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(Circulation. 1995;91:541-548.)
© 1995 American Heart Association, Inc.

Articles

Gene Therapy for Cardiovascular Disease

Elizabeth G. Nabel, MD

From the Cardiovascular Research Center and Department of Internal Medicine, University of Michigan (Ann Arbor).

Correspondence to Elizabeth G. Nabel, MD, University of Michigan, Cardiovascular Research Center, MSRB III, Rm 7301, 1150 W Medical Center Dr, Ann Arbor, MI 48109-0644.

Key Words: genes • cardiovascular diseases • myocardium

	Introduction

Top Introduction Methods of Gene Delivery... Animal Models of Gene... Clinical Trials References

 
The field of cardiovascular gene transfer has developed rapidly during the past 5 years. Important advances have been made in vector development, in vivo gene delivery, and definition of potential therapeutic targets. Despite substantial progress, a number of technical issues need to be addressed before gene therapy is applied safely and broadly to cardiovascular diseases. In this review, major advances in cardiovascular gene transfer are summarized. In addition, technical issues required for translation of preclinical studies of gene transfer into clinical protocols are discussed.

Advances in recombinant DNA technology, including gene transfer, have stimulated hope that this technology can be used to improve the practice of cardiovascular medicine. Applications of this technology that affect the clinical management of patients include the development of new therapeutic products, engineered by the overexpression of genes, such as recombinant tissue-type plasminogen activator. Recombinant DNA technology has also provided techniques that have been used to create animal models of cardiovascular diseases. These models permit definition of the role of specific gene products in the pathogenesis of cardiovascular diseases. Characterization of its molecular basis has led to a more precise definition of diseases and the potential for relevant clinical treatments. The development of molecular genetic interventions to treat cardiovascular diseases depends on technical advances in the development of methods of gene delivery; achievement of long-term, highly efficient, and targeted expression to relevant cells of the cardiovascular system; and design of vectors that are safe for long-term human administration.

	Methods of Gene Delivery

Top Introduction Methods of Gene Delivery... Animal Models of Gene... Clinical Trials References

 
The transduction and expression of genes in appropriate cell types represent important steps in the development of gene therapy. Therefore, investigations have focused on the development of methods to deliver and express genes in vascular cells and cardiac myocytes. Viral vectors (retroviruses and adenoviruses), viral conjugate vectors (adenovirus-augmented receptor-mediated vectors and hemagglutinating virus of Japan [HVJ] liposomes), and nonviral vectors (cationic liposomes, polymers, and injection of plasmid DNA) have been used. To optimize gene delivery to target cells, characteristics of the cell must be considered, such as proliferative capacity and location within the target tissue. Vascular cells (endothelial and smooth muscle cells) and cardiac myocytes differ particularly in proliferation features, and therefore different strategies have been used to transduce these cells.

Vascular Cells
Retroviral Vectors
Because vascular cells are accessible through the blood stream, percutaneous, site-specific gene delivery was developed for local arterial segments. Retroviral vectors were initially used in in vivo vascular gene transfer studies.^{1 2} Interest in retrovirus-mediated gene transfer was based on the use of these vectors in gene transfer to other organ systems, including bone marrow stem cells,³ liver,⁴ and the skin,⁵ where in some target cells, these vectors transduce a large proportion of target cells. In the case of retroviral vectors, the viral vector stably integrates into chromosomal DNA of the target cell, resulting in potentially stable gene expression, although integration into chromosomal DNA could potentially result in insertional mutagenesis.⁶ These vectors are most appropriate for ex vivo gene transfer for cardiovascular disease,^{1 2 7} which involves removal of the relevant target cells, ie, endothelial cells or hepatocytes from the host, transfection of the cells in vitro, and subsequent reintroduction of the modified cells into the animal or patient. It is unknown whether stable high-level gene expression in vivo will be achieved with retrovirally transduced cells.

Several features of retroviral gene transfer may limit its application to cardiovascular medicine, particularly with respect to direct in vivo gene therapy. Replication of target cells is necessary for proviral integration.⁸ Recent studies suggest that viral integration may depend on mitosis, not just DNA synthesis.⁹ Successful retrovirus-mediated gene transfer to vascular cells may require induction of proliferation in target endothelial or smooth muscle cells, at least for short periods of time. In uninjured arteries, endothelial and smooth muscle cell proliferation occurs slowly, and therefore retroviral integration would occur at a low frequency. After vascular injury or stimulation, such as with a balloon catheter, injured vascular cells may be transduced at higher rates. Previous studies have suggested that retroviral transduction of vascular cells can be used successfully in vivo. Limitations of this gene delivery vector include its low frequency of gene transfer and the relative lability of retroviral particles compared with other viruses. Retroviral particles are rapidly inactivated in vivo in primates, presumably by the presence of complement in serum.¹¹ Because of the instability of retroviral particles and the inability of retroviruses to integrate in nonreplicating cells, retroviral vectors may have limited use for direct gene transfer in vivo, although there may be a role for these vectors for ex vivo gene transfer to vascular stents or prosthetic grafts.

Adenoviral Vectors
To improve the frequency of direct gene transfer into arteries, recent investigations have focused on adenoviral vectors. The adenovirus genome is composed of linear, double-stranded DNA of approximately 36 kb in length (for a review, see Reference 12). The gene products are organized into early (E1-E4) and late (L1-L5) regions, based on expression before or after initiation of DNA replication. Expression of viral genes depends on cellular transcription factors and expression of the adenoviral E1 region, which encodes a transactivator of viral gene expression. The E3 region encodes viral proteins that regulate immunosurveillance in vivo. Adenoviruses have a lytic life cycle, characterized by attachment to an adenoviral glycoprotein receptor on mammalian cells and cell entry by receptor-mediated endocytosis. Adenoviruses escape degradation in lysosomes due to adenoviral capsid proteins, and viral DNA is transported to the nucleus. In the nucleus, adenoviral genome persists in an unintegrated form. During the lytic infection, viral genome replicates to several thousand copies per cell.

Adenovirus serotypes 2 (Ad-2) and 5 (Ad-5) have been developed as viral vectors for gene transfer (for a review, see Reference 13). These vectors are engineered to be replication incompetent by deleting the E1A and E1B genes from the viral genome. Vectors are produced by homologous recombination in 293 cells or any cell line that contains an integrated copy of the adenoviral E1 gene. A foreign cDNA with eukaryotic regulatory sequences is introduced into a bacterial plasmid containing a region of the left adenoviral genome that is deleted of the E1 gene. This plasmid is cotransfected into 293 cells with an incomplete adenoviral genome. Homologous recombination between the two DNAs generates a recombinant genome in which the E1 gene is replaced by the foreign DNA. Viral stock is propagated in 293 cells to high titer, approximately 10¹⁰ to 10¹² particles per milliliter.

Adenoviruses effectively infect mammalian cells, including nondividing cells in vitro and in vivo. Physiological levels of recombinant proteins have been secreted into the circulation after adenoviral infection of skeletal muscle.¹⁴ The virus particle is relatively stable and amenable to purification and concentration at a high titer. Integration of adenoviral DNA sequences into chromosomal DNA of the target cell occurs at a low frequency, and adenoviral DNA is maintained in an extrachromosomal form. Extrachromosomal replication of the vector reduces the likelihood of mutation by random integration and dysregulation of cellular genes.

Despite these advantages, there are limitations to current, or "first-generation," adenoviral vectors. In most models, gene expression is transient after adenoviral infection, generally less than 3 weeks, and inflammation is observed in organs expressing the transgene.^{15 16 17 18} Gene expression in vascular cells is transient as well, usually persisting for only several weeks.^{19 20 21 22 23} Although transient gene expression may be well suited to vascular therapies requiring expression of a gene product over a short period of time, development of an immune response to adenoviral proteins is a major limitation to the use of these vectors. Recent studies in genetically defined strains of mice have demonstrated that viral proteins, expressed from the E1-deleted adenoviral genome, are presented as foreign antigens and lead to the generation of cytolytic T lymphocytes that destroy adenovirus-infected cells.²⁴ Insertion of a temperature-sensitive mutation within the E2A region of E1-deleted adenoviral vectors results in lack of expression of late viral gene products at nonpermissive temperatures, resulting in prolonged gene expression (70 days) and blunted cytolytic T-cell infiltration in mouse liver.²⁵ In arterial gene transfer studies using first-generation adenoviral vectors, mononuclear cell infiltrates have been occasionally observed in the adventitia of peripheral²⁶ and pulmonary²³ arteries of pigs, but medial and intimal inflammation, necrosis, and aneurysm formation have not been observed. Further studies are required to examine the use of first-generation adenoviral vectors for vascular gene transfer studies. It is likely that further modifications in these vectors, including deletions in the E2 and E4 regions and modifications in the E3 region, will diminish host immune responses.

Adenovirus-Augmented, Receptor-Mediated Gene Delivery
Viral vector conjugate systems may have application in vascular gene therapy and include adenovirus-augmented, receptor-mediated gene delivery. This vector uses inactivated adenovirus complexed to a receptor ligand to facilitate entry of DNA to a cell.²⁷ This vector consists of two components. DNA condenses with polylysine, which in turn is bound to inactivated virus.^{28 29} The virus is coupled to a ligand such as transferrin. The transferrin ligand binds to a transferrin receptor in a cell, and the transferrin viral polylysine DNA complex enters the cell by receptor-mediated endocytosis. The inactivated adenovirus functions to disrupt lysosomes in the host cell, reducing DNA degradation and releasing DNA into the cytoplasm.³⁰ The use of these vectors for vascular gene transfer is being investigated.

Cationic Liposomes
Most nonviral methods of gene transfer rely on normal mechanisms used by cells for the uptake and cellular transport of macromolecules. These methods rely on receptor-mediated endocytotic pathways or fusion of cell membranes. One example is cationic liposomes, which are positively charged artificial lipid vesicles that incorporate negatively charged DNA and deliver nucleic acid to cells through fusion with cell membranes or receptor-mediated endocytosis.³¹ Plasmid DNA is released in the cytoplasm and transported to the nucleus where it is maintained in an unintegrated form. Cationic liposomes interact spontaneously and rapidly with polyanions, such as DNA and mRNA, to form liposome complexes. Cationic liposome reagents used in vascular gene transfer studies include DOTMA/DOPE (Lipofectin),³² DC-cholesterol,³² DOSPA/DOPE (Lipofectamine),²³ and DMRIE/DOPE.³³ Expression of recombinant genes in vivo after liposomal transfection has been reported in rats,³⁴ rabbits,³⁵ dogs,^{36 37} and pigs^{38 39 40 41} . Cationic liposomes produce more efficient gene delivery compared with neutrally charged or anionic liposomes,⁴² but current formulations, including DOSPA/DOPE, are less efficient than adenoviral vectors.²³ Further modifications in plasmids and chemical formulations of liposomes appear to have promise in improving transfection efficiency.⁴³ Cationic liposomes have a favorable safety profile for in vivo administration.^{32 44} Liposome vectors contain no viral sequences, and there are no cDNA size constraints in vector construction. In addition, this vector is straightforward to prepare for clinical use.⁴⁴ Cell division is not required for liposome transfection, although the efficiency appears to be increased in proliferating cells.⁴⁵

HVJ Liposome Conjugates
Recent studies suggest that complexing inactivated HVJ with liposomes improves transfection efficiencies of vascular smooth muscle cells in in vitro and in vivo models of vascular injury, including the injured rat carotid artery.³⁴ These vectors have also been successfully used for hepatic⁴⁶ and renal⁴⁷ in vivo gene transfer. It is likely that further modifications to vectors used for vascular gene transfer will include components of viral and nonviral vectors that optimize delivery, improve gene expression, and minimize toxic side effects.⁴⁸

Polymers
Additional strategies for the local delivery of therapeutic agents include impregnating oligonucleotides into polymer gels and applying the polymer to the external surfaces of arteries.^{49 50} Although pharmacokinetics of oligonucleotide delivery and retention have not been precisely defined, the data suggest that there is sufficient retention of oligonucleotide to inhibit c-myb⁴⁹ and PCNA⁵⁰ RNA expression within 24 hours after balloon injury and inhibit intimal thickening after 2 weeks. Plasmid DNA⁵¹ and adenoviral vectors⁵² have been applied directly to polyethylene balloons coated with a hydrogel polymer. Although there is some loss of plasmid DNA from the balloon during transit through the circulation, DNA is distributed transmurally after inflation of the balloon. Modifications in polymers to provide slow release of therapeutic agents hold promise for site-specific delivery of oligonucleotides and vectors to arterial segments.

Myocyte Gene Transfer
Dissection of molecular mechanisms governing myocardial differentiation has been performed in neonatal cardiac myocytes in culture, in part because these cells are relatively amenable to gene transfer with plasmid-based transfections.^{53 54 55 56 57} Recombinant gene expression in adult myocardium in vivo requires an expression vector with high-level activity in adult cardiac myocytes and a method for introducing this vector into myocardial cells. Because cardiac myocytes are terminally differentiated cells, they require a vector that is not dependent on cell replication for delivery and expression. The analysis of foreign genes within intact adult myocardium has been performed by direct injection of plasmid DNA.^{58 59} Although direct injection of genes is a simple procedure and permits examination of the behavior of genes in vivo, this technique is limited by transfection of a small number of cells within several millimeters around the injection site.⁶⁰ Expression of recombinant genes is temporally limited as well, with expression peaking within several weeks and declining rapidly thereafter.^{58 60} Episomal persistence of the introduced DNA and the postmitotic state of adult cardiac myocytes, which prevent integration of genes into chromosomes, limit stability of the transgene.

Some limitations of in vivo plasmid DNA injection have been addressed by adenoviral vectors. Adenoviruses effectively infect nonreplicating mammalian cells, including skeletal and cardiac myotubes. These viruses are grown and purified in high titer. These properties result in highly efficient gene transfer into adult cardiac myocytes in vitro^{61 62} and in vivo.^{22 62 63} Quantitative comparisons of chloramphenicol acetyltransferase (CAT) activity resulting from injection of a CAT plasmid or an adenoviral vector encoding CAT revealed that the amount of CAT activity resulting from adenovirus infection was 10- to 100-fold higher compared with plasmid DNA.⁶² Similar findings were observed comparing adenoviral vectors and plasmids encoding lacZ.^{22 63} Although adenoviral vectors produce efficient gene transfer into the myocardium, expression is transient, peaking at 1 to 2 weeks. Acute inflammatory responses have been observed in hearts injected with adenovirus,⁶³ although inflammation along the injection path has also been noted after injection of plasmid DNA.^{58 64} Further investigations will identify factors that account for the transient nature of gene expression and will characterize potential proinflammatory effects of this vector.

	Animal Models of Gene Transfer

Top Introduction Methods of Gene Delivery... Animal Models of Gene... Clinical Trials References

 
Vascular Gene Transfer
In the past 5 years, there has been great interest in expressing recombinant DNA and other nucleic acids in blood vessels in vivo. The goals of these studies have been to define gene function and to develop new therapeutic strategies for vascular diseases.

The feasibility of direct gene transfer to arteries in vivo was demonstrated using viral (retrovirus) and nonviral (liposomes) vectors in several animal species, including pigs,^{38 39} rabbits,³⁵ and dogs.^{36 37} These studies reported a low efficiency of gene transfer, generally 1% or less of vascular cells in vivo. More recent studies have suggested that the efficiency of gene transfer into arteries can be improved with adenoviral vectors; increased expression of reporter genes has been reported in sheep,¹⁹ rat,^{20 21} rabbit,^{22 52} and pig^{23 26} vessels. Endothelial cells of normal arteries and endothelial and smooth muscle cells in injured arteries have been transduced at efficiencies approximately 10- to 100-fold higher than reported for retroviral and liposome vectors. A major limitation to adenoviral gene transfer in the vasculature has been transient expression; in most studies, expression of reporter gene has been observed for 7 to 14 days and is diminished or lost by 28 days. Lack of persistence of gene expression may result from cytolytic responses directed against infected cells. Transient gene expression, however, may be desirable for vascular diseases, like restenosis after angioplasty, which are characterized by cellular proliferation peaking in the first several weeks after arterial injury.^{26 65 66 67}

Several observations concerning the delivery of recombinant genes and patterns of gene expression can be drawn from these studies. Infusion of vector into normal arteries with an intact endothelium results in transfection of intimal cells (primarily endothelial cells).^{19 22 23 52} Injury to the vessel and/or application of pressure to the vector infusate results in delivery of DNA transmurally and gene expression in the media.^{21 26 52 65} Several catheters have been used in gene transfer studies, including double-balloon catheters, porous balloon catheters, and hydrogel catheters, and the patterns of gene expression within an artery may differ depending on the design of the catheter, animal species, and type of artery transduced.^{51 52 68}

Direct gene transfer has been used to create somatic transgene models to define gene function in arteries. In this system, genes can be expressed within arterial segments, and their biological function can be investigated. This approach has proved useful for investigation of genes whose direct in vivo effects have been difficult to analyze. For example, transfection of a recombinant angiotensin-converting enzyme gene into rat arteries using HVJ liposomes promotes angiotensin II–mediated vascular hypertrophy.⁶⁹ After transfer of a recombinant endothelial cell–type nitric oxide (NO) synthase (ec-NOS) gene into balloon-injured rat carotid arteries, NO production was associated with a reduction in intimal thickening.⁷⁰ Gene transfer approaches have also proved useful in the analysis of atrial natriuretic peptide,⁷¹ type 2 angiotensin II receptor,⁷² and VCAM-1.⁷³

Growth factors and cytokines stimulate vascular cell proliferation and vessel formation in vivo. Although the genes encoding many factors have been cloned and their mechanism of action defined in vitro, definition of their role in vivo has been more difficult to analyze. Several recombinant growth factor genes, including platelet-derived growth factor–B (PDGF-B), a secreted form of acidic fibroblast growth factor (FGF-1), and an active form of transforming growth factor–ß₁ (TGF-ß₁) have been expressed by direct gene transfer in porcine arteries, and the function of these gene products has been analyzed. Expression of a PDGF-B gene in porcine arteries stimulated intimal hyperplasia characterized by smooth muscle cell proliferation.⁴⁰ Synthesis and secretion of FGF-1 were associated with expansion of the intima as well as intimal angiogenesis.⁴¹ Arteries transfected with a TGF-ß₁ gene demonstrated increased procollagen synthesis in the intima and media as early as 4 days after gene transfer compared with control arteries transfected with a reporter gene.⁷⁴ Although these recombinant genes stimulate vascular cell proliferation in vivo, they exert otherwise distinct effects on smooth muscle cell proliferation, angiogenesis, and extracellular matrix formation. These studies suggest that intimal thickening may represent a common response to gene expression of multiple growth factors, which in turn exert different effects on vessel repair.

Another approach to investigating the pathogenesis of vascular cell proliferation in vivo is to examine gene products that inhibit cell proliferation. Local delivery of an antiproliferative agent during the peak of smooth muscle cell proliferation or extracellular matrix synthesis after balloon injury might limit expansion of the intima. Several approaches have been explored in this setting, including recombinant chimeric toxins,^{75 76 77} antisense oligonucleotide strategies,^{49 50 78 79 80 81 82} and gene transfer.^{26 65 66 67}

One approach to the selective elimination of dividing cells is to express a herpes virus thymidine kinase (HSV-tk) gene in smooth muscle cells after balloon injury. Thymidine kinase, when expressed in transduced cells, converts ganciclovir, a nucleoside analogue, into an active toxic form, and subsequent incorporation of phosphorylated ganciclovir into cellular DNA induces chain termination in dividing cells, causing cell death.^{83 84} A bystander effect, demonstrated in smooth muscle and endothelial cells, confers susceptibility to ganciclovir in neighboring dividing cells, leading to inhibition of cell growth in nontransduced neighboring cells as well.⁸⁵ Adenoviral vectors encoding a HSV-tk gene or no cDNA insert were introduced into porcine arteries immediately after balloon injury, and a course of ganciclovir or saline was initiated. Three weeks after balloon injury and adenoviral infection, a significant reduction in intima-to-media area ratios (54% to 59%) was observed.²⁶ A reduction in intimal BrdC (5-bromo-deoxycytosine) incorporation of 40% was observed in HSV-tk ganciclovir-treated animals compared with HSV-tk saline-treated animals 7 days after gene transfer, indicating that inhibition of smooth muscle cell proliferation contributed to this effect. A significant reduction in intima-to-media area ratios in the HSV-tk ganciclovir-treated animals was observed 6 weeks after treatment, suggesting that the decrease in intimal hyperplasia was stable. In addition, no major systemic toxicities were observed associated with adenoviral infection and ganciclovir treatment. These data suggest that expression of an enzyme that catalyzes the formation of a cytotoxic drug locally within an artery may limit smooth muscle cell proliferation after balloon injury.

In balloon-injured rat carotid arteries, introduction of adenoviral vectors encoding HSV-tk immediately after balloon injury⁶⁶ or 7 days later⁶⁷ and treatment with ganciclovir also result in significant reductions in intima-to-media area ratios. Reendothelialization was present in rat and porcine arteries infected with HSV-tk adenoviral vectors and treated with ganciclovir, and significant toxicities were not observed in treated arteries or systemic organs. Additional approaches to limiting smooth muscle cell proliferation after vascular injury include targeting of nuclear cell cycle regulatory pathways, including the retinoblastoma gene product (Rb). Studies in injured rat carotid and porcine femoral artery models suggest that expression of a nonphosphorylatable, constitutively active form of Rb after adenoviral infection limits intimal smooth muscle proliferation for at least 3 weeks after vascular injury.⁶⁵

Myocardial Gene Transfer
Recent exciting developments hold promise for transduction of adult myocytes in vivo. Initial studies demonstrated the feasibility of expression of reporter genes in rat^{58 59 60 86} and canine⁸⁷ myocardium by direct injection of plasmid DNA, but these studies were limited by low efficiencies that hindered investigations of gene expression in myocytes. The observation that adenoviruses infect nondividing cells has heightened interest in these vectors for gene transfer to adult myocardium. Indeed, recent studies have demonstrated higher levels of gene expression in rat myocardium after direct injection of adenovirus vectors compared with injection of plasmid DNA alone.^{62 63} Adult myocardium in vivo has also been transduced by intravascular administration of adenoviral vectors encoding reporter genes.²² Gene expression was observed in both the coronary vasculature and the adjacent myocardium. Levels of gene expression in the myocardium were 10- to 50-fold higher compared with direct DNA plasmid injection. Although adenoviral vectors provide efficient gene transfer, gene expression in the myocardium is transient. In most studies, reporter gene expression peaked at 1 week, diminished at 2 weeks, and was present at low levels after 1 month in adult myocytes.^{22 62 63} The mechanisms for loss of gene expression, including immune responses, are not completely understood.

Gene transfer to the myocardium has proven to be a useful tool in understanding cardiac gene regulation in vivo. For example, transcriptional elements regulating basal and thyroid hormone–responsive cardiac -myosin heavy chain (-MHC) gene expression in adult rat hearts in vivo have been studied.⁵⁹ Sequences upstream of the rat -MHC gene linked to a luciferase reporter were injected into adult rat hearts, and thyroid hormone responsiveness was evaluated. The thyroid hormone–responsive element was necessary, but not sufficient, to confer positive and negative regulation of thyroid hormone. Direct injection of constructs into the myocardium is a model system for investigating DNA elements and regulatory pathways that control gene expression and growth in the heart.

An additional promising area is the direct injection of adenoviral vectors into skeletal muscle for production of secreted proteins. Myoblasts, transduced by retroviral vectors expressing human growth hormone, injected into skeletal muscle produced physiological levels of human growth hormone in the serum.^{88 89} Recent studies suggest that physiological levels of recombinant erythropoietin are secreted into the circulation after intramuscular injection of adenovirus into skeletal muscle of neonatal mice or adult SCID mice.¹⁴ Neonatal and adult SCID mice injected once with 10⁷ to 10⁹ plaque-forming units demonstrated significant dose-dependent elevations in serum human erythropoietin levels and increased hematocrit levels that were stable over the 4-month time course of the experiments, and no evidence of a localized inflammatory response or systemic infection was present. Intramuscular injection of adenoviral vectors may be useful for the treatment of inherited disorders of deficient serum proteins.

Lipoprotein Metabolism
Direct gene transfer has been a promising tool for investigations of hepatic regulation of lipoprotein metabolism. Initial approaches used ex vivo transduction of hepatocytes to express an LDL receptor (LDLR). Hypercholesterolemia in the Watanabe rabbit was reversed after infusion of LDLR-expressing hepatocytes into the liver.^{90 91} This approach has been used in a human gene therapy trial of familial hypercholesterolemia.⁷ Recently, adenoviral vectors have been used to reconstitute LDLR function in Watanabe rabbits⁹² and in homozygous mice lacking LDLRs produced by homologous recombination.⁹³ In this latter study, adenoviral vectors encoding a human LDL cDNA were injected intravenously into the tail vein of mice, and the hypercholesterolemic effects of the LDLR deficiency were reversed at 4 days. Adenoviral targeting to hepatocytes has also been demonstrated in healthy mice, where transient gene expression was observed.¹⁷

Hepatic chylomicron remnant uptake has been studied by adenovirus-mediated gene transfer. The hypothesis that LDLR-related protein (LRP) mediates the uptake of dietary lipoprotein into hepatocytes in concert with LDLR was tested by transferring a dominant negative regulator of LRP function into livers of homozygous mice lacking LDLR.⁹⁴ Inhibition of LRP was associated with accumulation of chylomicron remnants in these mice, suggesting a role for LRP and LDLR in chylomicron remnant clearance. Intravenous injection of an adenoviral vector encoding an apolipoprotein A-I gene results in transient hepatic production of HDL and total cholesterol.⁹⁵ These studies demonstrate the usefulness of genetically engineered animal models for the study of lipoprotein metabolism.

	Clinical Trials

Top Introduction Methods of Gene Delivery... Animal Models of Gene... Clinical Trials References

 
The treatment of human diseases by gene transfer has begun in the United States. Since 1989, more than 100 gene marking and gene therapy trials have been approved by the Recombinant DNA Advisory Committee (RAC) of the National Institutes of Health and the Food and Drug Administration.^{96 97} The majority of these trials have been directed toward high-risk patient populations with incurable diseases, such as single-gene–inherited disorders, cancer, and AIDS. Several trials have been initiated that are relevant to cardiopulmonary diseases, including catheter-mediated gene delivery in a cancer trial for metastatic melanoma,^{44 98} an ex vivo treatment of transduced hepatocytes for familial hypercholesterolemia,⁷ and direct in vivo treatment for cystic fibrosis.^{99 100 101} A cardiovascular gene therapy protocol to stimulate angiogenesis in patients with peripheral vascular disease has been initiated. The application of gene transfer to cardiovascular diseases has proceeded at a slower rate. The risk-to-benefit ratio for cardiovascular patients is higher than for patients with other diseases, such as cancer or AIDS, in which treatment options are more limited. Most cardiovascular diseases are polygenic disorders, and identification of candidate genes to treat vascular or myopathic disorders is complicated. However, with recent advances made in gene transfer and antisense oligonucleotides, potential candidate genes have been identified. Issues such as catheter design and delivery, gene expression, and vector pharmacokinetics are being considered. The safety of viral and nonviral vectors in the vasculature and myocardium is being addressed.

In summary, the field of cardiovascular gene transfer has progressed considerably in the past 5 years. Gene transfer, alone or in combination with other genetic technologies, is proving to be useful in creating complex animal models of cardiovascular diseases in which the in vivo function of gene products can be investigated. These technologies also show promise for the treatment of human cardiovascular diseases, despite remaining technical issues. The field of cardiovascular gene transfer holds tremendous promise and excitement for basic and clinical scientists to work together to explore the possibilities of molecular genetic technologies for the treatment of cardiovascular diseases.

	References

Top Introduction Methods of Gene Delivery... Animal Models of Gene... Clinical Trials References

 
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