OTCD Model

Otc^spf-ash mice were obtained from Jackson Laboratories (Stock 001811; Bar Harbor, ME, USA). Breeding took place at PhaseRx to generate hemizygous males containing the Otc^spf-ash mutation. In some cases, wild-type or heterozygous littermates were used as controls. A hyperammonemia model in Otc^spf-ash mice using AAV/Otc shRNA to knock down residual Otc expression was conducted essentially as described previously.29 The details of model optimization and testing are described in the Supplemental Materials and Methods and Figure S5.

Naive Otc^spf-ash male mice (7–10 weeks; n = 3) or CD1 female mice (6–8 weeks; n = 3) were given a single i.v. bolus of buffer or a formulation consisting of hOTC mRNA/HMT (3 mg/kg mRNA plus 25 mg/kg polymer; 10 mL/kg dosing volume by weight) on day 0. We selected a dose of 3 mg/kg mRNA based on initial studies where 1 mg/kg dosing did not show therapeutic effects. 25 mg/kg was selected as the polymer dose because maximal expression was achieved with luc mRNA/HMT at this dose level (Figure 3B). OTC protein expression (by western blot) and OTC enzyme activity were measured in the liver at 2, 4, 7, and 10 days after dosing. Detailed protocols for western blotting and OTC enzyme activity measurements can be found in the Supplemental Materials and Methods. Immunofluorescence was conducted at endpoint as described in the Supplemental Materials and Methods.

For the repeat-dose efficacy study, Otc^spf-ash male mice (8–12 weeks) were given 100 μL of AAV2/8/Otc shRNA at 1 × 10¹¹ genome copies (GCs) per mouse prepared by RegenXBio (Rockville, MD, USA) on day 0. The AAV2/8/Otc shRNA construct was a gift from I. Alexander. The mice were then given a formulation consisting of buffer (n = 12; twice per week), hOTC mRNA/HMT (n = 12; once per week or twice per week), or hOTC control mRNA/HMT (n = 12; once per week) (all at 3 mg/kg mRNA and 25 mg/kg polymer [10 mL/kg dosing volume by weight]) via i.v. bolus into the tail vein starting on day 4. Analyses included daily body weights, urinary orotic acid levels (days 5, 6, 7, 10, 13, 20, 27, and 34; see Supplemental Materials and Methods for procedural details), and plasma ammonia levels (days 14, 21, 28, and 35; see Supplemental Materials and Methods for procedural details). Mice were sacrificed if they had ≥20% body weight loss or signs of morbidity/ataxia. Animals removed at sacrifice were considered as deaths in survival analyses. One mouse in the buffer group and one mouse in the hOTC mRNA/HMT twice per week group were excluded from analysis because they succumbed to an anesthetic event during blood collection. Because mice treated with hOTC mRNA/HMT twice per week had extended survival, formulation dosing was stopped after day 35. Half of the hOTC mRNA/HMT mice were sacrificed on day 37 (48 hr after the last dose) to examine hOTC protein expression and endogenous mouse Otc mRNA in liver to confirm the knockdown activity of the Otc shRNA (see Supplemental Materials and Methods). The remaining half of the hOTC mRNA/HMT mice were continued to be monitored daily for body weight change and any signs of ataxia.