Adeno-associated Virus Capsid Proteins Research Articles

Isolation of Adeno-associated Viral Vectors Through a Single-step and Semi-automated Heparin Affinity Chromatography Protocol.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs. This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

Structural basis for nuclear import of bat adeno-associated virus capsid protein.

Adeno-associated viruses (AAV) are one of the world's most promising gene therapy vectors and as a result, are one of the most intensively studied viral vectors. Despite a wealth of research into these vectors, the precise characterisation of AAVs to translocate into the host cell nucleus remains unclear. Recently we identified the nuclear localization signals of an AAV porcine strain and determined its mechanism of binding to host importin proteins. To expand our understanding of diverse AAV import mechanisms we sought to determine the mechanism in which the Cap protein from a bat-infecting AAV can interact with transport receptor importins for translocation into the nucleus. Using a high-resolution crystal structure and quantitative assays, we were able to not only determine the exact region and residues of the N-terminal domain of the Cap protein which constitute the functional NLS for binding with the importin alpha two protein, but also reveal the differences in binding affinity across the importin-alpha isoforms. Collectively our results allow for a detailed molecular view of the way AAV Cap proteins interact with host proteins for localization into the cell nucleus.

Precise Manipulation of the Site and Stoichiometry of Capsid Modification Enables Optimization of Functional Adeno-Associated Virus Conjugates.

The ability to engineer adeno-associated virus (AAV) vectors for targeted transduction of specific cell types is critically important to fully harness their potential for human gene therapy. A promising approach to achieve this objective involves chemically attaching retargeting ligands onto the virus capsid. Site-specific incorporation of a bioorthogonal noncanonical amino acid (ncAA) into the AAV capsid proteins provides a particularly attractive strategy to introduce such modifications with exquisite precision. In this study, we show that using ncAA mutagenesis, it is possible to systematically alter the attachment site of a retargeting ligand (cyclic-RGD) on the AAV capsid to create diverse conjugate architectures and that the site of attachment heavily impacts the retargeting efficiency. We further demonstrate that the performance of these AAV conjugates is highly sensitive to the stoichiometry of capsid labeling (labels per capsid), with an intermediate labeling density providing optimal activity for cRGD-mediated retargeting. Finally, we developed a technology to more precisely control the number of attachment sites per AAV capsid by selectively incorporating an ncAA into the minor capsid proteins with high fidelity and efficiency, such that AAV conjugates with varying stoichiometry can be synthesized. Together, this platform provides unparalleled control over the site and stoichiometry of capsid modification, which will enable the development of next-generation AAV vectors tailored with desirable attributes.

Multiparametric domain insertional profiling of adeno-associated virus VP1

Several evolved properties of adeno-associated virus (AAV), such as broad tropism and immunogenicity in humans, are barriers to AAV-based gene therapy. Most efforts to re-engineer these properties have focused on variable regions near AAV's 3-fold protrusions and capsid protein termini. To comprehensively survey AAV capsids for engineerable hotspots, we determined multiple AAV fitness phenotypes upon insertion of six structured protein domains into the entire AAV-DJ capsid protein VP1. This is the largest and most comprehensive AAV domain insertion dataset to date. Our data revealed a surprising robustness of AAV capsids to accommodate large domain insertions. Insertion permissibility depended strongly on insertion position, domain type, and measured fitness phenotype, which clustered into contiguous structural units that we could link to distinct roles in AAV assembly, stability, and infectivity. We also identified engineerable hotspots of AAV that facilitate the covalent attachment of binding scaffolds, which may represent an alternative approach to re-direct AAV tropism.

N-glycomic profiling of capsid proteins from Adeno-Associated Virus serotypes.

Adeno-associated virus (AAV) vector has become the leading platform for gene delivery. Each serotype exhibits a different tissue tropism, immunogenicity, and in vivo transduction performance. Therefore, selecting the most suitable AAV serotype is critical for efficient gene delivery to target cells or tissues. Genome divergence among different serotypes is due mainly to the hypervariable regions of the AAV capsid proteins. However, the heterogeneity of capsid glycosylation is largely unexplored. In the present study, the N-glycosylation profiles of capsid proteins of AAV serotypes 1 to 9 have been systemically characterized and compared using a previously developed high-throughput and high-sensitivity N-glycan profiling platform. The results showed that all 9 investigated AAV serotypes were glycosylated, with comparable profiles. The most conspicuous feature was the high abundance mannosylated N-glycans, including FM3, M5, M6, M7, M8, and M9, that dominated the chromatograms within a range of 74 to 83%. Another feature was the relatively lower abundance of fucosylated and sialylated N-glycan structures, in the range of 23%-40% and 10%-17%, respectively. However, the exact N-glycan composition differed. These differences may be utilized to identify potential structural relationships between the 9 AAV serotypes. The current research lays the foundation for gaining better understanding of the importance of N-glycans on the AAV capsid surface that may play a significant role in tissue tropism, interaction with cell surface receptors, cellular uptake, and intracellular processing.

Characterization of Adeno-Associated Virus Capsid Proteins by Microflow Liquid Chromatography Coupled with Mass Spectrometry.

Adeno-associated virus (AAV) has been widely used to treat various human diseases as an important delivery vector for gene therapy due to its low immunogenicity and safety. AAV capsids proteins are comprised of three capsid viral proteins (VP; VP1, VP2, VP3). The capsid proteins play a key role in viral vector infectivity and transduction efficiency. To ensure the safety and efficacy of AAV gene therapy products, the quality of AAV vector capsid proteins during development and production should be carefully monitored and controlled. Microflow liquid chromatography coupled with mass spectrometry provides superior sensitivity and fast analysis capability. It showed significant advantages in the analysis of low- concentration and large numbers of AAV samples. The intact mass of capsid protein can be accurately determined using high-resolution mass spectrometry (MS). And MS also provides highly confident confirmation of sequence coverage and post-translational modifications site identification and quantitation. In this study, we used microflow liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the characterization of AAV2 capsid protein. we obtained nearly 100% sequence coverage of low-concentration AAV2 capsid protein (8 × 1011 GC/mL). More than 30 post-translational modifications (PTMs) sites were identified, the PTMs types included deamidation, oxidation and acetylation. From this study, the proposed microflow LC-MS/MS method provides a sensitive and high throughput approach in the characterization of AAVs and other biological products with low abundance.

Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches.

The purity of the three capsid proteins that make up recombinant adeno-associated virus (rAAV) is considered a critical quality attribute of gene therapy products. As such, there is a clear need to develop separation methods capable of rapidly characterizing these three viral proteins (VPs). In this study, the potential benefits and limitations of different electrophoretic and chromatographic methods were evaluated, including capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC), and hydrophobic interaction chromatography (HIC), for the analysis of VPs obtained from different serotypes (i.e., AAV2, AAV5, AAV8, and AAV9). CE-SDS is considered to be the reference method and provides a suitable separation of VP1-3 proteins using generic conditions and laser induced fluorescence detection. However, the characterization of post-translational modifications (i.e., phosphorylation, oxidation) remains difficult, and species identification is almost impossible due to the lack of compatibility between CE-SDS and mass spectrometry (MS). In contrast, RPLC and HILIC were found to be less generic than CE-SDS and require tedious optimization of the gradient conditions for each AAV serotype. However, these two chromatographic approaches are inherently compatible with MS, and were shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications. Finally, despite being non-denaturing, HIC offers disappointing performance for viral capsid proteins characterization.

Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity.

Post-translational modifications (PTMs) of adeno-associated virus (AAV) capsid proteins tune and regulate the AAV infective life cycle, which can impact the safety and efficacy of AAV gene therapy products. Many of these PTMs induce changes in protein charge heterogeneity, including deamidation, oxidation, glycation, and glycosylation. To characterize the charge heterogeneity of a protein, imaged capillary isoelectric focusing (icIEF) has become the gold standard method. We have previously reported an icIEF method with native fluorescence detection for denatured AAV capsid protein charge heterogeneity analysis. Although well suited for final products, the method does not have sufficient sensitivity for upstream, low-concentration AAV samples, and lacks the specificity for capsid protein detection in complex samples like cell culture supernatants and cell lysates. In contrast, the combination of icIEF, protein capture, and immunodetection affords significantly higher sensitivity and specificity, addressing the challenges of the icIEF method. By leveraging different primary antibodies, the icIEF immunoassay provides additional selectivity and affords a detailed characterization of individual AAV capsid proteins. In this study, we describe an icIEF immunoassay method for AAV analysis that is 90 times more sensitive than native fluorescence icIEF. This icIEF immunoassay provides AAV stability monitoring, where changes in individual capsid protein charge heterogeneity can be observed in response to heat stress. When applied to different AAV serotypes, this method also provides serotype identity with reproducible quantification of VP protein peak areas and apparent isoelectric point (pI). Overall, the described icIEF immunoassay is a sensitive, reproducible, quantitative, specific, and selective tool that can be used across the AAV biomanufacturing process, especially in upstream process development where complex sample types are often encountered.

Development of an icIEF assay for monitoring AAV capsid proteins and application to gene therapy products

Adeno-associated virus (AAV) gene therapy vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. Methods traditionally used in quality control labs, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), do not provide a complete understanding of capsid viral protein (VP) charge heterogeneity. In the present study, we developed simple, one-step sample preparation and charge-based VP separation using imaged capillary isoelectric focusing (icIEF) for monitoring AAV products. The robustness of the method was confirmed through a design of experiments (DoE) exercise. An orthogonal reverse-phase (RP) HPLC method coupled with mass spectrometry was developed to separate and identify charge species. Additionally, capsid point mutants demonstrate the capability of the method to resolve deamidation at a single site on the viral proteins. Finally, case studies using two different AAV serotype vectors establish the icIEF method as stability indicating and demonstrate that increases in acidic species measured by icIEF correlate with increased deamidation, which, we show, results in decreased transduction efficiency. The addition of a rapid and robust icIEF method to the AAV capsid analytical toolkit enables development and consistent manufacturing of well-characterized gene therapy products.

Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS

Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. With the skyrocketing rate of AAV research and the prevalence of many new engineered capsids being investigated in preclinical and clinical trials, capsid characterization plays a vital role in serotype confirmation and quality control. Further, peptide mapping the capsid proteins might inevitably be a future requirement by regulatory agencies since it is a critical step in good manufacturing practice (GMP) for biotherapeutic characterization. To overcome many challenges that traditional methods like SDS-PAGE and western blots carry, liquid chromatography and mass spectrometry (LC-MS) allows high resolution and sensitivity with great accuracy in characterizing the AAV capsid proteins. Our optimized LC-MS method provides quick sample preparation, a fast and high-throughput 4-min run, and high sensitivity, which allows for very efficient characterization of wild-type and engineered capsids. This study also reports the usage of LC-MS/MS peptide mapping of AAV capsid proteins to determine the most accessible lysine residues targeted by chemical modifications. Our detailed protocols are anticipated to promote the development and discovery of AAV variants with high accuracy and efficiency.

Directed evolution of adeno-associated virus for efficient gene delivery to microglia.

As the resident immune cells in the central nervous system (CNS), microglia orchestrate immune responses and dynamically sculpt neural circuits in the CNS. Microglial dysfunction and mutations of microglia-specific genes have been implicated in many diseases of the CNS. Developing effective and safe vehicles for transgene delivery into microglia will facilitate the studies of microglia biology and microglia-associated disease mechanisms. Here, we report the discovery of adeno-associated virus (AAV) variants that mediate efficient in vitro and in vivo microglial transduction via directed evolution of the AAV capsid protein. These AAV-cMG and AAV-MG variants are capable of delivering various genetic payloads into microglia with high efficiency, and enable sufficient transgene expression to support fluorescent labeling, Ca2+ and neurotransmitter imaging and genome editing in microglia in vivo. Furthermore, single-cell RNA sequencing shows that the AAV-MG variants mediate in vivo transgene delivery without inducing microglia immune activation. These AAV variants should facilitate the use of various genetically encoded sensors and effectors in the study of microglia-related biology.

Sample Preparation Matters for Peptide Mapping to Evaluate Deamidation of Adeno-Associated Virus Capsid Proteins Using Liquid Chromatography–Tandem Mass Spectrometry

Adeno-associated viral capsid proteins (AAV VP) are the major components that determine the tissue specificity and immunogenicity, and in vivo transduction performance of the vector. It was reported that asparagine deamidation of AAV capsid proteins leads to charge variants/heterogeneity and altered vector function, reduction of stability, and potency of AAV gene therapy products. Deamidation of asparagine residue is a common post-translational modification of proteins and is mostly detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based peptide mapping. However, deamidation can be spontaneously introduced during sample preparation before LC-MS/MS analysis. So far, no optimal sample preparations, instead, traditional sample preparation has been used for AAV VP peptide mapping, resulting in exaggerating the original deamidation levels. It is important to accurately monitor and provide true value of asparagine deamidation for development of AAV gene therapy products. In this study, we evaluated denaturation temperatures, digestion durations, and digestion temperatures using three different sample preparation formats for LC-MS/MS-based assessment of deamidation of AAV9 capsid proteins. The results demonstrated that the optimal sample preparation method for AAV9 VP peptide mapping minimized asparagine deamidation artifacts. Although AAV9 was used for method optimization, this study may also provide a guidance on how to control deamidation artifacts for other AAV serotypes.

Prokaryotic expression of a recombinant protein of adeno-associated virus capsid conserved regions and preparation of its polyclonal antibody

To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody. The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay. The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10. We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.

Cover Image

The cover image is based on the Research Article A novel protocol for in-depth analysis of recombinant adeno-associated virus capsid proteins using UHPLC-MS/MS by Mostafa Zarei et al., https://doi.org/10.1002/rcm.9247.

Functional roles of the membrane-associated AAV protein MAAP

With a limited coding capacity of 4.7 kb, adeno-associated virus (AAV) genome has evolved over-lapping genes to maximise the usage of its genome. An example is the recently found ORF in the cap gene, encoding membrane-associated accessory protein (MAAP), located in the same genomic region as the VP1/2 unique domain, but in a different reading frame. This 13 KDa protein, unique to the dependovirus genus, is not homologous to any known protein. Our studies confirm that MAAP translation initiates from the first CTG codon found in the VP1 ORF2. We have further observed MAAP localised in the plasma membrane, in the membranous structures in close proximity to the nucleus and to the nuclear envelope by co-transfecting with plasmids encoding the wild-type AAV (wt-AAV) genome and adenovirus (Ad) helper genes. While keeping VP1/2 protein sequence identical, both inactivation and truncation of MAAP translation affected the emergence and intracellular distribution of the AAV capsid proteins. We have demonstrated that MAAP facilitates AAV replication and has a role in controlling Ad infection. Additionally, we were able to improve virus production and capsid integrity through a C-terminal truncation of MAAP while other modifications led to increased packaging of contaminating, non-viral DNA. Our results show that MAAP plays a significant role in AAV infection, with profound implications for the production of therapeutic AAV vectors.

Fast and efficient digestion of adeno associated virus (AAV) capsid proteins for liquid chromatography mass spectrometry (LC-MS) based peptide mapping and post translational modification analysis (PTMs)

Adeno-associated virus (AAV) represent a widely used delivery mechanism for gene therapy treatments currently being developed. The size and complexity of these molecules requires the development of sensitive analytical methods for detailed product characterization. Among the quality attributes that need to be monitored, characterization of the AAV capsid protein amino acid sequences and any associated post translational modifications (PTM) present, should be performed. As commonly used for recombinant protein analysis, LC-MS based peptide mapping can provide sequence coverage and PTM information to improve product understanding and the development and deployment of the associated manufacturing processes. In the current study, we report a fast and efficient method to digest AAV5 capsid proteins in only 30 min prior to peptide mapping analysis. The performance of different proteases in digesting AAV5 was compared and the benefits of using nanoflow liquid chromatography for separation prior to high resolution mass spectrometry to obtain 100% sequence coverage are highlighted. Characterization and quantitation of PTMs on AAV5 capsid proteins when using pepsin as a single protease is reported, thereby demonstrating the potential of this method to aid with complete characterization of AAV serotypes in gene therapy development laboratories.

Characterization of Adeno-Associated Virus Capsid Proteins with Two Types of VP3-Related Components by Capillary Gel Electrophoresis and Mass Spectrometry.

Recombinant adeno-associated virus is a leading platform in human gene therapy. The adeno-associated virus (AAV) capsid is composed of three viral proteins (VPs): VP1, VP2, and VP3. To ensure the safety of AAV-based gene therapy products, the stoichiometry of VPs of AAV vector should be carefully monitored. In this study, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, capillary gel electrophoresis (CGE), and liquid chromatography-UV-mass spectrometry (LC-UV-MS) were performed to evaluate the VP components of AAV1, AAV2, and AAV6. Two types of VP3-related components, VP3 variant and VP3 fragment, were identified. The VP3 variant was the N-terminal shorter VP3, of which the translation started at M211, not at the conventional initiation codon, M203. The VP3 variant could be generated by leaky scanning of the first initiation codon of VP3. We also showed that the VP3 variant was identified in a minor peak before VP3 in CGE measurement. Meanwhile, the VP3 fragment was the C-terminal cleaved VP3, of which the sequence of VP3 ended at D590 or D626, indicating that cleavage occurred between D590 and P591, or D626 and G627. The cause of the cleavage of the DP or DG sequence was hydrolysis due to low pH of the mobile phase and high temperature of the column oven in the LC system, which was necessary to clearly separate the peak of VPs. VP3 fragments, detected only in LC-UV-MS in small amount account with less than 3% of total peak area, should be included in the quantification of VP3. Finally, the relationship of VP stoichiometry determined by the above three methods was discussed. From this study, we proposed that the VP components of AAV should be complementarily evaluated by CGE and LC-UV-MS.

Research Highlights

Research Highlights

Capillary Electrophoresis-Sodium Dodecyl Sulfate with Laser-Induced Fluorescence Detection as a Highly Sensitive and Quality Control-Friendly Method for Monitoring Adeno-Associated Virus Capsid Protein Purity.

The capsid protein purity of adeno-associated virus (AAV) is considered a critical quality attribute of AAV-based gene therapy products. However, the analytical methods currently available to monitor the viral capsid proteins, which are present in extremely low concentrations, have limited sensitivity and robustness, thus limiting their general applicability. As a result, there is an urgent need to develop robust separation methods with highly sensitive detection. In this article, we describe the first denaturation and fluorescence labeling procedure for AAV capsid proteins using the pyrylium dye Chromeo™ P503, enabling the establishment of the first capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method combined with laser-induced fluorescence (LIF) detection for AAV. Upon optimization using a quality-by-design approach, the newly developed method features a simple and robust one-step sample preparation workflow resulting in consistently labeled and denatured viral protein samples, which can subsequently be separated and quantified by CE-LIF. The method has been validated to be accurate and precise with a linear range of 50-150% of the nominal concentration of 2.0 × 1011 vector genomes per mL (vg/mL). The detection limit and quantitation limit were established to be 8.0 × 107 vg/mL (∼0.8 ng/mL) and 4.2 × 108 vg/mL (∼4 ng/mL), respectively, representing the highest sensitivity achieved for AAV capsid protein quantitation reported to date and a linear dynamic range of 8.0 × 107-3.0 × 1011 vg/mL. A comparison of the CE-SDS LIF method with existing methods, such as CE-SDS ultraviolet and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SYPRO Ruby stain, indicated that the new method has superior resolution and a significant increase in signal intensity. Capsid protein purity analysis of multiple AAV serotypes, including AAV5, scAAVrh10, AAV2, and AAV6, has been demonstrated for the first time using the same method, indicating the newly developed AAV labeling procedure and CE-LIF analysis could serve as a Quality Control-friendly platform and best-in-class analytical method for the control of AAV capsid protein purity.

SUMOylation Targets Adeno-associated Virus Capsids but Mainly Restricts Transduction by Cellular Mechanisms.

Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy due to its nonpathogenic nature, ease of production, and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the small ubiquitin-like modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study, we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated, as indicated by deletion and point mutations of VP2 or the obstruction of its N terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability than non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation; however, it reduced activation of gene transduction by shutdown of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular accumulation of AAV vectors. We also observe that the coexpression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly.IMPORTANCE Host factors within the cell are the major mode of restriction of adeno-associated virus (AAV) and keep it from fulfilling its maximum potential as a gene therapy vector. A better understanding of the intricacies of restriction would enable the engineering of better vectors. Via a genome-wide short interfering RNA screen, we identified that proteins of the small ubiquitin-like modifier (SUMO) pathway play an important role in AAV restriction. In this study, we investigate whether this restriction is targeted to the AAV directly or indirectly through host cell factors. The results indicate that both targets act in concert to restrict AAV.