Site-Specific Glycation and Chemo-enzymatic Antibody Sortagging for the Retargeting of rAAV6 to Inflamed Endothelium

Hannah A. Pearce, Hongwei Qian, Timothy U. Connell, Dexing Huang, Claudia Gottstein, Paul S. Donnelly, Karlheinz Peter, Paul Gregorevic, Christoph E. Hagemeyer

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Gene therapy holds great potential for conditions such as cardiovascular disease, including atherosclerosis and also vascular cancers, yet available vectors such as the adeno-associated virus (rAAV) transduce the vasculature poorly. To enable retargeting, a single-chain antibody (scFv) that binds to the vascular cell-adhesion molecule (VCAM-1) overexpressed at areas of endothelial inflammation was site specifically and covalently conjugated to the exterior of rAAV6. To achieve conjugation, the scFv was functionalized with an orthogonal click chemistry group. This conjugation utilized site-specific sortase A methodology, thus preserving scFv binding capacity to VCAM-1. The AAV6 was separately functionalized with 4-azidophenyl glyoxal (APGO) via covalent adducts to arginine residues in the capsid's heparin co-receptor binding region. APGO functionalization removed native tropism, greatly reducing rAAV6-GFP transduction into all cells tested, and the effect was similar to the inhibition seen in the presence of heparin. Utilizing the incorporated functionalizations, the scFv was then covalently conjugated to the exterior of rAAV6 via strain-promoted azide-alkyne cycloaddition (SPAAC). With both the removal of native heparin tropism and the addition of VCAM-1 targeting, rAAV6 transduction of endothelial cells was greatly enhanced compared to control cells. Thus, this novel and modular targeting system could have further application in re-directing AAV6 toward inflamed endothelium for therapeutic use.

Original languageEnglish
Pages (from-to)261-269
Number of pages9
JournalMolecular Therapy - Methods and Clinical Development
Volume14
DOIs
Publication statusPublished - 13 Sep 2019

Keywords

  • AAV
  • antibody
  • click chemistry
  • glyoxal
  • sortase
  • VCAM-1

Cite this

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title = "Site-Specific Glycation and Chemo-enzymatic Antibody Sortagging for the Retargeting of rAAV6 to Inflamed Endothelium",
abstract = "Gene therapy holds great potential for conditions such as cardiovascular disease, including atherosclerosis and also vascular cancers, yet available vectors such as the adeno-associated virus (rAAV) transduce the vasculature poorly. To enable retargeting, a single-chain antibody (scFv) that binds to the vascular cell-adhesion molecule (VCAM-1) overexpressed at areas of endothelial inflammation was site specifically and covalently conjugated to the exterior of rAAV6. To achieve conjugation, the scFv was functionalized with an orthogonal click chemistry group. This conjugation utilized site-specific sortase A methodology, thus preserving scFv binding capacity to VCAM-1. The AAV6 was separately functionalized with 4-azidophenyl glyoxal (APGO) via covalent adducts to arginine residues in the capsid's heparin co-receptor binding region. APGO functionalization removed native tropism, greatly reducing rAAV6-GFP transduction into all cells tested, and the effect was similar to the inhibition seen in the presence of heparin. Utilizing the incorporated functionalizations, the scFv was then covalently conjugated to the exterior of rAAV6 via strain-promoted azide-alkyne cycloaddition (SPAAC). With both the removal of native heparin tropism and the addition of VCAM-1 targeting, rAAV6 transduction of endothelial cells was greatly enhanced compared to control cells. Thus, this novel and modular targeting system could have further application in re-directing AAV6 toward inflamed endothelium for therapeutic use.",
keywords = "AAV, antibody, click chemistry, glyoxal, sortase, VCAM-1",
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Site-Specific Glycation and Chemo-enzymatic Antibody Sortagging for the Retargeting of rAAV6 to Inflamed Endothelium. / Pearce, Hannah A.; Qian, Hongwei; Connell, Timothy U.; Huang, Dexing; Gottstein, Claudia; Donnelly, Paul S.; Peter, Karlheinz; Gregorevic, Paul; Hagemeyer, Christoph E.

In: Molecular Therapy - Methods and Clinical Development, Vol. 14, 13.09.2019, p. 261-269.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Site-Specific Glycation and Chemo-enzymatic Antibody Sortagging for the Retargeting of rAAV6 to Inflamed Endothelium

AU - Pearce, Hannah A.

AU - Qian, Hongwei

AU - Connell, Timothy U.

AU - Huang, Dexing

AU - Gottstein, Claudia

AU - Donnelly, Paul S.

AU - Peter, Karlheinz

AU - Gregorevic, Paul

AU - Hagemeyer, Christoph E.

PY - 2019/9/13

Y1 - 2019/9/13

N2 - Gene therapy holds great potential for conditions such as cardiovascular disease, including atherosclerosis and also vascular cancers, yet available vectors such as the adeno-associated virus (rAAV) transduce the vasculature poorly. To enable retargeting, a single-chain antibody (scFv) that binds to the vascular cell-adhesion molecule (VCAM-1) overexpressed at areas of endothelial inflammation was site specifically and covalently conjugated to the exterior of rAAV6. To achieve conjugation, the scFv was functionalized with an orthogonal click chemistry group. This conjugation utilized site-specific sortase A methodology, thus preserving scFv binding capacity to VCAM-1. The AAV6 was separately functionalized with 4-azidophenyl glyoxal (APGO) via covalent adducts to arginine residues in the capsid's heparin co-receptor binding region. APGO functionalization removed native tropism, greatly reducing rAAV6-GFP transduction into all cells tested, and the effect was similar to the inhibition seen in the presence of heparin. Utilizing the incorporated functionalizations, the scFv was then covalently conjugated to the exterior of rAAV6 via strain-promoted azide-alkyne cycloaddition (SPAAC). With both the removal of native heparin tropism and the addition of VCAM-1 targeting, rAAV6 transduction of endothelial cells was greatly enhanced compared to control cells. Thus, this novel and modular targeting system could have further application in re-directing AAV6 toward inflamed endothelium for therapeutic use.

AB - Gene therapy holds great potential for conditions such as cardiovascular disease, including atherosclerosis and also vascular cancers, yet available vectors such as the adeno-associated virus (rAAV) transduce the vasculature poorly. To enable retargeting, a single-chain antibody (scFv) that binds to the vascular cell-adhesion molecule (VCAM-1) overexpressed at areas of endothelial inflammation was site specifically and covalently conjugated to the exterior of rAAV6. To achieve conjugation, the scFv was functionalized with an orthogonal click chemistry group. This conjugation utilized site-specific sortase A methodology, thus preserving scFv binding capacity to VCAM-1. The AAV6 was separately functionalized with 4-azidophenyl glyoxal (APGO) via covalent adducts to arginine residues in the capsid's heparin co-receptor binding region. APGO functionalization removed native tropism, greatly reducing rAAV6-GFP transduction into all cells tested, and the effect was similar to the inhibition seen in the presence of heparin. Utilizing the incorporated functionalizations, the scFv was then covalently conjugated to the exterior of rAAV6 via strain-promoted azide-alkyne cycloaddition (SPAAC). With both the removal of native heparin tropism and the addition of VCAM-1 targeting, rAAV6 transduction of endothelial cells was greatly enhanced compared to control cells. Thus, this novel and modular targeting system could have further application in re-directing AAV6 toward inflamed endothelium for therapeutic use.

KW - AAV

KW - antibody

KW - click chemistry

KW - glyoxal

KW - sortase

KW - VCAM-1

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U2 - 10.1016/j.omtm.2019.07.003

DO - 10.1016/j.omtm.2019.07.003

M3 - Article

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JO - Molecular Therapy - Methods and Clinical Development

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SN - 2329-0501

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