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Ann Thorac Surg 1996;62:1669-1676
© 1996 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Long-Term Gene Transfer in Porcine Myocardium After Coronary Infusion of an Adeno-Associated Virus Vector

Division of Neurosurgery, Department of Surgery, New York Hospital-Cornell University Medical College, New York, New York; Laboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, New York; Somatix Therapy Corp, Alameda, California; Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina; Division of Endocrinology, Department of Medicine, and Division of Cardiothoracic Surgery, Department of Surgery, North Shore University Hospital, Manhasset, New York; New York University School of Medicine, New York, New York; and Arizona Heart Institute, Phoenix, Arizona

Accepted for publication July 25, 1996.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. Viral vector-mediated gene transfer into the heart represents a potentially powerful tool for studying both cardiac physiology as well as gene therapy of cardiac disease. We report here the use of a defective viral vector, which expresses no viral gene products, for gene transfer into the mammalian heart. Previous studies have used recombinant viral vectors, which retained viral genes and yielded mostly short-term expression, often with significant inflammation.

Methods. An adeno-associated virus vector was used that contains no viral genes and is completely free of contaminating helper viruses. The adeno-associated virus vector was applied to rat hearts by direct intramuscular injection; adeno-associated virus was also infused into pig hearts in vivo via percutaneous intraarterial infusion into the coronary vasculature using routine catheterization techniques.

Results. Gene transfer into rat heart yielded no apparent inflammation, and expression was observed for at least 2 months after injection. Infusion into pig circumflex coronary arteries resulted in successful transfer and expression of the reporter gene in cardiac myocytes without apparent toxicity or inflammation; gene expression was observed for at least 6 months after infusion.

Conclusions. We report the use of adeno-associated virus vectors in the cardiovascular system as well as successful myocardial gene transfer after percutaneous coronary artery infusion of viral vectors in a large, clinically relevant mammalian model. These results suggest that safe and stable gene transfer can be achieved in the heart using standard outpatient cardiac catheterization techniques.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Gene therapy has great potential for influencing the natural course of many cardiovascular diseases. One of the most important issues facing practitioners of gene therapy is the choice of vehicle used for gene transfer. Modified viruses have become popular as vectors due to their high efficiency of receptor-mediated cellular uptake and efficient transfer of genetic material into the appropriate subcellular site. Retroviruses, which contain RNA as the genetic material, were historically the first viruses used as gene transfer vectors. Synthesis of a DNA copy of the RNA genome in target cells is necessary before expression of the desired gene. Because most nondividing cells do not permit efficient DNA synthesis, application of these vectors has concentrated on applications for dividing cells such as tumor therapy or modification of dividing cells in tissue culture before transplantation. Viruses with DNA genomes have become widely used for in vivo gene therapy, because the absence of a requirement for DNA synthesis permits gene transfer into nondividing as well as dividing cells with high efficiency.

For cardiovascular gene therapy, efforts have concentrated primarily on the use of adenovirus-based vectors for genetic modification of either liver cells or vascular endothelium [1, 2]. Adenoviral vectors have been used for gene transfer into myocardium of small mammals by direct, intramuscular injection [3, 4] and via infusion of vectors into the coronary arteries of rabbit [5]. Successful gene transfer has also been achieved after direct intramuscular injection of adenoviral vectors into myocardium of large mammals [6, 7]. These studies have found that foreign gene expression was often transient, and a significant cell-mediated immune response was observed [6, 7]. This has led to a search for improved vector systems.

Adeno-associated virus (AAV) vectors represent another approach to cardiovascular gene therapy. Adeno-associated virus is a defective DNA virus, which in nature requires coinfection by adenovirus for efficient replication and spread. Defective viral vectors contain no viral genes but permit packaging of foreign genes into a viral coat. This allows high-efficiency uptake and transfer of foreign genes into target cells while eliminating any immunologic or toxic side effects due to expression of viral genes (Fig 1). To generate AAV vectors, the foreign gene of interest is flanked by two AAV termini containing only recognition signals for replication and packaging into an AAV coat [8, 9]. All viral genes capable of expressing viral proteins are thereby eliminated from the vector. By contrast, adenoviral vectors are among the class of recombinant vectors in which the gene of interest is inserted in the viral chromosome and one or more genes are removed to prevent viral reproduction. Although pathology due to replication is eliminated using recombinant vectors, the vast majority of viral genes remain (see Fig 1). As a result, certain viral proteins will be expressed in target cells using current-generation adenoviral vectors [10]. Improved adenoviral vectors have been created in which several potentially toxic genes are eliminated [11]; here we report an adenovirus-based vector that does not contain any viral genes.

View larger version (87K): [in this window] [in a new window]   Fig 1. . (A) Myocardial gene therapy via adenovirus vectors. The majority of adenovirus vectors contain deletions within one or more essential genes that prevent viral replication and spread, thereby limiting toxicity by this mechanism. Recognition signals for DNA replication and packaging are present (ori, pac); however, numerous genes encoding viral proteins also remain. First, the vector DNA enters the nucleus of the cell (A); messenger RNAs for both the gene of interest (G) as well as certain residual viral genes (V) are synthesized in the nucleus and transported to the cytoplasm (B), which are then translated into proteins (C). The gene of interest may then perform the desired cellular function. As with most cellular proteins, the viral proteins attach to proteins from the major histocompatibility complex (M) for presentation on the cell surface to the immune system (D); cellular proteins are recognized as "self" whereas the viral proteins are recognized as foreign resulting in inflammatory/immune cells recruitment. (B) Myocardial gene therapy via adeno-associated virus vectors. Adeno-associated virus vectors possess only recognition signals for DNA replication and packaging (ori/pac); however, there are no genes remaining that encode viral proteins. Therefore, when the vector DNA enters the cell nucleus (A), only RNA for the gene of interest is synthesized (B), resulting in production of the desired protein in the cytoplasm (C). Because no viral proteins are present for presentation by major histocompatibility complex (M) proteins, the target cell is not seen as possessing foreign proteins and no inflammatory/immune cells are recruited.

This article describes the use of AAV vectors for gene transfer into mammalian myocardial cells. Adeno-associated virus-mediated gene transfer was achieved in rat myocardial cells in vivo after direct intramuscular injection into the left ventricle. We have also used the AAV vector to achieve successful transfer of a foreign gene into porcine myocardial cells in vivo after infusion into the circumflex coronary artery using selective coronary catheterization. Gene expression within muscle cells of the left ventricle was observed at 3 days, 2 months, and 6 months after vector infusion.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Plasmids and Viral Vectors
Construction of the plasmid pAAVlac has been described previously [14]. This plasmid contains a transcription unit consisting of the bacterial lacZ gene under the control of the human cytomegalovirus immediate-early promoter, as well as a downstream signal for messenger RNA polyadenylation from simian virus 40. The transcription unit is flanked by AAV terminal repeats containing the requisite replication and packaging signals. This plasmid served as the genome for the resulting defective AAV vectors used in the rodent studies. A similar construct was used for the pig studies; however, this vector additionally contained an SV40 splicing signal between the cytomegalovirus promoter and the lacZ gene [15]. The method for generation and propagation of packaged AAVlac, adenovirus inactivation, and purification of AAVlac have been described elsewhere [9, 12, 14, 15].

Direct Injection Into Rat Heart In Vivo
Male Sprague-Dawley rats were used for all studies. Animals were first anesthetized as described previously [16]. A 22-gauge needle on a Hamilton syringe containing 5 µL of AAV stock (2 x 10⁶ particles/mL) was then introduced by a transthoracic approach into the apex of the left ventricle. When the needle bounced at the approximate heart rate of the subject, the needle was believed to have entered the left ventricular wall. The needle was inserted slightly past this point and the plunger was withdrawn. An absence of blood indicated that the ventricle had not been entered yet, and the vector solution was then injected into the muscular wall. In some animals, a preparation was used in which the heart of a syngeneic animal was transplanted onto the abdominal aorta of a recipient littermate [17]. The heart could be kept alive in this fashion and could be injected by direct visualization of the apex of the myocardial wall, as previously described [17].

Percutaneous Infusions Into Porcine Coronary Vasculature
Access to the arterial system was obtained via cutdown to the right femoral artery. An 8F sheath was placed in the artery via the Seldinger technique. Systemic heparin (2,000 to 3,000 U) was administered. The left main coronary artery was engaged using an 8F hockey-stick guide catheter under fluoroscopic guidance. A Medtronic (Minneapolis, MN) transfer catheter was advanced into the mid-circumflex coronary artery over a 0.014-inch Hi-Torque (Advanced Cardiovascular Systems, Temecula, CA) floppy exchange length guidewire, which was then removed. One to 3 mL of an AAV stock containing 10⁷ to 10⁸ expressing units was then infused after verification of the catheter positions, followed by a flush with saline solution. During the entire study, 4 subjects were sacrificed 3 days after vector infusion, 2 subjects were sacrificed at 2 months, and 1 at 6 months after infusion. Two additional pigs served as sham controls for the study.

Tissue Preparation
All animals were cared for and sacrificed in accordance with the National Institutes of Health's "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985). Rodents were sacrificed by intraperitoneal injection of a lethal dose of sodium pentobarbital followed by intracardiac perfusion of fixative. Pigs were sacrificed by overdose of intravenous sodium pentobarbital followed by excision of the heart and perfusion with fixative. Pig hearts were divided into several equally sized pieces for sectioning, with each piece numbered and the orientation within the original heart noted. Rat hearts were sectioned whole. Hearts were then incubated overnight in 30% sucrose/2 mmol/L MgCl₂/phosphate-buffered saline solution (PBS), pH 7.3, which serves as a cryoprotectant before sectioning. Thirty-micrometer sections were taken from fixed tissue using a freezing microtome, which were then processed for either ß-galactosidase histochemistry or immunocytochemistry.

Histochemistry and Immunocytochemistry
Cells expressing enzymatically active ß-galactosidase can be detected by incubation with the chromogenic substrate X-gal, which yields an insoluble blue precipitate when cleaved. For X-gal histochemistry, hearts were first perfused with a fixative that has previously been shown to permit X-gal staining of positive cells while completely eliminating background staining due to endognous mammalian enzymes with ß-galactosidase–like activity [16]. Sections were then permeabilized and incubated in X-gal substrate solution as described previously [16].

For ß-galactosidase immunocytochemistry, tissue was fixed with 4% paraformaldehyde PBS, pH 7.3, by cardiac perfusion followed by overnight postfixation for 1 hour at 4°C. Tissue sections were taken as described above, then washed with PBS and blocked overnight with 10% goat serum in PBS at 4°C. The next day sections were treated rabbit polyclonal anti–ß-galactosidase antibody (5`-3`) at 1:500 dilution in PBS with 10% goat serum. After overnight incubation at 4°C and repeated PBS washes, the biotinylated goat anti-rabbit secondary antibody (Vector Labs, Burlingame, CA) in PBS with 10% goat serum was applied to sections for 1 to 2 hours at room temperature. Sections were again repeatedly washed with PBS. The ABC reagant consisting of avidin-biotin-horseradish peroxidase (Vector Labs) was prepared and applied to sections as per the manufacturer's instructions. After a final series of washes, bound horseradish peroxidase was detected by incubation for 3 to 5 minutes in the diaminobenzidine substrate solution described previously. Sections were then counterstained with hematoxylin and eosin to enable observation of the background tissue histology and architecture, and to permit detection of any infiltrating inflammatory cells.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Gene Transfer Into Rat Heart Cells In Vitro and In Vivo Using an Adeno-Associated Virus Vector
AAVlac was first tested in primary culture to demonstrate that AAV vectors are capable of transferring genes into neonatal rat myocardial cells (data not shown). AAVlac was then infused into rat left ventricular myocardium via two methods. A transthoracic approach was first taken as described above. To obtain greater control over the localization of vector injections, we used a model in which a heart was transplanted onto the abdominal aorta of a syngeneic animal [17]. This heart maintains spontaneous contractile activity and normal cardiac histology for extended periods. This permitted full exposure of the left ventricle before injections without necessitating a respirator, which would be requisite for equivalent exposure of the functional heart. Cells positive for ß-galactosidase histochemical activity were observed 3 days after injection by both methods, and animals sacrificed 2 months after injection also demonstrated positive cells (Fig 2). No positive cells were observed in uninjected control subjects. At both time points positive cells were observed local to the injection site, with minimal spread. Furthermore, in the AAVlac-injected subjects the surrounding tissue appeared to be healthy, with no evidence of significant inflammation or damage as a result of viral transduction.

View larger version (113K): [in this window] [in a new window]   Fig 2. . Positive rat cardiac muscle cell after histochemical assay for ß-galactosidase activity. This assay results in a blue color change in cells expressing the enzyme ß-galactosidase. These figures were taken from animals sacrificed 3 days (A) and 2 months (B) after direct, intramuscular injection of AAVlac into the myocardium of an adult rat. (x20 before 44% reduction.)

View larger version (108K): [in this window] [in a new window]   Fig 3. . Placement of the infusion catheter into the circumflex coronary artery. This is an image of the catheter under fluoroscopy after guidance to the coronary arteries. When the tip was believed to be at the opening of the circumflex artery (left; arrow), contrast dye was infused (right). The contrast dye can be seen enhancing only the circumflex coronary artery and its branches, which feeds the posterolateral wall of the left ventricle. The tip (arrow) was thereby determined to be correctly placed, and after flushing with saline solution, the adeno-associated virus vector was infused.

View larger version (2K): [in this window] [in a new window]   Fig 4. . Immunocytochemical detection of porcine heart cells expressing ß-galactosidase. (A, B) Positive myocardial cells 3 days after infusion of AAVlac. Note the elongated, muscular morphology of most of the positive cells. Section A and B were from the same animal but were separated by 450 sections. (C) Positive cells from an animal sacrificed 2 months after infusion of AAVlac. The morphology of the cells at 2 months was of slightly poor quality due to cutting artifact when these sections were taken. (D, E) Positive cells from the heart of a pig sacrificed 6 months after infusion of AAVlac into the circumflex coronary artery. Sections D and E were from the same animal but were separated by 240 sections. (F) A section of myocardium from a mock-infused control subject. This section was stained with substrate longer than all other sections to ensure the complete lack of positive staining. Although this resulted in a greater edge artifact seen as a slight increase in overall brown tone, there are clearly no genuine positive brown cells. Note the absence of any inflammatory cells or tissue damage (other than cutting artifact) in any of the tissue samples. (x10 before % reduction.)

Pigs sacrificed 2 months and 6 months after infusion also demonstrated positive cells (Figs 4C–E, 5). Numerous fully labeled muscle cells were again observed largely limited to the distribution of the circumflex coronary artery. The number of positive cells seen at 2 months after infusion appears to have decreased to approximately 25% of the number seen at 3 days after infusion. By 6 months the number of cells appears to have stabilized and was roughly equivalent to the number of immunoreactive cells observed at 2 months after infusion.

View larger version (120K): [in this window] [in a new window]   Fig 5. . High-power view of two cells expressing ß-galactosidase 6 months after infusion of AAVlac. Note the presence of the diaminobenzidine brown product within the borders of two individual muscle cells. This photomicrograph was taken with slight phase contrast, and intercalated discs can be appreciated upon close observation. Note the complete lack of inflammatory cells surrounding the positive muscle cells. (x40 before 46% reduction.)

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
This report demonstrates that AAV vectors can transfer genes into the mammalian heart. Previously adenoviral vectors have been used for gene transfer into the adult myocardium. As with any recominant vector, adenovirus can easily be grown to very high titers because vector reproduction occurs simply by viral replication. Thus, a large number of viral particles can be injected in a small volume. By contrast, AAV vector reproduction requires expression of proteins from the adenovirus genome, which then promote expression of AAV genes from a distinct plasmid. The resulting AAV proteins must then efficiently replicate and package the vector DNA from yet another plasmid. As a result AAV vectors do not grow to the same titers as adenovirus. Although we observed positive muscle cells at both 3 days and 2 months in rodent heart after direct needle injection of AAV vectors, it is clear that far fewer cells were observed in this study compared with prior reports using adenoviral vectors in rodents [3, 4]. However, these studies also used adenoviral stocks that were several orders of magnitude more concentrated than the stock used in our study. Thus, AAV-mediated myocardial gene transfer appears to be at least as efficient as adenoviral transduction of heart cells. This is consistent with prior studies that have documented highly efficient AAV-mediated gene transfer in other organ systems [12–15]. A more concentrated AAV stock was used for the porcine studies, and a far greater number of positive cells was also observed. The porcine data cannot readily be compared with prior reports of adenovirus gene transfer because we used a very different approach to gene delivery in the pig heart. However, if the number of positive cells is compared with the number of input vector particles, then it is clear that the AAV vector is at least as efficient at short-term delivery as other systems. Adeno-associated virus vectors can now achieve titers of greater than 10⁹ particles/mL in proficient hands, which is comparable with adenovirus titers that have been used to positively alter physiology in other organ systems. This suggests that physiologically meaningful numbers of positive cells can be achieved in the myocardium using the AAV approach.

Long-term expression of the bacterial lacZ gene was observed in pig hearts transduced with the AAV vector. Although gene transfer with adenoviral vectors has resulted in large numbers of positive cells for up to 7 to 10 days, expression has generally declined with time and appears to cease at 3 to 4 weeks after vector infusion [3–7]. In the current study, expression was observed to at least 6 months after AAV transduction of pig myocardium. Because relatively few positive cells were observed in rat hearts at short time points, it was not surprising that a small number of cells expressed lacZ at 2 months. In the pig heart, however, a large number of positive cells were observed at 2 and 6 months after AAV infusion, consistent with the larger number of positive cells seen at 3 days. The slight decline in the number of positive cells observed at longer time points is consistent with earlier observations in other organs. On a percentage basis, however, our data demonstrate that AAV yields stable expression in pig myocardium for at least 6 months after vector infusion.

Another feature of the current study was the absence of apparent inflammatory response to AAV transduction in the mammalian heart. Inflammation has been invoked as a possible explanation for the limited longevity of expression after adenoviral gene transfer into myocardial cells. Immune cell infiltrates have been observed surrounding adenovirus-transduced myocardial cells [6]. This is likely due to the presence of viral genes within the replication-incompetent vectors that continue to express adenovirus gene products within target cells. These products are then presented to the immune system, which recognizes them as foreign and reacts to the cell as if it were infected with a wild-type virus (see Fig 1). The resulting immune reaction may not only result in tissue damage, but also could play a role in limiting the length of gene expression either by inducing target cell degradation of vector DNA or by destruction of the transduced cell. Because the AAV vector does not contain any viral genes, the potential for immune reactions against transduced cells due to synthesis of viral proteins is eliminated (see Fig 1). This not only results in a vector with increased safety, but may actually improve longevity of gene expression.

In addition to the unusual vector used for this study, we used a different approach for delivery of viral vectors to the heart of a large mammal. Percutaneous infusion of fluids into the coronary vasculature via fluoroscopically guided cardiac catheterization is a routine procedure. Because this technique usually does not require hospital admission, it is attractive as a safe, simple, and cost-effective method for cardiac gene delivery. Furthermore, if a viral vector can penetrate effectively into the myocardium, this also presents the possibility of widespread gene delivery throughout the heart.

Previous studies in the rodent and porcine heart have used direct needle injection of adenovirus into a point within the left ventricular wall [3–6]. A percutaneous approach has been used in one canine study; however, the end of the catheter contained a needle that was floated into the lumen of the left ventricle and inserted into the myocardium [7]. Therefore, this approach also resulted in direct intramyocardial vector injection. Adenovirus has been infused into the rabbit coronary vasculature using a percutaneous approach [5]. Our data in a large, clinically relevant animal indicate that AAV vectors can penetrate into the myocardium to yield widespread gene delivery limited to the left ventricular region perfused by the injected artery.

Adeno-associated virus is presented here as an approach to in vivo gene transfer in the adult myocardium. The ability to genetically modify large numbers of myocardial cells in a safe and stable fashion suggests that this may have significant clinical utility for gene therapy of diseases previously refractory to conventional treatments. The use of a common, endovascular catheterization technique for introduction of vectors to a selective region of the myocardium further enhances the potential of this approach for application in a variety of clinical settings.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
We thank Barbara Robinson for her expert technical assistance in the care of the pig subjects and harvesting of heart tissue, Mike Austin and the graphic design team at The Arizona Heart Institute for assistance with figure production, Dr Jan Breslow and Dr. Thomas Shenk for helpful advice, Dr Donald Pfaff for use of small animal surgery and tissue processing equipment, and Dr Chull Hong for preparation of heterotopic transplanted hearts. The first two authors contributed equally to this project.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Address reprint requests to Dr Kaplitt, Division of Neurosurgery, Department of Surgery, The New York Hospital-Cornell University Medical Center, 525 E 70th St, New York, NY 10021.

Doctor Xiao, who assisted in this work while in Dr Samulski's laboratory, works for Somatix Therapy Corporation, which owns the rights to commercialize AAV vector therapy. Somatix Therapy Corporation provided no funding for or assistance with this project.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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