U.S. patent application number 11/449778 was filed with the patent office on 2006-11-09 for efficient and stable in vivo gene transfer to cardiomyocytes using recombinant adeno-associated virus vectors.
This patent application is currently assigned to Arch Development Corporation. Invention is credited to Jeffrey M. Leiden, Eric Svensson.
Application Number | 20060251626 11/449778 |
Document ID | / |
Family ID | 36659079 |
Filed Date | 2006-11-09 |
United States Patent
Application |
20060251626 |
Kind Code |
A1 |
Leiden; Jeffrey M. ; et
al. |
November 9, 2006 |
Efficient and stable in vivo gene transfer to cardiomyocytes using
recombinant adeno-associated virus vectors
Abstract
This invention relates to the use of recombinant
adeno-associated virus (rAAV) vectors to transduce cardiomyocytes
in vivo by infusing the rAAV into a coronary artery or coronary
sinus. 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.
Inventors: |
Leiden; Jeffrey M.; (Weston,
MA) ; Svensson; Eric; (Chicago, IL) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI;MARKET SQUARE
801 PENNSLYVANIA, N.W.
WASHINGTON
DC
200042604
US
|
Assignee: |
Arch Development
Corporation
Chicago
IL
|
Family ID: |
36659079 |
Appl. No.: |
11/449778 |
Filed: |
June 9, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09473830 |
Dec 28, 1999 |
7078387 |
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11449778 |
Jun 9, 2006 |
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60113923 |
Dec 28, 1998 |
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Current U.S.
Class: |
424/93.2 ;
435/456 |
Current CPC
Class: |
C12N 15/86 20130101;
C12N 2750/14143 20130101; A61K 48/00 20130101 |
Class at
Publication: |
424/093.2 ;
435/456 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/861 20060101 C12N015/861 |
Goverment Interests
[0002] 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.
Claims
1. A method of treating a cardiovascular condition which comprises:
infusing a recombinant adeno-associated virus (AAV) vector into a
coronary artery or a coronary sinus for a time and in an amount
sufficient to stably and efficiently transduce cardiomyocytes
perfused by said artery or said sinus, wherein said AAV vector
encodes at least one nucleic acid operably linked to a control
region, said nucleic acid encoding a therapeutically-effective
molecule; and expressing said therapeutically-effective molecule in
an amount effective to treat or ameliorate said cardiovascular
condition.
2. The method of claim 1, wherein said AAV transduces at least
about 10% of said cardiomyocytes.
3. The method of claim 1, wherein said AAV transduces at least
about 40% of said cardiomyocytes.
4. The method of claim 1, wherein said AAV transduces at least
about 50% of said cardiomyocytes.
5. The method of claim 1, wherein said AAV is infused for at least
about 2 minutes to about 30 minutes.
6. The method of claim 1, wherein said AAV is infused for at least
about 5 minutes to about 20 minutes.
7. The method of claim 1, wherein said AAV is infused for about 15
minutes.
8. The method of claim 1, wherein said amount of AAV is about
1.times.10.sup.5 IU AAV per gram body weight to about
1.times.10.sup.9 IU AAV per gram body weight.
9. The method of claim 9, wherein said amount of AAV is about
1.times.10.sup.6 IU AAV per gram body weight to about
1.times.10.sup.8 IU AAV per gram body weight.
10. The method of claim 9, wherein said amount of AAV is about
6.times.10.sup.7 IU AAV per gram body weight.
11. The method of claim 1, wherein about 1.times.10.sup.5 IU AAV
per gram body weight to about 1.times.10.sup.9 IU AAV per gram body
weight is infused for about 2 to about 30 minutes.
12. The method of claim 11, wherein about 1.times.10.sup.6 IU AAV
per gram body weight to about 1.times.10.sup.8 IU AAV per gram body
weight is infused.
13. The method of claim 11, wherein about 6.times.10.sup.7 IU AAV
per gram body weight is infused.
14. The method of any one of claims 11, 12 or 13, wherein said AAV
is infused for about 5 to about 20 minutes.
15. The method of any one of claims 11, 12 or 13, wherein said AAV
is infused for about 15 minutes.
16. The method of claim 11, wherein about 6.times.10.sup.7 IU AAV
per gram body weight is infused for about 15 minutes.
17. The method of claim 1, wherein said coronary artery is infused
ex vivo or in vivo.
18. The method of claim 1, wherein said therapeutically-effective
molecule is an anti-sense RNA or a protein.
19. The method of claim 1 wherein therapeutically-effective
molecule is an ion channel gene, a contractile protein, a
phospholamban, a .beta. adrenergic receptor, a .beta. adrenergic
kinase, a growth factor, an angiogenic factor, a protein or nucleic
acid capable of inducing angiogenesis, or a protein or nucleic acid
capable of inhibiting angiogenesis.
20. The method of claim 1, wherein said therapeutically-effective
molecule is FGF-1, FGF-2, FGF-5, VEGF, or HIF-1.
21. The method of claim 1, wherein said therapeutically-effective
molecule is thymidine kinase, p21, p27, p53, Rb or NF-.kappa.B.
22. The method of claim 1, wherein said cardiovascular condition is
restenosis, atherosclerosis, congestive heart failure, ischemic
cardiomyopathy, malignant arrhythmia, myocardial infarction,
congestive heart failure, or dilated and hypertrophic
cardiomyopathy.
23. The method of claim 1, wherein treating or ameliorating said
cardiovascular condition is for inducing angiogenesis, inhibiting
angiogenesis, stimulating or inhibiting cell proliferation,
treating restenosis, treating atherosclerosis, treating congestive
heart failure, treating ischemic cardiomyopathy or treating
malignant arrhythmia.
Description
[0001] This application is a continuation-in-part application of
provisional application U.S. Ser. No. 60/113,923, filed Dec. 28,
1998, which is incorporated by reference herein in its
entirety.
FIELD OF THE INVENTION
[0003] 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 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
[0004] 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 .gtoreq.6 months by cardiomyocytes 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).
[0005] 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).
[0006] Recently, rAAV vectors have been shown to program efficient
and stable recombinant 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. Pat. No.
5,858,351 to Podsakoff et al.). 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.
[0007] 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.sup.4 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 cardiomyocytes in amounts useful for treating or
ameliorating cardiac diseases or conditions.
SUMMARY OF THE INVENTION
[0008] 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.
[0009] 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
[0010] FIG. 1. Schematic of AAV.sub.CMV-LacZ. ITR indicates
inverted terminal repeats; BGH pA, bovine growth hormone
polyadenylation signal; CMV Pr, CMV immediate-early promoter; LacZ,
bacterial LacZ gene.
[0011] FIG. 2. Gene transfer into cardiomyocytes in vivo with
AAV.sub.CMV-LacZ. Gross sections (left) and photomicrographs
(right) of mouse hearts after coronary artery perfusion with
1.5.times.10.sup.9 IU of AAV.sub.CMV-LacZ and staining with X-gal.
Bar=25 microns.
DETAILED DESCRIPTION OF THE INVENTION
[0012] 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 cardiomyocytes by rAAV
depends upon the duration of the infusion period and the amount of
virus infused relative to body weight
[0013] 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
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.
[0014] 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.
[0015] 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. Pat.
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 5:793-801 or Berns, "Parvoviridae and their
Replication" in Fundamental Virology, 2nd Edition, (Fields &
Knipe, eds.)).
[0016] 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.
[0017] 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 .beta.
adrenergic receptors or .beta. adrenergic kinases.
[0018] 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-.kappa.B. 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.
[0019] 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 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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 CMV immediate early
promoter, a rous sarcoma virus (RSV) promoter. Alternatively the
promoter can be tissue-specific for expression in
cardiomyocytes.
[0024] 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.
[0025] 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.
[0026] 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 angiogenesis, stimulating or
inhibiting cell proliferation, treating restenosis, treating
atherosclerosis, treating congestive heart failure, treating
ischemic cardiomyopathies or treating malignant arrhythmias,
myocardial infarction, congestive heart failure, or dilated and
hypertrophic cardiomyopathy.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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 30 minutes, more
preferably from about 5 minutes to about 20 minutes and most
perferably is about fifteen minutes.
[0031] 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.times.10.sup.5 IU (infectious
units) of AAV per gram body weight to about 1.times.10.sup.9 IU AAV
per gram body weight, and preferably from about 1.times.10.sup.6 IU
AAV per gram body weight to about 1.times.10.sup.8 IU AAV per gram
body weight, and most preferably is about 5-6.times.10.sup.7 IU AAV
per gram body weight.
[0032] The example described below demonstrates efficient and
stable transduction of cardiac myocytes in vivo after intracoronary
infusion of an rAAV vector.
[0033] 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.
[0034] 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.
EXAMPLE
Intracoronary Infusion of rAAV
I. Methods:
Plasmids and Viruses
[0035] The structure of pAAV.sub.CMV-LacZ is shown in FIG. 1.
Ad.sub.CMV-LacZ and the E3-deleted adenovirus, Ad.sub.d1309, were
propagated and purified as described (Barr 1994).
Propagation and Purification of rAAV
[0036] 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 2.times.10.sup.11 genomes/mL, and 2 to
3.times.10.sup.9 infectious units (IU)/mL.
Intracoronary Perfusion with rAAV
[0037] 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 NaHCO.sub.3, 16 mmol/L MgCl.sub.2, 0.8 mmol/L
CaCl.sub.2, 40 mmol/L glucose) at 4.degree. C. until they stopped
beating. They were then perfused ex vivo for 15 minutes with
1.5.times.10.sup.9 IU of AAV.sub.CMV-LacZ in 0.5 mL of PBS at a
rate of 33 .mu.L/min at 4.degree. 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
[0038] 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.
.beta.-Galactosidase Activity
[0039] Cardiac homogenates were assayed for .beta.-galactosidase
(.beta.-gal) activity and protein concentrations. .beta.-Gal
activities were normalized for total protein and for the number of
infectious rAAV or RDAd particles injected.
II. Results
[0040] 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 (Barr 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.times.10.sup.9 IU of AAV.sub.CMV-LacZ for 15 minutes at
4.degree. 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 .beta.-gal-positive cardiomyocytes were
detected throughout the myocardium of the rAAV-perfused hearts
(FIG. 2). By 4 weeks after perfusion, .apprxeq.40% of the
cardiomyocytes were .beta.-gal positive. This high level of
transduction was stable at weeks after perfusion, with >50% of
the cardiomyocytes continuing to express .beta.-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.
* * * * *