337. Improved AAV1 and AAV9 Vectors for Gene Delivery

AAV VECTORS II 335. Directed Evolution of AAV Capsid for Targeted Cancer Therapy

Damien Marsic,1 Yuan Lu,2 Chunxia Cao,3 Dane Phelan,1 Lakshmanan Govindasamy,4 Frederic J. Kaye,3 Mavis AgbandjeMcKenna,4 Steve Ghivizzani,2 Sergei Zolotukhin.1 1 Department of Pediatrics, Division of Cellular & Molecular Therapy, University of Florida, College of Medicine, Gainesville, FL; 2Department of Orthopaedics and Rehabilitation, University of Florida, College of Medicine, Gainesville, FL; 3Department of Medicine, Division of Hematology & Oncology, University of Florida, College of Medicine, Gainesville, FL; 4Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville, FL. Adeno-associated virus (AAV) derived vectors are increasingly popular tools for human gene therapy applications because of their non-pathogenicity, low immunogenicity and stable transgene expression. Their tropism is determined by the capsid surface composition, and can therefore be altered by changes to the capsid genes. Directed evolution of the AAV capsid, combining the design of a highly complex combinatorial library with an efficient in vivo selection method, allows the development of gene therapy vectors capable of infecting target cells with high specificity. This approach has promising applications in cancer therapy, through the generation of tumor-specific variants that can be used to deliver an apoptoseinducing payload with high accuracy, minimizing off-target effects. We have developed a synthetic AAV capsid library, based on an AAV2 backbone in which only surface amino acid residues were changed. The library was subjected to several rounds of selection in human tumors growing as xenografts in immuno-compromised mice. Our directed evolution strategy produced several AAV variants with high affinity for metastatic osteosarcoma (OS156), lung mucoepidermoid carcinoma (H292) and salivary gland mucoepidermoid carcinoma (H3118). For OS156, a predominant sequence (33/56 clones) was obtained after only 2 rounds of selection and had 7 amino acid substitutions compared to AAV2, in loop I and V. For H292 and H3118, predominant sequences (3/19 and 5/18 respectively) were obtained after 4 rounds of selection and had 4 (Loop IV) and 7 (loops I and V) substitutions respectively. Evaluations of transduction efficiency and specificity are under way.

336. Structures of the Retargeted AAV-DJ Vector in the Presence and Not of Receptor-Analog Sucrose Octasulfate

Qing Xie,1 Nancy Meyer,1 Thomas F. Lerch,1 Dustin McCraw,1 Michael Spillman,2 Scott Stagg,2 Michael S. Chapman.1 1 Dept. Biochemistry & Molecular Biology, Oregon Health & Science University, Portland, OR; 2Inst. Molecular Biophysics, Florida State University, Tallahassee, FL.

AAV-DJ is a chimeric vector that was created through random homologous recombination of natural adeno-associated viruses followed by selection for improved transduction of liver cells and escape from human neutralizing sera [Grimm et al. J Virol, 2008. 82: 5887-911.]. Structures of the capsid have been determined at 4.5 and 4.8 Å resolution by cryo--electron microscopy respectively for the native state and for a complex with sucrose octasulfate (SOS), a small molecule analog of the natural receptor for AAV-2, heparan sulfate proteoglycan. The resolution is sufficient to trace the entire chain, see many of the side chains and resolve sulfates of the SOS. The SOS in AAV-DJ is fully consistent with the complex of AAV-2 with a 17 kDa heparin fragment that we had viewed at 9 Å resolution [O’Donnell et al. Virology, 2009. 385: 434-443.]. The difference is that only a single disaccharide is bound per subunit, suggesting that even when a polymer is bound, binding is strongest at arginines 587  590. The interactions are direct, contrary to a remote binding mode, S130

modeled for AAV-2 using lower resolution (18 Å) data [Levy et al. J Struct Biol, 2009. 165: 146-56]. Thus there is no need for proposed conformational changes to occur immediately on receptor-binding. Furthermore, comparisons of the SOS-bound and native states of AAV-DJ at 5 Å resolution show no evidence of conformational change. Although there is high similarity in the core -barrel, the detailed structure of functionally critical variable regions on the surface had not been correctly predicted by homology modeling, ahead of the AAV-9 crystal structure [Dimattia et al. J Virol, 2012. 86: 6947-58]. With this new structure, AAV-DJ can now be compared to an in silico hybrid of all 3 parental strains. AAV-DJ differs most from AAV-2 and -8 near Ser265 where locally the sequence is inherited from AAV-9. Here, the structure is closest to AAV-9, but it is distinct due to the influence of neighboring loops inherited from AAV-2  -8. The loop in AAV-DJ would overlap with the density of an Fab’ of monoclonal antibody A20, as seen in a 9 Å cryo-EM structure in complex with AAV-2. AAV-2 is neutralized by A20, but AAV-DJ is not recognized. The inference is that changes selected in this region might be those primarily responsible for AAV-DJ’s improved resistance to human polyclonal neutralizing serum.

337. Improved AAV1 and AAV9 Vectors for Gene Delivery

Balaji Balakrishnan,1 Akshaya Krishnagopal,2 Dwaipayan Sen,1 Alok Srivastava,1,2 Giridhara r Jayandharan.1,2 1 Hematology, Christian Medical College, Vellore, India; 2Center for Stem Cell Research, Christian Medical College, Vellore, India.

Gene delivery vectors based on adeno-associated virus serotype (AAV) 1 or 9 vectors share 80% identity with other known AAV serotypes (AAV1-10, except AAV5) and have remarkable transduction efficiency into the muscle or liver. Among the available serotypes, AAV1 and AAV9 have been shown to be efficient in treating diseases like Duchenne muscular dystrophy. It is also recognized that further improvement in their transduction efficiency and strategies to evade immune response directed against the AAV1 or AAV9 capsid are desirable for their successful use in human clinical trials. Recently, we have shown that targeted modification of AAV2 vector within phosphodegrons 1-3 at its specific serine(S), threonine(T) residues circumvents the host-cellular Ser/Thr kinase phosphorylation while modification of lysine(K) residues prevents ubiquitination of the viral capsid. These modifications also improved their hepatic transduction efficiency manifold. Since a majority of these residues are conserved in AAV1 and AAV9 and in anticipation improving their transduction efficiency, we performed targeted S/T>alanine (A), K>arginine (R) mutagenesis in AAV1 and AAV9 vectors. Thus we generated 7 AAV1 mutants (S277A, S663A, S669A, S499A, S526A, K137R and T251A) and 6 AAV9 mutants (S278A, S490A, S499A, S669A, T251A and K143R) in regions equivalent to that phosphodegrons 1-3 in AAV2 and based on in silico ubiquitination prediction algorithms. The wild type (WT) and all the mutant AAV1 and AAV9 were packaged with a self-complementary chicken beta-actin promoter driven luciferase vector. Subsequently, they were evaluated for their transduction efficiency at a multiplicity of infection of 5000 vgs/cell in CHO and HeLa cells in vitro. Of the seven AAV1 mutants tested, the transgene expression from three of the vectors, AAV1-S669A>K137R>S663A was significantly higher (3-4 fold) when compared to WT-AAV1 vectors 48hrs post-infection (Fig. 1A). The best performing mutant AAV1- S669A was then evaluated in comparison to WT-AAV1 vectors, at a dose of 5 x 1010 vgs/ C57BL6 mice in vivo. As seen in Fig. 1B, the transgene expression from AAV1-S669A vectors was 2.4 fold higher when compared to the WT vector, 2 weeks post gene transfer. With AAV9 mutants, we observed a modest increase of 20-25% luciferase expression with one of the AAV9 mutant vectors, T251A in HeLa or CHO cells in vitro. These data suggest that targeted modifications of S/T/K residues in alternate serotypes of AAV vectors Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

AAV VECTORS II in general and AAV1 in particular, at positions equivalent to AAV2 phosphodegrons improve their gene transfer efficiency. The AAV1S669A vector can also be potentially used for gene therapy of diseases affecting the muscle or the liver.

339. Short Hairpin RNA Delivered by AdenoAssociated Virus Vector Efficiently Reduced Target Gene Expression in Mouse Primary Cortical Neurons

Yun Lin,1 Holly Carlisle,2 Tom Wolfe,1 Danny Ortuno,2 Trish Amarante,2 Huiren Zhao,1 Steve Wood,2 Cherylene Plewa,1 Mike Wyszynski,2 Paul Kassner,1 Ki Jeong Lee.1 1 Department of Therapeutic Discovery, Amgen, Inc, Thousand Oaks, CA; 2Department of Neuroscience, Amgen, Inc, Thousand Oaks, CA.

Figure 1: Comparison of AAV1 wild-type and mutant vectors in vitro [A] and in vivo [B]. *BB/AK contributed equally to this work.

338. Plasmid Integration within AAVS1 in AAV Producer Cell Lines

Amy Frederick,1 Yuxia Luo,1 John Martin,1 Jeffery Ardinger,1 Donna Armentano,1 Sam Wadsworth,1 Karen Vincent.1 1 Gene Therapy, Genzyme, a Sanofi Company, Framingham, MA. AAV producer cell lines represent an effective method for largescale production of AAV vectors. To create producer cell lines, a single plasmid containing three components (the vector sequence, the AAV rep and cap genes and a selectable marker gene) is stably transfected into HeLaS3 cells. As this plasmid contains both cis (Rep binding elements) and trans (Rep) elements required for site-specific integration, it might be predicted that plasmid integration would occur within the AAVS1 locus on human chromosome 19 (ch19). Plasmid integration sites within several independent producer cell lines were investigated via both Southern and FISH. In the Southern analysis, the presence of fragments detected by both rep- and AAVS1-specific probes provided evidence of integration within the AAVS1 locus for several mid- and high-producing lines. Three highproducing AAV2-SEAP lines (MW82, MW156 and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1 and a ch19 marker. Though MW278 consistently showed two sites of integration, variability in the number of plasmid integration sites was observed among cells of MW82 and MW156 (1 or 2 sites). Where heterogeneity was observed, most cells shared a common integration site while an additional site was present in a subset of the cells. This finding suggests that plasmid and/or chromosomal rearrangements had resulted in gain/loss of one integration site. FISH analysis also revealed that the status of AAVS1 in HeLaS3 cells is complex; AAVS1 signals were located on ch19-positive chromosomes as well as ch19negative chromosomes and the number of chromosomes containing AAVS1 sequences was variable (ranged from 3 to 6). Plasmid and AAVS1 signal co-localization was observed in all (MW156), none (MW278) or some (MW82) of the cells in each line. These results were consistent with the Southern data. Because not all lines (or all cells within a line) exhibited integration events within AAVS1 though each produced vector at a high level (greater than or equal to 100,000 DNase-resistant genomes/cell), it may be concluded that plasmid integration within the AAVS1 locus is not an absolute requirement for high vector yield. Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

It has been well documented that adeno-associated virus (AAV) vector can be used for delivery of short hairpin RNA (shRNA) in vivo and in vitro to reduce the target gene expression. Mouse primary cortical neurons (PCNs) have proven difficult to transfect or transduce with siRNA or lentiviral and adenoviral vectors without altering viability or morphological characteristics of the cultures. However, we have shown that mouse PCNs can be transduced with AAV vector encoding enhanced green fluorescence protein (EGFP) with higher than 90% efficiency. Here we report that AAV vectormediated shRNA delivery could achieve high efficiency of target gene knock-down in mouse PCNs. We transduced mouse PCNs with AAV-shRNAs against mouse amyloid precursor protein (muApp) and mouse beta-site App cleaving enzyme 1 (muBace1), as well as nontargeting shRNAs as negative controls. Four days after AAV-shRNA transduction, levels of messenger RNA of muAPP and muBace1 were reduced by greater than 85% compared to cells treated with nontargeting AAV-shRNA vectors. Furthermore, there was reduction of protein function as measured by secreted amyloid beta 40 (A40) in conditioned medium. The result showed that mouse PCNs transduced with muApp and muBace1 shRNAs showed reduction in A40 by 94 % and 60%, respectively. Our results indicate that AAV vectormediated shRNA delivery provides a practical method for assessing the effect of knockdown of desired targets in mouse PCNs could be a valuable tool for target validation in mouse PCNs.

340. AAV Vectors Purified from Culture Medium with a Simple and Rapid Protocol Transduce Efficiently Murine Liver, Muscle and Retina

Monica Doria,1 Antonella Ferrara,1 Alberto Auricchio.1,2 1 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy; 2Medical Genetics, Department of Pediatrics, “Federico II” University, Naples, Italy. During the production of some AAV serotypes large amount of vectors are found in the medium of producing cells. Previous protocols used tangential flow-filtration (TFF) of medium followed by iodixanol-gradient centrifugation to purify these vectors. Taking advantage of the higher purity of medium than cell-derived material as source of AAV, we tested a simple method which combines production of large culture medium volumes containing AAV from cell-stacks with medium clarification+ TFF without further time-consuming and non-scalable centrifugation. We show that yields (n=6), purity (n=2) and in vitro infectivity (n=2) of AAV vectors produced from the culture medium using this method are similar to those of vector purified from the same cell lysate using a conventional CsCl gradient ultracentrifugation-based method. In addition, we found that the murine liver, muscle and retina transduction efficiency of AAV purified from medium was similar to that of AAV purified from the same cell lysate (n=1). Considering that the purification protocol from medium we describe requires 3 hours as opposed to 60 hours of the conventional 2-round CsCl-gradient ultracentrifugation+desalting, we conclude that TFF of medium containing AAV represents a quick and scalable method to purify research-grade vectors for use in small and large animal models. S131