Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs, and will be especially useful for ...co-delivery of coding genes and RNAi. A specific family of mirtrons - 3'-tailed mirtrons - has hairpins precisely defined on the 5' end by the 5' splice site and 3' end by the branch point. Here, we present design principles for artificial 3'-tailed mirtrons and demonstrate, for the first time, efficient gene knockdown with tailed mirtrons within eGFP coding region. These artificial tailed mirtrons, unlike canonical mirtrons, have very few sequence design restrictions. Tailed mirtrons targeted against VEGFA mRNA, the misregulation of which is causative of several disorders including cancer, achieved significant levels of gene knockdown. Tailed mirtron-mediated knockdown was further shown to be splicing-dependent, and at least as effective as equivalent artificial intronic miRNAs, with the added advantage of very defined cleavage sites for generation of mature miRNA guide strands. Further development and exploitation of this unique mirtron biogenesis pathway for therapeutic RNAi coupled into protein-expressing genes can potentially enable the development of precisely controlled combinatorial gene therapy.
Duchenne muscular dystrophy (DMD) is a systemic progressive muscular disease caused by frame‐disrupting mutations in the DMD gene. Although exon‐skipping antisense oligonucleotides (AOs) are ...clinically approved and can correct DMD, insufficient muscle delivery limits efficacy. If AO activity can be enhanced by safe dietary supplements, clinical trials for efficacy can be undertaken rapidly to benefit patients. We showed previously that intravenous glycine enhanced phosphorodiamidate morpholino oligomer (PMO) delivery to peripheral muscles in mdx mice. Here, we demonstrate that the combination of oral glycine and metformin with intravenous PMO enhances PMO activity, dystrophin restoration, extends lifespan, and improves body‐wide function and phenotypic rescue of dystrophin /utrophin double knock‐out (DKO) mice without any overt adverse effects. The DKO mice treated with the combination without altering the approved administration protocol of PMO show improved cardio‐respiratory and behavioral functions. Metformin and glycine individually are ineffective in DMD patients, but the combination of PMO with clinically‐approved oral glycine and metformin might improve the efficacy of the treatment also in DMD patients. Our data suggest that this combination therapy might be an attractive therapy for DMD and potentially other muscle diseases requiring systemic treatment with AOs.
Synopsis
The combination of oral glycine and metformin with clinically approved intravenous DMD exon‐skipping antisense oligonucleotides (AOs) enhances AO activity, dystrophin restoration, extends lifespan, and improves body‐wide function and phenotypic rescue of dystrophin/utrophin double knock‐out (DKO) mice.
Oral glycine augments efficacy of intravenous exon‐skipping PMO therapy in mdx and DKO mice.
Oral glycine and metformin with intravenous PMO enhances PMO activity, dystrophin restoration, extends lifespan, and improves body‐wide function and phenotypic rescue of dystrophic DKO mice without any overt adverse effects, most notably in the heart muscle.
The DKO mice treated with repurposed clinically‐approved glycine and metformin without altering the approved intravenous administration protocol of PMO results in improved cardio‐respiratory and behavioral functions.
The combination of oral glycine and metformin with clinically approved intravenous DMD exon‐skipping antisense oligonucleotides (AOs) enhances AO activity, dystrophin restoration, extends lifespan, and improves body‐wide function and phenotypic rescue of dystrophin/utrophin double knock‐out (DKO) mice.
The past decade has seen intense scientific interest in non-coding RNAs. In particular, the discovery and subsequent exploitation of gene silencing via RNA interference (RNAi) has revolutionized the ...way in which gene expression is now studied and understood. It is now well established that post-transcriptional gene silencing (PTGS) by the microRNA (miRNA) and other RNAi-associated pathways represents an essential layer of complexity to gene regulation. Gene silencing using RNAi additionally demonstrates huge potential as a therapeutic strategy for eliminating pathogenic gene expression. Yet despite the early promise and excitement of gene-specific silencing, several critical hurdles remain to be overcome before widespread clinical adoption. These include off-target effects, toxicity due to saturation of the endogenous RNAi functions, limited duration of silencing, and effective targeted delivery. In recent years, a range of novel strategies for producing RNA-mediated silencing have been developed that can circumvent many of these hurdles, including small internally segmented interfering RNAs, tandem hairpin RNAs, and pri-miRNA cluster mimics. This review discusses RNA-mediated silencing in light of this recent research, and highlights the benefits and limitations conferred by these novel gene-silencing strategies.
We evaluate a knockdown-replacement strategy mediated by mirtrons as an alternative to allele-specific silencing using spinocerebellar ataxia 7 (SCA7) as a model. Mirtrons are introns that form ...pre-microRNA hairpins after splicing, producing RNAi effectors not processed by Drosha. Mirtron mimics may therefore avoid saturation of the canonical processing pathway. This method combines gene silencing mediated by an artificial mirtron with delivery of a functional copy of the gene such that both elements of the therapy are always expressed concurrently, minimizing the potential for undesirable effects and preserving wild-type function. This mutation- and single nucleotide polymorphism-independent method could be crucial in dominant diseases that feature both gain- and loss-of-function pathologies or have a heterogeneous genetic background. Here we develop mirtrons against ataxin 7 with silencing efficacy comparable to shRNAs, and introduce silent mutations into an ataxin 7 transgene such that it is resistant to their effect. We successfully express the transgene and one mirtron together from a single construct. Hence, we show that this method can be used to silence the endogenous allele of ataxin 7 and replace it with an exogenous copy of the gene, highlighting the efficacy and transferability across patient genotypes of this approach.
We compared the performance of five assays for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) detection on nasopharyngeal swab samples: Roche “cobas,” Luminex “ARIES,” MiRXES ...“Fortitude,” Altona “RealStar,” and Thermo Fisher Scientific “TaqPath.” A total of 94 nasopharyngeal swab samples were obtained from 80 confirmed coronavirus disease 2019 cases in the first 2 weeks of illness (median, 7 days; range, 2–14 days) and 14 healthy controls. After collection, all samples were transported to the hospital clinical laboratory within 24 h. These samples were tested on all five assays within 3 days of sample receipt. Of the 94 samples, 69 yielded the same result on all platforms, resulting in an agreement of 73.4% (69 of 94). Of these, 14 were the healthy control swabs which all tested negative, demonstrating good specificity across all platforms. The ARIES assay had the lowest detection rate (68.8%), followed by Fortitude (85.0%), RealStar (86.3%), cobas (95.0%), and TaqPath (100%). Statistically significant differences were observed for ARIES, Fortitude, and RealStar when compared against the best performing TaqPath using McNemar's χ2 test. A consensus result was established based on the results obtained by the cobas, Fortitude, RealStar, and TaqPath. Six discrepancies had failed to reach a consensus and were adjudicated using the Cepheid Xpert Xpress SARS‐CoV‐2. Overall, the TaqPath and cobas assays were the most sensitive at detecting their designated SARS‐CoV‐2 gene targets. On the other hand, the ARIES assay was the least sensitive, thus warranting the need for assay re‐optimization before go‐live at the testing laboratory.
Antisense oligonucleotides (AOs) have the potential to induce functional dystrophin protein expression via exon skipping by restoring in-frame transcripts in the majority of patients suffering from ...Duchenne muscular dystrophy (DMD). AOs of morpholino phosphoroamidate (PMO) and 2′-O-methyl phosphorothioate RNA (2′Ome RNA) chemistry have been shown to restore dystrophin expression in skeletal muscle but not in heart, following high-dose systemic delivery in murine models of muscular dystrophy (mdx). Exploiting the cell transduction properties of two basic arginine-rich cell penetrating peptides, we demonstrate widespread systemic correction of dystrophin expression in body-wide muscles and cardiac tissue in adult dystrophic mdx mice, with a single low-dose injection of peptide-conjugated PMO AO. This approach was sufficient to restore uniform, high-level dystrophin protein expression in peripheral muscle and cardiac tissue, with robust sarcolemmal relocalization of the dystrophin-associated protein complex and functional improvement in muscle. Peptide-conjugated AOs therefore have significant potential for systemic correction of the DMD phenotype.
Mirtrons are a recently described category of microRNA (miRNA) relying on splicing rather than processing by the microprocessor complex to generate pre-miRNA precursors of the RNA interference (RNAi) ...pathway. Their discovery and subsequent verification provides important information about a distinct class of miRNA and inherent advantages that could be exploited to silence genes of interest. These include micro-processor-independent biogenesis, pol-II-dependent transcription, accurate species generation and the delivery of multiple artificial mirtrons as introns within a single host transcript. Here we determined the sequence motifs required for correct processing of the mmu-miR-1224 mirtron and incorporated these into artificial mirtrons targeting Parkinson's disease-associated LRRK2 and α-synuclein genes. By incorporating these rules associated with processing and splicing, artificial mirtrons could be designed and made to silence complementary targets either at the mRNA or protein level. We further demonstrate with a LRRK2 targeting artificial mirtron that neuronal-specific silencing can be directed under the control of the human synapsin promoter. Finally, multiple mirtrons were co-delivered within a single host transcript, an eGFP reporter, to allow simultaneous targeting of two or more targets in a combinatorial approach. Thus, the unique characteristics of artificial mirtrons make this an attractive approach for future RNAi applications.
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