Influence of Obesity on Factor IX Expression after Gene Therapy in a Mouse Model of Hemophilia B

Introduction:

Shire is developing a gene therapy to treat hemophilia B, using a recombinant adeno-associated virus 8 (rAAV8) vector and a FIX variant with increased specific activity under the control of a liver-specific promoter. To assess whether obesity could affect clinical therapy, the presented study investigated FIX expression after FIX gene therapy in normal-weight and adipose hemophilia B mice.

Methods:

Thirteen male FIX knockout (ko) mice were fed with standard maintenance or high-caloric diet ad libitum for 16 weeks from weaning until the time of sacrifice. Up to eight animals per feeding cohort were then treated with a single intravenous dose of 5.62x10¹¹ capside particles/kg of two different FIX gene therapy vector preparations. Two different lots of FIX gene therapy were tested per feeding cohort (n=5-8). FIX plasma levels were assessed using a 1-stage clotting assay (FIX activity) and an ELISA (FIX antigen) at days 7, 14, and 28. At the end of the observation period, the animals were humanely killed and livers were collected for histopathological examination.

Results:

Mean body weight after 16 weeks of standard or high-caloric diet was 30.7 vs. 43.0 g. The animals were subsequently treated with FIX gene therapy and the respective mean of resulting FIX levels were calculated for day 7, 14, and 28. In normal-weight animals, mean FIX activity levels were 1.75-3.42 IU/mL for Lot 1 and 1.55-2.65 IU/mL for Lot 2; mean FIX antigen levels were 0.77-1.78 µg/mL and 0.65-1.30 µg/mL. In adipose mice, mean FIX activity levels were 1.91-4.04 IU/mL for Lot 1 and 1.84-2.89 IU/mL for Lot 2; mean FIX antigen levels were 0.85-2.20 µg/mL and 0.80-1.41 µg/mL. The differences in FIX activity and FIX antigen between normal-weight and adipose mice were not statistically significant (p≥0.21 and p≥0.19, respectively).

Mean liver weight prior histological preparation was 2.58 g for adipose mice and 1.6 g for normal-weight mice. Histopathological examination showed moderate to marked lipidosis in the livers of adipose mice. Oil Red O staining revealed increased amounts of lipid above that diagnosed with H&E staining by one severity grade (grade 5 via Oil Red O). Normal-weight mice showed minimal lipidosis with H&E staining.

Conclusions:

Mean plasma FIX levels were slightly higher in obese mice at all time points analyzed; however, the difference to normal-weight mice was not statistically significant suggesting that obesity does not affect clinical therapy. As expected, the livers of obese mice showed increased lipidosis.