ArticleMedical science monitor basic research2013
A reliable and feasible qPCR strategy for titrating AAV vectors.
Article in Medical science monitor basic research, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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Who cites it
21 citing papers in PubMed, 30 citations in OpenAlex.
- Tracking Particle-Encapsulated DNA Across the Anion-Exchange Chromatography Fractions of Recombinant Adeno-Associated Virus Using Droplet Digital PCR and High-Throughput Sequencing.Biotechnology journal · 2025Article
- Development of a rabbit model for adrenoleukodystrophy: A pilot study on gene therapy using rAAV9.Molecular therapy. Nucleic acids · 2025Article
- Single-nuclei sequencing of skeletal muscle reveals subsynaptic-specific transcripts involved in neuromuscular junction maintenance.Nature communications · 2025Article
- An SDS-NaOH-based method to isolate genome of recombinant adeno-associated virus vectors for physical titer measurement.PloS one · 2025Article
- Evaluation of a rapid multi-attribute combinatorial high-throughput UV-Vis/DLS/SLS analytical platform for rAAV quantification and characterization.Molecular therapy. Methods & clinical development · 2024Article
- PCR-based analytics of gene therapies using adeno-associated virus vectors: Considerations for cGMP method development.Molecular therapy. Methods & clinical development · 2023Review
- Fast, multiplexable and efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9.Nature communications · 2023Article
- MiR133b-mediated inhibition of EGFR-PTK pathway promotes rAAV2 transduction by facilitating intracellular trafficking and augmenting second-strand synthesis.Journal of cellular and molecular medicine · 2023Article
- Gene Therapy Activates Retinal Pigment Epithelium Cell Proliferation for Age-related Macular Degeneration in a Mouse Model.Current medical science · 2023Article
- Gene therapy in a murine model of chronic eosinophilic leukemia-not otherwise specified (CEL-NOS).Leukemia · 2022Article
- PCR-Based Analytical Methods for Quantification and Quality Control of Recombinant Adeno-Associated Viral Vector Preparations.Pharmaceuticals (Basel, Switzerland) · 2021Review
- Quantitative analysis of genome packaging in recombinant AAV vectors by charge detection mass spectrometry.Molecular therapy. Methods & clinical development · 2021Article
- Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies.Molecular therapy. Methods & clinical development · 2021Review
- Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering.Scientific reports · 2021Article
- A qPCR Method for AAV Genome Titer with ddPCR-Level of Accuracy and Precision.Molecular therapy. Methods & clinical development · 2020Article
- [Optimized AAV package and experimental application of recombinant AAV8/hFⅧ for gene therapy on hemophilia A mice].Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi · 2020Article
- Filling Adeno-Associated Virus Capsids: Estimating Success by Cryo-Electron Microscopy.Human gene therapy · 2019Article
- Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors.Human gene therapy methods · 2019Article
- Accurate Quantification and Characterization of Adeno-Associated Viral Vectors.Frontiers in microbiology · 2019Article
- Optimized AAV rh.10 Vectors That Partially Evade Neutralizing Antibodies during Hepatic Gene Transfer.Frontiers in pharmacology · 2017Article
Corrections and comments
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
backgroundPrevious studies have revealed that traditional real-time quantitative PCR (qPCR) underestimates adeno-associated virus (AAV) titer. Because the inverted terminal repeat (ITR) exists in all AAV vectors, the only remaining element from the wild genome could form special configurations to interfere with qPCR titration. To solve this problem, a modified and universal qPCR method was tested and established. MATERIAL AND
methodsIn this work, there was a great variation in titration of ssAAV2-EGFP (Enhanced Green Fluorescence Protein) and scAAV2-EGFP genome by traditional qPCR. For ssAAV2-EGFP, the highest titer was found by using the targeting EGFP primers and the lowest titer was measured by those targeting bovine growth hormone polyA element (pBGH) primers.
resultsExperimental data were reverse for ssAAV2-EGFP and scAAV2-EGFP. Here we report an improved and universal SmaI qPCR method, based on cleaving all ITRs in AAV2 genome by SmaI with several advantages: (1) impact of all ITRs in ssAAV2 and scAAV2 was dismissed; (2) titers increased remarkably, up to 7-fold, especially for scAAV2; (3) the variation of titers was reduced when different primers were applied. A similar phenomenon was also observed in other ssAAV2 and scAAV2 products when the range of titration was at 3×107 to 7×109 V.G/µl in this study.
conclusionsThis modified qPCR strategy can increase rAAV' titer and reduce titration variance, possibly become a universal method for titrating AAV vectors.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.