Chapter 065. Gene Therapy in Clinical Medicine (Part 3) Long-Term Expression in Genetic Disease: In Vivo Gene Transfer with Recombinant Adeno-Associated Viral (AAV) Vectors Recombinant AAV vectors have emerged as attractive gene delivery vehicles for genetic disease. Engineered from a small replication-defective DNA virus, they are devoid of viral coding sequences and trigger very little immune response in experimental animals. They are capable of transducing nondividing target cells, and the donated DNA is stabilized primarily in an episomal form, thus minimizing risks associated with insertional mutagenesis. Because the vector has a tropism for certain long-lived cell types, such as skeletal muscle, the central nervous system (CNS), and hepatocytes, long-term expression can be achieved even in the absence of integration. Clinical trials using recombinant AAV vectors are now ongoing for muscular dystrophies, α 1 -antitrypsin deficiency, lipoprotein lipase deficiency, hemophilia B, and a form of congenital blindness called Leber's congenital amaurosis. Hemophilia is often considered a promising disease model for gene transfer, as the gene product does not require precise regulation of expression and biologically active clotting factors can be synthesized in a variety of tissue types, permitting latitude in choice of target tissue. Moreover, raising circulating factor levels from <1% (levels seen in those severely affected) into the range of 5% greatly improves the phenotype of the disease. Preclinical studies with recombinant AAV vectors infused into skeletal muscle or liver have resulted in long-term (>5 years) expression of factor VIII or factor IX in the hemophilic dog model. Administration to skeletal muscle of an AAV vector expressing factor IX in patients with hemophilia was safe and resulted in long-term expression as measured by muscle biopsy, but circulating levels never rose >1% for sustained periods, and a large number of IM injections (>80–100) was required to access a large muscle mass. Intravascular vector delivery has been employed to access large areas of skeletal muscle in animal models of hemophilia and will likely be tested in upcoming trials. Administration of an AAV vector expressing factor IX to the liver in humans with hemophilia resulted in therapeutic circulating levels at the highest dose tested, but expression at these levels (>5%) lasted for only 6–10 weeks before declining to baseline (<1%). A memory T cell response to viral capsid, present in humans but not in other animal species (which are not natural hosts for the virus), may be a contributing factor in the loss of expression. Fortunately, triggering of the memory T cell response appears tissue-specific, and it is possible that introduction of the vector into immunoprivileged sites, such as the CNS (e.g., for Parkinson's disease) or the retina, will avoid this complication. Leber's congenital amaurosis (LCA) is a form of retinal degenerative disease, characterized by severe early-onset blindness. This disease, not currently treatable, is caused by mutations in several different genes; ~15% of cases of LCA are due to a mutation in a gene, RPE65, encoding a retinal pigment epithelial protein. In dogs with a null mutation in RPE65, sight has been restored after subretinal injection of an AAV vector expressing RPE65. Transgene expression appears to be stable, with the first animals treated >5 years ago continuing to manifest electrophysiologic and behavioral evidence of visual function. As is the case for X-linked SCID, gene transfer must occur relatively early in life to achieve correction of the genetic disease, although the exact limitations imposed by age await clinical studies. AAV-RPE65 trials have now been approved in both the United States and Great Britain. Other inherited retinal degenerative disorders may also be amenable to correction by gene transfer, as are certain complex acquired disorders such as age-related macular degeneration, which affects several million people worldwide. The neovascularization that occurs in age-related macular degeneration can be inhibited by expression of vascular endothelial growth factor (VEGF) inhibitors such as angiostatin, or through the use of RNAi-mediated knockdown of VEGF. Early-phase trials of siRNAs that target VEGF RNA are underway, but these require repeated intravitreal injection of the siRNAs; an AAV vector–mediated approach might allow long-term knockdown of VEGF. Gene Therapy for Cancer The majority of clinical gene transfer experience has been in subjects with cancer (Fig. 65-1). As a general rule, a feature that distinguishes gene therapies from conventional cancer therapeutics is that the former are less toxic, in some cases because they are delivered locally (e.g., intratumoral injections), and in other cases because they are targeted specifically to elements of the tumor (immunotherapies, anti-angiogenic approaches). Cancer gene therapies can be divided into local and systemic approaches (Table 65-2). Some of the earliest cancer gene therapy trials focused on local delivery of a prodrug or a suicide gene that would increase sensitivity of tumor cells to cytotoxic drugs. A frequently used strategy has been intratumoral injection of an adenoviral vector expressing the thymidine kinase (TK) gene. Cells that take up and express the TK gene can be killed after the administration of gancyclovir, which is phosphorylated to a toxic nucleoside by TK. Because cell division is required for the toxic nucleoside to affect cell viability, this strategy was initially used in aggressive brain tumors (glioblastoma multiforme) where the cycling tumor cells were affected but the nondividing normal neurons were not. More recently, this approach has been explored for locally recurrent prostate, breast, and colon tumors, among others. Table 65-2 Gene Therapy Strategies in Cancer Local/regional approaches Suicide gene/prodrug Suppressor oncogene Oncolytic virus Systemic response Chemoprotection Immunomodulation Anti-angiogenesis . Chapter 065. Gene Therapy in Clinical Medicine (Part 3) Long-Term Expression in Genetic Disease: In Vivo Gene Transfer with Recombinant Adeno-Associated Viral (AAV) Vectors Recombinant. mutation in a gene, RPE65, encoding a retinal pigment epithelial protein. In dogs with a null mutation in RPE65, sight has been restored after subretinal injection of an AAV vector expressing RPE65 disease. Preclinical studies with recombinant AAV vectors infused into skeletal muscle or liver have resulted in long-term (>5 years) expression of factor VIII or factor IX in the hemophilic