71. The Structure of AAVrh32.33, a Novel Gene Delivery Vector

AAV VECTOR BIOLOGY 217 into the 3’ untranslated region of CVB3 genome. We quantified endogenous miR-1and miR-217 expression levels in a panel of cell lines and demonstrated that these miRNA expressions were very low in NSCLC cell lines while relatively high in HeLa cells and human normal lung cell lines. In vitro titration assays showed that infection with CVB3-miR-1217T in NSCLC cells retained its abundant viral replication in comparison with the parental CVB3, while its attenuated replication was observed in HeLa cells. On the other hand, gain of function assay showed that miR-1-transduced NSCLC cells became dramatically resistant to the CVB3-miR-1217T infection compared with parental CVB3, demonstrating the dependence of its infectivity on miR-1 level. Furthermore, the administrations into human NSCLC xenograft tumor preestablished in athymic nude mice with CVB3-miR-1217T, but not parental CVB3, dramatically decreased serum level of amylase and mitigated both pancreatitis and myocarditis with a significant tumor regression. Our data collectively demonstrated that our novel genetically modified attenuated strain of CVB3 could be a promising and safer modality for the treatment of NSCLC patients and such miRNA-based genetic modulation would provide us a versatile platform to enhance the intrinsic potentials of CVB3 in general cancer virotherapy.

by prodrug administration. Notably, both At- and PSES-targeted RRVs showed highly restricted systemic biodistribution in immunodeficient hosts, as compared to wild-type LTR-driven RRV. RRVs regulated by prostate-specific promoters thus exhibit transcriptionally targeted replication in prostate cancer models, achieving efficient tumor transduction and potent therapeutic efficacy in vitro and in vivo. In particular, PSES-targeted RRVs may be highly useful in castrationresistant prostate cancer. The ability to target RRVs specifically to cancer cells will further limit and control the replicative process, and minimize any potential risk to normal cells.

70. Retroviral Replicating Vector (RRV)Mediated Prodrug-Activator Gene Therapy Transcriptionally Targeted to Prostate Cancer

The Adeno-Associated viruses (AAVs) are being developed as clinical gene delivery vectors for therapeutic applications. However, the host immune response directed against the capsid, prevalent in a large proportion of general population, negatively impacts efficacy. AAVrh32.33, a novel vector developed from rhesus macaques isolates, has significantly lower seroprevalence in human populations compared to AAV2, the most widely studied and utilized human AAV serotype, as well as other non-human primate serotypes such as AAV7 and AAV8. To better understand the capsid determinants of this differential immune response to AAVrh32.33, its structure was determined X-ray crystallography to 3.5 Å resolution. The capsid viral protein (VP) structure conserves the eight-stranded -barrel core and -A helix reported for other parvoviruses and the distinct capsid surface topology of the AAVs: a depression at the icosahedral two-fold axis, three protrusions surrounding the threefold axis, and a depression surround a cylindrical channel at the fivefold axis. A comparison of this structure to AAV2, AAV4, and AAV8, to which AAVrh32.33 shares 61%, 81%, and 63% identity, respectively, identified differences in previously defined AAV VP structurally variable regions (VR1-VRIX) which function as receptor attachment, transduction efficiency, and/or antigenic determinants. This structure thus provides a platform for ongoing efforts to develop AAVrh32.33, as well as other AAV serotypes, for tissue targeted gene-therapy applications with vectors that can evade pre-existing antibody responses against the capsid. These features are required for full clinical realization of this promising gene delivery system.

Shuichi Kamijima,1 Takahiro Kimura,2 Kazunori Haga,3 Kei Hiraoka,1 Akihito Inagaki,1 Masamichi Takahashi,1 Christopher R. Logg,1 Chinghai Kao,4 Bernard H. Bochner,5 Noriyuki Kasahara.1,6 1 Medicine, University of California, Los Angeles, CA; 2Urology, The Jikei University School of Medicine, Tokyo, Japan; 3Sanriki Research Institute, Sanjukai Urological Hospital, Sapporo, Japan; 4Urology, Microbiology and Immunology, Indiana University, Indianapolis, IN; 5Urology, Memorial Sloan Kettering Cancer Center, New York City, NY; 6Molecular and Medical Pharmacology, University of California, Los Angeles, CA. Retroviral replicating vectors (RRVs) can propagate specifically within actively-dividing cancer cells, but are restricted in normal cells by innate immunity, and are currently being evaluated in first-in-human clinical trials for gene therapy of glioblastoma. To examine whether transcriptional targeting could further improve their efficiency and safety profile, we have developed and tested RRVs regulated by two different prostate-specific promoters. We therefore generated RRV-GFP (driven by the wild-type MLV promoter), RRV-GFP-At (regulated by ARR2PB, a probasin promoter variant which exhibits high specificity for androgen receptor (AR)-positive prostate cancer cells) and RRV-GFP-PSES (regulated by PSES, an androgen-independent promoter active in PSA/PSMA-positive prostate cancer cells, both in the presence and absence of androgen). We also generated wild-type RRV (RRV-CD) and prostate-targeted RRVs (RRV-CD-At and RRV-CD-PSES, respectively) expressing the yeast cytosine deaminase (CD) prodrug-activator gene, which converts the prodrug 5-fluorocytosine into 5-fluorouracil. Replication kinetics and cell type-specificity were first examined in prostate cancer cell lines (LNCaP, MDA PCa 2b, PC-3), non-prostate cells (293T) and primary human T cells in vitro. RRV-GFP-At showed high specificity and robust replication in AR-positive prostate cancer cells, but only in the presence of androgen. RRV-GFP-PSES showed high specificity for PSA/PSMA-positive prostate cancer cells, both in the presence and absence of androgen. In primary T cells, both prostate-targeted RRVs showed greatly reduced transgene expression compared to non-targeted RRV. In LNCaP subcutaneous tumors in vivo, both prostate-targeted RRVs achieved efficient transgene delivery upon progressive intratumoral replication. Even after initial inoculation at very low levels, RRV-mediated CD prodrug-activator gene delivery resulted in tumor regression after intratumoral virus spread followed Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

AAV Vector Biology 71. The Structure of AAVrh32.33, a Novel Gene Delivery Vector

Kyle Mikals,1 Hyun-Joo Nam,1 Kim Van Vliet,1 Luk H. Vandenberghe,2 Lauren E. Mays,2 Robert McKenna,1 James M. Wilson,2 Mavis Agbandje-McKenna.1 1 Biochemistry and Molecular Biology, University of Florida, Gainesville; 2Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia.

72. Identification of Nucleolar Localization Sequences in the C-Terminal Region of AssemblyActivating Protein Lauriel F. Earley,1 Xiao-Xin Sun,2 Kei Adachi,2 Mushui Dai,2 Hiroyuki Nakai.2 1 Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR; 2Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR.

Adeno-associated virus (AAV) has garnered considerable interest as a vector for gene therapy, but some of the fundamental properties of this virus, such as capsid assembly, are still not well understood. The discovery of Assembly-Activating Protein (AAP) shed some light on how AAV assembles in the nucleus when it was shown that this protein is required for the transport and assembly of the capsid subunits. AAP is produced by a second open reading frame (ORF) that overlaps the VP2/3 ORF and is known to accumulate in the S29