Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material

Abstract

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 x 10¹¹ particles/ml; 95% confidence interval [CI], 7.89 x 10¹¹ to 1.05 x 10¹² particles/ml), vector genomes ({X}, 3.28 x 10¹⁰ vector genomes/ml; 95% CI, 2.70 x 10¹⁰ to 4.75 x 10¹⁰ vector genomes/ml), transducing units ({X}, 5.09 x 10⁸ transducing units/ml; 95% CI, 2.00 x 10⁸ to 9.60 x 10⁸ transducing units/ml), and infectious units ({X}, 4.37 x 10⁹ TCID₅₀ IU/ml; 95% CI, 2.06 x 10⁹ to 9.26 x 10⁹ TCID₅₀ IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.

LRH-1–dependent glucose sensing determines intermediary metabolism in liver

“…AAV8 encoding the mouse SHP or GCK cDNAs (AAV8-SHP or AAV8-GCK), driven by the hybrid cytomegalovirus enhancer/chicken β-actin constitutive promoter or the liver-specific α1-antitrypsin promoter (hAAT; ref. 42), respectively, were generated and titrated as described previously to enable their use in vivo (43,44). Plasmids used to generate viral particles and the hAAT promoter were provided by K.A.…”

Impaired SUMOylation of nuclear receptor LRH-1 promotes nonalcoholic fatty liver disease

“…The Osbpl3-siRNA sequence (Supplemental Table 4) was engineered to create a pre-miRNA sequence targeting Osbpl3, which was cloned into an AAV vector construct driven by the liver-specific hAAT promoter. AAV8 mirOsbpl3 viral vectors were generated and titrated as described previously (48,49) and injected into jugular vein under isoflurane anesthesia (5 × 10 11 vg). Two weeks after the injection, mice were fasted for 24 hours followed by 6 hours refeeding, then sacrificed, and tissues were snap-frozen.…”

Soliciting Strategies for Developing Cell-Based Reference Materials to Advance Mesenchymal Stromal Cell Research and Clinical Translation

“…Indeed, physical standards are routinely used in many systems. For gene transfer vectors, recombinant adeno-associated virus 2 (AAV2) has been produced to provide a standard of reference for particle, vector genome, and infectious titer of AAV2 vectors [9]. In flow cytometry cell sorting or analysis, there are fluorescent beads to standardize the performance of the lasers and machines.…”

“…In general, a standard is perceived as impartial and is established through high-level international standardization programs [ [9] was developed over several years as a consortium effort. A similar consortium of groups, including governmental agencies such as the National Institutes of Health, (NIH), and FDA, or foundation, industry, or other groups including, for example, the California Institute of Regenerative Medicine (CIRM), the International Society for Stem Cell Research (ISSCR), the International Society for Cellular Therapy (ISCT), and nonprofit organizations such as the Drug Information Association (DIA) could take the lead in developing and validating such a ruler type of reference cell material.…”