72 Identification of Nucleolar Localization Sequences in the C Terminal Region of Assembly Activating Protein Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Ge[.]
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-1217T 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-1217T 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-1217T, 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 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 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 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 AAV VECTOR BIOLOGY nucleoli of AAV-infected cells In studying the evolution of these overlapping genes, we investigated a highly conserved region in the AAP coding sequence of AAV serotype that is translated into KSKRSRR (Basic amino acid-rich region 1, BR1) and found it to be one of two putative nuclear localization sequences (NLS) The second signal, RRIK (BR5), was 30 amino acids downstream By creating a series of mutant AAP proteins in which the charged amino acids in these two NLSs were replaced with alanine, we were able to show that mutation of either BR1 or BR5 still allowed for accumulation of AAP in the nucleus and nucleolus, with reduced accumulation, whereas mutation of both BR1 and BR5 resulted in exclusively cytoplasmic localization of AAP However, we were unable to find any mutants that entered the nucleus but were excluded from the nucleolus This raised a possibility that a nucleolar localization sequence (NoLS) resides outside the regions we had focused on The purpose of this study was to investigate the possibility that an NoLS is contained in any or all of the three small clusters of arginines in between the two NLSs (i.e., BR2, BR3 and BR4), which had not been investigated in our previous study We speculated that these regions have the potential to be involved in nucleolar localization due to their basic nature and proximity to the two NLS’s Characterization of AAP mutants with these positively charged small clusters replaced with alanines showed a wide range of mislocalization phenotypes, but importantly, many had nuclear accumulation with nucleolar exclusion of the mutant AAP Although further studies are required, the data we have obtained so far suggests that BR2, BR3, BR4, and BR5 are involved in nucleolar localization, with BR2 and BR5 having the most influence, but none of these four BRs by itself is sufficient for being a NoLS 73 Integrins Play a Pivotal Role in Transvascular Transport and Tissue Uptake of AAV9 In Vivo Shen Shen,1 Aravind Asokan.1 Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC Our lab and others previously proposed a “click-to-fit” model of AAV infection, wherein integrins V5 and 51 function as co-receptors along with heparan sulfate to mediate cellular uptake of AAV serotype However, the role of integrins in the infectious pathway(s) of other AAV serotypes and in animal models in vivo has not been characterized In the current study, we use AAV9 as a model to investigate how integrin co-receptors affect tropism in vivo Mutation of a highly conserved integrin ligand domain (NGR) on the AAV9 VP3 subunit yielded a mutant (AAV9/NGA) that is 100-fold less efficient than parental AAV9 in transducing all major organs (brain, heart, liver, lung, spleen, and skeletal muscle) following intravenous administration in balb/c mice Pharmacokinetic analysis of AAV9/NGA demonstrated low genome copy numbers in different tissues and rapid clearance of mutant virions from the blood circulation These results suggest that the putative integrin binding NGR motif on AAV9 is critical for transport across the vasculature and tissue uptake in general Further in vitro assays attribute the deficient transduction profile of AAV9/NGA to defective cellular uptake and not cell surface binding In addition, anti-integrin antibodies and shRNA-mediated silencing helped identify specific integrin subunits involved in direct interactions with AAV9 Our results highlight for the first time (i) a significant role for integrins in AAV transduction in vivo, (ii) new integrin co-receptors for AAV9, and (iii) potential mechanisms underlying transvascular transport and systemic spread of AAV9 in mammals 74 Adeno-Associated Virus Utilizes Host Cell Nuclear Import Machinery to Enter the Nucleus Sarah C Nicolson,1,2 Richard J Samulski.1,2 Gene Therapy Center, University of North Carolina Chapel Hill, Chapel Hill, NC; 2Pharmacology, University of North Carolina Chapel Hill, Chapel Hill, NC Adeno-associated viruses (AAV) have gained momentum as promising delivery vectors for gene therapy due to their lack of pathogenicity, ability to confer long term gene expression, and broad tissue tropism However, transduction efficiency of AAV remains relatively low, necessitating the use of high viral doses in clinical applications Studies of AAV biology reveal that AAV accumulates in the perinuclear region of cells, presumably unable traffic into the nucleus The mechanism by which AAV enters the nucleus is largely unknown Understanding this key process might lend insight into the rational design of novel viral vectors or pharmacological approaches that can overcome this barrier Cellular proteins and other viruses with nuclear localization sequences (NLSs) have been shown to utilize the classical nuclear import pathway Previous studies have suggested that AAV harbors three putative NLSs; therefore, we investigated the role of this pathway on AAV2 transduction We utilized Cy5-labeled viral particles coupled with confocal microscopy to visualize viral trafficking in HeLa cells Lectin inhibition of the nuclear pore via microinjection hindered AAV2 nuclear entry We employed an siRNA approach to investigate the role of different components of the nuclear import machinery in AAV2 transduction Silencing of several proteins related to the nuclear import pathway inhibited AAV2 transduction by 50-75 percent Confocal immunofluorescence microscopy of Cy5labeled AAV2 revealed that AAV capsids colocalized with several importin proteins as early as 1h post infection and endured 9h post infection both in the perinuclear region and inside the nucleus We confirmed these results with immunopreciptation analysis revealing that capsid proteins from wild type virions could interact with import proteins but capsid proteins from VP3-only particles could not Different regions of the unique N-terminal ends of VP1 and VP2 involved with nuclear entry and their interactions with different components of the nuclear import pathway were characterized in detail To our knowledge, this is the first report describing interactions between AAV and proteins of the classical nuclear import pathway These findings may lend insight into vector optimization or pharmacological strategies designed to enhance nuclear entry and transduction potential of AAV vectors 75 Functional Roles of the AAV2 Inverted Terminal Repeat in Messenger RNA Transport and Transgene Expression Yuan Wang,1,2,3,4 Yuan Lu,3 Lina Wang,1,2,3,4 Joon-Hyung Kim,3 George V Aslanidi,3,4 Changquan Ling,1,2 Arun Srivastava,3,4,5,6,7 Chen Ling.3,4 Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China; 2Shanghai University of Traditional Chinese Medicine, Shanghai, China; Division of Cellular and Molecular Therapy, Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL; 4Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, FL; 5Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL; 6Genetics Institute, College of Medicine, University of Florida, Gainesville; 7Shands Cancer Center, College of Medicine, University of Florida, Gainesville, FL The wild-type (WT) AAV2 genome containing the fundamental DNA elements, such as a promoter, a protein-encoding region, and a polyadenylation signal (polyA), is flanked by two 145 nucleotides cis-DNA sequences, termed inverted terminal repeats (ITRs) It is S30 Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy ... transduction efficiency of AAV remains relatively low, necessitating the use of high viral doses in clinical applications Studies of AAV biology reveal that AAV accumulates in the perinuclear region. ..AAV VECTOR BIOLOGY nucleoli of AAV-infected cells In studying the evolution of these overlapping genes, we investigated a highly conserved region in the AAP coding sequence of AAV serotype... Cy5-labeled viral particles coupled with confocal microscopy to visualize viral trafficking in HeLa cells Lectin inhibition of the nuclear pore via microinjection hindered AAV2 nuclear entry We employed