Serotype-specific detection of adeno-associated virus during laboratory preparation

Mitchell, Daniel A.; O’Donnell, Jason; Hare, Joan; Chapman, Michael S.

doi:10.1016/j.jviromet.2006.05.012

Cited by 6 publications

(6 citation statements)

References 25 publications

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“…Fraction aliquots of 2 – 8 μL were made up to 10 μL by mixing with DNA extraction buffer (2 μL proteinase K and 1 μL Tween-20 in 1mL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Capsid digestion proceeded at 56° C for 1.5 hours and was quenched for 1 hour at 90° C to denature proteinase K. PCR was performed using the DreamTaq Green Master Mix (Fermentas) (Mitchell et al, 2006). Amplified fragments were separated on 1.5% agarose gels which were scanned using the FluorChem 5500 (Alpha Innotech Inc.).…”

Section: Methodsmentioning

confidence: 99%

Structure–function analysis of receptor-binding in adeno-associated virus serotype 6 (AAV-6)

Xie¹,

Lerch²,

Meyer³

et al. 2011

Virology

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Crystal structures of the AAV-6 capsid at 3 Å reveal a subunit fold homologous to other parvoviruses with greatest differences in two external loops. The electrostatic potential suggests that receptor-attachment is mediated by four residues: Arg576, Lys493, Lys459 and Lys531, defining a positively charged region curving up from the valley between adjacent spikes. It overlaps only partially with the receptor-binding site of AAV-2, and the residues endowing the electrostatic character are not homologous. Mutational substitution of each residue decreases heparin affinity, particularly Lys531 and Lys459. Neither is conserved among heparin-binding serotypes, indicating that diverse modes of receptor attachment have been selected in different serotypes. Surface topology and charge are also distinct at the shoulder of the spike, where linear epitopes for AAV-2’s neutralizing monoclonal antibody A20 come together. Evolutionarily, selection of changed side-chain charge may have offered a conservative means to evade immune neutralization while preserving other essential functionality.

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Section: Methodsmentioning

confidence: 99%

Structure–function analysis of receptor-binding in adeno-associated virus serotype 6 (AAV-6)

Xie¹,

Lerch²,

Meyer³

et al. 2011

Virology

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“…In this mechanism, small double-stranded RNA molecules in cells induce sequence-specific degradation of homologous single-stranded RNA (6). It has been shown that expression and replication of some viral genes were suppressed by RNAi, including hepatitis C virus (7), human immunodeficiency virus (8,9), SARS-coronavirus (10,11) and HBV (12)(13)(14)(15). However, due to their short half-life and low in vivo transfection efficiency, the clinical use of synthetic siRNAs and plasmid-based shRNA have generally been limited.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of HBV replication and gene expression in vitro and in vivo with a single AAV vector delivering two shRNA molecules

Yao³

et al. 2009

BMB Reports

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Hepatitis B virus (HBV) infection is highly prevalent worldwide.The major challenge for current antiviral treatment is the elevated drug resistance that occurs via rapid viral mutagenesis. In this study, we developed AAV vectors to simultaneously deliver two or three shRNAs targeting different HBV-related genes. These vectors showed markedly better antiviral effects than ones that delivered a single shRNA in vitro. A dual shRNA expression vector (AAV-157i/1694i), which simultaneously expressed two shRNAs targeted the S and X genes of HBV, reduced HBsAg, HBeAg and HBV DNA levels by 87 ± 4, 80.3 ± 2.6 and 86.2 ± 7% respectively, eight days post-transduction. In a mouse model of prophylactic treatment, HBsAg and HBeAg were reduced to undetectable levels and the serum HBV DNA level was reduced by at least 100 fold. These results indicate that AAV-157i/1694i generates potent anti-HBV effects and that the strategy of constructing multi-shRNA expression vectors may lead to enhanced anti-HBV efficacy and overcome the evading mechanism of the virus and thus the development of drug resistance. [BMB reports 2009; 42(1): 59-64]

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“…Conventional methods are based on singleplex or low-density multiplex PCR and subsequent gel-electrophoresis or sequencing [Katano et al, 2004;Mitchell et al, 2006]. These assays are limited with regard to the number of AAV serotypes that can be detected simultaneously in one reaction.…”

Section: Discussionmentioning

confidence: 99%

“…However, no reliable, sensitive and specific high-throughput serotyping method is available for the detection of DNA cross-contaminations by these AAV plasmids. For example, a PCRbased assay using type-specific primers in the cap gene has been developed to identify a reduced number of serotypes (AAV2, AAV3B and AAV6) [Mitchell et al, 2006]. Due to the high degree of sequence homology between AAV1 and AAV6, the assay could not differentiate between these two serotypes.…”

Section: Introductionmentioning

confidence: 99%

Genotyping of AAV Plasmid Stocks: Quality Control in Adeno-Associated Virus Vector Production

Schmitt

Pawlita²,

Kleinschmidt³

2010

Microb Physiol

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Recombinant adeno-associated virus (rAAV) vectors are a promising tool for gene therapy. When multiple serotypes are handled in the same laboratory during the AAV vector production, it is essential to have means to identify the serotype in a sample and to confirm the absence of cross-contaminating AAV sequences in plasmid stocks as well as end products. Here, we describe the development of a Multiplex AAV Genotyping (MAG) assay to type sensitively and specifically DNA from AAV serotypes 1–12 and to detect AAV2 serotype DNA sequences encoding peptide insertions used to modify tissue tropism. MAG is based on multiplex PCR using type-specific primers and subsequent multiplex hybridization by Luminex. The assay is highly specific, and can easily identify plasmid cross-contaminations. Using 10-fold dilution series, the detection limit was below 10 AAV genomes per PCR. In artificial cross-contamination experiments with a 1,000-fold excess of one AAV serotype versus another one, the contaminating type could be still detected with 10–100 AAV genomes. In a first application, MAG identified successfully cross-contaminated AAV plasmid stocks. In conclusion, MAG is a powerful high-throughput tool in assessing the purity and identity of AAV DNA plasmids and other starting materials used for AAV vector production.

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Serotype-specific detection of adeno-associated virus during laboratory preparation

Cited by 6 publications

References 25 publications

Structure–function analysis of receptor-binding in adeno-associated virus serotype 6 (AAV-6)

Structure–function analysis of receptor-binding in adeno-associated virus serotype 6 (AAV-6)

Inhibition of HBV replication and gene expression in vitro and in vivo with a single AAV vector delivering two shRNA molecules

Genotyping of AAV Plasmid Stocks: Quality Control in Adeno-Associated Virus Vector Production

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