Biology Reference
In-Depth Information
Ad5 vector [
15
-
17
]. Gross modulation of the hexon hypervariable
regions may also be advantageous in terms of evading preexisting
anti Ad5 immunity, since 30-90 % (depending on ethnicity) of
individuals have previously been exposed to Ad5 and thus present
neutralizing antibodies (NAbs) against the vector [
18
-
20
]. The
majority of studies have implicated the hexon HVRs as the major
site of immunological recognition [
20
], at least following intra-
muscular challenge (the repertoire of immunological recognition
following natural infection of airway epithelia may differ signifi -
cantly [
10
,
21
]). Therefore, applications which entail repeated
administration of Ad5 will require the generation of genetically
modulated viruses that can evade the host acquired immune
response against the vector [
20
].
In addition to the well-documented interactions overviewed
above, a number of other dose limiting interactions between virus
and host proteins have been partially described in the literature
that may preclude effi cacious delivery via the systemic route. These
interactions include (but are not limited to) interactions with com-
plement proteins (C3, C1q, C4BP) [
4
,
22
,
23
], von Willebrand
Factor, p-selectin [
6
], lactoferrin (produced locally at sites of
infl ammation [
24
]), and other proteins involved in the blood clot-
ting cascade (FVII, FIX, Protein C, FX) [
10
]. It is therefore clear
that the production of safer and more effi cacious generations of
adenoviral vectors for systemic gene therapy applications will neces-
sitate protocols that permit the rapid genetic modifi cation of the
Ad5 capsid to prevent such dose limiting interactions and
toxicities.
In a previous topic [
25
] we have described detailed procedures
for the production and purifi cation of fi rst generation Ad5 vectors,
including methods for producing adenoviruses with point muta-
tions (to ablate CAR interactions) or peptide insertions (for retar-
geting strategies) in the fi ber protein, as well as point mutations
within the hexon protein (to ablate FX interactions). Here we
update and extend upon the protocols previously published to
include some new quality control procedures for validating viral
integrity (silver staining and use of NanoSight) as well as describ-
ing methods for ablating the penton base-integrin interaction, and
exchanging the hexon hypervariable regions from less seropreva-
lent adenoviral species into Ad5, to reduce recognition by preexist-
ing anti-hexon neutralizing antibodies and (potentially) to ablate
interactions with blood clotting factors. Our strategies are based
on “AdEasy-like” systems, necessitating the design of 4-8 kb shut-
tle plasmids containing the relevant gene, which are appropriate
for genetic manipulation, fl anked by ~500-1,500 bp fl anking
regions of homologous Ad5 sequence to permit homologous
recombination with the backbone Ad5 genome cosmid to gener-
ate a full Ad genome for propagation of genetically modulated
adenoviruses (overviewed in Fig.
1
).
