Adeno-associated Virus Type 2 Vectors Research Articles

Vasoinhibin is Generated and Promotes Inflammation in Mild Antigen-induced Arthritis.

Inflammatory arthritis defines a family of diseases influenced by reproductive hormones. Vasoinhibin, a fragment of the hormone prolactin (PRL), has antiangiogenic and proinflammatory properties. We recently showed that vasoinhibin reduces joint inflammation and bone loss in severe antigen-induced arthritis (AIA) by an indirect mechanism involving the inhibition of pannus vascularization. This unexpected finding led us to hypothesize that a severe level of inflammation in AIA obscured the direct proinflammatory action of vasoinhibin while allowing the indirect anti-inflammatory effect via its antiangiogenic properties. In agreement with this hypothesis, here we show that the intra-articular injection of an adeno-associated virus type-2 vector encoding vasoinhibin reduced joint inflammation in a severe AIA condition, but elevated joint inflammation in a mild AIA model. The proinflammatory effect, unmasked in mild AIA, resulted in joint swelling, enhanced leukocyte infiltration, and upregulation of expression of genes encoding proinflammatory mediators (Il1b, Il6, Inos, Mmp3), adhesion molecule (Icam1), and chemokines (Cxcl1, Cxcl2, Cxcl3, Ccl2). Furthermore, vasoinhibin induced the expression of proinflammatory mediators and chemokines in cultured synovial fibroblasts through nuclear factor-κB. Finally, matrix metalloproteases and cathepsin D, upregulated in the arthritic joint, cleaved PRL to vasoinhibin, and vasoinhibin levels increased in the circulation of mice subjected to AIA. We suggest that vasoinhibin is generated during inflammatory arthritis and acts on synovial fibroblasts and endothelial cells to initially promote and later inhibit inflammation, respectively. These opposite effects may work together to help keep joint inflammation under balance.

Viral vector purification with thermoresponsive-anionic mixed polymer brush modified beads-packed column

Viral vectors have attracted attention as a potential new therapeutic modality for gene therapy. In this study, we developed a temperature-modulated viral vector purification column using a mixed polymer brush composed of thermoresponsive and anionic polymers as packing material ligands. The mixed polymer brushes were modified on silica beads using a combination of reversible addition − fragmentation chain transfer (RAFT) polymerization of 2-acrylamido-2-methylpropanesulfonic acid (AMPS) and subsequent atom transfer radical polymerization (ATRP) of N-isopropylacrylamide (NIPAAm). The temperature-modulated zeta potential change in the prepared mixed polymer brush was attributed to the PNIPAAm shrinking and exposing of PAMPS. The prepared mixed polymer brush modified beads were used as packing materials, and the elution behavior of the adeno associated virus type 2 (AAV2) vector was observed. The AAV2 vector was adsorbed on the mixed polymer brush by electrostatic and hydrophobic interactions at 40 °C. By reducing the temperature to 5 °C, adsorbed AAV2 vector on the mixed polymer brush was desorbed and eluted from the column due to lowering the electrostatic and hydrophobic interactions between the AAV2 vector and mixed polymer brush. The AAV2 vector was separated from bovine serum proteins as a contaminant using the column. The ability to infect cells was maintained by the recovered AAV2 vectors from the column. Thus, the developed column would be beneficial for the simple purification of viral vectors.

Generation of Helper Plasmids Encoding Mutant Adeno-associated Virus Type 2 Capsid Proteins with Increased Resistance against Proteasomal Degradation.

Adeno-associated virus type 2 (AAV2) vectors are widely used for both experimental and clinical gene therapy. A recent research has shown that the performance of these vectors can be greatly improved by substitution of specific surface-exposed tyrosine residues with phenylalanines. In this study, a fast and simple method is presented to generate AAV2 vector helper plasmids encoding capsid proteins with single, double or triple Y→F mutations. A one-step, high-fidelity polymerase chain reaction (PCR) cloning procedure involving the use of two partially overlapping primers to amplify a circular DNA template was applied to produce AAV2 cap genes encoding VP1 mutants with Y→F substitutions in residues 444, 500 or 730. The resulting constructs were used to make the different double and triple mutant by another round of PCR (Y444500F mutant), subcloning (Y444730F and Y500730F mutants) or a combination of both techniques (Y444500730F mutant). Nucleotide sequence analysis revealed successful introduction of the desired mutations in the AAV2 cap gene and showed the absence of any unintended mutations in the DNA fragments used to assemble the final set of AAV2 vector helper plasmids. The correctness of these plasmids was further confirmed by restriction mapping. PCR-based, single-step site-directed mutagenesis of circular DNA templates is a highly efficient and cost-effective method to generate AAV2 vector helper plasmids encoding mutant Cap proteins for the production of vector particles with increased gene transfer efficiency.

Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences

Viral vectors based on various naturally occurring adeno-associated virus (AAV) serotypes are among the most promising tools in human gene therapy. For the production of recombinant AAV (rAAV) vectors, researchers are focusing predominantly on cross-packaging an artificial AAV genome based on serotype 2 (AAV2) into capsids derived from other serotypes. Within the packaged genome the inverted terminal repeats (ITRs) are the only cis-acting viral elements required for rAAV vector generation and depict the lowest common denominator of all AAV2-derived vector genomes. Up to now, no quantitative PCR (qPCR) for the detection and quantification of AAV2 ITRs could be established because of their extensive secondary hairpin structure formation. Current qPCR-based methods are therefore targeting vector-encoded transgenes or regulatory elements. Herein we establish a molecular biological method that allows accurate and reproducible quantification of AAV2 genomes on the basis of an AAV2 ITR sequence-specific qPCR. Primers and labeled probe are located within the ITR sequence and have been designed to detect both wild-type AAV2 and AAV2-based vectors. This method is suitable for detecting single-stranded DNA derived from AAV2 vector particles and double-stranded DNA derived from vector plasmids. The limit of detection has been determined as 50 ITR sequence copies per reaction, by comparison with a plasmid standard. In conclusion, this method describes the first qPCR system facilitating the detection and quantification of AAV2 ITR sequences. Because this method can be used universally for all AAV2 genome-based vectors, it will significantly simplify rAAV2 vector titrations in the future.

Tetradecanoylphorbol-13-acetate (TPA) significantly increases AAV2/5 transduction of human neuronal cells in vitro

Recombinant adeno-associated virus type 2 (AAV2) vectors have shown great promise in current ophthalmology clinical trials targeting gene delivery to the retinal pigment epithelium (RPE). To treat the majority of retinal diseases, however, gene delivery would need to be targeted to photoreceptor neurons of the outer retina. AAV2 pseudotyped with the AAV5 capsid (AAV2/5) has shown far greater transduction efficiency in photoreceptors compared to standard AAV2 vectors. For clinical trial applications using gene therapy, it is helpful to generate pre-clinical data in human cells wherever possible. There is however very little data, indeed some controversy, as to whether AAV2/5 can be used effectively in differentiated neurons in culture. In this study we show that transduction of the human neuroblastoma cell line SH-SY5Y with recombinant AAV2/5 expressing GFP is well tolerated. Furthermore, we explore the mechanism whereby exposure to retinoic acid (RA) and the phorbol ester 12-O-Tetradecanoylphorbol-13- acetate (TPA) can induce this cell line to differentiate into a stable population of human neurons, with significantly increased levels of AAV2/5 transduction. These observations may be helpful for assessing AAV2/5 vectors in vitro, particularly where it is necessary to generate pre-clinical data for clinical trials of gene therapy to the human central nervous system.

High-efficiency Transduction and Correction of Murine Hemophilia B Using AAV2 Vectors Devoid of Multiple Surface-exposed Tyrosines

Elimination of specific surface-exposed single tyrosine (Y) residues substantially improves hepatic gene transfer with adeno-associated virus type 2 (AAV2) vectors. Here, combinations of mutations in the seven potentially relevant Y residues were evaluated for further augmentation of transduction efficiency. These mutant capsids packaged viral genomes to similar titers and retained infectivity. A triple-mutant (Y444+500+730F) vector consistently had the highest level of in vivo gene transfer to murine hepatocytes, approximately threefold more efficient than the best single-mutants, and ~30-80-fold higher compared with the wild-type (WT) AAV2 capsids. Improvement of gene transfer was similar for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors, indicating that these effects are independent of viral second-strand DNA synthesis. Furthermore, Y730F and triple-mutant vectors provided a long-term therapeutic and tolerogenic expression of human factor IX (hF.IX) in hemophilia B (HB) mice after administration of a vector dose that only results in subtherapeutic and transient expression with WT AAV2 encapsidated vectors. In summary, introduction of multiple tyrosine-mutations into the AAV2 capsid results in vectors that yield at least 30-fold improvement of transgene expression, thereby lowering the required therapeutic dose and potentially vector-related immunogenicity. Such vectors should be attractive for treatment of hemophilia and other genetic diseases.

Real-time MR Imaging With Gadoteridol Predicts Distribution of Transgenes After Convection-enhanced Delivery of AAV2 Vectors

Gene therapies that utilize convention-enhanced delivery (CED) will require close monitoring of vector infusion in real time and accurate prediction of drug distribution. The magnetic resonance imaging (MRI) contrast agent, Gadoteridol (Gd), was used to monitor CED infusion and to predict the expression pattern of glial cell line-derived neurotrophic factor (GDNF) protein after administration of adeno-associated virus type 2 (AAV2) vector encoding human pre-pro-GDNF complementary DNA. The nonhuman primate (NHP) thalamus was utilized for modeling infusion to allow delivery of volumes more relevant to planned human studies. AAV2 encoding human aromatic L-amino acid decarboxylase (AADC) was coinfused with AAV2-GDNF/Gd to confirm regions of AAV2 transduction versus extracellular GDNF diffusion. There was a close correlation between Gd distribution and GDNF or AADC expression, and the ratios of expression areas of GDNF or AADC versus Gd were both close to 1. Our data support the use of Gd and MRI to monitor AAV2 infusion via CED and to predict the distribution of GDNF protein after AAV2-GDNF administration.

DNA Shuffling of Adeno-associated Virus Yields Functionally Diverse Viral Progeny

Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to engineer enhanced viral vectors. Here, we significantly extend this capability by performing high-efficiency in vitro recombination to create a large (10(7)), diverse library of random chimeras of numerous parent AAV serotypes (AAV1, 2, 4-6, 8, and 9). In order to analyze the extent to which such highly chimeric viruses can be viable, we selected the library for efficient viral packaging and infection, and successfully recovered numerous novel chimeras. These new viruses exhibited a broad range of cell tropism both in vitro and in vivo and enhanced resistance to human intravenous immunoglobulin (IVIG), highlighting numerous functional differences between these chimeras and their parent serotypes. Thus, directed evolution can potentially yield unlimited numbers of new AAV variants with novel gene-delivery properties, and subsequent analysis of these variants can further extend basic knowledge of AAV biology.

Site-specific Modification of AAV Vector Particles With Biophysical Probes and Targeting Ligands Using Biotin Ligase

We have developed a highly specific and robust new method for labeling adeno-associated virus (AAV) vector particles with either biophysical probes or targeting ligands. Our approach uses the Escherichia coli enzyme biotin ligase (BirA), which ligates biotin to a 15-amino-acid biotin acceptor peptide (BAP) in a sequence-specific manner. In this study we demonstrate that by using a ketone isotere of biotin as a cofactor we can ligate this probe to BAP-modified AAV capsids. Because ketones are absent from AAV, BAP-modified AAV particles can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage modification methodology in the context of a mammalian cell lysate for the labeling of AAV vector particles with various fluorophores, and for the attachment of a synthetic cyclic arginine-glycine-aspartate (RGD) peptide (c(RGDfC)) to target integrin receptors that are present on neovasculature. Fluorophore labeling allowed the straightforward determination of intracellular particle distribution. Ligand conjugation mediated a significant increase in the transduction of endothelial cells in vitro, and permitted the intravascular targeting of AAV vectors to tumor-associated vasculature in vivo. These results suggest that this approach holds significant promise for future studies aimed at understanding and modifying AAV vector-cellular interactions.

Adeno-Associated Virus Type 2 Contains an Integrin α5β1 Binding Domain Essential for Viral Cell Entry

Integrins have been implicated as coreceptors in the infectious pathways of several nonenveloped viruses. For example, adenoviruses are known to interact with alphaV integrins by virtue of a high-affinity arginine-glycine-aspartate (RGD) domain present in the penton bases of the capsids. In the case of adeno-associated virus type 2 (AAV2), which lacks this RGD motif, integrin alphaVbeta5 has been identified as a coreceptor for cellular entry. However, the molecular determinants of AAV2 capsid-integrin interactions and the potential exploitation of alternative integrins as coreceptors by AAV2 have not been established thus far. In this report, we demonstrate that integrin alpha5beta1 serves as an alternative coreceptor for AAV2 infection in human embryonic kidney 293 cells. Such interactions appear to be mediated by a highly conserved domain that contains an asparagine-glycine-arginine (NGR) motif known to bind alpha5beta1 integrin with moderate affinity. The mutation of this domain reduces transduction efficiency by an order of magnitude relative to that of wild-type AAV2 vectors in vitro and in vivo. Further characterization of mutant and wild-type AAV2 capsids through transduction assays in cell lines lacking specific integrins, cell adhesion studies, and cell surface/solid-phase binding assays confirmed the role of the NGR domain in promoting AAV2-integrin interactions. Molecular modeling studies suggest that NGR residues form a surface loop close to the threefold axis of symmetry adjacent to residues previously implicated in binding heparan sulfate, the primary receptor for AAV2. The aforementioned results suggest that the internalization of AAV2 in 293 cells might follow a "click-to-fit" mechanism that involves the cooperative binding of heparan sulfate and alpha5beta1 integrin by the AAV2 capsids.

Insertional Mutagenesis at Positions 520 and 584 of Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Generation of AAV2 Vectors with Eliminated Heparin- Binding Ability and Introduced Novel Tropism

Recombinant adeno-associated virus (AAV) vectors are promising in the context of gene therapy because of their ability to mediate efficient gene transfer and stable gene expression. AAV2 uses heparin sulfate as its primary receptor, which is widely expressed on the various tissues and organs. This limits the application of AAV2 in targeting specific tissues. To make an AAV2 vector with modified tropism, we constructed various AAV2 capsid mutants by inserting RGD-4C peptide at position 520 and/or at position 584. Eight mutants were generated, identified, and characterized. Heparin-binding ability was completely abrogated in five mutants, and partially reduced in three mutants. Solid-phase ELISA and gene transduction assays confirmed that the novel tropism is determined by the introduced RGD epitope, which binds to cellular integrin receptor. Our observations suggest that simultaneous modification at both sites, tentatively involved in heparin binding, results in altered tropism and improved transduction efficiency in vitro.

Insertional Mutagenesis at Positions 520 and 584 of Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Generation of AAV2 Vectors with Eliminated Heparin-Binding Ability and Introduced Novel Tropism

Insertional Mutagenesis at Positions 520 and 584 of Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Generation of AAV2 Vectors with Eliminated Heparin-Binding Ability and Introduced Novel Tropism

Convection-Enhanced Delivery of Adeno-Associated Virus Type 2 (AAV2) into the Striatum and Transport of AAV2 Within Monkey Brain

Adeno-associated virus type 2 (AAV2)-based vectors are promising transgene carriers for experimental gene therapy treatments of brain diseases. However, detailed evaluation of transgene distribution, trafficking, and transport within the brain is of the utmost importance before applying any type of gene therapy in humans. We examined the distribution of AAV2-thymidine kinase (AAV2-TK) and AAV2-aromatic L-amino acid decarboxylase (AAV2-AADC) in monkey brain after convection-enhanced delivery (CED). The AADC group consisted of two 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys that received unilateral infusions of AAV2-AADC into six sites in the right hemisphere. The TK group consisted of three monkeys that received bilateral CED infusion of AAV2-TK into the putamen; one side in all three monkeys was coinfused with heparin. Six weeks after AAV delivery, the brains were collected and processed for immunohistochemical staining. Volumetric measurement of TK distribution showed that at least 75% of the putamen could be covered by a single infusion of the vector; however, no effects of heparin coadministration were found, most likely because of the already robust gene transfer achieved by CED. Interestingly, TK- and AADCimmunoreactive cells were also present outside the striatum, in the globus pallidus, subthalamic nucleus, thalamus, and substantia nigra. CED proved to be an efficient method for delivery of the AAV2 vector. Detection of the transgenes in brain structures distant from the site of injection emphasizes the potential for gene transport, and the advantages and disadvantages of CED for gene therapy deserve further study.

Improved neuronal transgene expression from an AAV‐2 vector with a hybrid CMV enhancer/PDGF‐β promoter

Adeno-associated virus type 2 (AAV-2) vectors are highly promising tools for gene therapy of neurological disorders. After accommodating a cellular promoter, AAV-2 vectors are able to drive sustained expression of transgene in the brain. This study aimed to develop AAV-2 vectors that also facilitate a high level of neuronal expression by enhancing the strength of a neuron-specific promoter, the human platelet-derived growth factor beta-chain (PDGF) promoter. A hybrid promoter approach was adopted to fuse the enhancer of human cytomegalovirus immediately early (CMV) promoter to the PDGF promoter. In cultured cortex neurons, AAV-2 vectors containing the hybrid promoter augmented transgene expression up to 20-fold over that mediated by titer-matched AAV-2 vectors with the PDGF promoter alone and 4-fold over the CMV enhancer/promoter. Injection of AAV-2 vectors with the hybrid promoter into the rat striatum resulted in neuron-specific transgene expression, the level of which was about 10-fold higher than those provided by the two control AAV-2 expression cassettes at 4 weeks post-injection and maintained for at least 12 weeks. Gene expression in the substantia nigra through possible retrograde transport of the AAV-2 vectors injected into the striatum was not obvious. After direct injection of AAV-2 vectors into the substantia nigra, transgene expression driven by the hybrid promoter was observed specifically in dopaminergic neurons and its level was about 3 and 17 times higher than that provided by the PDGF promoter alone and the CMV enhancer/promoter, respectively. Enhanced transgene capacity plus neuron-specificity of the AAV-2 vectors developed in this study might prove valuable for gene therapy of Parkinson's disease.

522. Increased Expression of OCT4, SOX2 and FGF4 Genes Following Injection of Embryonic Stem Cell into Damaged Murine Brain

Many studies have shown that vascular endothelial growth factor (VEGF) plays a critical role in development of choroidal and retinal neovascularization in diseases such as age-related macular degeneration and proliferative diabetic retinopathy. Therefore, antagonizing VEGF is a potentially useful strategy for treating such disorders. In the current study we are evaluating the relative efficacy of various truncated and full-length versions of soluble VEGF receptors, VEGF-R1 (sFlt-1) and VEGF-R2 (sKDR), toward inhibiting neovascularization. Biological potency of various versions was ranked in vitro using Human Vascular Endothelial Cell (HUVEC) proliferation and migration assays. Candidate genes were inserted into adenovirus type 2 (Ad2) or adeno-associated virus type 2 (AAV2) gene transfer vectors. The efficacy of Ad2 and AAV2 vectors is being evaluated in vivo by intravitreal injection in the mouse retinopathy of prematurity (ROP) model. The extent of retinal neovascularization is measured both by fluorescein angiography and histology. Several versions of sFlt-1 were potent inhibitors of VEGF-dependent HUVEC proliferation and migration assays. Our preliminary results show that intravitreal delivery of 5x108 particles of Ad2/sFlt-1 significantly decreased retinal neovascularization in the ROP model. Evaluation of safety and efficacy following single and multiple vector administrations is underway. Viral vector mediated intra-ocular delivery of sFlt-1 appears to be a promising tool for the inhibition of retinal neovascularization.

536. Design and Production of an AAV2 Vector Encoding a Chimeric cDNA Allowing Efficient Secretion of Functional Human Neurturin from Human Cells

Neurturin (NTN) belongs to the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFL) and has been found to be a potent survival factor for midbrain dopaminergic neurons. Therefore, it is an attractive candidate protein for the gene therapy of Parkinson's disease. Like other GFLs, mature NTN is secreted from producer cells upon cleavage of a precursor protein at an RXXR site recognized by furin-like convertases. We have produced adeno-associated virus type 2 (AAV2)-based vectors encoding human NTN (hNTN) under the control of the CAG promoter and observed that upon transduction of cells in vitro or in vivo, hNTN expression remained essentially cell bound. Only low levels of secreted protein could be detected in the extra-cellular compartment in rat brain. In tissue culture supernatants, secreted protein was not detected. In contrast to these observations, rodent NTN has been shown to be efficiently secreted from cells.

Second generation adeno-associated virus type 2-based gene therapy systems with the potential for preferential integration into AAVS1.

Adeno-associated virus type 2 (AAV-2) is a non-pathogenic human parvovirus that is being developed as a gene therapy vector for the treatment of numerous diseases. One property of wild-type AAV-2, that is highly desirable in a gene therapy vector, is its ability to preferentially integrate its DNA into a 4 kilobase region of human chromosome 19, designated AAVS1. One disadvantage of AAV-2 is its relatively small packaging capacity, approximately 4.7 kilobases. Because of this size limitation, the AAV-2 rep and cap genes were removed from first-generation AAV-2-based gene therapy vectors to make room for the therapeutic or marker gene. It was later discovered that the rep gene, or at least one of its products, the Rep68 or Rep78 protein, is required for preferential integration of AAV-2. Recent developments in AAV-2 gene therapy vector construction allow the inclusion of the rep gene into a second generation of AAV-2-based gene therapy systems. These new systems fall into four major categories: plasmid-based systems, co-transduction with multiple AAV-2 vectors, incorporation of the AAV-2 vector into a larger virus, and in vitro packaging. These systems not only allow the inclusion of the rep gene, they also allow the delivery of larger therapeutic genes.

A genetic screen identifies a cellular regulator of adeno-associated virus.

Adeno-associated virus type 2 (AAV2) is a human parvovirus that has attracted attention as a vector for gene transfer. Replication and site-specific integration of the wild-type virus requires binding of the AAV2 Rep proteins to a cis-regulatory element named the Rep recognition sequence (RRS). RRS motifs are found within the cellular AAVS1 integration locus, the viral p5 promoter, and the inverted terminal repeats (ITRs). Here we report the design of a genetic screen based on the yeast one-hybrid assay to identify cellular RRS-binding proteins. We show that the human zinc finger 5 protein (ZF5) binds specifically to RRS motifs in vitro and in vivo. ZF5 is a highly conserved and ubiquitously expressed transcription factor that contains five C-terminal zinc fingers and an N-terminal POZ domain. Ectopic expression of ZF5 leads to an ITR-dependent repression of the autologous p5 promoter and reduces both AAV2 replication and the production of recombinant AAV2. By using deletion and substitution mutants we show that two different domains of ZF5 contribute to AAV2 repression. Negative regulation of the p5 promoter requires the POZ domain, whereas viral replication is inhibited by the zinc finger domain, likely by competing with Rep for binding to the ITR. Identification and characterization of proteins that bind the ITR, the only viral genetic element retained in AAV2 vectors, will lead to new insights into the unique life cycle of AAV2 and will suggest improvements important for its application as a gene therapy vector.

Insertional mutagenesis of the adeno-associated virus type 2 (AAV2) capsid gene and generation of AAV2 vectors targeted to alternative cell-surface receptors.

Recombinant adeno-associated virus (AAV) vectors are of interest in the context of gene therapy because of their ability to mediate efficient transfer and stable expression of therapeutic genes in a wide variety of tissues. However, AAV-mediated gene delivery to specific cell populations is often precluded by the widespread distribution of heparan sulfate proteoglycan (HSPG), the primary cellular receptor for the virus. Conversely, an increasing number of cell types are being identified that do not express HSPG and are therefore poor targets for AAV-mediated gene transfer. To address these issues, we have developed strategies to physically modify AAV vectors and allow efficient, HSPG-independent, receptor-targeted infection. We began by generating a series of 38 virus capsid mutants containing peptide insertions at 25 unique sites within the AAV capsid protein. The mutant viruses were characterized on the basis of their phenotypes and grouped into three classes: class I mutants (4 of 38) did not assemble particles; class II mutants (14 of 38) assembled noninfectious particles; and class III mutants (20 of 38) assembled fully infectious particles. We examined the HSPG-binding characteristics of the class II mutants and showed that a defect in receptor binding was a common reason for their lack of infectivity. The display of foreign peptide epitopes on the surface of the mutant AAV particles was found to be highly dependent on the inclusion of appropriate scaffolding sequences. Optimal scaffolding sequences and five preferred sites for the insertion of targeting peptide epitopes were identified. These sites are located within each of the three AAV capsid proteins, and thus display inserted epitopes 3, 6, or 60 times per vector particle. Modified AAV vectors displaying a 15-amino acid peptide, which binds to the human luteinizing hormone receptor (LH-R), were generated and assessed for their ability to target gene delivery to receptor-bearing cell lines. Titers of these mutant vectors were essentially the same as wild-type vector. The LH-R-targeted vector was able to transduce ovarian cancer cells (OVCAR-3) in an HSPG-independent manner. Furthermore, transduction was shown to proceed via the LH-R and therefore treatment of OVCAR-3 cells with progesterone, to increase LH-R expression, accordingly increased LH mutant-mediated gene transfer. This technology may have a significant impact on the use of AAV vectors for human gene therapy.

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

Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be beta-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.