Structure comparison of the chimeric AAV2.7m8 vector with parental AAV2 |
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| Institution: | 1. Department of Biochemistry and Molecular Biology, Center for Structural Biology, McKnight Brain Institute, College of Medicine, University of Florida, 1200 Newell Drive, Gainesville, FL 32610, USA;2. Adverum Biotechnologies, 1035 O’Brien Dr., Menlo Park, CA 94025, USA;3. Biological Science Imaging Resource, Department of Biological Sciences, The Florida State University, 89 Chieftan Way, Rm 119, Tallahassee, FL 32306, USA;1. Department of Biochemistry & Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, College of Medicine, University of Florida, 1600 SW Archer Road, P.O. Box 100245, Gainesville, FL 32610, USA;2. Gene Therapy Center and the Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA;3. German Cancer Research Center, Heidelberg, Germany;1. Ophthalmology Disease Area, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, USA;2. Ophthalmology Disease Area, Novartis Institutes for BioMedical Research, Fort Worth, Texas, USA;3. Preclinical Safety, Alcon, Fort Worth, Texas, USA;4. Preclinical Safety, Novartis Institutes for BioMedical Research, East Hanover, New Jersey, USA;1. Translational Vectorology Research Unit, Children’s Medical Research Institute, The University of Sydney, Westmead, NSW 2145, Australia;2. Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK;3. Gene Therapy Research Unit, Children’s Medical Research Institute & The Children’s Hospital at Westmead, University of Sydney, Westmead, NSW 2145, Australia;4. Commonwealth Scientific and Industrial Research Organisation (CSIRO), North Ryde, NSW 2113, Australia;5. Department of Biochemistry and Molecular Biology, Center for Structural Biology, University of Florida, Gainesville, FL 32610, USA;6. Discipline of Child and Adolescent Health, The University of Sydney, Sydney, NSW 2006, Australia;7. Vector and Genome Engineering Facility, Children’s Medical Research Institute, The University of Sydney, Westmead, NSW 2145, Australia;8. Military Institute of Hygiene and Epidemiology, Biological Threats Identification and Countermeasure Center, 24-100 Puławy, Poland;1. Department of Biochemistry and Molecular Biology, College of Medicine, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA;2. Biological Science Imaging Resource, Department of Biological Sciences, The Florida State University, 89 Chieftan Way, Rm 119, Tallahassee, FL 32306, USA;3. Department of Molecular Genetics and Microbiology and Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, FL 32610, USA;4. Department of Pharmacology, Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA;1. Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL, USA;2. Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA;1. Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL, USA;2. Department of Cardiology and Laboratory of Gene Therapy for Heart Diseases, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan, China |
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| Abstract: | The AAV2.7m8 vector is an engineered capsid with a 10-amino acid insertion in adeno-associated virus (AAV) surface variable region VIII (VR-VIII) resulting in the alteration of an antigenic region of AAV2 and the ability to efficiently transduce retina cells following intravitreal administration. Directed evolution and in vivo screening in the mouse retina isolated this vector. In the present study, we sought to identify the structural differences between a recombinant AAV2.7m8 (rAAV2.7m8) vector packaging a GFP genome and its parental serotype, AAV2, by cryo-electron microscopy (cryo-EM) and image reconstruction. The structures of rAAV2.7m8 and AAV2 were determined to 2.91 and 3.02 Å resolution, respectively. The rAAV2.7m8 amino acid side-chains for residues 219–745 (the last C-terminal residue) were interpretable in the density map with the exception of the 10 inserted amino acids. While observable in a low sigma threshold density, side-chains were only resolved at the base of the insertion, likely due to flexibility at the top of the loop. A comparison to parental AAV2 (ordered from residues 217–735) showed the structures to be similar, except at some side-chains that had different orientations and, in VR-VIII containing the 10 amino acid insertion. VR-VIII is part of an AAV2 antigenic epitope, and the difference is consistent with rAAV2.7m8′s escape from a known AAV2 monoclonal antibody, C37-B. The observations provide valuable insight into the configuration of inserted surface peptides on the AAV capsid and structural differences to be leveraged for future AAV vector rational design, especially for retargeted tropism and antibody escape. |
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| Keywords: | Adeno-associated virus AAV cryo-EM AAV2 7m8 Gene delivery |
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