118. The Histone Deacetylase Inhibitor Enhanced Recombinant Adeno-Associated Virus-Mediated Transgene Expression in Cancer Cells

BIOLOGY OF TRANSDUCTION 118. The Histone Deacetylase Inhibitor Enhanced Recombinant Adeno-Associated VirusMediated Transgene Expression in Cancer Cells Takashi Okada,1 Tatsuya Nomoto,1 Kuniko Shimazaki,2 Mayumi Iwata-Okada,3 Toru Yoshioka,1 Masafumi Takahashi,4 Rahim Ajalli,1 Yuhe Liu,1 Takashi Matsushita,1 Hiroaki Mizukami,1 Masashi Urabe,1 Yutaka Hanazono,1 Akihiro Kume,1 Keiya Ozawa.1,3 1 Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Minami-kawachi, Tochigi, Japan; 2 Department of Physiology, Jichi Medical School, Minamikawachi, Tochigi, Japan; 3Division of Hematology, Department of Medicine, Jichi Medical School, Minami-kawachi, Tochigi, Japan; 4 Division of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical School, Minami-kawachi, Tochigi, Japan. The recombinant adeno-associated virus (rAAV) has been used with considerable interest for the clinical gene therapy of inherited monogeneic diseases. However, the rAAV is not very efficient in transduction of a variety of tumor cell lines, partly due to a limited second strand synthesis. We and others have previously reported the enhanced transduction of rAAV in cancer in vitro and in vivo with DNA-damaging stress such as gamma-rays or chemotherapeutic agents. An alternative approach is to assist transcription in the target cells. Histone deacetylase inhibitors have been known to regulate the transcription of various genes and also increase the alpha v integrin levels on some cancer cell lines, although which mechanism was not fully defined. In this present work, we have studied whether the histone deacetylase inhibitor could enhance rAAV-mediated tumor transduction. FR901228 is a deacetylase inhibitor, inducing a hyperacetylation of the chromatin and an enhancement of transcription. Rat primary cortical neurons and astrocytes, the human glioblastoma cell lines U-251MG and U87MG, cervical adenocarcinoma cell line HeLa, hepatocellular carcinoma cell line HepG2, and laryngeal epidermoid carcinoma cell line HEp-2 were used to examine deacetylase inhibitor-assisted gene transfer. Target cells were treated with the AAV2 vector expressing the eGFP gene (AAVeGFP) along with various concentration of the FR901228. The FR901228 enhanced transduction of those cells with the rAAV. Flow cytometric analysis showed that the treatment improved the rAAV-mediated gene transfer in a dose-dependent manner, and the highest enhancement was observed in the U-251MG cells. Twenty-four hours after the AAVeGFP infection at 1 x 10 (4) genome copies/cell with 1 ng/ml of the FR901228, 48% of the U251MG cells were eGFP-positive, whereas very few cells were eGFP-positive in the absence of the FR901228. In contrast, the enhancement of the alpha v integrin level was modest under the same condition of the treatment. Besides, real-time quantitative PCR analysis showed that the copy number of the rAAV in the transduced cells was not significantly augmented by the FR901228 treatment. These results suggest that enhancement of transgene expression rather than that of the viral entry might be responsible for the enhanced transduction. Furthermore, epigenetic modification might be involved in the enhancement of the transduction. This is compatible with other reports describing an effect of butyrate, deacetylase inhibitor, on the reactivation of silenced transgene expression in stably transduced cells. The rAAV genome may be present in a histone-associated chromatin form in the cells shortly after the infection. The use of the histone deacetylase inhibitor may enhance a utility of rAAV-mediated transduction strategies for cancer therapy and epigenetic modifications should be taken into consideration for the development of the efficient cancer gene therapy.

Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

119. Monoclonal Antibodies Against the AdenoAssociated Virus Type 2 and Their Characteristics Z. H. Yuan,1 J. Q. Peng,2 S. P. Tan,2 X. B. Wu.2 State Key Lab. of Molecular Virology and Genetic Engineering, Beijing, China; 2AGTC Gene Technology Company Ltd, Beijing, China. 1

A series of monoclonal antibodies against adeno-associated virus type 2(AAV2) were generated and their biological characteristics were analyzed briefly. The purified recombinant AAV2 particles containing GFP gene (rAAV2-GFP) were used for intra-abdominal immunization and two boost injections and the plasma were screened by AAV2 particles ELISA. The rAAV2-GFP was generated by infecting BHK-21 cells carrying pSNAV-GFP plasmids with rHSV1rc, a recombinant herpes simplex virus type 1 carrying AAV2 rep and cap gene we produced previously. Polyethylene glycol precipitation and chloroform extraction were used in purification pretreatment followed by cation exchange and molecular-size chromatography. The purity of rAAV2-GFP was more than 98% analyzed by HPLC and SDFS-PAGE. Seven MAbs were isolated. Five of them were characterized as neutralizing antibodies (B10,B7,E1,H6,G4), with one of them( G4) showing strong inhibition of virus attachment to cells. Subtype assay demonstrates that one is IgG1 (B10), two are IgM (B7 and G10), two IgG3 (B9 and H6),one IgG2a(G4) and one IgG2b(E1). Western blotting assay demonstrated that all these seven antibodies recognize VP1,VP2 and VP3 of AAV2, suggested that the recognized epitopes were all resided in VP3. Further experiments need to be done to give more informations about these antibodies.

120. Protein Transduction of AAV Rep 78 Can Support AAV Vector Packaging Sanae Hisayasu,1 Kumi Adachi,1 Hiroyasu Kinoshita,1 Yukihiko Hirai,1 Takashi Shimada.1 1 Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan. Rep 78/68 of adeno-associated virus (AAV) are essential for both viral replication and site-specific integration of the AAV genome into a specific site of chromosome 19, called the AAVS1 region. It is known that constitutive expression of Rep 78/68 is cytostatic or cytotoxic for mammalian cells. Therefore, stable packaging cell lines for AAV vector production have not been established. We have previously shown liposome mediated Rep78 transduction is useful for site-specific integration of AAV plasmid in culture cells. In this study, we examined the feasibility of purified Rep78 protein for AAV vector production. We constructed expression vectors for His tag-Rep78 protein fused with or without the HIV Tat peptide (HRep and Tat-Rep). The fusion proteins were expressed in E.Coli and purified by an affinity column chromatography. To study the utility of the purified Rep78 proteins for AAV production, 293 cells were first transfected with a vector plasmid (pAAV/EGFP) along with plasmids for helper function (pHelper) and cap expression (pCMVCap) by the CaPO4 co-precipitation method. H-Rep and Tat-Rep were directly added in the culture medium together with plasmids or after medium change of transfection. Both of crude cell lysates from H-Rep and TAT-Rep were capable of transducing HeLa cells, indicating that AAV vectors were packaged in 293 cells. These results represent purified Rep 78 is able to support AAV packaging. Transduction with purified Rep 78 and recombinant adenoviruses containing other helper and packaging components may provide a new strategy for AAV vector packaging.

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