Lipid nanoparticles deliver messenger RNA for hemophilia B protein replacement therapy [PreClinical]

1. A lipid nanoparticle (LNP) delivery system was developed to introduce factor IX (FIX) messenger RNA (mRNA) into a FIX deficient mouse model of hemophilia B.

2. Treatment with the lipid-enabled and unlocked nucleic acid modified RNA (LUNAR) LNPs led to clotting and FIX restoration in the mice.

Evidence Rating Level: 2 (Good)

Study Rundown: Hemophilia B is characterized by a deficiency in the coagulation protein FIX. Because this condition is caused by a deficiency of a single protein, it is an ideal candidate for protein replacement therapy through the introduction of mRNA. There are currently many pitfalls to making RNA a stable and efficient therapy. This study designed LUNAR LNPs to deliver FIX mRNA, as a safer alternative to recombinant human FIX protein, the current therapy for hemophilia B.

LUNAR LNPs specifically delivered mRNA to the liver, the site of FIX protein synthesis. Following the administration of LUNAR LNPs in FIX-deficient mice, there was a significant increase in serum FIX protein concentration and an increase in clotting activity. In addition, mutated variants of FIX mRNA were generated and delivered via LUNAR LNPs, which enhanced clotting activity compared to wildtype mRNA. Compared to mice that received recombinant human FIX protein, mice administered LUNAR with the mutated mRNA showed increased FIX protein levels for a longer period of time. No significant adverse events or immune complications developed following the administration of LUNAR LNPs.

Although a more relevant clinical model will need to be used to further assess the safety of this therapy, the development of this LUNAR LNP mRNA delivery system may lead to improved therapeutics for clotting disorders and other monogenic conditions.

Click to read the article in PNAS

Relevant Reading: New developments in the management of moderate to severe hemophilia B

In-Depth [animal study]: The LUNAR formulation contained 4 different lipids. One of the lipids had a protonated amino group, allowing for the interaction with and encapsulation of RNA. Another lipid contained ester bonds in its backbone, increasing stability until cleavage at the delivery site. A LUNAR-encapsulated luciferase was injected into mice to visualize the delivery location of these nanoparticles. Although a weak signal was seen in the spleen, no signal was detected in other organs including the heart, lungs, and kidney. High luciferase expression was observed in the liver, demonstrating the high liver specificity of LUNAR LNPs.

FIX knockout mice were administered 2 mg/kg human FIX mRNA in LUNAR nanoparticles (LUNAR:hFIX). Following treatment, mice exhibited normal physiological serum levels of FIX protein of over 2,500 ng/mL, as assessed through an ELISA. A functional assay of the FIX protein demonstrated a rescue of clotting activity, with elevated activity even 48 hours post-injection (p<0.0395).

Two mutations were then introduced into the FIX mRNA, R338A and R338L, designed to enhance the catalytic activity of the resulting proteins. Mice treated with LUNAR containing the R338A and R338L variants showed enhanced clotting activity in serum at 115% and 88%, respectively, compared to the 20% activity seen with the wildtype FIX. When FIX knockout mice were treated with LUNAR:R338A or recombinant human FIX protein (Benefix), circulating levels of FIX in the LUNAR:R338A-treated mice were 8 –10 times higher than in the Benefix-treated mice. LUNAR:R338A-treated mice also showed increased clotting activity, with consistent results following repeated doses. These mice did not experience any clear adverse events or weight loss, and did not show significantly altered immune responses. 

Image: PD

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