Circular Intermediates of Recombinant Adeno-Associated Virus Have Defined Structural Characteristics Responsible for Long-Term Episomal Persistence in Muscle Tissue

Duan, Dongsheng; Sharma, Prerna; Yang, Jusan; Yue, Yongping; Dudus, Lorita; Zhang, Yulong; Fisher, Krishna J.; Engelhardt, John F.

doi:10.1128/jvi.72.11.8568-8577.1998

Cited by 447 publications

(188 citation statements)

References 33 publications

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“…This may further explain why a high rate of co-infection, or co-detection, between HBoV1 and other respiratory viruses has been reported [5]. Since recombinant AAV persists as an episome in transduced tissues, which prolongs gene expression [72,73], it is possible that also the HBoV1 genome can be presented as an episome [29,30] for long term expression and replication. Apparently, the mechanism underlying this feature of HBoV1 infection warrants further investigation.…”

Section: Discussionmentioning

confidence: 99%

Establishment of a Reverse Genetics System for Studying Human Bocavirus in Human Airway Epithelia

et al. 2012

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Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 293 cells. The HBoV1 virions generated from this cell line-based production system exhibits a typical icosahedral structure of approximately 26 nm in diameter, and is capable of productively infecting polarized primary human airway epithelia (HAE) from the apical surface. Infected HAE showed hallmarks of lung airway-tract injury, including disruption of the tight junction barrier, loss of cilia and epithelial cell hypertrophy. Notably, polarized HAE cultured from an immortalized airway epithelial cell line, CuFi-8 (originally derived from a cystic fibrosis patient), also supported productive infection of HBoV1. Thus, we have established a reverse genetics system and generated the first cell line-based culture system for the study of HBoV1 infection, which will significantly advance the study of HBoV1 replication and pathogenesis.

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Section: Discussionmentioning

confidence: 99%

Establishment of a Reverse Genetics System for Studying Human Bocavirus in Human Airway Epithelia

et al. 2012

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“…(3) Ability to transduce dividing and nondividing cells [10]. (4) Episomal maintenance: wild-type AAV exhibits site-specific integration of its genome into chromosome 19; however, the majority of vector genomes appear to be maintained episomally [11][12][13]. Therefore, the risk of integration is minimal, compared with retroviral vectors that require integration into the host genome and have the potential to activate proto-oncogenes.…”

Section: Introductionmentioning

confidence: 99%

The Role of the Adeno-Associated Virus Capsid in Gene Transfer

Vliet

Blouin

Brument

et al. 2008

Methods in Molecular Biology™

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

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“…Seven‐week‐old C57Bl/6 (BL6) male mice were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). Recombinant AAV was delivered to the TA muscle according to a previously described protocol 21. Briefly, a 2 mm incision was made on the skin covering the proximal end of the TA muscle.…”

Section: Methodsmentioning

confidence: 99%

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

Ghosh

Yue

Duan

2005

The Journal of Gene Medicine

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Background-The overlapping approach was developed recently to expand the adeno-associated viral (AAV) packaging capacity. In this approach, a gene is split into two partially overlapping fragments and separately packaged into an upstream and a downstream vector, respectively. Transgene expression is achieved in co-infected cells after homologous recombination. Despite the promising proof-of-principle results in the lung, the efficiency has been very disappointing in skeletal muscle. Here we examined two potential rate-limiting factors including AAV serotype and the transgene sequence.

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Circular Intermediates of Recombinant Adeno-Associated Virus Have Defined Structural Characteristics Responsible for Long-Term Episomal Persistence in Muscle Tissue

Cited by 447 publications

References 33 publications

Establishment of a Reverse Genetics System for Studying Human Bocavirus in Human Airway Epithelia

Establishment of a Reverse Genetics System for Studying Human Bocavirus in Human Airway Epithelia

The Role of the Adeno-Associated Virus Capsid in Gene Transfer

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

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