RNA therapeutics.

"RNA therapeutics" refers to a disease treatment or drug that utilizes RNA as a component. In this context, RNA may be the direct target of a small-molecule drug or RNA itself may be the drug, designed to bind to a protein, or to mimic or target another RNA. RNA has gained attention in the drug-development world, as recent clinical successes and breakthrough technologies have revolutionized the drug-like qualities of the molecule or its usefulness as a drug target. In this special issue of RNA, we gathered expert perspectives on the past, present, and future of the field, to serve as a primer and also a challenge to the broad scientific community to incorporate RNA into their experimental design and problem-solving process, and to imagine and realize the potential of RNA as a therapeutic drug or target.

Open reading frame correction using splice-switching antisense oligonucleotides for the treatment of cystic fibrosis.

CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR messenger RNA (mRNA) expression as a result of nonsense-mediated mRNA decay, as well as the production of a nonfunctional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs), designed to induce skipping of exons in order to restore the mRNA open reading frame, have shown therapeutic promise preclinically and clinically for a number of diseases. We hypothesized that ASO-mediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the fifth most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from a homozygote CFTR-W1282X patient. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein.

RNA localization and co-translational interactions control RAB13 GTPase function and cell migration.

Numerous RNAs exhibit specific distribution patterns in mammalian cells. However, the functional and mechanistic consequences are relatively unknown. Here, we investigate the functional role of RNA localization at cellular protrusions of migrating mesenchymal cells, using as a model the RAB13 RNA, which encodes a GTPase important for vesicle-mediated membrane trafficking. While RAB13 RNA is enriched at peripheral protrusions, the expressed protein is concentrated perinuclearly. By specifically preventing RAB13 RNA localization, we show that peripheral RAB13 translation is not important for the overall distribution of the RAB13 protein or its ability to associate with membranes, but is required for full activation of the GTPase and for efficient cell migration. RAB13 translation leads to a co-translational association of nascent RAB13 with the exchange factor RABIF. Our results indicate that RAB13-RABIF association at the periphery is required for directing RAB13 GTPase activity to promote cell migration. Thus, translation of RAB13 in specific subcellular environments imparts the protein with distinct properties and highlights a means of controlling protein function through local RNA translation.

Corticosteroid sensitization drives opioid addiction.

The global crisis of opioid overdose fatalities has led to an urgent search to discover the neurobiological mechanisms of opioid use disorder (OUD). A driving force for OUD is the dysphoric and emotionally painful state (hyperkatifeia) that is produced during acute and protracted opioid withdrawal. Here, we explored a mechanistic role for extrahypothalamic stress systems in driving opioid addiction. We found that glucocorticoid receptor (GR) antagonism with mifepristone reduced opioid addiction-like behaviors in rats and zebrafish of both sexes and decreased the firing of corticotropin-releasing factor neurons in the rat amygdala (i.e., a marker of brain stress system activation). In support of the hypothesized role of glucocorticoid transcriptional regulation of extrahypothalamic GRs in addiction-like behavior, an intra-amygdala infusion of an antisense oligonucleotide that blocked GR transcriptional activity reduced addiction-like behaviors. Finally, we identified transcriptional adaptations of GR signaling in the amygdala of humans with OUD. Thus, GRs, their coregulators, and downstream systems may represent viable therapeutic targets to treat the "stress side" of OUD.

Antisense oligonucleotide-mediated correction of CFTR splicing improves chloride secretion in cystic fibrosis patient-derived bronchial epithelial cells.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in >15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10 kb C>T), which creates a new 5' splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.

Fetal antisense oligonucleotide therapy for congenital deafness and vestibular dysfunction.

Disabling hearing loss impacts ∼466 million individuals worldwide with 34 million children affected. Gene and pharmacotherapeutic strategies to rescue auditory function in mouse models of human deafness are most effective when administered before hearing onset, after which therapeutic efficacy is significantly diminished or lost. We hypothesize that preemptive correction of a mutation in the fetal inner ear prior to maturation of the sensory epithelium will optimally restore sensory function. We previously demonstrated that transuterine microinjection of a splice-switching antisense oligonucleotide (ASO) into the amniotic cavity immediately surrounding the embryo on embryonic day 13-13.5 (E13-13.5) corrected pre-mRNA splicing in the juvenile Usher syndrome type 1c (Ush1c) mouse mutant. Here, we show that this strategy only marginally rescues hearing and partially rescues vestibular function. To improve therapeutic outcomes, we microinjected ASO directly into the E12.5 inner ear. A single intra-otic dose of ASO corrects harmonin RNA splicing, restores harmonin protein expression in sensory hair cell bundles, prevents hair cell loss, improves hearing sensitivity, and ameliorates vestibular dysfunction. Improvements in auditory and vestibular function were sustained well into adulthood. Our results demonstrate that an ASO pharmacotherapeutic administered to a developing organ system in utero preemptively corrects pre-mRNA splicing to abrogate the disease phenotype.

Identification of SRSF10 as a regulator of SMN2 ISS-N1.

Understanding the splicing code can be challenging as several splicing factors bind to many splicing-regulatory elements. The SMN1 and SMN2 silencer element ISS-N1 is the target of the antisense oligonucleotide drug, Spinraza, which is the treatment against spinal muscular atrophy. However, limited knowledge about the nature of the splicing factors that bind to ISS-N1 and inhibit splicing exists. It is likely that the effect of Spinraza comes from blocking binding of these factors, but so far, an unbiased characterization has not been performed and only members of the hnRNP A1/A2 family have been identified by Western blot analysis and nuclear magnetic resonance to bind to this silencer. Employing an MS/MS-based approach and surface plasmon resonance imaging, we show for the first time that splicing factor SRSF10 binds to ISS-N1. Furthermore, using splice-switching oligonucleotides we modulated the splicing of the SRSF10 isoforms generating either the long or the short protein isoform of SRSF10 to regulate endogenous SMN2 exon 7 inclusion. We demonstrate that the isoforms of SRSF10 regulate SMN1 and SMN2 splicing with different strength correlating with the length of their RS domain. Our results suggest that the ratio between the SRSF10 isoforms is important for splicing regulation.

Mitochondrial clearance and maturation of autophagosomes are compromised in LRRK2 G2019S familial Parkinson's disease patient fibroblasts.

This study utilized human fibroblasts as a preclinical discovery and diagnostic platform for identification of cell biological signatures specific for the LRRK2 G2019S mutation producing Parkinson's disease (PD). Using live cell imaging with a pH-sensitive Rosella biosensor probe reflecting lysosomal breakdown of mitochondria, mitophagy rates were found to be decreased in fibroblasts carrying the LRRK2 G2019S mutation compared to cells isolated from healthy subject (HS) controls. The mutant LRRK2 increased kinase activity was reduced by pharmacological inhibition and targeted antisense oligonucleotide treatment, which normalized mitophagy rates in the G2019S cells and also increased mitophagy levels in HS cells. Detailed mechanistic analysis showed a reduction of mature autophagosomes in LRRK2 G2019S fibroblasts, which was rescued by LRRK2 specific kinase inhibition. These findings demonstrate an important role for LRRK2 protein in regulation of mitochondrial clearance by the lysosomes, which is hampered in PD with the G2019S mutation. The current results are relevant for cell phenotypic diagnostic approaches and potentially for stratification of PD patients for targeted therapy.

Direct Delivery of Antisense Oligonucleotides to the Middle and Inner Ear Improves Hearing and Balance in Usher Mice.

Usher syndrome is a syndromic form of hereditary hearing impairment that includes sensorineural hearing loss and delayed-onset retinitis pigmentosa (RP). Type 1 Usher syndrome (USH1) is characterized by congenital profound sensorineural hearing impairment and vestibular areflexia, with adolescent-onset RP. Systemic treatment with antisense oligonucleotides (ASOs) targeting the human USH1C c.216G>A splicing mutation in a knockin mouse model of USH1 restores hearing and balance. Herein, we explore the effect of delivering ASOs locally to the ear to treat hearing and vestibular dysfunction associated with Usher syndrome. Three localized delivery strategies were investigated in USH1C mice: inner ear injection, trans-tympanic membrane injection, and topical tympanic membrane application. We demonstrate, for the first time, that ASOs delivered directly to the ear correct Ush1c expression in inner ear tissue, improve cochlear hair cell transduction currents, restore vestibular afferent irregularity, spontaneous firing rate, and sensitivity to head rotation, and successfully recover hearing thresholds and balance behaviors in USH1C mice. We conclude that local delivery of ASOs to the middle and inner ear reach hair cells and can rescue both hearing and balance. These results also demonstrate the therapeutic potential of ASOs to treat hearing and balance deficits associated with Usher syndrome and other ear diseases.

A mutation affecting polycystin-1 mediated heterotrimeric G-protein signaling causes PKD.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of renal cysts that ultimately destroy kidney function. Mutations in the PKD1 and PKD2 genes cause ADPKD. Their protein products, polycystin-1 (PC1) and polycystin-2 (PC2) have been proposed to form a calcium-permeable receptor-channel complex; however the mechanisms by which they function are almost completely unknown. Most mutations in PKD1 are truncating loss-of-function mutations or affect protein biogenesis, trafficking or stability and reveal very little about the intrinsic biochemical properties or cellular functions of PC1. An ADPKD patient mutation (L4132Δ or ΔL), resulting in a single amino acid deletion in a putative G-protein binding region of the PC1 C-terminal cytosolic tail, was found to significantly decrease PC1-stimulated, G-protein-dependent signaling in transient transfection assays. Pkd1ΔL/ΔL mice were embryo-lethal suggesting that ΔL is a functionally null mutation. Kidney-specific Pkd1ΔL/cond mice were born but developed severe, postnatal cystic disease. PC1ΔL protein expression levels and maturation were comparable to those of wild type PC1, and PC1ΔL protein showed cell surface localization. Expression of PC1ΔL and PC2 complexes in transfected CHO cells failed to support PC2 channel activity, suggesting that the role of PC1 is to activate G-protein signaling to regulate the PC1/PC2 calcium channel.

Rescue of peripheral vestibular function in Usher syndrome mice using a splice-switching antisense oligonucleotide.

Usher syndrome type 1C (USH1C/harmonin) is associated with profound retinal, auditory and vestibular dysfunction. We have previously reported on an antisense oligonucleotide (ASO-29) that dramatically improves auditory function and balance behavior in mice homozygous for the harmonin mutation Ush1c c.216G > A following a single systemic administration. The findings were suggestive of improved vestibular function; however, no direct vestibular assessment was made. Here, we measured vestibular sensory evoked potentials (VsEPs) to directly assess vestibular function in Usher mice. We report that VsEPs are absent or abnormal in Usher mice, indicating profound loss of vestibular function. Strikingly, Usher mice receiving ASO-29 treatment have normal or elevated vestibular response thresholds when treated during a critical period between postnatal day 1 and 5, respectively. In contrast, treatment of mice with ASO-29 treatment at P15 was minimally effective at rescuing vestibular function. Interestingly, ASO-29 treatment at P1, P5 or P15 resulted in sufficient vestibular recovery to support normal balance behaviors, suggesting a therapeutic benefit to balance with ASO-29 treatment at P15 despite the profound vestibular functional deficits that persist with treatment at this later time. These findings provide the first direct evidence of an effective treatment of peripheral vestibular function in a mouse model of USH1C and reveal the potential for using antisense technology to treat vestibular dysfunction.

Targeting Amyloid-ß Precursor Protein, APP, Splicing with Antisense Oligonucleotides Reduces Toxic Amyloid-ß Production.

Alterations in amyloid beta precursor protein (APP) have been implicated in cognitive decline in Alzheimer's disease (AD), which is accelerated in Down syndrome/Trisomy 21 (DS/TS21), likely due to the extra copy of the APP gene, located on chromosome 21. Proteolytic cleavage of APP generates amyloid-ß (Aß) peptide, the primary component of senile plaques associated with AD. Reducing Aß production is predicted to lower plaque burden and mitigate AD symptoms. Here, we designed a splice-switching antisense oligonucleotide (SSO) that causes skipping of the APP exon that encodes proteolytic cleavage sites required for Aß peptide production. The SSO induced exon skipping in Down syndrome cell lines, resulting in a reduction of Aß. Treatment of mice with the SSO resulted in widespread distribution in the brain accompanied by APP exon skipping and a reduction of Aß. Overall, we show that an alternatively spliced isoform of APP encodes a cleavage-incompetent protein that does not produce Aß peptide and that promoting the production of this isoform with an SSO can reduce Aß in vivo. These findings demonstrate the utility of using SSOs to induce a spliced isoform of APP to reduce Aß as a potential approach for treating AD.

Induction of antagonistic soluble decoy receptor tyrosine kinases by intronic polyA activation.

Alternative intronic polyadenylation (IPA) can generate truncated protein isoforms with significantly altered functions. Here, we describe 31 dominant-negative, secreted variant isoforms of receptor tyrosine kinases (RTKs) that are produced by activation of intronic poly(A) sites. We show that blocking U1-snRNP can activate IPA, indicating a larger role for U1-snRNP in RNA surveillance. Moreover, we report the development of an antisense-based method to effectively and specifically activate expression of individual soluble decoy RTKs (sdRTKs) to alter signaling, with potential therapeutic implications. In particular, a quantitative switch from signal transducing full-length vascular endothelial growth factor receptor-2 (VEGFR2/KDR) to a dominant-negative sKDR results in a strong antiangiogenic effect both on directly targeted cells and on naive cells exposed to conditioned media, suggesting a role for this approach in interfering with angiogenic paracrine and autocrine loops.

Splice-switching antisense oligonucleotides as therapeutic drugs.

Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials.

Antisense oligonucleotides delivered to the amniotic cavity in utero modulate gene expression in the postnatal mouse.

Congenital diseases account for a large portion of pediatric illness. Prenatal screening and diagnosis permit early detection of many genetic diseases. Fetal therapeutic strategies to manage disease processes in utero represent a powerful new approach for clinical care. A safe and effective fetal pharmacotherapy designed to modulate gene expression ideally would avoid direct mechanical engagement of the fetus and present an external reservoir of drug. The amniotic cavity surrounding the fetus could serve as an ideal drug reservoir. Antisense oligonucleotides (ASOs) are an established tool for the therapeutic modulation of gene expression. We hypothesize that ASOs administered to the amniotic cavity will gain entry to the fetus and modulate gene expression. Here, we show that an ASO targeting MALAT1 RNA, delivered by transuterine microinjection into the mouse amniotic cavity at embryonic day 13-13.5, reduces target RNA expression for up to 4 weeks after birth. A similarly delivered ASO targeting a causal splice site mutation for Usher syndrome corrects gene expression in the inner ear, a therapeutically relevant target tissue. We conclude that intra-amniotic delivery of ASOs is well tolerated and produces a sustained effect on postnatal gene expression. Transuterine delivery of ASOs is an innovative platform for developing fetal therapeutics to efficaciously treat congenital disease.

Drosha promotes splicing of a pre-microRNA-like alternative exon.

The ribonuclease III enzyme Drosha has a central role in the biogenesis of microRNA (miRNA) by binding and cleaving hairpin structures in primary RNA transcripts into precursor miRNAs (pre-miRNAs). Many miRNA genes are located within protein-coding host genes and cleaved by Drosha in a manner that is coincident with splicing of introns by the spliceosome. The close proximity of splicing and pre-miRNA biogenesis suggests a potential for co-regulation of miRNA and host gene expression, though this relationship is not completely understood. Here, we describe a cleavage-independent role for Drosha in the splicing of an exon that has a predicted hairpin structure resembling a Drosha substrate. We find that Drosha can cleave the alternatively spliced exon 5 of the eIF4H gene into a pre-miRNA both in vitro and in cells. However, the primary role of Drosha in eIF4H gene expression is to promote the splicing of exon 5. Drosha binds to the exon and enhances splicing in a manner that depends on RNA structure but not on cleavage by Drosha. We conclude that Drosha can function like a splicing enhancer and promote exon inclusion. Our results reveal a new mechanism of alternative splicing regulation involving a cleavage-independent role for Drosha in splicing.

SCRIB and PUF60 are primary drivers of the multisystemic phenotypes of the 8q24.3 copy-number variant.

Copy-number variants (CNVs) represent a significant interpretative challenge, given that each CNV typically affects the dosage of multiple genes. Here we report on five individuals with coloboma, microcephaly, developmental delay, short stature, and craniofacial, cardiac, and renal defects who harbor overlapping microdeletions on 8q24.3. Fine mapping localized a commonly deleted 78 kb region that contains three genes: SCRIB, NRBP2, and PUF60. In vivo dissection of the CNV showed discrete contributions of the planar cell polarity effector SCRIB and the splicing factor PUF60 to the syndromic phenotype, and the combinatorial suppression of both genes exacerbated some, but not all, phenotypic components. Consistent with these findings, we identified an individual with microcephaly, short stature, intellectual disability, and heart defects with a de novo c.505C>T variant leading to a p.His169Tyr change in PUF60. Functional testing of this allele in vivo and in vitro showed that the mutation perturbs the relative dosage of two PUF60 isoforms and, subsequently, the splicing efficiency of downstream PUF60 targets. These data inform the functions of two genes not associated previously with human genetic disease and demonstrate how CNVs can exhibit complex genetic architecture, with the phenotype being the amalgam of both discrete dosage dysfunction of single transcripts and also of binary genetic interactions.

Biogenesis of mammalian microRNAs by a non-canonical processing pathway.

Canonical microRNA biogenesis requires the Microprocessor components, Drosha and DGCR8, to generate precursor-miRNA, and Dicer to form mature miRNA. The Microprocessor is not required for processing of some miRNAs, including mirtrons, in which spliceosome-excised introns are direct Dicer substrates. In this study, we examine the processing of putative human mirtrons and demonstrate that although some are splicing-dependent, as expected, the predicted mirtrons, miR-1225 and miR-1228, are produced in the absence of splicing. Remarkably, knockout cell lines and knockdown experiments demonstrated that biogenesis of these splicing-independent mirtron-like miRNAs, termed 'simtrons', does not require the canonical miRNA biogenesis components, DGCR8, Dicer, Exportin-5 or Argonaute 2. However, simtron biogenesis was reduced by expression of a dominant negative form of Drosha. Simtrons are bound by Drosha and processed in vitro in a Drosha-dependent manner. Both simtrons and mirtrons function in silencing of target transcripts and are found in the RISC complex as demonstrated by their interaction with Argonaute proteins. These findings reveal a non-canonical miRNA biogenesis pathway that can produce functional regulatory RNAs.

MicroRNAs are exported from malignant cells in customized particles.

MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body.

Ensemble analysis of primary microRNA structure reveals an extensive capacity to deform near the Drosha cleavage site.

Most noncoding RNAs function properly only when folded into complex three-dimensional (3D) structures, but the experimental determination of these structures remains challenging. Understanding of primary microRNA (miRNA) maturation is currently limited by a lack of determined structures for nonprocessed forms of the RNA. SHAPE chemistry efficiently determines RNA secondary structural information with single-nucleotide resolution, providing constraints suitable for input into MC-Pipeline for refinement of 3D structure models. Here we combine these approaches to analyze three structurally diverse primary microRNAs, revealing deviations from canonical double-stranded RNA structure in the stem adjacent to the Drosha cut site for all three. The necessity of these deformable sites for efficient processing is demonstrated through Drosha processing assays. The structure models generated herein support the hypothesis that deformable sequences spaced roughly once per turn of A-form helix, created by noncanonical structure elements, combine with the necessary single-stranded RNA-double-stranded RNA junction to define the correct Drosha cleavage site.