Development of Emerin mRNA Lipid Nanoparticles to Rescue Myogenic Differentiation

Abstract

Emery-Dreifuss muscular dystrophy 1 (EDMD1) arises from mutations in EMD. Most EDMD1 patients lack detectable emerin expression. They experience symptoms such as skeletal muscle wasting, joint contractures, and cardiac conduction defects. Currently, physicians rely on treating patient symptoms without addressing the underlying cause-lack of functional emerin protein. Thus, there is a need for therapeutic approaches that restore emerin protein expression to improve patient outcomes. One way would be to deliver emerin mRNA or protein directly to affected tissues to restore tissue homeostasis. Here, we evaluated the utility of lipid nanoparticles (LNPs) to deliver emerin mRNA to diseased cells. LNPs have been studied for decades and have recently been used clinically for vaccination and treatment of a myriad of diseases. Here, we show that the treatment of emerin-null myogenic progenitors with LNPs encapsulating emerin mRNA causes robust emerin protein expression that persists for at least 4 days. The treatment of differentiating emerin-null myogenic progenitors with 2.5 pg/cell emerin LNPs significantly improved their differentiation. The toxicity profiling of emerin mRNA LNP (EMD-LNP) dosing shows little toxicity at the effective dose. These data support the potential use of EMD-LNPs as a viable treatment option and establishes its utility for studying EDMD pathology.

Keywords: EDMD; Emery–Dreifuss muscular dystrophy; emerin; lipid nanoparticle; myogenic differentiation.

Figure 1

EMD-LNPs efficiently deliver cargo in vitro. (A) Emerin-null myogenic progenitors dosed with 2.5 pg/cell EMD-LNPs (EMD^−/y + LNP) or PBS (EMD^−/y + PBS) for 22 h. Immunofluorescence microscopy was performed using emerin antibodies (green). DAPI (blue), DNA. (B) Greater than 97% of EMD-LNP-treated emerin-null progenitors retain emerin protein expression after 4 days. (C) Western blotting was performed on wildtype (WT) or emerin-null proliferative myogenic progenitors treated with EMD-LNPs (EMD^−/y + LNP) or PBS (EMD^−/y + PBS) over four days. (D) Quantitation of emerin protein expression normalized to endogenous emerin in wildtype progenitors. Scale bar represents 100 µm. Error bars represent S.D. (n = 4).

Figure 2

EMD-LNPs become cytotoxic at ≥15 pg/cell. PrestoBlue cell viability assays were used to measure cellular metabolism in proliferating myogenic progenitors treated with increasing concentrations of PBS (PBS), EMD-LNP (EMD), or luciferase mRNA LNP (LUC). Measurements were recorded every 24 h for 72 h, and relative light units (RLUs) were normalized to PBS: (A) 24 h after LNP incubation; (B) 48 h after LNP incubation; (C) 72 h after LNP incubation. Error bars represent S.D. (n = 3); * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 3

EMD-LNPs rescue differentiation of emerin-null myogenic progenitors. (A) Line graph showing the timing of LNP treatment (green dot), differentiation induction, and sample collection (pink dot). Wildtype myogenic progenitors treated with PBS (WT + PBS), emerin-null progenitors treated with PBS (EMD^−/y + PBS), or emerin-null progenitors treated with 2.5 pg/cell of EMD-LNPs (EMD^−/y + LNP) were incubated for 22 h and induced to differentiate. (B) After 24 h, the cells were incubated with EdU for 2 h. The cells were fixed and incubated with emerin antibodies. EdU, red; emerin, green; DAPI, DNA (blue). (C) Quantification of EdU-positive nuclei. (D,F) Immunofluorescence microscopy was performed after 48 h (D) or 72 h (F) to detect MyHC (red) or emerin (green). DAPI, DNA (blue). (E) The differentiation index was quantified by determining the percentage of MyHC-positive cells. (G) The fusion index represents the percentage of nuclei present in a shared (≥3 nuclei/cell) MyHC-positive cytoplasm. (H) Western blot images of myogenesis markers in differentiating myogenic progenitors. Representative images of normal exposure (i) or increased exposure (ii) are shown. Scale bars represent 100 µm. Error bars represent S.D. (n = 4; ≥100 nuclei per biological replicate); * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 4

EMD-LNPs partially rescue H4K5ac and H3K9me2 levels in emerin-null myogenic progenitors. Whole-cell lysates were collected from wildtype cells treated with PBS (WT + PBS), emerin-null cells were treated with 2.5 pg/cell of EMD-LNPs (EMD^−/y + LNP), and emerin-null cells were treated with PBS (EMD^−/y + PBS) 24 h after treatment. (A) Western blotting for emerin, H3K9me2, H4K5ac, and γ-tubulin. (B,C) Quantification of H3K9me2 and H4K5ac Western blots. Levels of each protein were normalized to γ-tubulin and then normalized to wildtype cells. Error bars represent S.D. (n = 3); # p ≤ 0.08; * p ≤ 0.05; ** p ≤ 0.01.