Abstract
Gene therapy product release requires reliable and consistent demonstration of biopotency. In hemophilia B vectors, this is usually determined in vivo by measuring the plasma levels of the expressed human factor IX (FIX) transgene product in FIX knockout mice. To circumvent this laborious assay, we developed an in vitro method in which the HepG2 human liver cell line was infected with the vector, and the resulting FIX activity was determined in the conditioned medium using a chromogenic assay. The initial low sensitivity of the assay, particularly toward adeno-associated viral serotype 8 (AAV8), increased approximately 100-fold and allowed linear measurement in a broad range of multiplicities of infection. Statistical parameters indicated high assay repeatability (relative standard deviation (RSD) < 5%) and intra-assay reproducibility (RSD < 20%). To compare the performance of the in vitro and in vivo biopotency assay, we applied statistical analyses including regression techniques and variation decomposition to the results obtained for 25 AAV8-FIX vector lots (BAX 335). These showed a highly significant correlation, with the cell culture-based assay demonstrating less variation than the in vivo test. The in vitro assay thus constitutes a viable alternative to using animals for lot release testing.
Highlights
Hemophilia B is caused by an X-linked mutation in the factor IX (FIX) (F9) gene, which encodes the circulating plasma coagulation FIX and occurs in approximately 1/25,000 male births.[1]
The efficacy of hemophilia B gene therapy could be improved upon employing the single amino acid exchange variant FIX Padua, where leucine is substituted for arginine at position 338 (R338L).[5]
We describe the development of a highly sensitive in vitro biopotency assay for BAX 335, an associated viral (AAV) serotype 8 (AAV8)-based hemophilia B gene therapy vector expressing FIX Padua that was explored in a clinical phase I/II study (ClinicalTrials.org: NCT01687608)
Summary
Hemophilia B is caused by an X-linked mutation in the factor IX (FIX) (F9) gene, which encodes the circulating plasma coagulation FIX and occurs in approximately 1/25,000 male births.[1]. Current therapy for hemophilia B involves regular infusions of FIX protein concentrates to prevent and treat bleeding events These concentrates must be administered intravenously on a regular basis throughout the patient’s lifetime.[2] In recent years, gene therapy using adeno-associated viral (AAV) vectors has shown promise in treating hemophilia B because of the viral vectors’ relative safety and long-term gene expression.[3] Applying AAV serotype 8 (AAV8) vectors carrying a codon-optimized and CpGdepleted human F9 gene in clinical studies has led to a substantial increase in FIX levels, reducing or even obviating the need for recombinant FIX administration.[4] The efficacy of hemophilia B gene therapy could be improved upon employing the single amino acid exchange variant FIX Padua, where leucine is substituted for arginine at position 338 (R338L).[5] This naturally occurring variant shows an up to 10-fold higher specific activity than wild-type (WT) FIX6 and yields adequate FIX activity despite low protein expression levels.[7]
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