Structure of AAV-DJ, a retargeted gene therapy vector: Cryo-electron microscopy at 4.5 Å resolution

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

doi:10.1016/j.str.2012.05.004

Structure of AAV-DJ, a retargeted gene therapy vector: Cryo-electron microscopy at 4.5 Å resolution

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

Research output: Contribution to journal › Article › peer-review

62 Scopus citations

Abstract

AAV-DJ, a leading candidate vector for liver gene therapy, was created through random homologous recombination followed by directed evolution, selecting for in vivo liver tropism and resistance to in vitro immune neutralization. Here, the 4.5 Å resolution cryo-EM structure is determined for the engineered AAV vector, revealing structural features that illuminate its phenotype. The heparan sulfate receptor-binding site is little changed from AAV-2, and heparin-binding affinity is similar. A loop that is antigenic in other serotypes has a unique conformation in AAV-DJ that would conflict with the binding of an AAV-2 neutralizing monoclonal antibody. This is consistent with increased resistance to neutralization by human polyclonal sera, raising the possibility that changed tropism may be a secondary effect of altered immune interactions. The reconstruction exemplifies analysis of fine structural changes and the potential of cryo-EM, in favorable cases, to characterize mutant or ligand-bound complexes.

Original language	English (US)
Pages (from-to)	1310-1320
Number of pages	11
Journal	Structure
Volume	20
Issue number	8
DOIs	https://doi.org/10.1016/j.str.2012.05.004
State	Published - Aug 8 2012
Externally published	Yes

ASJC Scopus subject areas

Structural Biology
Molecular Biology

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10.1016/j.str.2012.05.004

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title = "Structure of AAV-DJ, a retargeted gene therapy vector: Cryo-electron microscopy at 4.5 {\AA} resolution",

abstract = "AAV-DJ, a leading candidate vector for liver gene therapy, was created through random homologous recombination followed by directed evolution, selecting for in vivo liver tropism and resistance to in vitro immune neutralization. Here, the 4.5 {\AA} resolution cryo-EM structure is determined for the engineered AAV vector, revealing structural features that illuminate its phenotype. The heparan sulfate receptor-binding site is little changed from AAV-2, and heparin-binding affinity is similar. A loop that is antigenic in other serotypes has a unique conformation in AAV-DJ that would conflict with the binding of an AAV-2 neutralizing monoclonal antibody. This is consistent with increased resistance to neutralization by human polyclonal sera, raising the possibility that changed tropism may be a secondary effect of altered immune interactions. The reconstruction exemplifies analysis of fine structural changes and the potential of cryo-EM, in favorable cases, to characterize mutant or ligand-bound complexes.",

author = "Lerch, {Thomas F.} and O'Donnell, {Jason K.} and Meyer, {Nancy L.} and Qing Xie and Taylor, {Kenneth A.} and Stagg, {Scott M.} and Chapman, {Michael S.}",

note = "Funding Information: We thank Jay Nix and the staff at the MBC ALS beamline 4.2.2 for help in X-ray data collection. EM data processing and reconstruction was performed with help from the Florida State University shared High-Performance Computing facility. The research was supported by grants from the National Institutes of Health (R01GM66875 and R01GM78538 to M.S.C.), the Office of Naval Research (N000141010082 to John Carruthers at Portland State University), and a grant partially supporting the purchase of the microscope (S10-RR025080 to K.A.T.). T.F.L. was supported by the Interactions at the Microbe-Host Interface training grant from the National Institutes of Health (T32AI007472). ",

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AU - Meyer, Nancy L.

AU - Xie, Qing

AU - Taylor, Kenneth A.

AU - Stagg, Scott M.

AU - Chapman, Michael S.

N1 - Funding Information: We thank Jay Nix and the staff at the MBC ALS beamline 4.2.2 for help in X-ray data collection. EM data processing and reconstruction was performed with help from the Florida State University shared High-Performance Computing facility. The research was supported by grants from the National Institutes of Health (R01GM66875 and R01GM78538 to M.S.C.), the Office of Naval Research (N000141010082 to John Carruthers at Portland State University), and a grant partially supporting the purchase of the microscope (S10-RR025080 to K.A.T.). T.F.L. was supported by the Interactions at the Microbe-Host Interface training grant from the National Institutes of Health (T32AI007472).

PY - 2012/8/8

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N2 - AAV-DJ, a leading candidate vector for liver gene therapy, was created through random homologous recombination followed by directed evolution, selecting for in vivo liver tropism and resistance to in vitro immune neutralization. Here, the 4.5 Å resolution cryo-EM structure is determined for the engineered AAV vector, revealing structural features that illuminate its phenotype. The heparan sulfate receptor-binding site is little changed from AAV-2, and heparin-binding affinity is similar. A loop that is antigenic in other serotypes has a unique conformation in AAV-DJ that would conflict with the binding of an AAV-2 neutralizing monoclonal antibody. This is consistent with increased resistance to neutralization by human polyclonal sera, raising the possibility that changed tropism may be a secondary effect of altered immune interactions. The reconstruction exemplifies analysis of fine structural changes and the potential of cryo-EM, in favorable cases, to characterize mutant or ligand-bound complexes.

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Structure of AAV-DJ, a retargeted gene therapy vector: Cryo-electron microscopy at 4.5 Å resolution

Abstract

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