Delivery of A Chemically Modified Noncoding RNA Domain Improves Dystrophic Myotube Function

Abstract

Fast twitch muscle fibers are prone to degradation in skeletal muscle pathologies, such as sarcopenia and muscular dystrophies. We previously showed that the exercise-induced long noncoding RNA CYTOR promotes fast-twitch myogenesis. Here, we identify an independent functional element within human CYTOR, and optimize its RNA delivery. In human primary myoblasts exogenous CYTOR exon 2 recapitulates the effect of full-length CYTOR by boosting fast-twitch myogenic differentiation. Furthermore, chemically modified CYTOR exon 2 RNA^ΨU (N1-me-PseudoU, 7-methyl guanosine 5'Cap, polyA) enhances RNA stability and reduces immunogenicity to CYTOR^exon2 RNA. Viral- or chemically optimized RNA-mediated CYTOR^exon2 administration drives commitment toward myogenic maturation in Duchenne muscular dystrophy-derived primary myoblasts, myogenic progenitor cells, and mouse embryonic stem cells. Furthermore, CYTOR^{exon2, m1ΨU} improves key disease characteristics in dystrophic myotubes, including calcium handling and mitochondrial bioenergetics. In summary, we identify CYTOR exon 2 as the functional domain of CYTOR that can be delivered in a disease context using chemical modifications. This is of particular importance given the susceptibility of fast muscle fibers in different muscle pathologies such as aging and dystrophies, and the oncogenic effect of CYTOR exon 1. This study, therefore, highlights the potential of identifying functional domains in noncoding RNAs. Delivery, or targeting of RNA domains might constitute next-generation RNA therapeutics.

Keywords: CYTOR; RNA structure; aging; differentiation; dystrophy; functional domain; long noncoding RNA; myoblast; myogenesis; sarcopenia; skeletal muscle.

Figure 1

Lentivirus‐mediated stable exogenous overexpression of human CYTOR exons in human muscle cells. (A‐B) SHAPE‐directed RNA secondary structure prediction in vitro and in cellulo by selective 2′‐hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE‐MaP) of A) human CYTOR exon 1 and B) CYTOR exon 2 in K562 cells. C) Delta SHAPE reactivity scores from (A‐B) calculated from in vitro and in cellulo SHAPE reactivities. N = 2. D) Normalized gene expression in differentiating human primary myoblasts transduced with lentivirus capable to express GFP, CYTOR^exon1, CYTOR^exon2 or full‐length CYTOR. N = 3‐4. E) Immunocytochemistry of differentiating human primary myoblasts transduced with lentivirus expressing GFP, CYTOR^exon1, CYTOR^exon2 or full‐length CYTOR. N = 4. F) Quantification of myotube diameter in human primary myoblasts transduced with lentivirus expressing GFP, CYTOR^exon1, CYTOR^exon2 or full‐length CYTOR. N = 4. G) Quantification of surface area covered by myotubes in human primary myoblasts transduced with lentivirus expressing GFP, CYTOR^exon1, CYTOR^exon2 or full‐length CYTOR. N = 4. H) Quantification of number of nuclei per myotube in human primary myoblasts transduced with lentivirus expressing GFP, CYTOR^exon1, CYTOR^exon2 or full‐length CYTOR. N = 4. ^* p < 0.05, ^** p < 0.01.

Figure 2

Optimization of RNA‐based CYTOR exon 2 delivery. ​​A) Normalized CYTOR expression at different time points in HEK293 cells after modified and unmodified CYTOR exon 2 RNA or DNA plasmid delivery. N = 4. B–E) Time course of normalized gene expression of genes related to the innate immune response (TNFα, IL‐6) and genes induced by pattern recognition receptor activation (NFκB, IFNα) upon treatment with modified and unmodified CYTOR exon 2 RNA or DNA plasmid delivery. N = 4. F) CYTOR RNA abundance from (A) at the 24 h time point. N = 4. ^* p < 0.05, ^** p < 0.01.

Figure 3

Myogenic effect of optimized RNA gene therapy using the exon 2 myogenic CYTOR element in dystrophic human primary muscle cells. A) Normalized CYTOR RNA levels in primary muscle cells from healthy controls compared to Duchenne muscular dystrophy patients (E‐MTAB‐8321). N = 8‐9. B) Schematic showing culturing and treatment regimen of human primary myoblasts from Duchenne muscular dystrophy patients (DMD) with chemically optimized CYTOR exon 2. C) Normalized gene expression after chemically enhanced CYTOR^exon2 RNA administration in differentiating human primary myoblasts isolated from DMD. N = 4‐5. D) Immunocytochemistry of differentiating dystrophic human primary muscle cells transfected with chemically enhanced CYTOR^exon2 RNA. N = 4. E–G) Quantification of myotube diameter (E), surface area covered by myotubes (F), and number of nuclei per myotube in dystrophic human primary muscle cells treated with chemically enhanced CYTOR^exon2 RNA. N = 4. H) Representative traces of cytosolic, baseline‐normalized Ca²⁺ transients in dystrophic myotubes treated with CYTOR exon 2 upon 5 mm caffeine stimulation (SR Ca²⁺ store). I) Raw baseline Ca²⁺ signal in dystrophic myotubes treated with chemically enhanced CYTOR exon 2 RNA or a scramble control RNA as percentage of control. N = 8. J) Ca²⁺ amplitude (SR Ca²⁺ store) upon 5 mm caffeine stimulation as percentage of scramble control. N = 8. K) Ca²⁺ clearance as calculated from percentage decrease from the Ca²⁺ peak amplitude. N = 8. L) Mitochondrial respiration in chemically enhanced CYTOR exon 2 and scramble control RNA treated dystrophic muscle cells at baseline and upon addition of oligomycin (1 um) and CCCP (2.5 um). N = 8. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

Figure 4

Forced expression of human CYTOR exon 2 in mouse‐induced myogenic progenitor and embryonic stem cells. A) Normalized gene expression in mouse embryonic stem cells exogenously expressing a GFP control construct or CYTOR^exon2 from a DNA vector. N = 5‐6. B) Normalized gene expression in mouse embryonic stem cells after transfection of chemically modified CYTOR^exon2 RNA. N = 5‐6. C) Normalized gene expression in induced myogenic progenitors expressing a GFP control construct or CYTOR^exon2 from a DNA vector. N = 3. D) Normalized gene expression in induced myogenic progenitors after transfection of CYTOR^exon2,ΨU RNA. N = 3.^* p < 0.05, ^** p < 0.01.

Figure 5

Summary of functional elements of CYTOR driving myogenesis and oncogenesis. Schematic overview over the different effects exons 1 and 2 exert. CYTOR exon 1 has been shown to drive cancer cell proliferation.^{[ 34 ]} In this study we identify CYTOR exon 2 to promote muscle cell differentiation and function.