HUMAN GENE THERAPY 17:1–10 (December 2006)
© Mary Ann Liebert, Inc.
Phase I Trial of Intramuscular Injection of a Recombinant
Adeno-associated Virus Serotype 2 ?1-Antitrypsin (AAT)
Vector in AAT-Deficient Adults
MARK L. BRANTLY,1,2L. TERRY SPENCER,3MARGARET HUMPHRIES,2,3THOMAS J. CONLON,2,3
CAROLYN T. SPENCER,2,3AMY POIRIER,2,3WENDY GARLINGTON,2,3DAWN BAKER,2,3
SIHONG SONG,2,4KENNETH I. BERNS,2,5NICHOLAS MUZYCZKA,2,5RICHARD O. SNYDER,2,5
BARRY J. BYRNE,2,3,5and TERENCE R. FLOTTE2,3,5
A phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 (rAAV2) ?1-
antitrypsin (AAT) vector was performed in 12 AAT-deficient adults, 10 of whom were male. All subjects were
either homozygous for the most common AAT mutation (a missense mutation designated PI*Z) or compound
heterozygous for PI*Z and another mutation known to cause disease. There were four dose cohorts, ranging
from 2.1 ? 1012vector genomes (VG) to 6.9 ? 1013VG, with three subjects per cohort. Subjects were injected
sequentially in a dose-escalating fashion with a minimum of 14 days between patients. Subjects who had been
receiving AAT protein replacement discontinued that therapy 28 days before vector administration. There
were no vector-related serious adverse events in any of the 12 participants. Vector DNA sequences were de-
tected in the blood between 1 and 3 days after injection in nearly all patients receiving doses of 6.9 ? 1012VG
or higher. Anti-AAV2 capsid antibodies were present and rose after vector injection, but no other immune
responses were detected. One subject who had not been receiving protein replacement exhibited low-level ex-
pression of wild-type M-AAT in the serum (82 nM), which was detectable 30 days after receiving an injection
of 2.1 ? 1013VG. Unfortunately, residual but declining M-AAT levels from the washout of the protein re-
placement elevated background levels sufficiently to obscure any possible vector expression in that range in
most of the other individuals in the higher dose cohorts.
preclinical models as a potential means to deliver secreted gene
products to the serum (Kessler et al., 1996; Xiao et al., 1996;
Fisher et al., 1997; Song et al., 1998). In preclinical models,
intramuscular injection of rAAV2 was found to be generally
safe and associated with long-term, robust expression of the
transgene product, although immune responses have been re-
ported in some contexts (Manning et al., 1998; Xiao et al., 1999;
Herzog et al., 2002; Song et al., 2002; Gao et al., 2004; Wang
et al., 2005a,b). Clinical investigation of this approach has been
reported in the case of hemophilia B gene therapy (Kay et al.,
NTRAMUSCULAR INJECTION OF recombinant adeno-associated
virus serotype 2 (rAAV2) vectors has been investigated in
2000; Manno et al., 2003). In those studies, expression levels
were low, but there was evidence of a transgene product-spe-
cific interaction with collagen in the extracellular matrix.
?1-Antitrypsin (AAT) deficiency has been proposed as a po-
tential candidate for replacement therapy by intramuscular de-
livery of an rAAV2 vector. Although protein replacement ther-
apy has demonstrated sufficient safety and efficacy to garner
U.S. Food and Drug Administration (FDA) approval, it requires
weekly intravenous injections (Gadek et al., 1981; Gadek and
Crystal, 1983; Wewers et al., 1987b; Abusriwil and Stockley,
2006), which can be costly and difficult. Lessons from protein
replacement therapy indicate that AAT replacement, as com-
pared with factor IX, is less likely to elicit immune response
and has a broad therapeutic range (Hubbard et al., 1988; Crys-
1Department of Medicine, 2Powell Gene Therapy Center, 3Department of Pediatrics, 4Department of Pharmaceutics and 5Department of Mo-
lecular Genetics and Microbiology, University of Florida, Gainesville, FL 32611.
tal, 1989). The lack of an immune response may be due to the
high prevalence of the PI*Z genotype (a missense mutation),
although null patients have also been safely treated with AAT
protein replacement (Wewers et al., 1987a).
Preclinical studies with intramuscular injection of rAAV2-
AAT vectors reinforce the concept that this vector/route/trans-
gene product can result in safe, long-lasting, high-level ex-
pression of AAT in the serum in mice (Song et al., 1998, 2001b;
Xu et al., 2001; Poirier et al., 2004). Safety has also been con-
firmed in rabbit and nonhuman primate models (Song et al.,
2002; Poirier et al., 2004). AAT produced from muscle has been
shown to be functional in in vitro assays and in a mouse model
of the emphysema phenotype induced by a vascular endothe-
lial growth factor (VEGF) receptor antagonist (Lu et al., 2006;
Petrache et al., 2006). Finally, immunostaining has not indi-
cated any interaction between muscle-derived AAT and the ex-
tracellular matrix (Song et al., 2001a; Lu et al., 2006).
On the basis of these findings, we undertook a phase I trial
(the first in humans) of intramuscular injection of rAAV2-AAT
in AAT-deficient adults. Eligible, consenting subjects under-
went intramuscular injections of doses of rAAV2-AAT rang-
ing from 2.1 ? 1012vector genomes (VG) to 6.9 ? 1013VG.
The vector was generally safe and well tolerated, although sub-
jects had transient vector viremia and developed humoral im-
mune responses to the rAAV2 capsid. Wild-type (M)-specific
serum enzyme-linked immunosorbent assay (ELISA) data in-
dicated that expression was absent in most subjects, although
one subject in the third cohort demonstrated a transient modest
rise in M-AAT to a level of approximately 80 nM. It is hoped
that these general safety data will facilitate the performance of
later trials with the same vector DNA cassette pseudotyped into
an alternative AAV capsid serotype with greater efficiency for
gene transfer in muscle.
MATERIALS AND METHODS
Regulatory approvals and oversight
The clinical protocol and informed consent documents were
reviewed by the National Institutes of Health Office of Biotech-
nology Activities (OBA), the University of Florida Institutional
Biosafety Committee (IBC), the University of Florida Institu-
tional Review Board (IRB), the University of Florida Human
Use of Radiation and Radioactive Materials Committee, the
University of Florida General Clinical Research Center Advi-
sory Committee, and the National Heart, Lung, and Blood In-
stitute Data and Safety Monitoring Board (which also per-
formed ongoing review). An Investigational New Drug
application was filed with the FDA Center for Biologics Eval-
uation and Research and an initial clinical hold was removed
before enrolling the first subject in the trial.
Vector construct and cGMP production
The vector DNA cassette is shown diagrammatically in Fig.
1. The AAV2 inverted terminal repeats were present on either
end of the construct. The CBA enhancer/promoter sequence in-
dicates a cytomegalovirus immediate-early enhancer/chicken
?-actin promoter with a hybrid intron (chicken ?-actin/rabbit
?-globin). The AAT gene insert is the human cDNA. The
bovine growth hormone polyadenylation signal was also in-
cluded. Vector was packaged and purified according to clinical
Good Manufacturing Practices (cGMP), using a published co-
transfection technique in HEK-293 cells (adenovirus serotype
2 [Ad2], E1A?E1B?) with the pDG packaging plasmid, which
encodes the other three needed adenoviral genes (E2A, VA, and
E4), as well as the AAV2 rep and cap genes (Grimm et al.,
1998). Downstream vector purification was performed by an
all-column purification method (Snyder and Flotte, 2002; Zolo-
tukhin et al., 2002; Francis and Snyder, 2005).
Inclusion and exclusion criteria are indicated in Table 1. To
summarize, subjects included male and female adults with con-
firmed AAT deficiency and a genotype consisting either of ho-
mozygosity for the PI*Z allele or heterozygosity between PI*Z
and a second mutant AAT allele, known to be associated with
a deficient phenotype. Subjects were excluded for other condi-
tions that might confound the safety studies or unduly increase
their risk for participation.
Vector dose levels, cohorts, and escalation
The vector dose level for each cohort in the trial is shown
in Table 2. Four cohorts of three patients each were enrolled
sequentially, with at least 2 weeks allowed between each dos-
ing, to allow for the possibility of stopping further dosing if
BRANTLY ET AL.
TABLE 1. SELECTION CRITERIA FOR STUDY SUBJECTS
Inclusion criteriaExclusion criteria
Adult (?18 years old)
AAT deficiency (?11 ?M)
PI*ZZ or compound heterozygote of PI*Z
with a second disease allele
FEV1? 25% predicted
Willing to discontinue protein therapy
Recent antibiotics (?15 days)
LFTs ? twice upper normal
CK ? three times upper normal
Other investigational drug being used
Pregnant or nursing
Fertile and not using contraception
Cigarette smoking or substance abuse
Immune response to AAT replacement
Other conditions at discretion of principal investigator
Abbreviations: AAT, ?1-antitrypsin; CK, creatine kinase; FEV1, forced expiratory volume in 1 sec;
LFTs, liver function tests.
vector-related serious adverse events (SAEs) were observed.
The dose levels consisted of 2.1 ? 1012VG for cohort 1, 6.9 ?
1012VG for cohort 2, 2.1 ? 1013VG for cohort 3, and 6.9 ?
1013VG for cohort 4.
The deltoid muscle of the nondominant arm (left arm for
right-handed individuals and vice versa) was chosen as the site
of injection. Before vector injection, a baseline computed to-
mography (CT) scan of the chest and upper extremity was per-
formed in order to assess baseline morphology and rule out
underlying abnormalities of the upper extremity. Doppler
ultrasound was performed over the potential injection site im-
mediately before and during the injection in order to avoid vas-
cular structures with observable flow (Fig. 2). The needle was
also directly visualized and the outflow of vector was visual-
ized with the Doppler in real time. The dose was administered
in a constant 3.3-ml volume, regardless of VG dose. A diluent
identical to the excipient was used to dilute vector up to the
3.3-ml volume for each dose in cohorts 1, 2, and 3. Meanwhile
vector was injected in its original excipient for cohort 4. At the
time of the injection, the 3.3-ml volume was separated into three
separate injections of 1.1 ml each. Each 1.1-ml volume was in-
fused slowly over 1 min by the ultrasound guidance method de-
INTRAMUSCULAR INJECTION OF rAAV2 AAT VECTOR
peats; CBA hybrid promoter, CMV enhancer, chicken ?-actin promoter, ?-actin/rabbit ?-globin hybrid intron; pA, bovine growth
hormone polyadenylation signal.
Map of rAAV2-AAT vector. Key features of the vector DNA cassette are depicted. ITR, AAV2 inverted terminal re-
site. (A) Example of a computed tomography
(CT) scan of the chest and upper extremities
performed at baseline in a study subject. (B)
Color signal visualized during vector injection
within a well-visualized deltoid muscle mass.
The light blue line outlines the zone in which
Doppler flow is detected. The presence of red
and blue colors during infusion indicates flow
of vector into the muscle mass. The lack of ap-
parent flow-containing vascular structures in
the vicinity confirms that large arteries and
veins have been avoided.
Imaging of rAAV2-AAT injection
Overall design and safety outcomes
The overall time line and design of the study are depicted in
Fig. 3. Subjects underwent baseline screening and evaluation at
the University of Florida General Clinical Research Center
(GCRC), approximately 28 days before the day of vector in-
jection (day 0). Those patients who had been receiving intra-
venous AAT protein therapy discontinued therapy at the day ?28
time point and remained off therapy until day 75 after vector in-
jection. Baseline safety studies, blood and semen PCR assays,
and immune response and AAT assays were performed. The con-
formity of each potential subject with inclusion and exclusion
criteria was then confirmed. Subjects returned to the GCRC on
day ?1 for repeated safety studies. These safety studies included
complete blood counts, coagulation studies (partial thrombo-
plastin time [PTT], prothrombin time [PT], international nor-
malized ratio [INR], and a platelet function study), and chem-
istry panels (including transaminases). These assays were
repeated at the GCRC several times during the first 3 months,
whereas after that time point most studies were done remotely.
Blood were obtained and assayed during the course of the
study on days ?28, 1, 3, and 14. DNA was extracted and vec-
tor sequences were detected by TaqMan (Applied Biosystems,
Foster City, CA) real-time PCR techniques with a previously
published primer–probe set directed against the CBA promoter
sequences, which was vector specific (Song et al., 2002; Poirier
et al., 2004). Likewise, semen samples from all male subjects
were assayed at those time points, when they could be obtained.
No equivalent technique was available to sample germ line-de-
rived cells from females. All samples were done in triplicate.
The technique has as sensitivity of 100 copies per microgram
of input DNA.
Anti-AAV2 and anti-AAT antibodies were assayed accord-
ing to a previously published ELISA method, modified for use
on human serum by substituting an appropriate rabbit anti-hu-
man IgG secondary antibody. In the case of the anti-AAT an-
tibody assay, positive control serum was taken from a baboon
immunized against human AAT in a prior study of intramus-
cular injection (Song et al., 2002). The anti-human IgG sec-
ondary antibody gave a robust signal with the baboon serum,
and this signal diluted out in linear fashion as the standard curve
for each ELISA plate used in the assay.
Anti-AAV2 and anti-AAT antigen-specific lymphocyte pro-
liferation responses (ASRs) were assessed as previously de-
scribed (Hernandez et al., 1999; Poirier et al., 2004). A stimu-
lation index was defined as the ratio of [3H]thymidine uptake
in the presence of antigen to 3H uptake in the absence of anti-
gen. A stimulation index greater than 2 was taken to represent
a positive response. Both antibody and ASR assays were per-
formed on days ?28, 14, and 90.
Assays for total and M-specific AAT in serum
Total serum AAT was assayed on days ?28, ?1, 3, 14, 30,
45, 60, 75, 90, and 180 after vector injection, using a clinically
approved antibody-based nephelometry assay. M-AAT was de-
tected with an M-specific capture ELISA at the same time points.
Long-term follow-up plan
On completion of this study, each subject was enrolled in a
subsequent long-term follow-up study, to span an additional 14
years of postadministration follow-up (15 years total). Subjects
were to be contacted annually to determine whether additional
neurologic, immunologic, or hematologic toxicity had devel-
oped (Nyberg et al., 2004).
BRANTLY ET AL.
TABLE 2. DOSE COHORTS
Number of subjects
2.1 ? 1012
6.9 ? 1012
2.1 ? 1013
6.9 ? 1013
come measurements. The period of discontinu-
ation of protein therapy for those who had pre-
viously been on such therapy is indicated in the
upper shaded bar. See text for other details.
Time line for vector injection and out-
Characteristics of the 12 AAT-deficient subjects who com-
pleted the study are provided in Table 3. Subjects ranged in age
from 42 to 69 years. Ten males and two females were included.
Two other subjects were screened and failed to meet inclusion
criteria before the planned vector dosing, and so were excluded
from further analysis. A broad range of disease severity was
represented in the population studied, with a forced expiratory
volume in 1 sec (FEV1) ranging from 34.8 to 103.4% predicted.
No serious adverse events (SAEs) were reported during the
study. Potentially related adverse events (AEs) are listed in
Table 4. Potentially related AEs included mild erythema near
the injection site, although this was believed to be related to
tape adhesive, based on the physical distribution in at least one
instance. All potentially related AEs were minor in nature and
did not require significant intervention. Importantly, there was
no indication of muscle toxicity or inflammation, as indicated
by clinical examinations and creatine kinase (CK) levels in the
Real-time DNA PCR for vector DNA in the blood
Vector DNA sequences were detected in the blood on day 1
or 3 in most subjects receiving doses of 6.9 ? 1012VG or
greater (cohorts 2, 3, and 4) (Table 5). In only one instance did
vector DNA persist at the 14-day time point. Vector DNA was
not detected in the semen, although not all males were able to
produce sufficient semen for testing at all time points. The pres-
ence of vector DNA sequences in the peripheral blood samples
was not associated with adverse events.
Immune responses to AAV2 capsid components
Anti-AAV2 antibodies were detected by ELISA in all sub-
jects at their baseline, but preexisting anti-AAV2 antibody lev-
els spanned a nearly 100-fold range. Most subjects demon-
INTRAMUSCULAR INJECTION OF rAAV2 AAT VECTOR
TABLE 4. ADVERSE EVENTS IN PHASE I TRIAL
(VG)Event SeriousUnanticipated Outcome
Slight redness at outer
Muscle tenderness, left
Injection site, slight
Tenderness at injection
Warmth at upper
border of erythema
101 2.1 ? 1012
NY Cannot be
2046.9 ? 1012
205 6.9 ? 1012
3012.1 ? 1013
4016.9 ? 1013
401 6.9 ? 1013
Abbreviations: N, no; Y, yes.
TABLE 3. CHARACTERISTICS OF STUDY SUBJECTS
(years) Subject ID SexFEV1(% predicted)PriorSubsequent/study day
Protein replacement therapy
strated an increase in anti-AAV2 antibodies after vector ad-
ministration (Fig. 4A). The presence of anti-AAV2 antibodies
was not associated with adverse events.
Anti-AAT antibodies were all extremely low, more than
1,000,000-fold lower than the positive control serum (derived
from a nonhuman primate immunized against human AAT).
None of the subjects demonstrated a significant increase in anti-
AAT levels after intramuscular vector injection (Fig. 4B). Lym-
phocyte proliferation responses to both AAV2 and AAT were
low, and neither increased after intramuscular vector injection
Total and M-specific AAT levels
Total serum AAT levels were high at baseline in individuals
treated with AAT protein replacement, and decreased to a lower
BRANTLY ET AL.
Serum Anti-AAV2 antibodies, mU/ml
Time, Days post-injection
Serum Anti-AAT antibodies, mU/ml
?200 20 40
Time, Days post-injection
and AAT. Anti-AAV2 antibody titers are
shown in (A), summarized by cohort. Anti-
AAT titers are shown in (B). In each case,
the positive control serum used in the
ELISA was defined as 1 unit/ml, such that
the positive control level in (A) was 103
mU/ml and in (B) was 109nU/ml.
Antibody responses to AAV2
TABLE 5. VECTOR DNA SEQUENCES IN BLOOD OF
Subject Day ?28Day 1 Day 3 Day 14
level, demonstrating a plateau in the deficient range around
the time of vector dosing. M-specific AAT levels demon-
strated a similar pattern (Fig. 6), although residual M-AAT
levels from protein replacement remained substantially above
background in many 14- and 30-day samples. Only one sub-
ject, 303, demonstrated an elevation of M-AAT that was
clearly above background. This was a transient elevation on
day 30, to a maximal value of 82 nM (Fig. 7). Although this
value is above baseline, it remains approximately 125-fold be-
low the lower end of the therapeutic range. On the basis of
the residual declining levels of M-AAT in the individuals re-
ceiving protein replacement, this range and timing of eleva-
tion would likely have been obscured in the other patients in
this cohort, as well as in subjects 401 and 403 in the next co-
hort. However, subject 402 was not receiving protein re-
placement, and this individual failed to show evidence of an
M-AAT response. It should be noted that subject 402 was the
only subject from cohort 2 or beyond who also did not have
any positive blood DNA PCR results.
The data presented here support the general safety of intra-
muscular injection of rAAV2-AAT in AAT-deficient adults at
doses up to 6.9 ? 1013VG per patient. Vector DNA was pres-
ent in the blood on days 1 and 3 after intramuscular injection
of doses of 6.9 ? 1012VG or greater. Anti-AAV2 antibody re-
sponses were also observed in all subjects, but neither of these
findings was associated with adverse effects. On the basis of
the M-specific AAT ELISA, it appeared that a serum level of
the transgene product was detectable transiently in only one
INTRAMUSCULAR INJECTION OF rAAV2 AAT VECTOR
Stimulation Index (SI)
Time, Days post-injection
Stimulation Index (SI)
?200 20 40
Time, Days post-injection
liferation (ASR). (A) Stimulation index (ratio
of [3H]thymidine uptake in the presence of
antigen to 3H uptake in the absence of anti-
gen) for exposure of patient lymphocytes to
AAV2 whole capsid antigen; (B) the ASR af-
ter exposure to AAT.
Antigen-specific lymphocyte pro-
subject, and the level was approximately 125-fold below the
lower end of the therapeutic range in that individual. However,
residual levels of M-AAT from protein replacement could have
masked lower levels of transgene expression.
These data are similar in a number of respects to the pub-
lished results of intramuscular rAAV2-mediated gene transfer
in hemophilia B patients (Kay et al., 2000; Manno et al., 2003).
In that study, patients demonstrated transient vector viremia and
anti-AAV2 antibody responses. Likewise, transgene expression
levels in the serum were transiently in the detectable range and
were relatively low. There were a number of key differences
between the current study and the hemophilia B studies, how-
ever, including the promoter choice, and immunohistochemical
evidence of binding of vector-expressed factor IX to the extra-
cellular matrix (Herzog et al., 1997; Manno et al., 2003). The
dose–response data in the current study are also consistent with
preclinical data in mice, if normalized on the basis of body
weight (Song et al., 1998; Poirier et al., 2004). On the basis of
these data, one might have predicted that a dose greater than
1 ? 1012VG/kg would be required for consistent detectable
levels in humans, a dose readily achieved in the mouse studies,
but difficult to achieve in humans. This is in contrast to the sug-
gestion by Zhou et al., who, in a baboon study of intramuscu-
lar injection of rAAV2-epo, suggested that a lower dose per
body weight may be required in primates (Zhou et al., 1998).
It is also not clear whether one might have expected a local-
ized region of transduced muscle to be able to supply systemic
levels of AAT. It seems that this will be easier to study in later
studies using either higher doses or alternative serotypes, where
higher levels will allow for better comparisons between deliv-
One peculiarity in the dose–response data is that the one in-
dividual with a reading on the M-AAT assay that was clearly
above baseline was from the second highest dose group (2.1 ?
1013VG) and not the highest (6.9 ? 1013VG). An important
issue is that of the six subjects in the two highest dose cohorts,
there were only two who had not been receiving protein re-
placement, one from each cohort. For those undergoing protein
therapy, the residual declining M-AAT levels carried over from
incomplete washout of recombinant M-AAT effectively ele-
vated the background to levels that would have obscured re-
sponses in the range of 50 to 100 nM. Of the two individuals
for whom this was not a factor, one showed an increase in
M-AAT and the other did not. The one that did not was coin-
cidentally the only subject who failed to show evidence of vec-
tor DNA sequences in the blood at the 1- or 3-day time point.
This individual had low levels of preexisting anti-AAV2 anti-
bodies, significantly lower than those observed in subject 303,
so it does not seem likely that preexisting immunity was the
primary factor. Although it is impossible to draw any conclu-
sions from such small numbers of patients, it is possible that
there was some technical difficulty with the dose preparation,
dilution, or injection of that one individual. It would appear ad-
visable in future studies to either avoid patients receiving pro-
tein replacement or to lengthen the interval between discontin-
uation of protein therapy and vector injection. In retrospect, it
would have been helpful to have included an analysis of mus-
cle biopsy material from the injection site, both to document
vector expression and to determine whether the apparent low-
BRANTLY ET AL.
?500 50 100150
Time, days post-injection
and after injection of rAAV2-AAT. An M-specific
AAT ELISA was used to detect vector-expressed,
wild-type AAT in the serum of study subjects. In-
jection was given on day 0.
Serum M-AAT levels in subjects before
Serum M-AAT, nM
?200 20 40
Time post-injection, days
set of symbols represents a different subject. Triangles and solid
circles represent levels from subjects 301 and 302, respectively,
both of whom were receiving protein replacement before gene
transfer; open circles represent levels from subject 303, who
was not receiving protein replacement.
M-specific AAT levels in subjects in cohort 3. Each
level transient expression seen in subject 303 was associated with
cellular infiltration or other evidence of an immune response not
detectable in the systemic immune response assays.
Since this product development program was initiated, other
rAAV serotypes have emerged and have been shown to be more
efficient for transduction of skeletal muscle in mice and larger
mammals (Xiao et al., 1999; Louboutin et al., 2005). These find-
ings must be viewed cautiously, however, as studies have shown
that the dramatic advantage, in terms of efficiency, of certain
serotypes for liver delivery in mice is not directly mirrored in
larger animals. In any case, the current study provides both safety
and dosing data that could be compared with future results of a
study of intramuscular injection of an rAAV1-pseudotyped ver-
sion of this same vector DNA cassette (Flotte, 2005a–c; Flotte
and Berns, 2005; Lu et al., 2006; Petrache et al., 2006).
Finally, one must consider the possibility that a different route
of administration could be used for rAAV-mediated gene therapy
of AAT deficiency. Preclinical feasibility data have been pub-
lished with a wide range of routes of injection, including portal
vein, peripheral vein, intrapleural, intratracheal, salivary gland
duct, and others (Song et al., 2001a; Voutetakis et al., 2004; Con-
lon et al., 2005; Virella-Lowell et al., 2005). In addition, one other
clinical trial based on intranasal injection has been reported
(Brigham et al., 2000). Of these, the intrapleural injection of
rAAV5-AAT or of rAAVrh.10-AAT and the portal vein injection
of rAAV8 vectors appear to yield the highest levels (De et al.,
2004; Conlon et al., 2005; Heguy and Crystal, 2005). In addition,
one might consider molecular means to downregulate mutant Z-
AAT along with therapeutic M-AAT expression with rAAV8-me-
diated targeting of the liver (Zern et al., 1999). The former of
these could potentially be helpful in treating AAT-related liver
disease. Clearly, many more preclinical and clinical trials will be
required to evaluate the relative advantages and disadvantages of
each of these potential approaches.
This work was supported by grants from the NIH (HL4456),
NHLBI (HL69877), NIDDK (DK58327), and NCRR (RR00082),
and by support from Shands Hospital, the Alpha One Founda-
tion, and the University of Florida College of Medicine. The
University of Florida holds an equity interest in Applied Ge-
netic Technologies Corporation which has licensed the vector
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Address reprint requests to:
Dr. Terence R. Flotte
1600 SW Archer Road
University of Florida
Gainesville, FL 32610-0296
Received for publication July 19, 2006; accepted after revision
October 18, 2006.
Published online: November 21, 2006.
BRANTLY ET AL.