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PubMed1998

The use of heterologous promoters for adeno-associated virus (AAV) protein expression in AAV vector production.

Although adeno-associated virus (AAV) vectors are potentially useful gene transfer vehicles for gene therapy, the vector production system is currently at the developmental stage. We constructed AAV helper plasmids (Rep and Cap expression plasmids) by replacing a native AAV promoter, p5, with various heterologous promoters to examine whether the efficiency of AAV vector production was influenced by modulating the AAV protein expression pattern. The helper plasmids containing heterologous promoters (EF, CMV, SV40, B19p6, and CAG promoters, respectively) expressed Rep78/68 more efficiently than a conventional helper plasmid (pIM45), but the expression of Rep52/40 and Cap decreased, resulting in a significant reduction in AAV vector production. Furthermore, the efficiency of vector production never fully recovered even if the Cap proteins were supplied by an additional expression plasmid. A large amount of Rep78/68 and/or a reduced level of Rep52/40 may have deleterious effects on AAV vector production. The present findings will aid in the development of a more efficient AAV vector production system.

Capsid↗

PubMed2009

Reducing the risk of adeno-associated virus (AAV) vector mobilization with AAV type 5 vectors.

Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV type 2 (AAV2). Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to "mobilization" of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates. To investigate this possibility, we flanked a green fluorescent protein transgene with type 2 and, the most divergent AAV serotype, type 5 TRs (TR2 or TR5). Consistent with AAV clades, AAV5 specifically replicated TR5 vectors, while AAV2 and AAV6 replicated TR2-flanked vectors. To exploit this specificity, we created a TR5 vector production system for Cap1 to Cap5. Next, we showed that persisting recombinant AAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper. Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2-flanked vector but not a TR5-flanked vector upon adenoviral superinfection. Based on these data and the relative prevalence of wt AAV serotypes in the population, we propose that TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use.

Animals↗

PubMed2005

Serotype-specific replicating AAV helper constructs increase recombinant AAV type 2 vector production.

One of the major limitations of the use of adeno-associated virus (AAV) as a tool for gene therapy is the difficulty in providing sufficient quantities of the virus for pre-clinical and clinical trials. Here, we report a novel approach for amplifying AAV helper functions, which mimics the normal replication of wild type (wt) AAV resulting in a high yield of AAV vectors. Cotransfection of replicating but non-packaging AAV helper constructs in the presence of adenovirus (Ad) produces a high level of Rep and Cap proteins. Yield of AAV2/GFP vector obtained from this helper DNA replication system was approximately 20-fold higher than traditional methods. Molecular analysis suggested that virus yield was associated with capsid protein production. The transfection ratio was optimized using these novel helper constructs, resulting in an additional 2-fold increase in vector yield without presence of replication competent AAV (rcAAV). This strategy supports development of AAV packaging systems that retain normal virus replication capability without helper virus encapsidation.

Cell Line↗

PubMed2001

Adeno-associated virus (AAV) site-specific recombination does not require a Rep-dependent origin of replication within the AAV terminal repeat.

Adeno-associated virus (AAV) is the only known eukaryotic virus capable of targeted integration in human cells. An AAV Rep binding element (RBE) and terminal resolution site (trs) identical to the viral terminal repeats required for AAV DNA replication are located on chromosome (ch) 19. Both ch-19 RBE and trs elements have been shown to be essential for viral targeting to this locus. To characterize the role of the AAV inverted terminal repeat (ITR) cis-acting sequences in targeted integration an AAV trs mutant incapable of supporting viral replication was tested. Wild-type and mutant substrates were assayed for targeted integration after cotransfection in the presence or absence of Rep. Our results demonstrated that, in the presence of Rep78, both ITR substrates targeted to ch-19 with similar frequency. Molecular characterization of the mutant ITR integrants confirmed the presence of the trs mutation in the majority of samples tested. Complementation analysis confirmed that the mutant targeted viral genomes were unable to rescue and replicate. In addition, Rep78 induced extensive rearrangement and amplification of ch-19 sequences independent of wild-type or mutant targeting substrate. These studies demonstrate that Rep-dependent nicking of the viral cis-acting trs sequence is not a prerequisite for site-specific recombination and suggests AAV targeting is mediated by Rep78/68-dependent replication from the ch-19 origin of replication (ori). These studies have significant impact toward the understanding of AAV site-specific recombination and the development of targeting vectors.

Chromosomes, Human, Pair 19↗

PubMed1994

Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.

AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration.

Bacterial Proteins↗

PubMed2006

Prevalence of neutralizing antibodies against adeno-associated virus (AAV) types 2, 5, and 6 in cystic fibrosis and normal populations: Implications for gene therapy using AAV vectors.

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy directed to the lungs, in particular for treatment of cystic fibrosis (CF). In animal models of lung gene transfer, neutralizing antibodies in serum made in response to vector exposure have been associated with a partial to complete block to repeat transduction by vectors with the same capsid type, thus transduction by AAV vectors might be inefficient in humans previously exposed to the same AAV type. AAV type 2 (AAV2) has been used in clinical trials of lung gene transfer, but AAV5 and AAV6 have been shown to mediate more efficient transduction in rodent lungs and in cultured human airway epithelia compared with that of AAV2. Here we have measured neutralizing antibodies against AAV type 2, 5, and 6 vectors in serum from children and adults with CF, and from normal adults. About 30% of adults were seropositive for AAV2, 20-30% were seropositive for AAV6, and 10-20% were seropositive for AAV5. CF children were seropositive for AAV type 2, 5, or 6 at rates of 4-15%. All individuals seropositive for AAV6 were also seropositive for AAV2, and the AAV6 titer was low compared with the AAV2 titer. AAV5-positive sera were lower both in titers and rates than those seen for AAV6. The results indicate that AAV type 2, 5 or 6 exposure is low in CF and control populations and even lower in CF children.

Adolescent↗

PubMed2006

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

In this study, recombinant AAV vectors pseudotyped with viral capsids derived from AAV serotypes 7 and 8 were evaluated for gene transfer in the murine striatum relative to vectors pseudotyped with AAV serotypes 2, 5, and 6. In comparison with rAAV serotype 2, pseudotyped vectors derived from AAV-7 and AAV-8 have increased transduction efficiency in the murine CNS, with the rank order rAAV-7 > rAAV-8 > rAAV-5 > rAAV-2 = rAAV-6, with all vectors demonstrating a marked tropism for neuronal transduction. Pseudotyped rAAV vector gene transfer in the brain after preimplantation of a murine 4C8 glioblastoma tumor was also evaluated. Efficiency of gene transfer to the orthotopic tumor was increased when using AAV-6, -7, and -8 capsid proteins in comparison with serotype 2, with the order rAAV-8 = rAAV-7 > rAAV-6 > rAAV-2 > rAAV-5. The increased gene transfer efficiency of rAAV vectors pseudotyped with the rAAV-8 capsid also provided enhanced therapeutic efficacy in a mouse model of glioblastoma multiforme, using vectors encoding an inhibitor of the vascular endothelial growth factor pathway. These studies demonstrate that rAAV vectors pseudotyped with capsids derived from AAV serotypes 7 and 8 provide enhanced gene transfer in the murine CNS and may offer increased therapeutic efficacy in the treatment of neurological disease.

Animals↗

PubMed1995

Negative regulation of the adeno-associated virus (AAV) P5 promoter involves both the P5 rep binding site and the consensus ATP-binding motif of the AAV Rep68 protein.

Transcript levels from the P5 promoter of adeno-associated virus type 2 (AAV) are negatively regulated by the AAV Rep78 and Rep68 proteins in the absence of helper virus. We have identified a Rep-responsive negative cis element of the P5 promoter between the P5 TATA box and transcription start site by using 5' and 3' deletions of the P5 promoter fused to the chloramphenicol acetyltransferase gene. This element contains four imperfect GAGC repeats similar to the Rep recognition sequences (RRSs) in the AAV inverted terminal repeats and in the AAV preferred integration locus in chromosome 19. Band shift analyses showed that human 293 cell nuclear extracts containing Rep68 or Rep68/K340H, a putative nucleoside triphosphate (NTP)-binding-site mutant of Rep68, formed Rep-specific complexes with this P5 RRS DNA. Within the P5 RRS, mutation of a cytosine at position 273 in the AAV sequence to guanine abolished Rep68 binding to the DNA. A mutation in the P5 RRS within a full-length AAV genome, which abolished Rep binding, resulted in a 40 to 50% reduction in the ability of wild-type Rep68 to inhibit the accumulation of P5 transcripts in vivo. In contrast, the Rep68/K340H mutant was unable to down-regulate this mutated promoter. These results indicate that there are at least two mechanisms involved in the negative regulation of P5 transcript levels by Rep68; one involves Rep68 binding to the P5 RRS, and another requires the region of Rep68 containing the consensus NTP-binding motif. Furthermore, our studies of AAV genomes containing mutated RRS- and/or YY1-binding elements suggest that transcription factor YY1 binding to the transcription start site of P5 interferes with Rep68 repression of the P5 promoter.

Adenosine Triphosphate↗

PubMed2026

Suppression of AAV-Delivered Transgene Expression Using Artificial MicroRNAs Delivered by an Alternative AAV Serotype.

Adeno-associated virus (AAV) gene transfer vectors mediate long-term expression in nondividing cells, an advantage for treating chronic disorders. However, current platforms lack a way to selectively shut down transgene expression if adverse effects arise. To create an "off switch," we hypothesized that incorporating unique artificial microRNA (amiRNA) target sequences into an AAV expression cassette would allow subsequent suppression of transgene expression using a second AAV vector encoding the cognate amiRNA. We introduced 22-nt sequences absent from human and mouse transcriptomes into the 3' untranslated region (UTR) of a therapeutic AAV cassette. To identify optimal amiRNAs, two tandem copies of each amiRNA were cloned into the 3'UTR of an mCherry reporter gene. In vitro assessment of six amiRNA/target pairs using a dual luciferase assay identified four amiRNAs that efficiently suppressed reporter expression. Cells cotransfected with target site 3 (TS3) and amiRNA-T3B showed the greatest reduction in luciferase activity (80%, p < 0.0001) and were selected for further study. The "off-switch" system was then evaluated using an AAV5 therapeutic vector expressing a recombinant humanized anti-IgE monoclonal antibody (AAV5-TBG-anti-IgE-TS3), designed for long-term suppression of allergen-induced reactions. Co-transfection of HEK293T cells with anti-IgE-TS3 and amiRNA-T3B significantly reduced anti-IgE mRNA and protein levels relative to a control amiRNA (p < 0.0001). In vivo testing in Balb/c mice (n = 5) involved intravenous administration of AAV5-anti-IgE-TS3 (3.2 × 1010 gc), followed 4 weeks later by an AAVrh.10 amiRNA vector (AAVrh.10-TBG-amiRNA-T3B; 1 × 1011 gc). Control mice receiving only the therapeutic vector expressed 18.4 ± 13.8 µg/mL serum anti-IgE at 10 weeks. In contrast, mice receiving the amiRNA "off" vector showed marked suppression of anti-IgE (0.3 ± 0.15 µg/mL, p < 0.0001). These findings provide proof-of-concept that AAV-delivered amiRNAs can selectively switch off transgene expression, offering a strategy to improve the safety of AAV-mediated gene therapies.

Dependovirus↗

PubMed1999

Adeno-associated virus (AAV) site-specific integration: formation of AAV-AAVS1 junctions in an in vitro system.

An in vitro system to study the mechanism of site-specific integration of adeno-associated virus (AAV) was developed. This system is based on two substrates, a linear or circular AAV donor and a circular acceptor containing the preintegration locus AAVS1. In the presence of HeLa extract and the His-Tag-purified Rep68 protein, specific covalent junctions between AAV and AAVS1 were formed and detected by PCR. The majority of the junctions were located within the Rep binding site of both the AAV and the AAVS1 substrates, underlining the involvement of the Rep protein. A limited amount of replication and the presence of nuclear factors promoted the efficiency of the reaction. The process was ATP-dependent, indicating that the helicase activity of Rep may be important in the formation of the junctions. According to current models of integration, the formation of the junctions would represent a first step in the process of AAV integration. This step could be crucial for the site specificity of the recombination event that leads to the integration of AAV into human chromosome 19 in vivo.

Base Sequence↗

PubMed1996

Differential expression in human cells from the p6 promoter of human parvovirus B19 following plasmid transfection and recombinant adeno-associated virus 2 (AAV) infection: human megakaryocytic leukaemia cells are non-permissive for AAV infection.

Expression from the human parvovirus B19p6 promoter fused to the firefly luciferase ('Luc') reporter gene was evaluated in a non-erythroid human nasopharyngeal carcinoma cell line, KB, and a human megakaryocytic leukaemia cell line, MB-02, known to become permissive for B19 replication following erythroid-differentiation. The B19p6-Luc construct was introduced into KB and MB-02 cells, both in undifferentiated and differentiated states, either via DNA-mediated transfection, or via infection with recombinant adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus known to possess a broad host-range. Although Luc activity was readily detected in KB cells following transfection of the B19p6-Luc plasmid DNA, no expression from the B19p6 promoter was observed following infection with recombinant virus. In addition, transfection of the reporter plasmid resulted in high-level expression of Luc in differentiated but not in undifferentiated MB-02 cells. However, no Luc activity was detected, even in differentiated MB-02 cells, following infection with recombinant virus. Further studies with an additional recombinant as well as wild-type (wt) AAV revealed that MB-02 cells were non-permissive for AAV infection. A second human megakaryocytic leukaemia cell line, M07e, was likewise resistant to infection by recombinant as well as wt AAV. Taken together, these studies identify the first human cell type that cannot be infected by AAV. They indicate that expression from the B19p6 promoter, in the context of an AAV genome, is restricted to primary human haematopoietic cells, perhaps because parvoviral DNA replication and transcription are intrinsically coupled.

Cell Differentiation↗

PubMed1990

Evidence for covalent attachment of the adeno-associated virus (AAV) rep protein to the ends of the AAV genome.

We have demonstrated that when the covalently joined ends of linear adeno-associated virus (AAV) DNA are resolved in vitro, the virus-encoded Rep protein becomes covalently attached to the 5' ends of the DNA. The covalent bond is between a tyrosine residue of the AAV Rep protein and a 5' phosphate of a thymidine residue in the AAV genome. Only the Rep protein encoded by the AAV p5 promoter, Rep68, was capable of becoming covalently attached to the ends of the AAV genome; the Rep proteins encoded by the p19 promoter were not. We also investigated some of the requirements for the complete in vitro resolution reaction. Inhibitor studies suggested that terminal resolution required DNA polymerase delta, ATP, and the deoxyribonucleoside triphosphates but did not require the remaining ribonucleoside triphosphates, DNA polymerase alpha, RNA polymerase II, or topoisomerases I and II. Finally, purified AAV Rep68, when added to the crude cytosol from uninfected HeLa cells, was sufficient for resolution. This suggested that terminal resolution relies on host enzymes and the virus-encoded p5 Rep proteins.

Binding Sites↗

PubMed1997

Identification and elimination of replication-competent adeno-associated virus (AAV) that can arise by nonhomologous recombination during AAV vector production.

Adeno-associated virus (AAV) vector preparations are often contaminated with variable amounts of replication-competent AAV (rcAAV), which may influence the behavior of these vectors both in cultured cells and in animals. A packaging plasmid/vector plasmid system containing no significant homology and lacking the wild-type AAV p5 promoter was constructed to eliminate the production of wild-type AAV by recombination. Still, rcAAV was detected in vector produced by cotransfection of these plasmids at large scale. Sequence analysis revealed that nonhomologous recombination was responsible for the generation of these novel rcAAVs. A new AAV packaging plasmid carrying separate rep and cap expression cassettes in opposite transcriptional orientations was constructed. AAV vector preparations produced by using this packaging construct did not contain rcAAV.

Animals↗

PubMed1999

Adeno-associated virus (AAV) type 5 Rep protein cleaves a unique terminal resolution site compared with other AAV serotypes.

Adeno-associated virus (AAV) replication depends on two viral components for replication: the AAV nonstructural proteins (Rep) in trans, and inverted terminal repeat (ITR) sequences in cis. AAV type 5 (AAV5) is a distinct virus compared to the other cloned AAV serotypes. Whereas the Rep proteins and ITRs of other serotypes are interchangeable and can be used to produce recombinant viral particles of a different serotype, AAV5 Rep proteins cannot cross-complement in the packaging of a genome with an AAV2 ITR. In vitro replication assays indicated that the block occurs at the level of replication instead of at viral assembly. AAV2 and AAV5 Rep binding activities demonstrate similar affinities for either an AAV2 or AAV5 ITR; however, comparison of terminal resolution site (TRS) endonuclease activities showed a difference in specificity for the two DNA sequences. AAV2 Rep78 cleaved only a type 2 ITR DNA sequence, and AAV5 Rep78 cleaved only a type 5 probe efficiently. Mapping of the AAV5 ITR TRS identified a distinct cleavage site (AGTG TGGC) which is absent from the ITRs of other AAV serotypes. Comparison of the TRSs in the AAV2 ITR, the AAV5 ITR, and the AAV chromosome 19 integration locus identified some conserved nucleotides downstream of the cleavage site but little homology upstream.

Base Sequence↗

PubMed2006

Adeno-associated virus-2 (AAV-2) causes trophoblast dysfunction, and placental AAV-2 infection is associated with preeclampsia.

Shallow invasion by extravillous trophoblast cells into the uterine wall reduces placental perfusion and causes placental dysfunction, but the one or more causes of shallow placental invasion are unknown. We hypothesized that infection with adeno-associated virus-2 (AAV-2) inhibits trophoblast invasion and is associated with preeclampsia, which is a common obstetric complication resulting from placental dysfunction. We determined that transformed extravillous trophoblast (HTR-8/SVneo) cells were susceptible to AAV-2 infection in the presence or absence of adenovirus, which provides helper function for AAV-2 replication, and that AAV-2 infection reduced invasion of HTR-8/SVneo cells through an extracellular matrix before cytopathic effects were detected. In a case-control study, AAV-2 DNA was found more frequently in trophoblast cells from cases of severe preeclampsia (22/40) than from normal term deliveries (5/27, P = 0.002). These results indicate that AAV-2 infection is a previously unidentified cause of placental dysfunction. Additional studies to determine the susceptibility of extravillous trophoblast to other viruses, and the mechanisms by which viral infection impairs placental function, are warranted.

Adenoviridae Infections↗

PubMed1994

Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels.

The rep gene of adeno-associated virus type 2 (AAV) encodes four overlapping Rep proteins that are involved in gene regulation and replication of the virus. We studied here the regulation of mRNA transcribed from the AAV p5 and p19 promoters, using transient expression in human 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p5 transcript encodes the larger Rep proteins, Rep78 and Rep68, while the p19 transcript encodes the smaller proteins, Rep52 and Rep40. A plasmid (pNTC3) containing the entire AAV genome with an amber mutation in the rep gene accumulated higher levels of p5 and p19 mRNA than a plasmid containing the wild-type AAV genome. Addition of increasing amounts of the wild-type rep gene in trans from a heterologous promoter inhibited p5 and p19 mRNA accumulation from pNTC3, indicating that the levels of both transcripts were decreased by the Rep proteins. Cotransfections with plasmids producing individual wild-type Rep proteins in trans showed that p5 and p19 mRNA accumulation was inhibited 5- to 10-fold by Rep78 and Rep68 and 2- to 3-fold by Rep52 and Rep40. Analysis of carboxyl-terminal truncation mutants of Rep78 showed that the ability of Rep78 to decrease p5 and p19 mRNA levels was lost when 159 or more amino acids were deleted. Rep78 and Rep68 mutants deleted for the methionine at residue 225 showed decreased abilities to down-regulate both p5 and p19 transcript levels, while mutants containing a substitution of glycine for the methionine resembled the wild-type Rep78. A Rep78 protein with a mutation in the putative nucleoside triphosphate binding site inhibited expression from p5 but not from p19, suggesting that the regulation of p5 transcript levels by Rep78 and Rep68 differs from that of p19. A deletion analysis of AAV cis sequences revealed that an intact terminal repeat was not required for negative regulation of p5 and p19 transcript levels and that the regulation of p19 mRNA levels by Rep78 did not require the presence of the p5 promoter.

Blotting, Northern↗

PubMed1998

Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2.

Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, and AAV4 was 75 to 78% identical to these AAVs. Significant sequence variation was noted in portions of the capsid proteins that presumably are responsible for serotype-specific functions. Vectors produced from AAV3 and AAV6 differed from AAV2 vectors in host range and serologic reactivity. The AAV3 and AAV6 vector serotypes were able to transduce cells in the presence of serum from animals previously exposed to AAV2 vectors. Our results suggest that vectors based on alternative AAV serotypes will have advantages over existing AAV2 vectors, including the transduction of different cell types, and resistance to neutralizing antibodies against AAV2. This could be especially important for gene therapy, as significant immunity against AAV2 exists in human populations and many protocols will likely require multiple vector doses.

Amino Acid Sequence↗

PubMed2004

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

The driving interest in adeno-associated virus (AAV) has been its potential as a gene delivery vector. The early observation that AAV can establish a latent infection by integrating into the host chromosome has been central to this interest. However, chromosomal integration is a two-edged sword, imparting on one hand the ability to maintain the therapeutic gene in progeny cells, and on the other hand, the risk of mutations that are deleterious to the host. A clearer understanding of the mechanism and efficiency of AAV integration, in terms of contributing viral and host-cell factors and circumstances, will provide a context in which to evaluate these potential benefits and risks. Research to date suggests that AAV integration in any context is inefficient, and that the persistence of AAV gene delivery vectors in tissues is largely attributable to episomal genomes.

Cell Line↗