Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 9

Mitchell, M. Van; Nam, Hyun Joo; Carter, Adam; McCall, Angela L; Rence, Chelsea; Bennett, Antonette; Gurda, Brittney L.; McKenna, Robert; Porter, Mark L.; Sakai, Yoshihisa; Byrne, Barry J.; Muzyczka, Nicholas; Aslanidi, George; Zolotukhin, Sergei; Agbandje‐McKenna, Mavis

doi:10.1107/s1744309109021460

Cited by 24 publications

(18 citation statements)

References 28 publications

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“…In contrast, only preliminary X-ray crystallographic data of the AAV9 capsid structure were published recently and structure determination is still under progress. 31 Because insertion of heptapeptides behind arginineresidue 588 (R588), part of several basic amino-acid residues clustered within the threefold spike of the capsid surface involved in HSPG binding, 32 altered AAV2 tropism, we reasoned that peptide sequence alignment of AAV2 and AAV9 capsids would indicate the position of similar infection-relevant regions within the AAV9 capsid. AAV2 and AAV9 share about 83% of their capsid amino-acid sequences 33 and sequence comparison of VP3 subunits comprising the HSPG-binding domain revealed highly conserved regions (R484/R485 and R488/R489, (AAV2/AAV9)) as well as variable regions (R585/S586 and R588/A589), which might contribute to the different tissue tropism of these two serotypes.…”

Section: Discussionmentioning

confidence: 99%

Novel random peptide libraries displayed on AAV serotype 9 for selection of endothelial cell-directed gene transfer vectors

et al. 2011

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We have demonstrated the potential of random peptide libraries displayed on adeno-associated virus (AAV)2 to select for AAV2 vectors with improved efficiency for cell type-directed gene transfer. AAV9, however, may have advantages over AAV2 because of a lower prevalence of neutralizing antibodies in humans and more efficient gene transfer in vivo. Here we provide evidence that random peptide libraries can be displayed on AAV9 and can be utilized to select for AAV9 capsids redirected to the cell type of interest. We generated an AAV9 peptide display library, which ensures that the displayed peptides correspond to the packaged genomes and performed four consecutive selection rounds on human coronary artery endothelial cells in vitro. This screening yielded AAV9 library capsids with distinct peptide motifs enabling up to 40-fold improved transduction efficiencies compared with wild-type (wt) AAV9 vectors. Incorporating sequences selected from AAV9 libraries into AAV2 capsids could not increase transduction as efficiently as in the AAV9 context. To analyze the potential on endothelial cells in the intact natural vascular context, human umbilical veins were incubated with the selected AAV in situ and endothelial cells were isolated. Fluorescence-activated cell sorting analysis revealed a 200-fold improved transduction efficiency compared with wt AAV9 vectors. Furthermore, AAV9 vectors with targeting sequences selected from AAV9 libraries revealed an increased transduction efficiency in the presence of human intravenous immunoglobulins, suggesting a reduced immunogenicity. We conclude that our novel AAV9 peptide library is functional and can be used to select for vectors for future preclinical and clinical gene transfer applications.

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Section: Discussionmentioning

confidence: 99%

Novel random peptide libraries displayed on AAV serotype 9 for selection of endothelial cell-directed gene transfer vectors

et al. 2011

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“…Recombinant AAV9 virus-like particles (VLPs) were expressed using the Bac-to-Bac baculovirus-Sf9 insect cell expression system (Gibco/Invitrogen, Carlsbad, CA) and purified using a 20% sucrose cushion followed by a sucrose gradient (5 to 40% [wt/vol]) as previously reported (48). Purified AAV9 VLPs were concentrated to ϳ5 mg/ml (in 10 mM Tris-HCl [pH 7.5], 350 mM NaCl, and 2 mM MgCl 2 , buffer A) using Apollo concentrators (Orbital Biosciences, LLC, Topsfield, MA) (150,000-molecular-weight cutoff) at 2,372 ϫ g at 277 K and stored at the same temperature.…”

Section: Methodsmentioning

confidence: 99%

Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9

DiMattia

Nam

Vliet

et al. 2012

J Virol

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172

174

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Adeno-associated virus serotype 9 (AAV9) has enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood-brain barrier compared to other AAV serotypes. To help identify the structural features facilitating these properties, we have used cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction (cryo-reconstruction) and X-ray crystallography to determine the structure of the AAV9 capsid at 9.7- and 2.8-Å resolutions, respectively. The AAV9 capsid exhibits the surface topology conserved in all AAVs: depressions at each icosahedral two-fold symmetry axis and surrounding each five-fold axis, three separate protrusions surrounding each three-fold axis, and a channel at each five-fold axis. The AAV9 viral protein (VP) has a conserved core structure, consisting of an eight-stranded, β-barrel motif and the αA helix, which are present in all parvovirus structures. The AAV9 VP differs in nine variable surface regions (VR-I to -IX) compared to AAV4, but at only three (VR-I, VR-II, and VR-IV) compared to AAV2 and AAV8. VR-I differences modify the raised region of the capsid surface between the two-fold and five-fold depressions. The VR-IV difference produces smaller three-fold protrusions in AAV9 that are less “pointed” than AAV2 and AAV8. Significantly, residues in the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes of this serotype.

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“…The baculovirus/Sf9 protein expression system provides a tractable method for the production of large quantities of AAV virus-like particles (VLPs) shown to be structurally and antigenically similar to virus particles and virions produced in mammalian cell culture systems (34,35). For GMA binding studies, VLPs of AAV9 were prepared using the Bac-to-Bac baculovirus/Sf9 expression system (Invitrogen) as previously described (36) and screened in a high-throughput GMA developed by Cores D and H of the Consortium for Functional Glycomics (CFG; an NIH National Institute of General Medicine Science Initiative; ref. 37).…”

Section: Figurementioning

confidence: 99%

The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice

Bell

Vandenberghe²,

Bell³

et al. 2011

J. Clin. Invest.

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156

146

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Vectors based on adeno-associated virus (AAV) serotype 9 are candidates for in vivo gene delivery to many organs, but the receptor(s) mediating these tropisms have yet to be defined. We evaluated AAV9 uptake by glycans with terminal sialic acids (SAs), a common mode of cellular entry for viruses. We found, however, that AAV9 binding increased when terminal SA was enzymatically removed, suggesting that galactose, which is the most commonly observed penultimate monosaccharide to SA, may mediate AAV9 transduction.

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Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 9

Cited by 24 publications

References 28 publications

Novel random peptide libraries displayed on AAV serotype 9 for selection of endothelial cell-directed gene transfer vectors

Novel random peptide libraries displayed on AAV serotype 9 for selection of endothelial cell-directed gene transfer vectors

Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9

The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice

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