Preclinical evaluation of AGT mRNA replacement therapy for primary hyperoxaluria type I disease

Abstract

Primary hyperoxaluria type 1 (PH1) is a rare inherited liver disorder caused by alanine glyoxylate aminotransferase (AGT) dysfunction, leading to accumulation of glyoxylate which is then converted into oxalate. Excessive oxalate results in kidney damage due to deposition of oxalate crystals. We have developed an mRNA-based protein replacement therapy for PH1 to restore normal glyoxylate to glycine metabolism. Sequence optimized human AGT mRNA (hAGT mRNA) was encapsulated in lipopolyplex (LPP) and produced functional AGT enzyme in peroxisomes. Pharmacokinetics and pharmacodynamics (PK/PD) were evaluated in vitro and in vivo. PK demonstrated that AGT mRNA and AGT protein maintained high expression levels for up to 48 hours. A single 2 mg/kg dose in AgxtQ84^-/- rats achieved a 70% reduction in urinary oxalate. Toxicological assessment identified the highest nonserious toxic dose (HNSTD) as 2 mg/kg. These findings affirm the efficacy and safety of hAGT mRNA/LPP and support its clinical application in PH1 treatment.

Fig. 1.. Synthesis and characterization of optimized hAGT mRNA/LPP.

(A) Schematic diagram of optimization of hAGT mRNA, selection of lipid, synthesis, and work mechanism in vivo of hAGT mRNA/LPP. (B) Candidate mRNA sequences were generated on the basis of MFE and CAI. (C) Secondary structure of WT hAGT mRNA and 2770 hAGT mRNA. (D) Western blot of AGT expression in HEK293T cells transfected with candidate mRNAs 6 and 48 hours after the transfection. (E to G) Quantification of AGT immunoblot normalized to β-tubulin in HEK293T cells transfected with candidate mRNAs 6, 24, and 48 hours after the transfection. (H) Western blot of liver AGT expression of rats injected with original-UTR hAGT mRNA/LPP and optimized-UTR hAGT mRNA/LPPs, WT rats, and untreated AgxtQ84^−/− rats. (I) Quantification of AGT immunoblot normalized to vinculin. The protein level of WT group was set at 1. (J to L) Comparison of IVIS radiance between luciferase mRNA LNP and LPP 6 and 24 hours after the injection. The luciferase had better and longer expression in vivo when the mRNA was encapsulated with LPP compared to the LNP. (M and N) Comparison of IVIS radiance between different parenchymatous organs in BALB/c mice 6 hours after the injection with hAGT mRNA/LPP. (O) Cryo–transmission electron microscope (cryo-TEM) of hAGT mRNA/LPP. (P) Size distribution of hAGT mRNA/LPP by number (diameter peak = 89.15 nm, average = 87.2 nm). (Q) Encapsulation efficiency (n = 3) and zeta potential (n = 3) of hAGT mRNA/LPP. (R) PDI of hAGT mRNA/LPP at different temperatures within 4 weeks. (S) Change of particle size (n = 3) of hAGT mRNA/LPP after being stored at different temperatures for 4 weeks. (T) Change of particle size (n = 3), encapsulation efficiency (n = 3), and purity (n = 3) of hAGT mRNA/LPP through different numbers of freeze-thaw cycles. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Graphs show means ±SD.

Fig. 2.. Colocalization and enzyme activity of hAGT protein in HEK293T cells transfected with hAGT mRNA/LPP and the effect of hAGT mRNA/LPP on human PH1 liver 3D models.

(A) Experimental plan about immunofluorescent staining and enzyme activity detection in HEK293T after the transfection with hAGT mRNA/LPP. (B and C) Immunofluorescent staining of hAGT protein (green) and peroxin 14 (red), a peroxisomal marker, were colocated with each other (yellow). Nuclear was stained by DAPI (blue). The white box area in the merge was selected for colocalization analysis. (D and E) Mitochondria were stained with MitoTracker Red CMXRos (red), negative for hAGT staining. The white box area in the merge was selected for colocalization analysis. (F) Pearson’s R value comparation of colocalization of hAGT protein with peroxisome and mitochondria. (G) Under the same system conditions, the pyruvate concentration was elevated in groups transfected with hAGT mRNA/LPP relative to the group transfected with GFP mRNA/LPP. (H) Experimental plan about the isolation of liver and construction of liver 3D models. (I) Relative hAGT mRNA expression levels in PH1 liver 3D models with or without hAGT mRNA/LPP transfection. (J) There was a significant decrease in 3-day oxalate accumulation in the PH1 liver 3D models transfected with hAGT mRNA/LPP relative to the baseline value and the contents were close to donor liver 3D models. (K) Immunofluorescence staining showed hAGT protein expression levels in normal human liver 3D models, PH1 liver 3D models with or without hAGT mRNA/LPP transfection at various time points. (L) Hematoxylin-eosin (HE) staining showed no significant damages to the cells in 3D models. *P < 0.05, **P < 0.01, and ****P < 0.0001. Graphs show means ±SD.

Fig. 3.. Pharmacokinetics of hAGT mRNA/LPP in WT BALB/c mice, AgxtQ84^−/− rats, and WT cynomolgus monkeys.

(A) Experimental plan about pharmacokinetics of hAGT mRNA/LPP in WT mice. (B) qPCR detection of endogenous Agxt and hAGT mRNA levels in the WT mouse liver tissues at various time points after injection. The hAGT expression in 3 mg/kg group 6 hours after injection was set at 1. The exogenous hAGT mRNA level (high saturation red, blue, and yellow) showed a downtrend, while the mouse Agxt mRNA expressions (low transparency red, blue, yellow, and gray) have no apparent difference among various time points after injection. (C) Relative hAGT mRNA expression levels in the WT mouse blood at various time points after injection. The AGT expression in 3 mg/kg group 6 hours after injection was set at 1. (D) Experimental plan about pharmacokinetics of hAGT mRNA/LPP in AgxtQ84^−/− rats. (E) Relative hAGT mRNA expression levels in the AgxtQ84^−/− rat liver tissues at various time points after injection compared with the WT rats (n = 3). The Agxt mRNA expression in WT group 6 hours after injection was set at 1. (F and G) hAGT protein expression levels in the AgxtQ84^−/− rat liver tissues at various time points after injection compared with the WT rats (n = 3). The protein level of WT group was set at 1. (H and I) Immunohistochemistry staining to show hAGT protein expression levels in the AgxtQ84^−/− rat liver tissues at various time points after injection (n = 3). (J) Experimental plan about pharmacokinetics of hAGT mRNA/LPP in WT cynomolgus monkeys. Liver tissues were obtained via percutaneous puncture 48 and 144 hours after first and third injection. (K) Relative hAGT mRNA expression in WT monkey liver tissues after first and third injection. (L) The relative levels of anti-PEG IgG in the serum of rats injected with PBS or hAGT mRNA/LPP once a week for 3 weeks. The results were presented as absorbance at 450 nm (OD). *P < 0.05, **P < 0.01, and ****P < 0.0001. Graphs show means ±SD.

Fig. 4.. Pharmacodynamics of single-dose hAGT mRNA/LPP in Agxt^−/− mice and AgxtQ84^−/− rats.

(A) Experimental plan about pharmacodynamics of single-dose hAGT mRNA/LPP in Agxt^−/− mice. AgxtQ84^−/− mice (n = 4) were given doses of hAGT mRNA/LPP (0.5, 1, and 2 mg/kg) by tail vein. Urine was collected on D0 and D1, D3, D5, and D7 after injection for the oxalate detection. (B) Urinary oxalate levels were all reduced after injection in three groups. A 61.16% reduction was achieved with 2 mg/kg dose of hAGT mRNA/LPP. (C) Experimental plan about pharmacodynamics of single-dose hAGT mRNA/LPP in AgxtQ84^−/− rats. AgxtQ84^−/− rats (n = 4) were given doses of 0.5, 1, and 2 mg/kg of hAGT mRNA/LPP by tail vein. Urine was collected every 24 hours until 168 hours after injection for the oxalate detection. (D) Urinary oxalate levels were all reduced to a minimum 72 hours after injection in three groups. A 69% reduction to normal range was achieved with 2 mg/kg dose of hAGT mRNA/LPP. The 24-hour urine oxalate 72 hours after the 2 mg/kg dose was compared with the baseline (0 hour). *P < 0.05, **P < 0.01, and ****P < 0.0001. Graphs show means ± SD.

Fig. 5.. Pharmacodynamics of single-dose hAGT mRNA/LPP in AgxtQ84^−/− rats combined with vitamin B6, single-dose in AgxtQ84^−/− rats given 0.8% EG for a week and multidose in AgxtQ84^−/− rats.

(A) Experimental plan about pharmacodynamics of single-dose hAGT mRNA/LPP in AgxtQ84^−/− rats combined with vitamin B6. AgxtQ84^−/− rats (n = 4) were given doses of 0.5 and 1.0 mg/kg of hAGT mRNA/LPP by tail vein with or without vitamin B6 intake. Serums were collected every 24 hours until 168 hours after injection for the oxalate detection. (B) Serum oxalate concentrations decreased in all groups (n = 3) of rats within 96 hours of hAGT mRNA/LPP injection with or without vitamin B6 intake. (C) Change of oxalate concentration in every group compared with 0 hour. (D) Experimental plan about pharmacodynamics of single-dose hAGT mRNA/LPP in AgxtQ84^−/− rats given 0.8% EG for a week. Urine was collected every 24 hours until 168 hours after injection for the oxalate detection. (E) The 24-hour urinary oxalate level in each group decreased to the lowest 48 hours after hAGT mRNA/LPP injection. (F) Change of 24-hour urine oxalate content in every group compared with 0 hour. (G) Experimental plan about pharmacodynamics of multi-dose hAGT mRNA/LPP in AgxtQ84^−/− rats. (H) The 24-hour urine oxalate levels decreased in all groups after every dose. (I) The maximum decrease of every group after every dose. *P < 0.05, ***P < 0.001, and ****P < 0.0001. Graphs show means ± SD.

Fig. 6.. Toxicology of hAGT mRNA/LPP in SD rats.

(A) Schematic illustration of experimental plan. SD rats (n = 6) were given weekly doses of hAGT mRNA/LPP (1 or 2 mg/kg), vehicle/LPP (2 mg/kg), or 0.9% NaCl from day 1 up to 3 weeks. There was a convalescence for 4 weeks after the last injection. Heart, liver, spleen, lung, and kidney tissues were collected for the pathology after the experiment. (B to D) Food consumptions (B) and body weights (C and D) of rats in each group were not obviously different compared with the control. (E) Body temperature temporarily ascended in groups given hAGT mRNA/LPP 6 hours after third dose. (F and G) Serum immune markers like IL-6 and CXCL1 were not obviously different compared with the control. Significant difference was reported in detail as follows: (H) Serum ALT, AST, T-Bil, CK, C3, and C4 showed significant difference 24 hours after every dose and 1 week after the last dose. (I) Hematological indicators showed significant difference in each group 24 hours after second and third dose and 1 week after the last dose. (J) Hematoxylin-eosin staining showed no significant damages to every parenchymal organ of rats. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Graphs show means ± SD.

Fig. 7.. Toxicology of hAGT mRNA/LPP in WT cynomolgus monkeys.

(A) Schematic illustration of experimental plan. WT cynomolgus monkeys (n = 8) were given weekly doses of hAGT mRNA/LPP (1 or 2 mg/kg), vehicle/LPP (2 mg/kg), or 0.9% NaCl from day 1 up to 3 weeks. Each group had one male and one female. There was a convalescence for 4 weeks after the last injection. (B and C) Body weights and temperatures of WT cynomolgus monkeys in each group had no significant changes during the whole experiment. The only exception happened due to anesthesia for liver perforation. (D to H) Summary of abnormal hematological (D), plasma biochemical (E), hemagglutination (F), immunoglobulin and complement (G), and abnormal cytokine (H) indicators in WT cynomolgus monkeys injected with hAGT mRNA/LPP during the whole experiment.