Phase 2 Clinical Trial of a Recombinant Adeno-Associated
Viral Vector Expressing a1-Antitrypsin: Interim Results
Terence R. Flotte,1Bruce C. Trapnell,2Margaret Humphries,1Brenna Carey,2Roberto Calcedo,3
Farshid Rouhani,4Martha Campbell-Thompson,4Anthony T. Yachnis,4Robert A. Sandhaus,5
Noel G. McElvaney,6Christian Mueller,1Louis M. Messina,1James M. Wilson,3Mark Brantly,4
David R. Knop,7Guo-jie Ye,7and Jeffrey D. Chulay7
Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of a1-antitrypsin (AAT)
deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained,
vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a
herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intra-
muscular injection at doses of 6.0·1011, 1.9·1012, and 6.0·1012vector genomes/kg (n=3 subjects/dose).
Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and
persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions
and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in
the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies
and interferon-c enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies
to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated
with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and
moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were
primarily of the CD8+subtype. These results support the feasibility and safety of AAV gene therapy for AAT
deficiency, and indicate that serum levels of vector-derived normal human AAT >20lg/ml can be achieved.
However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve
therapeutic target serum AAT concentrations.
cretion of AAT from the liver and consequent impaired anti-
protease activity in the lung, resulting in early-onset
pulmonary emphysema (Silverman and Sandhaus, 2009).
More than 95% of AAT-deficient individuals have the Z-type
of AAT instead of the normal M-type AAT (Brantly et al.,
1991). Gene therapy approaches to treatment of AAT defi-
ndividuals with a1-antitrypsin (AAT) deficiency have
mutations in the SERPINA1 gene that cause reduced se-
ciency, using recombinant adeno-associated viral (rAAV)
vectors expressing AAT, have been evaluated in preclinical
and clinical studies (Song et al., 1998, 2002; Poirier et al., 2004;
Brantly et al., 2006, 2009; De et al., 2006; Liqun Wang et al.,
2009; Halbert et al., 2010; Chulay et al., 2011). We previously
conducted a phase 1 clinical trial with an rAAV vector ex-
pressing human AAT (rAAV1-CB-hAAT), produced by a
plasmid transfection method, in which sustained expression
of AAT was achieved, but serum levels were substantially
below the levels considered to be therapeutic (Brantly et al.,
1University of Massachusetts Medical School, Worcester, MA 01655.
2Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229.
3University of Pennsylvania, Philadelphia, PA 19104.
4University of Florida School of Medicine, Gainesville, FL 32610.
5National Jewish Health, Denver, CO 80206.
6Beaumont Hospital, Dublin, 9 Ireland.
7Applied Genetic Technologies Corporation, Alachua, FL 32615.
Registration: This study has been registered at Clinical Trials.gov (NCT01054339).
HUMAN GENE THERAPY 22:1239–1247 (October 2011)
ª Mary Ann Liebert, Inc.
2009). Production of rAAV1-CB-hAAT using a recombinant
herpes simplex virus (HSV) complementation system (Kang
et al., 2009; Thomas et al., 2009) can generate much higher
yields, enabling a substantial increase in dose in clinical
studies, and the HSV-produced vector is more potent when
given by intramuscular injection in mice (Chulay et al., 2011).
We report here preliminary results from a phase 2 clinical trial
of rAAV1-CB-hAAT produced by the HSV-based method.
Research Design and Methods
Individuals of either gender were eligible for study in-
clusion if they had a diagnosis of AAT deficiency, a serum
AAT level <11lM, a forced expiratory volume in 1sec
(FEV1) >25% of predicted, and had not received AAT aug-
mentation therapy in the 3 months before enrollment and
planned not to receive it for 12 months after enrollment.
Vector production and characterization
The rAAV1-CB-hAAT vector was identical to the vector
used in a phase 1 clinical trial (Brantly et al., 2009) except that it
was made using a recombinant HSV complementation system
in suspension baby hamster kidney (BHK) cells (Kang et al.,
2009; Thomas et al., 2009), based on the method of Conway
and colleagues (1999) and purified by Convective Interaction
Media (CIM) QA Monolith anion-exchange chromatography
(BIA Separations, Villach, Austria) followed by AVB Sephar-
ose affinity chromatography (GE Healthcare Life Sciences,
Piscataway, NJ). It was produced in compliance with current
Good Manufacturing Practice at SAFC Pharma (Carlsbad, CA)
and characterized in compendial assays or product-specific
assays as described previously (Chulay et al., 2011).
Study design and conduct
This is a nonrandomized, open-label, multicenter, se-
quential, three-arm, phase 2 clinical trial evaluating the
safety and efficacy of administration of rAAV1-CB-hAAT
conducted under an investigational new drug (IND) appli-
cation with approval by institutional review boards and in
accordance with the tenets of the Declaration of Helsinki.
Written informed consent was obtained before any study
procedures were performed. Three cohorts of three subjects
each received rAAV1-CB-hAAT at dose levels of 6·1011,
1.9·1012, or 6·1012vector genomes (VG)/kg body weight
by intramuscular injection on a single occasion. Subjects in
cohort 1 received 10 intramuscular injections distributed
across a single muscle site, subjects in cohort 2 received 32
intramuscular injections distributed across three muscle sites,
and subjects in cohort 3 received 100 intramuscular injections
distributed across 10 muscle sites. Each injection was given
in a volume of 1.35ml, at the appropriate vector concentra-
tion to achieve the desired total vector dose. The clinical
protocol specified that subjects could choose to have injec-
tions administered using topical anesthetic cream or con-
scious sedation with intravenous midazolam.
A data and safety monitoring board reviewed safety data
for the first two dose level cohorts before the next higher
dose cohort was enrolled. Safety is being monitored by
evaluation of adverse events, hematology (complete blood
count with white cell differential) and clinical chemistry
parameters (electrolytes, glucose, albumin, globulin, blood
urea nitrogen, creatinine, creatine kinase, bilirubin, and he-
patic enzymes), histological examination of muscle biopsies
at 3 and 12 months, and measurement of serum antibodies to
AAT. Efficacy is being evaluated by measurement of serum
concentrations of M-type AAT and total AAT. Additional
information being collected includes changes in antibody
responses to AAV and T cell responses to AAV and AAT.
Antibodies to AAT were measured by ELISA, using a
modification of a previously described method (Brantly et al.,
2006) in which serum from a cynomolgus macaque immu-
nized against human AAT by injection with rAAV1-CB-
hAAT was used as the reference standard. All other assays
were performed as previously described (Brantly et al., 2009),
including measurement of antibodies to AAV1 using a
neutralization assay, T cell responses by ex vivo interferon
(IFN)-c enzyme-linked immunospot (ELISPOT) assay and
polychromatic flow cytometry of peripheral blood mono-
nuclear cells (PBMCs) after stimulation with AAV or AAT
peptides, and M-specific AAT by ELISA. Epitope mapping of
the ELISPOT response to an AAT peptide pool was per-
formed with a matrix of subpools to identify the single
peptide that was reactive.
Muscle biopsies were performed with a disposable Price
muscle biopsy clamp (V. Mueller catalog #SU20910; Cardinal
Health Medical Products, McGaw Park, IL) to obtain two ap-
proximately 2·1·0.5cm specimens. Half of each specimen
was immediately frozen and the other half was prepared for
4 to 24hr and then transferred to 70% ethanol until processing.
Paraffin serial sections (4lm) were stained with hematoxylin
and eosin (H&E) and by immunohistochemistry for human
cell immunophenotype was determined with monoclonal an-
tibodies that recognize CD3, CD20, CD68 (Dako, Carpinteria,
CA), and CD4 (Cellmarque, Rocklin, CA) following standard
clinical validation on an autostainer (Dako).
Characterization of rAAV1-CB-hAAT
The four 25-liter batches of crude cell lysate yielded a total
of 1.0·1016VG, with an overall product recovery during
purification of 23%. The study drug met all release criteria,
including sterility, endotoxin (<0.3 EU/ml), and absence of
detectable mycoplasma, adventitious viruses, replication-
competent AAV, and replication-competent HSV. Residual
BHK DNA and protein were 4.1 and 15.9ng/ml, respec-
tively, and residual HSV DNA and protein were 76 and
834ng/ml, respectively. Vector concentration measured by
quantitative PCR was 5.0·1012VG/ml, vector infectivity
measured as median tissue culture infective dose (TCID50)
was 4.7·1010IU/ml, and hAAT expression measured by
ELISA in infected HEK 293 cell supernatant was 2.2lg/ml.
Preliminary clinical trial results
Nine subjects were enrolled and received intramuscular
injections of rAAV1-CB-hAAT between June 2010 and
October 2010. All were white and seven were female, with a
1240FLOTTE ET AL.
mean age of 51.1 (range, 20–68) years and mean weight of
71.2 (range, 55–90) kg. The AAT phenotype was ZZ for eight
subjects and SZ for one.
Administration of rAAV1-CB-hAAT by multiple intramus-
subject preferences, injections were performed with topical
anesthetic cream for the three subjects in cohort 1 and for two
subjects in each of cohorts 2 and 3, and under conscious se-
dation using intravenous midazolam for one subject in each of
cohorts 2 and 3.
All subjects reported at least one adverse event of mild to
moderate intensity. The most frequently reported adverse
events were injection site reactions (discomfort, erythema,
bruising, or pain) of mild intensity, which occurred in eight
of nine subjects. A list of all adverse events is provided in
Table 1. No serious adverse events were reported.
Table 1. Summary of Adverse Events
Mild Moderate SevereTotal
Events related to study agent or its administration
General disorders and administration site conditions
Injection site discomfort
Injection site erythema
Injection site hemorrhage
Injection site pain
Blood creatine phosphokinase increased
Musculoskeletal and connective tissue disorders
Events not related to study agent or its administration
General disorders and administration site conditions
Infections and infestations
Urinary tract infection
Injury, poisoning, and procedural complications
Metabolism and nutrition disorders
Musculoskeletal and connective tissue disorders
Pain in extremity
Nervous system disorders
Respiratory, thoracic and mediastinal disorders
Upper respiratory tract infection
Skin and subcutaneous tissue disorder
Postmastectomy lymphedema syndrome
Data represent the number of subjects who reported the listed adverse event on one or more occasions.
PHASE 2 CLINICAL TRIAL OF rAAV1-CB-hAAT1241
Measurement of serum M-specific AAT levels demon-
strated a dose-dependent increase after injection of rAAV1-
CB-hAAT, which peaked on day 30 (mean value of 572nM in
the highest dose cohort) and then declined on day 45 with
little change thereafter (mean value of 240nM on day 90 in
the highest dose cohort). The average peak serum M-AAT
level in the lowest dose cohort in this study was more than 2-
fold higher than the average peak serum M-AAT level in the
highest dose cohort in the previous phase 1 study with
transfection-produced vector (Fig. 1). Serum M-AAT levels
for individual subjects are shown in Fig. 2.
Serum creatine kinase (CK) was transiently elevated on
day 30 in two of three subjects in cohort 2 and in three of
three subjects in cohort 3, and had normalized by day 45 in
four subjects and by day 60 in one subject (Fig. 2). There were
no clinically significant changes in any other clinical chem-
istry or hematology parameter.
As expected on the basis of previous preclinical and clin-
ical testing, all subjects in all three dose level cohorts de-
veloped neutralizing antibodies against AAV (Table 2) and
IFN-c ELISPOT responses to AAV peptides (Fig. 3). There
was no apparent relationship between the dose of vector
administered and the magnitude of the IFN-c ELISPOT re-
sponses to AAV peptides. Both AAV1-specfic CD8+and
CD4+T cell responses were detected and had a cytokine
profile similar to that seen in the previous clinical trial with
this vector (Brantly et al., 2009).
None of the subjects has developed antibodies to AAT, but
one subject in the mid-dose cohort (subject 401) developed
IFN-c ELISPOT responses to a pool of AAT peptides at
month 1 that persisted at months 2 and 3 (baseline and
screening samples were negative). Epitope mapping identi-
fied a single peptide (peptide 46, DTEEEDFHVDQVTTV)
distant from the site of the PI*Z mutation that was the target
of the ELISPOT response. Cytokine flow cytometry after
stimulation with the AAT peptide pool showed that this
subject had a response to AAT mediated by CD4+T cells
expressing IFN-c but not tumor necrosis factor (TNF)-a and
by CD8+T cells expressing both IFN-c and TNF-a. On the
basis of review of history and physical examination findings
at each visit and hematology and clinical chemistry data,
there was no evidence that the T cell response to this
AAT peptide was associated with any untoward clinical
Histological examination of muscle biopsies, obtained on
day 90 from eight subjects, showed moderate to marked
mononuclear endomysial and perivascular inflammatory
infiltrates composed primarily of mature lymphocytes and
smaller populations of monocytes and plasma cells (Fig. 4).
There was also prominent myofiber regeneration, as evi-
denced by numerous basophilic myofibers with large vesic-
ular nuclei containingprominent
myofibers undergoing active necrosis with phagocytosis by
macrophage-like cells were identified. No significant en-
domysial fibrosis was seen. Immunohistochemical staining
indicated that CD3-immunoreactive T lymphocytes com-
prised the most abundant single subset of mononuclear cells,
with CD8+cells accounting for slightly more of the total
inflammatory cell population than CD4-reactive cells. Scat-
tered CD20-immunoreactive B lymphocytes and CD68-
reactive macrophages were also seen. There was strong
immunostaining of AAT within endomysial and perimysial
blood vessels, not associated with cell components, and in
perimysial and endomysial connective tissue. Focal individ-
ual myofibers showed weaker AAT immunoreactivity of the
sarcoplasm, with some myofibers having a dispersed gran-
ular pattern of reactivity.
The most significant finding in this study was a clear
demonstration of a linear dose–response relationship. This is
the first time, to the authors’ knowledge, that such a linear
relationship between physical dose of a gene therapy prod-
uct and expression of a therapeutic protein has been shown
in humans. Previous clinical trials with the rAAV2-CB-hAAT
vector (Brantly et al., 2006, 2009) and with an AAV2 vector
expressing clotting factor IX (Manno et al., 2003, 2006) did
not have sufficient numbers of subjects with sustained ex-
pression at various doses to enable demonstration of a linear
dose response, and in clinical trials with an AAV1 vector
expressing lipoprotein lipase (Stroes et al., 2008) the end
point was indirect, and thus cannot be compared directly
with our results. Our observation indicates that human
rAAV gene therapy for AAT deficiency behaves in a pre-
dictable fashion and is an important step in the ability to
design and conduct appropriate safety and efficacy studies in
support of product licensure.
However, AAT is one of the most abundant serum pro-
teins (normal concentration, 20 to 50lM or about 1,000 to
2,500lg/ml), and the peak serum AAT levels achieved after
delivery of 6·1012VG/kg by multiple intramuscular injec-
tions (between 412 and 694nM, equivalent to 21 to 36lg/ml)
were below the target therapeutic concentration (>11lM,
equivalent to 572lg/ml) required to reduce the risk of
emphysema. Further improvements in product delivery
or product design will therefore be required to achieve
tion after injection of rAAV1-CB-hAAT produced by plasmid
transfection (TFX) or the herpes simplex virus (HSV) meth-
od. Values shown represent means–SD. The dose of vector
administered to subjects is indicated in the figure legend.
Values for the TFX group are from a previous study (Brantly
et al., 2009). Values for the 6·1011VG/kg HSV group do not
include results for subject 303, who had an AAT phenotype
of SZ; the monoclonal antibody used to determine serum
M-specific AAT concentrations has little cross-reactivity with
Z-type AAT but cross-reacts strongly with S-type AAT, causing
results for this assay in this subject to be spuriously high. Color
images available online at www.liebertonline.com/hum
Serum M-specific a1-antitrypsin (AAT) concentra-
1242FLOTTE ET AL.
symbols) after injection of rAAV1-CB-hAAT in individual subjects. Subject 303 had an AAT phenotype of SZ, and results for
the M-specific AAT ELISA in this subject are spuriously high. Subjects 305 and 307 were the two male subjects.
Serum M-specific a1-antitrypsin (AAT) concentration (solid symbols) and serum creatine kinase (CK) levels (open
Table 2. Neutralizing Antibody Responses to AAV
Low dose Middle dose High dose
rAAV-lacZ vectors mixed with serial dilutions of serum were used to infect Huh7 cells. Results are expressed as the reciprocal of the
highest serum dilution that inhibited b-galactosidase expression by 50%.
PHASE 2 CLINICAL TRIAL OF rAAV1-CB-hAAT 1243
therapeutic target serum AAT concentrations. For example,
administration of an rAAV1 vector expressing a CTLA4Ig
transgene by a regional intravenous method achieved serum
concentrations 5- to 8-fold higher than multiple intramus-
cular injections of the same vector in cynomolgus macaques
(Toromanoff et al., 2008), and suggests that regional vascular
delivery of rAAV1-CB-hAAT might result in higher serum
AAT concentrations. It is also possible that regional vascular
delivery may elevate expression levels by reducing anti-
AAV immune responses (Toromanoff et al., 2010), or that a
similar result could be achieved by short-term administra-
tion of immunosuppressive drugs. The use of alternative
AAV serotypes should also be considered. AAV1 was se-
lected for use in the current clinical trial on the basis of
evidence of improved transduction efficiency with AAV1
compared with AAV2 after intramuscular injection in mice
(Xiao et al., 1999; Chao et al., 2000; Gao et al., 2002; Rabino-
witz et al., 2002; Lu et al., 2006). However, more recent data
ELISPOT responses to pools of
AAV1 capsid peptides or con-
trols. PBMCs were obtained at
screening, baseline, and 1, 2, and
3 months after vector adminis-
with each of three pools (A, B,
and C) of AAV1 capsid peptides
amino acids) or with a positive
control peptide pool (CEF). SFC,
Time course of IFN-c
1244FLOTTE ET AL.
indicate that other serotypes, including recombinant sero-
types and other nonnaturally occurring serotypes, may
transduce muscle cells more efficiently than AAV1 (Rodino-
Klapac et al., 2007; Asokan et al., 2010; Qiao et al., 2010; Pu-
licherla et al., 2011). A combination of these approaches may
be necessary in order to ultimately achieve the goal of ef-
fective gene therapy for AAT deficiency. Results of the cur-
rent study provide a foundation for designing rational
Results of this study provide additional evidence of the
safety of AAV gene therapy. In the highest dose cohort
(6·1012VG/kg), subjects received a total of between
3.3·1014and 4.3·1014VG, administered in a total of 135ml
distributed over 100 intramuscular injections, with only mild
and transient discomfort at the injection sites.
As expected, all subjects developed anti-AAV antibodies
and IFN-c ELISPOT responses to AAV peptides. In a previ-
ous clinical trial with the same vector, anti-AAV immune
responses were not associated with any significant decline in
peak AAT expression; expression rose irregularly during the
first 30 to 180 days and was then sustained at similar levels
through day 365. In the present study, serum CK levels were
elevated in most subjects in the higher two dose level cohorts
on day 30 after injection, which corresponded to the time of
peak serum AAT expression, and there was histological
evidence of inflammatory cells in muscle biopsy samples 3
months after injections, but no clinical symptoms suggestive
of ongoing myositis. It is not known if the T cells seen in
muscle biopsies are AAV specific, or if antivector immune
responses are responsible for the observed decline in AAT
expression after day 30.
We documented T cell response to a single AAT peptide in
one subject but found no evidence of untoward clinical
effects (no symptoms, no antibody response to AAT, no
abnormal liver function tests, and no change in total AAT
concentration). The fact that the epitope eliciting this T cell
response was distant from the site of the missense mutation
is puzzling. Although there is no evidence that the glyco-
sylation pattern of AAT expressed from muscle is different
from that of AAT expressed from liver, it is possible that
altered glycosylation of AAT may break tolerance and trig-
ger adaptive immune responses in some individuals under
certain circumstances. Alternatively, this subject may have
had low levels of preexisting reactive T cells that were not
detected in the peripheral blood before vector administra-
tion. It is reassuring that the three subjects in the highest dose
cohort did not mount any detectable T cell responses to AAT
In summary, results from this clinical trial support the
feasibility and safety of AAV gene therapy of AAT
chemical study of skeletal muscle. (A)
H&E-stained section showing a moder-
ate endomysial inflammatory reaction
composed primarily of mononuclear
cells. (B) H&E-stained section showing
a marked endomysial inflammatory
for AAT, showing individual weak to
moderate granular reactivity in indi-
vidual myofibers on cross-section. (D)
Immunohistochemistry for AAT, show-
ing individual weak to moderate gran-
ular reactivity in individual myofibers
cut longitudinally. (E) Immunohisto-
chemistry for CD3, showing a high
proportion of T lymphocytes compris-
ing the inflammatory infiltrate. (F)
CD8-immunoreactive T cells comprise a
significant subset of the total lympho-
Histology and immunohisto-
PHASE 2 CLINICAL TRIAL OF rAAV1-CB-hAAT1245
deficiency, although further improvements in the design or
delivery of rAAV-AAT vectors will be required to achieve
therapeutic target serum AAT concentrations.
This study was supported in part by grant R01FD003896
from the Office of Orphan Products Development, U.S.
Food and Drug Administration, and by grant R01HL069877
from the National Heart, Lung, and Blood Institute
(NHLBI), National Institutes of Health. The authors thank
the technical staff of SAFC Pharma (Carlsbad, CA) for
contract manufacture of the study drug, the NHLBI DSMB
for review of clinical safety data, and Dr. Ann Fu for his-
Author Disclosure Statement
D.R.K., G.Y., and J.D.C. are employees of and hold share
options in Applied Genetic Technologies Corporation, and
have a conflict of interest to the extent that this work po-
tentially increases their personal financial interests. J.M.W. is
a consultant to ReGenX Holdings, and is a founder of, holds
equity in, and receives a grant from affiliates of ReGenX
Holdings; in addition, he is an inventor on patents licensed
to various biopharmaceutical companies, including affiliates
of ReGenX Holdings. None of the other authors has a com-
peting financial interest.
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Address correspondence to:
Dr. Terence R. Flotte
University of Massachusetts Medical School
55 Lake Avenue North
Worcester, MA 01655
Received for publication March 31, 2011;
accepted after revision May 23, 2011.
Published online: May 24, 2011.
PHASE 2 CLINICAL TRIAL OF rAAV1-CB-hAAT1247
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