Gudrun Schiedner

Although adenoviral vectors provide prolonged gene expression in the brain by comparison to peripheral organs, expression is eliminated by a severe inflammatory infiltration (i.e., activated macrophages/microglia and T-lymphocytes) after peripheral infection with adenovirus. Here, we demonstrate that high-capacity adenoviral (HC-Ad) vectors succeed in maintaining long-term transgene expression in the brain, even in the presence of an active peripheral immunization with adenovirus that completely eliminates expression from first-generation vectors within 60 days. Importantly, even 60 days after the peripheral infection, brains injected with first-generation vectors exhibited evidence of a chronic infiltration of CD8(+) cells, macrophage/microglial activation, and up-regulation of brain MHC-I expression. No inflammation was observed in the brains injected with the HC-Ad vector. Thus, these results demonstrate that HC-Ad vectors will allow safe, stable, and long-term transgene expression in the brain, even in the presence of peripheral infection with adenovirus. This markedly improves the prospects for the use of adenoviral vectors for long-term gene therapy of neurological disorders.

To regulate expression of a transferred gene in response to an exogenous compound, we have combined a high capacity adenoviral vector devoid of all viral coding sequences with a regulatory system that can be used to express a target gene in vivo in a selected site and at a desired time. This system uses a chimeric transactivator, GLp65, which consists of a mutated progesterone receptor-ligand binding domain fused to the GAL4 DNA binding domain and part of the activation domain of the human p65 protein, a component of the NF-kappaB complex. In the presence of the antiprogestin mifepristone, this chimeric regulator binds to a target gene containing the 17-mer GAL4 binding site, resulting in an efficient ligand-inducible transactivation of the target gene. We inserted the regulator GLp65 and a regulable human growth hormone target gene containing the 17-mer GAL4 binding site into the same adenoviral vector. To obtain tissue-specific expression of the target gene, we coupled the regulator to a liver-specific promoter. Infection of HepG2 cells and experimental mice with the adenovirus resulted in consistently high induction levels of human growth hormone in the presence of mifepristone whereas the transgene expression was undetectable in the absence of the ligand. Taken together, our regulable adenoviral vector represents an important tool for transgene regulation that can be used for potentially diverse applications, ranging from tissue-specific gene expression in transgenic animals to human gene therapy.

Optimal gene therapy for many disorders will require efficient transfer to cells in vivo, high-level and long-term expression, and tissue-specific regulation, all in the absence of significant toxicity or inflammatory responses. While recombinant adenoviral vectors are efficient for gene transfer to hepatocytes, their usefulness is limited by short duration of expression related, at least in part, to immune responses to viral proteins and by a low capacity for foreign DNA. A number of systems have been developed for producing adenoviral vectors devoid of all viral coding sequences. Using AdSTK109, a vector lacking all viral coding sequences and carrying the complete human α1-antitrypsin (hAAT) genomic DNA locus, we have demonstrated sustained expression for longer than 10 months in mice. Utilizing high doses of this vector for hepatic gene transfer in mice, we find that supraphysiological levels of hAAT can be achieved without hepatotoxicity. Limited data are available to assess the efficacy and toxicity of helper-dependent adenoviral vectors with all viral coding sequences deleted. Using intravenous administration of high doses of a helper-dependent vector expressing hAAT in mice, we found long-term and high-level expression with minimal hepatic toxicity. The data suggest that helper-dependent vectors have substantially less acute and chronic, nonimmune-related toxicity likely related to the elimination of leaky viral gene expression. It may be possible to administer higher doses of helper-dependent adenoviral vectors with less toxicity compared with earlier generations of vectors.

Primary human cells are relatively refractory to transformation by adenoviral E1 functions. For almost two decades, human embryonic kidney (HEK)-derived 293 cells have been the only E1-complementing cell line suitable for production of E1-deleted adenoviral vectors. More recently, new vector production cell lines have been derived from human embryonic retina (HER) cells, a cell type that is difficult to obtain. We were surprised to find that readily available primary human amniocytes are efficiently transformed by adenoviral E1 functions. We selected cell lines that allow high-titer production of recombinant adenoviral vectors. The generation of replication-competent adenovirus (RCA) during production, caused by homologous recombination between vector and cellular DNA, was excluded by designing the transforming plasmid to lack sequence overlap with current adenoviral vectors. In addition, we generated an infectious plasmid that can be used for convenient generation of first-generation adenoviral vectors in Escherichia coli and that matches the E1 complementation in the new production cell lines.