From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Blood First Edition Paper, prepublished online August 23, 2012; DOI 10.1182/blood-2012-06-440123
The efficacy and the risk of immunogenicity of FIX Padua (R338L) in
hemophilia B dogs treated by AAV muscle gene therapy
Jonathan D. Finn1, Timothy C. Nichols2, Nikolaos Svoronos1, Elizabeth P. Merricks2,
Dwight A. Bellenger2, Zhangshen Zhou1, Paolo Simioni3, Katherine A. High 1,4,5,
Valder R. Arruda1,5
1
The Children’s Hospital of Philadelphia, Philadelphia-USA, 2University of North
Carolina at Chapel Hill, Chapel Hill-USA, 3University of Padua, Padua-Italy, 4Howard
Hughes Medical Institute, Philadelphia-USA, 5University of Pennsylvania Perelman
School of Medicine, Philadelphia-USA
Short title: Expression of FIX Padua in hemophilia B dogs
Key words: factor IX, hemophilia, inhibitor, gene therapy, skeletal muscle, AAV
Correspondence:
Valder R. Arruda, MD
The Children’s Hospital of Philadelphia
3501 Civic Center Blvd
5056 Colket Translational Research Center
Philadelphia, PA 19104
Phone: (215) 590-4907
Fax: (215) 590-3660
[email protected]
Copyright © 2012 American Society of Hematology
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Abstract
Studies on gene therapy for hemophilia B (HB) using adeno-associated viral (AAV)
vectors showed that the safety of a given strategy is directly related to the vector dose. To
overcome this limitation, we sought to test the efficacy and the risk of immunogenicity of
a novel factor IX (FIX) R338L associated with ~8-fold increased specific activity.
Muscle-directed expression of canine FIX-R338L by AAV vectors was carried out in HB
dogs. Therapeutic levels of circulating canine FIX activity (3.5-8%) showed 8-9 fold
increased specific activity, similar to humans with FIX-R338L. Phenotypic improvement
was documented by the lack of bleeding episodes for a cumulative 5-year observation.
No antibody formation and T cell responses to FIX-R338L were observed even upon
challenges with FIX-wild type protein. Moreover, no adverse vascular thrombotic
complications were noted. Thus, FIX-R338L provides an attractive strategy to safely
enhance the efficacy of gene therapy for HB.
2
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Introduction
Hemophilia B (HB) is an X-linked bleeding disorder resulting from a deficiency of
coagulation factor IX (FIX). Gene therapy is an attractive strategy for the treatment of the
disease, since continuous maintenance of FIX levels as low as 1-5% of normal have been
shown to substantially ameliorate the bleeding phenotype in both preclinical and clinical
models1-6.
Studies using adeno-associated viral (AAV) vectors showed that the safety profile is
vector dose-dependent3,4. In a liver-directed approach, immune responses to AAV-capsid
proteins at the highest dose tested (2 x1012 vg/kg) required transient immunosuppression
for sustained transgene expression3,4. In a study on direct intramuscular (IM) AAV-FIX,
the safety profile was excellent2 and the local transgene expression of FIX in the injected
muscle persisted for 3.7 and 10 years in two human subjects tested7,8. However, all doses
tested in the IM study resulted in subtherapeutic circulating FIX levels2. The use of FIX
variants with gain-of-function offers the opportunity to enhance the efficacy of genebased approaches for HB without increasing the vector doses. In an early study we
demonstrated that replacement of arginine 338 by alanine (R338A) was associated with a
~3-fold increase in the protein specific activity in murine models of HB receiving AAVFIX-R338A9 as later confirmed in other models10,11. Recently, we described a naturally
occurring gain-of-function mutation in humans characterized by leucine at position 338
(R338L), that exhibits normal antigen levels, but an ~8-fold higher specific activity12.
Notably, the arginine at position 338 in FIX is conserved among mammals and this
region of the enzyme appears to be part of the substrate exosite for factor X13. Here we
3
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
report for the first time the use of the homologous FIX-R338L in a large and
immunocompetent canine model of severe HB (< 1% of normal).
Materials and Methods
AAV vector production and administration
Recombinant AAV6 vectors were produced by a triple transfection protocol as previously
described using an expression cassette containing canine FIX-R338L (cFIX-Padua) under
control of a cytomegalovirus promoter1,2. The R338L mutation was generated using a
QuickChange™ II-XL site-directed mutagenesis kit (Stratagene). All animal experiments
were approved by the Institutional Animal Care and Use Committee at The Children's
Hospital of Philadelphia and the University of North Carolina (UNC) at Chapel Hill.
Three adult male HB dogs were administered between 2.5 to 3 x 1012 vg/kg of AAV6cFIX-R338L by peripheral transvenular delivery to the skeletal muscle as previously
described1.
Systemic and local toxicity
Hematologic and comprehensive biochemical analyses of blood and serum samples for
liver and kidney function tests were performed as previously described1,14. Thrombin/
anti-thrombin complex (TAT) levels were measure at baseline and at day 50, 100 and
>400 post-AAV injection using Enzygnost TAT kit (Siemens Healthcare Diagnostic).
Normal plasma challenges
Two cFIX-R338L expressing dogs (N07 and M59) were treated 4 times/week with 100
4
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
cc of normal canine plasma intravenously. Plasma samples were collected pre and post
administration of the pooled normal plasma and peripheral blood mononuclear cells
(PBMC) were collected after the 4th challenge.
Canine FIX antigen, activity and antibody assays
The whole blood clotting time (WBCT), cFIX antigen and activity levels, neutralizing
antibodies to cFIX (Bethesda assay), and non-neutralizing antibodies against cFIX were
measured as previously described1,14.
Enzyme-linked immunosorbent spot (ELISpot) analysis
One color ELISpot assays were used to measure IFN-γ T cell responses to AAV capsid,
cFIX protein, or overlapping peptides spanning the 338 region of the cFIX protein
(RATCLR/LSTKFTIYNM, LKVPVDRATCLR/LST).
All proteins and peptides were
used at a final concentration of 10 μg/mL as previously described14. Concanavalin
stimulated PBMC’s were used as a positive control and media alone was the negative
control.
PBMC’s collected after the 4th challenge with canine plasma were used for
analysis.
Results and Discussion
Skeletal muscle is an ideal target for the expression of therapeutic transgenes in genetic
diseases that are associated with primary or iatrogenic underlying liver disease. In adults
with hemophilia, the high rates of viral hepatitis due to blood transfusion preclude the
enrolment of many patients in liver-directed gene therapy. We sought to test whether
5
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
cFIX-R338L could be safely expressed in HB dogs by regional transvenular delivery of
AAV vector to skeletal muscle of a single limb. We generated an AAV-6 vector encoding
cFIX-338L and delivered it to three adult HB dogs. We chose to use an AAV-6 vector
given the excellent tropism for systemic transduction of striated muscle15, and our own
experience using AAV-6 for muscle transduction in HB dogs1. There was a continuous
increase in the circulating cFIX levels that reached plateau levels after day 100 (Figure
1). The kinetics and magnitude of expression in cFIX-338L dogs were similar to those
observed in our earlier studies in dogs using cFIX wild-type (WT) via transvenular
route1. In a series of previous studies in HB dogs following liver, IM or intravascular
delivery to skeletal muscle of AAV vectors of distinct serotypes expressing cFIX-WT,
there was a linear correlation between cFIX antigen and activity levels (ratio 0.81.1)1,3,5,16. Here, the ratio of antigen and activity clearly demonstrated that cFIX-338L
exhibits an 8-9 fold higher specific activity of the protein (Table 1) as observed in
humans with R338L12.
The expression of cFIX-338L was accompanied by
normalization of the WBCT one week post-vector delivery; this was stable throughout
the follow up (Table 1) and comparable to values observed in hemostatically normal
dogs17. Moreover, no spontaneous bleeding episodes requiring treatment occurred for a
cumulative period of ~5 years after vector delivery (ongoing observations). The
frequency of spontaneous bleeding episodes in untreated HB dogs is 5.5 bleeds per
dog/year18; thus the phenotypic improvement supported by cFIX-338L is clinically
evident.
One important consideration in any novel therapeutic approach for hemophilia is
the risk of induction of neutralizing antibodies to the clotting factor. The success of gene
6
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
therapy strategies to cure disease relies on the control of unwanted immune responses to
transgene products, genetically modified cells, and/or to the vector19. In early studies, we
showed that transient immunosuppression (up to 4-weeks) of post-skeletal muscle
delivery of AAV-cFIX is required to prevent antibodies to cFIX-WT transgene 1,6,19. One
potential risk of introducing FIX with a variant sequence distinct from the WT protein is
the immune response to the neotransgene product. The underlying mutation in the HB
model from UNC is a missense mutation that results in a G379E substitution in cFIX20.
Notably, there was no formation of inhibitory antibodies to cFIX-R338L upon multiple
challenges with cFIX-WT protein even >1 year after stopping immunosuppression
(Figure 1, B-C). These data were further supported by the lack of IFN-γ secretion by T
cells after exposure to FIX-WT protein, peptides spanning the 338 residue with (R or L),
and AAV-6 capsid (Figure 1, D). Thus, the FIX-R338L variant shows no detectable
immunogenicity in this approach. There was also no evidence of local or systemic
toxicity, changes in TAT levels (data not shown) or clinically evident thrombotic
complications, however these data should be interpreted cautiously given the small
number of animals used in this study. The latter is consistent with limited observation in
humans where only patients with supraphysiological levels of FIX-R338L (>700% of
normal) developed thrombosis12 and unpublished data.
The expression of FIX-R338L variant has the potential to lower the therapeutic
vector dose and is an attractive strategy to overcome limitations in performing critical
post-translational modifications for a fully biologically active transgene in ectopic
targets6 as we previously reported in murine models of HB9. Moreover, the superior
specific activity of FIX-R338L was also exhibited following infusion of recombinant
7
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
FIX-R338L in HB dogs (V.R.A., unpublished data). In conclusion, our findings in HB
dogs support the use of FIX-338L for clinical translational studies of HB in a variety of
therapeutic settings such as protein replacement or gene-based approaches targeting the
liver or ectopic sites.
Acknowledgements
We thank Scott Ashley and Nicholas Martin for technical assistance. This work was
funded by the National Institutes of Health (P01 HL64190, Project 1) to V.R.A., PO1
HL64190 and Howard Hughes Medical Institute to K.A.H, and HL63098 to T.C.N.
Authorship Contributions and Disclosure of Conflicts of Interest
J.D.F. designed and executed the experiments and drafted the paper. T.C.N, E.P.M., and
D.A.B. performed the vector administration, provided care to the animals, and collected
laboratory samples. N.S. executed the T cell assays. S.Z. provided the recombinant AAV
vectors used in this study. P.S. and K.A.H provided insights on experimental design and
edited the manuscript. V.R.A directed experimental design, conducted data analysis and
interpretation, and drafted the manuscript. K.A.H holds patents related to AAV
manufacture and purification. She holds patents related to AAV-F.IX, in which she has
waived any financial interest. The remaining authors declare no competing financial
interests.
8
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Reference
1.
Arruda VR, Stedman HH, Haurigot V, et al. Peripheral transvenular delivery of
adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B.
Blood. 2010;115(23):4678-4688.
2.
Manno CS, Chew AJ, Hutchison S, et al. AAV-mediated factor IX gene transfer
to skeletal muscle in patients with severe hemophilia B. Blood. 2003;101(8):2963-2972.
3.
Manno CS, Pierce GF, Arruda VR, et al. Successful transduction of liver in
hemophilia by AAV-Factor IX and limitations imposed by the host immune response.
Nat Med. 2006;12(3):342-347.
4.
Nathwani AC, Tuddenham EG, Rangarajan S, et al. Adenovirus-associated virus
vector-mediated gene transfer in hemophilia B. N Engl J Med. 2011;365(25):2357-2365.
5.
Niemeyer GP, Herzog RW, Mount J, et al. Long-term correction of inhibitorprone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene
therapy. Blood. 2009;113(4):797-806.
6.
High KA. Gene therapy for haemophilia: a long and winding road. J Thromb
Haemost. 2011;9 Suppl 1:2-11.
7.
Buchlis G, Podsakoff GM, Radu A, et al. Factor IX expression in skeletal muscle
of a severe hemophilia B patient 10 years after AAV-mediated gene transfer. Blood.
2012;119(13):3038-3041.
8.
Jiang H, Pierce GF, Ozelo MC, et al. Evidence of multiyear factor IX expression
by AAV-mediated gene transfer to skeletal muscle in an individual with severe
hemophilia B. Mol Ther. 2006;14(3):452-455.
9.
Schuettrumpf J, Herzog RW, Schlachterman A, Kaufhold A, Stafford DW,
Arruda VR. Factor IX variants improve gene therapy efficacy for hemophilia B. Blood.
2005;105(6):2316-2323.
10.
Brunetti-Pierri N, Grove NC, Zuo Y, et al. Bioengineered factor IX molecules
with increased catalytic activity improve the therapeutic index of gene therapy vectors for
hemophilia B. Hum Gene Ther. 2009;20(5):479-485.
11.
Kao CY, Lin CN, Yu IS, et al. FIX-Triple, a gain-of-function factor IX variant,
improves haemostasis in mouse models without increased risk of thrombosis. Thromb
Haemost. 2010;104(2):355-365.
12.
Simioni P, Tormene D, Tognin G, et al. X-linked thrombophilia with a mutant
factor IX (factor IX Padua). N Engl J Med. 2009;361(17):1671-1675.
13.
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(20):12089-12094.
14.
Haurigot V, Mingozzi F, Buchlis G, et al. Safety of AAV factor IX peripheral
transvenular gene delivery to muscle in hemophilia B dogs. Mol Ther. 2010;18(7):13181329.
15.
Gregorevic P, Allen JM, Minami E, et al. rAAV6-microdystrophin preserves
muscle function and extends lifespan in severely dystrophic mice. Nat Med.
2006;12(7):787-789.
9
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
16.
Arruda VR, Stedman HH, Nichols TC, et al. Regional intravascular delivery of
AAV-2-F.IX to skeletal muscle achieves long-term correction of hemophilia B in a large
animal model. Blood. 2005;105(9):3458-3464.
17.
Nichols TC, Franck HW, Franck CT, De Friess N, Raymer RA, Merricks EP.
Sensitivity of whole blood clotting time and activated partial thromboplastin time for
factor IX: relevance to gene therapy and determination of post-transfusion elimination
time of canine factor IX in hemophilia B dogs. J Thromb Haemost. 2012;10(3):474-476.
18.
Nichols TC, Dillow AM, Franck HW, et al. Protein replacement therapy and gene
transfer in canine models of hemophilia A, hemophilia B, von willebrand disease, and
factor VII deficiency. ILAR J. 2009;50(2):144-167.
19.
Arruda VR, Favaro P, Finn JD. Strategies to modulate immune responses: a new
frontier for gene therapy. Mol Ther. 2009;17(9):1492-1503.
20.
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(24):10095-10099.
10
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Table 1: Summary of results in hemophilia B dogs following skeletal muscle delivery
of AAV-canine FIX R338L
Plateau cFIX expression
Dog
activity (%)
antigen (%)
Ratio
activity/antigen *
Age/weight
IFN-γ ELISpot
WBCT**
(min)
Inhibitors
AAV
cFIX
338
peptid
M55
7 month male
(23 kg)
8
1.5
8.58 (± 0.46)
11.4 ± 0.7
ND
-
-
-
M59
7 month male
(16.5 kg)
3.5
0.35
9.24 (± 0.44)
13.7 ± 0.7
ND
ND
ND
ND
N07
6 month male
(13 kg)
5.5
0.65
9.0 (± 0.37)
14.4 ± 0.7
ND
ND
ND
ND
*Average ratio of activity/antigen ± standard error from all time points
** Whole blood clotting time: average values ± standard error (normal values: 812 minutes)
ND: not detected
- : not done
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Figure Legend
Figure 1: cFIX-R338L antigen and activity levels over time in HB dogs treated with
AAV6-cFIX-R338L.
Three adult male HB dogs (M55 [A], M59 [B] and N07 [C]) were administered between
2.5-3 x 1012 vg/kg of AAV6-cFIX-R338L by peripheral transvenular delivery to the
skeletal muscle. cFIX antigen levels were measured over time by ELISA (open squares)
and cFIX activity levels were measured by aPTT (filled circle). Black arrows indicate
weekly challenges with pooled normal canine plasma (100 cc). D) PBMC’s collected
from M59 and N07 following the 4th challenge were subjected to IFN-γ ELISpot analysis
after stimulation with AAV6 capsid, cFIX protein, or overlapping peptides spanning the
338 region (RATCLR/LSTKFTIYNM, LKVPVDRATCLR/LST).
12
A
B
50
50
Figure 1
cFIX Antigen
% normal
% normal
20
20
cFIX Activity
20
20
15
15
10
10
5
5
0
0
0
100
200
300
400
500
600
700
0
800
100
200
500
600
317
113
D
50
100
90
20
20
80
spots/1e6 PBMCs
% normal
400
day
day
C
300
15
10
5
70
60
N07
50
40
M59
30
20
10
0
0
0
100
200
300
day
400
500
600
ConA
AAV capsid
cFIX
R338 peptides
Media
700
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Prepublished online August 23, 2012;
doi:10.1182/blood-2012-06-440123
The efficacy and the risk of immunogenicity of FIX Padua (R338L) in
hemophilia B dogs treated by AAV muscle gene therapy
Jonathan D. Finn, Timothy C. Nichols, Nikolaos Svoronos, Elizabeth P. Merricks, Dwight A. Bellenger,
Zhangshen Zhou, Paolo Simioni, Katherine A. High and Valder R. Arruda
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
Advance online articles have been peer reviewed and accepted for publication but have not yet
appeared in the paper journal (edited, typeset versions may be posted when available prior to
final publication). Advance online articles are citable and establish publication priority; they are
indexed by PubMed from initial publication. Citations to Advance online articles must include
digital object identifier (DOIs) and date of initial publication.
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.