ArticleHuman gene therapy2020
A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts.
Article in Human gene therapy, 2020. 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, 37 citations in OpenAlex.
- Generation and characterization of a HEK293 cell line optimized for recombinant adeno-associated virus production.Biotechnology reports (Amsterdam, Netherlands) · 2026Article
- Article
- Quantifying DNA point mutations in commercially available AAV reporter vectors and plasmids.Molecular therapy. Methods & clinical development · 2025Article
- E2A, VA RNA I, and L4-22k adenoviral helper genes are sufficient for AAV production in HEK293 cells.Molecular therapy. Methods & clinical development · 2024Article
- Characterization of AAV vectors: A review of analytical techniques and critical quality attributes.Molecular therapy. Methods & clinical development · 2024Review
- Article
- A Comprehensive Study of the Effects by Sequence Truncation within Inverted Terminal Repeats (ITRs) on the Productivity, Genome Packaging, and Potency of AAV Vectors.Microorganisms · 2024Article
- Stowaways in the cargo: Contaminating nucleic acids in rAAV preparations for gene therapy.Molecular therapy : the journal of the American Society of Gene Therapy · 2023Review
- Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis.Analytical chemistry · 2023Article
- Quality control for Adeno-associated viral vector production.Neuromethods · 2023Article
- Cryptic resolution sites in the vector plasmid lead to the heterogeneities in the rAAV vectors.Journal of medical virology · 2023Article
- Article
- Evaluating the state of the science for adeno-associated virus integration: An integrated perspective.Molecular therapy : the journal of the American Society of Gene Therapy · 2022Review
- Human and Insect Cell-Produced Recombinant Adeno-Associated Viruses Show Differences in Genome Heterogeneity.Human gene therapy · 2022Article
- Biodistribution and Tolerability of AAV-PHP.B-CBh-Human gene therapy · 2022Article
- PCR-Based Analytical Methods for Quantification and Quality Control of Recombinant Adeno-Associated Viral Vector Preparations.Pharmaceuticals (Basel, Switzerland) · 2021Review
- Analytical methods to characterize recombinant adeno-associated virus vectors and the benefit of standardization and reference materials.Current opinion in biotechnology · 2021Review
- Mechanistic model for production of recombinant adeno-associated virus via triple transfection of HEK293 cells.Molecular therapy. Methods & clinical development · 2021Article
- Contamination Issue in Viral Metagenomics: Problems, Solutions, and Clinical Perspectives.Frontiers in microbiology · 2021Review
- Nanopore sequencing of native adeno-associated virus (AAV) single-stranded DNA using a transposase-based rapid protocol.NAR genomics and bioinformatics · 2020Article
Corrections and comments
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Authors and funding
14 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Recombinant adeno-associated virus (rAAV) vectors are increasingly popular gene delivery tools in biological systems. They are safe and lead to high-level, long-term transgene expression. rAAV are available in multiple serotypes, natural or engineered, which enable targeting to a wide array of tissues and cell types. In addition, rAAVs are relatively easily produced in a well-equipped lab or obtained from a viral vector core facility. Unfortunately, there is no standardization of quality control assays beyond titering and purity assessments. Next-generation sequencing (NGS) can be used to identify rAAV preparations. Because the rAAV genome is single stranded, previous studies have assumed that rAAV genomes must be converted to double strands before NGS. We demonstrate that rAAV DNA extracts exist primarily as double-stranded species. We hypothesize that these molecules form from the natural base pairing of complementary [+] and [-] strands after DNA extraction and show that rAAV DNA extracts are sufficient templates for downstream NGS without the labor-intensive double-stranding step. Here, we provide a detailed protocol for the simple and rapid NGS of rAAV genomes from DNA extracts. With this protocol, users can quickly confirm the identity of an rAAV preparation and detect the presence of contaminating rAAV DNA. In addition, we share custom Python scripts that allow users to accurately determine the serotype and detect Cre-independent DNA recombination events in rAAV containing Lox sites within minutes. We have used these scripts to analyze more than 100 rAAV preparations. Although we focused on the detection of cross-contaminating rAAV DNA and recombination events, our Python scripts can be customized to detect other sequences or events, such as reverse packaging of plasmid backbone or DNA from the packaging cell line. We find that the NGS of rAAV DNA extracts, termed viral genome sequencing, is a simple and powerful method for rAAV validation.
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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.