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Efficient and Stable Transduction of Cardiomyocytes After
Intramyocardial Injection or Intracoronary Perfusion With
Recombinant Adeno-Associated Virus Vectors
Eric C. Svensson, MD, PhD; Deborah J. Marshall, PhD; Karen Woodard, BS; Hua Lin, MD;
Fang Jiang, MD; Lein Chu, MS; Jeffrey M. Leiden, MD, PhD
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Background—The delivery of recombinant genes to cardiomyocytes holds promise for the treatment of a variety of
cardiovascular diseases. Previous gene transfer approaches that used direct injection of plasmid DNA or replicationdefective adenovirus vectors have been limited by low transduction frequencies and transient transgene expression due
to immune responses, respectively. In this report, we have tested the feasibility of using intramyocardial injection or
intracoronary infusions of recombinant adeno-associated virus (rAAV) vectors to program transgene expression in
murine cardiomyocytes in vivo.
Methods and Results—We constructed an rAAV containing the LacZ gene under the transcriptional control of the
cytomegalovirus (CMV) promoter (AAVCMV-LacZ). We then injected 13108 infectious units (IU) of this virus into the left
ventricular myocardium of adult CD-1 mice. Control hearts were injected with the AdCMV-LacZ adenovirus vector. Hearts
harvested 2, 4, and 8 weeks after AAVCMV-LacZ injection demonstrated stable b-galactosidase (b-gal) expression in large
numbers of cardiomyocytes without evidence of myocardial inflammation or myocyte necrosis. In contrast, the
AdCMV-LacZ-injected hearts displayed transient b-gal expression, which was undetectable by 4 weeks after injection.
Explanted C57BL/6 mouse hearts were also perfused via the coronary arteries with 1.53109 IU of AAVCMV-LacZ and
assayed 2, 4, and 8 weeks later for b-gal expression. b-Gal expression was detected in ,1% of cardiomyocytes at 2
weeks after perfusion but was detected in up to 50% of cardiomyocytes 4 to 8 weeks after perfusion.
Conclusions—Direct intramyocardial injection or coronary artery perfusion with rAAV vectors can be used to program
stable transgene expression in cardiomyocytes in vivo. rAAV appears to represent a useful vector for the delivery of
therapeutic genes to the myocardium. (Circulation. 1999;99:201-205.)
Key Words: myocardium n genes n molecular biology
M
yocardial gene therapy holds promise for the treatment
of a number of cardiovascular diseases, including
ischemic cardiomyopathies, congestive heart failure, and
malignant arrhythmias.1 The ideal vector for myocardial gene
delivery would allow the efficient and stable transduction of
cardiomyocytes with a variety of transgenes after either direct
intramyocardial injection or infusion into the coronary arteries. Plasmid DNA vectors injected directly into the left
ventricular myocardium are expressed for $6 months by
cardiomyocytes adjacent to the area of injection.2– 4 To date,
however, the therapeutic usefulness of this approach has been
limited by the low efficiency of cardiomyocyte transduction
(0.1% to 1.0% of cardiomyocytes in the area of injection).
Both intramyocardial injection and intracoronary infusion of
replication-defective adenovirus (RDAd) vectors can be used
to efficiently transduce cardiomyocytes in rodents, rabbits,
and pigs in vivo.4 – 6 However, the feasibility of adenovirus-
mediated gene transfer has been limited by immune responses
to viral and foreign transgene proteins, which cause significant myocardial inflammation, eliminate virus-transduced
cells within 30 days of infection, and thereby result in
transient recombinant gene expression in immunocompetent
hosts.6
Recently, recombinant adeno-associated virus (rAAV) vectors have been shown to program efficient and stable recombinant gene expression in skeletal muscle and liver in both
rodents and primates.7–9 Unlike RDAd, rAAV vectors do not
encode viral proteins and have not been associated with
immune responses to foreign transgene proteins. A previous
report has shown that rAAV can transduce cardiomyocytes in
vivo.10 However, in that study, the efficiency of rAAVmediated transgene expression in the heart was low ('0.2%).
In the present report, we have assessed the efficiency and
stability of rAAV-mediated gene transfer in the heart after
Received September 24, 1998; revision received November 9, 1998; accepted November 12, 1998.
From the Departments of Medicine (E.C.S., D.J.M., K.W., H.L., F.J., L.C.) and Pathology (J.M.L.), University of Chicago, Chicago, Ill.
Correspondence to Jeffrey M. Leiden, MD, PhD, University of Chicago, Room B608 MC 6080, 5841 S Maryland Ave, Chicago, IL 60637. E-mail
[email protected]
© 1999 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
201
202
AAV-Mediated Transduction of Cardiomyocytes
Figure 1. rAAV-mediated gene transfer into primary cardiomyocytes in vitro. A, Schematic of
AAVCMV-LacZ. ITR indicates inverted terminal repeat;
BGH pA, bovine growth hormone polyadenylation
signal; CMV Pr, CMV immediate-early promoter;
LacZ, bacterial LacZ gene. B and C, LacZ expression in primary rat neonatal cardiomyocytes 48
hours after infection in vitro with 100 IU/cell of
AAVCMV-LacZ (B) or 24 hours after infection in vitro
with 100 IU/cell of AAVCMV-LacZ plus 2 pfu/cell
Addl309 (C).
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both intramyocardial injection and intracoronary infusion.
Our results suggest that rAAV vectors may be useful vectors
for myocardial gene delivery.
Methods
Plasmids and Viruses
The structure of pAAVCMV-LacZ is shown in Figure 1A. AdCMV-LacZ and
the E3-deleted adenovirus, Addl309, were propagated and purified as
described previously.6
Propagation and Purification of rAAV
rAAV was prepared as described.11 We determined viral titer by
using a dot blot hybridization assay to determine the number of viral
genomes per milliliter and by infecting HeLa cells with the virus and
staining with X-gal 24 hours after infection. All viral preparations
had titers of 1 to 231011 genomes/mL and 2 to 33109 infectious
units (IU)/mL.
In Vitro Infection of Neonatal Rat Cardiomyocytes
Cultures of primary neonatal rat cardiomyocytes12 (43106 cells)
were infected with 43108 IU of rAAV with or without 33107
plaque-forming units (pfu) of Addl309 (n53 for each group). After 24
to 48 hours, cells were fixed and stained with X-gal.
Intramyocardial Injection of rAAV or RDAd
Six- to 8-week-old CD-1 mice were anesthetized, intubated, and
mechanically ventilated. AAVCMV-LacZ (13108 IU) or AdCMV-LacZ
(23109 pfu) in a volume of 50 mL was injected into the apex of the
left ventricle with a 30-gauge needle (n53 for each time point).
Intracoronary Perfusion With rAAV
Adult C57BL/6 mouse hearts were perfused via the left carotid artery
with cardioplegia solution (110 mmol/L NaCl, 25 mmol/L KCl,
22 mmol/L NaHCO3, 16 mmol/L MgCl2, 0.8 mmol/L CaCl2, 40 mmol/L
glucose) at 4°C until they stopped beating. They were then perfused ex
vivo for 15 minutes with 1.53109 IU of AAVCMV-LacZ in 0.5 mL of PBS
at a rate of 33 mL/min at 4°C. After perfusion, the hearts were
transplanted into the neck of a syngeneic host with anastomosis of the
donor aorta to the right common carotid artery of the host and
anastomosis of the donor pulmonary artery to the right external jugular
vein13 (n53 for each time point).
X-Gal Staining
Freshly isolated hearts were fixed in PBS plus 1.25% glutaraldehyde
for 10 minutes at room temperature, stained overnight with X-gal,2
and counterstained with eosin.
b-Galactosidase Activity
Cardiac homogenates were assayed for b-galactosidase (b-gal)
activity and protein concentration.2 b-Gal activities were normalized
for total protein and for the number of infectious rAAV or RDAd
particles injected.
Results
Efficient In Vitro Transduction of Cardiomyocytes
by rAAV Vectors
To test the capacity of rAAV vectors to transduce cardiomyocytes in vitro, we constructed an rAAV (AAVCMV-LacZ) containing the LacZ gene under the transcriptional control of the
cytomegalovirus (CMV) immediate-early promoter (Figure
1A). This virus was used to infect cultures of primary
neonatal rat cardiomyocytes at a multiplicity of infection
(MOI) of 100 IU/cell. Duplicate cultures were infected with
AAVCMV-LacZ (MOI5100 IU/cell) plus 2 pfu/cell Addl309. In
other cell types, coinfection with adenovirus has been reported to facilitate the conversion of rAAV genomes to
double-stranded DNA and to thereby increase the efficiency
of rAAV-mediated transgene expression.14 Twenty-four to 48
hours after infection, b-gal expression was detected by
staining with X-gal. In the absence of added adenovirus,
'10% of cardiomyocytes expressed b-gal (Figure 1B). The
addition of adenovirus increased transduction frequencies to
'50% (Figure 1C). These results demonstrate that rAAV
vectors can be used to transduce primary cardiomyocytes in
vitro.
Stable Transduction of Cardiomyocytes In Vivo
After Direct Intramyocardial Injection of
rAAV Vectors
To determine if rAAV could be used to stably transduce
cardiomyocytes in vivo, 13108 IU of AAVCMV-LacZ was
Svensson et al
January 19, 1999
203
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Figure 2. Gene transfer into cardiomyocytes in vivo after intramyocardial injection or coronary artery perfusion with AAVCMV-LacZ. A,
Gross sections (left) and photomicrographs (right) of mouse hearts after intramyocardial injection with 13108 IU of AAVCMV-LacZ and
staining with X-gal. B, b-Gal activity in heart lysates at different times after intramyocardial injection of AAVCMV-LacZ (n53) or AdCMV-LacZ
(n53). Data are presented as mean6SEM. C, Gross sections (left) and photomicrographs (right) of mouse hearts after coronary artery
perfusion with 1.53109 IU of AAVCMV-LacZ and staining with X-gal. Bar525 mm.
204
AAV-Mediated Transduction of Cardiomyocytes
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injected directly into the left ventricular apical myocardium
of 8-week-old immunocompetent CD-1 mice. Mice were
killed 2, 4, and 8 weeks after injection, and b-gal expression
in the myocardium was assayed by X-gal staining. As shown
in Figure 2A, b-gal expression was detected in a small
number of cells surrounding the site of injection at 2 weeks
after injection and in a larger number of cells at 4 and 8 weeks
after injection. The majority of the b-gal–positive cells were
cardiomyocytes, as evidenced by their readily identifiable
myofibers (Figure 2A). Furthermore, the rAAV-injected
hearts did not display detectable myocardial inflammation
(assessed by hematoxylin and eosin staining) or myocyte
necrosis (Figure 2A and data not shown), as has been seen
after myocardial injection of RDAd.3– 6 To directly compare
the efficiency and stability of rAAV-mediated gene transfer
with those of RDAd, adult CD-1 mouse hearts were injected
with either AAVCMV-LacZ or AdCMV-LacZ and quantitatively
assayed for b-gal activity at different times after injection
(Figure 2B). Consistent with previous reports, direct intramyocardial injection of RDAd resulted in transient transgene expression, with peak levels of b-gal activity seen 1
week after injection.3– 6 By 4 weeks after injection, transgene
expression was undetectable in the AdCMV-LacZ-injected hearts.
In contrast, b-gal activity in the AAVCMV-LacZ-injected hearts
exceeded that seen in the AdCMV-LacZ-injected hearts at both 2
and 4 weeks after injection. Peak levels of b-gal activity in
the AAVCMV-LacZ-injected hearts were '25% of those seen
with AdCMV-LacZ. Thus, rAAV vectors can be used to stably
transduce cardiomyocytes in vivo without significant myocardial inflammation and with an efficiency of at least 25%
relative to adenovirus vectors.
Stable and Efficient Transduction of
Cardiomyocytes After Intracoronary Perfusion
With rAAV
Many clinical applications of myocardial gene therapy will
require the stable and efficient transduction of cardiomyocytes distributed throughout large areas of myocardium.
Coronary artery infusions of RDAd have been shown to result
in the efficient transduction of cardiomyocytes throughout the
region of perfused myocardium.6 To test whether rAAV is
similarly capable of transducing cardiomyocytes after coronary artery perfusion, we explanted hearts from C57BL/6
mice and perfused them with 1.53109 IU of AAVCMV-LacZ for
15 minutes at 4°C via a catheter placed in the left common
carotid artery. These perfused hearts were then transplanted
into syngeneic hosts, and the arterial circulation was reestablished by anastomosis of the transplanted aorta to the recipient carotid artery. Such transplanted and revascularized
hearts resumed beating and continued to do so until the
recipient mice were killed 2, 4, or 8 weeks after perfusion.
Two weeks after perfusion, small numbers (,1%) of b-gal–
positive cardiomyocytes were detected throughout the myocardium of the rAAV-perfused hearts (Figure 2C). By 4
weeks after perfusion, '40% of the cardiomyocytes were
b-gal positive. This high level of transduction was stable at 8
weeks after perfusion, with .50% of the cardiomyocytes
continuing to express b-gal. Similar increases in recombinant
gene expression over the first several weeks after rAAV
infection have been observed in skeletal muscle.7,8 It has been
postulated that such increases may reflect the gradual process
of conversion of the single-stranded AAV genome into a
double-stranded DNA molecule that is competent for transcription of the transgene.14 Thus, rAAV delivered by coronary artery perfusion can be used to stably transduce cardiomyocytes throughout the myocardium.
Discussion
In this report, we have demonstrated efficient and stable
transduction of cardiac myocytes in vivo after intramyocardial injection or intracoronary infusions of rAAV vectors.
Our results suggest that rAAV displays significant advantages for myocardial gene transfer compared with either
plasmid DNA or adenovirus vectors. Plasmid DNA vectors
produce low-efficiency (albeit stable) cardiomyocyte transduction and can only be administered by direct intramyocardial injection.2–5 In contrast, rAAV allows efficient transduction of cardiomyocytes and can be administered by either
intramyocardial injection or intracoronary infusion. Adenovirus vectors allow highly efficient cardiomyocyte gene
transfer but produce intense intramyocardial inflammation
and myocyte necrosis, thereby resulting in transient recombinant gene expression in immunocompetent hosts in vivo.3– 6
In contrast, rAAV vectors program stable expression of
foreign transgenes in immunocompetent hosts. The stability
of transgene expression observed with rAAV even after
expression of a foreign transgene protein likely reflects the
fact that rAAV vectors, unlike their adenovirus counterparts,
do not express any viral gene products and are therefore
significantly less immunogenic. This lack of immunogenicity
represents a major advantage of rAAV for myocardial gene
transfer.
Despite these advantages, there are several issues that
might limit the utility of rAAV for cardiovascular gene
therapy. First, this vector can only accept transgenes less than
'4.5 kb in length. Second, current techniques do not allow
the convenient production of large amounts of rAAV. Finally,
although our studies have demonstrated efficient transduction
of cardiomyocytes after 15 minutes of coronary artery perfusion with rAAV, it remains unclear if similar high-efficiency
transduction can be obtained by catheter-mediated intracoronary infusions of this vector. Despite these caveats, our
results suggest that rAAV vectors will significantly enhance
our ability to stably express recombinant genes in cardiomyocytes in vivo and, as such, may represent an important vector
system for myocardial gene therapy.
Acknowledgments
This work was supported in part by grants from the National
Institutes of Health (DK-48987, AR-42885, and HL-54592) to
Dr Leiden.
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Efficient and Stable Transduction of Cardiomyocytes After Intramyocardial Injection or
Intracoronary Perfusion With Recombinant Adeno-Associated Virus Vectors
Eric C. Svensson, Deborah J. Marshall, Karen Woodard, Hua Lin, Fang Jiang, Lein Chu and
Jeffrey M. Leiden
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Circulation. 1999;99:201-205
doi: 10.1161/01.CIR.99.2.201
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
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