The gene therapy field has its origin in molecular biology and recombinant DNA technology. DNA was first discovered in 1944 as the genetic material in bacterial cells (1). Watson and Crick determined the double helical structure of DNA in 1953 (2). Leder and Nirenberg elucidated the genetic code by 1964 (3), and the first recombinant DNA molecules were synthesized using molecular biology techniques by 1972 (4). The scientific basis for gene therapy was developed in the 1970s, based on knowledge of the mechanisms of cell transformation by tumor viruses. The principles of gene transfer are based on the entry of foreign recombinant DNA into a host cell. Cells are resistant to the uptake of foreign genes, and viruses have adapted methods to gain entry into the cell. Early gene transfer methods adapted viruses (modified to be replication incompetent) to carry recombinant genes into cells. Basic investigations of tumor viruses, particularly retroviruses, laid the scientific foundation for the development of gene therapy in the 1980s. In the early 1990s, investigators used their knowledge of viral vectors and gene delivery to develop animal models of human disease (5). These experimental models evolved into the development of human gene therapy protocols in the United States. The first approved trial of gene transfer in humans was performed in May 1989 (6). A human gene therapy trial was initiated in 1990 for adenosine deaminase deficiency, which used ex vivo delivery of retroviral vectors encoding the human ADA gene into lymphocytes (7). Since this time, approximately 150 phase I–II clinical gene therapy trials have been performed in the United States, but no gene therapy treatments have been approved for clinical use by the Food and Drug Administration (FDA).

Gene therapy is a set of approaches to the treatment of human disease based on the transfer of genetic material into an individual. Gene delivery can be achieved either by direct introduction of vector or DNA to blood or tissues, or indirectly through the modification of cells that have been transduced to carry the foreign DNA. Gene therapy treats a disease in an individual by the administration of DNA rather than a drug. Somatic cells, not germ cells, are the target of these efforts. Therefore, gene therapy affects only individuals and not their offspring.

An initial step in the delivery of genes to individuals is the construction of gene transfer vectors (Fig. 97.1). A vector carries the recombinant gene (or DNA or RNA) into cells within a tissue. Vectors are used to transfer genes into cells because most cells are resistant to the uptake of foreign DNA. Vectors penetrate host cells with varying efficiency, depending on the cell type and the vector. Viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated vectors. Nonviral vectors include plasmid DNA, cationic liposomes, and interfering RNA.

After construction of a vector, the vector is generally tested in the laboratory in cultured target cells. If the gene is adequately expressed in these target cells, then animal models are developed in vivo. Gene delivery can be achieved by two methods: ex vivo and in vivo approaches. Ex vivo gene transfer involves the removal of autologous cells from the individual, genetically modifying or transducing the cells in culture, and returning the genetically modified autologous cells to the individual (8). Ex vivo gene transfer permits the introduction of recombinant genetic material into a particular cell type (e.g., a smooth muscle cell) and analysis of gene expression within that cell type (9). Ex vivo gene transfer, however, is a cumbersome and costly procedure, requiring the establishment and modification of cells in the laboratory. In vivo gene transfer, on the other hand, is a relatively straightforward approach by which the vector harboring the foreign DNA is directly inserted into host cells in vivo. This might be achieved by direct injection (10) or catheter-mediated gene delivery (11). After in vivo gene transfer, recombinant genes are generally expressed in multiple cell types.

The fate of recombinant DNA, once inside host cells, has been difficult to regulate and control. Considerable efforts have been devoted to developing methods for retaining the correct function of the gene and for regulating the gene so that the physiologic effects of gene expression can be evaluated.

As the gene therapy field has grown and seasoned, several important concepts have emerged. First, genetic defects contribute to the pathophysiology of many human diseases. In addition to monogenic or single gene disorders, many acquired diseases are characterized by normal genetic structure but abnormal expression of genes. For example, in atherosclerotic arteries, normal repair of an injured artery leads to abnormal expression of cytokine and growth factor genes (12). Many human diseases, both inherited and acquired, are characterized by genetic mutations that result in abnormal gene expression and pathophysiology.

Second, the fields of genetics and gene therapy, in the past, have developed in parallel, but there is increasing recognition of the interdependence of the two areas. The human genome has been mapped (13). The fields of genomics and proteomics
are emerging. Studies of gene expression and protein mapping will influence the diagnosis and treatment of cardiovascular diseases. Genomics, proteomics, and gene therapy complement one another and must be developed together.

Gene transfer vectors have relied on normal mechanisms of viral infection of human cells. For example, viruses have evolved mechanisms for receptor-mediated entry into human cells. Once within, a cell may reside latent for long periods. On viral activation by cytokines or other host genes, active viral replication occurs. In a similar manner, researchers have modified viruses to become vectors that carry a recombinant gene into a cell but cannot replicate within the cell. The recombinant gene resides within the nucleus, where it either integrates into host genome or undergoes episomal replication.

Viral and nonviral vectors have been developed and evaluated for cardiovascular gene therapy (Table 97.1). Viral vectors (retroviruses, lentiviruses, adenoviruses, adeno-associated viruses) have been the major methods for delivery of genes into cells. In response to concerns about the safety of viral vectors, nonviral vectors (plasmid DNA, DNA liposome complexes, interfering RNA) have also been developed. Each vector has advantages and disadvantages, which are dependent on the particular application, including cell type and organ system. Selection of a vector should consider the following factors. First, specificity to target tissues is critical. The vector should achieve local gene expression within an organ without systemic shedding and widespread toxicity. The vector should be delivered to host cells in sufficient quantity to result in high-level gene expression. Expression of the gene should persist for a sufficient period of time to treat the disease. Gene expression preferably is regulated through tissue-specific promoters or inducible or repressible promoters, or both. Finally, regulatory concerns about possible vector and gene toxicity should be kept in mind.

Several approaches have been developed to achieve site-specific gene expression. These include ligand-directed vectors, targeted gene expression using cell-specific promoters, catheter-mediated gene delivery, and direct injection. In the cardiovascular system, catheter-mediated gene delivery has been used most often to achieve site-specific gene expression within focal segments of blood vessels (11). Direct injection may be a feasible approach for gene delivery into the myocardium (14). Cell-specific gene expression may be desirable for selected approaches to vascular gene transfer, such as inhibition of
inflammatory cells or smooth muscle cell proliferation. Several laboratories have identified smooth muscle and endothelial cell-specific promoters, which have useful features for gene transfer vectors (15,16).