Blood, 15 April 2003, Vol. 101, No. 8, pp. 3338-3338
CORRESPONDENCE
To the editor:
The liver sieve and gene therapy
We were intrigued by the article by Mount et
al1 on the reversal of hemophilia B following the
intraportal injection of an adeno-associated viral vector encoded with
the coagulation factor IX gene into hemophiliac dogs. Mount et al
reported substantial correction of hemophilia B in 3 of the 4 animals.
However, we think that the fourth dog, Beech, is more interesting. He
obtained only temporary reversal and died. Necropsy revealed early
cirrhosis thought to be associated with iron overload secondary to
concurrent pyruvate kinase deficiency.1 Furthermore, at 12 years of age, Beech was the oldest dog studied.
Cirrhosis and old age both are associated with loss of the liver
sieve.2,3 The "liver sieve" is a term used to describe the endothelial cells that line the hepatic sinusoids. These cells are
perforated by multiple fenestrae of approximately 100-nm diameters, within very thin cytoplasmic extensions (Figure
1).
Lacking a basal lamina, sinusoidal fenestrae are truly discontinuous
and thus allow unimpeded passage of macromolecules up to those with a
diameter of about 100 nm including, potentially, viral vectors such as
those involved in gene therapy. Indeed, we suggested 25 years ago that
the normal liver sieve allows circulating viruses smaller than 100 nm
to contact hepatocytes,4 and this has largely been
substantiated since.5,6 The adeno-associated virus used by
Mount et al has a diameter of about 20 nm.7
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| Figure 1.
Scanning electron micrograph of the liver sieve,
demonstrating fenestrations clustered into liver sieve plates.
The scale bar indicates 1 µm.
|
|
Mount and colleagues suggested that in Beech's case the usual hepatic
tolerance of the viral vector and gene was decreased secondary to a
change of the normal tolerance-inducing liver endothelial cell
phenotype that might occur in cirrhosis. We agree, and posit that loss
of fenestrations hinders contact between hepatocytes and circulating
lymphocytes, which also is known to induce tolerance.8 We
therefore suggest that loss of fenestrations in the hepatic sinusoids,
by hindering interactions between both the lymphocytes and the viral
vector in the portal blood with the hepatocytes, decreased the uptake,
expression, and tolerance of the introduced viral vector and its gene
in Beech.
Conversely, it is of interest that interventions undertaken to improve
success of gene therapy, such as partial hepatectomy,9 are
associated with increased fenestration of the liver
sieve.10 Therefore, modulation of the liver sieve may
prove to have an important role in the outcome of gene therapy, either
through influencing hepatocyte-lymphocyte interactions or,
alternatively, the physical uptake of the viral vector.
Robin Fraser, David
G. Le Couteur, Alessandra Warren, Victoria C. Cogger, Patrick Bertolino, and Mark Smith
Correspondence: David Le Couteur, CERA, Concord RG
Hospital, NSW 2139 Australia; e-mail:
dlecouteur{at}med.usyd.edu.au
References
1.
Mount JD, Herzog RW, Tilson DM, et al.
Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy.
Blood.
2002;99:2670-2676[Abstract/Free Full Text].
2.
Fraser R, Dobbs BR, Rogers GW.
The role of the fenestrated sinusoidal endothelium in lipoprotein metabolism, atherogenesis and cirrhosis.
Hepatol.
1995;21:863-874[CrossRef][Medline]
[Order article via Infotrieve].
3.
Le Couteur DG, Fraser R, Cogger VC, McLean AJ.
Hepatic pseudocapillarisation and atherosclerosis in aging.
Lancet.
2002;359:1612-1615[CrossRef][Medline]
[Order article via Infotrieve].
4.
Fraser R.
Thoughts on the liver sieve.
Bull Kupffer Cell Found.
1978;1:46-47.
5.
Steffan AM, Pereira A, Bingen M, et al.
Mouse hepatitis virus type 3 infection provokes a decrease in the number of sinusoidal endothelial fenestrae both in vivo and in vitro.
Hepatol.
1995;22:395-401[CrossRef][Medline]
[Order article via Infotrieve].
6.
Pereira CA, Steffan AM, Kirn A.
Kupffer cell and endothelial liver cell damage renders A/J mice susceptible to mouse hepatitis virus type 3.
Virus Res.
1984;1:557-563[CrossRef][Medline]
[Order article via Infotrieve].
7.
Flotte TR.
Size does matter: overcoming the adeno-associated virus packaging limit.
Respir Response.
2000;1:16-18.
8.
Bertolino P, McCaughan GW, Bowen DG.
Role of primary intrahepatic T-cell activation in the "liver tolerance effect."
Immunol Cell Biol.
2002;80:84-92[CrossRef][Medline]
[Order article via Infotrieve].
9.
Hirano T, Fujimoto J, Ueki T, et al.
HVJ-liposome mediated gene transfer into hepatocytes in vivo.
J Hepatol.
1998;29:910-914[CrossRef][Medline]
[Order article via Infotrieve].
10.
Wack KE, Ross MA, Zegarra V, Sysko LR, Watkins SC, Stolz DB.
Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat.
Hepatol.
2001;33:363-378[CrossRef][Medline]
[Order article via Infotrieve].
Response:
AAV-mediated gene transfer to liver
Fraser et al raise the interesting possibility that loss of
fenestrations in the endothelial cell lining of the hepatic sinusoids might, in animals with cirrhosis, result in decreased uptake and expression of vector administered via portal vein and poor induction of
tolerance to the transgene product on that basis. While we agree that
the role of the "liver sieve"
and indeed the role of portal vein
versus hepatic artery administration in induction of tolerance to the
transgene producthas not been thoroughly studied yet, we do not
concur that our data support a hypothesis based on reduced transduction
in the case of the animal with early cirrhotic changes. As noted in
Figure 1 of Mount et al,1 this animal initially showed a
rapid rise in circulating levels of factor IX, reaching a level of 600 ng/mL at 3 weeks after vector administration and 2500 ng/mL at 4 weeks.
Note that these levels are 4-fold to 10-fold higher than levels seen at
the same time point in dogs receiving a 3-fold lower dose. Thus, uptake
and expression of vector were excellent in the cirrhotic animal
initially. Subsequently, this animal developed an inhibitory antibody;
although we cannot exclude reduced interaction between lymphocytes and hepatocytes as a factor in failure of tolerance induction, the role of
such an interaction is entirely speculative. We suspect that other
factors besides this anatomic one are involved in the difference in
immune response to the transgene product seen in the cirrhotic versus
the normal animals. Differences in the hepatic microenvironment in the
cirrhotic liver (eg, local cytokine concentrations and number and
identity of antigen-presenting cells) and/or differences in the level
of antigen (factor IX) produced and presented
(since the cirrhotic animal received a 3-fold higher dose) are both
potential factors in the different immune response to the transgene
product in the cirrhotic animal versus animals with normal livers.
In the ongoing clinical trial based on these findings,2
vector is delivered via the hepatic artery rather than the portal vein
(since the former can be accessed via percutaneous cannulation of the
femoral artery). In preclinical studies in dogs with hemophilia B
caused by a missense mutation,3 levels of transgene
expression are equivalent using these 2 routes of administration
(K.A.H., V.A., unpublished data, May 2002), and neither route
results in inhibitory antibody formation at doses up to
1 × 1012 vector genomes (vg)/kg. Mingozzi et
al4 have shown that portal vein administration of AAV-F.IX
vector in hemophilic mice induces tolerance through the induction of
antigen-specific CD4+ T regulatory cells and that such
tolerance can be adoptively transferred. Whether a similar mechanism
accounts for lack of inhibitor development in hemophilic dogs and
humans is currently under investigation. If the mechanism of tolerance
induction is similar whether vector is administered via the portal vein
or the hepatic artery, this would argue against a role for the "liver sieve" in this phenomenon, since the fenestrated sinusoidal
endothelium is a characteristic of the portal circulation but not of
the hepatic arterial vasculature.
Katherine A. High, Roland Herzog, and Valder Arruda
Correspondence: Katherine High, Children's
Hospital of Philadelphia, Hematology Division, 34th St and Civic Center
Blvd, Philadelphia, PA 19104; e-mail:
high{at}emailchop.edu
References
1.
Mount MJ, Herzog RW, Tillson M, et al.
Sustained high-level correction of inhibitor-prone hemophilia B dogs by liver-directed gene therapy.
Blood.
2002;99:2670-2676[Abstract/Free Full Text].
2.
Kay MA, High K, Glader B, et al.
A phase I/II clinical trial for liver-directed AAV-mediated gene transfer for severe hemophilia B.
Blood.
2002;100:115.
3.
Evans JP, Brinkhous KM, Brayer GD, Reisner HM, High KA.
Canine hemophilia B resulting from a point mutation with unusual consequences.
Proc Natl Acad Sci U S A.
1989;86:10095-10099[Abstract/Free Full Text].
4.
Mingozzi F, Liu Y-L, Dobrzynski E, Arruda VR, High KA, Herzog RW.
AAVmediated hepatic gene transfer induces regulatory CD4+ T cells promoting tolerance to the coagulation factor IX transgene product.
Blood.
2002;100:116.
