448 Using AAV5 Vectors To Reduce the Risk of AAV Vector Mobilization Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S174 BIOLOGY OF AAV AND VECTOR[.]
BIOLOGY OF AAV AND VECTOR DEVELOPMENT II Biology of AAV and Vector Development II 446 AAV-rDNA Mediated Site-Specific Integration in Human Cell and Safety Analysis in Long-Term AAV-rDNA Treated Mice Zhongya Wang,1 Jyoti Mathur,2 Leszek Lisowski,2 Mark Kay,2 Markus Grompe.1 Oregon Stem Cell Center, Oregon Health & Sciences University, Portland, OR; 2Pediatrics and Genetics, Stanford University, Palo Alto, CA Recombinant adeno-associated virus (AAV) vectors remain mostly episomal and hence are lost in dividing cells over time We have previously reported new vectors, AAV-rDNAs, which contain ribosomal DNA homology flanking the expression cassette These vectors are able to mediate site-specific integration 10 times more frequently than regular AAV vectors in vivo In a Hereditary Tyrosinemia I (HT1) mice model, Fah-/- mice were rescued with 10-30 fold lower vector dose (1x109 vg for AAV8-rDNA-Fah) than the identical vector without rDNA homology In the hemophilia B mice model, more than 20% of hFIX levels persisted at even after 2/3 partial hepatectomy and two subleathal CCl4 treatments, which produce massive liver damage and induce liver regeneration, while the control AAV-stuffer-hFIX mice only had 1-5% of hFIX left after the same treatments As integrating vectors, AAV-rDNAs will persist in the host genome for lifetime or until the host cells die An important issue to be addressed before clinical application is if the vector will cause pathological change to the host while integrating in the genome To address this question, high doses of AAV-rDNAhFAH (up to 2x10e12 for serotype and 3x10e11 for serotype 2) were administrated to WT mice After 12 to 14 months, these mice were sacrificed for further analysis There was no detectable pathological appearance or tumors in the liver and other organs from the AAV-rDNA-hFah long term treated mice except that 2/6 had lymphoma hFah PCR was performed on genomic DNA from these lymphoma samples to see if AAV-rDNA-hFah integration was involved in the tumorgenesis process Importantly, the hFah gene was undetectable in these lymphoma samples by two independent hFah PCRs, suggesting that AAV-rDNA-hFah is not involved in the tumorgenesis Large scale analysis of the integration sites is being performed to further pursue the mechanisms and safety concerns of AAV-rDNA mediated integration Another important issue to be addressed is if AAV-rDNAs also work in human cells Two new constructs, pAAV-hrDNA-neo and pAAV-neo, were made to produce the AAV virus of serotype DJ Human fibroblasts were transduced with different MOIs of AAV-hrDNA-neo and AAV-neo After G418 selection, plates infected with AAV-hrDNA-neo had about 10 times more clones than plates infected with the same dose of AAV-neo Southern blotting and PCR were applied to detect the integration sites The presence of junction fragments in the population of G418 resistant cells derived from AAV-hrDNA-neo infection suggests a high frequency of site-specific integration These results indicate AAV-rDNAs also mediate higher frequency of integration in human cells and that a large percentage of integration events are site-specific in ribosomal DNA Fah-/-/Rag2-/-/Il2rg-/- mouse repopulated with human hepatocytes will be used to test whether AAV-hrDNA also functions in human cells in vivo Together, these results suggest that AAV-rDNAs can integrate and provide therapeutic level gene expression without any pathological damage to the host, suggesting a promising future for this new vector S174 447 Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelia Wuping Li,1 Liqun Zhang,2 Jarrod S Johnson,1 Raymond J Pickles,2 Jude R Samulski.1 Gene Therapy Center, University of North Carolina, Chapel Hill, NC; 2Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC Recombinant adeno-associated virus type (AAV-2) vectors have been used to deliver cystic fibrosis transmembrane regulator (CFTR) gene to airway of cystic fibrosis (CF) patients in clinical trials Although the vectors were found to be safe and well tolerated, no significant efficacy was achieved due to the lack of efficient transduction in human airway epithelia To improve the efficiency of AAV transduction in these cells, we applied a chimeric AAV library, generated by DNA shuffling in vitro, to ciliated human airway epithelial cells (HAE), to enrich and select for AAV variants with enhanced transduction efficiency Virions that successfully infected HAE were amplified with helper adenovirus, and the recovered chimeric AAV pool was subjected to further rounds of screening on HAE After five successive screening cycles, we have identified two novel AAV variants (dubbed chimeric HAE-1 and HAE-2), which are comprised of capsid components from serotypes AAV1, AAV6, & AAV9 The transduction efficiencies of these two novel AAV variants in airway epithelia were compared to numerous AAV serotypes (1, 2, 5, 6, & 9) While AAV scored best of the natural isolates, HAE variants were three times higher for airway transduction using reporter gene (GFP & Luc) expression Binding assays determined that this enhancement was post entry, and these observations were further collaborated by co-administration of proteasome inhibitors or anthracycline compounds that enhanced chimeric vector transduction efficiencies even higher Structural analysis determined that two key amino acids (584 and 598) of HAE-1 where responsible for increased transduction while HAE-2, the better chimeric capsid, involved 13 aa changes Finally chimeric vector carrying mini-CFTR expression cassette (4.9kb), exhibited a significant increase in forskolin-inducible chloride transport (up to 23-31%) following apical inoculations to human CF airway epithelia These studies provide a first step in testing unique AAV variants that may lead to successful clinical gene delivery in human CF airway epithelia 448 Using AAV5 Vectors To Reduce the Risk of AAV Vector Mobilization F Curtis Hewitt,1,2 Chengwen Li,1 Steven J Gray,1 Shelley Cockrell,4 Michael Washburn,2 R Jude Samulski.1,2,3 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; 2Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC; 3Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC; 4Interdisciplinary Biomedical Graduate Program, University of Pittsburgh, Pittsburgh, PA Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV2 Although these vectors are replication deficient, wild type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a TR2-flanked transgene in trans during superinfection by a helper virus, leading to “mobilization” of the vector genome from treated cells in a wt AAV capsid More importantly, it appears likely that the majority of currently characterized AAV serotypes (as well as the majority of new novel isolates) are capable of rescuing and replicating AAV2 vector templates To investigate this possibility, we flanked a GFP transgene with type and the most divergent AAV serotype, type TRs (TR2 or TR5) Consistent with AAV clades and previous reports, AAV5 specifically replicated TR5 vectors while Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy BIOLOGY OF AAV AND VECTOR DEVELOPMENT II AAV serotypes and replicated TR2-flanked vectors To exploit this specificity we created a TR5 vector production system for Cap1-5 which produced roughly equivalent titers and transduction potential compared to the TR2 vectors currently used Next, we showed that persisting, presumably integrated, rAAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2 flanked vector but not a TR5 flanked vector upon adenoviral superinfection Based on this data and the relative prevalence of wt AAV serotypes in the population, we propose TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use 449 Transcytosis of AAV8 and AAV9 across Endothelial Barrier Bo He,1 Zhenhua Yuan,1 Chunping Qiao,1 Victoria Madden,2 Dhiren Thakker,1 Juan Li,1 Xiao Xiao.1 Molecular Pharmaceutics, Eshelman Pharmacy School, University of North Carolina at Chapel Hill, Chapel Hill, NC; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC Adeno-Associated Virus (AAV) based vectors have shown great promise for human gene therapy In the previous studies of systemic gene delivery into muscle, we have demonstrated that AAV8 was the most efficient vector for crossing blood vessel barrier to attain systemic gene transfer and prolonged transduction in skeletal and cardiac muscles Other studies further revealed that AAV9 vector could also efficiently cross vascular endothelial cell barriers The potential mechanisms, however, remain elusive Elucidation of AAV transendothelial movement pathways will facilitate the improvement of AAV systemic gene delivery In this study, we hypothesized that transcytosis maybe the major pathway involved in AAV8 and AAV9 crossing the microcapilaries of the vasculature In transcytosis experiment, we employed Caco-2 cells, which are originated form intestine epithelium and commonly used to study transcellular transport of macromolecule drugs We also employed human umbilical vein endothelial cells (Huvec) as a model cell system to test the capability of AAV8, AAV9 and AAV2 penetrating cell monolayer on transwell membrane The viral DNA in the medium of basolateral chamber was measured by real-time PCR Cross-section of fixed cells was examined under transmission electron microscope (TEM), which showed direct evidence of transcytosis that AAV8 and AAV9, but not AAV2, particles underwent: 1) endocytosis with membrane pit formation on the apical side of cell monolayer; 2) intracellular vesicles trafficking in the cytoplasm, and 3) exocytosis on the basolateral side of cell monolayer The real-time PCR results demonstrated that AAV9 and AAV8 had much better transcytosis capability than AAV2 in both Huvec and Caco-2 cell models We detected 5.5x105 vector genome(vg) of AAV9, 4.4x105vg of AAV8 and 1.1x105 vg of AAV2, respectively, when 2x108 vg particles were loaded on the Huvec cell monolayer This pattern is in general agreement with the results of in vivo systemic gene delivery Together, the in vitro findings along with our previous studies suggest that AAV8 and AAV9 vectors appear to exhibit efficient transcytosis capability to cross the blood vessel endothelial barriers, which may contribute to their superiority in systemic gene delivery to the muscle, heart and other tissues and organs Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy 450 Overcoming Barriers to AAV Mediated Retinal Transduction from the Vitreous Deniz Dalkara,1,3 Kate D Kolstad,1,2 Ryan R Klimczak,1,2 Meike Visel,1 Natalia Caporale,1,2 John G Flannery,1,2 David V Schaffer.1,3 HWNI, UCB, Berkeley, CA; 2MCB, UCB, Berkeley, CA; 3ChemE, UCB, Berkeley, CA Adeno-associated viral gene therapy has shown great promise in treating inherited and acquired retinal disorders, with three clinical trials in progress this year Mutations in genes specific to photoreceptors and the retinal pigment epithelium (RPE) comprise the great majority of defects leading to inherited blindness; however, the majority of AAV serotypes, such as AAV1 and AAV5, can infect these cell types with subretinal delivery The one exception is AAV2, which can infect the inner retina from the vitreous Unfortunately, retinal detachment occurs during subretinal injection, causing cellular changes that impact the microenvironment and survival of retinal neurons and can also affect the return of vision The purpose of this study is to understand and overcome barriers to AAV mediated retinal transduction from the vitreous while minimizing any deleterious effect on retinal function To examine physical barriers to viral infection, we have fluorescently labeled virions of numerous relevant AAV serotypes (1, 2, 5, and 9) and followed their distribution in the retina after intravitreal injection AAV serotypes and show accumulation at the vitreoretinal junction while AAV8 exhibits only weak binding at the same location We did not observe accumulation of labeled AAV1 and capsids at the vitreoretinal junction, suggesting a lack of receptor binding sites for these serotypes at the inner limiting membrane (ILM) We hypothesized that the inner limiting membrane could pose a barrier to AAV-mediated gene delivery, and we therefore attempted to utilize a non-specific protease to mildly digest the ILM We were able to substantially enhance viral access to multiple retinal cell types as judged by robust GFP expression after intravitreal injection of each serotype The use of enzymatic treatment had the greatest effect on AAV5 transduction, producing the most remarkable expression profile Specifically, GFP could be detected in a variety of retinal cell types including retinal ganglion cells, Müller glia, inner nuclear layer cells, photoreceptors, and the RPE In addition, the protease treatment, at a low dose, has no deleterious effect on retinal function as demonstrated by electroretinogram and visual cortex cell population responses to visual stimuli presented to enzyme treated eyes and untreated contralateral eyes To our knowledge, this is the first time broad, efficient outer retinal cell transduction has been achieved through an intravitreal injection We anticipate this finding will help avoid the limitations, risks, and damage associated with subretinal injections currently necessary for gene therapy in the clinic 451 Comparison of Transduction Efficiency of Tyrosine-Mutant AAV Vectors in Muscle Chunping Qiao,1 Hui Zheng,1 Zhenhua Yuan,1 Jianbin Li,1 Li Zhong,2 Arun Srivastava,2 Juan Li,1 Xiao Xiao.1 Department of Molecualr Pharmaceutics, UNC Eshelman School of Pharmacy, Chapel Hill, NC; 2Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL Mutations of the surface-exposed tyrosine in AAV2 vectors led to dramatic improvement of transduction efficiency both in vitro and in vivo The increased transduction efficiency of tyrosine-mutant AAV2 vector is due to the lack of capsid ubiquitination and improved intracellular trafficking to the nucleus In this study, we were interested in studying the transduction efficiency of different serotypes of tyrosine-mutant AAV vectors in muscle The identification of high efficient AAV vectors in muscle tissue will result in better therapeutic effect for muscle-directed gene therapy We investigated three AAV serotypes, namely AAV8, and 6, with two tyrosine residue mutation for each AAV serotype Expression of luciferase reporter gene under the control of cytomegalovirus (CMV) promoter packaged by different S175 ... studying the transduction efficiency of different serotypes of tyrosine-mutant AAV vectors in muscle The identification of high efficient AAV vectors in muscle tissue will result in better therapeutic... and AAV9 vectors appear to exhibit efficient transcytosis capability to cross the blood vessel endothelial barriers, which may contribute to their superiority in systemic gene delivery to the. .. serotypes in the population, we propose TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use 449 Transcytosis of AAV8 and AAV9 across Endothelial Barrier