+Open data
-Basic information
Entry | Database: PDB / ID: 7rl1 | |||||||||
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Title | AAVrh.10-7x capsid | |||||||||
Components |
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Keywords | VIRUS / Icosahedral Capsid / AAVrh.10 / capsid engineering / Adeno-associated virus / Parvovirus / Gene Therapy | |||||||||
Function / homology | Function and homology information | |||||||||
Biological species | Adeno-associated virus synthetic construct (others) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.71 Å | |||||||||
Authors | Mietzsch, M. / McKenna, R. | |||||||||
Funding support | United States, 1items
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Citation | Journal: J Virol / Year: 2021 Title: Structural Study of Aavrh.10 Receptor and Antibody Interactions. Authors: Mario Mietzsch / Jennifer C Yu / Jane Hsi / Paul Chipman / Felix Broecker / Zhang Fuming / Robert J Linhardt / Peter H Seeberger / Regine Heilbronn / Robert McKenna / Mavis Agbandje-McKenna / Abstract: Recombinant adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, ...Recombinant adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, the AAV vectors have to circumvent potential preexisting neutralizing host antibodies and bind to the receptors of the target cells. Both of these aspects have not been structurally analyzed for AAVrh.10. Here, cryo-electron microscopy and three-dimensional image reconstruction were used to map the binding site of sulfated -acetyllactosamine (LacNAc; previously shown to bind AAVrh.10) and a series of four monoclonal antibodies (MAbs). LacNAc was found to bind to a pocket located on the side of the 3-fold capsid protrusion that is mostly conserved to AAV9 and equivalent to its galactose-binding site. As a result, AAVrh.10 was also shown to be able to bind to cell surface glycans with terminal galactose. For the antigenic characterization, it was observed that several anti-AAV8 MAbs cross-react with AAVrh.10. The binding sites of these antibodies were mapped to the 3-fold capsid protrusions. Based on these observations, the AAVrh.10 capsid surface was engineered to create variant capsids that escape these antibodies while maintaining infectivity. Gene therapy vectors based on adeno-associated virus rhesus isolate 10 (AAVrh.10) have been used in several clinical trials to treat monogenetic diseases. However, compared to other AAV serotypes little is known about receptor binding and antigenicity of the AAVrh.10 capsid. Particularly, preexisting neutralizing antibodies against capsids are an important challenge that can hamper treatment efficiency. This study addresses both topics and identifies critical regions of the AAVrh.10 capsid for receptor and antibody binding. The insights gained were utilized to generate AAVrh.10 variants capable of evading known neutralizing antibodies. The findings of this study could further aid the utilization of AAVrh.10 vectors in clinical trials and help the approval of the subsequent biologics. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7rl1.cif.gz | 5.2 MB | Display | PDBx/mmCIF format |
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PDB format | pdb7rl1.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 7rl1.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7rl1_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 7rl1_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 7rl1_validation.xml.gz | 618 KB | Display | |
Data in CIF | 7rl1_validation.cif.gz | 1011.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/rl/7rl1 ftp://data.pdbj.org/pub/pdb/validation_reports/rl/7rl1 | HTTPS FTP |
-Related structure data
Related structure data | 24513MC 7s1wC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 58323.359 Da / Num. of mol.: 60 / Fragment: UNP residues 219-738 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Adeno-associated virus / Gene: cap / Variant: rh.10-7x / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q6JC62 #2: DNA chain | Mass: 557.431 Da / Num. of mol.: 60 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Adeno-associated virus / Type: VIRUS / Entity ID: #1 / Source: RECOMBINANT |
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Source (natural) | Organism: Adeno-associated virus / Strain: rh.10-7x |
Source (recombinant) | Organism: Homo sapiens (human) / Cell: HEK293 |
Details of virus | Empty: NO / Enveloped: NO / Isolate: OTHER / Type: VIRION |
Buffer solution | pH: 7.4 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Cs: 2.7 mm |
Image recording | Electron dose: 75 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.10-2155_2155: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17861 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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