An investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the treatment of hereditary angioedema

Abstract

Hereditary angioedema (HAE) is a genetic disorder mostly caused by mutations in the C1 esterase inhibitor gene (C1INH) that results in poor control of contact pathway activation and excess bradykinin generation. Bradykinin increases vascular permeability and is ultimately responsible for the episodes of swelling characteristic of HAE. We hypothesized that the use of RNA interference (RNAi) to reduce plasma Factor XII (FXII), which initiates the contact pathway signaling cascade, would reduce contact pathway activation and prevent excessive bradykinin generation. A subcutaneously administered GalNAc-conjugated small-interfering RNA (siRNA) targeting F12 mRNA (ALN-F12) was developed, and potency was evaluated in mice, rats, and cynomolgus monkeys. The effect of FXII reduction by ALN-F12 administration was evaluated in two different vascular leakage mouse models. An ex vivo assay was developed to evaluate the correlation between human plasma FXII levels and high-molecular weight kininogen (HK) cleavage. A single subcutaneous dose of ALN-F12 led to potent, dose-dependent reduction of plasma FXII in mice, rats, and NHP. In cynomolgus monkeys, a single subcutaneous dose of ALN-F12 at 3 mg/kg resulted in >85% reduction of plasma FXII. Administration of ALN-F12 resulted in dose-dependent reduction of vascular permeability in two different mouse models of bradykinin-driven vascular leakage, demonstrating that RNAi-mediated reduction of FXII can potentially mitigate excess bradykinin stimulation. Lastly, ex vivo human plasma HK cleavage assay indicated FXII-dependent bradykinin generation. Together, these data suggest that RNAi-mediated knockdown of FXII by ALN-F12 is a potentially promising approach for the prophylactic treatment of HAE.

Keywords: Factor XII; GalNAc-siRNA; HK cleavage; bradykinin; hereditary angioedema; vascular permeability.

FIGURE 1.

ALN-F12 for HAE prophylaxis: therapeutic hypothesis. (A) ALN-F12 silences hepatic F12 gene expression. N-acetylgalactosamine (GalNAc) conjugated siRNA duplex ALN-F12 specifically binds hepatocytes via the asialoglycoprotein receptor (ASGPR). Following calthrin-mediated endocytosis and endosomal escape, ALN-F12 is unwound and loaded into RISC (RNA-induced silencing complex) and specifically recognizes and degrades F12 mRNA, leading to a reduction of secreted Factor XII (FXII) protein levels in plasma. (B) Conversion of FXII to FXIIa initiates an enzymatic signaling cascade that results in the production of bradykinin, a peptide signal that controls vascular permeability. C1 esterase inhibitor (C1INH), which binds and inhibits FXIIa and plasma kallikrein (PKa), negatively regulates this process. In HAE patients, loss of C1INH activity leads to excess bradykinin generation, resulting in increased vascular permeability and subsequent edema. RNAi-mediated knockdown of F12 gene expression by ALN-F12 would decrease circulating FXII protein levels and therefore preventing excess bradykinin generation.

FIGURE 2.

ALN-F12 dose-dependent knockdown of circulating FXII in mice and rats. (A) Female C57BL/6J mice were subcutaneously administered a single dose of ALN-F12 (n = 3 per group). Plasma FXII levels were measured by ELISA and normalized to PBS control group. (B) Female Sprague–Dawley rats were subcutaneously administered a single dose of ALN-F12 (n = 3 per group). Plasma FXII levels were measured by ELISA and normalized to the average of PBS control group. Error bars are SD.

FIGURE 3.

Inhibition of ACE inhibitor-induced vascular permeability following a single dose subcutaneous administration of ALN-F12. Female C57BL/6J mice were subcutaneously administered a single dose of ALN-F12 at dose levels of 0, 0.1, 0.3, 1, or 3 mg/kg (n = 10 per group). Seven days after administration, animals were IV injected with the ACE inhibitor captopril (2.5 mg/kg); the vascular permeability tracer, Evans blue dye (30 mg/kg), was IV injected 15 min after captopril injection. Animals were euthanized 15 min after Evans blue dye injection. Blood (A) and intestine (B) were collected to evaluate Evans blue extravasation; livers were collected for F12 mRNA analysis (C). In panels A, B, and C, symbols denote data from individual animals, the group mean ± SD are also plotted. Statistical analysis performed using one-way ANOVA with Dunnett's post-hoc test with respect to PBS-treated animals in the captopril treatment group. (**) P < 0.01; (****) P < 0.0001. A correlation of intestinal Evans blue uptake and F12 mRNA levels (D). Panels E and F are the correlation of intestinal Evans blue extraction and PK (and HK) mRNA levels (Supplemental Figs. S2, S3).

FIGURE 4.

ALN-F12-rescued mustard oil-induced vascular permeability in a C1INH knockdown mouse model. Female CD1 mice were subcutaneously dosed with a siRNA targeting C1INH (10 mg/kg) and ALN-F12 at different dose levels (n = 10 per group). Seven days post-administration, Evans blue dye (30 mg/kg) was given IV, and 5% mustard oil was topically applied to the right ear, followed 15 min later by a second application. The animals were euthanized 15 min after the second application of mustard oil. Both right (mustard oil-treated) and left (control) ears were collected for dye extraction (A). Livers were collected for mRNA measurements (B). Group mean ± SD are plotted. Statistical analysis in Panel A performed using two-way ANOVA with Tukey's post-hoc test; asterisks indicate statistical comparisons to mustard oil-treated ears in the PBS control group. Statistical analysis in Panel B performed using one-way ANOVA with Dunnett's post-hoc test; statistical comparisons to PBS-treated animals. For both analyses: (**) P < 0.01; (****) P < 0.0001.

FIGURE 5.

ALN-F12 dose-dependent reduction of circulating FXII in female cynomolgus monkeys. Female cynomolgus monkeys were subcutaneously administered a single dose of ALN-F12 (n = 3 per group). Plasma FXII levels were measured by ELISA and normalized to the average of individual animal's pre-dose average of Days −5, −3, and −1 (error bars = SD). Panel A represents the relative plasma FXII level on Day 28 post-dosing; Panel B depicts the relative FXII levels at all collected time points for all dosing groups.

FIGURE 6.

FXII dose-dependent HK cleavage in human plasma. Normal human pooled plasma was mixed with FXII-deficient (<1%) human plasma at different ratios to generate different concentration of FXII in the mixture. The plasma mixtures were activated under mild conditions (0.2 µg/mL Kaolin, 37°C, 10 min) and loaded into SDS-PAGE for western blot analysis. The anti-HK antibody detects the intact, full-length HK. The uncleaved full-length HK signal ratio of kaolin treatment to no kaolin treatment is represented in Table 1 below.