63 Effects of DNA Methylation on AAV Integration and Transgene Expression Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S24 AAV VECTORS I[.]
AAV VECTORS I Cytoskeletons including microtubules and actin laments have been shown to facilitate several viruses’ infection, such as adenovirus, HIV and CPV By disrupting microtubules using anti-microtubule (ATM) drugs such as Nocodazole and Vinblastine, we recently observed a several-fold enhancement of AAV2 transduction in both Hela cells and Normal Human Fibroblasts (NHF) Using Aphidicolin (APH), we were able to rule out that this enhancement of AAV2 transduction was cell cycle arrest related The above studies suggested that disruption of microtubules in the cells can facilitate AAV transduction Use of microtubule stabilizing drug (Taxol) simultaneously further support this hypothesis Previous studies have shown that proteosome inhibitor (PI) such as MG132 enhance AAV transduction Our data showed a synergistic effect on the enhancement of AAV transduction when the cells were simultaneously treated with PI and ATM drugs Given the fact that disassembly of microtubules activates RhoA, we further tested the effect of RhoA protein on the AAV2 transduction Our preliminary data showed that increased level of active RhoA proteins enhanced AAV2 transduction and knock-down of RhoA protein reduced AAV2 transduction Based on these results, we hypothesize that activation of RhoA by microtubule disassembly promotes the efciency of AAV2 intracellular processing such as subcellular trafcking and nuclear targeting We are starting to explore the intracellular processes of AAV2 and corresponding molecular basis underlying the above ndings The data collected will advance our knowledge regarding AAV biology, and will provide experimental basis for enhancing AAV2 performance using anti-cancer reagents such as ATM drugs layer and Muller glia cells The quadruple Y-F mutant (730-500-444272) exhibited the most diverse range of retinal cell transduction, with signicant transduction of all retinal cell layers, including the pigmented epithelial cell layer Conclusions: We have found that introducing multiple Y-F mutations can alter vector properties in unanticipated but potentially very useful ways Many of the mutant vectors exhibited the ability to very efciently transduce retinal cells and a subset were also able to penetrate the entire thickness of the retina to transduce retinal cells separated from the site of administration by many cell layers, a property never previously seen with conventional AAV vectors Particularly the possibility to transduce all retinal cell types in the outer retina without detachment after an intravitreal delivery represents a potentially much safer paradigm for retinal gene therapy, mostly when the genetic defect or degenerative process has left the retina fragile and prone to further damage upon retinal detachment 61 Tyrosine Mutations in the Capsid of AAV Vectors Alters Transduction Properties in the Retina Cytoskeletal proteins and Rab GTPases are involved in a spectrum of intracellular trafficking events, such as vesicle formation, trafcking, sorting, and recycling Such events are critical regulators of viral transport within host cells, as demonstrated for hepatitis B and C, dengue virus, foot-and-mouth disease virus etc Adeno-associated virus type (AAV2) is known to enter cells through clathrin-dependent receptor-mediated endocytosis, following which AAV2 capsids have been shown to travel through late (Rab7) and recycling (Rab11) endosomal compartments in a dose-dependent manner These earlier studies suggest the potential involvement of a wide variety of dynamin and Rab GTPases in the intracellular transport of AAV serotypes In order to test the aforementioned hypotheses, we have studied the effect of over-expressing wild type/dominant negative dynamin-GFP and different Rab-GFP fusion proteins on the transduction efciency and vesicular trafcking of different AAV serotypes Wild type dynamin appears to enhance transduction efciency of several AAV serotypes in vitro Further, dual color image analysis of AAV-mediated RFP gene expression in cells transfected earlier with dominant negative dynamin/Rab-GFP fusion constructs suggests that Rab5/Rab7/Rab11, but not Rab4/Rab9 might play signicant roles in vesicle transport of AAV2 Further studies outlining the effect of dynamin and Rab GTPases on transduction efciency and vesicular trafcking of other AAV serotypes will also be presented Hilda Petrs-Silva,1 Astra Dinculescu,1 Seok H Min,1 Li Zhong,1 Sergei Zolotukhin,1 Arun Srivastava,1 Alfred Lewin,1 William W Hauswirth.1 Ophthalmology, University of Florida, Gainesville, FL; 2Pediatric, University of Florida, Gainesville, FL; 3Molecular genetics and Microbiology, University of Florida, Gainesville, FL Purpose: Surface exposed capsid amino acid residues are key elements in AAV-mediated transduction since they dene not only cell tropism, but perhaps also intracellular trafcking and hence the onset and intensity of passenger gene expression Recently we showed that single tyrosine to phenylalanine (Y-F) mutations in AAV serotype can greatly improve transduction in retinal ganglion cells Seven tyrosine residues map to the capsid surface in the AAV Therefore, to more completely analyze the AAV tropism of Y-F mutant vectors, different sets of multiple Y-F mutants were made and tested in murine retina Methods: Intravitreal injections of AAV type wild type (WT) and Y-F capsid mutant vectors with identical small CBA promoters expressing green uorescent protein (GFP) were performed in adult mice GFP expression was evaluated in retinal whole mounts and in retinal sections by GFP immunohistochemistry weeks after vector treatment Results: All the AAV2 Y-F mutants have the capacity to transduce retinal ganglion cells like WT-AAV-2 Triple Y-F mutant (730-500-444) and sextuple Y-F mutant (730-704-700-500-444-272) F both showed enhanced transduction in ganglion cells upon analysis of at mount whole retinas Analysis of retinal cross sections showed a very distinct patter of transduction through the all cell layers for some mutants For double Y-F mutants (444 -730) and (704-730), pentuple Y-F mutant (730-704-500-444-272), sextuple Y-F mutant (730-704-700-500-444272) and septuple Y-F mutant (730+704+700+500+444+272+250), GFP expression was concentrated on the ganglion cell layer as seen in WT-AAV The triple Y-F mutant (730-500-444) exhibited a signicant transduction of both photoreceptors within the inner cell Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy 62 Riding Along with AAV: Mechanistic Studies on Vesicular Trafcking Pathways of AAV Serotypes Shen Shen,1,2 Xin Ming,3 Aravind Asokan,1,4 Rudy L Juliano.3 Gene Therapy Center, University of North Carolina, Chapel Hill, NC; 2Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC; 3Division of Molecular Pharmaceutics, School of Pharmacy, University of North Carolina, Chapel Hill, NC; 4Department of Genetics, University of North Carolina, Chapel Hill, NC 63 Effects of DNA Methylation on AAV Integration and Transgene Expression Diptiman Chanda,1 Jonathan A Hensel,1 Jerome T Higgs,1 Niroop Kaza,1 Rajat Grover,1 Selvarangan Ponnazhagan.1 Pathology, University of Alabama, Birmingham, AL Site-specic integration of wild-type adeno-associated virus (AAV) in the AAVS1 site of human chromosome 19 is well known Induction of site-specic integration of recombinant AAV (rAAV) into AAVS1 may result in long-term expression of therapeutic transgene(s) Previous studies have demonstrated that the leading 510 nucleotides at the 5’ end of AAVS1 sequence are key to AAV integration and containing homology to the Rep binding site (RBS) and the terminal resolution site (TRS) of AAV AAVS1 sequence analysis also indicated S25 AAV VECTORS I the presence of signicant number of CpG motifs in this region CpG islands are widely distributed in the mammalian genome and typically methylated at the cytosine bases Unmethylated CpGs are located mainly in the 5’ regulatory region of active genes, and addition of methyl group to the cytosine may lead to silencing of gene expression Hypermethylation of CpGs in the promoter region of tumor suppressor genes is common in majority of cancers and activation of such genes using demethylating agents offer better prognosis Interestingly, wild-type AAV has been known to have anti-oncogenic effects, in particular in HPV-related carcinogenesis In the present study we have examined if accumulation of methyl groups in the AAVS1 sequence has effects on AAV transgene expression and integration To this end, EBV based shuttle vector p220.2 containing the 8.2 kb AAVS1 site was in vitro methylated using CpG methylase and methyl donor S-adenosyl methionine (SAM) and transfected into C18 cells (derivative of 293 cells expressing EBNA-1) using lipofectamine reagent Control C18 cells were transfected with unmodied p220.2AAVS1 plasmid The cells were then infected with wild-type AAV and low molecular weight DNA was extracted by HIRT method 96 hours after the infection To determine the effect on integration, a nested PCR was performed using primers anking the AAV R-ITR region and AAVS1 sequence, to compare AAV integration between unmethylated and methylated AAVS1 Genomic DNA isolated from Detroit-6 cells (containing integrated AAV sequence) was used as a PCR control The PCR products were run on 1.5% agarose gel and transferred to a nylon membrane followed by Southern Hybridization using probes complementary to AAV and AAVS1 Results indicated a lack of AAV integration into methylated p220.2-AAVS1, which may have been due to inhibition of Rep binding to AAVS1-RBS due to CpG methylation To determine the effect of methylation status on AAV transgene expression, various human cancer cell lines; prostate cancer, PC3 & Du145; Colon Cancer, HT-29; breast cancer, MDA-MB-435 were tested for rAAV transduction in presence of different concentrations of demethylating agent, 5-Aza-2’-deoxycytidine Results of these studies indicated a signicant enhancement of transgene expression following demethylation of genomic DNA, suggesting the potential of altering the methylation status of target cells to achieve enhanced AAV transduction/transgene expression 64 Efcient Transduction of Mouse Liver and Other Organs Following Intravenous Administration of a Recombinant Porcine AAV Alexander J Bello,1,2 Allan R Chand,1,2 Monica Doria,3 Kaylie Tran,1 Mariacarmela Allocca,3 Alberto Auricchio,3 Gary Kobinger.1,2 Special Pathogens, Public Health Agency of Canada, Winnipeg, MB, Canada; 2Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada; 3Telethon Institute of Genetics and Medicine, Naples, Italy The discovery of Adeno-associated virus (AAV) was first described as a contaminant in Adenoviral stocks As more AAVs were discovered from natural sources, each serotype was found to have differing phenotypic properties such as tissue tropism, immunogenicity, and transduction efciency Ongoing AAV vector development includes engineering AAV capsids to incorporate properties from various serotypes which has been described as ‘Directed Evolution’ However, this strategy is limited to the number of AAVs with similar genetic sequences belonging to similar clades Divergent AAVs such as AAV5 have been found difcult to ‘shufe’ with other existing AAVs One solution to this is to isolate new AAVs with similar genetic sequences We recently described AAVpo1, a new AAV serotype isolated from porcine tissues which belongs to the same clade as AAV5 A recently isolated novel porcine AAV (AAVpo4) was found to belong to a divergent clade, but presumably with genetic sequences allowing DNA shufing with AAV5 Our primary research S26 objectives were to isolate new AAV sequences from porcine tissues, produce vectors based on these novel AAVs, and characterise them in vitro and in vivo We report here the isolation of novel AAVs based on genetic make up and alignments with existing AAVs One of them, AAVpo4, was used to generate AAV2/po4-LacZ vector by triple transfection which was evaluated in vivo Biodistribution of AAV2/po4-LacZ in intravenous tail-vein injected mice showed highly efcient transduction of the liver, with increased targeting to various organs in comparison to AAV2/5 DNA shufing with AAV5 as well as assessment of transduction efciency in the eye, muscle and lungs following subretinal, intramuscular or intranasal administration will be presented AAVpo4 can be added as a new candidate for liver gene transfer as well as for other target tissues of interests for different applications including inherited or acquired disorders and vaccine development 65 Rational Design Meets Directed Evolution for Tailoring the AAV Capsid Luk H Vandenberghe,1 Ru Xiao,1 Christie L Bell,1 Kim Van Vliet,2 Jing Lu,1 Mavis Agbandje-McKenna,2 James M Wilson.1 Gene Therapy Program, Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2Center for Structural Biology, Department of Biochemistry and Molecular Biology, University of Florida, Gainesville Vectors based on AAV are attractive vehicles for in vivo gene transfer The many available variants of the AAV capsid, the main determinant of its biology, distinguish themselves in tropism, gene transfer efciency, receptor use and interaction with the host immune system These features can either be desired or to be avoided and therefore determine the selection of capsid for a particular application As the field evolves, more hurdles for safe and efficient gene transfer are elucidated, novel therapeutic targets are dened, and the requirements of the vector to perform in specic applications are expanded Here we propose a novel methodology, named Combinatorial Domain Diversication (CDD) to engineer AAV particles by uncoupling functional determinants from a single particle structure in order to combine or transfer phenotypes from one AAV capsid to another The generation of engineered capsid variants has been achieved in either one of two ways: directed evolution or rational design Rational design strategies are limited in that they require a full understanding of the structure-function interplay, are high risk and are performed iteratively Biopanning strategies are possibly able to overcome these concerns yet suffer from signicant interference of non-functional diversity within its libraries and limitations in the available, mostly in vitro, selection systems Our methodology incorporates the available structural and functional understanding of the AAV particle while advancing the power of purifying selection based on functional transduction, rather than viral replication Practically, CDD is a PCR-based methodology in which a full length AAV capsid VP1 molecule is assembled from individual building blocks These building blocks are delineated based on functional mapping and structural information of known AAV biology CDD permits certain building blocks, such as conserved domains, to remain untouched, whereas other domains, e.g hypervariable domains, to be diversied Next to this positional control, the diversity itself can be controlled by varying individual sets of residues within domains and/or modifying their serotype origin with as primary objective maximizing functional diversity and thereby increasing the efciency of the library Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... lack of AAV integration into methylated p220.2-AAVS1, which may have been due to inhibition of Rep binding to AAVS1-RBS due to CpG methylation To determine the effect of methylation status on AAV. .. if accumulation of methyl groups in the AAVS1 sequence has effects on AAV transgene expression and integration To this end, EBV based shuttle vector p220.2 containing the 8.2 kb AAVS1 site was... region of active genes, and addition of methyl group to the cytosine may lead to silencing of gene expression Hypermethylation of CpGs in the promoter region of tumor suppressor genes is common