incorporated into the packaging cells to yield AAV vector producer cell lines that need
only be infected with adenovirus to generate vector. Producer cell lines provide a scal-
able AAV vector production system that does not require manufacture of DNA.
A modification of the packaging cell line method is to use a similar cell line con-
taining a rep-cap gene cassette that is infected with an Ad/AAV hybrid virus having
an E1 gene-deleted adenovirus containing the AAV ITR vector cassette [119,120] .
This modification allows the same packaging cell line to be used for the produc-
tion of different AAV vectors simply by changing the Ad/AAV hybrid virus; this
is because after the infection of cells containing the rep-cap genes, the AAV-ITR
cassette is excised from the Ad/AAV and amplified and packaged into AAV parti-
cles. Reuse of the same packaging cell lines for production of different AAV vectors
requires coinfection with adenovirus to provide the E1 gene function. A packaging
cell system was described in which the packaging cell contains both a rep - cap cas-
sette and the AAV-ITR vector cassette, with both cassettes attached to a semian virus
40 (SV40) replication origin [121] .
Another modification of the cell-based approach utilizes an HSV/AAV hybrid
virus in which the AAV rep-cap genes are inserted into the HSV genome. This HSV/
AAV rep-cap virus can generate AAV vector when infected into cell lines along with
a transfected AAV vector plasmid, or into cell lines carrying an AAV vector provirus
[122,123] .
Hybrid Ad/AAV viruses can also be used as delivery vehicles for AAV vectors
[124,125] , but this modification suffers from some disadvantages such as induction of
innate immune responses characteristic of the adenoviral capsid interaction with cells.
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