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6. Topology of Wildtype AAV Integration
AAV VECTOR BIOLOGY 6.
Topology of Wildtype AAV Integration
Karl Petri,1 Richard Gabriel,1 Leticia Agundez Cortes,2 Raffaele Fronza,1 Christine Kaeppel,1 Saira Afzal,1 Christof von Kalle,1 R. Michael Linden,2 Els Henckaerts,2 Manfred Schmidt.1 1 Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ, Heidelberg, Germany; 2Department of Infectious Diseases, King’s College London School of Medicine, London, United Kingdom. Wildtype adeno-associated virus (wtAAV) has the ability to integrate its genome site-specifically into a region on chromosome 19 called AAVS1, which is considered one of the most promising “safe harbors” for transgene insertion. This process is mediated by the viral Rep protein and involves a mechanism that causes partial duplication of the target site. However, large-scale high-resolution integration site analyses have been missing. Three reports of wtAAV integration site (IS) distribution exist that showed IS clustering near Rep binding site (RBS)-motifs. These analyses however present few IS numbers or map IS with only a low nucleotide resolution, and they have exclusively been performed in HeLa cells. To avoid the bias that might arise from HeLa cell aneuploidy and the associated high incidence of non-Rep-mediated AAV genome insertion in double strand breaks, we analyzed efficiency and specificity of Repmediated wtAAV integration in HeLa cells and diploid human dermal fibroblasts (HDF). We obtained a total of 3659 unique IS (HeLa: 2403 IS; HDF: 1256 IS) and identified 276 common IS (CIS) besides AAVS1. Notably, only few CIS were shared between HeLa cells and HDFs (Shared CIS: 23; HeLa CIS: 162; HDF CIS: 91). 16.5% of HeLa IS and 9.1% of HDF IS were located in the AAVS1 region. wtAAV IS were almost exclusively distributed at one side of the RBSmotifs, while the viral genome is mostly integrated in one orientation. We subsequently validated CIS from our IS analysis by performing electrophoretic mobility shift assay (EMSA). All analyzed secondary RBS that induced CIS formation showed binding to Rep with equal or greater affinity than the canonical AAVS1 RBS. In a second step we are analyzing the nicking ability of Rep at these off-target loci. Up until now most IS reported for wtAAV were derived from vectorgenome fusion sequences, where the viral sequence fragment was mapped near the ITRs or near the p5 region of the viral genome. Most state-of-the-art methods for IS pattern analysis rely on amplification of the vector-genome junction with primers that bind closely to the ends of viral genomes. Knowledge about potential vector integration at other sites of the wtAAV genome however remains limited. We investigated wtAAV concatemeric structures to analyze the stability of the wtAAV genome and to identify potential alternative breakpoints throughout the wtAAV genome. We found that breakpoints can occur at any position of the wtAAV genome. Apart from the ITRs and the p5 promoter there is a number of other preferred breakpoints throughout the wtAAV genome. We could also find evidence that junctions inside the wtAAV genome not only occur in wtAAV concatemeric structures but can also be observed in wtAAV integration events in the human genome. This suggests that comprehensive analysis of the wtAAV IS profile should include the analysis of viral-genome junctions throughout the whole viral genome.
7. Syntaxin 5-Dependent Retrograde Transport to the Golgi is Required for AAV Transduction
Mathieu E. Nonnenmacher,1 Thomas Weber.1 1 Cardiovascular Research Center, Mount Sinai School of Medicine, New York, NY. Intracellular transport of Adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm—allowing subsequent nuclear import and the Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
initiation of gene expression—remain to be elucidated fully. We and others previously showed that a significant proportion of viral particles are transported to the Golgi apparatus, and that Golgi disruption caused by the drug brefeldin A efficiently blocks recombinant AAV (rAAV) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of Golgi transport for rAAV transduction remains to be established definitively. Here we show that rAAV trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus strongly correlates with transduction efficiency and relies on a non-classical retrograde transport pathway that is independent of late endosomes, recycling endosomes and the retromer complex. Transport of rAAV2 from the plasma membrane to the Golgi is unaffected by siRNAmediated knock-down of syntaxins 6 and 16, which are required for retrograde trafficking of cholera and shiga toxin. Knock-down of syntaxin 5, on the other hand, strongly inhibits rAAV transduction. Similarly, rAAV transduction is blocked by the compound Retro2cycl and its derivative RN3-122, both potent inhibitors of syntaxin 5. This inhibition of transduction is observed with several AAV serotypes and in a variety of primary and immortalized cells. Together, these data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi is a highly conserved feature of AAV trafficking that is independent of the identity of the receptors used for viral attachment.
8. An Intriguing Rep and VP-Dependent Fate of Assembly-Activating Protein During AdenoAssociated Virus Capsid Formation
Lauriel F. Earley,1 Hiroyuki Nakai.2 1 Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR; 2Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR.
The increasing popularity of adeno-associated virus (AAV) as a laboratory tool and a clinical gene delivery vector has emphasized the need for efficient production of this virus with high titer yields. To explore new methods for improving AAV vector production, it is imperative to substantially understand how the viral and host cellular proteins are involved in the capsid assembly process. One of the key players in this process is the assembly-activating protein (AAP), a newly-discovered nonstructural viral protein encoded by a second open reading frame within the cap gene. Previous studies have shown that the AAP transports VP capsid proteins to the nucleolus, and promotes assembly of VP proteins into a capsid in the nucleolus. However, there still remains a substantial gap in our knowledge about the interplay between AAP, VP, Rep and cellular proteins. Here we show evidence indicating that Rep and capsid proteins downregulate AAP, which is otherwise stably expressed. To examine the role of AAP in capsid formation, we transiently transfected HEK293 cells with 1) a plasmid expressing a FLAG-tagged, codon-modified (optimized) AAP (cmAAP) derived from either AAV serotype 2, 8, or 9 (pCMVFLAG-cmAAP2, 8 or 9), 2) a plasmid expressing Rep and VP3 from AAV2, and 3) an adenovirus helper plasmid. The cells were fixed and analyzed at various time points by immunofluorescence microscopy using antibodies against fully assembled AAV2 capsids and FLAG. At the 18-hour time point, some cells expressing assembled capsids showed decreased FLAG staining compared to cells not expressing capsids. By 48 hours post-transfection, a decrease or loss of FLAG staining in the assembled capsid-producing cells became a frequent observation irrespective of the serotype from which each AAP is derived. This observation, together with western blot data, indicates that AAP levels are diminished during the viral capsid assembly process. To investigate the mechanism of the AAP downregulation in the AAV capsid assembly, HEK293 cells were transfected with pCMV-FLAG-cmAAP2 and a plasmid expressing either Rep40, Rep52, Rep68, Rep78, VP3, or GFP or a control empty plasmid; and protein levels were assessed by a western blot analysis 48 hours S3
Topology of Wildtype AAV Integration
Karl Petri,1 Richard Gabriel,1 Leticia Agundez Cortes,2 Raffaele Fronza,1 Christine Kaeppel,1 Saira Afzal,1 Christof von Kalle,1 R. Michael Linden,2 Els Henckaerts,2 Manfred Schmidt.1 1 Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ, Heidelberg, Germany; 2Department of Infectious Diseases, King’s College London School of Medicine, London, United Kingdom. Wildtype adeno-associated virus (wtAAV) has the ability to integrate its genome site-specifically into a region on chromosome 19 called AAVS1, which is considered one of the most promising “safe harbors” for transgene insertion. This process is mediated by the viral Rep protein and involves a mechanism that causes partial duplication of the target site. However, large-scale high-resolution integration site analyses have been missing. Three reports of wtAAV integration site (IS) distribution exist that showed IS clustering near Rep binding site (RBS)-motifs. These analyses however present few IS numbers or map IS with only a low nucleotide resolution, and they have exclusively been performed in HeLa cells. To avoid the bias that might arise from HeLa cell aneuploidy and the associated high incidence of non-Rep-mediated AAV genome insertion in double strand breaks, we analyzed efficiency and specificity of Repmediated wtAAV integration in HeLa cells and diploid human dermal fibroblasts (HDF). We obtained a total of 3659 unique IS (HeLa: 2403 IS; HDF: 1256 IS) and identified 276 common IS (CIS) besides AAVS1. Notably, only few CIS were shared between HeLa cells and HDFs (Shared CIS: 23; HeLa CIS: 162; HDF CIS: 91). 16.5% of HeLa IS and 9.1% of HDF IS were located in the AAVS1 region. wtAAV IS were almost exclusively distributed at one side of the RBSmotifs, while the viral genome is mostly integrated in one orientation. We subsequently validated CIS from our IS analysis by performing electrophoretic mobility shift assay (EMSA). All analyzed secondary RBS that induced CIS formation showed binding to Rep with equal or greater affinity than the canonical AAVS1 RBS. In a second step we are analyzing the nicking ability of Rep at these off-target loci. Up until now most IS reported for wtAAV were derived from vectorgenome fusion sequences, where the viral sequence fragment was mapped near the ITRs or near the p5 region of the viral genome. Most state-of-the-art methods for IS pattern analysis rely on amplification of the vector-genome junction with primers that bind closely to the ends of viral genomes. Knowledge about potential vector integration at other sites of the wtAAV genome however remains limited. We investigated wtAAV concatemeric structures to analyze the stability of the wtAAV genome and to identify potential alternative breakpoints throughout the wtAAV genome. We found that breakpoints can occur at any position of the wtAAV genome. Apart from the ITRs and the p5 promoter there is a number of other preferred breakpoints throughout the wtAAV genome. We could also find evidence that junctions inside the wtAAV genome not only occur in wtAAV concatemeric structures but can also be observed in wtAAV integration events in the human genome. This suggests that comprehensive analysis of the wtAAV IS profile should include the analysis of viral-genome junctions throughout the whole viral genome.
7. Syntaxin 5-Dependent Retrograde Transport to the Golgi is Required for AAV Transduction
Mathieu E. Nonnenmacher,1 Thomas Weber.1 1 Cardiovascular Research Center, Mount Sinai School of Medicine, New York, NY. Intracellular transport of Adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm—allowing subsequent nuclear import and the Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
initiation of gene expression—remain to be elucidated fully. We and others previously showed that a significant proportion of viral particles are transported to the Golgi apparatus, and that Golgi disruption caused by the drug brefeldin A efficiently blocks recombinant AAV (rAAV) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of Golgi transport for rAAV transduction remains to be established definitively. Here we show that rAAV trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus strongly correlates with transduction efficiency and relies on a non-classical retrograde transport pathway that is independent of late endosomes, recycling endosomes and the retromer complex. Transport of rAAV2 from the plasma membrane to the Golgi is unaffected by siRNAmediated knock-down of syntaxins 6 and 16, which are required for retrograde trafficking of cholera and shiga toxin. Knock-down of syntaxin 5, on the other hand, strongly inhibits rAAV transduction. Similarly, rAAV transduction is blocked by the compound Retro2cycl and its derivative RN3-122, both potent inhibitors of syntaxin 5. This inhibition of transduction is observed with several AAV serotypes and in a variety of primary and immortalized cells. Together, these data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi is a highly conserved feature of AAV trafficking that is independent of the identity of the receptors used for viral attachment.
8. An Intriguing Rep and VP-Dependent Fate of Assembly-Activating Protein During AdenoAssociated Virus Capsid Formation
Lauriel F. Earley,1 Hiroyuki Nakai.2 1 Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR; 2Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR.
The increasing popularity of adeno-associated virus (AAV) as a laboratory tool and a clinical gene delivery vector has emphasized the need for efficient production of this virus with high titer yields. To explore new methods for improving AAV vector production, it is imperative to substantially understand how the viral and host cellular proteins are involved in the capsid assembly process. One of the key players in this process is the assembly-activating protein (AAP), a newly-discovered nonstructural viral protein encoded by a second open reading frame within the cap gene. Previous studies have shown that the AAP transports VP capsid proteins to the nucleolus, and promotes assembly of VP proteins into a capsid in the nucleolus. However, there still remains a substantial gap in our knowledge about the interplay between AAP, VP, Rep and cellular proteins. Here we show evidence indicating that Rep and capsid proteins downregulate AAP, which is otherwise stably expressed. To examine the role of AAP in capsid formation, we transiently transfected HEK293 cells with 1) a plasmid expressing a FLAG-tagged, codon-modified (optimized) AAP (cmAAP) derived from either AAV serotype 2, 8, or 9 (pCMVFLAG-cmAAP2, 8 or 9), 2) a plasmid expressing Rep and VP3 from AAV2, and 3) an adenovirus helper plasmid. The cells were fixed and analyzed at various time points by immunofluorescence microscopy using antibodies against fully assembled AAV2 capsids and FLAG. At the 18-hour time point, some cells expressing assembled capsids showed decreased FLAG staining compared to cells not expressing capsids. By 48 hours post-transfection, a decrease or loss of FLAG staining in the assembled capsid-producing cells became a frequent observation irrespective of the serotype from which each AAP is derived. This observation, together with western blot data, indicates that AAP levels are diminished during the viral capsid assembly process. To investigate the mechanism of the AAP downregulation in the AAV capsid assembly, HEK293 cells were transfected with pCMV-FLAG-cmAAP2 and a plasmid expressing either Rep40, Rep52, Rep68, Rep78, VP3, or GFP or a control empty plasmid; and protein levels were assessed by a western blot analysis 48 hours S3