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Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls

Although the clinical use of recombinant adeno-associated virus (rAAV) vectors is constantly increasing, the development of suitable quality control methods is still needed for accurate vector characterization. Among the quality criteria, the titration of infectious particles is critical to determin...

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Autores principales: François, Achille, Bouzelha, Mohammed, Lecomte, Emilie, Broucque, Frédéric, Penaud-Budloo, Magalie, Adjali, Oumeya, Moullier, Philippe, Blouin, Véronique, Ayuso, Eduard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090651/
https://www.ncbi.nlm.nih.gov/pubmed/30112419
http://dx.doi.org/10.1016/j.omtm.2018.07.004
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author François, Achille
Bouzelha, Mohammed
Lecomte, Emilie
Broucque, Frédéric
Penaud-Budloo, Magalie
Adjali, Oumeya
Moullier, Philippe
Blouin, Véronique
Ayuso, Eduard
author_facet François, Achille
Bouzelha, Mohammed
Lecomte, Emilie
Broucque, Frédéric
Penaud-Budloo, Magalie
Adjali, Oumeya
Moullier, Philippe
Blouin, Véronique
Ayuso, Eduard
author_sort François, Achille
collection PubMed
description Although the clinical use of recombinant adeno-associated virus (rAAV) vectors is constantly increasing, the development of suitable quality control methods is still needed for accurate vector characterization. Among the quality criteria, the titration of infectious particles is critical to determine vector efficacy. Different methods have been developed for the measurement of rAAV infectivity in vitro, based on detection of vector genome replication in trans-complementing cells infected with adenovirus, detection of transgene expression in permissive cells, or simply detection of intracellular vector genomes following the infection of indicator cells. In the present study, we have compared these methods for the titration of infectious rAAV8 vector particles, and, to assess their ability to discriminate infectious and non-infectious rAAV serotype 8 particles, we have generated a VP1-defective AAV8-GFP vector. Since VP1 is required to enter the cell nucleus, the lack of VP1 should drastically reduce the infectivity of rAAV particles. The AAV8 reference standard material was used as a positive control. Our results demonstrated that methods based on measurement of rAAV biological activity (i.e., vector genome replication or transgene expression) were able to accurately discriminate infectious versus non-infectious particles, whereas methods simply measuring intracellular vector genomes were not. Several cell fractionation protocols were tested in an attempt to specifically measure vector genomes that had reached the nucleus, but genomes from wild-type and VP1-defective AAV8 particles were equally detected in the nuclear fraction by qPCR. These data highlight the importance of using suitable controls, including a negative control, for the development of biological assays such as infectious unit titration.
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spelling pubmed-60906512018-08-15 Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls François, Achille Bouzelha, Mohammed Lecomte, Emilie Broucque, Frédéric Penaud-Budloo, Magalie Adjali, Oumeya Moullier, Philippe Blouin, Véronique Ayuso, Eduard Mol Ther Methods Clin Dev Article Although the clinical use of recombinant adeno-associated virus (rAAV) vectors is constantly increasing, the development of suitable quality control methods is still needed for accurate vector characterization. Among the quality criteria, the titration of infectious particles is critical to determine vector efficacy. Different methods have been developed for the measurement of rAAV infectivity in vitro, based on detection of vector genome replication in trans-complementing cells infected with adenovirus, detection of transgene expression in permissive cells, or simply detection of intracellular vector genomes following the infection of indicator cells. In the present study, we have compared these methods for the titration of infectious rAAV8 vector particles, and, to assess their ability to discriminate infectious and non-infectious rAAV serotype 8 particles, we have generated a VP1-defective AAV8-GFP vector. Since VP1 is required to enter the cell nucleus, the lack of VP1 should drastically reduce the infectivity of rAAV particles. The AAV8 reference standard material was used as a positive control. Our results demonstrated that methods based on measurement of rAAV biological activity (i.e., vector genome replication or transgene expression) were able to accurately discriminate infectious versus non-infectious particles, whereas methods simply measuring intracellular vector genomes were not. Several cell fractionation protocols were tested in an attempt to specifically measure vector genomes that had reached the nucleus, but genomes from wild-type and VP1-defective AAV8 particles were equally detected in the nuclear fraction by qPCR. These data highlight the importance of using suitable controls, including a negative control, for the development of biological assays such as infectious unit titration. American Society of Gene & Cell Therapy 2018-07-27 /pmc/articles/PMC6090651/ /pubmed/30112419 http://dx.doi.org/10.1016/j.omtm.2018.07.004 Text en © 2018 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Article
François, Achille
Bouzelha, Mohammed
Lecomte, Emilie
Broucque, Frédéric
Penaud-Budloo, Magalie
Adjali, Oumeya
Moullier, Philippe
Blouin, Véronique
Ayuso, Eduard
Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls
title Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls
title_full Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls
title_fullStr Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls
title_full_unstemmed Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls
title_short Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls
title_sort accurate titration of infectious aav particles requires measurement of biologically active vector genomes and suitable controls
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090651/
https://www.ncbi.nlm.nih.gov/pubmed/30112419
http://dx.doi.org/10.1016/j.omtm.2018.07.004
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