Good Manufacturing Practice Production of Self-Complementary Serotype 8 Adeno-Associated Viral Vector for a Hemophilia B Clinical Trial

Abstract

To generate sufficient clinical-grade vector to support a phase I/II clinical trial of adeno-associated virus serotype 8 (AAV8)-mediated factor IX (FIX) gene transfer for hemophilia B, we have developed a large-scale, good manufacturing practice (GMP)-compatible method for vector production and purification. We used a 293T-based two-plasmid transient transfection system coupled with a three-column chromatography purification process to produce high-quality self-complementary AAV2/8 FIX clinical-grade vector. Two consecutive production campaigns using a total of 432 independent 10-stack culture chambers produced a total of *2Â10 15 vector genomes (VG) by dot-blot hybridization. Benzonase-treated microfluidized lysates generated from pellets of transfected cells were purified by group separation on Sepharose beads followed by anion-exchange chromatography. The virus-containing fractions were further processed by gel filtration and ultrafiltration, using a 100-kDa membrane. The vector was formulated in phosphate-buffered saline plus 0.25% human serum albumin. Spectrophotometric analysis suggested *20% full particles, with only low quantities of nonviral proteins were visible on silver-stained sodium dodecyl sulfate-polyacrylamide gels. A sensitive assay for the detection of replication-competent AAV was developed, which did reveal trace quantities of such contaminants in the final product. Additional studies have confirmed the long-term stability of the vector at À808C for at least 24 months and for at least 24 hr formulated in the clinical diluent and stored at room temperature within intravenous bags. This material has been approved for use in clinical trials in the United States and the United Kingdom.

The gene therapy journey for hemophilia: are we there yet?

“…In many manufacturing procedures, these can constitute a majority of the initial product (Ͼ 90%). [29][30][31] Manufacturing processes vary in terms of whether these "empties" are removed or not. Evidence has accumulated, though, 32 to suggest that empty AAV capsids can gain access to a target cell and thus contribute to the intracellular capsid burden that will eventually be processed and presented on the surface of the transduced cell, flagging these cells for destruction by capsid-specific CD8 ϩ T cells.…”

AAV Delivery of Endothelin-1 shRNA Attenuates Cold-Induced Hypertension

“…[35][36] AAV has been approved by the Food and Drug Administration as a vector for clinical gene therapy. [37][38][39][40][41][42][43][44] In this study we found that the virus had no effect on growth rate, food consumption (data not shown), or heart function. There were no deaths following the tail vein injection and the rats developed no abnormalities for the duration of the treatment.…”

Intrabodies as Neuroprotective Therapeutics

“…AAV currently appears to be the safest human gene therapy vector for central nervous system clinical trials as it transduces nondividing cells without genomic integration, leading to long-term expression, and there are multiple different capsid variants available to improve cell-specific transduction [99]. Genes encoding intrabodies are small enough (750 nucleotides for scFvs and 360-420 for singledomain nanobodies) that the coding regions can be readily accommodated within either single or double-stranded AAV vectors [100], leaving space for bispecific or bifunctional constructs. A localized immune response to AAV9 that induced a response against the transgene was recently reported in rats [101].…”