(W1530-02-06) Age-Dependent Reduction in Hepatic Vector Genome Loss in Juvenile OTC^spf-ash Mice Following a Single Dose of DTX301 and Partial Compensation Achieved with a Higher Dose

Purpose: Ornithine transcarbamylase (OTC) deficiency is a X-linked urea cycle disorder resulting from mutations in the OTC gene that affect the expression or activity of the OTC protein in the liver. OTC deficiency presents as a severe form in males shortly after birth (neonatal onset; ≤ 30 days of age) or later in life for both males and females (late onset; > 30 days of age) with disease that ranges from mild to severe depending on the residual activity of OTC. As a result, treatment in young pediatric population may be required. DTX301 is a nonreplicating, self-complementary, recombinant adeno-associated virus (AAV) serotype 8 (AAV8) vector that contains a codon-optimized, wild type human OTC coding sequence for the treatment of OTC deficiency by restoring OTC activity in the liver. However, liver proliferation during the developmental stage in children may result in loss of the transgene and efficacy achieved by AAV therapy in the liver over time, and the long-term effects of time-dependent decrease in vector genome is currently unknown in the pediatric population. In this study, juvenile OTC deficient (OTC^spf-ash) mice were used as a model to investigate age- and dose-dependent changes in the magnitude of vector genome decreases over time and pharmacological consequences following a single administration of DTX301.

Methods: Male OTC^spf-ash mice were administered a single intravenous dose of DTX301 at 1×10¹³ or 7×10¹³ GC/kg on either post-natal day (PND) 2, 11, 14, 21, 42, or 64. The duration of the study was 12 weeks with interim timepoint at 4 weeks after DTX301 administration. Liver samples were collected to analyze for DNA vector genome copies and human OTC mRNA by quantitative PCR (qPCR) and OTC activity by LC-MS/MS method.

Results: In general, the magnitude of vector genome loss in the liver decreased as the age of mice increased at the time of DTX301 administration. Relative to mice dosed at PND 42, hepatic vector genome copies at 4-weeks post dose were reduced by 155-fold for mice dosed at PND 2, 217-fold for mice dosed at PND 11, 108-fold for mice dosed at PND 14, and 17-fold for mice dosed at PND 21 (Figure 1). A similar trend was also captured in the 12-weeks post dose group. At 12-weeks post dose, comparable hepatic vector genome was observed in mice dosed at PND 42 and PND 64, suggesting that negligible vector genome loss could be achieved when AAV is dosed as early as PND 42. With a 7-fold higher dose (7×10¹³ GC/kg), vector decreases over time in the liver were still observed in juvenile mice dosed at ≤ PND 21 at 4- and 12-weeks post dose similar to those administered with low dose of 1×10¹³ GC/kg. Although administration of a 7-fold higher dose could not completely prevent the decreases in hepatic vector genome in juvenile mice, higher amounts of vector genome copies did remain in the liver compared to animals dosed at 1×10¹³ GC/kg. Notably in the PND 21 group, 7x10¹³ GC/kg could overcome the expected vector loss and reach comparable vector genome levels compared to PND 42 group dosed at 1×10¹³ GC/kg. Age-dependent changes in human OTC mRNA expression (Figure 2) and OTC activity (Figure 3) in the liver were similar to the trend observed in vector genome. When the dose was increased by 7-fold to 7×10¹³ GC/kg, no additional gain in OTC activity was found in the PND 42 group despite a dose-proportional increase in the hepatic vector genome copies, suggesting a saturation in the response. Nonetheless, the higher dose did provide a consistent boost in OTC activity at both 4 and 12 weeks in mice dosed at PND 11, PND 14, and PND 21. The resulting enzyme activities at 7×10¹³ GC/kg at these age groups were nearly comparable or having less than 50% deficit (95 - 192 pmol/mg/min) when compared to the levels observed in PND 42 animals dosed at 1×10¹³ GC/kg (152 - 168 pmol/mg/min) and WT animals (129 – 134 pmol/mg/min).

Conclusion: Due to the extensive cell division in the growing liver, the levels of hepatic vector genome decreased over time in juvenile OTC^spf-ash mice following a single administration of DTX301. The loss in vector genome decreased with increasing age (PND < 42) at the time of DTX301 administration. A similar trend was observed with human OTC mRNA expression and OTC activity in the liver. By increasing the dose level, transgene product as measured by OTC activity could be recovered at least partially in mice dosed as early as PND 11, suggesting that a higher dose may be a strategy to compensate for the expected loss of vector genome and pharmacology in the liver during developmental age.

Acknowledgements: Method for measuring OTC activity in the liver was developed by Edward Brewer from Pyxant Labs.

Figure 1. Decrease in the magnitude of hepatic vector genome DNA loss in juvenile OTCspf-ash mice with increase in age of DTX301 administration and a higher dose


Figure 2. Decrease in the loss of hepatic human OTC mRNA in juvenile OTCspf-ash mice with increase in age of DTX301 administration and a higher dose


Figure 3. Decrease in hepatic OTC activity loss in juvenile OTCspf-ash mice with increase in age of DTX301 administration and a higher dose