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In this article, the authors explore prospective elementary teachers' engagement with and reflection on activities they conducted to learn about a single child from their practicum classroom. Through these activities, prospective teachers learned about their child's mathematical thinking and the interests, competencies, and resources she or he brought to the mathematics classroom, and then wrote reports that included instructional suggestions as to next steps to further the child's growth in mathematics. The authors' analyses of these reports indicate that there were a variety of ways which prospective teachers made connections to one or more of their child's knowledge bases. In a high percentage of cases, prospective teachers attended to one of these knowledge bases, indicating that they were attending to particularities about their child and developing the dispositions to continue to do so. Implications for research and practice are discussed.

The recent Nobel Prizes awarded to Ambros and Ruvkun have refocused attention on microRNAs (miRNAs). The importance of miRNAs for basic science has always been clear, but the application to therapy has lagged behind. This delay has been made even more apparent by the accelerating pace of successful programs using duplex RNAs and antisense oligonucleotides to target mRNA. Why has progress been slow? A clear understanding of how miRNAs function in mammalian cells is obscured by the fact that miRNAs can exert their effects through multiple complex mechanisms. This gap in our knowledge has complicated progress in drug discovery. Better insights into the mechanism of miRNAs, more rigorous definitions of miRNAs, and more powerful tools for establishing the physical contacts necessary for miRNA action are now available. These advances lead to a central question for nucleic acid therapy-can miRNAs be productive targets for drug discovery and development?

Argonaute protein is associated with post-transcriptional control of cytoplasmic gene expression through miRNA-induced silencing complexes (miRISC). Specific cellular and environmental conditions can trigger AGO protein to accumulate in the nucleus. Localization of AGO is central to understanding miRNA action, yet the consequences of AGO being in the nucleus are undefined. We show nuclear enrichment of AGO2 in HCT116 cells grown in two-dimensional culture to high density, HCT116 cells grown in three-dimensional tumor spheroid culture, and human colon tumors. The shift in localization of AGO2 from cytoplasm to nucleus de-represses cytoplasmic AGO2-eCLIP targets that were candidates for canonical regulation by miRISC. Constitutive nuclear localization of AGO2 using an engineered nuclear localization signal increases cell migration. Critical RNAi factors also affect the localization of AGO2. Knocking out an enzyme essential for miRNA biogenesis, DROSHA, depletes mature miRNAs and restricts AGO2 localization to the cytoplasm, while knocking out the miRISC scaffolding protein, TNRC6, results in nuclear localization of AGO2. These data suggest that AGO2 localization and miRNA activity can be regulated depending on environmental conditions, expression of mature miRNAs, and expression of miRISC cofactors. Localization and expression of core miRISC protein machinery should be considered when investigating the roles of miRNAs., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Synthetic antisense oligonucleotides (ASOs) and duplex RNAs (dsRNAs) are an increasingly successful strategy for drug development. After a slow start, the pace of success has accelerated since the approval of Spinraza (nusinersen) in 2016 with several drug approvals. These accomplishments have been achieved even though oligonucleotides are large, negatively charged, and have little resemblance to traditional small-molecule drugs-a remarkable achievement of basic and applied science. The goal of this review is to summarize the foundation underlying recent progress and describe ongoing research programs that may increase the scope and impact of oligonucleotide therapeutics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

One advantage of antisense oligonucleotides (ASOs) for drug development is their long-lasting gene knockdown after administration in vivo . In this study, we examine the effect on gene expression after intraocular injection in target tissues in the eye. We examined expression levels of the Malat1 gene after intracameral or intravitreal (IV) injection of an anti- Malat1 ASO in corneal epithelium/stroma, corneal endothelium, lens capsule epithelium, neurosensory retina, and retinal pigment epithelium/choroid of the mouse eye. We assessed potency of the compound at 7 days as well as duration of the gene knockdown at 14, 28, 60, 90, and 120 days. The ASO was more potent when delivered by IV injection relative to intracameral injection, regardless of whether the tissues analyzed were at the front or back of the eye. For corneal endothelium, inhibition was >50% after 120 days for ASO at 50 μg. At IV dosages of 6 μg, we observed >75% inhibition of gene expression in the retina and lens epithelium for up to 120 days. ASOs have potential as long-lasting gene knockdown agents in the mouse eye, but efficacy varies depending on the specific ocular target tissue and injection protocol.

Fuchs' corneal endothelial dystrophy (FECD) is a major cause of vision loss. Corneal transplantation is the only effective curative treatment, but this surgery has limitations. A pharmacological intervention would complement surgery and be beneficial for many patients. FECD is caused by an expanded CUG repeat within intron 2 of the TCF4 RNA. Agents that recognize the expanded repeat can reverse the splicing defects associated with the disease. Successful drug development will require diverse strategies for optimizing the efficacy of anti-CUG oligomers. In this study, we evaluate anti-CUG morpholinos conjugated to cyclic cell penetrating peptides. The morpholino domain of the conjugate is complementary to the repeat, while the peptide has been optimized for import across cell membranes. We show that morpholino conjugates can enter corneal endothelial cells and block the CUG RNA foci associated with the disease. These experiments support morpholino peptide conjugates as an approach for developing anti-CUG therapies for FECD., Competing Interests: The authors declare the following competing financial interest(s): X.S., M.K., M.D.S., and Z.Q. are employees of Entrada Therapeutics., (© 2023 The Authors. Published by American Chemical Society.)

The potential for microRNAs (miRNAs) to regulate gene expression remains incompletely understood. DROSHA initiates the biogenesis of miRNAs while variants of Argonaute (AGO) and trinucleotide repeat containing six (TNRC6) family proteins form complexes with miRNAs to facilitate RNA recognition and gene regulation. Here we investigate the fate of miRNAs in the absence of these critical RNAi protein factors. Knockout of DROSHA expression reduces levels of some miRNAs annotated in miRBase but not others. The identity of miRNAs with reduced expression matches the identity of miRNAs previously identified by experimental approaches. The MirGeneDB resource offers the closest alignment with experimental results. In contrast, the loss of TNRC6 proteins had much smaller effects on miRNA levels. Knocking out AGO proteins, which directly contact the mature miRNA, decreased expression of the miRNAs most strongly associated with AGO2 as determined from enhanced crosslinking immunoprecipitation (AGO2-eCLIP). Evaluation of miRNA binding to endogenously expressed AGO proteins revealed that miRNA:AGO association was similar for AGO1, AGO2, AGO3, and AGO4. Our data emphasize the need to evaluate annotated miRNAs based on approximate cellular abundance, DROSHA-dependence, and physical association with AGO when forming hypotheses related to their function., (© 2023 Johnson et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Purpose: In the United States, 70% of Fuchs' endothelial corneal dystrophy (FECD) cases are caused by an intronic trinucleotide repeat expansion in the TCF4 gene. CUG repeat RNA transcripts from this expansion accumulate as nuclear foci in the corneal endothelium. In this study, we sought to detect foci in other anterior segment cell types and assess their molecular impact., Methods: We examined CUG repeat RNA foci appearance, expression of downstream affected genes, gene splicing, and TCF4 RNA expression in corneal endothelium, corneal stromal keratocytes, corneal epithelium, trabecular meshwork cells, and lens epithelium., Results: CUG repeat RNA foci, the hallmark of FECD in corneal endothelium (found in 84% of endothelial cells), are less detectable in trabecular meshwork cells (41%), much less prevalent in stromal keratocytes (11%) or corneal epithelium (4%), and absent in lens epithelium. With few exceptions including mis-splicing in the trabecular meshwork, differential gene expression and splicing changes associated with the expanded repeat in corneal endothelial cells are not observed in other cell types. Expression of the TCF4 transcripts including full-length isoforms containing the repeat sequence at the 5' end is much higher in the corneal endothelium or trabecular meshwork than in the corneal stroma or corneal epithelium., Conclusions: Expression of the CUG repeat containing TCF4 transcripts is higher in the corneal endothelium, likely contributing to foci formation and the large molecular and pathologic impact on those cells. Further studies are warranted to examine any glaucoma risk and impact of the observed foci in the trabecular meshwork of these patients.

RNA interference is almost always associated with post-transcriptional silencing in the cytoplasm. MicroRNAs (miRNAs) and critical RNAi protein factors like argonaute (AGO) and trinucleotide repeat binding containing 6 protein (TNRC6), however, are also found in cell nuclei, suggesting that nuclear miRNAs may be targets for gene regulation. Designed small duplex RNAs (dsRNAs) can modulate nuclear processes such as transcription and splicing, suggesting that they can also provide leads for therapeutic discovery. The goal of this Perspective is to provide the background on nuclear RNAi necessary to guide discussions on whether nuclear RNAi can play a role in therapeutic development programs., (© 2023 Johnson and Corey; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

After decades overcoming difficult problems, antisense oligonucleotide (ASO), duplex RNA (siRNA), and messenger RNA (mRNA) nucleic acid therapeutic strategies are finally demonstrating clinical benefits. This success presents new challenges. What goals remain for basic research? Will there be an explosion of clinical applications that benefit many patients with different diseases, or will success be restricted to diseases that are ideal for the application of current technologies? The aim of this perspective is to describe a selection of the major goals for the next decade.

MicroRNAs (miRNAs) are short endogenously expressed RNAs that have the potential to regulate the expression of any RNA. This potential has led to the publication of several thousand papers each year connecting miRNAs to many different genes and human diseases. By contrast, relatively few papers appear that investigate the molecular mechanism used by miRNAs. There is a disconnect between rigorous understanding of mechanism and the extraordinary diversity of reported roles for miRNAs. Consequences of this disconnect include confusion about the assumptions underlying the basic science of human miRNAs and slow development of therapeutics that target miRNAs. Here, we present an overview of investigations into miRNAs and their impact on gene expression. Progress in our understanding of miRNAs would be aided by a greater focus on the mechanism of miRNAs and a higher burden of evidence on researchers who seek to link expression of a particular miRNA to a biological phenotype., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Friedreich's ataxia (FA) is an inherited neurodegenerative disorder caused by decreased expression of frataxin (FXN) protein. Previous studies have shown that antisense oligonucleotides (ASOs) and single-stranded silencing RNAs can be used to increase expression of frataxin in cultured patient-derived cells. In this study, we investigate the potential for oligonucleotides to increase frataxin expression in a mouse model for FA. After confirming successful in vivo delivery of oligonucleotides using a benchmark gapmer targeting the nuclear noncoding RNA Malat1, we tested anti- FXN oligonucleotides designed to function by various mechanisms. None of these strategies yielded enhanced expression of FXN in the model mice. Our inability to translate activation of FXN expression from cell culture to mice may be due to inadequate potency of our compounds or differences in the molecular mechanisms governing FXN gene repression and activation in FA model mice.

Friedreich's ataxia (FRDA) is a severe multisystem disease caused by transcriptional repression induced by expanded GAA repeats located in intron 1 of the Frataxin (FXN) gene encoding frataxin. FRDA results from decreased levels of frataxin; thus, stabilization of the FXN mRNA already present in patient cells represents an attractive and unexplored therapeutic avenue. In this work, we pursued a novel approach based on oligonucleotide-mediated targeting of FXN mRNA ends to extend its half-life and availability as a template for translation. We demonstrated that oligonucleotides designed to bind to FXN 5' or 3' noncoding regions can increase FXN mRNA and protein levels. Simultaneous delivery of oligonucleotides targeting both ends increases efficacy of the treatment. The approach was confirmed in several FRDA fibroblast and induced pluripotent stem cell-derived neuronal progenitor lines. RNA sequencing and single-cell expression analyses confirmed oligonucleotide-mediated FXN mRNA upregulation. Mechanistically, a significant elongation of the FXN mRNA half-life without any changes in chromatin status at the FXN gene was observed upon treatment with end-targeting oligonucleotides, indicating that transcript stabilization is responsible for frataxin upregulation. These results identify a novel approach toward upregulation of steady-state mRNA levels via oligonucleotide-mediated end targeting that may be of significance to any condition resulting from transcription downregulation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

TNRC6 is a scaffolding protein that bridges interactions between small RNAs, argonaute (AGO) protein, and effector proteins to control gene expression. There are three paralogs in mammalian cells, TNRC6A , TNRC6B , and TNRC6C These paralogs have ∼40% amino acid sequence identity and the extent of their unique or redundant functions is unclear. Here, we use knockout cell lines, enhanced crosslinking immunoprecipitation (eCLIP), and high-throughput RNA sequencing (RNA-seq) to explore the roles of TNRC6 paralogs in RNA-mediated control of gene expression. We find that the paralogs are largely functionally redundant and changes in levels of gene expression are well-correlated with those observed in AGO knockout cell lines. Splicing changes observed in AGO knockout cell lines are also observed in TNRC6 knockout cells. These data further define the roles of the TNRC6 isoforms as part of the RNA interference (RNAi) machinery., (© 2021 Johnson et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Mammalian RNA interference (RNAi) is often linked to the regulation of gene expression in the cytoplasm. Synthetic RNAs, however, can also act through the RNAi pathway to regulate transcription and splicing. While nuclear regulation by synthetic RNAs can be robust, a critical unanswered question is whether endogenous functions for nuclear RNAi exist in mammalian cells. Using enhanced crosslinking immunoprecipitation (eCLIP) in combination with RNA sequencing (RNA-seq) and multiple AGO knockout cell lines, we mapped AGO2 protein binding sites within nuclear RNA. The strongest AGO2 binding sites were mapped to micro RNAs (miRNAs). The most abundant miRNAs were distributed similarly between the cytoplasm and nucleus, providing no evidence for mechanisms that facilitate localization of miRNAs in one compartment versus the other. Beyond miRNAs, most statistically significant AGO2 binding was within introns. Splicing changes were confirmed by RT-PCR and recapitulated by synthetic miRNA mimics complementary to the sites of AGO2 binding. These data support the hypothesis that miRNAs can control gene splicing. While nuclear RNAi proteins have the potential to be natural regulatory mechanisms, careful study will be necessary to identify critical RNA drivers of normal physiology and disease., (© 2021 Chu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Purpose: Fuchs' endothelial corneal dystrophy (FECD) is the leading indication for corneal transplantation. Seventy percent of cases are caused by an intronic CTG triplet repeat expansion in the TCF4 gene that results in accumulation of pathogenic expanded CUG repeat RNA (CUG exp ) as nuclear foci in corneal endothelium. A catalytically dead Cas9 (dCas9) can serve as an effective guide to target genomic DNA or RNA transcripts. Here, we examined the utility of the clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 system to effectively target and reduce CUG exp ., Methods: We delivered dCas9 and repeat-targeting single guide RNA (sgRNA) expression plasmids to patient-derived endothelial cells using lipofection or lentiviral transduction. We used fluorescence in situ hybridization (FISH) and RNA dot-blot hybridization to quantify CUG exp foci and repeat RNA levels, respectively. TCF4 expression levels were assessed using quantitative PCR (qPCR)., Results: Using FISH, we found that expression of both dCas9 and a (CAG) n sgRNA complementary to CUG exp are necessary to reduce foci. We observed a reduction in percentage of cells with foci from 59% to 5.6% and number of foci per 100 cells from 73.4 to 7.45 ( P < 0.001) in cells stably expressing dCas9-(CAG) n sgRNA but saw no decrease in cells expressing dCas9-(CUG) n sgRNA or nontargeting control sgRNA. In cells with dCas9-(CAG) n sgRNA, we detected a reduction in CUG exp RNA by dot-blot without any reduction in TCF4 mRNA levels using qPCR., Conclusions: Using CRISPR-dCas9 to target the trinucleotide repeat is a promising treatment for FECD contingent on effective in vivo delivery., Translational Relevance: This work advances a gene therapy for a common age-related degenerative disorder., Competing Interests: Disclosure: Z. Rong, None; X. Gong, None; J.D. Hulleman, None; D.R. Corey, None; V.V. Mootha (P), (Copyright 2020 The Authors.)

Antisense oligonucleotides (ASOs) are synthetic nucleic acids that recognize complementary RNA sequences inside cells and modulate gene expression. In this study, we explore the feasibility of ASO delivery to the cornea. We used quantitative polymerase chain reaction to test the efficacy of a benchmark ASO targeting a noncoding nuclear RNA, Metastasis-Associated Lung Adenocarcinoma Transcript 1 ( MALAT1 ), in a human corneal endothelial cell line, ex vivo human corneas, and in vivo in mice. In vivo delivery was via intravitreal or intracameral injections as well as topical administration. The anti- MALAT1 ASO significantly reduced expression of MALAT1 in a corneal endothelial cell line. We achieved a dose-dependent reduction of target gene expression in endothelial tissue from ex vivo human donor corneas. In vivo mouse experiments confirmed MALAT1 reduction in whole corneal tissue via intravitreal and intracameral routes, 82% and 71% knockdown, respectively ( P  < 0.001). Effects persisted up to at least 21 days, 32% ( P  < 0.05) and 43% ( P  < 0.05) knockdown, respectively. We developed protocols for the isolation and analysis of mouse corneal endothelium and observed reduction in MALAT1 expression upon both intravitreal and intracameral administrations, 64% ( P  < 0.05) and 63% ( P  < 0.05) knockdown, respectively. These data open the possibility of using ASOs to treat corneal disease.

Despite two decades of study, the full scope of RNAi in mammalian cells has remained obscure. Here we combine: (i) Knockout of argonaute (AGO) variants; (ii) RNA sequencing analysis of gene expression changes and (iii) Enhanced Crosslinking Immunoprecipitation Sequencing (eCLIP-seq) using anti-AGO2 antibody to identify potential microRNA (miRNA) binding sites. We find that knocking out AGO1, AGO2 and AGO3 together are necessary to achieve full impact on steady state levels of mRNA. eCLIP-seq located AGO2 protein associations within 3'-untranslated regions. The standard mechanism of miRNA action would suggest that these associations should repress gene expression. Contrary to this expectation, associations between AGO and RNA are poorly correlated with gene repression in wild-type versus knockout cells. Many clusters are associated with increased steady state levels of mRNA in wild-type versus knock out cells, including the strongest cluster within the MYC 3'-UTR. Our results suggest that assumptions about miRNA action should be re-examined., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

How genetic defects trigger the molecular changes that cause late-onset disease is important for understanding disease progression and therapeutic development. Fuchs' endothelial corneal dystrophy (FECD) is an RNA-mediated disease caused by a trinucleotide CTG expansion in an intron within the TCF4 gene. The mutant intronic CUG RNA is present at one-two copies per cell, posing a challenge to understand how a rare RNA can cause disease. Late-onset FECD is a uniquely advantageous model for studying how RNA triggers disease because: (i) Affected tissue is routinely removed during surgery; (ii) The expanded CTG mutation is one of the most prevalent disease-causing mutations, making it possible to obtain pre-symptomatic tissue from eye bank donors to probe how gene expression changes precede disease; and (iii) The affected tissue is a homogeneous single cell monolayer, facilitating accurate transcriptome analysis. Here, we use RNA sequencing (RNAseq) to compare tissue from individuals who are pre-symptomatic (Pre&#95;S) to tissue from patients with late stage FECD (FECD&#95;REP). The abundance of mutant repeat intronic RNA in Pre&#95;S and FECD&#95;REP tissue is elevated due to increased half-life in a corneal cells. In Pre&#95;S tissue, changes in splicing and extracellular matrix gene expression foreshadow the changes observed in advanced disease and predict the activation of the fibrosis pathway and immune system seen in late-stage patients. The absolute magnitude of splicing changes is similar in pre-symptomatic and late stage tissue. Our data identify gene candidates for early drivers of disease and biomarkers that may represent diagnostic and therapeutic targets for FECD. We conclude that changes in alternative splicing and gene expression are observable decades prior to the diagnosis of late-onset trinucleotide repeat disease., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Friedreich's Ataxia (FRDA) is an incurable genetic disease caused by an expanded trinucleotide AAG repeat within intronic RNA of the frataxin (FXN) gene. We have previously demonstrated that synthetic antisense oligonucleotides or duplex RNAs that are complementary to the expanded repeat can activate expression of FXN and return levels of FXN protein to near normal. The potency of these compounds, however, was too low to encourage vigorous pre-clinical development. We now report testing of "gapmer" oligonucleotides consisting of a central DNA portion flanked by chemically modified RNA that increases binding affinity. We find that gapmer antisense oligonucleotides are several fold more potent activators of FXN expression relative to previously tested compounds. The potency of FXN activation is similar to a potent benchmark gapmer targeting the nuclear noncoding RNA MALAT-1, suggesting that our approach has potential for developing more effective compounds to regulate FXN expression in vivo., Competing Interests: Declaration of Competing Interest DRC has filed a patent application related to early work on this topic., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Trinucleotide repeats are responsible for many genetic diseases. Previous studies have shown that duplex RNAs (dsRNAs) can be used to target expression of a mutant repeat allele while leaving expression of the wild-type allele untouched, creating opportunities for allele-selective inhibition and better therapeutic outcomes. In contrast to successes with other genes, we report here that we cannot achieve allele-selective inhibition when targeting the expanded CAG repeat within Ataxin-1 (ATXN1), the cause of spinal cerebellar ataxia-1 (SCA1). The most likely explanation for this unfavorable outcome is that the mean CAG repeat number within wild-type ATXN1 is relatively high compared to other trinucleotide repeat diseases. Because the wild-type repeat number is high, it is likely that there is poor discrimination between the mutant and wild-type repeat and less opportunity for allele-selective inhibition across the entire spectrum of mutations found in SCA1 patients. Our data support the conclusion that the potential for multiple cooperative binding interactions is a critical factor governing allele-selective recognition of trinucleotide repeat genes by duplex RNAs. These results should be helpful in predicting which diseases and which patients are most likely to benefit from allele-selective targeting of expanded repeats.

The trinucleotide repeat containing 6 (TNRC6) family of proteins are core components of RNA interference (RNAi) and consist of three paralogs (TNRC6A, TNRC6B, and TNRC6C). The TNRC6 paralogs associate with argonaute (AGO) protein, the core RNAi factor, and bridge its interactions with other proteins. We obtained TNRC6A and TNRC6B single and double knockout cell lines to investigate how the TNRC6 paralogs contribute to RNAi. We found that TNRC6 proteins are not required for gene silencing when duplex RNAs are fully complementary. TNRC6 expression was necessary for regulation by a microRNA. TNRC6A, but not TNRC6B, expression was necessary for transcriptional activation by a duplex RNA targeting a gene promoter. By contrast, AGO2 is required for all three gene expression pathways. TNRC6A can affect the Dicer localization in cytoplasm versus the nucleus, but none of the three TNRC6 paralogs was necessary for nuclear localization of AGO2. Our data suggest that the roles of the TNRC6 paralogs differ in some details and that TNRC6 is not required for clinical therapeutic silencing mechanisms that involve fully complementary duplex RNAs.

Purpose: Fuchs' endothelial corneal dystrophy (FECD) is a major cause of vision loss and the most common nucleotide repeat disorder, affecting 4% of United States population greater than 40 years of age. Seventy percent of FECD cases are due to an intronic CTG expansion within the TCF4 gene, resulting in accumulation of CUG repeat RNA nuclear foci in corneal endothelium. Each endothelial cell has approximately two sense foci, and each focus is a single RNA molecule. This study aimed to obtain a better understanding of how rare repeat RNA species lead to disease., Methods: We quantitatively examined muscleblind-like (MBNL) proteins and their interaction with foci in both patient-derived corneal endothelial cell lines and human corneal endothelial tissue., Results: Using fluorescent in situ hybridization and immunofluorescence, we found that depletion of both MBNL1 and MBNL2 reduces nuclear RNA foci formed by the repeat, suggesting that both are necessary for foci. Quantitative studies of RNA and protein copy number revealed MBNLs to be abundant in the total cellular pool in endothelial cell lines but are much lower in human corneal endothelial tissue. Studies using human tissue nuclear and cytoplasmic fractions indicate that most MBNL proteins are localized to the cytoplasm., Conclusions: The low levels of MBNL1/2 in corneal tissue, in combination with the small fraction of protein in the nucleus, may make corneal endothelial cells especially susceptible to sequestration of MBNL1/2 by CUG repeat RNA. These observations may explain how a limited number of RNA molecules can cause widespread alteration of splicing and late-onset degenerative FECD.

Oligonucleotide drugs are experiencing greater success in the clinic, encouraging the initiation of new projects. Resources are insufficient to develop every potentially important project, and persuasive experimental data using cell lines close to disease target tissue is needed to prioritize candidates. Friedreich's ataxia (FRDA) is a devastating and currently incurable disease caused by insufficient expression of the enzyme frataxin (FXN). We have previously shown that synthetic nucleic acids can activate FXN expression in human patient-derived fibroblast cells. We chose to further test these compounds in induced pluripotent stem cell-derived neuronal progenitor cells (iPSC-NPCs). Here we describe methods to deliver oligonucleotides and duplex RNAs into iPSC-NPCs using electroporation. Activation of FXN expression is potent, easily reproducible, and potencies parallel those determined using patient-derived fibroblast cells. A duplex RNA and several antisense oligonucleotides (ASOs) with different combinations of 2'-methoxyethyl (2'-MOE), 2'-fluoro (2'-F), and constrained ethyl (cEt) were active, providing multiple starting points for further development and highlighting improved potency as an important goal for preclinical development. Our data support the conclusion that ASO-mediated activation of FXN is a feasible approach for treating FRDA and that electroporation is a robust method for introducing ASOs to modulate gene expressions in neuronal cells., (© 2019 Shen et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

After decades of research and development, synthetic nucleic acids are beginning to enjoy significant success in the clinic. Approved drugs have increased interest in the field, and many basic research studies have focused on synthetic nucleic acids to control the action of messenger RNA and noncoding RNAs. Unfortunately, experimental designs are often inadequate, resulting in misleading interpretation of data and unconvincing work that wastes resources and does little to advance the field. The goal of this commentary is to outline the problems facing many researchers, especially those new to the use of synthetic oligonucleotides. We describe the minimum control experiments necessary to build a strong case for real effects that are likely due to interactions at the intended molecular target. A common set of standards for preparing and judging experiments should facilitate better interpretation of data and publications that contribute positively to using synthetic nucleic acids as tools and drugs.

Fuchs' endothelial corneal dystrophy (FECD) leads to vision loss and is one of the most common inherited eye diseases. Corneal transplants are the only curative treatment available, and there is a major unmet need for treatments that are less invasive and independent of donor tissue. Most cases of FECD are associated with an expanded CUG repeat within the intronic region of TCF4 and the mutant RNA has been implicated as the cause of the disease. We previously presented preliminary data suggesting that single-stranded antisense oligonucleotides (ASOs) can inhibit CUG RNA foci in patient-derived cells and tissue. We now show that duplex RNAs and single-stranded silencing RNAs (ss-siRNAs) reduce the number of cells with foci and the number of foci per cells. Potencies are similar to those that are achieved with chemically modified ASOs designed to block foci. These data widen the potential for synthetic nucleic acids to be used to treat a widely prevalent and debilitating disease.

Friedreich's ataxia (FRDA) is an incurable neurodegenerative disorder caused by reduced expression of the mitochondrial protein frataxin (FXN). The genetic cause of the disease is an expanded GAA repeat within the FXN gene. Agents that increase expression of FXN protein are a potential approach to therapy. We previously described anti-trinucleotide GAA duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) that activate FXN protein expression in multiple patient derived cell lines. Here we test two distinct series of compounds for their ability to increase FXN expression. ASOs with butane linkers showed low potency, which is consistent with the low T m values and suggesting that flexible conformation impairs activity. By contrast, single-stranded siRNAs (ss-siRNAs) that combine the strengths of dsRNA and ASO approaches had nanomolar potencies. ss-siRNAs provide an additional option for developing nucleic acid therapeutics to treat FRDA., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

GW182 and argonaute 2 (AGO2) are core proteins of the RNA interference complex. GW182 is a scaffolding protein that physically associates with AGO2 and bridges its interactions with other proteins. A fundamental problem in biology is how scaffolding proteins adapt or contribute to differing functional demands within cells. Here we test the necessity for human GW182 proteins (paralogs TNRC6A, TNRC6B, and TNRC6C) for several mechanisms of small duplex RNA-mediated control of gene expression, including translational silencing by miRNAs, translational silencing by siRNAs, transcriptional silencing, transcriptional activation, and splicing. We find that GW182 is required for transcriptional activation and for the activity of miRNAs but is dispensable for the regulation of splicing, transcriptional silencing, and the action of siRNAs. AGO2, by contrast, is necessary for each of these processes. Our data suggest that GW182 does not alter AGO2 to make it active. Instead, GW182 organizes protein complexes around AGO2. Sometimes this higher level of organization is necessary, and sometimes it is not. AGO2 and GW182 offer an example for how a partnership between a scaffolding protein and a functional protein can be powerful but not obligatory.

Objective: To conduct a more robust examination of perchlorate exposure on iodide uptake inhibition (IUI) using pooled data from four clinical studies of perchlorate exposure., Methods: To establish a response threshold for IUI, data were analyzed using segmented linear regression and benchmark dose (BMD) analysis., Results: Segmented linear regression applied to data for 69 subjects representing nine doses identified a breakpoint corresponding to a change in the slope of the dose-response relationship of 3.0 mg/d perchlorate. The estimated BMD for a 20% decrease in iodine uptake was 2.3 mg/d, with a lower 95% confidence interval limit of 1.6 mg/d., Conclusions: A threshold dose for IUI from perchlorate exposure of 1.6 to 3.0 mg/d (0.021 to 0.038 mg/kg d) was estimated using two modeling approaches. These estimates are slightly higher than the lowest observed effect level of 0.02 mg/kg d from the Greer Study.

Fuchs' endothelial corneal dystrophy (FECD) is the most common repeat expansion disorder. FECD impacts 4% of U.S. population and is the leading indication for corneal transplantation. Most cases are caused by an expanded intronic CUG tract in the TCF4 gene that forms nuclear foci, sequesters splicing factors and impairs splicing. We investigated the sense and antisense RNA landscape at the FECD gene and find that the sense-expanded repeat transcript is the predominant species in patient corneas. In patient tissue, sense foci number were negatively correlated with age and showed no correlation with sex. Each endothelial cell has ∼2 sense foci and each foci is single RNA molecule. We designed antisense oligonucleotides (ASOs) to target the mutant-repetitive RNA and demonstrated potent inhibition of foci in patient-derived cells. Ex vivo treatment of FECD human corneas effectively inhibits foci and reverses pathological changes in splicing. FECD has the potential to be a model for treating many trinucleotide repeat diseases and targeting the TCF4 expansion with ASOs represents a promising therapeutic strategy to prevent and treat FECD.

RNA plays a central role in the expression of all genes. Because any sequence within RNA can be recognized by complementary base pairing, synthetic oligonucleotides and oligonucleotide mimics offer a general strategy for controlling processes that affect disease. The two primary antisense approaches for regulating expression through recognition of cellular RNAs are single-stranded antisense oligonucleotides and duplex RNAs. This review will discuss the chemical modifications and molecular mechanisms that make synthetic nucleic acid drugs possible. Lessons learned from recent clinical trials will be summarized. Ongoing clinical trials are likely to decisively test the adequacy of our current generation of antisense nucleic acid technologies and highlight areas where more basic research is needed.

Friedreich's Ataxia (FA) is an inherited neurologic disorder caused by an expanded GAA repeat within intron 1 of the frataxin (FXN) gene that reduces expression of FXN protein. Agents that increase expression of FXN have the potential to alleviate the disease. We previously reported that duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) complementary to the GAA repeat could enhance expression of FXN protein. We now explore the potential of a diverse group of chemically modified dsRNAs and ASOs to define the breadth of repeat-targeted synthetic nucleic acids as a platform for therapeutic development for FA. ASOs and dsRNAs can activate FXN protein expression in FA patient-derived cell lines that possess varied numbers of GAA repeats. Increased FXN protein expression was achieved by ASOs incorporating diverse chemical modifications with low nanomolar potencies, suggesting substantial flexibility in choosing compounds for further chemical optimization and animal studies. Our data encourage further development of ASOs as agents to treat FA.

In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations within the chromosome 9 open reading frame 72 (c9orf72) gene are associated with both familial amyotrophic lateral sclerosis and frontotemporal dementia. The mutation leads to an expanded GGGGCC hexanucleotide repeat within the first intron of c9orf72 and an expanded CCCCGG repeat within a corresponding antisense transcript. Both the mutant intronic and antisense RNAs have been implicated in disease. We have previously reported that duplex RNAs complementary to the repeats can recognize disease-causing RNA and block detection of nuclear foci formed by the mutant transcripts. Here, we test the hypothesis that inhibition can also be achieved by single-stranded silencing RNAs (ss-siRNAs). ss-siRNAs are single-stranded antisense oligonucleotides (ASOs) that function through RNAi interference (RNAi) to silence gene expression. ss-siRNAs can block the expanded repeats within both intronic RNA and the antisense transcripts. Inhibition is more potent than by analogous duplex RNAs. Our data suggest that the potent effects on foci are caused by a combination of mechanisms including RNAi and direct binding of the ss-siRNA to the target transcripts. These findings reinforce the suggestion that ss-siRNAs combine the favorable properties of duplex RNA and single-stranded ASOs.

Most of the human genome encodes RNAs that do not code for proteins. These non-coding RNAs (ncRNAs) may affect normal gene expression and disease progression, making them a new class of targets for drug discovery. Because their mechanisms of action are often novel, developing drugs to target ncRNAs will involve equally novel challenges. However, many potential problems may already have been solved during the development of technologies to target mRNA. Here, we discuss the growing field of ncRNA - including microRNA, intronic RNA, repetitive RNA and long non-coding RNA - and assess the potential and challenges in their therapeutic exploitation.

The chromosome 9 open reading frame 72 (c9orf72) gene contains a hexanucleotide (GGGGCC) repeat expansion responsible for many cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The mutant intronic RNA forms "foci" within nuclei, but the connection between transcript expression, foci, and biochemical disease mechanisms is unclear. Knowing the absolute numbers of cellular RNAs, in any system, is important for understanding the molecular mechanisms of natural physiology, disease, and drug action. Absolute numbers, however, are rarely determined, and this absence is a major impediment to understanding complex systems. Using quantitative methods, we demonstrate that foci are single RNA molecules. Most cells have no foci while 1%-2% have more than ten. Knowing the number of disease-causing molecules may contribute to understanding ALS and FTD pathology and successful drug discovery. More broadly, our data suggest that small numbers of RNA molecules may have a sizable impact on disease., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The ability to activate gene expression would provide new opportunities for drug development for diseases caused by inadequate or nonexistent expression of a therapeutic protein. Duplex RNAs that target gene promoters are one strategy for enhancing the expression of target genes. This chapter summarizes our understanding of mechanism behind gene activation by small RNAs that bind noncoding transcripts at gene promoters. We describe a path for choosing candidate genes for therapeutic development.

Importance: The ability to control gene expression with antisense oligonucleotides (ASOs) could provide a new treatment strategy for disease., Objective: To review the use of ASOs for the treatment of neurological disorders., Evidence Review: Articles were identified through a search of PubMed references from 2000 to 2016 for articles describing the use of ASOs to treat disease, with specific attention to neurological disease. We concentrated our review on articles pertaining to activation of frataxin expression (Friedreich's ataxia) and production of active survival motor neuron 2 (SMN2, spinal muscular atrophy)., Findings: Many neurological diseases are caused by inappropriate expression of a protein. Mutations may reduce expression of a wild-type protein, and strategies to activate expression may provide therapeutic benefit. For other diseases, a mutant protein may be expressed too highly and methods that reduce mutant protein expression might form the basis for drug development. Synthetic ASOs can recognize cellular RNA and control gene expression. Antisense oligonucleotides are not a new concept, but successful clinical development has proceeded at a slow pace. Advances in ASO chemistry, biological understanding, and clinical design are making successful applications more likely., Conclusions and Relevance: Both laboratory and clinical studies are demonstrating the potential of ASOs as a source of drugs to treat neurological disease.

Recent studies suggest that the microprocessor (Drosha-DGCR8) complex can be recruited to chromatin to catalyze co-transcriptional processing of primary microRNAs (pri-miRNAs) in mammalian cells. However, the molecular mechanism of co-transcriptional miRNA processing is poorly understood. Here we find that HP1BP3, a histone H1-like chromatin protein, specifically associates with the microprocessor and promotes global miRNA biogenesis in human cells. Chromatin immunoprecipitation (ChIP) studies reveal genome-wide co-localization of HP1BP3 and Drosha and HP1BP3-dependent Drosha binding to actively transcribed miRNA loci. Moreover, HP1BP3 specifically binds endogenous pri-miRNAs and facilitates the Drosha/pri-miRNA association in vivo. Knockdown of HP1BP3 compromises pri-miRNA processing by causing premature release of pri-miRNAs from the chromatin. Taken together, these studies suggest that HP1BP3 promotes co-transcriptional miRNA processing via chromatin retention of nascent pri-miRNA transcripts. This work significantly expands the functional repertoire of the H1 family of proteins and suggests the existence of chromatin retention factors for widespread co-transcriptional miRNA processing., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with Flag-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners., (© 2016 Kalantari et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

MicroRNAs (miRNAs) are small noncoding transcripts that regulate gene expression. Aberrant expression of miRNAs can affect development of cancer and other diseases. Synthetic miRNA mimics can modulate gene expression and offer an approach to therapy. Inside cells, mature miRNAs are produced as double-stranded RNAs and miRNA mimics typically retain both strands. This need for two strands has the potential to complicate drug development. Recently, synthetic chemically modified single-stranded silencing RNAs (ss-siRNA) have been shown to function through the RNAi pathway to induce gene silencing in cell culture and animals. Here, we test the hypothesis that single-stranded miRNA (ss-miRNA) can also mimic the function of miRNAs. We show that ss-miRNAs can act as miRNA mimics to silence the expression of target genes. Gene silencing requires expression of argonaute 2 (AGO2) protein and involves recruitment of AGO2 to the target transcripts. Chemically modified ss-miRNAs function effectively inside cells through endogenous RNAi pathways and broaden the options for miRNA-based oligonucleotide drug development.

Expansion of a GGGGCC/CCCCGG repeat sequence in the first intron of the C9ORF72 gene is a leading cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In this combined disorder, called c9FTD/ALS, the expansion is bidirectionally transcribed into sense and antisense repeat RNA associated with disease. To better understand the role of C9ORF72 repeat RNA in molecular disease pathology, we determined crystal structures of a [(CCCCGG)3(CCCC)] model antisense repeat RNA to 1.47 Å resolution. The RNA structure was an A-form-like double helix composed of repeating and regularly spaced tandem C:C mismatch pairs that perturbed helical geometry and surface charge. Solution studies revealed a preference for A-form-like helical conformations as the repeat number increased. Results provide a structural starting point for rationalizing the contribution of repeat RNA to c9FTD/ALS molecular disease mechanisms and for developing molecules to target C9ORF72 repeat RNA as potential therapeutics.

Friedreich's ataxia is an incurable genetic disorder caused by a mutant expansion of the trinucleotide GAA within an intronic FXN RNA. This expansion leads to reduced expression of frataxin (FXN) protein and evidence suggests that transcriptional repression is caused by an R-loop that forms between the expanded repeat RNA and complementary genomic DNA. Synthetic agents that increase levels of FXN protein might alleviate the disease. We demonstrate that introducing anti-GAA duplex RNAs or single-stranded locked nucleic acids into patient-derived cells increases FXN protein expression to levels similar to analogous wild-type cells. Our data are significant because synthetic nucleic acids that target GAA repeats can be lead compounds for restoring curative FXN levels. More broadly, our results demonstrate that interfering with R-loop formation can trigger gene activation and reveal a new strategy for upregulating gene expression.