Gene Delivery and Gene Therapy for Alzheimer’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 93

1.3 Advancements
in rAAV Production

As a complement to innovations in vector design, improvements in
methods for rAAV manufacturing have advanced AAV gene ther-
apy for human use. These include the engineering of plasmids car-
rying adenovirus helper functions without the complete adenovirus
genome, stable “producer” cell lines providing helper functions,
and purifi cation methods resulting in large rAAV yields appropriate
for clinical use.
Recombinant AAV was historically produced after gene con-
struction by using wild-type adenovirus as a helper virus. This
method involves transfecting cell lines that stably harbor AAV rep/
cap genes with wild-type adenovirus and the AAV vector
DNA. Although this can be scaled up to produce vectors with high
titers, it is diffi cult to avoid adenovirus contamination in this
method even with the use of repeated cesium chloride (CsCl) gra-
dients. This has been overcome by the development of the “mini-
Ad genome,” plasmids carrying only the adenovirus helper genes
(which include the E1a, E1b, E2a, E4, and VA RNA genes). This
adenovirus-free method involves transient transfection of all ele-
ments required for AAV production into host cells (such as
HEK293 cells): an rAAV ITR-containing plasmid carrying the
gene of interest, a plasmid that carries AAV rep and cap genes, and
a helper plasmid with only the adenovirus helper genes. This triple
transfection “helper-free” method was fi rst used with the mini-Ad
genome plasmid PXX6 [ 60 ], and since then similar plasmids have
been developed, such as pFD13, which has an 8-kb deletion in the
adenovirus E2B region and most of the late genes [ 37 ]. The mini-
Ad genome preceded the development of pDG, a plasmid with
AAV2 packaging functions and adenovirus helper functions (AAV2
rep and cap genes and VA, E2A, and E4 genes of adenovirus 5),
allowing for transfection of only two plasmids [ 61 ]. Additional
helper viruses based on pDG, with its original AAV2 cap genes
replaced by cap genes from AAV1 and AAV3-6, were engineered
by the same group, in order to achieve increased effi cacy in varied
cell types [ 62 ].
Other innovations have been developed for maximized AAV
growth and scalability of production. “Producer” stable cell lines,
such as the AAV293 cell line, provide Ad genes to support AAV
growth and produce higher viral titers (Agilent #24003). AAV293
cells are human embryonic kidney cells derived from the com-
monly used HEK293 cell line that have been transformed by
sheared adenovirus type 5 DNA. AAV293 cells produce the adeno-
virus E1 gene in trans , allowing the production of infectious AAV
particles when cells are cotransfected with three AAV helper-free
system plasmids (an ITR-containing plasmid, pAAV-RC which
supplies rep and cap genes in trans , and an E1-deleted helper plas-
mid). Another producer cell line, 84-31 cells (Human Applications
Laboratory, University of Pennsylvania), are a subclone of HEK293
cells that stably express E4 of the adenovirus genome and are used