113 Characterization of Porcine Adeno Associated Viruses Belonging to Unique Clades Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy S46 AAV[.]
AAV VECTORS I 110 Persistent Transgene Expression from AAVHSC15 in Mice Pre-Treated with High Dose Human IVIG Laura J Smith,1 Erin A Miller,1 Taihra B Ul-Hasan,1 Etana Koplin,1 K K Wong,2 Saswati Chatterjee.1 Virology, City of Hope, Duarte, CA; 2Hematology/Stem Cell Transplantation, City of Hope, Duarte, CA Adeno-associated virus (AAV) has been shown to be an exciting and promising vector for gene therapy due to its lack of pathogenicity and the array of various serotypes with different tissue tropisms, for use in different applications We have recently isolated a panel of novel AAV capsids from CD34+ hematopoietic stem cells (HSC) isolated from healthy donors of cytokine-primed human peripheral blood stem cells Of these, AAV isolates such as AAVHSC15, which only differs from AAV9 by two residues, appear to have unique gene transfer capabilities Intramuscular injection of 1e10 particles of AAVHSC15 led to approximately 15-fold and 50-fold higher levels of long-term muscular transgene expression compared to AAV8 and AAV9, respectively This study was designed to test for the presence of pre-existing antibodies against AAVHSC15 in humans, and their potential effects on in vivo transduction We injected 12 mg of pooled human intravenous immunoglobulin (IVIG) from four separate batches into the tail veins of NOD/SCID mice Two hours later, 1E10 particles of either AAVHSC15, AAV8, or AAV9 luciferase vectors were injected into the same mice by intramuscular injection Transgene expression was assessed following quantitative serial bioluminescent imaging (BLI) from to 35 days post-injection Expression was observed in the primary injection site, the muscle, as well as secondarily in the liver This is possibly due to escape of virus particles which then trafficked to the liver At days post-injection, AAVHSC15 muscular transgene expression in mice pre-treated with IVIG was 140-fold and 3000-fold higher in the liver when compared to AAV8 or AAV9 muscular transgene expression, respectively Long term, 28 to 35 days post-injection, transgene expression in the muscle in IVIG pre-treated mice was 300-fold higher than AAV8 and 18,000fold higher than AAV9 Additionally, secondary transgene expression in the liver of IVIG pre-treated mice at day post vector transduction was 17-fold and 30-fold higher than AAV8 and AAV9, respectively Long term secondary transgene expression in the liver of AAVHSC15 mice was 50-fold higher than AAV8 and 65-fold higher than AAV9 From these results we conclude that AAVHSC15 transduces muscle more efficiently than AAV8 and AAV9; Intravenous pretreatment with pooled human IVIG reduces transduction to different degrees in all three AAV strains tested; AAVHSC15 is neutralized to a much lower extent than either AAV8 or AAV9; Specific transgene expression is readily detectable following AAVHSC15 transduction both short and long term in IVIG-treated mice Thus, AAVHSC15 may be less prone to neutralization by naturally occurring human antibodies and thus may prove to be superior for gene transfer applications transduction In this study we focused on AAV6 to identify how Cav1 can regulate AAV transduction in 3T3 MEF lines from both normal and Cav1-/- mice RESULTS: AAV6 transduction of Cav1-/- 3T3 MEFs is elevated 10 fold over wild type cells and can be reduced back to wild type levels by a peptide consisting of the Cav1 scaffolding domain Western blot results of EGFR and phospho-ERK indicate that EGFR expression and EGF-mediated signaling are elevated in Cav1-/- lines However, AAV6 transduction was unaffected by the EGFR kinase inhibitor AG1478 or by competition assays using EGF AAV6 was unable to stimulate EGFR mediated ERK phosphoralation, EGFR translocation to the perinuclear space, or compete with EGF for receptor activation, indicating that AAV6 and EGFR not directly interact in these cells Significantly increased AAV6 transduction in the presence of the dynamin inhibitor dynosore demonstrates that endocytosis via clatherin and caveolae likely occurs but may not lead to a productive infection Inhibition of AAV6 transduction by PI3K inhibitor LY294002 suggests a dynamin independent mechanism as the optimum endocytic route for AAV6 in 3T3 MEFs We demonstrate that AAV6 binding and internalization, which results in functional transduction in Cav-/- MEFs is independent of EGFR binding and clathrin and caveolin mediated endocytosis 112 Design and Characterization of New Generation Synthetic AAV Capsid Libraries Damien Marsic,1 Lakshaman Govindasamy,2 Mavis AgbandjeMcKenna,2 Sergei Zolotukhin.1 Department of 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) derived vectors are promising tools for human gene therapy because of AAV’s non-pathogenicity, low immunogenicity, episomal localization and stable transgene expression Limitations include limited specificity and susceptibility to neutralization by antibodies, both of which are determined by the capsid surface composition Therefore, capsid libraries represent an attractive approach to develop AAV variants capable of effectively targeting specific tissues or organs Our gene synthesis-based approach allows to alter only the amino acid residues that have side chains exposed to the capsid surface, as well as to restrict diversity to naturally occurring variants, thus improving compatibility with capsid structure and function and increasing the probability of generating useful variants In addition, gene synthesis allows virtual recombination between naturally-occurring sequences at the nucleotide level, producing unique combinations that can not be obtained through other methods We present here the design, assembly and characterization of several capsid libraries, as well as preliminary results of in vivo selection in mice 113 Characterization of Porcine AdenoAssociated Viruses Belonging to Unique Clades 111 AAV6 Functional Endocytosis in Mouse Embryo Fibroblast Is Independent of Clathrin and Caveolin Mechanisms and Does Not Require EGFR Activation Jonathan Peterson,1 Joseph E Rabinowitz.1 Department of Medicine, Center for Translational Medicine, Philadelphia, PA Alexander J Bello,1,2 Allan R Chand,1,2 Alberto Auricchio,3 Gary P Kobinger.1,2 Special Pathogens, Public Health Agency of Canada, Winnipeg, MB, Canada; 2Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada; 3Molecular Therapy Research Programme, Telethon Institute of Genetics and Medicine, Naples, Italy Mouse embryonic fibroblasts (MEFs) derived from Caveolin-1 homozygous knockout mice (Cav1-/-) are highly permissive to transduction of AAV serotypes 1-9 (with the exception of AAV5), in comparison to MEFS from normal littermates Cav-1 is known to negatively regulate a number of surface receptors including Epidermal growth factor receptor (EGFR) a putative co-receptor in AAV6 Despite the growing list of clinical trials involving the use of Adeno-associated viruses (AAV), only a few AAV capsid genotypes have been selected to target and treat several diseases of interest Previously observations have demonstrated the benefit of using some AAV serotypes rather than others The identification of specific AAVs capable of efficient transduction mainly in the target tissue of S46 Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy AAV VECTORS I interest could help optimize therapeutic dosing while minimising undesirable side-effects From a pool of over 20 new porcine-derived AAV isolates spanning across four different clades, this study focuses on the characterization of AAVpo2 and –po6 in parallel to AAVpo1, -po4, -po5 AAV2/5 and 2/8 were also added to the study and used as controls for comparative analysis with all other AAVs At least one porcine AAV representing each clade and/or a new serotype was produced by triple transfection generating AAV2/po1, -po2, -po4, -po5, -po6, -po7, and -po8 Of the isolates identified, production of viable particles was unsuccessful for AAVpo7 and – po8 AAVpo2 and -po6 demonstrated distinct biodistribution profile despite relatively high sequence homology While AAVpo2 showed low transduction efficiency in most tissues, AAVpo6 efficiently targeted spleen, heart, lung, kidney, ovary, muscle, and liver following intravenous administration Intramuscular or intranasal administration of AAVpo6-LacZ showed relatively high transduction efficiency in murine muscle fibers or airway epithelia respectively Transduction in the central nervous system (CNS) after intracranial delivery and detailed biodistribution following intravenous injections of porcine AAV vectors will be presented The results presented in this study will contribute to the larger effort of characterising a multitude of AAV vectors with the goal of identifying genotypes best suited for specific gene therapy applications with a maximum risk/benefit ratio 114 Limitations of Encapsidation of Recombinant scAAV2 Genomes in Different Serotype Capsids and Their Quantitation Chen Ling,1 Yuan Wang,1,2 Liujiang Song,1,3 George V Aslanidi,1 Mengqun Tan,2 Changquan Ling,3 Arun Srivastava.1 University of Florida, Gainesville; 2Second Military Medical University, Shanghai, China; 3Shenzhen Institute of Xiangya Biomedicine, Shenzhen, China We have reported that self-complementary AAV2 genomes of up to 3.3 kb can be successfully encapsidated into AAV2 serotype capsids (Human Gene Therapy, 18: 171-182, 2007) Here we report that such oversized AAV2 genomes fail to undergo packaging in other AAV serotype capsids, such as AAV1, AAV3, AAV6, and AAV8, as determined by Southern blot analyses of the vector genomes, although hybridization signals on quantitative DNA slot-blots could still be obtained DNA slot-blot hybridization and quantitative real-time PCR (qPCR) are most frequently used methods to determine the titers of recombinant AAV vector genomes, and it has been reported that qPCR assays may result in substantial differences in determining titers of scAAV vectors depending on the length between the primer sets and the terminal hairpin structure in the scAAV genomes (Human Gene Therapy, 2011 Sep 23 [Epub ahead of print]; PMID: 21846188] Although it is generally thought that the terminal hairpin does not affect methods based on hybridization, we determined that the observed hybridization signal was due to packaging of fragmented viral genomes We also observed that the vector titers determined by the standard slot-blot assays are highly dependent on the specific probe being used, with probes hybridizing to the ends of viral genomes being significantly over-represented compared with the probes hybridizing close to the middle of the viral genomes These differences among various probes were not observed using Southern blot assays This over-estimation of titer is a systemic error during scAAV genome quantification, regardless of viral genome sequences and capsid serotypes Furthermore, different serotypes capsid and modification of capsid sequence may affect the ability of packaging intact, full-length AAV genomes Although the discrepancy is modest with WT serotypes capsid and short viral genomes, the measured titer could be as much as 5-fold different with capsid mutant vectors and large genomes Using such a quantitation method, which is more reliable, we further demonstrate that site-directed mutagenesis of surface-exposed serine 459 and 663 residues on AAV3 capsids leads Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy to improved transduction efficiency of recombinant AAV3 serotype vectors, and suggest that Southern blot analyses should be performed to more accurately determine the titers of recombinant AAV vectors CL and YW contributed equally to this work 115 Bio-Engineering of Adeno-Associated Virus Serotype (AAV)-2 Capsid at Serine/Threonine Residues Improves Its Transduction Efficiency Both In Vitro and In Vivo Sangeetha Hareendran,1 Mansoor Hussain,1 Rupali A Gadkari,2 Sudha Govindarajan,2 Dwaipayan Sen,3 Balaji Balakrishnan,3 Yesupatham SathishKumar,1 Vaani Kalaivani,3 Banumathi Ramakrishna,4 Arun Srivastava,5 Alok Srivastava,1,3 Giridhara Rao Jayandharan.1,3 Centre for Stem Cell Research, Vellore, India; 2Indian Institute of Science, Bangalore, India; 3Dept of Hematology, Christian Medical College, Vellore, India; 4Dept of Pathology, Christian Medical College, Vellore, India; 5University of Florida, Gainesville The success of AAV2 mediated hepatic gene transfer in human trials for diseases such as hemophilia has been hampered by a combination of low transduction efficiency and a robust immune response directed against these vectors We hypothesized that AAV2 is targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery and modification of the serine/threonine kinase targets on AAV capsid may improve its transduction efficiency To test this, we first assessed the effect of various inhibitors of cellular serine/threonine kinases (JNK, IKK, p38 MAPK, mTOR, CaMKII) on AAV2 mediated gene expression in HeLa cells in vitro Indeed, a 4-6 fold increase in AAV2 mediated EGFP expression was seen upon pharmacological inhibition of JNK and MAPK pathways In-silico structural analysis of the AAV2 capsid enabled the identification of three protein motifs (phosphodegrons) which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases We generated point-mutations in each of the ten conserved and surface-exposed serine/threonine (S276A, S498A, S668A, T251A, T671A, T701A, T713A, T716A, T503A, T454A) residues on AAV2 capsid protein (VP3) Self-complementary (sc) AAV2-CBAp-EGFP vectors containing the wild-type (WT) and each one of the ten serine/threonine-mutant capsids were evaluated for their transduction in HeLa cells at a dose of or x103 vgs/cell The transduction efficiency of four of the serine/threonine-mutant vectors was significantly higher [6-8 fold] compared to WT- AAV2 vectors All these four residues were either within (S498) or in close proximity (S668, S276, T251) to a phosphodegron In our preliminary studies in C57BL/6 mice, a similar increase in transduction efficiency (∼ fold) was observed upon hepatic gene transfer of ∼5x1010 vgs of AAV2 mutant vectors [S276A>S498A>T251A>S668A] (Fig.1) Further on-going studies with the optimal serine/threonine-mutant scAAV2 vectors expressing human coagulation factor IX in murine models of hemophilia B, will demonstrate the feasibility of the use of these novel vectors for potential gene therapy of hemophilia B S47 ... injections of porcine AAV vectors will be presented The results presented in this study will contribute to the larger effort of characterising a multitude of AAV vectors with the goal of identifying... performed to more accurately determine the titers of recombinant AAV vectors CL and YW contributed equally to this work 115 Bio-Engineering of Adeno- Associated Virus Serotype (AAV)-2 Capsid at Serine/Threonine... American Society of Gene & Cell Therapy to improved transduction efficiency of recombinant AAV3 serotype vectors, and suggest that Southern blot analyses should be performed to more accurately