Gene Delivery and Gene Therapy for Alzheimer’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 94

in the same manner as AAV293 [ 37 ]. Production technologies
have recently been improved to allow production methods that are
more scalable than using adherent HEK293 cells for rAAV produc-
tion. These include HEK293 cell lines that can grow in serum-free
suspension, the dual rHSV infection system of suspension baby
hamster kidney cells, and the baculovirus expression vector system
(BEVS) utilizing Spodoptera frugiperda 9 (Sf9) insect cells ( see ref. 28 ).
These systems are especially appropriate for clinical applications,
where scalability of rAAV production is essential.
In addition to the preparation of rAAV in large quantities, it
must also be purifi ed in large quantities for clinical use. Purifi cation
rids the preparation of contaminants such as “empty shells” (AAV
particles lacking viral nucleic acid) or adenoviral particles if adeno-
virus is used as a helper virus. The traditionally used method of
sedimentation through CsCl gradients is not scalable and has been
replaced by nonionic iodixanol gradient purifi cation or ammonium
sulfate precipitation, followed by ion exchange or heparin/agarose
column chromatography [ 63 ]. This method is based on the fi nd-
ing that AAV2 binds to the heparin sulfate proteoglycan surface
molecule [ 64 ]. AAV6 is also known to bind heparin with moderate
affi nity, but despite its high sequence homology with AAV1
(~99.2 %), AAV1 does not exhibit this quality [ 65 ] and must there-
fore be purifi ed using other methods such as ion exchange chro-
matography [ 63 ]. Wu et al. identifi ed a single amino acid (K531)
that is essential for conferring the heparin binding characteristics of
AAV6 and that introduction of an E531K change in AAV1 yields a
heparin binding ability similar to that of AAV6 [ 30 ].

1.4 Gene Delivery
to the Central Nervous
System

There are many potential risks associated with viral gene delivery
in humans, and therefore certain considerations must be made
for studies involving CNS gene delivery. Unless AAV9 is used in
neonates, therapeutic vectors must be administered directly into
the CNS. This requires bilateral stereotaxic surgery on anesthe-
tized subjects, which can lead to signifi cant side effects [ 66 , 67 ].
In this case, gene transfer must be locally restricted to minimize
the risks of serious adverse effects that can result from broad dis-
tribution. Thus, intracranial injections must be optimized, and
vectors must be designed in order to maximize effi ciency and
specifi city of transduction to minimize required dose. Though
the fi rst rAAV clinical trials used AAV2, most recent clinical trials
use AAV2 ITRs pseudotyped with capsids of other serotypes such
as AAV5 or 8 [ 49 , 68 , 69 ], allowing increased transduction effi -
ciency of targeted cell types, as well as avoidance of AAV2-
neutralizing antibodies. Increased transduction effi ciency is
important for minimizing dose.
Although simple injection methods using a constant fl ow rate
have proven to work well for rAAV delivery, some researchers have
developed methods that may improve brain delivery of rAAV.