- Email: [email protected]
44. Unique Properties of AAV5 Assembly-Activating Protein (AAP) and Its Role in Capsid Assembly
AAV Biology acid composition on the tropism and biodistribution of various AAV serotypes. Post systemic administration in humanized mice, modest differences in transduction were noted in case of serotypes 4 and 6, without changes to endogenous tissue tropism. Interestingly, AAV9, which utilizes galactose (Gal) showed an increase in transduction across multiple organs. Further, AAV9, which tends to transduce both neuronal and glial cell populations upon intracranial injection in normal mice, showed a transduction pattern restricted to neurons in humanized mice. These results suggest AAV vector biology may differ across species at both the cellular and tissue levels. These findings could have important implications towards the design and interpretation of pre-clinical studies/data leading to clinical translation.
43. Distinct Intracellular Localization of AAPs Derived from AAV Serotypes 1-11 and Their Ability To Cross-Complement in Heterologous Capsid Assembly
John Powers,1 Lauriel Earley,2 Kei Adachi,1 Hiroyuki Nakai.1 Molecular & Medical Genetics, Oregon Health & Science University, Portland, OR; 2Microbiology & Immunology, Oregon Health & Science University, Portland, OR. 1
AAV expresses a unique non-structural viral protein that exhibits no homology to any known protein, called assembly-activating protein (AAP). It has been shown that AAP derived from AAV serotype 2 (AAP2) is a nucleolar localizing protein that transports VP proteins to the nucleolus and promotes capsid assembly. Although understanding the AAV capsid assembly process is key to developing novel AAV vectors for improved and targeted gene delivery, little is known about the roles and functions of AAPs, in particular AAPs derived from alternative serotypes. Here, by analyzing AAPs derived from 11 serotypes (AAP1 to 11), we show that strong nucleolar enrichment, the hallmark of the prototype AAP2, is not the case in other serotype AAPs, and AAP5 exhibits an unambiguous nucleolar exclusion pattern. We also show that, although all AAPs can promote assembly of many heterologous VP3 proteins into a capsid, particular VP3-AAP combinations do not effectively cross-complement. To elucidate intracellular localization of AAP1 to 11 and their functional roles, we constructed plasmids expressing VP3s or FLAGtagged codon-optimized AAPs derived from AAV1 to 11 under the control of the CMV promoter. Confocal immunofluorescence (IF) microscopy revealed that all AAPs translocate exclusively in the nucleus with strong nucleolar enrichment (e.g., AAP2), nucleolar and nucleoplasmic retention (e.g., AAP9), or clear nucleolar exclusion (e.g., AAP5). To investigate the cross-complementation abilities of AAP1 to 11 with AAV1 to 11 VP3s (11 x 11=121 combinations) in a biologically triplicated experiment, we utilized our lab’s novel AAV Barcode-Seq approach. In this approach, HEK 293 cells were transfected with a mixture of VP3, AAP, AAV2-Rep and adenovirus helper plasmids together with a unique barcode plasmid for each combination. An empty plasmid backbone was used as a no AAP control. Five days after transfection, cells and culture media were harvested and pooled for each replicate, viral DNA was extracted, and barcodes were PCR amplified and sequenced by multiplexed Illumina sequencing. This approach allowed for titering AAV vector production in ∼400 different cell culture wells in a simple manner. The data demonstrated that, although the AAPs from the 11 serotypes are capable of assembling and packaging most heterologous serotype VP3 capsids, some combinations showed a large reduction in the ability to assemble capsids compared to the native AAP + VP3 pairings. Some of the incompatible combinations, including AAP11 + AAV8 VP3, were confirmed by IF. We also applied the same Illumina-based approach to investigate whether overexpression of heterologous AAPs can enhance production of infectious AAV serotype vector particles, Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
and identified combinations showing >1.5 x enhancement, although the majority of combinations did not exhibit such an enhancing effect. Thus, this study provides the first comprehensive dataset on the biological properties of AAPs derived from 11 serotypes and significantly helps understand the functional roles of AAP in AAV vector production.
44. Unique Properties of AAV5 AssemblyActivating Protein (AAP) and Its Role in Capsid Assembly
Lauriel F. Earley,1 John Powers,2 Kei Adachi,2 Hiroyuki Nakai.2 Microbiology & Immunology, Oregon Health & Science University, Portland, OR; 2Molecular & Medical Genetics, Oregon Health & Science University, Portland, OR. 1
Adeno-associated virus (AAV) has now been used successfully as a vector for in vivo gene therapy and continues to hold great promise for future AAV-based therapies, in part because if its numerous, naturally occurring variants including the widely appreciated AAV serotypes 1 through 9. Each variant exhibits distinct biological properties; and therefore, their mechanistic basis should be thoroughly understood in each variant for successful vector development and gene therapy. In this respect, assembly-activating protein (AAP), a non-structural AAV protein that was discovered in 2010 and remains not well characterized, has gained increasing attention for its pivotal role in the AAV life cycle, particularly in promoting the assembly of VP proteins into a full capsid. AAV2 AAP (AAP2) has a joint nuclear-nucleolar localization signals (NLS-NoLS) near its C terminus and the nucleolar localization of AAP2 mediated by the NLS-NoLS is a prerequisite for effective capsid assembly, which takes place predominantly in the nucleolus and nucleostemin-positive nuclear bodies. Here we report previously unknown biological properties and functions of AAV5 AAP (AAP5) that are distinct from those of AAP2. To examine the role of AAP5 in AAV5 capsid assembly, we transiently transfected HeLa cells with a plasmid expressing a CMV-driven AAV5 VP3 and a plasmid expressing a FLAG-tagged, codon-optimized AAP5. The cells were fixed and immunostained for FLAG, VP3, and assembled AAV5 capsids using anti-FLAG, B1, and ADK5a monoclonal antibodies, respectively. Unlike AAP2, AAP5 and AAV5 capsids were nucleolar excluded, demonstrating that the AAP5 does not have an NoLS and the nucleolus is not the site for AAV5 capsid assembly. Surprisingly, regardless of the presence of AAP5, ADK5a-positive signals indicative of assembled AAV5 capsids could be easily detected at 48 hours post transfection. To support this observation, vector genome-packaged AAV5 particles could be produced at a high titer using an AAV5 helper plasmid that expresses AAV2 Rep and AAV5 VP1, 2 and 3 proteins but does not express fully functional AAP5 due to introduction of a premature stop codon, S133X, resulting in truncation of the C terminus containing the functionally important basic-rich region. We also find that, in a cross-complementation study using AAV2VP3, AAP2 and AAP5, the localization of capsids is determined by AAV serotype, not by AAP localization, indicating that host protein-capsid interactions both determine the site of assembly and are different for each serotype. Indeed, we have observed a strong colocalization of AAV2 capsids with nucleostemin that is absent with AAV5 capsids. These observations, together with the fact that AAV5 is phylogenetically the most divergent serotype, indicate that the role of AAP and host-proteins in the AAV5 life cycle may be different from that for AAV2 and potentially other serotypes. Our study provides new insights into the functional roles of AAP derived from AAV5 and shows that intracellular host-virus interactions are not necessarily the same for all AAV serotypes.
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43. Distinct Intracellular Localization of AAPs Derived from AAV Serotypes 1-11 and Their Ability To Cross-Complement in Heterologous Capsid Assembly
John Powers,1 Lauriel Earley,2 Kei Adachi,1 Hiroyuki Nakai.1 Molecular & Medical Genetics, Oregon Health & Science University, Portland, OR; 2Microbiology & Immunology, Oregon Health & Science University, Portland, OR. 1
AAV expresses a unique non-structural viral protein that exhibits no homology to any known protein, called assembly-activating protein (AAP). It has been shown that AAP derived from AAV serotype 2 (AAP2) is a nucleolar localizing protein that transports VP proteins to the nucleolus and promotes capsid assembly. Although understanding the AAV capsid assembly process is key to developing novel AAV vectors for improved and targeted gene delivery, little is known about the roles and functions of AAPs, in particular AAPs derived from alternative serotypes. Here, by analyzing AAPs derived from 11 serotypes (AAP1 to 11), we show that strong nucleolar enrichment, the hallmark of the prototype AAP2, is not the case in other serotype AAPs, and AAP5 exhibits an unambiguous nucleolar exclusion pattern. We also show that, although all AAPs can promote assembly of many heterologous VP3 proteins into a capsid, particular VP3-AAP combinations do not effectively cross-complement. To elucidate intracellular localization of AAP1 to 11 and their functional roles, we constructed plasmids expressing VP3s or FLAGtagged codon-optimized AAPs derived from AAV1 to 11 under the control of the CMV promoter. Confocal immunofluorescence (IF) microscopy revealed that all AAPs translocate exclusively in the nucleus with strong nucleolar enrichment (e.g., AAP2), nucleolar and nucleoplasmic retention (e.g., AAP9), or clear nucleolar exclusion (e.g., AAP5). To investigate the cross-complementation abilities of AAP1 to 11 with AAV1 to 11 VP3s (11 x 11=121 combinations) in a biologically triplicated experiment, we utilized our lab’s novel AAV Barcode-Seq approach. In this approach, HEK 293 cells were transfected with a mixture of VP3, AAP, AAV2-Rep and adenovirus helper plasmids together with a unique barcode plasmid for each combination. An empty plasmid backbone was used as a no AAP control. Five days after transfection, cells and culture media were harvested and pooled for each replicate, viral DNA was extracted, and barcodes were PCR amplified and sequenced by multiplexed Illumina sequencing. This approach allowed for titering AAV vector production in ∼400 different cell culture wells in a simple manner. The data demonstrated that, although the AAPs from the 11 serotypes are capable of assembling and packaging most heterologous serotype VP3 capsids, some combinations showed a large reduction in the ability to assemble capsids compared to the native AAP + VP3 pairings. Some of the incompatible combinations, including AAP11 + AAV8 VP3, were confirmed by IF. We also applied the same Illumina-based approach to investigate whether overexpression of heterologous AAPs can enhance production of infectious AAV serotype vector particles, Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
and identified combinations showing >1.5 x enhancement, although the majority of combinations did not exhibit such an enhancing effect. Thus, this study provides the first comprehensive dataset on the biological properties of AAPs derived from 11 serotypes and significantly helps understand the functional roles of AAP in AAV vector production.
44. Unique Properties of AAV5 AssemblyActivating Protein (AAP) and Its Role in Capsid Assembly
Lauriel F. Earley,1 John Powers,2 Kei Adachi,2 Hiroyuki Nakai.2 Microbiology & Immunology, Oregon Health & Science University, Portland, OR; 2Molecular & Medical Genetics, Oregon Health & Science University, Portland, OR. 1
Adeno-associated virus (AAV) has now been used successfully as a vector for in vivo gene therapy and continues to hold great promise for future AAV-based therapies, in part because if its numerous, naturally occurring variants including the widely appreciated AAV serotypes 1 through 9. Each variant exhibits distinct biological properties; and therefore, their mechanistic basis should be thoroughly understood in each variant for successful vector development and gene therapy. In this respect, assembly-activating protein (AAP), a non-structural AAV protein that was discovered in 2010 and remains not well characterized, has gained increasing attention for its pivotal role in the AAV life cycle, particularly in promoting the assembly of VP proteins into a full capsid. AAV2 AAP (AAP2) has a joint nuclear-nucleolar localization signals (NLS-NoLS) near its C terminus and the nucleolar localization of AAP2 mediated by the NLS-NoLS is a prerequisite for effective capsid assembly, which takes place predominantly in the nucleolus and nucleostemin-positive nuclear bodies. Here we report previously unknown biological properties and functions of AAV5 AAP (AAP5) that are distinct from those of AAP2. To examine the role of AAP5 in AAV5 capsid assembly, we transiently transfected HeLa cells with a plasmid expressing a CMV-driven AAV5 VP3 and a plasmid expressing a FLAG-tagged, codon-optimized AAP5. The cells were fixed and immunostained for FLAG, VP3, and assembled AAV5 capsids using anti-FLAG, B1, and ADK5a monoclonal antibodies, respectively. Unlike AAP2, AAP5 and AAV5 capsids were nucleolar excluded, demonstrating that the AAP5 does not have an NoLS and the nucleolus is not the site for AAV5 capsid assembly. Surprisingly, regardless of the presence of AAP5, ADK5a-positive signals indicative of assembled AAV5 capsids could be easily detected at 48 hours post transfection. To support this observation, vector genome-packaged AAV5 particles could be produced at a high titer using an AAV5 helper plasmid that expresses AAV2 Rep and AAV5 VP1, 2 and 3 proteins but does not express fully functional AAP5 due to introduction of a premature stop codon, S133X, resulting in truncation of the C terminus containing the functionally important basic-rich region. We also find that, in a cross-complementation study using AAV2VP3, AAP2 and AAP5, the localization of capsids is determined by AAV serotype, not by AAP localization, indicating that host protein-capsid interactions both determine the site of assembly and are different for each serotype. Indeed, we have observed a strong colocalization of AAV2 capsids with nucleostemin that is absent with AAV5 capsids. These observations, together with the fact that AAV5 is phylogenetically the most divergent serotype, indicate that the role of AAP and host-proteins in the AAV5 life cycle may be different from that for AAV2 and potentially other serotypes. Our study provides new insights into the functional roles of AAP derived from AAV5 and shows that intracellular host-virus interactions are not necessarily the same for all AAV serotypes.
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