Defining strategies to extend duration of gene expression from targeted compacted DNA vectors.

Abstract

Gene transfer complexes containing poly-L-lysine (poly-K) and DNA with ligands directed at the serpin enzyme complex receptor (sec-R) deliver reporter genes to receptor-bearing cells in vivo. Expression lasts for about 30 days, when complexes containing long-chain poly-K are used. Extending the duration of expression would be desirable if correction of genetic defects is the goal. To test whether the mechanism by which expression is extinguished was due to an immune response to the transgene, or the loss of the transgene, we conducted two experiments. In the first, we injected sec-R-targeted lacZ complexes intravenously (i.v.) into mice genetically engineered to express this gene briefly during development. These mice, who should recognize the protein as 'self', also extinguished lacZ expression after 30 days. In a second experiment, we injected immunodeficient animals with sec-R-targeted human factor IX complexes. A similar temporal pattern of expression was observed in Rag-1 -/- mice, in whom expression also extinguished by 40 days. Moreover, factor IX plasmid DNA was detected in the lung and spleen 50 days after injection of complexes, suggesting that not all cells which had taken up the transgene had been destroyed. Thus, the host's immune response to the transgene may not account for the loss of reporter gene expression from these molecular conjugates. We further tested whether repeat administration of sec-R-targeted complexes will be limited by host immune responses. Mice were pre-dosed twice with sec-R-targeted complexes containing lacZ over a 40-day period. We then injected the animals i.v. with sec-R-targeted human factor IX complexes and measured gene expression and antibody production. Although 14 of 36 animals displayed low-titer antibodies to the ligand in targeted complex, expression levels were unaffected compared with virgin dosing. When the complexes were administered three times intranasally (n=10), no antibodies against the complex were detected in blood. Plasma from mice dosed with saline, nontargeted complex or naked DNA did not react with the ligand, ligand-poly K conjugate or targeted complex. All animals exhibiting human factor IX expression developed antibodies to that transgene by 21 days. Thus, at least three repeat administrations of sec-R-directed molecular conjugates are possible, provided that immune responses to the transgene itself are not limiting.