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404 Systematical Evaluation of Tropism of Different AAV Serotype Vectors in Adult and Neonatal Mice Following I V Injections Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The America[.]
AAV CAPSIDS & TRAFFICKING resulted in rapid (within one week), specific T-cell clone proliferation and generation of robust CTLs An in vitro CTL assay was set up, and specific cell lysis was determined by fluorescence-activated cell sorting (FACS) analysis of live/dead cell ratios (Fig 1B) These results suggest that high-efficiency transduction of moDCs by serinemodified AAV vectors is feasible, which supports the potential utility of these vectors for future human DC vaccine studies liver-specific human Alpha Anti-Trypsin (hAAT) promoter was packaged using normal or VP1.5 variant capsids and the vectors were purified using an optimized double cesium gradient ultracentrifugation purification method (Grimm et al, 2005; Ayuso et al, 2010) Based on titering by two methods (protein based and qPCR) to ensure comparability, the respective vectors were injected via tail vein in B57/Bl6 mice at a dose of 2.5E10 vg/mouse (five mice per dose) Both vectors resulted in indistinguishable, high level expression of hFIX at all time points tested (2, and weeks), with > 75ug/mL of circulating hFIX measured at week Animals receiving the variant capsid showed a lower anti-capsid IgG response than AAV8-injected mice These data characterize a novel capsid composition observed in recombinant AAV8, caused by a novel translational start codon in Cap, that retains normal structure and functional activity Manipulation of capsid protein VP length and composition may be exploited to further optimize recombinant vectors 404 Systematical Evaluation of Tropism of Different AAV Serotype Vectors in Adult and Neonatal Mice Following I.V Injections Fig (A) Transduction efficiency of scAAV2 vectors in primary human moDCs following pre-treatment of cells with JNK and MAPK inhibitors, or following site-directed mutagenesis of the surfaceexposed serine (S) to valine (V) at position 662 (S662V) (B) Killing curve for specific lysis by S662V-AAV2-hTERT vector-transduced moDCs against Huh7 cells 403 A Capsid Composition Variant Containing four VP Proteins Caused by a Novel Start Codon at Position 219 of the Nucleotide Sequence of AAV8 Cap Olga Zelenaia,1 Federico Mingozzi,1 Bernd Hauck,1 Shangzhen Zhou,1 Katherine A High,1,2 J Fraser Wright.1,3 Center for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia, Philadelphia, PA; 2Howard Hughes Medical Institute, Children’s Hospital of Philadelphia, Philadelphia, PA; 3Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA Recombinant adeno-associated virus serotype vectors generated by transient transfection of HEK293 cells and highly purified by combined column chromatography and gradient ultracentrifugation steps were observed to contain a VP band in addition to the canonical VP1, and 3, when assessed by SDS-PAGE / silver staining The additional band migrated at a position intermediate between the VP1 and VP2 as determined by Western blotting using monoclonal antibody B1, and was designated ‘VP1.5’ Sequencing of the packaging plasmid used to generate this vector indicated a single nucleotide difference from the expected AAV8 Cap sequence, with G replacing C at position 219 This nucleotide exchange is not predicted to change the amino acid sequence of the encoded proteins, but results in a novel CTG start codon at a position intermediate between the VP1 and VP2 start codons Estimated by band intensity using silver staining, or Coomassie blue staining (with higher loading) of SDS-PAGE gels, the anomalous VP1.5 band was present at a comparable quantity to VP1 and VP2 Dynamic light scattering analysis indicated that highly purified AAV8 particles containing VP1.5 were indistinguishable from normal recombinant AAV8, both demonstrating a radius in the range 13-15nm Examination of normal and VP1.5-containing AAV8 particles by negative staining electron microscopy indicated similar morphology; however, a slight loss of symmetry in the variant particles was noted In a side by side experiment to characterize the functional activity of the VP1.5 variant, a human coagulation FIX expression cassette driven by the S156 Li Zhong,1,3 Mengxin Li,1,2,4 Xin Mu,1,4 Christopher Boisvert,1,4 Dmitry Ratner,1,4 Qin Su,1 Ran He,1 Sheng Chen,1,2,4 Jun Xie,1,4 Yu Zhang,1,4 Hongwei Zhang,1,4 Daniel Lucking,5 Terry Flotte,3 Guangping Gao.1,4 Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA; 2Division of Hematology/Oncology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA; 3Department of Pediatrics, University of Massachusetts Medical School, Worcester, MA; 4Department of Microbiology and Physiology Systems, University of Massachusetts Medical School, Worcester, MA; 5Olympus America, Inc, Center Valley, PA Twelve adeno-associated virus (AAV) serotypes and more than 100 AAV variants have been characterized in human and non human primate (NHP) populations, which have gained much attention as potentially useful vector for gene transfer and gene therapy Due to their different capsid surface properties, these new vectors present novel tropism profile in vivo In this study, we compared the transgene expression in different organs after intravenously injecting different self-complementary (sc)AAV serotypes and variants - EGFP vectors at doses of 4x1012 GC/animal in 10-week old adult mice (AAV1, 6, 6.2, 7, 9, rh10 and rh39) and 4x1011 GC/animal in postnatal 1-day old newborn mice (AAV1, 2, 5, 6, 6.2, 7, 9, rh10, rh39 and rh43) The organs and tissues were collected, sectioned and examined under fluorescence microscopy after weeks post-injection In adult mice, we found that AAVrh10 led to dramatical GFP expression in adipose tissues, renal tubules in kidney, pancreas, keratinocytes in skin and stomachs, although it was the least efficient vector to transduce liver and also was modest in transducing cardiac muscles The AAV mediated-GFP expression is low in the lung, and is high in the skeletal muscles without much difference among all the vectors In neonatal mice, we found that the transduction profile was different The AAVmediated GFP expression was rare in pancreas and gastric intestines The transduction efficiency was low in liver and stomachs But all the vectors led to strong GFP expression in the skeletal muscles and cardiac muscles In the kidney, the AAV2 vector transduced renal tubules and glomeruli very efficiently, whereas AAVrh10 led to highly-efficient GFP expression in renal papilla In the skin, AAVrh39 and AAVrh10 led to high GFP expression in keratinocytes In the lung, AAVrh10 resulted in highly efficient GFP expression, and both alveolar and airway epithelia cells were targeted AAV7 and AAV9 also transduced the lung well, however, primarily targeted alveolar cells Overall, our data not only provided the AAV tropism information after high dose I.V administration, but also indicated some AAV vectors have unique tropism, such as AAVrh10, which targeted kidney, Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy AAV CAPSIDS & TRAFFICKING lung, skin and adipose tissue efficiently after i.v administration The result of this study should be informative for selecting different AAV vectors for different applications in human gene therapy 405 A Rationally Designed Synthetic Combinatorial AAV Capsid Library as a Gene Therapy Tool specific surface-exposed tyrosine residues on the AAV3 capsid leads to improved transduction efficiency; and (v) a specific combination of two tyrosine-mutations further improves the extent of transgene expression (Fig 1D) These optimized AAV3 vectors may be useful for the potential gene therapy of liver cancer in humans Damien Marsic,1 Sergei Zolotukhin,1 Lakshaman Govindasamy,2 Babak Moghimi,1 Daniela Hurtado.1 Pediatrics - Division of Cell & Molecular Therapy, University of Florida, Gainesville, FL; 2Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL Adeno-associated virus (AAV) is a promising vector for human gene therapy applications because of its absence of pathogenicity, low immunogenicity, episomal localization and stable transgene expression However, major limitations include limited tropism and susceptibility to neutralization by human antibodies, both of which are determined by the capsid surface composition Therefore, capsid libraries present an attractive approach to overcome those limitations Various strategies have been described to generate AAV capsid libraries, including random peptide display, error-prone PCR or DNA shuffling We describe a novel approach based on gene synthesis technology, which allows to specifically vary the amino acid residues which side chains are exposed to the capsid surface, and to limit diversity to naturally occurring variants Compatibility with capsid structure and function is therefore optimized, resulting in a higher probability of generating useful clones In addition, recombination between naturally-occurring sequences is at the nucleotide level The resulting libraries therefore contain unique combinations that could not be obtained through other methods We present here the design, assembly and characterization of such a synthetic AAV capsid library, as well as preliminary data on in vivo selection of targeted AAV vectors using rodent models 406 Development of Optimized AAV3 Serotype Vectors: Mechanism of High-Efficiency Transduction of Human Liver Cancer Cells Binbin Cheng,1,2 Chen Ling,1 Yao Dai,3 Lyudmyla G Glushakova,4 Yuan Lu,1 Samantha W Y Gee,1 Katherine E McGoogan,1 George V Aslanidi,1 Morag Park,5 Peter W Stacpoole,4 Dietmar Siemann,3 Arun Srivastava,1 Changquan Ling.2 Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL; 2Department of Traditional Chinese Medicine, Second Military Medical School, Shanghai, China; Department of Radiation Oncology, University of Florida College of Medicine, Gainesville, FL; 4Department of Medicine, University of Florida College of Medicine, Gainesville, FL; 5Department of Biochemistry, McGill University, Montreal, QC, Canada We have reported that AAV serotype vectors transduce human hepatoblastoma (HB) and hepatocellular carcinoma (HCC) cell lines as well as primary human hepatocytes extremely efficiently (Mol Genet Metabol., 98: 289-299, 2009) We have also documented that AAV3 utilizes human HGFR as a cellular co-receptor for viral entry (Hum Gene Ther., 21: 1741-1747, 2010) Here, we provide further evidence that both extracellular as well as intracellular kinase domains of hHGFR are involved in AAV3 vector entry and AAV3-mediated transgene expression We report here that: (i) AAV3 vector-mediated transduction is significantly increased in T47D cells, a human breast cancer cell line that expresses undetectable levels of the endogenous hHGFR, following stable transfection and over-expression of hHGFR (Fig 1A); (ii) the tyrosine kinase activity associated with hHGFR negatively affects the transduction efficiency of AAV3 vectors (Fig 1B,C); (iii) the use of proteasome inhibitors significantly improves AAV3 vector-mediated transduction; (iv) site-directed mutagenesis of Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy Fig 1: AAV3-mediated transgene expression in T47D and T47D+hHGFR cells (A)Cells were infected with various indicated multiplicity-of-infection of scAAV3-CBAp-EGFP vectors under identical conditions Transgene expression was determined by fluorescence microscopy 72 hrs post-infection (B) Cells were transduced with 2,000 vgs/cell of scAAV3 vectors in the absence or presence of µg/ml of hHGF Transgene expression was determined as above (C) The effect of HGFR tyrosine kinase-specific inhibitor, BMS-777607 (BMS), on AAV3-mediated transgene expression Cells were mock-treated or pretreated with BMS for hrs Whole-cell lysates were prepared and analyzed on Western blots using various indicated primary antibodies.(D) Transduction efficiency of WT and single, double, and triple tyrosine-mutant AAV3 vectors Cells were transduced with WT or various indicated Y-F mutant scAAV3-CBApEGFP vectors under identical conditions 407 Development of a Simple and SerotypeIndependent System To Assess the AAV AssemblyActivating Protein (AAP) Function Tatsuji Enoki,1,2 Kei Adachi,1 Christopher S Naitza,1 Hiroyuki Nakai.1 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Center for Cell and Gene Therapy, Takara Bio Inc., Otsu, Shiga, Japan Recombinant adeno-associated virus (rAAV) is an attractive platform for gene delivery vectors due to its non-pathogenicity and ability to mediate long-term therapeutic gene expression in vivo However, challenges remain in controlling in vivo tropism and creating next generation rAAV vectors with the most desirable properties in each application, due to the lack of sufficient knowledge about the AAV capsid biology Recently, an alternative open reading frame, ORF2, has been identified within the AAV cap gene by Kleinschmidt and his colleagues Their study has shown that assembly-activating protein (AAP) encoded by the ORF2 plays an indispensable role in AAV capsid assembly assumingly by providing a scaffold for capsid formation in nucleoli It is presumed that the coincidental functional conservation of the two frame-shifted ORFs for VP and AAP proteins encoded by a shared viral genomic region have constrained natural evolution of the AAV capsids derived from various serotypes Therefore, capsid reverse genetics studies with the AAP function being supplied in trans would be of great significance Here we report our novel and simple system to assess S157 .. .AAV CAPSIDS & TRAFFICKING lung, skin and adipose tissue efficiently after i. v administration The result of this study should be informative for selecting different AAV vectors for different. .. efficiency of AAV3 vectors (Fig 1B,C); (iii) the use of proteasome inhibitors significantly improves AAV3 vector-mediated transduction; (iv) site-directed mutagenesis of Molecular Therapy Volume... Military Medical School, Shanghai, China; Department of Radiation Oncology, University of Florida College of Medicine, Gainesville, FL; 4Department of Medicine, University of Florida College of