In Vitro and In Vivo Potency Differences between AAVRh74var and AAV5 Vectors Encoding the Same High-Activity Human Factor IX Variant, FIX-R338L, Expression Cassette: Implications for Hemophilia B Gene Therapy

Background

Fidanacogene elaparvovec is a non-replicating, recombinant adeno-associated virus (AAV)-based gene therapy vector that utilizes AAVRh74var (derived from a naturally occurring AAVRh74), to transfer a high-activity variant of human factor IX (FIX) FIX-R338L for the treatment of hemophilia B (HB). Data from the ongoing phase 3 BENEGENE-2 study demonstrated FIX activity levels in the mild hemophilia to normal range and a 71% reduction in annualized bleeding rate in participants with HB treated with fidanacogene elaparvovec. Fidanacogene elaparvovec 5×10¹¹ vg/kg was recently approved for use in Canada, the United States and Europe. The only other approved HB gene therapy (etranacogene dezaparvovec) utilizes the AAV5 capsid and is administered at a 40-fold higher dose (2×10¹³ gc/kg). In vitro and in vivo studies evaluated whether the AAVRh74var capsid confers a higher transduction efficiency, relative to AAV5, thus potentially allowing for lower overall AAV capsid dosing to achieve comparable FIX activity levels in patients with HB.

Methods

The vector genome of fidanacogene elaparvovec (including the FIX-R338L transgene, AAV2 inverted terminal repeats [ITRs], genetic control elements, intron and polyA signal) was packaged into either the AAVRh74var or AAV5 capsid using an HEK-293 cellular production system. A molecular assessment demonstrated AAVRh74var FIX-R338L and AAV5 FIX-R338L preparations were similar in empty/full capsid ratio, viral protein (VP)1/2/3 ratio, and purity. In an in vitro study, the Huh7 human-derived hepatocellular carcinoma cell line was transduced with increasing concentrations of AAVRh74var FIX-R338L or AAV5 FIX-R338L ranging from 1.6×10⁴ to 1.0×10⁶ vg/cell. FIX activity in conditioned cell growth medium was assessed 96 and 120 hours post transduction. Each concentration was tested in duplicate in 2 independent experiments. After removal of the conditioned medium for FIX activity determination at 120 hours post transduction, cells from a single transduction were lysed and RNA was extracted. In an in vivo mouse model of HB, male mice (10 mice/group) received a single intravenous injection of AAV5 FIX-R388L or AAVRh74var FIX-R388L diluted in PBS to achieve 4×10¹⁰ or 1×10¹¹ vg/kg or PBS alone (control). Blood and tissues were harvested at Weeks 1 and 4 post treatment. In the in vitro and in vivo studies, FIX activity was determined using the one-stage activated partial thromboplastin time (aPTT) clotting assay. FIX mRNA expression and vector genomes were determined by digital droplet PCR. All procedures performed in animals were reviewed and approved by an Institutional Animal Care and Use Committee.

Results

In Huh7 cells, a dose-dependent increase in FIX activity was observed with both AAV vectors, with similar trends in FIX activity observed at 96 and 120 hours post transduction. AAVRh74var FIX-R338L exhibited an increase in potency at all concentrations tested (potency difference of AAVRh74var FIX-R338L vs AAV5 FIX-R338L at 1.0×10⁶ vg/cell: 6.66- to 10.57-fold). FIX mRNA expression levels were greater with AAVRh74var FIX-R338L vs AAV5 FIX-R338L. HB mice dosed with 4×10¹⁰ vg/kg had a mean FIX activity of 0.119 IU/mL vs 0.007 IU/mL with AAVRh74var FIX-R338L vs AAV5 FIX-R338L. Mean FIX activity was >100x higher with AAVRh74var FIX-R338L vs AAV5 FIX-R338L in mice dosed with 1×10¹¹ vg/kg when assessed at both timepoints (Weeks 1 and 4). Liver mRNA levels were consistent with FIX activity. FIX expression was consistently higher in mice that received AAVRh74var FIX-R338L compared with AAV5 FIX-R338L. FIX expression was very low in the AAV5 FIX-R338L-treated mice. Liver vector genome copies at 4 weeks post treatment were higher in the AAVRh74var FIX-R338L compared with AAV5 FIX-R338L treated mice.

Conclusions

In this head-to-head potency comparison in the Huh-7 human liver cell line and male HB mice, AAVRh74var had a higher transduction potency compared with AAV5. FIX-R338L activity and liver mRNA expression levels were higher in mice that received AAVRh74var FIX-R338L vs AAV5 FIX-R338L. These results demonstrate capsid selection can impact intended infectivity and the mechanism of cellular uptake. Overall, the results demonstrate differences between the AAVRh74var and AAV5 capsids. This is consistent with fidanacogene elaparvovec (AAVRh74var) achieving clinically robust efficacy, despite a several-fold lower dose than AAV5-based gene therapies.