Haematologica 2000;85:supplement to no. 10 - Supplements ...

110
D. Lillicrap
a transgene in tissues such as skin, where APCs
are abundant, may significantly increase the risk
of effective antigen presentation. Another way in
which this process can be influenced is through
the use of transgene constructs in which tissuespecific
regulatory elements are employed to preclude
transgene expression in APCs. The benefit
of such a strategy has recently been demonstrated
using an early generation, E1 deleted,
adenoviral vector delivering a human α1 antitrypsin
(hAAT) transgene delivered by tail vein
injection to C3H/HeJ mice. 4 In these studies,
long-term transgene expression was documented
with the use of a regulatory element derived
from the murine albumin promoter and
enhancer, whereas, in contrast, using an ubiquitous
mouse phosphoglycerate kinase (PGK) promoter,
serum hAAT levels returned to baseline
within three weeks after an initial period of
expression. These observations coincided with
the development of high titer antibodies to hAAT
in the mice injected with the PGK-regulated
transgene construct. These results add further
strength to the hypothesis that limiting expression
of the transgene product to cells that do not
present antigen efficiently significantly reduces
the risk of subsequent inhibitor formation.
Finally, transient administration of agents that
interfere with the immune response have also
shown some promise in reducing the likelihood
of a subsequent humoral response. Standard
immunosuppressive agents such as cyclophosphamide
and cyclosporin A have been delivered
as a single high dose pulse at the time of vector
administration and a similar strategy using the
inhibitor of APC-mediated T-cell co-stimulation,
CLA4Ig, has also been documented to reduce
the risk of a productive humoral response.
Role of the transgene product
Most of the current gene therapy strategies for
hemophilia involve the ultimate synthesis of a
transgene product that will differ from the native
coagulation protein. In studies of factor VIII
gene delivery, all of the transgene constructs to
date have utilized B domain deleted cDNAs,
while in the case of factor IX gene therapy protocols,
cell types other than hepatocytes have
proven to be effective targets. In all of these situations,
the secretion of a transgene product
which is either grossly or subtly different in structure
to the native protein may result in the development
of inhibitory antibodies. With regards
to the B domain deleted factor VIII transgene
products, previous experience from both
immunogenicity studies in mice 5 and from clinical
observations in hemophiliacs treated with
the recombinant factor VIII product, ReFacto,
suggest that at least when administered as an
exogenous protein, this molecule is no more
immunogenic than native factor VIII. In the
ReFacto studies, after a median of 12 exposure
days, 30 of 101 previously untreated patients
developed an inhibitor (13≥10 B.U.: 4 between
5-10 B.U. and 13 ≤ 5 B.U.) and the inhibitors
have persisted in 11 patients. 6 These figures for
inhibitor incidences are comparable to those
obtained with other full-length recombinant factor
VIII products. Similarly, only one of 113 previously
treated patients has developed an
inhibitor after 93 exposure days to a ReFacto
further indicating the relative lack of neo-antigenicity
associated with this B domain deleted
product. 7 Finally, with reference to recent comparative
studies of factor IX synthesized from
myotubes, significant differences have been documented
for several post-translational modifications
present in native, hepatocyte-derived
factor IX including reduced tyrosine sulphation,
serine phosphorylation and different patterns of
glycosylation. 8 It remains to be seen whether
these more subtle alterations in structure will
produce a greater risk of inhibitor development.
Enhanced immune activation
Optimal initiation of the immune response to
antigen requires that the antigen-presenting cells
become activated by some form of pathophysiologic
danger signal. 9 This signal is most effectively
delivered in the form of an inflammatory
state or through cell death and tissue damage.
In the context of hemophilia gene therapy, examples
already exist in which increased activation of
the immune response has followed different
forms of gene delivery with the subsequent
development of an inhibitor response. In the first
of these cases, adenoviral delivery of a B
domain-deleted canine factor VIII transgene to
two hemophilic dogs resulted in acute hepatotoxicity
and, in one of the dogs (from the
inhibitor prone part of the pedigree), a factor
VIII inhibitor response peaking at 1,200 B.U. 10 In
the second instance in which an enhanced state
of immunologic activation may have played a
role in inhibitor development, a neutralizing
anti-canine factor IX antibody developed transiently
in one of five hemophilic dogs in which a
canine factor IX AAV transgene had been delivered
intramuscularly. 11 This animal was distinct
from the four others in the study because of the
occurrence of a clinically evident skin infection.
These findings suggest that hemophilia gene
therapy in subjects in whom the immune system
is already primed by an inflammatory state or by
tissue damage, or in whom the transgene delivery
protocol is likely to incite such danger signals
may be more likely to mount a productive
response to the transgene product.
Haematologica vol. 85(supplement to n. 10):October 2000