Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material

Abstract

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.

FIG. 1.

Flow chart showing the steps of rAAV8RSM manufacturing. Note the pictures on the right showing the empty and full vector particle bands at the first and second CsCl gradient step. MCB, master cell bank; PEG, polyethylene glycol; QC, quality control; rAAV8, recombinant adeno-associated virus serotype 8; RSM, Reference Standard Materials.

FIG. 2.

The rAAV8RSM was run on SDS-PAGE gels under both denaturating and native conditions and then stained with (a) silver stain and (b) Sypro Ruby. In-house rAAV standard was run as a positive control and buffer as a negative control. The lanes for each gel are (1) benchmark ladder (unstained or prestained)—reduced; (2) negative control—reduced; (3) AAV8 reference material—reduced; (4) empty; (5) positive control—reduced; (6) benchmark ladder (unstained or prestained)—native; (7) negative control—native; (8) AAV reference material—native; (9) empty; and (10) positive control—native. SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

FIG. 3.

The rAAV2RSM and rAAV8RSM were run on SDS-PAGE gels under denaturating conditions and then stained with Sypro Ruby. Several decreasing amounts of vector particles (pt) of a reference rAAV8 vector (WL217S) were loaded as standard curve (lanes 2–6). Different amounts (10¹⁰ pt or 5×10⁹ pt, lanes 7 and 8 or 9 and 10, respectively) of rAAV2RSM (lane 7 and 9) and rAAV8RSM (lane 8 and 10) were loaded on a gel according to a particle titer of 9.1×10¹¹ pt/ml for rAAV2RSM (mean titer published by Lock et al., 2010) and 4.2×10¹¹ pt/ml for the rAAV8RSM (titer calculated by this lab using the ELISA AAV8).