From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
should be considered. In addition, although
there are various approaches to activate innate
immunity components, including NK cells, it
is important to select those approaches that
not only activate innate immunity but also
connect innate immunity to adaptive immunity. As demonstrated in this study, interleukin-12, a potent activator of NK cells, is less
potent in the eradication of tumor dormancy
and it is likely that this is partially due to the
inability of this cytokine to induce B7-H1 on
NK cells. ■
REFERENCES
1. Chen L. Co-inhibitory molecules of the B7-CD28 family
in the control of T-cell immunity. Nat Rev Immunol.
2004;4:336-347.
2. Wang SD, Bajorath J, Flies DB, Dong H, Honjo T,
Chen L. Molecular modeling and functional mapping of
B7-H1 and B7-DC uncouple costimulatory function from
PD-1 interaction. J Exp Med. 2003;197:1083-1091.
3. Dong H, Strome SE, Salomao DR, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002;8:793-800.
4. Saudmont A, Quesnel B. In a model of tumor dormancy,
long-term persistent leukemic cells have increased B7-H1
and B7.1 expression and resist CTL-mediated lysis. Blood.
2004;104:2124-2133.
● ● ● GENE THERAPY
Comment on Schuettrumpf et al, page 2316
Gain-of-function proteins for gene
transfer
in hemophilia
---------------------------------------------------------------------------------------------------------------Francesco Bernardi
FERRARA UNIVERSITY ITALY
Schuettrumpf and colleagues report in this issue of Blood an innovative mouse model of
gene therapy for hemophilia B that could substantially improve the efficacy of gene transfer.
emophilia B, an X-linked bleeding disorder characterized by a deficiency of coagulation factor IX (F.IX), is a preferred
model for gene-based therapy because, in patients lacking this factor, 1% of normal levels
could substantially reduce spontaneous bleeding. In past years, several groups1 have demonstrated sustained expression of clotting factors
in animal models of hemophilia. The goal of
the ongoing clinical studies is to determine
whether these results can safely and efficiently
H
be extended to humans. However, achieving
and maintaining therapeutic F.IX levels is still
a difficult task. In hemophilia B patients, intramuscular injection of adeno-associated viral
(AVV) vectors, encoding F.IX driven by viraland tissue-specific promoters, has produced
only subtherapeutic doses.
Schuettrumpf and colleagues have devised a
mouse model of gene therapy for hemophilia B
based on transduction by AAV vectors of mutant
F.IX molecules characterized by advantageous
biologic properties. Basic molecular biology and
biochemistry studies conducted in the 1990s
have found that lysine 5 and valine 10 are essential for interaction of F.IX with endothelial cells
(collagen IV),2 and that the R338A change
modulates the catalytic efficiency (3-fold increase in kcat and a 2-fold decrease in Km) of activated F.IX bound to its cofactor, factor VIII.3
This could improve F.IX biodistribution and
availability to procoagulant macromolecular
complexes by impairing F.IX binding to useless
targets in the extracelluar space.
The optimized release of F.IX into the circulation and the gain of biologic activity were tested
in mice and the results obtained (see figure) exceeded expectations. The variant with lowaffinity to extracellular matrix (L5A/V10K)
reached levels 2- to 5-fold higher than wild type,
whereas the catalytically improved variant
(R388A) showed a 2- to 6-fold-higher specific
activity. Gene transfer of these mutant F.IX
molecules, particularly with the catalytic variant,
provided effective hemostasis in vivo upon a very
crude challenge, the tailclipping assay. Moreover,
injection of AAV-F.IX variants in mice tolerant to
wild-type F.IX did not cause antibody formation, a major problem in treatment of hemophilia
patients with null mutations.
The potential of this approach has not been
fully exploited: the amino acid changes improving biodistribution and catalytic properties, located in different F.IX domains, could be combined with each other in the same F.IX molecule,
eventually producing additive effects. More robust
AAV vectors,4 able to increase the number of transduced cells in the liver, could further boost the
efficiency of gene transfer. It remains to be established whether new F.IX molecules and AAV
vectors will be so powerful in humans.
The combination of protein molecular biology and biochemistry with viral vector technology provides a promising strategy to improve the
efficacy for a variety of gene-based therapies in
hematology. ■
REFERENCES
1. High KA. Gene transfer as an approach to treating hemophilia. Semin Thromb Hemost. 2003;29:107-120.
2. Cheung W-M, Hamaguchi N, Smith K, Stafford DW.
The binding of human F.IX to endothelial cells is mediated
by residues 3-11. J Biol Chem. 1992;267:20529-20531.
3. Chang J, Jin J, Lollar P, et al. Changing residue 338 in
human factor IX from arginine to alanine causes an increase
in catalytic activity. J Biol Chem. 1998;273:12089-12094.
On the left, F.IX structure derived from 1RFN PDB (Protein Data Bank) entry. Residue R338 (spacefilling) was mutated to A for gene transfer. On the right, F.IX clotting activity and blood loss in a tailclipping assay 4 weeks after
delivery of AAV–wild type F.IX or AAV-R338A F.IX to hemophilia B mice. Activity is reported as percentage of normal
mice. OD indicates absorbance of hemoglobin in the saline solution in which the tail was placed.
blood 1 5 M A R C H 2 0 0 5 I V O L U M E 1 0 5 , N U M B E R 6
4. Nakai H, Fuess S, Storm TA, Muramatsu S, Nara Y, Kay
MA. Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice. J Virol. 2005;79:214-224.
2243
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2005 105: 2243
doi:10.1182/blood-2004-12-4912
Gain-of-function proteins for gene transfer in hemophilia
Francesco Bernardi
Updated information and services can be found at:
http://www.bloodjournal.org/content/105/6/2243.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.