Dominique Dreyer

Gene therapy with recombinant adenovirus vectors: evaluation of the host immune response

E1, E3-deleted, replication-deficient recombinant adenoviruses are widely studied as vectors for their capacity to transfer therapeutic genes in vivo. They can infect a wide variety of dividing and quiescent cells from different organs and possess a large packaging capacity. One of the major limitations in the use of these vectors for gene therapy is the transient expression of the transgene in vivo and the poor transduction efficiency when re-administered. Despite the deletion of the viral E1 region, low level of early and late viral genes are expressed in vivo. Thus, viral antigens plus those derived from transgene expression in transduced cells contribute to cellular immune responses leading to the destruction of these cells. Production of anti-adenovirus antibodies, the cellular immune response as well as the early non-specific clearance of the vectors, constitute barriers to successful gene therapy. New vectors have been derived with additional deletions in the E2a or the E4 regions. Such second generation vectors were evaluated in vivo. These studies have revealed the complexity of the immune mechanisms elicited by these vectors and the importance of several parameters in these evaluations (i.e. mouse strains, nature of the transgene, route of administration...). In order to inhibit the production of neutralizing antibodies to adenovirus that prevent from further readministration of the vectors, immunosuppressive strategies were undertaken. Treatment regimens with immunosuppressive drugs (cyclophosphamide, FK506) or with monoclonal antibodies that block either the T cell receptor or costimulation pathways allow prolonged transgene expression and/or readministration of adenoviral vectors. In addition, transduction efficiencies may be increased by transiently inhibiting non-specific immune mechanisms that lead to the dramatic early clearance of the vectors. Taken together, these strategies may improve further gene therapy protocols by decreasing the host immune response to adenoviral vectors.

Modulation of the Inflammatory Properties and Hepatotoxicity of Recombinant Adenovirus Vectors by the Viral E4 Gene Products

Liver toxicity and inflammation were assessed in C57BL/6, CBA, and BALB/c mice injected intravenously with a series of recombinant adenoviruses deleted simultaneously in E1/E3, in E1/E3/E2A, or in E1/E3/E4. All vectors were either devoid of transgenes or carried in E1 the human CFTR cDNA under the control of the CMV promoter. Injection of the E1/E3-deleted vector induced a significant liver dystrophy and inflammatory responses that were accompanied by an increased serum transaminase concentration. The vector toxicity remained elevated on additional deletion of the E2A gene and was further enhanced when hCFTR was expressed. In contrast, additional deletion of E4 led to a reduction in hepatotoxicity, suggesting an active role of E4 gene products in liver injury. However, deletion of E4 also led to a loss of transgene expression. To identify the individual E4 product(s) involved in liver toxicity and in the regulation of transgene expression, a series of isogenic E1/E3-deleted vectors, with or without the hCFTR transgene, and containing various combinations of functional E4 open reading frames (ORFs), were evaluated in vitro and in vivo. We demonstrate that liver injury was markedly reduced with vectors containing either ORF3 alone or ORF3,4 while vectors containing ORF4, ORF6,7 or ORF3,6,7 still displayed elevated hepatotoxicity and inflammatory responses. Moreover, transgene expression was restored when ORF3,4 or ORF3,6,7 was retained in the vector. These results highlight the importance of the E4 gene products in the design of improved in vivo gene transfer vectors.

Differential influence of the E4 adenoviral genes on viral and cellular promoters.

Strong and stable transgene expression is fundamental to the success of recombinant adenovirus vectors in human gene therapy. However, control of transgene expression is a complex process, involving both viral and cellular factors. In this study, the influence of the E4 adenoviral region on the activity of various promoters was investigated in vitro and in vivo.

Immunocytochemical analysis reveals differences between the subcellular localization of normal and ΔPhe508 recombinant cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation responsible for CF is the deletion of amino acid residue Phe508, with an average allelic frequency of 70%. We have isolated an anti-CFTR monoclonal antibody which specifically recognizes recombinant normal and delta Phe508-CFTR produced by a vaccinia virus expression system. Immunocytochemical analysis of L cells expressing either normal or delta Phe508-CFTR showed a marked difference in subcellular distribution. Normal CFTR had a distinct localization in the perinuclear area and was also associated with the plasma membrane. delta Phe508-CFTR essentially lacked the membrane-associated distribution and was present throughout the cytoplasm. This heterologous expression system thus provides a model system for studying the subcellular localization of different mutant forms of CFTR.

Characterization of trans-immortalized hepatic cell lines established from transgenic mice

Hepato-specific regulatory (promoter/enhancer) DNA sequences were used for targeting the expression of onc genes, such as murine c-myc and Simian Virus 40 T Antigen, to hepatocytes of transgenic mice which subsequently developed hepatocellular carcinomas after a variable period of time (depending on the type of onc gene employed). Several trans-immortalized cell lines were established and compared with respect to the expression of adult hepatic markers and response to growth factors. Despite the morphological differences observed between trans-hepatomas, owing to the expression of the two different onc genes, all tumor-derived cell lines behaved in a comparable fashion during long-term culture displaying an adult hepatic phenotype for at least 40 passages. They differed, however, in response to epidermal growth factor. When the gene coding for human alpha 1-antitrypsin was placed under the control of the same hepato-specific promoter/enhancer, high levels of the human recombinant protein could be harvested from the supernatants of trans-hepatoma-derived cell lines.