Transient expression has long been another defining characteristic of synthetic vectors, which has until recently limited potential clinical applications to acute therapies. However, there are now examples of long-term expression from synthetic vectors lasting several weeks to greater than 1 year in vivo. A key aspect has been to identify promoters that remain transcriptionally active over an extended period in the major target organs, such as muscle, lung, and liver. Part of that challenge has been to identify promoters or promoter hybrids that are as transcriptionally robust as that of the widely used human cytomegalovirus (CMV) promoter, while not being prone to the known observed inactivation of CMV. Combinations of liver-specific promoter and enhancer elements have been identified that confer sustained expression in the liver with levels equivalent or superior to that of CMV (8,9). Hybrid promoters containing viral enhancer sequences linked to cellular promoters such as p-actin or ubiquitin also have been demonstrated to have significantly increased duration of expression (10,11).
Other advances in increasing the persistence of expression include the use of integrating elements to facilitate the retention of pDNA in tissues, for example, transposons (12) and phage integrases that promote integration of the vector (13,14). In addition, pDNA vectors that have been largely depleted of CpG motifs confer sustained expression through a mechanism that is presently unknown (15). Taken together, synthetic vectors are no longer limited to acute therapies and can be considered for diseases requiring long-term correction.
One of the more serious problems of synthetic vectors that are composed of a complex of DNA and a cationic liposome, polyamine, or other polycation is the acute inflammatory response observed in vivo. Intratracheal or intranasal delivery of cationic lipid-pDNA complexes induces an acute, dose-dependent inflammatory response characterized by an influx of neutrophils and the elevation of proinflammatory cytokines such as IFN-7, TNF-a, IL-6, and IL-12 (16). Intravenous injection of complex causes a similar induction of cytokines as well as a dose-dependent activation of complement, loss of lymphocytes and platelets, and elevated alanine aminotrans-ferase (ALT) and aspartate aminotransferase (AST) levels indicative of liver damage (17). Injecting higher doses of complex can lead to significant morbidity. Several groups have shown that immunostimulatory CpG motifs within the pDNA vector play a significant role in the acute inflammatory and toxic response to cationic lipid-pDNA complexes (18-20). Methylation of the CpGs in the pDNA can markedly reduce cytokine induction, but interestingly cannot eliminate other toxic parameters such as lymphocyte and platelet loss or elevation of liver enzymes (21). Only synthetic pDNA vectors that have been largely depleted of CpG motifs can reduce but not totally obviate the acute toxic response (15).
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