Biology Reference
In-Depth Information
derived from human serotypes 2 and 5 [
1
,
2
]. Strategies to circum-
vent this problem include the use of less prevalent human serotypes
or adenovirus vectors derived from nonhuman adenoviruses.
Canine adenovirus vectors type 2 (CAV-2) are most likely the
best-studied representative of nonhuman adenovirus vectors.
These vectors retain key advantages of human Ad5; yet, despite
intimate cohabitation of humans and dogs, CAV-2 has not been
shown to cross the species barrier and cannot replicate in human
cells [
3
]. Upon injection in the brain, CAV-2 vectors exhibit pref-
erential tropism for neurons and have shown a high level of retro-
grade axonal transport that allows them to reach cells located at
distant brain regions [
4
]. These characteristics make them particu-
larly attractive for targeting neurodegenerative diseases using gene
therapy approaches [
5
]. In addition, CAV-2 vectors are being eval-
uated as oncolytic agents in virotherapy approaches [
6
-
8
] and as
oral vaccines for the control of rabies in wild and domestic animals
[
9
-
13
].
The rising interest in CAV-2 vectors for gene therapy, onco-
lytic therapy and vaccine approaches calls for the development of
scalable GMP compliant production and purification strategies as
significant amounts of clinical-grade material are required for pre-
clinical and clinical trials [
14
]. Recently, scalable downstream pro-
cessing strategies specifically tailored for CAV-2 have been reported
[
15
,
16
]. These strategies combine multiple membrane filtration
and chromatography purification steps.
A detailed step-by-step protocol for the purification of CAV-2
vectors is reported here. Primary recovery of CAV-2 particles is
achieved by microfiltration and ultrafiltration-diafiltration. A nucle-
ase digestion step is carried out between ultrafiltration and diafiltra-
tion operations. Chromatography purification is accomplished by
capture of CAV-2 vectors using hydrophobic interaction chroma-
tography followed by CAV-2 vector polishing by anion exchange
chromatography. A representative scheme indicating the yield that
can be expected along the process is presented (
see
Fig.
1
). Of note,
the higher the number of downstream processing steps, the higher
the purity achieved but the lower the virus yield. Thus, the required
level of purity needs to be determined in advance according to the
application being considered. The complete strategy renders high-
quality CAV-2 vector preparations with low contamination with
empty viral capsids and other inactive vector forms as judged by
total and infective particle quantitation assays, OD
260/280
chroma-
tography absorbance ratios, electron microscopy and electropho-
retic analyses [
16
]. The complete process overall yield was estimated
to be between 38 and 45 % [
16
], which is well in line with those
reported for other viral vectors [
17
,
18
].
