Adeno-associated Virus Entry Research Articles

AAV-Containing Exosomes as a Novel Vector for Improved Gene Delivery to Lung Cancer Cells.

Lung carcinoma is the most common type of cancer and the leading cause of cancer-related death worldwide. Among the numerous therapeutic strategies for the treatment of lung cancer, adeno-associated virus (AAV)-mediated gene transfer has been demonstrated to have the potential to effectively suppress tumor growth or reverse the progression of the disease in a number of preclinical studies. AAV vector has a safety profile; however, the relatively low delivery efficacy to particular subtypes of lung carcinoma has limited its prospective clinical translation. Exosomes are nanosized extracellular vesicles secreted from nearly all known cell types. Exosomes have a membrane-enclosed structure carrying a range of cargo molecules for efficient intercellular transfer of functional entities, thus are considered as a superior vector for drug delivery. In the present study, we developed a novel strategy to produce and purify AAV-containing exosomes (AAVExo) from AAV-packaging HEK 293T cells. The cellular uptake capacity of exosomes assisted and enhanced AAV entry into cells and protected AAV from antibody neutralization, which was a serious challenge for AAV in vivo application. We tested a list of lung cancer cell lines representing non-small-cell lung cancer and small-cell lung cancer and found that AAVExo apparently improved the gene transfer efficiency compared to conventional AAV vector. Our in vitro results were supported in vivo in a lung cancer xenograft rodent model. Additionally, we evaluated the gene delivery efficiency in the presence of neutralizing antibody on lung cancer cells. The results demonstrated that AAVExo-mediated gene transfer was not impacted, while the AAV vectors were significantly blocked by the neutralizing antibody. Taken together, we established an efficient methodology for AAVExo purification, and the purified AAVExo largely enhanced gene delivery to lung cancer cells with remarkable resistance to antibody neutralization.

Structural and cellular biology of adeno-associated virus attachment and entry.

Adeno-associated virus (AAV) is a nonenveloped, ssDNA virus in the parvovirus family, which has become one of the leading candidate vectors for human gene therapy. AAV has been studied extensively to identify host cellular factors involved in infection, as well as to identify capsid variants that confer clinically favorable transduction profiles ex vivo and in vivo. Recent advances in technology have allowed for direct genetic approaches to be used to more comprehensively characterize host factors required for AAV infection and allowed for identification of a critical multi-serotype receptor, adeno-associated virus receptor (AAVR). In this chapter, we will discuss the interactions of AAV with its glycan and proteinaceous receptors and describe the host and viral components involved in AAV entry, which requires cellular attachment, endocytosis, trafficking to the trans-Golgi network and nuclear import. AAV serves as a paradigm for entry of nonenveloped viruses. Furthermore, we will discuss the potential of utilizing our increased understanding of virus-host interactions during AAV entry to develop better AAV-based therapeutics, with a focus on host factors and capsid interactions involved in in vivo tropism.

GPR108 Is a Highly Conserved AAV Entry Factor.

Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells invitro. These findings are further validated invivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. Invitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both invitro and invivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.

GluA4-Targeted AAV Vectors Deliver Genes Selectively to Interneurons while Relying on the AAV Receptor for Entry

Selective gene delivery into subtypes of interneurons remains an important challenge in vector development. Adeno-associated virus (AAV) vector particles are especially promising for intracerebral injections. For cell entry, AAV2 particles are supposed to attach to heparan-sulfate proteoglycans (HSPGs) followed by endocytosis via the AAV receptor (AAVR). Here, we assessed engineered AAV particles deficient in HSPG attachment but competent in recognizing the glutamate receptor 4 (GluA4, also known as GluRD or GRIA4) through a displayed GluA4-specific DARPin (designed ankyrin repeat protein). When injected into the mouse brain, histological evaluation revealed that in various regions, more than 90% of the transduced cells were interneurons, mainly of the parvalbumin-positive subtype. Although part of the selectivity was mediated by the DARPin, the chosen spleen focus-forming virus (SFFV) promoter had contributed as well. Further analysis revealed that the DARPin mediated selective attachment to GluA4-positive cells, whereas gene delivery required expression of AAVR. Our data suggest that cell selectivity of AAV particles can be modified rationally and efficiently through DARPins, but expression of the AAV entry receptor remains essential.

Adeno-associated virus 2 bound to its cellular receptor AAVR.

Adeno-associated virus (AAV) is a leading vector for virus-based gene therapy. The receptor for AAV (AAVR; also named KIAA0319L) was recently identified, and the precise characterization of AAV-AAVR recognition is in immediate demand. Taking advantage of a particle-filtering algorithm, we report here the cryo-electron microscopy structure of the AAV2-AAVR complex at 2.8 Å resolution. This structure reveals that of the five Ig-like polycystic kidney disease (PKD) domains in AAVR, PKD2 binds directly to the spike region of the AAV2 capsid adjacent to the icosahedral three-fold axis. Residues in strands B and E, and the BC loop of AAVR PKD2 interact directly with the AAV2 capsid. The interacting residues in the AAV2 capsid are mainly in AAV-featured variable regions. Mutagenesis of the amino acids at the AAV2-AAVR interface reduces binding activity and viral infectivity. Our findings provide insights into the biology of AAV entry with high-resolution details, providing opportunities for the development of new AAV vectors for gene therapy.

An essential receptor for adeno-associated virus infection.

Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity. They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2), and have been approved for treatment of lipoprotein lipase deficiency in Europe. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration, yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans-Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr(-/-) (also known as Au040320(-/-) and Kiaa0319l(-/-)) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection.

Prolonged Susceptibility to Antibody-mediated Neutralization for Adeno-associated Vectors Targeted to the Liver

Adeno-associated virus (AAV) vectors demonstrate highly efficient gene transfer to hepatocytes in vivo. One of the remaining obstacles to the treatment of hemophilia B patients with AAV vectors is the sensitivity of these vectors to antibody-mediated neutralization following systemic delivery. Testing and implementation of strategies to circumvent pre-existing antibodies requires knowledge of the clearance kinetics of AAV from circulation. In this study, AAV clearance kinetics were established for serotypes 2 and 8 in cell culture and in mice. Administration of pooled neutralizing serum subsequent to administration of the vector was used to define the time period in which the vector is susceptible to antibody-mediated neutralization. These experiments defined the in vivo clearance rates for both AAV2 and AAV8 vectors to be between 2 and 4 hours. In mice, portal vein and tail vein administration of each vector was tested with similar results. Cell culture studies in W162 cells established that cellular attachment and internalization both contribute to the clearance kinetics of AAV vectors. These studies characterize the in vivo clearance rates of AAV vectors for the first time and guide the development of future strategies for the avoidance of antibody-mediated AAV vector neutralization.

1084. A Screen for Host Cellular Proteins That Interact with Adeno-Associated Virus Capsid Proteins

Top of pageAbstract Vectors from adeno-associated virus (AAV) appear very promising for use in some gene therapy applications, and have even shown success in animal models. Different serotypes of AAV have been found, each having different properties. For example, it has been shown that AAV-8 uncoats more readily than AAV-2 in the nuclei of hepatocytes in mouse liver, possibly explaining the more robust AAV-8 |[ndash]|transduction efficiencies (Thomas et al., Journal of Virology, 2004, 78 (6) p. 3110|[ndash]|3224). Our goal is to better understand the mechanisms of AAV capsid uncoating in vivo. One approach is to identify cellular proteins that interact with the capsid, and perhaps establish if certain proteins interact with the capsids in a serotype-specific manner. Towards this end, we used a yeast two-hybrid screen to identify cellular proteins that interact with different domains of the AAV-8 capsid. In one of the screens, using the middle portion of the AAV capsid protein VP3 as bait against a mouse liver cDNA library, we found that over half of the library clones recovered encoded part of the same hypothetical ORF AK014478. These clones did interact with the corresponding portion of the AAV-2 capsid, indicating this interaction is not serotype-specific. However, they did not interact with the C- or N-terminal portions of the capsid protein. Homologues for this hypothetical protein have been found only in certain vertebrates and insects, and the human homologue has been shown to localize to the mitochondria. Additional clones isolated from these and related screens are currently being identified, and their association with AAV-capsid proteins are being verified by independent assays. By identifying these cellular proteins such as the one encoded by AK014478 we can better understand the mechanisms behind AAV entry and uncoating in the cell, and design better vectors for gene transfer.

Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors.

We have investigated the infectious entry pathway of adeno-associated virus (AAV) and recombinant AAV vectors by assessing AAV-mediated gene transfer and by covalently conjugating fluorophores to AAV and monitoring entry by fluorescence microscopy. We examined AAV entry in HeLa cells and in HeLa cell lines which inducibly expressed a dominant interfering mutant of dynamin. The data demonstrate that AAV internalizes rapidly by standard receptor-mediated endocytosis from clathrin-coated pits (half-time <10 min). The lysosomotropic agents ammonium chloride and bafilomycin A(1) prevent AAV-mediated gene transfer when present during the first 30 min after the onset of endocytosis, indicating that AAV escapes from early endosomes yet requires an acidic environment for penetration into the cytosol. Following release from the endosome, AAV rapidly moves to the cell nucleus and accumulates perinuclearly beginning within 30 min after the onset of endocytosis. We present data indicating that escape of AAV from the endosome and trafficking of viral particles to the nucleus are unaffected by the presence of adenovirus, the primary helper virus for a productive AAV infection. Within 2 h, viral particles could be detected within the cell nucleus, suggesting that AAV enters the nucleus prior to uncoating. Interestingly, the majority of the intracellular virus particles remain in a stable perinuclear compartment even though gene expression from nuclear AAV genomes can be detected. This suggests that the process of nuclear entry is rate limiting or that AAV entry involves multiple pathways. Nevertheless, these data establish specific points in the AAV infectious entry process and have allowed the generation of a model for future expansion to specific cell types and AAV vector analysis in vivo.