What can cardiovascular gene transfer learn from genomics: and vice versa?

Tae Ho Kim2, Kimberly A. Skelding1, Elizabeth G. Nabel3 and Robert D. Simari1

1 Division of Cardiovascular Diseases, Department of Biochemistry and Molecular Biology, Molecular Medicine Program, Mayo Clinic and Foundation, Rochester, Minnesota 55905
2 Division of Cardiovascular Disease, Department of Internal Medicine, Chung-Ang University Hospital, Seoul 140-757, Korea
3 National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 The Current State of...
 What Can Cardiovascular Gene...
 What Can Genomics Learn...
 Conclusion
 REFERENCES
 
The field of gene transfer has developed in an era of expanding biomedical knowledge. The potential for gene transfer to treat cardiovascular disease is great, yet identified and unidentified barriers remain. Gene transfer and its ultimate application, gene therapy, require extensive details of not only the mechanism of disease but the biological implications of the vectors used to deliver the therapeutic genes as well. Many of these details are becoming available via the study of genomics. Genomics, the study of complete genetic sequences, holds the potential for enabling and amplifying the therapeutic hopes for gene transfer. Identification of new therapeutic genes, new regulatory sequences, and establishing the patterns of gene expression from tissues exposed to vectors and transgenes will rapidly advance the application of gene transfer. Finally, there are historical and ongoing lessons learned from the development of gene transfer that may be applicable to the challenging field of genomics and may enable its future success.

gene therapy; vectors


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 The Current State of...
 What Can Cardiovascular Gene...
 What Can Genomics Learn...
 Conclusion
 REFERENCES
 
GENE TRANSFER is the introduction of foreign or native genetic material into target cells to modulate gene expression with the ultimate goal of treating human disease. Gene transfer has outstanding potential to treat a variety of acquired and genetic human diseases and is currently being tested in over 500 clinical trials in the United States. Of these, 10% test strategies to treat cardiac and vascular diseases. The primary cardiovascular disease targets are therapeutic angiogenesis for chronic ischemia and the inhibition of vasculoproliferative disorders such as restenosis and vein graft occlusion.

The inherent complexity of gene transfer vectors and their associated pharmacokinetics and pharmacodynamics necessitates an intricate knowledge of the biology that regulates the interface between vector and patient (24). The study of whole genomes or genomics will impact vector development, define novel transgenes, and identify new diseases to be treated by gene transfer. These findings may ultimately be crucial in the establishment of widespread and effective use of gene therapy for cardiovascular diseases. Furthermore, the challenges that the field of gene transfer has faced and its responses to those challenges may provide insights into similar challenges faced by the field of genomics.


    The Current State of Cardiovascular Gene Transfer
 TOP
 ABSTRACT
 INTRODUCTION
 The Current State of...
 What Can Cardiovascular Gene...
 What Can Genomics Learn...
 Conclusion
 REFERENCES
 
Enabled by the introduction of recombinant DNA technologies in the 1970’s, the concept of the use of genetic material as an alternative to standard therapies emerged in the 1980’s. Originally conceived as a means to treat monogenetic diseases, the initial clinical application of gene transfer technology was in patients with combined immunodeficiency syndrome (1, 2, 9). Early studies aimed to treat monogenetic diseases including cystic fibrosis (3, 15, 32, 33) were disappointing to those expecting clinical improvement. However, these early clinical studies and many preclinical studies began to define the limitations and potential of viral and nonviral vectors.

With this growing base of knowledge and the inability to transduce an adequate number of cells to treat many systemic monogenetic diseases, approaches to acquired diseases were developed. Initial attempts to deliver vectors to the vasculature included plasmid and retroviral-based strategies (21). These studies demonstrated the ability to transduce vascular cells, albeit with low efficiency. The generation of recombinant adenoviral vectors allowed for enhanced gene transfer efficiency in normal and atherosclerotic arteries compared with nonviral and retroviral vectors (6). The enhanced efficiency of adenoviral vectors allowed for the testing of therapeutic strategies to inhibit neointimal formation following arterial injury (22). Although initial attempts to translate these early antiproliferative studies were stymied by concerns regarding the safety of adenoviral vectors and the lack of effective delivery catheters, clinical trials have recently begun in the US and Europe testing antiproliferative vascular gene transfer strategies delivering genes encoding for isoforms of nitric oxide synthase, a family of pluripotent cytostatic transgenes (12, 13, 31). Clinical targets for these initial studies include arteriovenous fistulae stenosis and restenosis following coronary stenting.

Concurrent with attempts to modulate arterial remodeling following arterial injury, investigators hypothesized that delivery of angiogenic peptides via gene transfer might enhance blood flow into ischemic tissue. Vascular endothelial growth factor (VEGF), a potent and specific secreted mitogen for endothelium, was demonstrated to induce angiogenesis in a series of animal models (28, 29). These pioneering studies by Jeffrey Isner and colleagues (10) took advantage of the potency and the secreted nature of the transgene product and used "naked" DNA delivery without adjuvants. This approach was quickly advanced to clinical trials using plasmid delivery of a gene encoding for VEGF165 into ischemic limbs. Similar trials began to define the feasibility and safety of this approach. Other angiogenic transgenes have also been used in clinical trials, including VEGF121, VEGF-2, and fibroblast growth factor-4 (FGF-4) (8, 19, 25). In general, these studies have demonstrated feasibility and promising but unproven efficacy. Safety concerns, particularly concerns regarding the effects of angiogenesis on preexisting tumors, await larger studies. An ongoing phase 3 trial of intracoronary adenoviral-mediated delivery of FGF-4 in chronic ischemic coronary artery disease is the first randomized multicenter phase 3 trial of gene transfer for cardiovascular disease and represents the potential for a gene transfer "product" in cardiovascular disease.

A great deal of effort is aimed at identifying novel targets for gene transfer in cardiovascular disease. Novel targets include disorders without effective standard therapies, such as pulmonary hypertension (11) as well as more prevalent disorders such as systemic hypertension and congestive heart failure (5, 17, 18). Ultimately, the prevention of atherosclerosis, the proximate cause of myocardial infarction, limb ischemia, and stroke may be the most important target for gene transfer.


    What Can Cardiovascular Gene Transfer Learn from Genomics?
 TOP
 ABSTRACT
 INTRODUCTION
 The Current State of...
 What Can Cardiovascular Gene...
 What Can Genomics Learn...
 Conclusion
 REFERENCES
 
Effective gene transfer requires a delivery system capable of efficiently transducing target cells resulting in expression of a potent transgene product. This transgene product must be expressed within a therapeutic range over a specified temporal pattern defined by the disease to be treated. The components required for such a system include sufficient genetic and biochemical insights into the disease, whether genetic or acquired, to select an appropriate transgene. Transgene expression is regulated by genetic regulatory elements. The host’s response to vector delivery and transgene expression ultimately determine the extent and the duration of transgene expression. The study of genomics will enable and enhance each of these essential components of gene transfer: knowledge of the disease, identification of novel transgenes and regulatory elements, and understanding the response of host cells to vector delivery.

Understanding the molecular and biochemical basis for disease.
An absolute requirement for gene transfer in genetic diseases is a complete understanding of the biochemical nature of the disease and the identity of potentially therapeutic transgenes. Analysis of global gene expression using microarrays will provide for new insights into the biochemical nature of cardiovascular disease. Microarray studies from activated macrophages and endothelial cells have identified patterns of gene expression associated with cellular activation (4, 26). In shear stress-exposed endothelium, a unique transcription factor, lung Kruppel-like factor (LKLF), was upregulated suggesting a potential molecular basis for shear-induced phenotypic changes (4). Although these studies have identified scores of gene products that are up- and downregulated, patterns of expression will eventually lead to unifying conclusions as to key regulators of activation and identify novel targets for genetic regulation.

Gene expression profiling of neointima formed within coronary stents has been performed and compared with samples of normal media (34). This study identified over 200 differentially regulated genes. Of those, FK506 binding protein-12 (FKBP-12) was identified as novel gene product upregulated in neointima. As FKBP-12 is a receptor for sirolimus (Rapamycin), a therapeutic agent for restenosis (7, 20), these findings implicate the potential impact of the analysis on therapeutics. Furthermore, genes that are downregulated in neointima, such as desmin, may be potential candidates for overexpression with gene transfer.

A logical step following analysis of gene expression will be the evaluation of the identified gene products that can be performed with protein analysis. Proteomics, while still in its infancy, will allow the characterization of proteins on a large scale. The proteome, once defined, is a complete view of the phenotype of the cell studied and can lead to disease definition and ultimately therapeutic modalities.

Identifying novel transgenes to be delivered may be the most obvious benefit of genomic analyses to gene transfer. In acquired and polygenic disorders such as hypertension, novel target regions within the genome have been identified using comparative genomics (27). Through studies like these, novel transgenes will be identified. As these transgenes products may be potent and specific, their use may be effective with the current vector systems in spite of their limitations.

Genomics also has the potential for individualizing gene therapy. Genotyping may be a prerequisite for identifying characteristics of patients who would be best suited for gene transfer studies. For example, in patients with hypertrophic cardiomyopathy, a clinically and genetically heterogenous disorder, specific identification of the mutated gene (and the specific mutation) may be necessary prior to enrollment in a gene transfer study. Ultimately, it may be important to define the genotype of distinct disease modifying genes as well.

Developing better vectors and transgenes.
The information gained from genomic studies will provide an opportunity to improve vectors and transgenes for gene transfer studies. These improvements include new regulatory elements identified in genomic studies. Originally, transgenes are regulated in a simple manner via constitutive promoters and enhancers, often viral. This strategy could be summarized as optimizing the level of expression without regard to temporal or tissue specificity. Tissue-specific regulatory elements may be an improvement over constitutive viral regulatory elements in that they may prevent transgene expression in nontarget cells (14, 16). In the vasculature, the SM22{propto} promoter has been used to limit transgene expression to vascular smooth muscle cells. Further modification of these elements to include inducibility currently require small molecule or peptides to be given exogenously. An ideal regulatory system would take advantage of the novel regulatory elements currently being defined in large-scale genomic analyses. Complex genomic regulatory sequences are being defined that may provide for both temporal and spatial regulation of transgene expression (23). These sequences, which regulate the complex pattern of native gene expression, might be used to drive transgene expression. Thus genomic analysis may lead to the development of novel ways to regulate transgene expression.

Defining host response to vectors.
The host response to vector and transgene product exposure plays a major role in the limiting transgene expression. Host cellular responses to viral exposure include direct toxic and cytopathic effects as well as presentation of viral epitopes in antigen presenting cells. Global analyses of gene and protein expression using microarray analysis has the potential for providing unique insights into vector/host interactions.

Opportunities to utilize microarray analyses to dissect problems in gene transfer include defining a target cell’s response to both vector and transgene product. For example, viral vectors are known to stimulate a host response including activation of the immune system and inflammatory mediators. If the response to the vector was fully characterized, then it might allow for strategies to be developed that might attenuate these responses. Thus the microarray approach should be used to identify the global patterns of gene expression following vector exposure.

In addition to vector-induced patterns of expression, certain transgenes act via transcriptional regulation of target genes. For instance, hypoxia inducible factor-1{alpha} (HIF-1{alpha}) is a transcription factor known to regulate transcription of hypoxia-inducible genes. Gene transfer of HIF-1{alpha} has been used to induce angiogenesis at sites of ischemia (30). Determining the global patterns of gene expression induced by overexpression of HIF-1{alpha} would provide data that might be used to define the mechanisms and risks of such an approach. This approach might not be limited to transgenes that are transcription factors but may be of use in any gene transfer study that targets an intracellular protein or signaling pathway.


    What Can Genomics Learn from Gene Transfer?
 TOP
 ABSTRACT
 INTRODUCTION
 The Current State of...
 What Can Cardiovascular Gene...
 What Can Genomics Learn...
 Conclusion
 REFERENCES
 
Clinical gene transfer studies and the expectations for gene therapy have generated serious concerns and review within the past 5 years. Excessive hyperbole and expectations have led some to question the future of gene-based medicines. Although the history of gene transfer is relatively brief, it has generated lessons from which genomics might benefit. First and foremost, expectations for revolution must be managed. As with gene transfer, many expect everyday life to change due to genomics. These expectations are often increased by proponents with agendas of self-interest. Neither the practitioners nor the public benefit from unrealistic expectations, and they should be avoided assiduously. Second, self-interest must be avoided where it may impede scientific rigor. This is not to downplay the importance of intellectual property in the advancement of the fields but does suggest that when managed carefully, and in coordination with public efforts, the field and the individual may gain. Efforts between private and public consortia in the Human Genome Project may be an example of such cooperation. Third, public scrutiny is critical to the success of such important endeavors. Early efforts from the National Institutes of Health and federal government to explore gene therapy in the public domain, such as the Recombinant DNA Advisory Committee, have provided opportunities for public input, albeit perhaps by increasing the burden of regulatory oversight of investigators. Finally, standardization of techniques and technologies among investigators will ease the assimilation of the data generated by diverse labs. In gene transfer, attempts to standardize the quantification and purification of viral vectors has been long overdue and has stifled comparisons within the field. Similar issues are important in comparing various microarray platforms available currently.


    Conclusion
 TOP
 ABSTRACT
 INTRODUCTION
 The Current State of...
 What Can Cardiovascular Gene...
 What Can Genomics Learn...
 Conclusion
 REFERENCES
 
Gene transfer is a relatively young field that has undergone extreme scrutiny in the recent years. Gene transfer that achieves important therapeutic goals will require improvements in vector technology. The field of gene transfer can benefit greatly form the emerging science of genomics. The study of genomics will provide investigators with new opportunities to deliver more potent transgenes, in a more safe and efficient manner. Similarly, the field of genomics would benefit by managing public expectations in this technology.


    ACKNOWLEDGMENTS
 
We thank Traci Paulson for assistance with preparation of the manuscript.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: R. D. Simari, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905 (E-mail: simari.robert{at}mayo.edu).

10.1152/physiolgenomics.00063.2002.


    REFERENCES
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 INTRODUCTION
 The Current State of...
 What Can Cardiovascular Gene...
 What Can Genomics Learn...
 Conclusion
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