Mitochondrion-targeted RNA therapies as a potential treatment strategy for mitochondrial diseases

Abstract

Mitochondrial diseases are one of the largest groups of neurological genetic disorders. Despite continuous efforts of the scientific community, no cure has been developed, and most treatment strategies rely on managing the symptoms. After the success of coronavirus disease 2019 (COVID-19) mRNA vaccines and accelerated US Food and Drug Administration (FDA) approval of four new RNAi drugs, we sought to investigate the potential of mitochondrion-targeting RNA-based therapeutic agents for treatment of mitochondrial diseases. Here we describe the causes and existing therapies for mitochondrial diseases. We then detail potential RNA-based therapeutic strategies for treatment of mitochondrial diseases, including use of antisense oligonucleotides (ASOs) and RNAi drugs, allotopic therapies, and RNA-based antigenomic therapies that aim to decrease the level of deleterious heteroplasmy in affected tissues. Finally, we review different mechanisms by which RNA-based therapeutic agents can be delivered to the mitochondrial matrix, including mitochondrion-targeted nanocarriers and endogenous mitochondrial RNA import pathways.

Keywords: MT: Oligonucleotides, Therapies and Applications; RNA therapeutics; RNA therapy; mitochondrial DNA; mitochondrial RNA import; mitochondrial disease; mitochondrial therapy; mitochondrion-targeted nanocarrier.

Graphical abstract

Figure 1

Schematic of mitochondrial diseases and heteroplasmic load of mtDNA mutations (A and B) Mutations of mtDNA may lead to translational deficiencies, Oxidative phosphorilation deficiencies, and global mitochondrial functional decline. When the heteroplasmic load of the pathogenic mutation surpasses the threshold in tissues, symptoms of mitochondrial disease may manifest. Homoplasmic WT, 0%; heteroplasmic (unaffected), 0%–70%; heteroplasmic (affected), >70%–80% threshold.

Figure 2

Potential RNA-based strategies for treatment of mitochondrial diseases (A) Single-stranded ASOs may specifically bind to mitochondrial transcripts, inducing degradation or inhibiting translation (a) siRNA or mitomiRNA mimics may bind to mitochondrial transcripts (b). However, whether RNAi or RNAi-like mechanisms are functional in the mitochondrial matrix is still inconclusive, and mixed findings have been reported regarding gene silencing mechanisms in the mitochondrial matrix. (B) WT mitochondrial mRNAs, tRNAs, and rRNAs can be delivered to the mitochondrial matrix to substitute their defective or missing counterparts.^,, (C) Antigenomic RNA molecules capable of specifically binding to the mutant locus of mtDNA and stalling its replication can be delivered to the mitochondrial matrix to allow a replicative advantage for the WT mtDNA, decreasing the level of pathogenic heteroplasmy. The figure only describes the RNA-based approaches that have been at least tested in vitro using human cell lines. Therefore, although therapeutic agents such as miRNAs and ASOs can be used to not only repress but also upregulate the translation of some mRNAs and even affect the transcription of mtDNA, these effects are not described here because the feasibility of these approaches has yet to be defined.

Figure 3

Strategies for mitochondrial delivery of RNA-based therapeutic agents (A) The RNA import complex (RIC) from L. tropica can be taken up by cells in vitro and targeted to mitochondria by itself.^, Localization of the RIC on mitochondrial membranes induces import of some cytosolic tRNAs (not shown). 8 nuclear-encoded subunits of RIC, bound to an exogenous pcRNA, are also taken up by the cell and targeted to the mitochondrial membrane, where the pcRNA is released into the mitochondrial matrix. This strategy allows import of several tRNAs, antisense RNAs, and polycistronic mRNA to the mitochondrial matrix of cells in vitro.^, (B) Mitochondrion-targeted RNA nanocarriers can enter the cell and actively target mitochondria, where they accumulate (e.g., RP/β-MEND and PAMAM(G5)-TPP, not shown) or fuse with the mitochondrial membranes (MITO-porter); then their cargo is released into the mitochondrial matrix.^,,, Using such nanocarriers, ASOs, miRNAs, mRNAs, pre-tRNA, and 12S rRNA can be imported into mitochondria of cells in vitro.^,,, (C–F) Specific endogenous RNA import determinants can be attached to exogenous RNA molecules that, upon delivery to the cytosol of a target cell by means of a nanocarrier or RNA conjugate, are released and targeted for mitochondrial import via an endogenous pathway. Cytosol-targeted vesicles include liposome- and polymer-based nanocarriers and an RNA conjugate, but other types of cytosol-targeted carriers can also be used to deliver RNAs to the cytosol. (C) S. cerevisiae tRNA^Lys (tRKs), tRK1, tRK3, and mutant tRK2 (tRK93, described below), are imported into human mitochondria in vivo. Hence, modified tRKs that mimic human mitochondrial tRNAs are imported into the mitochondrial matrix from the cytosol. This approach has been successful for recovering normal mitochondrial function in MERRF and MELAS cybrid cell lines and patient-derived fibroblasts.^, (D) Attachment of a 20-nt stem-loop structure of RPPH1 to the exogenous RNAs directs them for PNPase-mediated mitochondrial import. This approach has been shown to be effective for delivering mRNA and tRNA to the mitochondria of cells in vitro. (E and F) Attachment of import determinants of human 5S rRNA (E) or yeast tRK1 (F) to the antireplicative RNA therapeutic agents directs them for import into the mitochondrial matrix, where they hybridize with the mutant mtDNA and stall its replication, providing a replicative advantage for WT mtDNA.^,, This decreases the level of deleterious heteroplasmy in a cell and recovers mitochondrial function. ∗ represents direct RNA import strategies. ǂ represents indirect RNA import strategies. ∗ǂ represents the potential for the strategy to be indirect or direct. RNA structures in the figure are only a schematic representation of the actual molecules. Some components of the figure are not to scale.

Figure 4

The RNA importome of human mitochondria Some nucleus-encoded RNAs are actively imported into the mitochondrial matrix. They play a variety of roles in mitochondria, ranging from cytosol-mitochondrion signaling to participation in mitochondrial essential processes such as translation or mtRNA metabolism.^, All of the known imported RNA species are non-coding, and no mRNA has been reported to be naturally targeted for import into human mitochondria. Most of the mitochondrial functions of the imported RNAs remain undefined and can be different from the functions of these RNAs in the cytosol. At the top, the types of imported RNA species, their mechanism of import, and their function in mitochondria are shown. From left to right, these are as follows. tRNA (tRNA^Gln_UUG, tRNA^Gln_CUG, and tRNA^Leu_UAA).^, Cytosolic function: translation. Mitochondrial function: mitochondrial translation; could play a role in conditional adaptation in mitochondrial protein synthesis. Import mechanism: not well defined in humans; ATP dependent; may require cytosolic factors and be similar to that in S. cerevisiae (reviewed here: Kamenski et al.¹⁴⁸). 5S rRNA. Cytosolic function: structural component of a large subunit of cytosolic ribosomes. Mitochondrial functions: still to be defined. Does not act as a scaffold for mitochondrial ribosomes. Disruption of 5S rRNA import leads to a decrease in mitochondrial translation. Import mechanism: pre-MRP-L18 binds the ƴ-domain of 5S rRNA in the cytosol; this exposes the α domain of 5S rRNA, which binds nascent rhodanese, displacing pre-MRP-L18 in the process. The rhodanese-5S rRNA complex is then imported into mitochondria. PNPase may be involved in transfer of 5S rRNA because its knockdown leads to decreased import of 5S rRNA. Import determinants: ƴ-domain (specifically loop E and helix IV) and α domain (helix I). RPPH1 (RNA component of RNAse P; H1 RNA). Cytosolic function: component of RNAse P, 5′ tRNA maturation. Mitochondrial function: mitochondrial localization induces replicative cellular senescence. May play a role in mt-tRNA maturation as a part of mitochondrial RNAse P. Import mechanism: mediated by PNPase.^, Import determinants: 20-nt stem-loop, nucleotides 115–134 (NR_002312.1). RMRP (RNA component of RNAse MRP). Cytosolic function: component of the nuclear RNAse MRP; 5′ 5.8S rRNA maturation; participates in generation of double-stranded RNA (dsRNA) precursors of siRNA; cell cycle progression.^, Mitochondrial function: not firmly defined. May participate in mitochondrial RNA metabolism in a complex with GRSF1.^, Import mechanism: exported from the nucleus in a complex with HuR. The precise mechanism of RMRP transport through the mitochondrial membrane is unknown, although it has been hypothesized that it may cross the OMM via the TOMM40 complex and intramembrane space (IMS) in a PNPase-dependent manner. It has also been shown that levels of GRSF1 (associated with RMRP in the mitochondrial matrix) in mitochondria can affect RMRP localization. Import determinants: 20-nt stem-loop, Nucleotides 151–170 (NR_003051.3). GAS5 (growth arrest-specific 5). Cytosolic function: RNA sponge to buffer miRNAs; regulates INSR transcription in adipocytes; acts as a decoy hormone response element for glucocorticoid receptor (GR).^,, Mitochondrial function: modulates mitochondrial tricarboxylic acid (TCA) cycle flux. Import mechanism: Unknown. Import determinants: GAS5-loop 2. hTERC (human telomerase RNA gene). Cytosolic function: telomerase-related functions; Cellular senescence and cognitive decline in mice as hTERC-53.^, Mitochondrial function: mitochondrion-cytosol communication (hTERC-53 export is affected by mitochondrial function). Import mechanism: mediated by PNPase. Import determinants: stem-loop structure, similar to the ones found in RPPH1 and RMRP. miRNA (various miRNAs; e.g., miR-1, miR-181c, miR-378, and others); pre-miRNAs).^,,,,,, Cytosolic function: repress gene expression at the post-transcriptional level. Mitochondrial function: stimulate or inhibit mitochondrial gene expression, suppress mtDNA transcription, cytosol-mitochondrion crosstalk.^,,, Import mechanism: It is unknown whether all miRNAs share the same import mechanism. PNPase participates in import of miR-378. Import determinants: unknown. SAMMSON (survival-associated mitochondrial melanoma-specific oncogenic non-coding RNA). Cytosolic function: facilitates p32 trafficking to mitochondria in melanoma cells.^, Mitochondrial function: unknown. Import mechanism: unknown. Import determinants: unknown. Straight green arrows indicate that the import mechanism is still unknown. Arrows going to PNPase mean that the import pathway involves PNPase in the IMS. OMM, outer mitochondrial matrix; IMS, intramembrane space; IMM, inner mitochondrial membrane; matrix, mitochondrial matrix. Question marks mean that the function/import mechanism is plausible based on the current state of knowledge but has not yet been definitively proven.