Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 228

appear to retain specificity [102,103] , and a number of other regulated expression
systems have now been used successfully in AAV vectors. Introns function in AAV
vectors and may enhance expression, and more than one promoter and gene cassette
can be inserted in the same vector. Transcription from AAV does not seem to be sus-
ceptible to in vivo silencing, as shown by expression for over a year after intramuscu-
lar delivery in rodents [104,105] .
5.3.4.2 Production
It is noted that the production of AAV is highly efficient because AAV has one of the
largest burst sizes of any virus. Following infection of cells with AAV and adenovirus
as a helper, the burst size of AAV may be well in excess of 100,000 particles per cell
[98] . In development of vector production systems, the burst size of AAV yielding a
high number of AAV vector particles per cell is theoretically attainable. High spe-
cific productivity is crucially important in developing scaled-up production, because
the ability to obtain maximum yields ideally requires either high specific productiv-
ity (yield of particles per cell) or a large biomass (total number of cells). For max-
imization, the specific production may avoid unnecessary increases in biomass. In
AAV vectors production, the cytostatic properties of the AAV rep protein presented
a difficulty for generating stable packaging cell lines [106] . AAV vector production
is based on the transfection of a vector plasmid and a second plasmid, to provide
complementing rep and cap functions, into adenovirus-infected cells, usually the
transformed human 293 cell line. But these original vector production systems have
relatively low specific productivity. Moreover, recombination between the vector
plasmid and complementing plasmids generates wild-type AAV (wtAAV), pseudo-
wild-type or replication-competent AAV (rcAAV) or other recombinant AAV species
[107,108] . Production of AAV vectors can also be increased by using two further
improved methods such as DNA transfection-based methods, as well as development
of new cell-based AAV production systems (that do not require DNA transfection).
Also, an important advantage is that, the transfection systems in which adenoviral
infection is replaced by DNA transfection with the relevant adenoviral genes are now
available. Both the transfection and cell-based approaches can give much higher pro-
ductivity in excess of 104 vector particles per cell. But, the cell-based systems are
possibly more acceptable to scale-up for commercial production than are DNA trans-
fection systems. Moreover, significant improvements in downstream of AAV vectors
have been made by using chromatographic procedures [107,108] .
5.3.4.2.1 Complementation Systems
For enhancement in the upstream production of AAV, various modifications have
been made to the complementing rep-cap cassette in an attempt to enhance specific
productivity and to decrease production of rcAAV. One study suggested that expres-
sion of rep and cap proteins may be limiting [109] ; some other reports [110,111]
also indicated that production of cap proteins were limiting owing to downregulation
of cap by increased production of rep. This is very surprising, because in the AAV
replication cycle, rep protein increases expression of cap. Perhaps these apparent