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ABSTRACT Patients with neutralizing antibodies (Nabs) against adeno-associated virus (AAV) are usually excluded from the treatment with AAV vectors. To develop a standard assay for detecting

Nab inhibition activity, we systematically studied current AAV Nab assays _in vitro_ and _in vivo_. Several factors were found that influence the Nab titers based on the _in vitro_ assay,

including sera volume, AAV dose per cell, cell number and choice of transgenes. When the Nab titer assay was performed _in vivo_ via intramuscular (IM) or systemic administration, a fourfold

increase in sensitivity for measurement of Nab titers was observed compared with an identical _in vitro_ test. To better mimic the clinical setting, after passively transferring human Nabs

into mice, blood was collected before systemic injection of AAV vector and used for Nab titer analysis _in vitro_ or via IM injection. The results showed that AAV delivered via IM injection

had a similar inhibition pattern to systemic administration. These studies indicate critical parameters necessary for optimizing Nab sensitivity and that an _in vivo_ Nab assay is more

sensitive than an _in vitro_ assay for inclusion/exclusion criteria. The variables identified by this study may explain some of the compounding clinical data seen to date with respect to

efficiency of AAV transduction in various phase I clinical trials. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution

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about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS CLINICAL ENROLLMENT ASSAY TO DETECT PREEXISTING NEUTRALIZING ANTIBODIES TO

AAV6 WITH DEMONSTRATED TRANSGENE EXPRESSION IN GENE THERAPY TRIALS Article Open access 01 July 2022 ANTIVIRAL NEUTRALIZING ANTIBODIES: FROM IN VITRO TO IN VIVO ACTIVITY Article 17 April

2023 SEROPREVALENCE OF ADENO-ASSOCIATED VIRUS TYPES 1, 2, 3, 4, 5, 6, 8, AND 9 IN A BASQUE COHORT OF HEALTHY DONORS Article Open access 10 July 2024 REFERENCES * Bennett J, Ashtari M,

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1 (rAAV-1) and rAAV-6 in mice. _J Virol_ 2013; 87: 10784–10791. Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful to Xiaojing Chen

and Karen Hogan for their excellent technical assistance, as well as Marc Weinberg for his critical reading of the manuscript. We acknowledge the UNC Biomedical Research Imaging Center

(BRIC) Small Animal Imaging (SAI) facility for assistance of mouse imaging. This work was supported by National Institutes of Health Grants R01DK084033 (to CL and RJS), P01HL112761,

R01AI072176, R01AR064369 (to RJS), P30-CA016086-35-37, U54-CA151652-01-04 (to the BRIC SAI facility) and a research grant from Asklepios BioPharmaceutical (to CL). AUTHOR INFORMATION AUTHORS

AND AFFILIATIONS * Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin, China M Wang * Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC,

USA, M Wang, A Crosby, E Hastie, G Joshua, R J Samulski & C Li * Asklepios BioPharmaceutical Inc., Chapel Hill, NC, USA J J Samulski & S McPhee * Department of Pharmacology,

University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, R J Samulski * Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA C Li Authors * M

Wang View author publications You can also search for this author inPubMed Google Scholar * A Crosby View author publications You can also search for this author inPubMed Google Scholar * E

Hastie View author publications You can also search for this author inPubMed Google Scholar * J J Samulski View author publications You can also search for this author inPubMed Google

Scholar * S McPhee View author publications You can also search for this author inPubMed Google Scholar * G Joshua View author publications You can also search for this author inPubMed 

Google Scholar * R J Samulski View author publications You can also search for this author inPubMed Google Scholar * C Li View author publications You can also search for this author

inPubMed Google Scholar CORRESPONDING AUTHORS Correspondence to R J Samulski or C Li. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL

INFORMATION Supplementary Information accompanies this paper on Gene Therapy website SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION (DOC 289 KB) RIGHTS AND PERMISSIONS Reprints and

permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Wang, M., Crosby, A., Hastie, E. _et al._ Prediction of adeno-associated virus neutralizing antibody activity for clinical application. _Gene

Ther_ 22, 984–992 (2015). https://doi.org/10.1038/gt.2015.69 Download citation * Received: 22 April 2015 * Revised: 18 June 2015 * Accepted: 23 June 2015 * Published: 30 June 2015 * Issue

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