848. Helper Dependent Adenovirus-Epstein- Barr Virus Hybrid Vector for Long Term Persistance in Hepatocytes
Authors: Jose S. Gil, Sean D. Gallaher, Arnold J. Berk
Journal: Molecular Therapy (2006)

Helper Dependent Adenoviral (HDA) vectors are an effective method for in vivo gene transfer. The lack of viral genes and replication competent virus greatly diminishes the immune response and cytotoxicity of transduced cells. They are relatively easy to grow to high titers, appropriate for in vivo gene therapy. Though, like typical Adenoviral vectors, HDAs have no mechanism to persist in dividing cells.Epstein-Barr Viral (EBV) episomes, confer stability with the use of the Epstein-Barr Nuclear Antigen-1 (EBNA-1), acting upon the OriP region. EBNA-1 induces episomal replication at S phase, ensuring dilution does not occur, and tethers the episome to metaphasic chromosomes during mitosis, to minimizing episome lost during mitosis and cell division.Our previous work demonstrated effective recombination of our HDAs into EBV episomes in vivo, through optical imaging using a dual (binary) vector system. This work required co-infection of the both the HDA target vector, to be recombined into an EBV episome, and the recombinase expressing HDA vector. The separation of the vectors has been necessary to prevent premature recombination of the target vector during propagation.To circumvent the problem of premature self recombination in this newer single vector system, Cre recombinase must be restricted during viral propagation, and expressed upon infection of the target tissue. As we are targeting hepatocytes, we have opted to use a liver specific promoter/enhancer, consisting of the minimal Alpha Anti- trypsin promoter and 3 repeats of the ApoE enhancer.Endogenous promoters are also known to be less susceptible to silencing, compared to constitutive viral promoters. As the binary system employs a CMV promoter, silencing could limit persistence due to a loss of EBNA-1 expression. The endogenous promoter in the single vector system may circumvent this issue.The single vector is propagated in 293FLP cells, expressing FLP recombinase to restrict packaging of the helper virus, through the FRT flanked packaging region. After several rounds of serial infection with addition of helper virus, the vector is purified through cesium chloride buoyant density centrifugation.Cell culture experiments have shown recombination using the single vector system occurs in liver derived cells, as expected. Preliminary in vivo data supports these findings with detectable transgene expression in living mice.Long term persistence assay will be performed to determine the dynamics of this system. The single vector system promises to prove more practical, as coinfection is not required, reducing the MOI required for efficient coinfection. Helper Dependent Adenoviral (HDA) vectors are an effective method for in vivo gene transfer. The lack of viral genes and replication competent virus greatly diminishes the immune response and cytotoxicity of transduced cells. They are relatively easy to grow to high titers, appropriate for in vivo gene therapy. Though, like typical Adenoviral vectors, HDAs have no mechanism to persist in dividing cells. Epstein-Barr Viral (EBV) episomes, confer stability with the use of the Epstein-Barr Nuclear Antigen-1 (EBNA-1), acting upon the OriP region. EBNA-1 induces episomal replication at S phase, ensuring dilution does not occur, and tethers the episome to metaphasic chromosomes during mitosis, to minimizing episome lost during mitosis and cell division. Our previous work demonstrated effective recombination of our HDAs into EBV episomes in vivo, through optical imaging using a dual (binary) vector system. This work required co-infection of the both the HDA target vector, to be recombined into an EBV episome, and the recombinase expressing HDA vector. The separation of the vectors has been necessary to prevent premature recombination of the target vector during propagation. To circumvent the problem of premature self recombination in this newer single vector system, Cre recombinase must be restricted during viral propagation, and expressed upon infection of the target tissue. As we are targeting hepatocytes, we have opted to use a liver specific promoter/enhancer, consisting of the minimal Alpha Anti- trypsin promoter and 3 repeats of the ApoE enhancer. Endogenous promoters are also known to be less susceptible to silencing, compared to constitutive viral promoters. As the binary system employs a CMV promoter, silencing could limit persistence due to a loss of EBNA-1 expression. The endogenous promoter in the single vector system may circumvent this issue. The single vector is propagated in 293FLP cells, expressing FLP recombinase to restrict packaging of the helper virus, through the FRT flanked packaging region. After several rounds of serial infection with addition of helper virus, the vector is purified through cesium chloride buoyant density centrifugation. Cell culture experiments have shown recombination using the single vector system occurs in liver derived cells, as expected. Preliminary in vivo data supports these findings with detectable transgene expression in living mice. Long term persistence assay will be performed to determine the dynamics of this system. The single vector system promises to prove more practical, as coinfection is not required, reducing the MOI required for efficient coinfection.