Adenovirus-associated virus vector-mediated gene transfer in hemophilia B.

Abstract

Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder.
We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months.
AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values.
Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.).

Full text

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n engl j med 365;25 nejm.org december 22, 2011
2357
The new england
journal of medicine
established in 1812
december 22, 2011 vol. 365 no. 25
Adenovirus-Associated Virus Vector–Mediated Gene Transfer
in Hemophilia B
Amit C. Nathwani, M.B., Ch.B., Ph.D., Edward G.D. Tuddenham, M.B., B.S., M.D., Savita Rangarajan, M.B., B.S.,
Cecilia Rosales, Ph.D., Jenny McIntosh, Ph.D., David C. Linch, M.B., B.Chir., Pratima Chowdary, M.B., B.S.,
Anne Riddell, B.Sc., Arnulfo Jaquilmac Pie, B.S.N., Chris Harrington, B.S.N., James O’Beirne, M.B., B.S., M.D.,
Keith Smith, M.Sc., John Pasi, M.D., Bertil Glader, M.D., Ph.D., Pradip Rustagi, M.D., Catherine Y.C. Ng, M.S.,
Mark A. Kay, M.D., Ph.D., Junfang Zhou, M.D., Yunyu Spence, Ph.D., Christopher L. Morton, B.S., James Allay, Ph.D.,
John Coleman, M.S., Susan Sleep, Ph.D., John M. Cunningham, M.D., Deokumar Srivastava, Ph.D.,
Etiena Basner-Tschakarjan, M.D., Federico Mingozzi, Ph.D., Katherine A. High, M.D., John T. Gray, Ph.D.,
Ulrike M. Reiss, M.D., Arthur W. Nienhuis, M.D., and Andrew M. Davidoff, M.D.
ABSTRACT
From the Department of Haematology,
University College London Cancer Insti-
tute (A.C.N., C.R., J.M., D.C.L.); the Kath-
arine Dormandy Haemophilia Centre and
Thrombosis Unit (A.C.N., E.G.D.T., P.C.,
A.R., A.J.P., C.H.) and the Liver Unit (J.O.),
Royal Free National Health Service (NHS)
Trust; NHS Blood and Transplant (A.C.N.,
C.R., J.M., K.S.); and the Centre for Hae-
matology, Barts and the London, Queen
Mary’s School of Medicine (J.P.) — all in
London; Basingstoke and North Hamp-
shire NHS Foundation Trust, Basingstoke,
United Kingdom (S.R.); the Stanford Uni-
versity School of Medicine, Palo Alto, CA
(B.G., P.R., M.A.K.); the Departments of
Surgery (C.Y.C.N., J.Z., Y.S., C.L.M., A.M.D.),
Biostatistics (D.S.), and Hematology
(J.T.G., U.M.R., A.W.N.), St. Jude Children’s
Research Hospital; and Children’s GMP
(J.A., J.C., S.S.) — both in Memphis, TN;
the Department of Pediatrics, University
of Chicago, Chicago (J.M.C.); the Center
for Cellular and Molecular Therapeutics
at Children’s Hospital of Philadelphia,
Philadelphia (E.B.-T., F.M., K.A.H.); and
Howard Hughes Medical Institute, Chevy
Chase, MD (K.A.H.). Address reprint re-
quests to Dr. Nathwani at the UCL Can-
cer Institute, Paul O’Gorman Bldg., Uni-
versity College London, 72 Huntley St.,
London WC1E 6BT, United Kingdom, or
at a.nathwani@ucl.ac.uk.
This article (10.1056/NEJMoa1108046)
was published on December 10, 2011, at
NEJM.org.
N Engl J Med 2011;365:2357-65.
Copyright © 2011 Massachusetts Medical Society.
Background
Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated
the use of a new gene therapy in patients with the disorder.
Methods
We infused a single dose of a serotype-8–pseudotyped, self-complementary adeno-
virus-associated virus (AAV) vector expressing a codon-optimized human factor IX
(FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe
hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled se-
quentially in one of three cohorts (given a high, intermediate, or low dose of vector),
with two participants in each group. Vector was administered without immunosuppres-
sive therapy, and participants were followed for 6 to 16 months.
Results
AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all par-
ticipants. Four of the six discontinued FIX prophylaxis and remained free of sponta-
neous hemorrhage; in the other two, the interval between prophylactic injections was
increased. Of the two participants who received the high dose of vector, one had a tran-
sient, asymptomatic elevation of serum aminotransferase levels, which was associated
with the detection of AAV8-capsid–specific T cells in the peripheral blood; the other
had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of
these two participants received a short course of glucocorticoid therapy, which rap-
idly normalized aminotransferase levels and maintained FIX levels in the range of
3 to 11% of normal values.
Conclusions
Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expres-
sion at levels sufficient to improve the bleeding phenotype, with few side effects.
Although immune-mediated clearance of AAV-transduced hepatocytes remains a con-
cern, this process may be controlled with a short course of glucocorticoids without
loss of transgene expression. (Funded by the Medical Research Council and others;
ClinicalTrials.gov number, NCT00979238.)
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n engl j med 365;25 nejm.org december 22, 2011
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H
(FIX), a serine protease that is critical for blood
clotting. Persons with severe hemophilia B have
functional FIX levels that are less than 1% of nor-
mal values and have frequent bleeding episodes,
which are associated with crippling arthropathy
and early death.1,2 Current treatment involves fre-
quent intravenous injections of FIX protein con-
centrate (i.e., two to three times a week). However,
this treatment is prophylactic rather than curative,
is extremely expensive, and is associated with in-
hibitor formation. Somatic gene therapy for he-
mophilia B offers the potential for a cure through
continuous endogenous production of FIX after a
single administration of vector, especially since a
small rise in circulating FIX to at least 1% of nor-
mal levels can substantially ameliorate the bleed-
ing phenotype.
At present, gene transfer mediated by an ade-
novirus-associated virus (AAV) vector shows the
greatest promise for long-term correction of he-
mophilia B in the preclinical setting.3-7 However,
a combined phase 1 and 2 study that involved se-
rotype 2–based AAV vectors (AAV2) showed only
transient expression of FIX and suggested that
stable expression of therapeutic levels of FIX may
be limited by a capsid-specific cytotoxic T-cell
response against the transduced hepatocytes.8,9
We have tested an approach to treating patients
with severe hemophilia B that is distinct from the
approaches used in previous clinical trials of AAV-
mediated gene transfer in three important re-
spects. First, we developed a codon-optimized FIX
(FIXco) expression cassette that is packaged as
complementary dimers within a single virion.
These self-complementary AAV (scAAV) vectors
mediate transgene expression at substantially
higher levels than do single-stranded AAV vec-
tors.6,10 Second, to circumvent the possibility of
humoral immunity to AAV, we pseudotyped these
vectors with a capsid of serotype 8 (AAV8), which
has a lower seroprevalence in humans than does
AAV2.11,12 Finally, since AAV8 has a strong tro-
pism for the liver, we were able to administer the
vector in the peripheral vein — a simple, nonin-
vasive approach that is safe for patients with a
bleeding diathesis. On the basis of our preclinical
safety and efficacy data, we conducted a com-
bined phase 1 and 2 clinical trial of scAAV2/8-
LP1-hFIXco–mediated gene transfer in patients
with hemophilia B.6,7,10
emophilia B is an X-linked bleed-
ing disorder that results from a defect in
the gene encoding coagulation factor IX
Methods
Study Design
Patients who met the entry criteria and did not have
neutralizing antibodies to AAV8, as determined by
an in vivo transduction-inhibition assay (see Table
1 and the Methods section in the Supplementary
Appendix, available with the full text of this ar-
ticle at NEJM.org), were enrolled after providing
written informed consent. Participants 1 through
5 were recruited in 2010 and Participant 6 was
recruited early in 2011. All were admitted to the
Royal Free Hospital for vector administration and
subsequent observation overnight. Participants
were enrolled sequentially into one of three co-
horts according to dose level: low (2×1011 vector
genomes [vg] per kilogram of body weight), in-
termediate (6×1011 vg per kilogram), and high
(2×1012 vg per kilogram).
The study was sponsored by St. Jude Children’s
Research Hospital and was conducted indepen-
dently by the authors in accordance with the
protocol, which is available at NEJM.org, and
with the provisions of the Good Clinical Practice
guidelines.
Vector Production and Formulation
The viral vector, scAAV2/8-LP1-hFIXco, has been
described previously.6,10 It was manufactured and
formulated in phosphate-buffered saline (PBS) sup-
plemented with 0.25% human serum albumin by
the Children’s GMP, in Memphis, Tennessee, in ac-
cordance with Good Manufacturing Practice and
regulatory requirements.13 The vector titer initially
reported was determined by means of a validated,
quantitative real-time polymerase-chain-reaction
(qPCR) assay.14 Subsequent studies have shown that
with the qPCR assay, the presence of the covalently
closed hairpin at one end of the scAAV-vector ge-
nome results in an underestimate of the titer by a
factor of almost 10; therefore, the doses reported
here are based on titers determined by a method
that circumvents this issue and results in more ac-
curate and reliable vector-genome titration of the
scAAV vector (see Methods in the Supplementary
Appendix).15 Preparation and infusion of the vector
are described in the protocol.
Safety and Efficacy Assessments
Assessments performed after vector infusion in-
cluded vital signs, as well as plasma FIX activity,
anti-capsid and anti-FIX antibody levels, vector
shedding, and cellular immune response.8-10,16 Ef-
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Vector-Mediated Gene Transfer in Hemophilia B
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ficacy was defined as the persistence of biologi-
cally active FIX at 3% or more of normal levels.
Results
Characteristics of the Study Participants
Six men with severe hemophilia B (FIX activity,
<1% of normal values) were enrolled under a pro-
tocol approved by the relevant ethics boards and
regulatory agencies, after providing written in-
formed consent. All except one participant re-
ceived regular prophylaxis with FIX concentrates
(two or three times per week) before gene transfer
(Table 1). Participant 4 was receiving targeted pro-
phylaxis (about once weekly), which was tailored
to his sports activities and the associated risk of
traumatic injury. Participant 2 had a null mutation
in the FIX gene, and Participant 6 had a promoter
mutation; consequently, neither had FIX protein
expression. The other four participants had mis-
sense mutations, which resulted in normal plasma
levels of FIX antigen but less than 1% clotting ac-
tivity. All participants had modest levels (>5 rela-
tive units) of anti-AAV2 IgG antibodies before gene
transfer.
Safety and Efficacy of Peripheral-Vein
Infusion of Vector
No immediate changes in vital signs were noted
during or after vector infusion in any participant,
despite the persistence of the vector in bodily flu-
ids (including plasma) for up to 15 days after gene
transfer (Fig. 1 in the Supplementary Appendix).
There were no clinically significant changes in se-
rum chemical values (including liver enzyme lev-
els) in any participant for the first 6 weeks after
vector infusion (Fig. 2 in the Supplementary Ap-
pendix). Three adverse events were reported: ane-
mia developed in Participants 1 and 2 at 5 and 7
weeks, respectively, after gene transfer, and Par-
ticipant 3 had a transient period of bradycardia at
16 weeks after gene transfer, when he was being
prepared for surgery on his left knee (see the Sup-
plementary Appendix for details).
Low-Dose Group
In Participant 1, after the initial clearance of exog-
enously administered FIX protein, plasma FIX ac-
tivity stabilized at 2% of normal levels on day 14
after the infusion of 2×1011 vg of scAAV2/8-LP1-
hFIXco per kilogram. FIX activity remained rela-
tively stable at this level for more than 16 months
after gene transfer, despite the discontinuation
of twice-weekly FIX prophylaxis (Fig. 1A). This
level was substantially higher than his baseline
FIX activity level (<1% of normal levels) and is con-
sistent with endogenous synthesis of FIX. He had
no spontaneous hemorrhages during this period,
but FIX prophylaxis was required on five occasions
to provide protection in the event of accidental
injuries and during elective surgery.
In Participant 2, the plasma FIX level was 1% of
normal levels 18 days after gene transfer, which
was 3 weeks after his last injection of FIX con-
centrate. Although this level was consistent with
endogenous synthesis of transgenic FIX, he con-
tinued to have spontaneous hemorrhages and
resumed regular FIX prophylaxis. Over time, he
gradually increased the interval between prophy-
lactic FIX injections to 2 weeks, with no sponta-
neous hemorrhages. At 23 weeks after gene trans-
fer and 3 weeks after the last FIX concentrate
injection, his plasma FIX level was 2% of normal
values, which suggested ongoing endogenous FIX
synthesis (Fig. 1B).
Intermediate-Dose Group
In Participant 3, the level of FIX activity in plasma
consistently drifted downward to below 1% of
normal values in the absence of FIX concentrate
during the first 5 weeks after the infusion of
6×1011 vg of scAAV2/8-LP1-hFIXco per kilogram.
This change raised the possibility that the trans-
duction of hepatocytes was blocked because the
level of anti-AAV8 antibody before gene transfer
was higher in this participant than in the other
participants (Table 1). However, over time, Partici-
pant 3 extended the period between prophylactic
FIX injections to 2 weeks. Between weeks 20 and
33, he did not receive FIX prophylaxis and remained
free of spontaneous bleeding. During this period,
his plasma FIX level was between 1 and 3% of
normal values, which is consistent with endoge-
nous synthesis of transgenic protein (Fig. 1C).
Plasma FIX levels in Participant 4 increased
from a baseline value of less than 1% of normal
values to a peak of 4% 4 weeks after gene trans-
fer. FIX expression remained at this level for almost
3 months before declining to 2 to 3% of normal
values (Fig. 1D). The reason for this decline is un-
clear, since the results of liver-function tests re-
mained in the normal range, and assays for neu-
tralizing anti-FIX antibodies were consistently
negative. Despite this drop in FIX activity, he had
no bleeding episodes, even though he engaged in
physical activities that had previously provoked
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The new england journal of medicine
n engl j med 365;25 nejm.org december 22, 2011
2360
Table 1. Characteristics of the Six Study Participants at Baseline and after Gene Transfer, According to Dose of Vector.*
Characteristic
Vector Dose, 2×1011 vg/kg
Vector Dose, 6×1011 vg/kg
Vector Dose, 2×1012 vg/kg
Participant 1
Participant 2
Participant 3
Participant 4
Participant 5
Participant 6
At baselineAge (yr)
31
64
43
29
32
27
Mutation in FIX gene
31280G→A E387K
2bp deletion, frame shift
30097G→T W215C
31290G→A A309T
20518C→T R180W
−52 del C
FIX prophylaxis
Twice weekly
Twice weekly
Twice weekly
Targeted (average, weekly)
Twice weekly
Thrice weekly
Hepatitis C status
Negative
Positive, spontaneous
clearance
Positive, clearance with
interferon plus anti-
viral therapy
Positive, spontaneous
clearance
Positive, clearance with
interferon plus anti-
viral therapy
Negative
Antibody titer (relative units)†
AAV2 IgG
5
20
77
12
10
22
AAV8 IgG
1
12
37
1
5
8
After gene transfer
Maximum FIX level (IU/dl)‡
2
2
3
4
8
12
Duration of FIX expression (mo)
>15
>11
>9
>8
>6
>5
FIX expression on in vivo
transduction-inhibition assay
(% of control value)§
138
116
42
109
92
93
Peak alanine aminotransferase value
(IU/liter)¶
36
20
39
34
202
36
* FIX denotes factor IX, and vg vector genomes.
† The lower limit of detection for serotype-2 adenovirus-associated virus (AAV2) IgG antibodies was 3 relative units; the lower limit of detection for serotype-8 adenovirus-associated virus
(AAV8) IgG antibodies was 1 relative unit.
‡ The lower limit of detection was 1%.
§ The lower limit of detection was 0.7%.
¶ The upper limit of the normal range is 41 IU per liter.
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Vector-Mediated Gene Transfer in Hemophilia B
n engl j med 365;25 nejm.org december 22, 2011
2361
such episodes (e.g., playing soccer and cricket
without having received targeted prophylaxis). Ap-
proximately 9 weeks after gene transfer, however,
he did receive a bolus of FIX concentrate to prevent
severe bleeding after a fall.
High-Dose Group
Fourteen days after peripheral-vein infusion of
2×1012 vg of scAAV2/8-LP1-hFIXco per kilogram
(17 days after the last infusion of FIX concentrate),
the plasma FIX level in Participant 5 was 7% of
FIX (IU/dl)
Weeks after Vector Infusion
30
20
8
6
4
2
0
10
03020 1040
50
B Participant 2
FIX
F Participant 6
FIX (IU/dl)
Weeks after Vector Infusion
15
10
5
0
0
ALT (IU/liter)
2010
200
150
100
50
0
FIX (IU/dl)
Weeks after Vector Infusion
7
6
5
4
3
2
1
0
0 3020104050 60
70
A Participant 1
FIX
FIX (IU/dl)
Weeks after Vector Infusion
8
6
4
2
0
03020 10
40
C Participant 3
FIX
D Participant 4
FIX (IU/dl)
Weeks after Vector Infusion
9
8
7
6
5
4
3
2
1
0
0 2010
30
FIX
E Participant 5
FIX (IU/dl)
ALT (IU/liter )
Weeks after Vector Infusion
12
8
6
2
10
4
0
02010
30
200
150
100
50
0
60 mg
0
Pred
FIX
FIX
ALT
FIX
Pred
FIX
ALT
60 mg
0
Figure 1. Factor IX (FIX) Activity after Peripheral-Vein Infusion of the Adenovirus-Associated Virus (AAV) Vector
in the Six Study Participants.
A one-stage clotting assay was used to determine FIX coagulation activity at prespecified time points (arrows) after
the administration of the AAV vector (scAAV2/8-LP1-hFIXco). Panels A and B show FIX levels in Participants 1 and 2,
respectively, who received the low dose of vector (2×1011 vector genomes [vg] per kilogram of body weight). Panels C
and D show FIX levels in Participants 3 and 4, respectively, who received the intermediate dose of vector (6×1011 vg
per kilogram). Panels E and F show both FIX levels and alanine aminotransferase (ALT) levels in Participants 5 and 6,
respectively, who received the high dose of vector (2×1012 vg per kilogram). The duration of treatment with pred-
nisolone (Pred), which was initiated at a dose of 60 mg per day and then gradually tapered, is also shown.
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normal values, and this level was maintained un-
til week 7 without the administration of concen-
trate. On day 49 after the vector infusion, aspartate
aminotransferase and alanine aminotransferase
levels were elevated, reaching peaks of 143 and
202 IU per liter, respectively, at 58 days (upper limit
of the normal range, 37 and 41 IU per liter, respec-
tively). He was asymptomatic, with normal biliru-
bin levels and prothrombin time, but the plasma
FIX level dropped to 3% of normal values (Fig. 1E).
After ruling out possible causes of this reduction,
such as autoimmune responses, toxic factors (al-
cohol, drugs, or toxins), or infectious viral causes
(hepatitis A, B, C, or E virus, herpes simplex virus,
Epstein–Barr virus, cytomegalovirus, human im-
munodeficiency virus, or human T-lymphotrophic
virus type I or II), we considered an immunologic
response to scAAV2/8-LP1-hFIX as a potential ex-
planation for the liver injury. This was reported as
a grade 3 adverse event related to the study agent.
Treatment with prednisolone was begun on day
58 at a dose of 60 mg per day, with subsequent ta-
pering of the dose, rapidly reducing aminotransfer-
ase levels. Nine weeks after glucocorticoid therapy
was discontinued, transgene expression persisted
at the 3% level, and the results of liver-function
tests remained in the normal range. The patient
was free of spontaneous hemorrhage for more than
6 months after the gene transfer but required
three boluses of FIX concentrate to prevent bleed-
ing due to traumatic injuries sustained during a
recent geologic field trip.
In Participant 6, plasma FIX expression re-
mained between 8 and 12% of normal levels dur-
ing an 8-week period after vector administration.
He was able to stop FIX prophylaxis even while he
was training for a marathon. Clinical and labora-
tory measures remained within the normal range
until week 9 (day 62 after gene transfer), when
aspartate aminotransferase and alanine amino-
transferase values roughly doubled, as compared
with baseline levels (with both reaching 36 IU per
liter). This change followed a weekend of intensive
physical activity, which included participation in a
half-marathon, and was associated with a rise in
the level of lactate dehydrogenase (to 523 IU per
liter; normal range, 240 to 480). When FIX activity
declined to 5% of normal levels, Participant 6 also
began a short (4-week) course of prednisolone for
suspected immune-mediated hepatocyte clearance
after other possible causes had been ruled out.
Aminotransferase levels subsequently returned to
baseline values, and FIX expression remained at a
level that was between 8 and 12% of normal val-
ues (Fig. 1F). However, even at their peak, liver
enzyme levels in Participant 6 remained below
the upper limit of the normal range and therefore
did not meet the criteria for an adverse event.
Immune Responses to Vector and Transgene
Neutralizing antibodies to FIX were not detected at
any time point in any participant. The kinetic char-
acteristics of the humoral immune response to the
AAV8 capsid were similar in all six participants and
were consistent with a primary immune response
to AAV8 (Fig. 2 and Table 1, and Results in the
Supplementary Appendix).
T-cell–mediated immune responses to the FIX
transgene or putative products of translation from
alternative open reading frames in the codon-opti-
mized FIX complementary DNA17 were not detect-
able in any of the participants (data not shown), as
determined with the use of an interferon-γ en-
zyme-linked immunosorbent spot (ELISPOT) as-
say. The same assay showed that participants in
the low-dose cohort did not have significant AAV8-
capsid–specific T-cell responses after gene trans-
fer. At the intermediate-dose level, a significant
increase in the frequency of AAV8-capsid–specific
T cells was observed. In particular, Participant 3
had T-cell reactivity to the AAV capsid until week
33 after the gene transfer (Fig. 3), and Participant
4 had a very strong capsid-specific T-cell response
Anti-AAV8 IgG Antibodies
(relative units)
Anti-AAV8 IgM Antibodies
(relative units)
Days after Vector Injection
2500
1500
1000
2000
500
0
0100 50
150
100
50
0
AAV8-specific IgM
Total IgG
IgG1
Figure 2. Humoral Immune Response in Participant 1.
The profile of the humoral immune response in Participant 1 is representa-
tive of the responses seen in the other participants. Plasma samples ob-
tained after peripheral-vein infusion of the scAAV2/8-LP1-hFIXco vector
were analyzed with the use of an enzyme-linked immunosorbent assay for
the presence of AAV8-specific IgM antibodies, total IgG antibodies, and
IgG isotypes. Data for the IgG1 isotype are shown. IgG2 and IgG4 levels
did not increase above baseline (data not shown).
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Vector-Mediated Gene Transfer in Hemophilia B
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2363
at week 2 (about 1700 spot-forming units [SFU]
per 1 million peripheral-blood mononuclear cells
[PBMCs]). At the highest dose level, an increase in
circulating AAV8-capsid–specific T cells was ob-
served in Participant 5 starting at week 5 after
transduction and reaching a peak of more than
500 SFU per 1 million PBMCs by week 8, which
was concomitant with the increase in liver enzyme
SFU per 106 PBMCs
700
600
400
300
100
500
200
0
0123465
7
Weeks
A Participant 1
SFU per 106 PBMCs
700
600
400
300
100
500
200
0
012365
10987
Weeks
B Participant 2
SFU per 106 PBMCs
700
600
400
300
100
500
200
0
012346
3378911 12
Weeks
C Participant 3
SFU per 106 PBMCs
1700
600
400
300
100
500
200
0
0123465
2812987
Weeks
D Participant 4
SFU per 106 PBMCs
700
600
400
300
100
500
200
0
123468910 12 135
2120
Weeks
E Participant 5
SFU per 106 PBMCs
700
600
400
300
100
500
200
0
0123465
1510 11 12 14987
Weeks
F Participant 6
Figure 3. Cellular Immune Response in the Six Study Participants.
Results of the interferon-γ enzyme-linked immunosorbent spot assay for capsid-specific T-cell responses are shown as the maximum
number of spot-forming units (SFU) per 1 million peripheral-blood mononuclear cells (PBMCs) in response to AAV8-capsid peptide
pools. The horizontal lines in each panel represent the threshold for positivity, defined as three times the T-cell response for the nega-
tive control (medium only) and as at least 50 SFU per 1 million PBMCs. Liver-enzyme levels were elevated in Participant 5 at weeks 8
and 9 and in Participant 6 at week 9. In Participant 6, poor cell recovery and viability were noted at weeks 4 through 8.
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The new england journal of medicine
n engl j med 365;25 nejm.org december 22, 2011
2364
levels. In this participant, broad reactivity was
observed across the six capsid peptide pools as-
sessed (Fig. 3 in the Supplementary Appendix).
By week 10, capsid-specific T cells were once again
undetectable. No T-cell reactivity to the AAV cap-
sid was detectable in PBMCs from Participant 6
until week 8 after the gene transfer, although this
may have been due in part to reduced cell recovery
and viability. At weeks 9 and 10, capsid-specific
T cells became detectable, with levels of up to ap-
proximately 200 SFU per 1 million PBMCs, a find-
ing that was concomitant with the minor increase
in liver enzyme levels.
Discussion
The development and widespread use of clotting
factor concentrates for the treatment of hemophil-
ia in the early 1970s dramatically improved the life
expectancy for patients with the disease. Subse-
quent development of recombinant clotting factor
concentrates has improved their safety profile, but
there remains a strong interest in treatment strate-
gies that would eliminate the need for long-term
intravenous infusions and that would be available
to the hemophilia population throughout the
world. This study documents a critical step toward
that goal and shows that sustained therapeutic
expression of a transferred factor IX gene can be
achieved in humans. The increase in FIX levels in
our study participants was roughly dose-dependent,
with the high dose of the vector scAAV2/8-LP1-
hFIX (2×1012 vg per kilogram) mediating peak
expression at 8 to 12% of normal levels. After
peripheral-vein administration of scAAV2/8-LP1-
hFIXco, four of the six participants were able to
stop using prophylaxis with FIX concentrate with-
out having spontaneous hemorrhage, even when
they undertook activities that had provoked bleed-
ing in the past. For the other two participants, the
interval between prophylactic FIX concentrate injec-
tions was extended, but prophylaxis was not com-
pletely discontinued. These two participants had
severe preexisting hemophilic arthropathy. Maxi-
mal protection from bleeding in this subgroup may
require higher endogenous expression of FIX.18
None of the participants had an immunologic
response to the FIX transgene product. As reported
in previous studies, the kinetics and magnitude
of capsid-specific T-cell responses in PBMCs were
highly variable in our study, with the highest num-
ber of capsid-specific T cells detected in Partici-
pant 4 at 2 weeks after gene transfer.8,19,20 There
was no evidence of hepatocellular injury in this
participant, suggesting that the T-cell repertoire
in peripheral blood may be distinct from that in
the liver or that levels of AAV8-capsid–antigen pre-
sentation on major-histocompatibility-complex
class I hepatocytes were insufficient to drive the
immune-mediated clearance of transduced cells.
A scenario consistent with AAV8-capsid–specific
T-cell–mediated destruction of transduced hepato-
cytes was observed 7 weeks after gene transfer in
Participant 5. It is less certain whether the small
increase in liver enzyme levels 9 weeks after gene
transfer in Participant 6 was due to immune-
mediated destruction of transduced hepatocytes.
This increase occurred after a period of intense
physical exertion, which is known to increase ami-
notransferase and lactate dehydrogenase levels.21,22
In both Participant 5 and Participant 6, the initia-
tion of prednisolone therapy was followed by a
return of the aminotransferase levels to baseline
values and the disappearance of capsid-specific
T cells from peripheral blood. Arguably, this could
have happened without glucocorticoid treatment,
since at 7 weeks after gene transfer, many of the
transduced hepatocytes will have cleared the pro-
voking AAV8 capsid peptide through natural pro-
teolytic degradation and would therefore escape
elimination by effector T cells.
The different outcomes of gene transfer in the
participants in this study and in the previous trial
of AAV2 hemophilia B gene therapy8 suggest that
in addition to AAV vector dose, other factors such
as individual variations in antigen processing and
presentation, as well as exposure to wild-type AAV,
seem to influence the type and magnitude of the
immunological response to AAV-vector particles.23
It is very important to identify these immuno-
modifying factors, as well as to determine the
frequency of clinically significant elevations in
aminotransferase levels after gene transfer. How-
ever, this will require a larger number of partici-
pants treated at the high-dose level. Currently, we
do not think that routine use of a course of pro-
phylactic glucocorticoids is indicated, since the
incidence and timing of immune-mediated ele-
vations in aminotransferase levels are uncertain,
whereas early intervention, when needed, appears
to be effective at curbing the immune response to
transduced hepatocytes.
In summary, we have found that a single pe-
ripheral-vein infusion of our scAAV2/8-LP1-hFIXco
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Page 9

Vector-Mediated Gene Transfer in Hemophilia B
n engl j med 365;25 nejm.org december 22, 2011
2365
vector consistently leads to long-term expression of
the FIX transgene at therapeutic levels, without
acute or long-lasting toxicity in patients with severe
hemophilia B. Immune-mediated, AAV-capsid–
induced elevations in aminotransferase levels re-
main a concern, but our data suggest that this
process may be controlled by a short course of
glucocorticoids, without loss of transgene expres-
sion. Follow-up of larger numbers of patients for
longer periods of time is necessary to fully define
the benefits and risks and to optimize dosing.
However, this gene-therapy approach, even with
the associated risk of transient hepatic dysfunc-
tion, has the potential to convert the severe bleed-
ing phenotype into a mild form of the disease or
to reverse it entirely.
The views expressed in this article are those of the authors and
not necessarily those of the National Health Service (NHS), the
National Institute of Health Research (NIHR), or the Department
of Health in the United Kingdom.
Supported by grants from the NIHR (RP-PG-0310-1001), the
Medical Research Council, the Katharine Dormandy Trust, the U.K.
Department of Health, NHS Blood and Transplant, the NIHR
Biomedical Research Centers (to University College London Hos-
pital and University College London), the ASSISI Foundation of
Memphis, the American Lebanese Syrian Associated Charities,
the Howard Hughes Medical Institute, the National Heart, Lung,
and Blood Institute (HL094396), the Royal Free Hospital Charity
Special Trustees Fund 35, the Royal Free Hospital NHS Trust, and
St. Jude Children’s Research Hospital.
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
We thank all the study participants for their interest and partici-
pation in this study and Patricia Lilley, Anja Griffioen, Cheng-Hock
Toh, Paul Eddlemon, Alison Evans, Paul Lloyd-Evans, and Keith
Williams for their help with trial-related activities, including vector
importation and qualification, data entry, and regulatory affairs.
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