Systemic delivery of factor IX messenger RNA for protein replacement therapy

Abstract

Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4-6 h) that remains stable for up to 4-6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA-LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable.

Keywords: hemophilia B therapy; hepatic diseases; lipid nanoparticles; nonviral mRNA delivery; systemic delivery.

Fig. 1.

Lipid-enabled and unlocked nucleic acid modified RNA (LUNAR) is safe and effectively delivers RNA to the liver. (A) Comparing the knockdown efficiencies of LUNAR:FVII siRNA formulation with MC3 (a lipid formulation currently approved for clinical use) and vehicle (PBS). At a dose of 0.3 mg/kg, LUNAR:FVII siRNA gave up to 97% knockdown of FVII protein levels in mouse serum, which is up to five times more than the levels achieved by MC3 (n = 6). (B) The LUNAR:FVII siRNA treatment of doses up to 10 mg/kg in five male and five female CD-1 mice did not cause significant elevations in AST/ALT levels, compared with saline controls. Serum was collected 48 h postdose for clinical chemistry (performed on a clinical chemistry analyzer at Contract Research Lab BTS Research, Inc.). (C) LUNAR:GFP mRNA complexes were i.v. (tail vein) administered to 6- to 8-wk-old male C57Bl6 mice at doses of 0.1, 0.5, 2.5, and 10 mg/kg (n = 3 per group). At 24 h posttreatment, the animals were killed and livers were flash frozen for immunofluorescence analysis. Tissue sections imaged at 4× magnification are shown. (D) In vitro transcribed hFIX mRNA, packaged in the LUNAR and MC3 formulations, was administered once to 7-wk-old female balb/c mice at 2 mg/kg (n = 3 per group). The animals were bled at 6-h postdosing and the serum FIX levels were assessed by an ELISA (Assay Pro: EF1009-1) at 1:200 dilution as per the manufacturer’s instructions. As can be seen, the LUNAR formulation was up to two times more efficient than the clinically approved MC3 lipid formulation. (E) Luc mRNA encapsulated in LUNAR LNPs was administered to mice at a dose of 0.5 mg/kg (n = 3). Mice were imaged on an IVIS system 5-h postinjection following which they were killed and tissues were also imaged ex vivo. LUNAR-delivered mRNA was concentrated in the liver.

Fig. 2.

LUNAR-delivered human FIX-mRNA can restore normal clotting activity in hemophilic mice. (A) FIX activity for the WT and hemophilic mice measured by clotting time in an APTT assay (percentage of activity calculated based on a standard curve generated from serial dilutions of pooled normal serum). Panel below shows the Western blot for FIX protein in the WT and hemophilic mouse serum. FIX^−/− mice had less than 5% of WT FIX activity. (B) ELISA for circulating hFIX in FIX^−/− animals after i.v. delivery of hFIX mRNA encapsulated in LUNAR LNPs (dose = 2 mg/kg; n = 3). Significance was tested using a two-tailed Student’s t test. (C) FIX activity upon injection of hFIX mRNA:LUNAR LNPs as measured by clotting time in an APTT assay. Significance was tested using a two-tailed Student’s t test (dose = 2 mg/kg; n = 3). (D) Circulating hFIX levels in the serum of hemophilic mice over a 24-h time course after i.v. delivery of hFIX mRNA:LUNAR LNPs at a dose of 2 mg/kg.

Fig. 3.

Hyperfunctional variants of mRNA extend the therapeutic efficacy. (A) ELISA for circulating hFIX, hFIX R338A, and hFIX R338L in FIX^−/− animals after i.v. delivery of the three different mRNAs encapsulated in LUNAR LNPs (dose = 4 mg/kg; n = 3). An ANOVA and post hoc Tukey’s were used to test for significant differences between groups. (B) Clotting efficiency of the three variant hFIX mRNAs (WT, R338A, and R338L) in mouse serum were determined by an APTT assay at 48 h after i.v. delivery of the mRNA-LUNAR LNP formulation. The hyperfunctional variants R338A and R338L exhibited greater therapeutic efficiency than the WT protein, i.e., they restored clotting efficiency to 100% with lower amounts of circulating protein (based on Western, C and ELISA, A). An ANOVA and post hoc Tukey’s were used to test for significant differences between groups. (C) Western blot to examine hFIX levels both in the liver (where they are synthesized) and in the serum (where they are functional). Upon i.v. administration of the WT and variant hFIX mRNAs, hFIX protein can be detected in protein lysates from the liver (Uppermost vs. Middle) and from the serum (Lowermost). The WT variant was produced and secreted into circulation at amounts significantly higher than that of the R338A variant.

Fig. 4.

R338A FIX mRNA:LUNAR is therapeutically more effective than recombinant human FIX protein, which is the current standard of care. (A) Animals were administered the rhFIX or LUNAR:hFIX mRNA every 10 d for a total of three repetitions (with two males and two females in each group). Animals showed higher levels of FIX protein in the serum, as measured by an ELISA; when administered, the LUNAR:R338A hFIX mRNA LNPs compared with recombinant human FIX protein. The exogenously administered FIX was cleared from the system in 10 d (or 240 h) at which point the next dose was administered. (B) Administration of LUNAR R338A hFIX mRNA LNPs gave higher and longer therapeutic levels of functional FIX protein in the serum. Over the three rounds of injections, the LUNAR:R338A hFIX mRNA LNPs formulation gave higher therapeutic efficiency at both 24 and 96 h. The clotting activity was restored to baseline levels at 10 d postadministration, suggesting that the LUNAR LNP formulation remained therapeutically efficient for 4–9 d in vivo, whereas the recombinant protein remained effective for 1–3 d. (C) We also analyzed the cumulative APTT activity over the three rounds of repeat administrations and compared the therapeutic efficacy of the rhFIX protein with our LUNAR-mRNA LNP formulation. At 24 h, the LUNAR-mRNA administration resulted in dramatically high levels of therapeutic clotting activity compared with the rhFIX protein (130% vs. 20%). Even at 96 h, while the rhFIX protein was no longer therapeutically efficient, the hFIX mRNA:LUNAR LNP formulation gave significantly higher clotting efficiency (∼20%), enough to rescue the phenotypic defect. (D) Additionally, over the course of the 30 d, as animals were given repeated doses of the rhFIX or the LUNAR:R338A hFIX, the animals remained healthy and their body weights remained stable.

Fig. 5.

Repeated dosing over a 3- to 4-mo period does not elicit adverse immune reactions. A small cohort of FIX^−/− animals (n = 3 per group) was dosed three times with the mRNA:LUNAR LNP formulation over a period of 20 wk. The first two injections were 2 wk apart (WT hFIX mRNA:LUNAR LNP), whereas the last injection was after a 3-mo interval (and a 1:1 mix of FIX and luciferase mRNAs at a final dose of 4 mg/kg). At the third injection, the animals were examined for biodistribution and kinetics of the hFIX mRNA using the luciferase signal as a proxy for the localization of the LUNAR-hFIX. (A) Intravital imaging system (IVIS) from Xenogen was used to image animals at 7 h after LUNAR-hFIX + Luc mRNA administration. Animals were imaged at 15 min after administration of 15 mg/mL luciferin. As can be seen, most of the delivered LNPs enter the liver and are expressed there at this time point. (B) Hemophilic animals injected with the Luc + hFIX mRNA:LUNAR LNP complexes were bled at the indicated time points and the serum was assayed for the presence of human FIX protein by a Western blot. Circulating levels of FIX protein peak early by 4–7 h postinjection. (C) Levels of circulating hFIX in the mouse serum were measured by an ELISA. Levels are reported in nanograms per milliliter based on a standard curve generated from serial dilution of a known standard. (D) The serum from these animals was assayed for clotting efficiency by an APTT assay. The level of FIX protein produced at 4 h is enough to achieve therapeutic efficacy at up to 20% of normal levels. Despite repeat dosing, therapeutic levels of FIX are attained reproducibly and consistently, suggesting no antibody or cell-mediated neutralization of the LUNAR-hFIX mRNA LNPs or the FIX protein. (E) As a proxy for any toxicity, body weights were tracked during this period and the animals, being young, demonstrated normal weight gain. (F) At the end of the third administration, mouse livers were fixed, sectioned, stained with H&E, and examined for any histopathological abnormalities. Normal tissue architecture was seen in most cases, suggesting no adverse events.