Productive life cycle of adeno-associated virus serotype 2 in the complete absence of a conventional polyadenylation signal

Wang, Lina; Yin, Zifei; Wang, Yuan; Lü, Yuan; Zhang, Dongke; Srivastava, Arun; Ling, Changquan; Aslanidi, George; Chen, Ling

doi:10.1099/jgv.0.000229

Cited by 5 publications

(6 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The negative controls of this assay included HEK293 cells that were triple transfected with pITR2/3-EGFP-Neo, pHelper, and a plasmid containing the AAV2 rep gene but no cap genes. 22 In the negative control cells, viral genome replication occurs, but no viral capsid proteins are expressed. The results of viral encapsidation assays revealed that the encapsidated viral genomes in the negative control group were below the detection limitation by qPCR assays (data not shown).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors

Chen

Yin

et al. 2016

Molecular Therapy - Methods & Clinical Development

Self Cite

View full text Add to dashboard Cite

Although recombinant adeno-associated virus serotype 3 (AAV3) vectors were largely ignored previously, owing to their poor transduction efficiency in most cells and tissues examined, our initial observation of the selective tropism of AAV3 serotype vectors for human liver cancer cell lines and primary human hepatocytes has led to renewed interest in this serotype. AAV3 vectors and their variants have recently proven to be extremely efficient in targeting human and nonhuman primate hepatocytes in vitro as well as in vivo. In the present studies, we wished to evaluate the relative contributions of the cis-acting inverted terminal repeats (ITRs) from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the AAV3 vector production and transduction. To this end, we utilized two helper plasmids: pAAVr2c3, which carries rep2 and cap3 genes, and pAAVr3c3, which carries rep3 and cap3 genes. The combined use of AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids led to the production of recombinant vectors, AAV3-Rep3/ITR3, with up to approximately two to fourfold higher titers than AAV3-Rep2/ITR2 vectors produced using AAV2 ITRs, AAV2 Rep proteins, and AAV3 capsids. We also observed that the transduction efficiency of Rep3/ITR3 AAV3 vectors was approximately fourfold higher than that of Rep2/ITR2 AAV3 vectors in human hepatocellular carcinoma cell lines in vitro. The transduction efficiency of Rep3/ITR3 vectors was increased by ~10-fold, when AAV3 capsids containing mutations in two surface-exposed residues (serine 663 and threonine 492) were used to generate a S663V+T492V double-mutant AAV3 vector. The Rep3/ITR3 AAV3 vectors also transduced human liver tumors in vivo approximately twofold more efficiently than those generated with Rep2/ITR2. Our data suggest that the transduction efficiency of AAV3 vectors can be significantly improved both using homologous Rep proteins and ITRs as well as by capsid optimization. Thus, the combined use of homologous Rep proteins, ITRs, and capsids should also lead to more efficacious other AAV serotype vectors for their optimal use in human gene therapy.

show abstract

Section: Resultsmentioning

confidence: 99%

“…Mice were injected subcutaneously with 5 million human liver cancer cells on the ventral side of the neck between shoulder blades. 22 Animals were kept in sterile cages until the end of the experiments.…”

Section: Methodsmentioning

confidence: 99%

Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors

Chen

Yin

et al. 2016

Molecular Therapy - Methods & Clinical Development

Self Cite

View full text Add to dashboard Cite

show abstract

“…Thereby a monomer turnaround structure (mT) is generated with the ITR connected to both complementary strands ( Fig 7A ). RNA polymerase can proceed through the ITR as shown recently for AAV mutants lacking the authentic polyA signal [ 48 ]. By continuing transcription on the opposite AAV strand double-stranded RNA can be formed ( Fig 7A ) as shown recently for the related parvovirus, minute virus of mice (MVM) [ 49 ].…”

Section: Discussionmentioning

confidence: 97%

“…An additional hairpin loop is generated at the terminal resolution site ( trs ) during Rep-dependent, strand-specific nicking of the AAV genome required to resolve the ssDNA genomes during AAV DNA replication [ 54 , 55 ]. Recently, AAV2 Rep was shown to also bind to ITRs composed of RNA [ 48 ]. Nicking of the trs RNA-bound Rep, would result in the here-described small RNAs starting at nucleotide position 1 or 125, respectively ( Fig 3 , sR-1 and sR-125).…”

Section: Discussionmentioning

confidence: 99%

Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation

et al. 2016

View full text Add to dashboard Cite

Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic.

show abstract

“… 30 The physical particle titers highly purified of rAAV2 vector stocks were determined by quantitative DNA slot-blot and Southern blot analyses, as previously described. 31 …”

Section: Methodsmentioning

confidence: 99%

Liposome Lipid-Based Formulation Has the Least Influence on rAAV Transduction Compared to Other Transfection Agents

Guo

Wang

et al. 2018

Molecular Therapy - Methods & Clinical Development

View full text Add to dashboard Cite

Recombinant adeno-associated virus (rAAV) vectors are considered ideal vehicles for human gene therapy. Meanwhile, non-viral strategies, such as transfection agents (TAs), have also shown promise to deliver genetic materials, such as siRNA. Transduction with the rAAV vector is performed concurrently with transfection with plasmid DNA or RNA. In the present study, we report that various TAs inhibited rAAV-mediated transgene expression at diverse levels. Overall, cationic polymers and dendrimers dramatically blocked rAAV transduction, while lipid-based liposomes displayed the least effect. The inhibitory effect was dependent on the dose of TAs and the timing of infection, suggesting that the early stages of viral infection were involved. In addition, the present results indicate that the transgene expression of rAAV vectors was significantly increased by liposome-mediated transfection with adenoviral helper genes. At the same time, this was dramatically inhibited by liposome-mediated transfection with the trichosanthin gene encoding a type I ribosome-inactivating protein isolated from traditional Chinese medicine. Furthermore, liposomes also have little effect on rAAV-mediated transgene expression in vivo. Taken together, these findings suggest liposome as the best choice of TAs, which should be used in combination with rAAV-mediated gene therapy.

show abstract

Productive life cycle of adeno-associated virus serotype 2 in the complete absence of a conventional polyadenylation signal

Cited by 5 publications

References 35 publications

Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors

Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors

Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation

Liposome Lipid-Based Formulation Has the Least Influence on rAAV Transduction Compared to Other Transfection Agents

Contact Info

Product

Resources

About