759. The DNA Minor Groove Binding Agents Hoechst 33258 and

AAV VECTORS: GENOMES AND BIOLOGY rAAV concatemer in transduced cells. The use of this HDAC inhibitor would greatly enhance the utility of rAAV-mediated transduction strategies for cancer gene therapy.

759. The DNA Minor Groove Binding Agents Hoechst 33258 and 33342 Enhance Recombinant Adeno-Associated Virus (rAAV) Transgene Expression Lina Li,1 Linda Yang,1 Robert M. Kotin.1 1 Laboratory of Biochemical Genetics, National Institutes of Health, Bethesda, MD. Recombinant adeno-associated viruses (rAAV) are commonly used in pre-clinical and clinical gene transfer studies. The relatively slow kinetics of rAAV transgene expression, however, complicates in vitro and in vivo experiments. Accelerating the kinetics of rAAV transgene expression would extend the applications of rAAV for gene transfer and facilitate the determination of vector biological activity in vitro. We found that the DNA minor groove binding agents, Hoechst 33258 and 33342, enhance rAAV gene expression. Hoechst 33258 or 33342 increase both the level of reporter gene expression and the population of transgene expressing cells. We found that Hoechst 33258 and 33342 augment rAAV gene expression in different cell types and in a concentration dependent manner. Hoechst 33258 or 33342 enhanced gene expression for rAAV-1, rAAV-2 and rAAV-5. Hoechst 33258 and 33342 mediated enhancement of rAAV gene expression correlated with an increase in S phase and G2/M phases of the cell cycle. Our data revealed that two different, although related, DNA binding drugs effect rAAV gene expression in a similar manner, suggesting that a common pathway is involved. Our results suggest that Hoechst 33258 and 33342 are potential enhancer drugs for the gene expression and might be useful for gene therapy applications.

760. Dual Therapeutic Utility of Proteasome Modulating Agents for Pharmico-Gene Therapy of the Cystic Fibrosis Airway Liang N. Zhang,1 Phil Karp,2 Christopher J. Gerard,4 Eric Pastor,4 Keith Munson,4 Ziying Yan,1 Simon Godwin,4 Joseph Zabner,2,3 Richard Peluso,4 Barrie Carter,4 John F. Engelhardt.1,2,3 1 Department of Anatomy and Cell Biology, The University of Iowa, Iowa City, IA; 2Department of Internal Medicine, The University of Iowa, Iowa City, IA; 3The Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, The University of Iowa, Iowa City, IA; 4Targeted Genetics Corporation, Seattle, WA. Pharmacologic- and gene-based therapies have historically been developed as two independent therapeutic platforms for cystic fibrosis (CF) lung disease. Inhibition of the dysregulated epithelial Na-channel (ENaC) is one pharmacologic approach to enhance airway clearance in CF. We investigated pharmacologic approaches to enhance CFTR gene delivery with recombinant adeno-associated virus (rAAV) and identified compounds that significantly improved viral transduction while simultaneously inhibiting ENaC activity through an unrelated mechanism. Treatment of human CF airway epithelia with proteasome modulating agents (LLnL and doxorubicin) at the time of rAAV-2 or rAAV-2/5 infection dramatically enhanced CFTR gene delivery and correction of CFTRmediated short-circuit currents (Isc). rAAV-2 provided significantly better functional correction and mRNA expression than rAAV-2/5 in the presence of proteasome inhibitor treatment. Surprisingly, these agents also facilitated long-term (15 day) functional inhibition of amiloride-sensitive ENaC currents independent of CFTR vector administration. Using quantitative RT-PCR, we found a 16-fold decrease in the ratio of γ-ENaC subunit to β-actin mRNA in the Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright  The American Society of Gene Therapy

doxorubicin treatment group (0.014+/-0.0046, n=9) as compared to the non-treated control (0.222+/-0.096, n=12). After treatment with doxorubicin, MspI/HpaII sites in CpG islands of the γ-ENaC promoter and Exon 1 were also protected from digestion with HpaII, a methylation-sensitive enzyme, indicating an increase in methylation at these regions. Hence, inhibition of ENaC activity was predominantly attributed to a doxorubicin-dependent decreased in γ-ENaC subunit mRNA expression caused by an increase in γ-ENaC promoter methylation. This report describes the first identification of compounds with dual therapeutic action able to enhance the efficacy of CFTR gene therapy to the airway while concurrently ameliorating primary aspects of CF disease pathophysiology. The identification of such compounds mark a new area for drug development, not only for CF, but also other gene therapy disease targets.

761. Targeting DNA-PK by siRNA To Study AAV Replication Young-Kook Choi, Barry J. Byrne, Sihong Song. 1 Pharmaceutics, Powell Gene Therapy Center, University of Florida, Gainesville, FL. It has been observed that DNA-dependent protein kinase (DNAPK) inhibits adeno-associated virus (AAV) DNA integration in vitro and in vivo (Song et al PNAS 2004). In order to test the effect of DNA-PK on AAV replication, we have infect MO59J (DNA-PK negative) and MO59K (DNA-PK positive) cells with a recombinant virus (UF5) in the presence or absence of a helper virus (carrying AAV rep and cap genes). Hirt DNA were isolated 2 days after infection, and subjected to southern blot analysis. Data showed that the abundance of replicated monomer of the vector DNA was lower in MO59K cells than in MO59J suggesting DNA-PK may inhibit AAV replication. To avoid the effect from the difference other than DNA-PK between the two cell lines, we have employed the small interfering RNA (siRNA) technology to decrease DNA-PK expression in MO59K cells. Synthetic siRNAs for human DNAPKcs were transfected into MO59K cells. Total RNA were extracted at 2 or 4 days after transfection. RT-PCR analysis showed that DNA-PKcs mRNA was nearly undetectable after 25, and 35 cycles. These results indicate that siRNAs can efficiently target DNAPKcs, thus provide a powerful tool for creating cell or animal models to study the cellular effects on AAV replication and integration. This work was supported by grants from the Juvenile Diabetes Research Foundation, NIDDK (DK 62652) and The Alpha One Foundation. Sihong Song and Young-Kook CHoi

762. Human Papillomavirus Type 16 Helper Functions for Adeno-Associated Virus Type 2 Replication Paul L. Hermonat,1 Yong Liu,1 Jawahar L. Mehta,1 Hong You.1 Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, AR.

1

Human papillomavirus (HPV) is known to “help” adenoassociated virus (AAV) to replicate to high levels during skin coinfection. However, unlike AAV’s other helper viruses adenovirus and herpes simplex, very little is known about the HPV genes which help AAV. In this study we surveyed four HPV-16 early genes, E1, E2, E6, and E7, for their ability to increase the basal level of AAV replication in a stratifying squamous epithelium. It was found that the HPV-16 E1, E2, and E6 genes were able to stimulate higher AAV replication in keratinocytes cultures upon co-transfection during wild type AAV-2 infection. E1 had the strongest enhancing effect upon AAV DNA replication (Southern blot), RNA expression (RTPCR), protein expression (Western blot) and AAV virion production S289