Arginase I mRNA therapy - a novel approach to rescue arginase 1 enzyme deficiency

Abstract

Arginase I (ARG1) deficiency is an autosomal recessive urea cycle disorder, caused by deficiency of the enzyme Arginase I, resulting in accumulation of arginine in blood. Current Standard of Care (SOC) for ARG1 deficiency in patients or those having detrimental mutations of ARG1 gene is diet control. Despite diet and drug therapy with nitrogen scavengers, ~25% of patients suffer from severe mental deficits and loss of ambulation. 75% of patients whose symptoms can be managed through diet therapy continue to suffer neuro-cognitive deficits. In our research, we demonstrate in vitro and in vivo that administration of ARG1 mRNA increased ARG1 protein expression and specific activity in relevant cell types, including ARG1-deficient patient cell lines, as well as in wild type mice for up to 4 days. These studies demonstrate that ARG1 mRNA treatment led to increased functional protein expression of ARG1 and subsequently an increase in urea. Hence, ARG1 mRNA therapy could be a potential treatment option to develop for patients.

Keywords: Arginase 1; mRNA therapy; urea cycle disorder.

Figure 1.

qPCR analysis for constitutive Arg1 protein expression in four different cell lines. HeLa and HepG2 cells express low levels of Arg1 protein, while primary human hepatocytes (PHH) and human liver sample express high levels of Arg1 protein.

Figure 2.

Arg1 protein expression profile across multiple relevant cell types in upper panel and fold change of protein expression when normalized to eGFP vehicle control in lower panel. A. HeLa, B. HepG2 and C. GM00954 (Arg1 deficient patient fibroblasts) show increased protein expression when transfected with Arg1 mRNA (WT and FLAG).

Figure 3.

Specific activity of Arg1 measured in terms of urea synthesized in A. HepG2 and B. GM00954 (Arg1 deficient patient fibroblasts). Note cells only and GFP vehicle controls do not show any urea synthesized due to lack of Arg1 enzyme; when cells are transfected with Arg1 mRNA (WT and FLAG), urea synthesis is increased.

Figure 4.

LNP formulated Arg1 mRNA (WT and FLAG) expresses protein in mouse liver. Arg1 protein expression increases statistically in treated animals at 24h (A) and 48h (B).

Figure 5.

Arg1 immunostaining showed protein biodistribution in hepatocyte cytoplasm, nucleus and sinusoidal spaces at 24h (A) and 48h (B) timepoint in C57Bl/6 wild type mice post Arg1 mRNA treatment (WT and FLAG). 40x magnification.

Figure 6.

In situ hybridization (ISH) to detect Arg1 mRNA in mouse liver treated with Arg1 WT or FLAG-tagged mRNA. Arg1 mRNA detected in hepatocyte cytoplasm (single red dots) and as mRNA clusters (larger red dots) in sinusoidal spaces (40x magnification).

Figure 7.

Arg1 mRNA demonstrated extended duration of protein expression in mouse liver. Arg1 protein expression increases in treated animals at 24h and 48h with lasting protein expression seen up to 168h.

Figure 8.

Extended time course of LNP-formulated Arg1 mRNA (WT) in mouse liver. Upon mRNA treatment, Arg1 mRNA level is high at 24h and is detectable until 7d post-injection.