Adeno-Associated Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 6

or regeneration of tissues [ 38 ]. Several approaches are being taken
to overcome this limitation. Targeted integration of rAAV genomes
with resulting long-term transgene expression has been achieved by
co-infecting animals with the therapeutic AAV alongside an rAAV
encoding a suitably designed zinc finger protein (ZFN) [ 39 ]. In
cases where direct control over the integration site is not required,
the need for this co-infection with a ZFN-encoding virus may be
removed by incorporating rDNA sequences in the rAAV genome,
resulting in an upregulation of integration events to the 26srDNA
locus of the human genome and increased longevity of transgene
expression [ 40 ]. In the near future, it seems likely that Scaffold
Matrix Attachment Regions (S/MAR) [ 41 ] and Tal Effector
Nucleases (TALEN) [ 42 ] will be added to the toolkit available to
researchers in this field, to improve episomal maintenance and to
facilitate genome editing and integration, respectively.

1.4 Production,
Purification, and
Titration of AAV

The aim of this section is to introduce and describe the techniques
involved in the production, purification, and titration of high
purity and high titer AAV vectors, suitable for preclinical studies.
While many variations of the central concepts exist, the techniques
described in this chapter have been derived through optimization
at each step along the process to establish a protocol yielding high-
quality virus in a relatively easily reproducible manner, utilizing
standard laboratory equipment.
We present here two different approaches of AAV vector pro-
duction. The classical approach employs calcium phosphate-
mediated triple transfection of HEK293T cells and vector harvest
after 48 h. The relative abundance of virus in cells and in the
medium at this time point varies between serotypes. In our experi-
ence, laboratories vary in their approach to using pellet and media
for various preps, discarding one or the other or combining the
two fractions with specialist homogenization equipment. However,
simple postponing of the vector collection to 120 h after transfec-
tion could double the total vector yield from the culture media.
Because supernatant is quite a pure source of the virus, in contrast
with cell-derived lysate which contains the bulk of cellular con-
taminants, vectors present there can be more efficiently separated
by well-described ultracentrifugation in iodixanol gradient [ 43 ,
44 ]. Additionally, it has been shown that NaCl addition to the
culture media to the final concentration of 500 mM at 2 h before
vector harvest encourages the release of AAV vectors to the culture
media. This is presumed to be a consequence of cellular stress and
has variable efficacy between serotypes [ 43 ]. Here we will focus on
two easier and more reproducible calcium phosphate and PEI-
based transfections with purification of an AAV9-CMV-GFP vector
from cells and supernatant, respectively.