Structure of AAV-DJ, a Retargeted Gene Therapy Vector: Cryo-Electron Microscopy at 4.5 Å Resolution

Lerch, Thomas F.; O’Donnell, Jason; Meyer, Nancy; Xie, Qing; Taylor, Kenneth A.; Stagg, Scott M.; Chapman, Michael S.

doi:10.1016/j.str.2012.05.004

Cited by 72 publications

(96 citation statements)

References 74 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Liver transduction had selected hybrids from five of eight starting serotypes (2, 4, 5, 8, and 9); then IVIg-escape selected a single hybrid of serotypes 2, 8, and 9. The most prominent difference between AAV-DJ and AAV-2 is in VR-I at the epitope of neutralizing mouse monoclonal antibody (mAb) A20 8, 12, 31, 32. This explains the lack of recognition of AAV-DJ by mAb A20, in spite of 93% capsid sequence identity with AAV-2 11 (Table 1; Figure S2).…”

Section: Discussionmentioning

confidence: 99%

The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparinoid Pentasaccharide

Xie

Spear

Noble

et al. 2017

Molecular Therapy - Methods & Clinical Development

Self Cite

View full text Add to dashboard Cite

Atomic structures of adeno-associated virus (AAV)-DJ, alone and in complex with fondaparinux, have been determined by cryoelectron microscopy at 3 Å resolution. The gene therapy vector, AAV-DJ, is a hybrid of natural serotypes that was previously derived by directed evolution, selecting for hepatocyte entry and resistance to neutralization by human serum. The structure of AAV-DJ differs from that of parental serotypes in two regions where neutralizing antibodies bind, so immune escape appears to have been the primary driver of AAV-DJ’s directed evolution. Fondaparinux is an analog of cell surface heparan sulfate to which several AAVs bind during entry. Fondaparinux interacts with viral arginines at a known heparin binding site, without the large conformational changes whose presence was controversial in low-resolution imaging of AAV2-heparin complexes. The glycan density suggests multi-modal binding that could accommodate sequence variation and multivalent binding along a glycan polymer, consistent with a role in attachment, prior to more specific interactions with a receptor protein mediating entry.

show abstract

Section: Discussionmentioning

confidence: 99%

The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparinoid Pentasaccharide

Xie

Spear

Noble

et al. 2017

Molecular Therapy - Methods & Clinical Development

Self Cite

View full text Add to dashboard Cite

show abstract

“…Given that the beam diameter is required to be greater than 1.3 μm in order to maintain parallel illumination with our imaging conditions, three exposures per hole is the maximum we can attain. The structures associated with these A data are being published elsewhere, but the AAV data reconstructed to 4.5 resolution, which shows that the data collection conditions are sufficient for high-resolution (Lerch et al, 2012). The images are 4k × 4k pixels and are stored as 16 bit signed floats in MRC format that results in an image that takes up 64 MB of disk space.…”

Section: Quantitation Of Throughputmentioning

confidence: 92%

Creating an infrastructure for high-throughput high-resolution cryogenic electron microscopy

Shrum

Woodruff

Stagg

2012

Journal of Structural Biology

View full text Add to dashboard Cite

New instrumentation for three-dimensional electron microscopy is facilitating an increase in the throughput of data collection and reconstruction. The increase in throughput creates bottlenecks in the workflow for storing and processing the image data. Here we describe the creation and quantify the throughput of a high-throughput infrastructure supporting collection of three-dimensional data collection.

show abstract

“…Defined structures for 11 naturally occurring or modified AAV serotype/variants have been resolved in recent years using X-ray crystallography and/or cryo-electron microscopy. These include 6 AAV-1 15 , AAV-2 [16][17][18][19][20][21][22] , AAV-3B 23, 24 , AAV-4 25, 26 , AAV-5 27-30 , AAV-6 31, 32 , AAV-7 33 , AAV-8 34-38 , AAV-9 39-41 , AAVrh32.33 42 , and AAV-DJ 43,44 . The structural information of these AAV capsids as well as their interaction with respective cellular receptor/co-receptors have provided the necessary framework to logically design tailored vectors.…”

Section: Aav Capsid Engineering By Rational Designmentioning

confidence: 99%

Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy

Nance

Duan

2015

Human Gene Therapy

View full text Add to dashboard Cite

Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy.

show abstract

Structure of AAV-DJ, a Retargeted Gene Therapy Vector: Cryo-Electron Microscopy at 4.5 Å Resolution

Cited by 72 publications

References 74 publications

The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparinoid Pentasaccharide

The 2.8 Å Electron Microscopy Structure of Adeno-Associated Virus-DJ Bound by a Heparinoid Pentasaccharide

Creating an infrastructure for high-throughput high-resolution cryogenic electron microscopy

Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy

Contact Info

Product

Resources

About