ArticleHuman gene therapy methods2014
Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.
Article in Human gene therapy methods, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 103 papers.
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Who cites it
103 citing papers in PubMed.
- Fecal AAV8 shedding and co-housing reduces EAE disease severity: implications for preclinical study design.Frontiers in medicine · 2026Article
- A transcriptomic atlas of astrocyte heterogeneity across space and time in mouse and marmoset.Neuron · 2025Article
- Plasmonic Nanopore Sensing to Probe the DNA Loading Status of Adeno-Associated Viruses.Chemosensors (Basel, Switzerland) · 2025Article
- Method validation and assessment of the biodistribution and shedding for adenovirus vector-based vaccine using qPCR and dPCR.Molecular therapy. Methods & clinical development · 2025Article
- Safety and efficacy of CRISPR-mediated genome ablation of VEGFA as a treatment for choroidal neovascularization in nonhuman primate eyes.Molecular therapy : the journal of the American Society of Gene Therapy · 2025Article
- Astrocyte regional specialization is shaped by postnatal development.bioRxiv : the preprint server for biology · 2025Article
- Gene therapy with feline anti-Müllerian hormone analogs disrupts folliculogenesis and induces pregnancy loss in female domestic cats.Nature communications · 2025Article
- Three-dimensional linkage analysis with digital PCR for genome integrity and identity of recombinant adeno-associated virus.Scientific reports · 2025Article
- Biomechanical characteristics of frozen bone allografts after gene therapy-induced revascularization.Academia biology · 2025Article
- Implementing a robust platform analytical procedure for measuring adeno-associated virus vector genome titer.Molecular therapy. Methods & clinical development · 2024Article
- Physicochemical and biological impacts of light stress on adeno-associated virus serotype 6.Molecular therapy. Methods & clinical development · 2024Article
- Chronic Activation of Tubulin Tyrosination Improves Heart Function.Circulation research · 2024Article
- 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
- Characterization of AAV vectors: A review of analytical techniques and critical quality attributes.Molecular therapy. Methods & clinical development · 2024Review
- An amplification-free CRISPR-Cas12a assay for titer determination and composition analysis of the rAAV genome.Molecular therapy. Methods & clinical development · 2024Article
- Assessment of Key Factors Impacting Variability in AAV Vector Genome Titration by Digital PCR.International journal of molecular sciences · 2024Article
- High-dose systemic adeno-associated virus vector administration causes liver and sinusoidal endothelial cell injury.Molecular therapy : the journal of the American Society of Gene Therapy · 2024Article
- Quantification of low-level human cytomegalovirus and Epstein-Barr virus DNAemia by digital PCR.Journal of virological methods · 2024Article
- Modular vector assembly enables rapid assessment of emerging CRISPR technologies.Cell genomics · 2024Article
- Prednisolone and rapamycin reduce the plasma cell gene signature and may improve AAV gene therapy in cynomolgus macaques.Gene therapy · 2024Article
43 more citing papers are in PubMed but not listed here.
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Authors and funding
4 authors.
Funding
Abstract
Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution analysis in inhibitory tissues.
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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.