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Find video protocols related to scientific articles indexed in Pubmed.
Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material.
Hum. Gene Ther.
PUBLISHED: 10-03-2014
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Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
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Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale.
Hum. Gene Ther.
PUBLISHED: 05-26-2010
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Adeno-associated viral (AAV) manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. Although scalable systems based on AAV-adenovirus, AAV-herpesvirus, and AAV-baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate-scale preclinical studies in large animals, in which several combinations of serotype and genome may need to be tested. We observed that during production of many AAV serotypes, large amounts of vector are found in the culture supernatant, a relatively pure source of vector in comparison with cell-derived material. Here we describe a high-yielding, recombinant AAV production process based on polyethylenimine (PEI)-mediated transfection of HEK293 cells and iodixanol gradient centrifugation of concentrated culture supernatant. The entire process can be completed in 1 week and the steps involved are universal for a number of different AAV serotypes. Process conditions have been optimized such that final purified yields are routinely greater than 1?x?10(14) genome copies per run, with capsid protein purity exceeding 90%. Initial experiments with vectors produced by the new process demonstrate equivalent or better transduction both in vitro and in vivo when compared with small-scale, CsCl gradient-purified vectors. In addition, the iodixanol gradient purification process described effectively separates infectious particles from empty capsids, a desirable property for reducing toxicity and unwanted immune responses during preclinical studies.
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Analysis of particle content of recombinant adeno-associated virus serotype 8 vectors by ion-exchange chromatography.
Hum Gene Ther Methods
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Advances in adeno-associated virus (AAV)-mediated gene therapy have brought the possibility of commercial manufacturing of AAV vectors one step closer. To realize this prospect, a parallel effort with the goal of ever-increasing sophistication for AAV vector production technology and supporting assays will be required. Among the important release assays for a clinical gene therapy product, those monitoring potentially hazardous contaminants are most critical for patient safety. A prominent contaminant in many AAV vector preparations is vector particles lacking a genome, which can substantially increase the dose of AAV capsid proteins and lead to possible unwanted immunological consequences. Current methods to determine empty particle content suffer from inconsistency, are adversely affected by contaminants, or are not applicable to all serotypes. Here we describe the development of an ion-exchange chromatography-based assay that permits the rapid separation and relative quantification of AAV8 empty and full vector particles through the application of shallow gradients and a strong anion-exchange monolith chromatography medium.
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Absolute Determination of Single-Stranded and Self-Complementary AAV Vector Genome Titers by Droplet Digital PCR.
Hum Gene Ther Methods
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Accurate titration of AAV vector genome copies is critical for ensuring correct and reproducible dosing in both pre-clinical and clinical settings. qPCR is the current method of choice for titrating AAV genomes due to the simplicity, accuracy and robustness of the assay. Recently 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 DI genomes have been reported. Alternate 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 which directly quantifies DNA copies with an un-paralleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties which appear to 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 increased sensitivity over a standard qPCR titration but with equivalent sensitivity to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-fold, 1.9-fold and 2.3-fold higher than those determined by standard qPCR, optimized qPCR and agarose gel assays respectively. ddPCR-based genome titering was superior to qPCR in terms of both intra- and inter-assay precision and is more resistant to PCR reaction inhibitors, a desirable feature for in-process monitoring of early stage production and for vector genome biodistribution analysis in inhibitory tissues.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.