key: cord-0876370-cjon7ihb
authors: Van Vliet, Kim M.; Blouin, Veronique; Brument, Nicole; Agbandje-McKenna, Mavis; Snyder, Richard O.
title: The Role of the Adeno-Associated Virus Capsid in Gene Transfer
date: 2008
journal: Drug Delivery Systems
DOI: 10.1007/978-1-59745-210-6_2
sha: 53b1df4f0b08025e9eccc0fa371619884a191b70
doc_id: 876370
cord_uid: cjon7ihb

Adeno-associated virus (AAV) is one of the most promising viral gene transfer vectors that has been shown to effect long-term gene expression and disease correction with low toxicity in animal models, and is well tolerated in human clinical trials. The surface of the AAV capsid is an essential component that is involved in cell binding, internalization, and trafficking within the targeted cell. Prior to developing a gene therapy strategy that utilizes AAV, the serotype should be carefully considered since each capsid exhibits a unique tissue tropism and transduction efficiency. Several approaches have been undertaken in an effort to target AAV vectors to specific cell types, including utilizing natural serotypes that target a desired cellular receptor, producing pseudotyped vectors, and engineering chimeric and mosaic AAV capsids. These capsid modifications are being incorporated into vector production and purification methods that provide for the ability to scale-up the manufacturing process to support human clinical trials. Protocols for small-scale and large-scale production of AAV, as well as assays to characterize the final vector product, are presented here. The structures of AAV2, AAV4, and AAV5 have been solved by X-ray crystallography or cryo-electron microscopy (cryo-EM), and provide a basis for rational vector design in developing customized capsids for specific targeting of AAV vectors. The capsid of AAV has been shown to be remarkably stable, which is a desirable characteristic for a gene therapy vector; however, recently it has been shown that the AAV serotypes exhibit differential susceptibility to proteases. The capsid fragmentation pattern when exposed to various proteases, as well as the susceptibility of the serotypes to a series of proteases, provides a unique fingerprint for each serotype that can be used for capsid identity validation. In addition to serotype identification, protease susceptibility can also be utilized to study dynamic structural changes that must occur for the AAV capsid to perform its various functions during the virus life cycle. The use of proteases for structural studies in solution complements the crystal structural studies of the virus. A generic protocol based on proteolysis for AAV serotype identification is provided here.

Adeno-associated virus (AAV) is a single-stranded DNA virus that is currently being utilized for gene therapy applications. AAV is a member of the family Parvoviridae, genus Dependovirus. Several features of AAVs that make them promising candidates as gene transfer vectors are as follows: (1) Good safety profile: the members of this genus are not associated with disease in humans. The virus is replication-deficient, and, except under special circumstances, it will not replicate or spread [1] , and AAV has been well tolerated in human clinical trials. (2) Stable long-term expression of the transgene: AAV has successfully been used to express transgenes in several tissues, including brain, muscle, liver, lung, vascular endothelium, and hematopoietic cells [2] [3] [4] [5] [6] [7] [8] [9] . (3) Ability to transduce dividing and nondividing cells [10] . (4) Episomal maintenance: wild-type AAV exhibits site-specific integration of its genome into chromosome 19; however, the majority of vector genomes appear to be maintained episomally [11] [12] [13] . Therefore, the risk of integration is minimal, compared with retroviral vectors that require integration into the host genome and have the potential to activate proto-oncogenes. (5) Low immunogenicity: unlike adenoviral vectors, a robust T cell response to the vector is not generated by AAV [14] , although humoral immune responses are generated, which may result in viral neutralization [15] . The use of other AAV serotypes may circumvent this response and allow for repeated treatments [16] . Alternatively, immunosuppressive therapy may be used during treatment with AAV to avoid the immune response [17] . (6) Physiochemical stability: AAV virions are highly stable over a wide range of pH and temperature, a feature that is important for production and purification methods for clinical-grade AAV vectors, as well as for stability of the final vector product [18] .

The genome of AAV is ∼4,700 bases of linear, single-stranded DNA. There are two genes, rep and cap, which are flanked by the inverted terminal repeats (ITRs). The ITRs consist of 145 nucleotides, which form a characteristic T-shaped hairpin. These are the only sequences required in cis for viral DNA replication and packaging. The rep gene encodes four nonstructural proteins, Rep78, Rep68, Rep52, and Rep40, which play a role in viral genome replication and transcription, as well as packaging. Rep78 and Rep68 are translated from mRNAs transcribed from the p5 promoter, while Rep52 and Rep 40 are derived from mRNAs transcribed from the p19 promoter. Alternative splicing replaces a 92 amino acid C-terminal element in Rep78 and Rep52 with a 9 amino acid element in Rep68 and Rep40 [19] . The cap gene encodes the three structural proteins of the AAV capsid, VP1 (87 kDa), VP2 (72 kDa), and VP3 (63 kDa), translated from mRNA transcribed from the p40 promoter. Differential splicing yields major and minor spliced products. VP1 is translated from the minor spliced mRNA, yielding less VP1 protein. VP2 and VP3 are both translated from the more abundant major spliced mRNA; however, VP2 is translated less efficiently because it initiates at an ACG codon, while VP3 is translated very efficiently because of a favorable Kozak context [20] . As a result, the AAV capsid proteins, which differ only in their N-terminal region, are present in the mature virion in a ratio of 1:1:10 (VP1:VP2:VP3). The singlestranded DNA genome of AAV and its products are depicted in Fig. 2 .1.

In 1980, the work of Myers and Carter provided evidence that the structural proteins for AAV assemble into empty capsids, and then the genome is packaged into these preformed capsids [21] . Pulse-chase experiments showed that empty particles rapidly accumulate (10-20 min), but that mature "full" virions accumulated more slowly (4-8 h) . They also showed that the number of empty viral particles decreases at the same rate as the number of DNA-containing mature virions increases during the course of infection. Additionally, de la Maza and Carter showed that DI particles, particles with deletions in the AAV genome, are packaged into apparently normal capsids, indicating that full-length viral genomic DNA is not required for the assembly or structural integrity of the AAV capsid [22] . In the absence of capsid assembly, ssDNA does not accumulate, further suggesting that empty capsids form first. Interactions between the preformed AAV capsid and Rep52 provides a mechanism where the nonstructural protein's helicase activity inserts the viral DNA [23] . Within the cell, capsid assembly occurs at centers within the nucleus where there is colocalization of Rep proteins, capsid proteins, and DNA [24] . Empty AAV capsids can be produced by expressing the AAV cap gene in insect cells using a baculovirus expression system [25] or in mammalian cells utilizing a recombinant adenovirus expressing the AAV capsid proteins. These systems have advanced the structural studies of the AAV capsid as a result of the large amount of empty capsids or virus-like particles (VLPs) that can be produced. Studies of AAV assembly have demonstrated that VP3 alone is sufficient to form VLPs [26] , but VP1 is required for infectivity [27] . Subsequently, it was shown that the unique N-terminus of VP1 has phospholipase A2 activity and contains a nuclear localization signal (NLS) [28] . The N-terminus of VP2 also has a NLS and may play a role in transporting VP3 into the nucleus; however, it has been shown that the N-terminus of VP2 is nonessential and that infectious virus can be produced that lack VP2 entirely [29] . Additionally, the N-terminus of VP2 has been replaced with green fluorescent protein and these capsids still assemble and maintain infectivity. This demonstrates that VP2 can tolerate peptide insertions and may be useful for incorporating peptides into the capsid for cell-specific targeting of AAV.

The adeno-associated virus capsid is ∼25 nm in diameter and is composed of 60 subunits arranged in T = 1 icosahedral symmetry. Because AAV has a number of features that make it attractive as a gene transfer vector, many studies have focused on the basic biology of the virus, including studies that address the structural characteristics. Cryo-EM or crystal structures for AAV2, AAV4, and AAV5 have been determined [30] [31] [32] [33] [34] , and the crystal structure for AAV8 is currently in progress [35] . Dependoviruses share the same subunit fold as the other members of the family Parvoviridae, including the insect densoviruses, and the autonomously replicating parvoviruses such as canine parvovirus (CPV) and minute virus of mice (MVM), even though AAV shares low capsid primary sequence identity (7-22%) [36] [37] [38] . The monomeric subunit of AAV has a conserved β-barrel core that is common in viral capsid proteins. Figure 2 .2 depicts the structure of a monomeric subunit of AAV2 as determined by Xie et al. [31] . The motif is an eight-stranded antiparallel β-barrel motif (jelly-roll β-barrel), with the β strands labeled B-I [36] . The β-strand labeled A is present in some parvoviruses, including AAV. Not surprisingly, genetic capsid mutants of AAV2 in the conserved core β-barrel, such as mut19 (βA mutation), mut20 (βB mutation), mut25 (βD mutation), and mut46 (βI mutation), are unable to assemble into capsids [40] . AAV has long loop insertions between the strands of the core β-barrel that are labeled according to the β strands that they flank. These long interstrand loops contain β ribbons and elements of secondary structure that form much of the outer surface features of the AAV capsid. The GH loop is the longest interstrand loop, and three VPs interact extensively at each three-fold axis of symmetry, forming a prominent spike. Five DE loops each form an antiparallel β ribbon at the five-fold axis of symmetry that results in a cylindrical structure surrounding a canyon-like depression. At the two-fold axis of symmetry there is a small depression, often referred to as the two-fold dimple [41, 42] . Analysis of newly discovered AAV genotypes identified a total of 12 hypervariable regions on the AAV capsid [43] . Overlaying these regions onto the X-ray crystallographic model of AAV2 showed that these regions are exposed on the capsid surface. Most of the variability is located between the G and H β strands, which are implicated in the formation of the valley and the peaks of the protruding three-fold axis of symmetry. These surface features of the virus are responsible for the interactions of the capsid with cellular receptors, as well as antibodies.

Motifs on the capsid surface are critical for attachment to the host cell, which is the first step required for infection. Different serotypes of AAV utilize unique cellular receptors. The primary receptor for AAV2 is heparin sulfate proteoglycan (HSPG). After binding the cell surface, AAV2 can utilize secondary receptors, such as αVβ5 integrin, human fibroblast growth factor receptor-1 (FGFR-1), or hepatocyte growth factor (c-met), which mediate entry [44] [45] [46] [47] . Recently, it was demonstrated that AAV2 utilizes α5β1 as an alternative co-receptor in HEK293 cells which lack αVβ5 integrin. The integrin recognition sequence, NGR, is at amino acid 511-513 within VP3, which, in assembled capsids, is located at the three-fold axis of symmetry,

The structure of a monomeric subunit of AAV2 as determined by Xie et al. [31] . This image was produced using the AAV2 coordinates from the Protein Databank, (PDB Accession no. 1lp3), with the molecular modeling software PyMOL (www.pymol.org) provided by DeLano Scientific, Palo Alto, CA [39] adjacent to R585 and R588 of AAV2, which have previously been implicated in heparin binding [48] . The NGR motif is conserved among AAV serotypes 1-11, with the exception of AAV4, AAV5, and AAV11. Similar to AAV2, AAV3H has also been reported to bind heparin, heparan sulfate, as well as FGFR-1 [49] . The receptor for AAV5 has been shown to be PDGFR [50] , and AAV5 binds to α-2-3-N-linked sialic acids [51] , while AAV4 binds to α2-3-O-linked sialic acids [52] . AAV1 and AAV6 utilize α-2-3-N-linked sialic acids, as well as α-2-6-N-linked sialic acids; however, unlike AAV4, they are unable to utilize O-linked sialic acids [53] . Domains on the surface of the AAV capsid interact with the cellular receptors found on specific cells, resulting in the range of tissue tropism seen for the various AAV serotypes.

There are 11 known serotypes of AAV with different cellular targets and antigenic properties. Recently, about 100 genomic variants of these primary AAV serotypes have been discovered [54] [55] [56] . These new variants may provide expanded tropism and unique cellular targets for AAV-mediated gene delivery. Utilizing the natural differences in tropism of the AAV serotypes provides one strategy to efficiently deliver AAV vectors to specific target tissues, and selecting the appropriate capsid serotype for the target tissue is an important consideration. The rAAV genome can be packaged into capsids of its own serotype, "isotype," or alternatively the rAAV genome can be "cross-packaged" into capsids derived from another serotype, a process called pseudotyping [57] [58] [59] . For pseudotyped vectors, the capsid gene of each serotype can be cloned with AAV2 rep, and the transgene is flanked by ITRs of AAV2. The resulting vector has a capsid of some serotype other than AAV2 and the packaged transgene flanked by the ITRs of AAV2. This provides a method to compare the transduction efficiency of various serotypes and the ability to choose the most efficient one for the specific application [58] . Table 2 .1 lists several target tissues and the comparative transduction efficiency of selected AAV serotypes studied in animal models. Efficient transduction could involve cell entry associated with receptor binding and internalization, or post-entry events such as cellular processing pathways, intracellular transport, nuclear entry, or processing of virions and vector genomes. Thomas et al. showed that AAV6 and AAV8 vectors uncoat faster and, as a result, transgene expression is seen sooner than AAV2 [90, 91] .

Repeated vector delivery might be needed in order to increase transgene expression or to deliver two genes in a coordinated fashion. For these applications, the same transgene can be packaged into capsids from different AAV serotypes for readministration and to circumvent an antibody neutralization response [92] . Differences in the amino acid composition of the viral capsid present unique epitopes to the host's immune system. Studies with rAAV2 and rAAV8 vectors AAV8-AAV9 > AAV2 [54] Pancreas AAV8 > AAV2 [91] AAV1 > AAV2 [68] Kidney AAV1 = AAV2 = AAV5 [69] AAV2 transduces tubular epithelium but not glomerular, blood vessel or interstitial cells Lung AAV5 and AAV6 [70] AAV9 > AAV5 [71] Retina AAV5 > AAV4 > AAV1 = AAV2 = AAV3 [58] Photoreceptor cells AAV5 > AAV2 [72] Hematopoietic stem cells AAV1 > AAV2-AAV5 [73] Dendritic cells AAV6 [74] (continued)

Cochlear inner ear cells AAV1 and AAV2 > AAV5 [75] Solid tumors and melanoma AAV2 > AAV1 and AAV3 [76] Glioblastoma AAV8 = AAV7 > AAV6 > AAV2 > AAV5 [77] Glioma cells AAV2 > AAV4 and AAV5 [78] Brain AAV5 > AAV1 > AAV2 [58] AAV7 > AAV8 > AAV5 > AAV2 = AAV6 [77] AAV1 and AAV5 > AAV2; AAV1 and AAV5 transduced pars reticulate; AAV1 transduced entire midbrain; AAV1 and AAV5 transduced pyramidal cell layers; AAV2 transduced the dentate gyrus [79] AAV5 > AAV4 > AAV2 [80] AAV1 > AAV2 [81, 82] AAV8 > AAV1 or AAV2 [83] AAV5 > AAV2 [84] AAV2 transduces neurons [85] AAV5 and AAV1 transduce neurons and glial cells [85] AAV4 transduces ependymal cells [85] AAV1 transduces glial cells and ependymal cells [82] Cardiac tissue AAV8 > AAV2 [64] AAV1 > AAV2 > AAV5 > AAV4 > AAV3 [86] AAV9 > AAV8 [67] AAV9 > AAV8 > AAV1 [87] AAV1 > AAV2 [88] AAV6 > AAV2

[89] [50] [51] [52] [53] [54] [55] [56] [57] [58] can elicit a cytotoxic T-lymphocyte (CTL) response [93] . In mice, the AAV8 capsid amino acids 126-140 elicited a Th1 response, whereas a Th2 response was elicited by AAV2 amino acids 475-489. The magnitude of the immune response depends on several factors, including vector dose, as well as the route and site of administration. Antibodies formed after transduction with one AAV serotype are likely to show only weak interaction with other serotypes, or may not cross-react. In animals that are transduced with AAV6 and then transduced with AAV2, delivery of the transgene was not hampered; however, animals transduced with AAV2 and then retransduced with AAV2 developed a neutralizing antibody response [92] . The region of the AAV2 capsid that has been shown to be responsible for generating a CD8 + T cell response is the RXXR motif in VP1, VP2, and VP3, which is involved in heparin binding for AAV2. This region was identified as being involved in uptake into dendritic cells, as well as the activation of capsid-specific T cells [94] . Alternative serotypes may also provide for treating cells that have heretofore been refractory to AAV infection, such as stem cells. It has been demonstrated that AAV2 is not efficient at transducing hematopoietic stem cells because of suboptimal levels of expression of the cell surface receptor for the viral vector (HSPG), impaired cellular trafficking of the vector, inefficient vector uncoating, and the lack of viral second-strand DNA synthesis [7] . However, AAV1 is able to transduce hemotopoietic stem/progenitor cells when evaluated in shortterm colony assays, as well as in long-term bone marrow transplantation assays in vivo [73] . There is also a low transduction efficiency for AAV2 in the airway epithelium of the lung due to a low abundance of HSPG on the apical surface; however, sialic acid is an abundant sugar on the apical surface of airway epithelium and pseudotyped AAV5 and AAV6 vectors have been shown to be efficient at gene transfer to murine airway epithelia in vivo [95, 96] . Encapsidated AAV6 vectors achieve transduction rates that should be sufficient for treating lung diseases, such as cystic fibrosis (CF). Recently, it was determined that the transduction efficiency of rAAV2 for the in vivo treatment of pancreatic and colon carcinoma is insufficient; however, other AAV serotypes, which have not yet been tested, may result in better transduction of this target tissue [97] . AAV2 and AAV1 bind to different cellular receptors, resulting in better transduction of muscle tissue for AAV1, and since the vectors in these studies contained the same cis elements, the AAV capsid is responsible for the differences in transduction efficiency between serotypes [98, 99] . Hauck and Xiao produced a series of capsid mutants to investigate the major regions of the AAV1 capsid responsible for the increased transduction efficiency of AAV1 in muscle tissue [100] ; the major tissue tropism determinants were located in the surface region of VP1 (amino acid 350-423). Similar functional studies of the AAV capsid will provide a better understanding of the surface features and the specific requirements for engineering AAV capsids to efficiently target specific cells.

A receptor, like HSPG, which is present on a variety of cell types poses difficulties for targeting AAV2 vectors to a specific tissue. One approach to limiting gene expression in the target tissue is to utilize a viral vector that harbors a tissue-specific promoter [4, 72, 101] . Genetic modifications of the AAV capsid also provide an alternative approach for retargeting AAV2. Regions of the capsid have been identified that will accept insertion of heterologous peptides for retargeting. Girod et al., first demonstrated that the insertion of a 14 amino acid integrin binding peptide at amino acid 587 allowed for retargeting of AAV2 [102] . To obtain a genetic map of the AAV2 capsid, Wu et al. constructed alanine scanning mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis [103] . Studies of these mutants showed that the capsid can tolerate some modifications and still maintain infectivity. A 34 amino acid IgG binding domain was inserted into rAAV2 capsids at amino acid 587, and this, coupled with antibodies against B1 integrin, CXCR4, or the c-kit receptor, mediated the targeting of these rAAV vectors to specific cell surface receptors [104] . Endothelial cell binding peptides were identified using phage display and inserted into the AAV2 capsid at amino acid 587 to transduce venous endothelial cells and significantly reduce hepatocyte transduction [105] . Recently, it was demonstrated that AAV2 could be retargeted to the heart utilizing a capsid with mutations at amino acids 484 and 585 to eliminate heparin binding, which is required for AAV2 to infect the liver [106] . Perabo et al. also demonstrated that insertion at amino acid 587 with peptides that confer a net negative charge will ablate binding to HSPG, and this correlated with liver and spleen detargeting for AAV2. Insertion of peptides between 585 and 588 can either cause spatial separation or sterically block heparin binding of AAV2 (using bulky amino acids), and the insertion of positively charged peptides reconstitutes the ability to bind HSPG [107] . This provides a strategy to improve AAV targeting in other tissues. For retargeting AAV to tumor cells, an RGD-4C motif can be inserted at amino acids 520 and 584, or 588 to confer a novel tropism and eliminate heparin binding. The RGD motif binds the cellular integrin receptor, which is expressed on tumor cells, including ovarian cancer cells, but these cells express only low levels of HSPG and are nonpermissive for AAV2 transduction [108, 109] .

Introduction of a peptide into the AAV2 capsid is a viable strategy for retargeting AAV2 to additional cell types; however, modifications in the AAV2 capsid often result in a reduction in vector yields, especially if the insertion is large. The unique N-terminal region of VP2 allows for peptide insertions without a loss of titer, when VP1 and VP3 are supplied in trans. AAV2 mosaics that have ligand insertions in a subset of VP1, VP2, and VP3 molecules result in increased vector yields and transducing titers, compared with viruses that carry the insertion in all 60 capsid protein subunits [110] . Insertion of an HA epitope (YPVDVPDYA) at amino acid 522 or 533 of VP1 results in noninfectious particles [103] ; however, the inclusion of wildtype capsid proteins can restore viral infectivity. Ried et al. [104] inserted an immunoglobulin-binding fragment of protein A (Z34C) that resulted in at least a tenfold reduction in particle titers, most likely due to its large size (34 amino acids). Compared with wild-type virus, infectious titers were 4 orders of magnitude lower. Gigout et al. generated mosaic viruses that contained between 25 and 75% of Z34C capsid proteins, with the rest of the capsid being composed of wild-type subunits to produce AAV2 mosaics that were infectious. Compared with wild-type virus, the Z34C mosaics showed up to a tenfold increase in titer, and those containing 25% Z34C capsid protein (an average of 15 subunits per capsid) were 4-5 orders of magnitude more infectious than all mutant viruses. By mutating R585 and R588 to alanine, they were able to eliminate HSPG binding and then the insertion of Z34C resulted in retargeting of AAV2 [110] . This work demonstrated for the first time that a combined approach of generating AAV2 mosaics to alter tropism and mutating two residues to reduce HSPG binding could be used to retarget AAV2 to specific cell types. For AAV1, incorporating an RGD4C motif in VP1 at amino acid 590 enables targeting to integrin receptors which are present on vascular endothelial cells [111] . It is also possible to incorporate a small biotin acceptor peptide (BAP) in this position for the purpose of metabolically biotinylating the AAV1 capsid for purification using a commercially available avidin affinity column. Mosaics with both of these modifications have been generated, which enable retargeting of the AAV1 capsid, and simplify purification.

Another strategy to broaden tissue tropism is to generate AAV with mixed or chimeric capsids [112] . This is accomplished by supplying the capsid gene from two distinct AAV serotypes during production. By varying the ratio of the two capsid genes, the resulting mixed virus may exhibit altered tropism. Rabinowitz et al. showed that mixed capsids of AAV2/5 at a ratio of 3:1 resulted in a loss of heparin binding. The ability of these virions to transduce HeLa cells, which have high levels of heparin sulfate, decreased, while the ability to transduce the heparin-sulfatedeficient cell line CHO pgsD increased from 2% to > 30% as the composition of the virion changed from AAV2-like to AAV5-like. Chimeras also may show combined tropism of both serotypes, broadening the tropism for these virions. Kohlbrenner et al. utilized chimeric capsids to improve infectivity when expressing AAV5 and AAV8 in insect cells [113] . It was determined that AAV5 and AAV8 vectors generated using the baculovirus system had low infectivity, which was due to insufficient phospholipase A2 activity. Substituting the entire VP1 protein from AAV2 in chimeric AAV5 or AAV8 capsids resulted in increased phospholipase activity levels and enhanced transduction. Generating chimeras also provides a method to functionally define structural relatedness for newly discovered serotypes. The ability to generate stable chimeric capsids suggests that the subunits from these different serotypes are structurally compatible. Conversely, AAV2 when mixed with AAV4 inefficiently packages genomes. The inability to generate stable virions may reflect a failure of essential structural subunit interactions at one or more axes of symmetry. Alternatively, the nonstructural Rep protein interactions that occur on the surface of the capsid for packaging may not be compatible in the AAV2/AAV4 chimeras, as suggested by the structures for AAV2 and AAV4 [30, 31] .

The AAV capsid provides a potent gene delivery vehicle and has shown great promise in animal models, as well as in human clinical trials conducted to date. AAV vectors have been developed for a multitude of diseases, including disorders of the central nervous system for which other vectors and methods of treatment have been inadequate. Examples include Parkinson's disease and Alzheimer's disease. Gene therapy approaches utilizing AAV have also addressed classic genetic disorders, such as lysosomal storage disorders, hemophilia, and cystic fibrosis. Many diseases that are amenable to AAV gene therapy approaches are listed in Table 2 .2. AAV vectors to treat these diseases have shown significant and persistent gene transfer without toxicity [3-5, 117-124, 138-141, 159-160] in human clinical trials. Vectors are administered to effect intracellular expression of proteins such as dystrophin or expression of secreted therapeutic proteins that result in cross correction of cells. AAV may also be utilized as a vaccine to deliver specific antigens; for example, AAV5 targets dendritic cells, allowing for antigen presentation, and AAV vectors have been produced that express components of papillomavirus, the causative agent of cervical cancer, as well as the receptor for the spike protein of SARS coronavirus [181, 182] . AAV-based gene transfer has the therapeutic potential to arrest or reverse the course of these inherited and acquired diseases.

An important consideration for the development of a suitable gene delivery vector is the ability to produce the quantities of vector required to treat human patients. Historically, production methods have been a limitation in the development of AAV vectors. Improvements in production and purification methods have resulted in better yields, increased purity, and higher titers through the development of scalable systems [113, [183] [184] [185] [186] [187] . Recombinant AAV vectors can be manufactured using a two-plasmid system, as shown in Fig. 2. 3. One plasmid harbors the therapeutic Vector expressing adiponectin Rat AAV1, AAV2, AAV3, AAV4, AAV5 [180] gene flanked by the ITRs and the other one contains the AAV rep and cap genes, as well as the helper functions for AAV2 replication. The adenovirus helper functions are provided by expression of E2A, E4ORF6, and VA RNAs in the helper plasmid, while the adenovirus E1A and E1B gene products are expressed in the HEK293 cells utilized for production [188] . The two plasmids are co-transfected into 293 cells, and this results in production of an AAV virion that contains the therapeutic gene flanked by the ITRs [189] . The ITRs are the only viral sequences remaining after purification of rAAV vectors. Protocol 1 (see Appendix) describes the small-scale production and purification of rAAV vectors as described by Zolotukhin et al. [190] . Generally, HEK293 cells are expanded, transfected, and harvested at 72 h post-transfection. The cells are then lysed, and loaded onto either a cesium chloride or an iodixanol gradient to separate infectious virions from empty capsids and other cellular proteins. Virus is then purified using either heparin affinity chromatography or ion-exchange chromatography. The chromatography method used is dependent on the natural receptors for AAV, as well as the charge characteristics of the viral particle, as shown in Fig. 2 .4. Following column chromatography, the virus is concentrated and characterized. These protocols yield vector stocks that are relatively pure; however, owing to the density gradient requirement, these methods are not easily scalable. Protocols 2 and 3 describe the large-scale production and purification of rAAV vectors as described by Snyder et al. [18] . Protocol 3 provides a method suitable for large-scale production of rAAV2 vectors that is scalable for the production of vectors under cGMP conditions for clinical trials [191] . Cell factories of HEK293 cells are transfected, incubated for 72 h, and then harvested. The cell pellet is resuspended in lysis buffer containing 0.5% deoxycholate to reduce viral particle aggregation and 50 U Benzonase/ml to degrade cellular, plasmid, and nonpackaged nucleic acid. A microfluidizer is utilized to lyse the cells in order to form a fine suspension that can be loaded directly onto a Streamline Heparin column. The column is washed and the virus is eluted from the column with phosphate-buffered saline (PBS) containing 0.2 M NaCl (for a total ionic strength of 350 mM). The peak fraction from the Streamline Heparin column is brought to 1 M NaCl and loaded on a Phenyl 

Several assays to characterize the final product with respect to titer, purity, identity, potency, particle to infectious ratio, and stability are listed in Table 2. 3. In addition, vector product safety testing should be conducted prior to animal or human studies. Assays used for product characterization and safety testing are included in Table 2 .3. [192, 193] Determine titer of infectious particles produced Serial Dilution Replication Assay (dRA) [194] Vector genome Dot-blot hybridization Determine genome-containing vector concentration PCR [195] Capsid ELISA [24] Determine total capsid protein concentration -enables a determination of empty particles in the prep Bradford Western Electron microscopy [196] Optical density (OD) [197] 

In the near future, customized AAV gene therapy vectors may consist of modified capsids that allow for specific targeting to treat patients with various diseases. The 3D structures of the AAV capsids will provide a basis for rational vector design; however, the 3D structures available for autonomous parvoviruses and dependoviruses provide only a "snapshot" of the capsid topology in a low energy conformation. Our knowledge about the AAV viral capsid structure in solution is limited; however, this structure must be dynamic to carry out the various functions required for viral attachment and entry, as well as trafficking within the cell. Cryo-EM studies have shown that the unique N-terminus of VP1 is internal to the capsid based on additional density at the two-fold axis of symmetry [198] . In vitro, upon heat treatment of AAV capsids, it has been shown that this region can be externalized. Mutagenesis experiments have shown that this externalization occurs through the pore at the five-fold axis of symmetry [199] . Previously, antibodies have been produced that detect various regions of the capsid proteins [200] . The B1 antibody epitope is on the C-terminal end of the capsid protein, and is primarily internal at the two-fold axis of symmetry in assembled capsids. This antibody is useful in detecting denatured AAV proteins and the epitope is highly conserved among the AAV serotypes. A1 antibody recognizes the unique N-terminal region of VP1, while the A69 epitope is in the N-terminal region of VP2 for AAV2. Polyclonal antibodies have been produced to AAV2 capsids, as well as to other serotypes. In addition, antibodies have been produced that recognize conformational epitopes that are present only on assembled capsids for AAV2, as well as AAV1. Capsid antibodies can be utilized for serotype identity testing of the final vector product; however, many of these antibodies cross-react with multiple serotypes. Historically, AAV has been shown to be remarkably stable and is generally resistant to proteases. However, we have demonstrated that when exposed to proteases, specific regions of the capsid are susceptible to proteolysis [201] . Owing to differences in the primary sequence of the capsid proteins of different serotypes, as well as the resulting differences in capsid structure, proteolysis can be utilized for AAV serotype determination. Different serotypes provide unique fragmentation patterns when cleaved with protease, with some serotypes such as AAV5 being relatively resistant, while others such as AAV2 are more susceptible to trypsin and chymotrypsin. Figure 2 .5 depicts protease mapping of the AAV capsid for capsid serotype determination. A generic assay is described in Protocol 4 where proteolysis is used together with specific antibodies to provide a powerful mapping technique and a method to identify and confirm the serotype of the AAV capsid. T0 T12  T0  T0  T0  T0  T0  T12  T12  T12  T12  T12   87 kDa  72 kDa  63 kDa  45 kDa   30 kDa   25 kDa  20 kDa A.

B.

Proteinase K Trypsin Chymotrypsin Serotype Fig. 2.5 Protease mapping of the AAV capsid for capsid serotype determination. A Samples are digested with a protease, in this case trypsin for one set of samples and chymotrypsin for the other set. A Western blot is performed using polyclonal antisera to AAV capsids, and based on the fragmentation pattern, a serotype determination can be made. Undigested sample (T 0 ) and samples digested for 12 h (T 12 ) for AAV2, AAV1, and AAV5 are shown. AAV5 is resistant to these proteases, while AAV1 and AAV2 exhibit differences in their fragmentation patterns. T 0 samples represent the undigested capsid proteins VP1, VP2, and VP3. B Different AAV serotypes demonstrate different susceptibilities to proteases due to the differences in their primary amino acid sequences. This differential susceptibility provides a unique signature for each serotype and allows for capsid serotype identification

In summary, the AAV capsid is the major determinant in gene transfer efficiency and targeting. Several approaches are available to target AAV viral vectors to specific cell types, including utilizing natural serotypes that target a desired cellular receptor, producing pseudotyped vectors, as well as engineering chimeric and mosaic capsids. Genetic mutations of the AAV capsid have identified regions that will accommodate peptide insertions and modifications for specific targeting. The use of modified capsids may enhance efficiency and target specificity to make a more potent and safe vector. Several genetic diseases are amenable to treatment with AAV vectors, making AAV a valuable gene delivery vehicle, especially for disorders for which current therapies are inadequate or non-existent. Current production methods allow for the generation of quantities of virus needed for clinical trials, and purification methods have been developed for several serotypes. In addition, assays have been developed for final product characterization to ensure patient safety.

1. Seed [20] 15-cm plates with 293 cells so that they will be 75-80% confluent the following day (12-16 h prior to transfection). 293 cells are cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin. 2. Check plates prior to transfection to ensure optimal confluency, and change media to ensure cells are in a volume of 20 ml. Be careful not to disrupt the monolayer. 3. For transfection, prepare the DNA mixture at room temperature in a 50-ml conical tube as follows: Mix 900 µg of helper plasmid with 300 µg rAAV vector plasmid. This is 60 µg DNA per plate in a 1:1 molar ratio. Bring the final volume of the DNA mixture to 22.5 ml with sterile dH 2 O. Add 2.5 ml of 2.5 M CaCl 2 to the DNA for a CaCl 2 concentration of 0.250 M. 4. Prepare ten 15-ml conical tubes each with 2.5 ml 2× HBS to transfect the 20 plates (25 ml 2× HBS total). Transfect two plates at a time by adding 2.5 ml of DNA mixture to a tube of 2.5 ml 2× HBS, mix, and incubate for 1 min at room temperature to allow precipitate to form. Add 5 ml of media to the tube and mix.

(Total volume in the tube is 10 ml.) Pipette 5 ml onto each of the two plates.

Repeat for the remainder of the plates. 5. Incubate at 37°C for 72 h.

and centrifuge at 1,000 × g, for 10 min at 4°C to pellet the cells. Discard the media. Resuspend pellet in PBS to wash the pellet. Centrifuge at 1,000 × g, for 10 min at 4°C. Discard PBS and freeze pellet. Frozen pellets can be stored at −20°C for processing at a later time. Adjust the pH to 7.05 with 0.5 N NaOH, bring to a final volume of 1 l with dH 2 O, and sterile filter. Store in 50-ml aliquots at −20°C. (Note: the optimal pH range for 2× HBS is 7.05-7.12. The pH of the 2× HBS is a key factor that influences precipitate formation.) 7 . Resuspend pellet in 30 ml lysis buffer. Lyse cells by three freeze/thaw cycles.

Thaw pellet in 37°C water bath and freeze cells in EtOH/dry ice bath; repeat twice. 8. Transfer the lysate to a 40-ml Dounce homogenizer. Homogenize the lysate with 20 strokes to shear cellular DNA. 9. For each 10 ml of lysate, add 5 g CsCl and homogenize until the CsCl is dissolved completely. 10. Fill six 12.5 ml ultra clear ultracentrifuge tubes (Beckman) with 10 ml of lysate and underlay with 0.5 ml each of CsCl (1.5 g/ml). Balance the tubes using CsCl (1.37 g/ml) prior to ultracentrifugation.

11. Centrifuge for 24 h at 265,000 × g (40,000 rpm in SW 41 rotor), 21°C. 12. Collect AAV from the gradient by dripping 1-ml fractions, and verify the density of the fractions by refractometry. The density of infectious AAV virions is 1.40-1.42 g/cm 3 . Empty AAV particles and DI particles have a density of 1.32-1.35 g/cm 3 . 13. Dialyze AAV-containing fractions into the buffer that will be used for column chromatography (1× TD buffer for heparin column or 15 mM NaCl, 20 mM Tris (pH 8.5) for Q Sepharose purification). 14. Purify using either heparin affinity chromatography (for AAV2, AAV3, or AAV6) or ion-exchange Q Sepharose chromatography (for AAV1, AAV4, AAV5, AAV8, or AAV9). Use a 1-ml column for 20-40 plates, as described in steps 1-7 under "AAV Purification by Heparin Chromatography" or "AAV Purification by Q Sepharose Chromatography." The purification method is as described by Zolotukhin et al. [190] .

Lysis buffer (prepare 1 l):

-150 mM NaCl (stock 58.44 g/mol) -8.766 g.

-50 mM Tris (stock 121.14 g/mol) -6.055 g.

-Adjust pH to 8.5 with HCl, bring to a final volume of 1 l with dH 2 O, and filter sterilize. Store at room temperature.

1.37 g/ml CsCl in PBS -Dissolve 50 g CsCl in PBS and adjust the final volume to 100 ml. Weigh 1 ml to verify the density is 1.37 g/ml. Filter sterilize. Store at room temperature.

1.5 g/ml CsCl in PBS -Dissolve 67.5 g CsCl in PBS and adjust the final volume to 100 ml. Weigh 1 ml to verify the density is 1.5 g/ml. Filter sterilize. Store at room temperature.

5× TD buffer (prepare 1 l):

-5× PBS −500 ml of 10× PBS (Invitrogen). . 12a. Collect the 40% iodixanol band and the interface between the 60% and the 40% bands. This is done by setting up a ring stand and placing the tube in a clamp. Swab the top and bottom of the tube with an alcohol swab. Vent the top of the tube with a needle and collect fractions from bottom of the tube. 13a. Dialyze virus-containing fractions into the buffer that will be used for column chromatography (1× TD buffer for heparin column or 15 mM NaCl, 20 mM Tris (pH 8.5) for Q Sepharose purification). 14a. Purify using either heparin affinity chromatography (for AAV2, AAV3, or AAV6) or ion-exchange Q Sepharose chromatography (for AAV1, AAV4, AAV5, AAV8, or AAV9). Use a 1-ml column for 20-40 plates, as described in steps 1-7 under "AAV Purification by Heparin Chromatography" or "AAV Purification by Q Sepharose Chromatography." The purification method is as described by Zolotukhin et al. [190] . 3. Re-equilibrate the column by washing with ten column volumes of 1× TD. 4. Apply the dialyzed virus from either the cesium gradient or iodixanol gradient to the column. 5. Wash the column with ten volumes of 1× TD. 6. Elute with five column volumes of elution buffer using either a continuous or step gradient. AAV2 can be eluted in 1× TD/0.5 M NaCl [190] and collect [5] 1-ml fractions. 7. Dialyze the virus preparation into storage buffer or suitable formulation and freeze at −20°C. 

1. Resuspend the cell pellet from one cell factory in 60 ml large-scale lysis buffer (20 mM Tris-Cl, 150 mM NaCl, 0.5% deoxycholate, and Benzonase (50 U/ml) ). Deoxycholate is used in the lysis buffer to reduce aggregation. 2. For ten cell factories, lyse the cells in a single-pass by using a microfluidizer (Microfluidics M-110S). This will form a fine suspension that can be loaded onto the column. 3. Load the lysate onto a 150-ml Streamline Heparin column (Pharmacia) at a flow rate of 20 ml/min using an AKTA-FPLC (Pharmacia). 4. Wash the column with four column volumes of lysis buffer, followed by five column volumes of PBS. 5. Elute the virus with PBS containing 0.2 M NaCl (0.350 M total ionic strength) and monitor the absorbance at 280 λ. The peak fraction will be ∼90 ml.

6. Bring the NaCl concentration of the peak fraction to 1 M NaCl and load this onto a Phenyl Sepharose column (5 ml column, Pharmacia). This is a hydrophobic interaction column and the flow-through that contains the virus is collected. 7. The flow-through is diluted to 150 mM NaCl with sterile water. 8. The virus is loaded onto a 5 ml SP Sepharose column (Pharmacia) at a flow rate of 5 ml/min. 9. Wash the column with ten column volumes of PBS. 10. Elute the virus with PBS containing 0.135 M NaCl (285 mM total ionic strength) and monitor the absorbance at 280 λ. Aliquot and store at −20°C. 11. Dialyze the virus prep into storage buffer (50 mM Tris-Cl, 100 mM NaCl, pH 8.0), aliquot, and freeze at −20°C.

1. Dialyze AAV vector into protease digestion reaction buffer, 50 mM Tris-Cl, 100 mM NaCl, pH 8.0, if needed. 2. Digest 0.8 µg (∼1.2 × 10 11 ) capsids with either 5 µg (0.02% final concentration) of trypsin, 1 µg of proteinase K, or 80 µg of α-chymotrypsin in a 25 µl reaction at 37°C for up to 24 h. For each serotype, an undigested sample should be included as a control. These proteases are commercially available from Sigma. 3. Add an equal volume of Laemmli sample buffer containing 1% sodium dodecyl sulfate (SDS) and 655 mM β-mercaptoethanol and boil the samples at 100°C for 5 min. 4. Separate the proteolytic fragments on a 10% SDS-PAGE gel for 90 min at 125 V (constant voltage) until the dye front reaches the bottom of the gel. 5. Transfer the proteins to nitrocellulose (Western blot) in transfer buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS, and 20% methanol) for 2 h at 0.5 A (constant amperes). 6. Probe the membrane with rabbit polyclonal antisera to AAV capsids. Duplicate samples can be run to probe with other AAV antibodies to determine the origin of the fragments that are produced. For example, a signal with monoclonal B1 antibody demonstrates that the fragment is from the common C-terminal end of VP1, VP2, and VP3. Alternatively, by probing with monoclonal A1 antibody, a signal on the Western blot would demonstrate that the origin of the fragment is the unique N-terminus of VP1. The B1 epitope is conserved among most AAV serotypes and polyclonal antibodies have been developed for a few AAV serotypes. Anti-AAV capsid antibodies are available from Progen or American Research Products, Inc. 

Adeno-associated viruses: an update

Long-term clinical improvement in MPTP-lesioned primates after gene therapy with AAV-hAADC

Clinical protocol. Administration of a replication-deficient adeno-associated virus gene transfer vector expressing the human CLN2 cDNA to the brain of children with late infantile neuronal ceroid lipofuscinosis

Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response

Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation

Phase I clinical trial utilizing gene therapy for limb girdle muscular dystrophy: alpha-, beta-, gamma-, or delta-sarcoglycan gene delivered with intramuscular instillations of adeno-associated vectors

Hematopoietic stem cell transduction by recombinant adeno-associated virus vectors: problems and solutions

Vascular bed-targeted in vivo gene delivery using tropism-modified adeno-associated viruses

High levels of persistent expression of alpha1-antitrypsin mediated by the nonhuman primate serotype rh.10 adeno-associated virus despite preexisting immunity to common human adeno-associated viruses

Cell proliferation in the mammalian lung

Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue

Integration of adeno-associated virus (AAV) and recombinant AAV vectors

Characterization of adeno-associated virus genomes isolated from human tissues

Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers

Immune responses to adeno-associated virus vectors

Circulating anti-wild-type adeno-associated virus type 2 (AAV2) antibodies inhibit recombinant AAV2 (rAAV2)-mediated, but not rAAV5-mediated, gene transfer in the brain

Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy

Adeno-associated viral vectors for clinical gene transfer studies

Mechanisms of adeno-associated virus genome encapsidation

Synthesis of adeno-associated virus structural proteins requires both alternative mRNA splicing and alternative initiations from a single transcript

Assembly of adeno-associated virus

Molecular structure of adeno-associated virus variant DNA

DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsids

Subcellular compartmentalization of adeno-associated virus type 2 assembly

Assembly of viruslike particles by recombinant structural proteins of adeno-associated virus type 2 in insect cells

Assembly of adeno-associated virus type 2 capsids in vitro

Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants

The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity

Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus

Agbandje-McKenna M. Structurally mapping the diverse phenotype of adeno-associated virus serotype 4

The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy

Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5

Structure of adeno-associated virus type 4

Structure of adeno-associated virus serotype 5

Production, purification, crystallization and preliminary X-ray analysis of adeno-associated virus serotype 8

Structure, sequence, and function correlations among parvoviruses

Canine parvovirus capsid structure, analyzed at 2.9 A resolution

Structure determination of minute virus of mice

The PyMOL Molecular Graphics System

Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism

Correlating structure with function in the viral capsid

Atomic structure of viral particles

Adeno-associated viruses undergo substantial evolution in primates during natural infections

Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2

Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions

AlphaVbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection

Hepatocyte growth factor receptor is a coreceptor for adeno-associated virus type 2 infection

Adeno-associated virus type 2 contains an integrin alpha5beta1 binding domain essential for viral cell entry

Attachment of adeno-associated virus type 3H to fibroblast growth factor receptor 1

Identification of PDGFR as a receptor for AAV-5 transduction

Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer

Adeno-associated virus serotype 4 (AAV4) and AAV5 both require sialic acid binding for hemagglutination and efficient transduction but differ in sialic acid linkage specificity

Alpha2,3 and alpha2,6 N-linked sialic acids facilitate efficient binding and transduction by adeno-associated virus types 1 and 6

New recombinant serotypes of AAV vectors

Molecular characterization of adeno-associated viruses infecting children

Identification and characterization of novel adeno-associated virus isolates in ATCC virus stocks

Production methods for gene transfer vectors based on adeno-associated virus serotypes

Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity

Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6

Efficient AAV1-AAV2 hybrid vector for gene therapy of hemophilia

Viral serotype and the transgene sequence influence overlapping adeno-associated viral (AAV) vector-mediated gene transfer in skeletal muscle

Enhancement of muscle gene delivery with pseudotyped adeno-associated virus type 5 correlates with myoblast differentiation

Adeno-associated virus (AAV)-7 and -8 poorly transduce vascular endothelial cells and are sensitive to proteasomal degradation

Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart

Improved hepatic gene transfer by using an adeno-associated virus serotype 5 vector

Adeno-associated virus vector serotypes mediate sustained correction of bilirubin UDP glucuronosyltransferase deficiency in rats

Efficacy and safety of adeno-associated viral vectors based on serotype 8 and 9 versus lentiviral vectors for hemophilia B gene therapy

Targeting recombinant adeno-associated virus vectors to enhance gene transfer to pancreatic islets and liver

Successful gene transfer using adeno-associated virus vectors into the kidney: comparison among adeno-associated virus serotype 1-5 vectors in vitro and in vivo

Adeno-associated virus types 5 and 6 use distinct receptors for cell entry

Adeno-associated virus serotype 9 vectors transduce murinealveolar and nasal epithelia and can be readministered

Human blue-opsin promoter preferentially targets reporter gene expression to rat s-cone photoreceptors

Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5

Enhanced transduction of mouse bone marrow-derived dendritic cells by repetitive infection with self-complementary adeno-associated virus 6 combined with immunostimulatory ligands

Adeno-associated virus-mediated gene transfer to hair cells and support cells of the murine cochlea

Adeno-associated virus serotypes 1 to 5 mediated tumor cell directed gene transfer and improvement of transduction efficiency

Enhanced gene transfer efficiency in the murine striatum and an orthotopic glioblastoma tumor model, using AAV-7-and AAV-8-pseudotyped vectors

Adeno-associated virus (AAV) serotypes 2, 4 and 5 display similar transduction profiles and penetrate solid tumor tissue in models of human glioma

Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system

Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system

Recombinant adeno-associated viral vector encoding human VEGF165 induces neomicrovessel formation in the adult mouse brain

Recombinant AAV serotype 1 transduction efficiency and tropism in the murine brain

Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or -2 serotypes for widespread gene delivery to the neonatal mouse brain

AAV2/5-mediated NGF gene delivery protects septal cholinergic neurons following axotomy

Recombinant AAVmediated gene delivery to the central nervous system

Differential myocardial gene delivery by recombinant serotype-specific adeno-associated viral vectors

Recombinant adeno-associated virus serotype 9 leads to preferential cardiac transduction in vivo

AAV Serotype-1 mediates early onset of gene expression in mouse hearts and results in better therapeutic effect

Widespread and early myocardial gene expression by adeno-associated virus vector type 6 with a beta-actin hybrid promoter

Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno-associated virus vectors

Comparison of adenoviral and adenoassociated viral vectors for pancreatic gene delivery in vivo

Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes

Determination of specific CD4 and CD8 T cell epitopes after AAV2-and AAV8-hF.IX gene therapy

Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid

Adenoassociated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer

Adeno-associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors

Recombinant adenoassociated and adenoviral vectors for the transduction of pancreatic and colon carcinoma

Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors

Liver transduction with recombinant adeno-associated virus is primarily restricted by capsid serotype not vector genotype

Characterization of tissue tropism determinants of adeno-associated virus type 1

Cardiomyocyte-specific gene expression following recombinant adeno-associated viral vector transduction

Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2

Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism

Adeno-associated virus capsids displaying immunoglobulin-binding domains permit antibody-mediated vector retargeting to specific cell surface receptors

Targeted gene delivery to vascular tissue in vivo by tropism-modified adeno-associated virus vectors

Improved cardiac gene transfer by transcriptional and transductional targeting of adeno-associated viral vectors

Heparan sulfate proteoglycan binding properties of adeno-associated virus retargeting mutants and consequences for their in vivo tropism

Insertional mutagenesis at positions 520 and 584 of adenoassociated virus type 2 (AAV2) capsid gene and generation of AAV2 vectors with eliminated heparin-binding ability and introduced novel tropism

Mutations in the carboxy terminus of adeno-associated virus 2 capsid proteins affect viral infectivity: lack of an RGD integrin-binding motif

Altering AAV tropism with mosaic viral capsids

Mosaic vectors comprised of modified AAV1 capsid proteins for efficient vector purification and targeting to vascular endothelial cells

Crossdressing the virion: the transcapsidation of adeno-associated virus serotypes functionally defines subgroups

Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system

Enhancing rAAV vector expression in the lung

A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia

Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector

Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus

A phase II, double-blind, randomized, placebo-controlled clinical trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with antrostomies

Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated virus serotype 2 (rAAV2)-CFTR vector in adult cystic fibrosis patients: a two-part clinical study

A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease

A phase I/II study of tgAAV-CF for the treatment of chronic sinusitis in patients with cystic fibrosis

Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebo-controlled trial

Maxillary sinusitis as a surrogate model for CF gene therapy clinical trials in patients with antrostomies

A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease

Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy

Comparison of the ability of adeno-associated viral vectors pseudotyped with serotype 2, 5, and 8 capsid proteins to mediate efficient transduction of the liver in murine and nonhuman primate models

Sustained correction of bleeding disorder in hemophilia B mice by gene therapy

Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors

Sustained correction of disease in naive and AAV2-pretreated hemophilia B dogs: AAV2/8-mediated, liver-directed gene therapy

Sustained expression of therapeutic level of factor IX in hemophilia B dogs by AAV-mediated gene therapy in liver

Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy

Therapeutic levels of factor IX expression using a muscle-specific promoter and adeno-associated virus serotype 1 vector

Sustained and complete phenotype correction of hemophilia B mice following intramuscular injection of AAV1 serotype vectors

Safety and efficacy of factor IX gene transfer to skeletal muscle in murine and canine hemophilia B models by adeno-associated viral vector serotype 1

Muscle-directed gene transfer and transient immune suppression result in sustained partial correction of canine hemophilia B caused by a null mutation

Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia

Persistent expression of canine factor IX in hemophilia B canines

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector

Gene therapy for hemophilia B: AAV-mediated transfer of the gene for coagulation factor IX to human muscle

AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B

Evidence of multiyear factor IX expression by AAV-mediated gene transfer to skeletal muscle in an individual with severe hemophilia B

Adeno-associated virus 2-mediated transduction and erythroid lineage-restricted long-term expression of the human beta-globin gene in hematopoietic cells from homozygous beta-thalassemic mice

Regulated expression of erythropoietin from an AAV vector safely improves the anemia of beta-thalassemia in a mouse model

Down-regulation of alpha-synuclein expression can rescue dopaminergic cells from cell death in the substantia nigra of Parkinson's disease rat model

Convection-enhanced delivery of adeno-associated virus type 2 (AAV2) into the striatum and transport of AAV2 within monkey brain

A dose-ranging study of AAV-hAADC therapy in Parkinsonian monkeys

AAV2-mediated gene delivery to monkey putamen: evaluation of an infusion device and delivery parameters

In vivo expression of therapeutic human genes for dopamine production in the caudates of MPTP-treated monkeys using an AAV vector

Practical aspects of the development of ex vivo and in vivo gene therapy for Parkinson's disease

Neonatal intramuscular injection with recombinant adeno-associated virus results in prolonged beta-glucuronidase expression in situ and correction of liver pathology in mucopolysaccharidosis type VII mice

Correction of the enzymatic and functional deficits in a model of Pompe disease using adeno-associated virus vectors

Correction of glycogen storage disease type II by an adeno-associated virus vector containing a musclespecific promoter

Intrathecal administration of AAV vectors for the treatment of lysosomal storage in the brains of MPS I mice

Intracranial delivery of CLN2 reduces brain pathology in a mouse model of classical late infantile neuronal ceroid lipofuscinosis

AAV8-mediated expression of glucocerebrosidase ameliorates the storage pathology in the visceral organs of a mouse model of Gaucher disease

Correction of Hunter syndrome in the MPSII mouse model by AAV2/8-mediated gene delivery

Adeno-associated virus-mediated aspartoacylase gene transfer to the brain of knockout mouse for Canavan disease

Aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for Canavan disease

Immune responses to AAV in a phase I study for Canavan disease

Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain

Widespread and stable pancreatic gene transfer by adeno-associated virus vectors via different routes

Efficient gene delivery to human and rodent islets with double-stranded (ds) AAV-based vectors

Anti-Abeta single-chain antibody delivery via adeno-associated virus for treatment of Alzheimer's disease

Development of a safe oral Abeta vaccine using recombinant adeno-associated virus vector for Alzheimer's disease

A novel recombinant adeno-associated virus vaccine reduces behavioral impairment and beta-amyloid plaques in a mouse model of Alzheimer's disease

Chronic recurrent myocardial ischemic injury is significantly attenuated by pre-emptive adeno-associated virus heme oxygenase-1 gene delivery

One year transgene expression with adeno-associated virus cardiac gene transfer

Capsid modifications overcome low heterogeneous expression of heparan sulfate proteoglycan that limits AAV2-mediated gene transfer and therapeutic efficacy in human ovarian carcinoma

Efficient gene transfer of CD40 ligand into ovarian carcinoma cells with a recombinant adeno-associated virus vector

Adeno-associated virus-mediated delivery of a mutant endostatin suppresses ovarian carcinoma growth in mice

Recombinant AAV-mediated HSVtk gene transfer with direct intratumoral injections and Tet-On regulation for implanted human breast cancer

Recombinant adeno-associated virus (rAAV) expressing TFPI-2 inhibits invasion, angiogenesis and tumor growth in a human glioblastoma cell line

Adeno-associated virus 2-mediated intratumoral prostate cancer gene therapy: long-term maspin expression efficiently suppresses tumor growth

Anti-angiogenic therapy subsequent to adeno-associated-virus-mediated immunotherapy eradicates lymphomas that disseminate to the liver

Oral adeno-associated virus-sTRAIL gene therapy suppresses human hepatocellular carcinoma growth in mice

Overexpression of soluble TRAIL induces apoptosis in human lung adenocarcinoma and inhibits growth of tumor xenografts in nude mice

Attenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery

Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin

AAV-mediated leptin receptor installation improves energy balance and the reproductive status of obese female Koletsky rats

Sustained peripheral expression of transgene adiponectin offsets the development of diet-induced obesity in rats

Induction of robust immune responses against HIV is supported by the inherent tropism of AAV5 for DC

Intranasal vaccination with recombinant adeno-associated virus type 5 against human papillomavirus type 16 L1

Improving rAAV production and purification: towards the definition of a scaleable process

Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids

High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap

Development and characterization of a cell line for large-scale, serum-free production of recombinant adeno-associated viral vectors

Insect cells as a factory to produce adeno-associated virus type 2 vectors

Characteristics of a human cell line transformed by DNA from human adenovirus type 5

Adeno-associated virus vectors can be efficiently produced without helper virus

Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors

Production of research and clinical grade recombinant adeno-associated viral vectors

Infectious titer assay for adenoassociated virus vectors with sensitivity sufficient to detect single infectious events

Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses

High throughput production, screening and analysis of adeno-associated viral vectors

Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks

Purification of recombinant adeno-associated virus vectors by column chromatography and its performance in vivo

Quantification of adeno-associated virus particles and empty capsids by optical density measurement

Electron cryo-microscopy and image reconstruction of adeno-associated virus type 2 empty capsids

Impact of capsid conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging

Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection

Proteolytic mapping of the adeno-associated virus capsid

Acknowledgments We thank Dr. Philippe Moullier at the Laboratoire de Therapie Genique, INSERM U649, CHU Hotel Dieu, Nantes Cedex 1, France. R.S. owns equity in a gene therapy company that is commercializing AAV for gene therapy applications. This project was funded by