Chapter 2 - Adeno-Associated Viral Vectors and Their Redirection to Cell-Type Specific Receptors

https://doi.org/10.1016/S0065-2660(09)67002-4Get rights and content

Abstract

Efficient and specific delivery of genes to the cell type of interest is a crucial issue in gene therapy. Adeno-associated virus (AAV) has gained particular interest as gene vector recently and is therefore the focus of this chapter. Its low frequency of random integration into the genome and the moderate immune response make AAV an attractive platform for vector design. Like in most other vector systems, the tropism of AAV vectors limits their utility for certain tissues especially upon systemic application. This may in part be circumvented by using AAV serotypes with an in vivo gene transduction pattern most closely fitting the needs of the application. Also, the tropism of AAV capsids may be changed by combining parts of the natural serotype diversity. In addition, peptides mediating binding to the cell type of interest can be identified by random phage display library screening and subsequently be introduced into an AAV capsid region critical for receptor binding. Such peptide insertions can abrogate the natural tropism of AAV capsids and result in detargeting from the liver in vivo. In a novel approach, cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids or serotype-shuffling libraries in vitro and in vivo for optimized transduction of the cell type or tissue of interest.

Introduction

The biological safety and the unsatisfactory efficacy of gene transfer vectors pose significant challenges for gene therapy (Somia and Verma, 2000, Thomas et al., 2003). Problems of current gene therapy vectors include unintended transduction of certain tissues, adverse immune reactions, and lack of efficient transduction of the cells of interest (Trepel et al., 2000a). Targeting vectors to certain cell types or tissues may have a very high impact on all these parameters. Recombinant adeno-associated virus (AAV) vectors have become very popular as potential gene therapy vectors because of their ability to mediate stable and efficient gene expression, combined with a favorable biological safety profile. Like in other vector systems, however, their lack of specificity poses a serious problem that has been challenged during the last decade by a plethora of different technical approaches to change their tropism. These will be reviewed here and comprise the use of the various AAV serotypes, the insertion of targeting ligands into the AAV capsid, as well as the development of various kinds of combinatorial AAV capsid libraries for selection of targeted and transduction-optimized AAV vectors. All of these approaches have their unique advantages and disadvantages, and their specific use depends very much on the intended application the vectors are needed for.

AAV is a small, nonenveloped DNA virus which belongs to the parvovirus family. To date, 12 different serotypes have been isolated from primate or human tissues (Grimm and Kay, 2003, Schmidt et al., 2008, Wu et al., 2006a). From all serotypes described so far, AAV-2 is the one that has been best characterized. As a wild-type virus, AAV is nonpathogenic, only mildly immunogenic, and has the potential to integrate site-specifically into the host genome. Its broad host tropism allows for efficient gene delivery in various target tissues.

AAV harbors a single-stranded genome of approximately 4.7 kb that comprises two open reading frames (rep and cap) flanked by inverted terminal repeats (ITRs). The palindromic nucleotide sequence of the ITRs forms a characteristic T-shaped hairpin structure serving as structural cis-acting elements that are required for viral genome replication, packaging, and rescue from the integrated state. In addition, the ITRs have regulatory influence on viral gene expression and host genome integration (Goncalves, 2005).

The nonstructural Rep proteins Rep78 and Rep52, and their respective splice variants Rep68 and Rep40 are under transcriptional control of two promoters (p5 and p19) and play an essential role in viral DNA replication and packaging, regulation of gene expression, and site-specific integration (Goncalves, 2005). The cap gene encodes for three structural capsid proteins VP1, VP2, and VP3 (90, 72, 62 kDa, respectively) that share the same C-terminal domain, while VP2 and VP1 contain additional N-terminal sequences. The N terminus of VP1 contains a phospholipase A2 (PLA2) domain necessary for endosomal escape and nuclear entry during the viral infection (Bleker et al., 2005, Girod et al., 2002, Sonntag et al., 2006). The N terminus of VP1, VP2, and VP3 contains four basic regions (BR) that constitute putative nuclear localization sequences (NLS) presumably involved in the nuclear transfer of the virus (Grieger et al., 2006, Grieger et al., 2007, Vihinen-Ranta et al., 2002).

The AAV capsid is composed of 60 subunits of the VP proteins VP1, VP2, and VP3 at a molar ratio of approximately 1:1:10 and generates a T = 1 icosahedral capsid with approximately 25 nm in diameter. The atomic structure of various serotypes has been resolved by X-ray crystallography (AAV-1, -2, -4, and -8) (Govindasamy et al., 2006, Miller et al., 2006, Nam et al., 2007, Xie et al., 2002) or by cryoelectron microscopy (AAV-5) (Walters et al., 2004), and the crystal structures for AAV-6, -7, and -9 are currently being resolved (Mitchell et al., 2009, Quesada et al., 2007, Xie et al., 2008). All serotypes share a common capsid structure comprising a conserved eight-stranded antiparallel β-barrel (labeled B-I) motif with large loop insertions between the β-strands that constitute the basic structure of the capsid (Fig. 2.1). In intact AAV capsids, three capsid subunits contribute to the loops between the subunits G and H, leading to the formation of characteristic protrusions which are arranged in groups of three (“threefold spikes”) and cluster around the threefold axis of symmetry. Another characteristic feature is the cylindrical pore structure which is clustered around the fivefold axis of symmetry and is surrounded by characteristic depressions termed as canyon, plateau, and dimple (Kern et al., 2003, Lochrie et al., 2006, Opie et al., 2003). Differences in amino acid sequences within the variable loop regions characterize the different AAV serotypes (ranging from ~ 1% to 45%) (Gao et al., 2002, Mori et al., 2004). These loop regions presumably mediate most if not all of the interactions of the AAV capsid with cellular receptors. In addition, they can serve as immune epitopes.

Mutagenesis-based approaches and structural data facilitated the identification of receptor-binding sites within the capsid. The amino acids involved in binding of AAV serotype 2 (AAV-2) to its primary cellular receptor heparan sulfate-proteoglycan (HSPG) are presented within two adjacent VP protein subunits forming protrusions on the threefold spike. This binding site comprises a cluster of five basic amino acids at amino acid positions R484, R487, K532, R585, and R588 (VP numbering) (Fig. 2.1). Positively charged amino acid residues bind negatively charged sulfate and carboxyl groups of HSPG mainly via electrostatic interactions (Kern et al., 2003, Opie et al., 2003, Xie et al., 2002). Of note, amino acids adjacent to the heparan sulfate binding site can play a crucial role for immune recognition by the host organism. Point mutations at position R471A or N587A or insertion of a small peptide ligand at the latter position enabled AAV capsids to escape binding and neutralizing by antibodies (Lochrie et al., 2006, Wobus et al., 2000). Further, the peptide string RXXR at positions 585–588 is involved in the cellular uptake into dendritic cells and the activation of capsid-specific T-cells, thereby contributing to the generation of host cytotoxic T-cell responses (Vandenberghe et al., 2006). The cryo-EM structure of AAV and the heparin complex suggest that the footprint that generates the HSPG binding motif is wider than apparent through mutagenesis-based approaches and extends into the “dead zone” region—a site that has been proposed to be a potential binding site for AAV co-receptors (Lochrie et al., 2006, O'Donnell et al., 2009). These observations make it difficult to confine particular capsid domains that interact with cellular co-receptors, since mutations in the HSPG binding region may also affect possible co-receptor interactions.

The amino acids NGR at positions 511–513 adjacent to the HSPG binding site is highly conserved in several serotypes (except for AAV serotypes -4, -5, and -11) and has been proposed to serve as a binding site for integrin α5β1. (Asokan et al., 2006) (Fig. 2.1).

The cellular transduction by AAV is initiated by binding of the viral capsid to cell surface receptors. Various glycan motifs that are widely expressed on various cell types and tissues can be used as attachment receptor for different AAV serotypes. The primary receptor for AAV-2 and -3 is cellular HSPG (Summerford and Samulski, 1998), while serotypes AAV-1, -4, -5, and -6 use different derivates of O- or N-linked sialic acid, respectively (Kaludov et al., 2001, Seiler et al., 2006, Wu et al., 2006a, Wu et al., 2006b). However, for AAV-2 and -3, cellular transduction of AAV has also been demonstrated in the absence of HSPG (Boyle et al., 2006, Handa et al., 2000). Furthermore, mutations of the AAV-2 capsid in the HSPG binding domain result in an heparin binding deficient AAV phenotype, leading to an increased transduction of the heart in vivo (Kern et al., 2003). Both findings suggested that other receptors than HSPG are involved in the viral attachment. To date, integrin αvβ5, integrin α5β1, hepatocyte growth factor receptor (c-Met), and CD9 have been described as co-receptors for AAV-2 infection (Asokan et al., 2006, Kashiwakura et al., 2005, Kurzeder et al., 2007, Summerford et al., 1999), while the fibroblast growth factor receptor-1 (FGFR1) is utilized both by AAV-2 and -3 (Qing et al., 1999). The 37/67-kDa laminin receptor (LamR) can serve as co-receptor for AAV-2, -3, -8, and -9 (Akache et al., 2006a, Akache et al., 2006b) and platelet derived growth factor receptor (PDGFR) mediates AAV-5 transduction (Di Pasquale et al., 2003).

For cellular entry, AAV depends on the interaction with various co-receptors. It has been proposed that HSPG binding to AAV-2 leads to structural rearrangements of the viral capsid providing the conformational basis for interaction with integrin α5β1 required for receptor-mediated endocytosis (Asokan et al., 2006). However, structural analysis of the AAV-2 heparin complex did not provide evidence for receptor-induced conformational changes (O'Donnell et al., 2009). Nevertheless, AAV-2 interaction to cellular co-receptors initiates cell entry of the virions via clathrin-coated pits in a dynamin-dependent process into endosomes (Bartlett et al., 2000, Di Pasquale et al., 2003). AAV binding to cell surface receptors is required to activate the phosphatidylinositol-3-kinase pathway via Rac1 that triggers the intracellular trafficking of AAV to the nucleus along microtubules and microfilaments (Sanlioglu et al., 2000).

Vesicular trafficking to the nuclear area is a slow and rate-limiting step in AAV gene transduction in some if not most cell types (Duan et al., 2000, Hansen et al., 2000, Hansen et al., 2001). Six potential pathways have been described for AAV-2 and -5. Following viral internalization, virions are taken up into early endosomes and can move either to late endosomal compartments where AAV accumulates in early lysosomes or the trans-Golgi network. Depending on the cell type and the viral dose, AAV can also accumulate in perinuclear recycling endosomes (PNRE) or traveling from PNRE to the trans-Golgi network, or even exit from very early endosomes (Ding et al., 2005). Endosomal proteases like Cathepsin B and L have significant influence on AAV gene transfer efficiency, possibly through selective cleavage of capsid proteins for further uncoating steps (Akache et al., 2006b). As AAV-5 internalization can also occur via the caveolar pathway, virions have been detectable in caveosomes (Bantel-Schaal et al., 2009).

Endosomal release of the virions to the cytoplasm occurs when conformational changes of the VP1/VP2 N-termini leads to the exposure of a phospholipase A2 (PLA2) domain within the capsid through pores located at the fivefold axis of symmetry. Acidification of the endosomal compartment seems likely to trigger this process but is not sufficient for the conformational change of VP1/VP2, suggesting that further, so far unknown, mediators are involved (Kronenberg et al., 2005, Sonntag et al., 2006). Interestingly, sequence analyses of the N-terminal amino acids of VP1 among different serotypes may suggest that the phopholipase A2 is a current feature of AAV (Ding et al., 2005). After endosomal escape and release into the cytosol, AAV accumulates in a perinuclear pattern before its translocation into the nucleus occurs. During this step of post-entry processing, ubiquitylation and ubiquitin-dependent degradation of the viral capsid by proteasomes can influence the level of gene transduction in different cell types. Consequently, proteasome inhibitors have been used to increase the rate of AAV-2 transduction in certain cell types and tissues (Duan et al., 2000, Grieger and Samulski, 2005, Jennings et al., 2005, Yan et al., 2002). Mutations of surface-exposed tyrosine phosphorylation sites can lead to a protection from proteasomal-mediated degradation of the capsid thereby increasing transduction efficiency of AAV-2 (Zhong et al., 2008). While it has been shown that the cytoplasmic movement of AAV is mediated via cytoskeletal ATP-dependent motor proteins and/or Brownian diffusion (Ding et al., 2005), the mechanism of nuclear translocation remains unclear. Possibly, the N terminus of VP1/VP2 serves as putative nuclear translocation signal that mediates viral entry via the nuclear pore complex (NPC) (Grieger et al., 2006). However, alternative pathways for nuclear translocation are also under discussion (Hansen et al., 2001, Xiao et al., 2002). The presence of intact virions in the nucleus suggests that uncoating of AAV occurs in the nucleus. It is likely that mobilization of intact AAV capsids from the nucleolus to nucleoplasmic sites permits uncoating and might determine the ratio of single-stranded genomes that become double stranded (Johnson and Samulski, 2009). Depending on the host cell DNA synthesis machinery, second strand synthesis is another major rate-limiting step in AAV gene transduction. The conversion of single-stranded AAV genome to double-stranded DNA is required for viral gene expression, stabilization of the genome, and prevention of degradation. When latent, AAV-2 persists either by Rep protein-mediated site-specific integration into the q-arm of chromosome 19 (AAVS1), a region adjacent to muscle-specific genes or as circular extrachromosal episomes (Kotin et al., 1992, Samulski et al., 1991, Schnepp et al., 2005). For productive replication, AAV requires helper viral proteins delivered by adenovirus (Ad) or herpes simplex virus (HSV) that enables the rescue of the AAV genome, DNA replication, and gene expression of the viral proteins. Capsid assembly takes place in the nucleoli of infected cells that are finally redistributed to the nucleoplasm (Hunter and Samulski, 1992, Wistuba et al., 1997). There, virions are colocalized with Rep 78/68-tagged viral DNA. Rep 52/40 proteins are involved in unwinding and transfer of the viral DNA into the empty capsid through pores located at the fivefold axes of symmetry (Bleker et al., 2006, King et al., 2001). Finally, replicated viruses are released by lysis of the host cell.

Recombinant AAV (rAAV) vectors are constructed by replacement of the viral DNA containing the two open reading frames rep and cap with an expression cassette encoding the gene of interest under transcriptional control of a suitable promoter. The ITR sequences required for replication and packaging are the sole remainder of the wild-type virus. For vector production, the structural and nonstructural Rep and Cap proteins must be provided in trans. Today, vectors are usually produced by transfection of a suitable cell line with three vector plasmids: (i) the expression cassette flanked by the ITRs, (ii) the rep cap helper sequences, and (iii) the adenoviral helper plasmid that encodes for the adenoviral E2a, E4, and VA helper genes (Grimm et al., 1999, Xiao et al., 1998a). This allows for the production of replication-deficient, wild-type-free, and adenovirus-free rAAV vectors stocks at adequate titers. To facilitate upscaling of vector production and to generate good manufacturing practice (GMP) compliant rAAV vector stocks for clinical or commercial use, several novel techniques have been proposed (Durocher et al., 2007, Zolotukhin, 2005). Such approaches are based on the generation of stably transfected producer cell lines (Blouin et al., 2004, Clark et al., 1995), suspension cell transfection and transduction techniques (Meghrous et al., 2005, Park et al., 2006, Smith et al., 2009), and even cell-free production of rAAV (Zhou and Muzyczka, 1998). Innovative purification protocols using gradient centrifugation and chromatography (Hermens et al., 1999, Zolotukhin et al., 1999) have contributed to making production and purity of stable rAAV vector stocks feasible even for large-scale production.

AAV vector transduction in most tissues is characterized by a delayed onset of gene expression. Since second strand synthesis is a rate-limiting step in AAV-mediated gene transfer, self-complementary AAV (scAAV) vectors have been generated by deletion of the D-sequence or mutation of one terminal resolution site (trs) sequence located within the ITR's. This results in the production of a high percentage of self-complementary vectors and allows for rapid and increased expression of the transgene. A serious drawback of this approach is a loss of reduced packing capacity (Duque et al., 2009, Fu et al., 2003, McCarty, 2008, McCarty et al., 2003, Zhong et al., 2004).

Vectors based on AAV-2 have been the best studied of all AAV serotype derivatives. Therefore, they have been used not only for countless experimental in vitro transductions but also for a very large number of preclinical studies in animal models. Such in vivo studies yielded promising results ranging from substantial correction to complete cure in models of hemophilia, α1-anti-trypsin deficiency, cystic fibrosis, Duchenne muscular dystrophy, rheumatoid arthritis, and others. Furthermore, AAV has been employed for a variety of anticancer gene therapy approaches. Toward this end, common strategies are based on the delivery of cytotoxic genes, reconstitution of tumor suppressor genes, inhibition of drug resistance, immunotherapy, and antiangiogenesis. AAV vectors have also been used in clinical trials in human patients. So far, at least 70 clinical trials have been approved or completed with AAV-based vectors (Carter, 2005, Coura Rdos and Nardi, 2007, Mueller and Flotte, 2008, Park et al., 2008). However, their broad host tropism and inefficient transduction of many, if not most potential, target tissues after systemic application continue to limit the utility of AAV vector systems for the majority of potential clinical applications in which topical administration of the vector to the target tissue is not feasible.

AAV-2 vectors have the potential to efficiently deliver genes to a broad spectrum of dividing and nondividing cell types and tissues in vitro and in vivo, including skeletal muscle, cardiac muscle, airway epithelium, hepatocytes, brain tissue, and several cancer cell lines (Arruda et al., 2005, Bartlett et al., 1998, Fisher et al., 1997, Flotte et al., 1992, Foust et al., 2009, Hacker et al., 2005, Herzog, 2004, Kaplitt et al., 1994, Palomeque et al., 2007, Snyder et al., 1997, Vassalli et al., 2003, Xiao et al., 1996, Xiao et al., 1997, Xiao et al., 1998a, Xiao et al., 1998b).

The exploitation of the distinct tissue tropism of the various AAV serotypes provides an opportunity to improve the efficiency of gene delivery to specific target tissues. Using alternative serotypes compared to AAV-2, numerous studies have shown superior transduction rates in certain tissue. AAV-1, -3, -5, -6, -7, -8, and -9 seem to be the more efficient for transduction of various tissues including the muscle, liver, heart, the central nervous system, vascular endothelium, arthritic joints, pancreas, cochlear inner hair cells, and the retina, while AAV-5 and -6 seem to be more appropriate for transduction of the lung or the airway epithelium upon local application (Apparailly et al., 2005, Blankinship et al., 2004, Burger et al., 2004, Chen et al., 2005, Halbert et al., 2001, Halbert et al., 2007, Limberis et al., 2009, Liu et al., 2005, Loiler et al., 2003, Riviere et al., 2006, Surace and Auricchio, 2008, Taymans et al., 2007, Vandendriessche et al., 2007, Wang et al., 2004).

If used for systemic administration, AAV-8 and -9 are more sufficient for gene transduction than other serotypes as they can efficiently cross vascular endothelial cell barriers to transduce liver hepatocytes, cardiac and skeletal muscle cells, and various other tissues (Inagaki et al., 2006, Nakai et al., 2005, Pacak et al., 2006, Paneda et al., 2009, Wang et al., 2005, Zincarelli et al., 2008). AAV-9 has some particularly intriguing features if applied systemically. This serotype can deliver genes both to neuronal and nonneuronal central nervous system cells (Foust et al., 2009). The latter finding is of special importance as nonneuronal cells were considered inaccessible to AAV gene transfer before. Further, work by Foust et al. (2009) suggested that AAV-9 has the unique property to cross the blood–brain barrier (BBB) and that the transduction of astrocytes is a receptor-mediated process that occurs via astrocytic endfeet.

Pseudotyping AAV vectors by cross-packaging of an AAV genome into the capsid of another serotype improve transduction of certain tissues in vivo while circumventing problems of preexisting immunity (Kwon and Schaffer, 2008, Wu et al., 2006a, Wu et al., 2006b). However, alternative serotypes or pseudotyped AAV vectors per se are not capable to mediate cell-type specific transduction of AAV or any other gene therapy vector. Some of the specificity problems may be overcome by the use of tissue-specific promoters (Halbert and Miller, 2004, Müller et al., 2003, Ruan et al., 2001, Wang et al., 2008). However, for many cell types, specific promoters that generate adequate expression levels are not available and do not allow for gene transduction in cells that are nonpermissive for AAV infection. Therefore, a plethora of strategies have been developed to manipulate the capsid for redirection of the vector to alternative, cell-type specific receptors.

Section snippets

Chimeric and mosaic capsids

One possible approach to alter the tropism of AAV-derived vectors is the generation of chimeric or mosaic vectors. Chimeric vectors are generated by exchange of certain capsid domains or sequences by such domains or sequences of different serotypes (Wu et al., 2000). Mosaic capsids are generated by mixing the capsid subunits from two different serotypes or capsid mutants. This yields vectors that combine the beneficial features of the originating vector capsids that synergistically enhance gene

Principle of random AAV-display libraries

As described above, detailed functional and structural examination of AAV serotypes has yielded gene delivery vectors with novel tropism and functional properties. However, the functional diversity of serotypes is limited. Therefore, it is pertinent to develop complementary vector engineering tools that can create novel vehicles with a desired set of properties. Even though rational approaches to designing AAV vectors that target specific cells, for example, by insertion of targeting ligands,

Perspectives

The multitude of approaches for AAV targeting is based on the assumption that differences in capsid sequence (and hence structure) can affect binding to cell surface receptors, intracellular trafficking, nuclear transport, uncoating, and even evasion of the AAV-immune responses, thus circumventing neutralizing immunity in the human population and allowing readministration.

Combinatorial and serotype-shuffled capsid library techniques have proved to be powerful strategies in establishing

References (196)

  • A. Hajitou

    A hybrid vector for ligand-directed tumor targeting and molecular imaging

    Cell

    (2006)
  • B. Hauck et al.

    Generation and characterization of chimeric recombinant AAV vectors

    Mol. Ther.

    (2003)
  • K. Inagaki

    Robust systemic transduction with AAV9 vectors in mice: Efficient global cardiac gene transfer superior to that of AAV8

    Mol. Ther.

    (2006)
  • K. Jennings

    Proteasome inhibition enhances AAV-mediated transgene expression in human synoviocytes in vitro and in vivo

    Mol. Ther.

    (2005)
  • P.D. Khare et al.

    Epitope selection from an uncensored peptide library displayed on avian leukosis virus

    Virology

    (2003)
  • P.D. Khare et al.

    Avian leukosis virus is a versatile eukaryotic platform for polypeptide display

    Virology

    (2003)
  • M.P. Limberis et al.

    Transduction efficiencies of novel AAV vectors in mouse airway epithelium in vivo and human ciliated airway epithelium in vitro

    Mol. Ther.

    (2009)
  • Y. Liu

    Specific and efficient transduction of cochlear inner hair cells with recombinant adeno-associated virus type 3 vector

    Mol. Ther.

    (2005)
  • S. Marchio

    Aminopeptidase A is a functional target in angiogenic blood vessels

    Cancer Cell

    (2004)
  • D.M. McCarty

    Self-complementary AAV vectors; advances and applications

    Mol. Ther.

    (2008)
  • S. Michelfelder

    Vectors selected from adeno-associated viral display peptide libraries for leukemia cell-targeted cytotoxic gene therapy

    Exp. Hematol.

    (2007)
  • B. Akache et al.

    The 37/67-kilodalton laminin receptor is a receptor for adeno-associated virus serotypes 8, 2, 3, and 9

    J. Virol.

    (2006)
  • B. Akache et al.

    A two-hybrid screen identifies cathepsins B and L as uncoating factors for adeno-associated virus 2 and 8

    Mol. Ther.

    (2006)
  • F. Apparailly

    Adeno-associated virus pseudotype 5 vector improves gene transfer in arthritic joints

    Hum. Gene Ther.

    (2005)
  • W. Arap et al.

    Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model

    Science (New York, NY)

    (1998)
  • W. Arap

    Steps toward mapping the human vasculature by phage display

    Nat. Med.

    (2002)
  • W. Arap

    Targeting the prostate for destruction through a vascular address

    Proc. Natl. Acad. Sci. USA

    (2002)
  • A. Asokan et al.

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

    J. Virol.

    (2006)
  • U. Bantel-Schaal et al.

    Adeno-associated virus type 5 exploits two different entry pathways in human embryo fibroblasts

    J. Gen. Virol.

    (2009)
  • J.S. Bartlett et al.

    Selective and rapid uptake of adeno-associated virus type 2 in brain

    Hum. Gene Ther.

    (1998)
  • J.S. Bartlett et al.

    Targeted adeno-associated virus vector transduction of nonpermissive cells mediated by a bispecific F(ab′ gamma)(2) antibody

    Nat. Biotechnol.

    (1999)
  • J.S. Bartlett et al.

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

    J. Virol.

    (2000)
  • M. Binder

    Identification of their epitope reveals the structural basis for the mechanism of action of the immunosuppressive antibodies basiliximab and daclizumab

    Cancer Res.

    (2007)
  • S. Bleker et al.

    Mutational analysis of narrow pores at the fivefold symmetry axes of adeno-associated virus type 2 capsids reveals a dual role in genome packaging and activation of phospholipase A2 activity

    J. Virol.

    (2005)
  • S. Bleker et al.

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

    J. Virol.

    (2006)
  • V. Blouin

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

    J. Gene Med.

    (2004)
  • E.T. Boder et al.

    Yeast surface display for screening combinatorial polypeptide libraries

    Nat. Biotechnol.

    (1997)
  • M.P. Boyle

    Membrane-associated heparan sulfate is not required for rAAV-2 infection of human respiratory epithelia

    Virol. J.

    (2006)
  • K. Bupp et al.

    Targeting a retroviral vector in the absence of a known cell-targeting ligand

    Hum. Gene Ther.

    (2003)
  • B.J. Carter

    Adeno-associated virus vectors in clinical trials

    Hum. Gene Ther.

    (2005)
  • S. Chen

    Efficient transduction of vascular endothelial cells with recombinant adeno-associated virus serotype 1 and 5 vectors

    Hum. Gene Ther.

    (2005)
  • K.R. Clark et al.

    Cell lines for the production of recombinant adeno-associated virus

    Hum. Gene Ther.

    (1995)
  • S. Coura Rdos et al.

    The state of the art of adeno-associated virus-based vectors in gene therapy

    Virol. J.

    (2007)
  • W. Ding et al.

    Intracellular trafficking of adeno-associated viral vectors

    Gene Ther.

    (2005)
  • G. Di Pasquale

    Identification of PDGFR as a receptor for AAV-5 transduction

    Nat. Med.

    (2003)
  • D. Duan et al.

    Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus

    J. Clin. Invest.

    (2000)
  • H.M. Ellerby

    Anti-cancer activity of targeted pro-apoptotic peptides

    Nat. Med.

    (1999)
  • K.J. Fisher

    Recombinant adeno-associated virus for muscle directed gene therapy

    Nat. Med.

    (1997)
  • T.R. Flotte

    Gene expression from adeno-associated virus vectors in airway epithelial cells

    Am. J. Respir. Cell Mol. Biol.

    (1992)
  • K.D. Foust

    Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes

    Nat. Biotechnol.

    (2009)
  • Cited by (0)

    View full text