Note: Descriptions are shown in the official language in which they were submitted.
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EFFICIENT AND STABLE IN VIVO GENE TRANSFER TO CARDIOMYOCYTES
USING RECOMBINANT ADENO-ASSOCIATED VIRUS VECTORS
This application is a continuation-in-part application of provisional
application
U.S. Ser. No. 60/113,923, filed December 28, 1998, which is incorporated by
reference
herein in its entirety.
This work was supported in part by grants from the National Institutes of
Health
(DK-48997, AR-42895, and HL-54592) to Jeffrey M. Leiden.
FIELD OF THE INVENTION
The ability to stably and efficiently program recombinant gene expression in
cardiomyocytes facilitates gene therapy approaches for a variety of
cardiovascular
diseases and conditions. Accordingly, this invention relates to the use of
recombinant
adeno-associated virus (rAAV) vectors to transduce cardiomyocytes in vivo by
infusing
I S the rAAV into a coronary artery or coronary sinus. For example, coronary
artery
perfusion of mouse hearts with a rAAV encoding the LacZ gene produced
efficient
transduction of cardiomyocytes which was stable for at least 8 weeks.
Moreover, rAAV
infection is not associated with detectable myocardial inflammation or myocyte
necrosis.
Thus, rAAV is a useful vector for the stable expression of therapeutic genes
in the
myocardium and can be used to deliver genes for inducing angiogenesis,
inhibiting
angiogenesis, stimulating cell proliferation, inhibiting cell proliferation
and/or treating or
ameliorating other cardiovascular conditions.
BACKGROUND OF THE INVENTION
Myocardial gene therapy can be used for the treatment of a number of
cardiovascular diseases, including ischemic cardiomyopathies, congestive heart
failure,
and malignant arrhythmias (Nabel (1995) Circulation 91:541-548). A useful
vector for
myocardial gene delivery will allow efficient and stable transduction of
cardiomyocytes
with a variety of transgenes after either direct intramyocardial injection or
infusion into
the coronary arteries or sinuses. For example, plasmid DNA vectors injected
directly into
the left ventricular myocardium have been expressed for z 6 months by
cardiomyocytes
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adjacent to the area of injection (Lin et al. (1990a) Circulation 82:2217-
2221; Kass et al.
(1993) Proc. Natl. Acad. Sci.USA 90:11498-11502; and Guzman et al. (1993)
Circ.
Res.73:1202-1207). 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 have been used to efficiently transduce
cardiomyocytes in
rodents, rabbits, and pigs in vivo. 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 (Guzman et al. (1993) Circ. Res.73:1202-1207; French et
al.(1994) Circulation 90:2414-2424; and Barr et al.(1994) Gene Ther. 1:51-58).
Recently, rAAV vectors have been shown to program efficient and stable recom-
1 S binant gene expression in skeletal muscle and liver in both rodents and
primates (Fisher et
al.( 1997) Nat. Med. 3:306-312; Kessler et al. ( 1996) Proc. Natl. Acad. Sci.
USA
93:14082-14087; and Snyder et al. ( 1997) Nat. Genet. 16: 270-276) and in
cardiac muscle
directly injected with rAAV (U.S. Patent No. 5,858,351 to Podsakoff et a1.).
However,
since rAAV vectors used in gene therapy applications, unlike RDAd, do not
encode viral
proteins, the rAAV vectors have not been associated with immune responses to
foreign
transgene proteins.
While a previous report showed that rAAV can transduce cardiomyocytes in vivo,
the efficiency of rAAV-mediated transgene expression in the heart was both low
(about
0.2%) and localized (Kaplitt et al. ( 1996) Ann. Thorac. Surg. 62:1669-1676).
In that
study, pigs hearts were rapidly perfused with a low titer of rAAV (less than
10~
expressing units AAV per gram of body weight). Based on those results,
infusing rAAV
into the heart would have severely limited use as a vector for myocardial gene
therapy.
However, as demonstrated herein, this invention establishes that by infusing
rAAV in
much higher amounts proportional to body weight of the animal and for
particular time
periods, then rAAV provides unexpected efficient and stable gene transfer into
the heart,
opening up use of rAAV vectors to deliver therapeutically-effective molecules
to
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cardiomyocytes in amounts useful for treating or ameliorating cardiac diseases
or
conditions.
SUMMARY OF THE INVENTION
The present invention is directed to a method of treating a cardiovascular
condition by infusing an rAAV vector into a coronary artery or a coronary
sinus for a time
and in an amount sufficient to stably and efficiently transduce the
cardiomyocytes
perfused by the artery or sinus. The rAAV vector encodes at least one nucleic
acid which
is operably linked to a control region and which encodes a therapeutically-
effective
molecule. After infusion and transduction of the cardiomyocytes, the
therapeutically-
effective molecule is expressed in an amount effective to treat or ameliorate
the
cardiovascular condition.
Thus, this method provides a means of delivering AAV vectors in a stable and
efficient manner. The vector can be infused by any convenient means and in
conjunction
with surgery or other cardiac procedure, if desired.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure I . Schematic of AAV~MV-,~~z. ITR indicates inverted terminal repeats;
BGH pA, bovine growth hormone polyadenylation signal; CMV Pr, CMV immediate-
early promoter; LacZ, bacterial LacZ gene.
Figure 2. Gene transfer into cardiomyocytes in vivo with AAV~MV-~a~z. Gross
sections (left) and photomicrographs (right) of mouse hearts after coronary
artery
perfusion with 1.5 x 1 O9 IU of AAVcMV-~~ and staining with X-gal. Bar=25
microns.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to treating cardiovascular conditions using rAAV
vectors.
In accordance with the invention, an rAAV vector encoding a therapeutically
effective
molecule is infused into a coronary artery or a coronary sinus to deliver the
vector to the
heart in a manner which stably and efficiently transduces cardiomyocytes. It
has
unexpectedly been found that the ability to obtain stable and efficient
transduction of
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cardiomyocytes by rAAV depends upon the duration of the infusion period and
the
amount of virus infused relative to body weight.
Moreover, rAAV displays significant advantages for myocardial gene transfer
compared with plasmid DNA or adenovirus vectors. For example, rAAV, when
delivered
as described herein, allows efficient transduction of cardiomyocytes. Further,
rAAV vec-
tors 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.
Hence, the invention is directed to a method of treating a cardiovascular
condition
which comprises infusing an rAAV vector into a coronary artery or sinus of an
animal for
a time and in an amount sufficient to stably and efficiently transduce
cardiomyocytes
perfused by the artery or sinus, wherein that vector encodes at least one
nucleic acid, i.e.,
the transgene, encoding a therapeutically-effective molecule; and expressing
the
therapeutically-effective molecule in an amount effective to treat or
ameliorate the
cardiovascular condition. Further, the nucleic acid is operably linked to a
control region,
e.g., promoters, enhancers, termination signals and the like, to permit
expression of the
molecule. When more than one nucleic acid is present on the rAAV vector, each
can be
controlled separately by individual control regions or, any group of them, or
all of them,
can be controlled in an operon, i.e, with one control region driving
expression of multiple
genes on a single transcript.
rAAV vectors useful in the present invention can be any rAAV vector with one
or
more transgenes (or nucleic acids of interest) inserted therein in a manner
allowing
expression of the transgene under control of appropriate regulatory elements
such as
promoters, enhancers, transcription terminators and the like. rAAV vectors are
well
known in the art and can be prepared by standard methodology know to those of
ordinary
skill in the art. For example, U.S. Patent No. 5,858,351 and the references
cited therein
describe a variety of rAAV vectors suitable for use in gene therapy as well as
how to
make and propagate those vectors (see, e.g., Kotin (1994) Human Gene Therapy
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5:793-801 or Berns, "Parvoviridae and their Replication" in Fundamental
Virology, 2nd
Edition, (Fields & Knipe, eds.)).
A "transgene" or "nucleic acid of interest" or the "nucleic acid encoded in
the
rAAV vector" as used herein refers to any nucleotide sequence which encodes a
therapeutically-effective molecule that can be used to treat a cardiovascular
condition.
Such transgenes may normally be foreign to the animal being treated or may be
a gene
normally found in that animal for which altered expression (e.g., temporal,
spatial or
amount of expression) is desired to achieve a particular therapeutic effect.
The
therapeutically-effective molecule encoded by the transgene is protein or an
anti-sense
RNA that imparts a benefit to the animal or subject undergoing treatment or
amelioration
of a cardiac condition or disease in accordance with this invention.
Proteins that can be administered to treat or ameliorate cardiovascular
conditions
are numerous and include, but are not limited to, molecules competent to
induce
angiogenesis, e.g., angiogenesis factors; anti-angiogenesis factors; proteins
capable of
inhibiting vascular smooth muscle cell proliferation; proteins useful for
treating
atherosclerosis; proteins useful for treating restenosis, proteins useful for
stimulating
cardiomyocyte activity; proteins capable of secretion from cardiomyocytes that
exert their
effect in the heart or capable of transport to other locales for treatment of
a cardiovascular
condition or disease; hormones, cytokines or growth factors useful for
treating cardiac
conditions or diseases; and proteins capable of stimulating vascular smooth
muscle cell
proliferation. Other genes encoding proteins useful in this invention include
ion channel
genes, contractile protein genes, phospholamban encoding genes and genes
encoding (3
adrenergic receptors or (3 adrenergic kinases.
Angiogenic factors include, but are not limited to FGF-1, FGF-2, FGF-5, VEGF,
HIF-1 and the like. Proteins useful for treating restenosis include thymidine
kinase,
cytosine deaminase, p21, p27, p53, Rb, and NF-tcB. Hence, this invention can
be used to
deliver any protein via an rAAV vector that has a therapeutic benefit for
treating or
ameliorating a cardiovascular condition or disease.
A protein competent to induce angiogenesis or an "angiogenesis factor" as used
herein is a protein or substance that causes proliferation of new blood
vessels and includes
fibroblast growth factors, endothelial cell growth factors or other proteins
with such
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biological activity. Particular proteins known to induce angiogenesis are FGF-
1, FGF-2,
FGF-5, VEGF and active fragment thereof, and HIF-1. Proteins competent to
inhibit
angiogenesis or "anti-angiogenesis factors" are proteins or substances that
inhibit the
formation of new blood vessels.
Anti-sense RNA that can be administered to treat or ameliorate cardiovascular
conditions have one of the same activities as proteins useful in the
invention. Such RNA
include, but are not limited to, c-myb, c-myc and others. Anti-sense RNA
molecules,
including how to design and use such molecules in expression vectors are well
know in
the art and can be contructed by routine methodology. Thus a strand of RNA
whose
sequence of bases is complementary to the sense, or translated, RNA strand can
form a
duplex to block translation or degradation of a particular mRNA or otherwise
control or
alter expression of the desired mRNA.
As used herein, a "control region" or "regulatory element" refers to
polyadenylation signals, upstream regulatory domains, promoters, enhancers,
transcription
termination sequences and the like which regulate the transcription and
translation of a
nucleic acid sequence.
The term "operably linked" refers to an arrangement of elements wherein the
components are arranged so as to perform their usual function. Thus, control
regions or
regulatory elements operably linked to a coding sequence are capable of
effecting the
expression of the coding sequence. The control elements need not be contiguous
with the
coding sequence, so long as they function to direct the expression thereof.
Thus, for
example, intervening untranslated yet transcribed sequences can be present
between a
promoter sequence and the coding sequence and the promoter sequence can still
be
considered "operably linked" to the coding sequence.
The regulatory elements of the invention can be derived from any source, e.g.,
viruses, mammals, insects or even synthetic, provided that they function in
cardiomyocytes. For example, any promoter can used to control expression of
the
transgene. Such promoters can be promiscous, i.e., active in many cell types,
such as the
SV40 early promoter, the mouse mammary tumor virus LTR promoter, the
adenovirus
major late promoter (Ad MLP), a herpes simplex promoter, a CMV promoter such
as the
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CMV immediate early promoter, a rous sarcoma virus (RSV) promoter.
Alternatively the
promoter can be tissue-specific for expression in cardiomyocytes.
The rAAV is delivered to cardiac myocytes by infusion into a coronary artery
or
coronary sinus. This mode of delivery has also been referred to as
intraluminal delivery
through a coronary artery, intracoronary delivery or intraarterial delivery.
As used herein,
infusion into a coronary artery includes intracoronary perfusion. In
accordance with the
invention, the rAAV vector can be infused when the heart is in situ, i.e., in
the body
cavity or when the heart or heart tissue (cardiac tissue) has been removed
from the body
as might occur when the heart is being donated for transplant into a
recipient. In the case
of a heart being prepared for transplantation or for heart tissue, the vector
can be infused
through any artery or vein attached thereto, by contacting with or soaking the
heart in an
appropriately concentrated solution of the vector, or by a combination of
both. Thus as
described herein, infusion includes delivering rAAV to a heart or heart tissue
ex vivo by
the means disclosed herein. If necessary, the infusion can be repeated at
intervals such as
3 months, 6 months, one year, or as appropriately determined.
As used herein, treating cardiac conditions include treating cardiac or
cardiovascular diseases. Examples of cardiac conditions subject to treatment
or
amelioration according to the method of the present invention include, but are
not limited
to, myocardial ischemia, myocardial infarction, congestive heart failure,
dilated and
hypertrophic cardiomyopathy, cardiac arrythmia, cardiac hypertrophy, cardiac
transplantation and rejection. For example, if the cardiac condition, such as
ischemia, can
be treated or improved by inducing angiogenesis, then the rAAV vector used in
accordance with the method of this invention would encode an angiogenesis
factor.
Thus the rAAV vector is infused into a coronary artery for a time and in an
amount sufficient to stably and efficiently transduce cardiac tissue perfused
by the artery,
wherein the AAV vector encodes a therapeutically-effective molecule which is
expressed
in the cardiac tissue in an amount effective to treat or ameliorate a
cardiovascular
condition including, but not limited to, inducing angiogenesis, inhibiting
angiagenesis,
stimulating or inhibiting cell proliferation, treating restenosis, treating
atherosclerosis,
treating congestive heart failure, treating ischemic cardiomyopathies or
treating malignant
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arrhythmias, myocardial infarction, congestive heart failure, or dilated and
hypertrophic
cardiomyopathy.
The method of the present invention can be used with any animal, including but
not limited to, mammals such as rodents, dogs, cats, cattle, primates and
humans.
Preferably the method is used for gene therapy to treat human acquired or
inherited
cardiac conditions or diseases.
The present invention thus provides a method of treating and/or ameliorating a
cardiovascular condition by infusing an rAAV vector for a time and in and
amount
sufficient to stable and efficiently transduce cardiomyocytes which was
heretofore
unachievable by methods known in the art. For this invention, stable and
efficient
transduction means that significant number of cardiomyocytes are transduced
and are
capable of expressing the protein for a prolonged period of time. Stable and
efficient
transduction occurs over a period of time and can actually be observed over
time as an
increase in the percentage of transduced cardiomyocytes, as continued
expression of the
transgene, or as continued observation of the therapeutic effect at a
molecular,
microscopic or macroscopic level. For example, with angiogenesis, stable and
efficient
transduction can be manifested by ongoing development and or growth of new
blood
vessels, by observing the improved blood flow to the heart, or by determining
measuring
the level of ischemia in the heart tissue.
Alternatively, efficient transduction occurs when at least about 10%, and
preferably more, of the cardiomyocytes have been transduced, i.e., infected
by, the rAAV.
By following the methods of the invention and by observing at particular times
after
transduction ranging over a few to many weeks, about 25%, about 40% or even
about
50% of the cardiomyocytes will be transduced. While about 10% of the
cardiomyocytes
can be transduced using only rAAV, this percentage can be increased by co-
infusing
adenovirus as a helper virus without adverse effects.
The time of infusion contributes to acheiving stable and efficient
transduction of
the cardiomyocytes as well. Thus the infusion time ranges from about 2 minutes
to about
minutes, more preferably from about 5 minutes to about 20 minutes and most
30 perferably is about fifteen minutes.
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The amount of rAAV infused into the animal is proportional to the body weight
of
the animal. Hence in accordance with the invention, stable and efficient
transduction
occurs when the amount of rAAV infused ranges from about 1 x 105 IU
(infectious units)
of AAV per gram body weight to about 1 x 109 IU AAV per gram body weight, and
preferably from about 1 x 106 IU AAV per gram body weight to about 1 x 108 IU
AAV
per gram body weight, and most preferably is about 5-6 x 107 IU AAV per gram
body
weight.
The example described below demonstrates efficient and stable transduction of
cardiac myocytes in vivo after intracoronary infusion of an rAAV vector.
Throughout this application, various publications, patents, and patent
applications
have been referred to. The teachings and disclosures of these publications,
patents, and
patent applications in their entireties are hereby incorporated by reference
into this
application to more fully describe the state of the art to which the present
invention
pertains.
1 S It is to be understood and expected that variations in the principles of
invention
herein disclosed in an exemplary embodiment may be made by one skilled in the
art and it
is intended that such modifications, changes, and substitutions are to be
included within
the scope of the present invention.
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EXAMPLE
Intracoronary Infusion of rAAV
I. Methods:
Plasmids and Viruses
The structure of pAAVcMV-~~z is shown in Figure 1. Ad~MV-,.~z and the E3-
deleted
adenovirus, Add,3~, were propagated and purified as described (Ban 1994).
Propagation and Purification of rAAV
rAAV was prepared as described (Rolling et al. (1995) Mol. Biotechnol. 3:9-15)
and purified by cesium chloride gradient centrifugation. Viral titer was
assessed by 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 2X 10" genomes/mL, and 2 to 3X 109
infectious units
(IU)/mL.
Intracoronary Perfusion With rAAV
Adult C57BL/6 mouse hearts were perfused via the left carotid artery with
cardioplegia solution ( 110 mmol/L NaCI, 25 mmol/L KCI, 22 mmol/L NaHC03, 16
mmol/L MgCI,, 0.8 mmol/L CaClz, 40 mmol/L glucose) at 4°C until they
stopped
beating. They were then perfused ex vivo for 15 minutes with 1.5 X 109 IU of
AAV~MV-
LacZ in 0.5 mL of PBS at a rate of 33 ~cL/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 vein (Lin et al. (1990b) J. Heart Transplant. 9:720-
723) (n=3 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 (Lin 1990a), and
counterstained with eosin.
(3-Galactosidase Activity
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Cardiac homogenates were assayed for ~i-galactosidase ((3-gal) activity and
protein
concentrations. (3-Gal activities were normalized for total protein and for
the number of
infectious rAAV or RDAd particles injected.
II. Results
Many clinical applications of myocardial gene therapy may 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
(Barn 1994). To test whether rAAV is similarly capable of transducing
cardiomyocytes
after coronary artery perfusion, hearts from C57BL/6 mice were explanted and
perfused
with 1.5 x 109 IU of AAVcMV-,~~z 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 ~i-gal-positive
cardiomyocytes were detected throughout the myocardium of the rAAV-perfused
hearts
(Figure 2). By 4 weeks after perfusion, ~40% of the cardiomyocytes were (3-gal
positive.
This high level of transduction was stable at weeks after perfusion, with >50%
of the
cardiomyocytes continuing to express ~3-gal. Similar increases in recombinant
gene
expression over the first several weeks after rAAV infection have been
observed in
skeletal muscle (Fisher 1997; Kessler 1998). 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
(Ferrari et al. (1996) J. virol. 70:3227-3234). Thus, rAAV delivered by
coronary artery
perfusion can be used to stably transduce cardiomyocytes throughout the
myocardium.
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