RNA regulation in brain function and disease 2022 (NeuroRNA): A conference report.

Recent research integrates novel technologies and methods from the interface of RNA biology and neuroscience. This advancing integration of both fields creates new opportunities in neuroscience to deepen the understanding of gene expression programs and their regulation that underlies the cellular heterogeneity and physiology of the central nervous system. Currently, transcriptional heterogeneity can be studied in individual neural cell types in health and disease. Furthermore, there is an increasing interest in RNA technologies and their application in neurology. These aspects were discussed at an online conference that was shortly named NeuroRNA.

A CAG repeat-targeting artificial miRNA lowers the mutant huntingtin level in the YAC128 model of Huntington's disease.

Among the many proposed therapeutic strategies for Huntington's disease (HD), allele-selective therapies are the most desirable but also the most challenging. RNA interference (RNAi) tools that target CAG repeats selectively reduce the mutant huntingtin level in cellular models of HD. The purpose of this study was to test the efficacy, selectivity, and safety of two vector-based RNAi triggers in an animal model of HD. CAG repeat-targeting short hairpin RNA (shRNA) and artificial miRNA (amiRNA) were delivered to the brains of YAC128 mice via intrastriatal injection of AAV5 vectors. Molecular tests demonstrated that both the shRNA and amiRNA reduced the mutant huntingtin level by 50% without influencing endogenous mouse huntingtin. In addition, a concentration-dependent reduction in HTT aggregates in the striatum was observed. In contrast to the shRNA, the amiRNA was well tolerated and did not show signs of toxicity during the course of the experiment up to 20 weeks post injection. Interestingly, amiRNA treatment reduced the spleen weight to values characteristic of healthy (WT) mice and improved motor performance on the static rod test. These preclinical data demonstrate that the CAG-targeting strategy and amiRNA could make an original and valuable contribution to currently used therapeutic approaches for HD.

Advances in Modeling Polyglutamine Diseases Using Genome Editing Tools.

Polyglutamine (polyQ) diseases, including Huntington's disease, are a group of late-onset progressive neurological disorders caused by CAG repeat expansions. Although recently, many studies have investigated the pathological features and development of polyQ diseases, many questions remain unanswered. The advancement of new gene-editing technologies, especially the CRISPR-Cas9 technique, has undeniable value for the generation of relevant polyQ models, which substantially support the research process. Here, we review how these tools have been used to correct disease-causing mutations or create isogenic cell lines with different numbers of CAG repeats. We characterize various cellular models such as HEK 293 cells, patient-derived fibroblasts, human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs) and animal models generated with the use of genome-editing technology.

Paving the way towards precise and safe CRISPR genome editing.

As the possibilities of CRISPR-Cas9 technology have been revealed, we have entered a new era of research aimed at increasing its specificity and safety. This stage of technology development is necessary not only for its wider application in the clinic but also in basic research to better control the process of genome editing. Research during the past eight years has identified some factors influencing editing outcomes and led to the development of highly specific endonucleases, modified guide RNAs and computational tools supporting experiments. More recently, large-scale experiments revealed a previously overlooked feature: Cas9 can generate reproducible mutation patterns. As a result, it has become apparent that Cas9-induced double-strand break (DSB) repair is nonrandom and can be predicted to some extent. Here, we review the present state of knowledge regarding the specificity and safety of CRISPR-Cas9 technology to define gRNA, protein and target-related problems and solutions. These issues include sequence-specific off-target effects, immune responses, genetic variation and chromatin accessibility. We present new insights into the role of DNA repair in genome editing and define factors influencing editing outcomes. In addition, we propose practical guidelines for increasing the specificity of editing and discuss novel perspectives in improvement of this technology.

Artificial miRNAs as therapeutic tools: Challenges and opportunities.

RNA interference (RNAi) technology has been used for almost two decades to study gene functions and in therapeutic approaches. It uses cellular machinery and small, designed RNAs in the form of synthetic small interfering RNAs (siRNAs) or vector-based short hairpin RNAs (shRNAs), and artificial miRNAs (amiRNAs) to inhibit a gene of interest. Artificial miRNAs, known also as miRNA mimics, shRNA-miRs, or pri-miRNA-like shRNAs have the most complex structures and undergo two-step processing in cells to form mature siRNAs, which are RNAi effectors. AmiRNAs are composed of a target-specific siRNA insert and scaffold based on a natural primary miRNA (pri-miRNA). siRNAs serve as a guide to search for complementary sequences in transcripts, whereas pri-miRNA scaffolds ensure proper processing and transport. The dynamics of siRNA maturation and siRNA levels in the cell resemble those of endogenous miRNAs; therefore amiRNAs are safer than other RNAi triggers. Delivered as viral vectors and expressed under tissue-specific polymerase II (Pol II) promoters, amiRNAs provide long-lasting silencing and expression in selected tissues. Therefore, amiRNAs are useful therapeutic tools for a broad spectrum of human diseases, including neurodegenerative diseases, cancers and viral infections. Recent reports on the role of sequence and structure in pri-miRNA processing may contribute to the improvement of the amiRNA tools. In addition, the success of a recently initiated clinical trial for Huntington's disease could pave the way for other amiRNA-based therapies, if proven effective and safe. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action RNA in Disease and Development > RNA in Disease.

Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and translation inhibition of mutant transcripts.

Polyglutamine (polyQ) diseases are incurable neurological disorders caused by CAG repeat expansion in the open reading frames (ORFs) of specific genes. This type of mutation in the HTT gene is responsible for Huntington's disease (HD). CAG repeat-targeting artificial miRNAs (art-miRNAs) were shown as attractive therapeutic approach for polyQ disorders as they caused allele-selective decrease in the level of mutant proteins. Here, using polyQ disease models, we aimed to demonstrate how miRNA-based gene expression regulation is dependent on target sequence features. We show that the silencing efficiency and selectivity of art-miRNAs is influenced by the localization of the CAG repeat tract within transcript and the specific sequence context. Furthermore, we aimed to reveal the events leading to downregulation of mutant polyQ proteins and found very rapid activation of translational repression and HTT transcript deadenylation. Slicer-activity of AGO2 was dispensable in this process, as determined in AGO2 knockout cells generated with CRISPR-Cas9 technology. We also showed highly allele-selective downregulation of huntingtin in human HD neural progenitors (NPs). Taken together, art-miRNA activity may serve as a model of the cooperative activity and targeting of ORF regions by endogenous miRNAs.

Computational Tools and Resources Supporting CRISPR-Cas Experiments.

The CRISPR-Cas system has become a cutting-edge technology that revolutionized genome engineering. The use of Cas9 nuclease is currently the method of choice in most tasks requiring a specific DNA modification. The rapid development in the field of CRISPR-Cas is reflected by the constantly expanding ecosystem of computational tools aimed at facilitating experimental design and result analysis. The first group of CRISPR-Cas-related tools that we review is dedicated to aid in guide RNA design by prediction of their efficiency and specificity. The second, relatively new group of tools exploits the observed biases in repair outcomes to predict the results of CRISPR-Cas edits. The third class of tools is developed to assist in the evaluation of the editing outcomes by analysis of the sequencing data. These utilities are accompanied by relevant repositories and databases. Here we present a comprehensive and updated overview of the currently available CRISPR-Cas-related tools, from the perspective of a user who needs a convenient and reliable means to facilitate genome editing experiments at every step, from the guide RNA design to analysis of editing outcomes. Moreover, we discuss the current limitations and challenges that the field must overcome for further improvement in the CRISPR-Cas endeavor.

Generation of New Isogenic Models of Huntington's Disease Using CRISPR-Cas9 Technology.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the expansion of CAG repeats in exon 1 of the huntingtin gene (HTT). Despite its monogenic nature, HD pathogenesis is still not fully understood, and no effective therapy is available to patients. The development of new techniques such as genome engineering has generated new opportunities in the field of disease modeling and enabled the generation of isogenic models with the same genetic background. These models are very valuable for studying the pathogenesis of a disease and for drug screening. Here, we report the generation of a series of homozygous HEK 293T cell lines with different numbers of CAG repeats at the HTT locus and demonstrate their usefulness for testing therapeutic reagents. In addition, using the CRISPR-Cas9 system, we corrected the mutation in HD human induced pluripotent stem cells and generated a knock-out of the HTT gene, thus providing a comprehensive set of isogenic cell lines for HD investigation.

Universal RNAi Triggers for the Specific Inhibition of Mutant Huntingtin, Atrophin-1, Ataxin-3, and Ataxin-7 Expression.

The expansion of CAG repeats within the coding region of associated genes is responsible for nine inherited neurodegenerative disorders including Huntington's disease (HD), spinocerebellar ataxias (SCAs), and dentatorubral-pallidoluysian atrophy (DRPLA). Despite years of research aimed at developing an effective method of treatment, these diseases remain incurable and only their symptoms are controlled. The purpose of this study was to develop effective and allele-selective genetic tools for silencing the expression of mutated genes containing expanded CAG repeats. Here we show that repeat-targeting short hairpin RNAs preferentially reduce the levels of mutant huntingtin, atrophin-1, ataxin-3, and ataxin-7 proteins in patient-derived fibroblasts and may serve as universal allele-selective reagents for polyglutamine (polyQ) diseases.

Gene Therapy for Huntington's Disease Using Targeted Endonucleases.

Huntington's disease (HD) is a hereditary neurological disorder caused by expansion of the CAG repeat tract in the huntingtin gene (HTT). The mutant protein with a long polyglutamine tract is toxic to cells, especially neurons, leading to their progressive degeneration. Similar to many other monogenic diseases, HD is a good target for gene therapy approaches, including the use of programmable endonucleases. Here, we describe a protocol for HTT gene knock out using a modified Cas9 protein (nickase, Cas9n) and a pair of sgRNAs flanking the repeats. Recently, we showed that excision of the CAG repeat tract resulted in a frameshift mutation and premature translation termination. As a model, we used HD patient-derived fibroblasts electroporated with a pair of plasmid vectors expressing CRISPR-Cas9n tools. Efficient HTT inactivation independent of the CAG tract length was confirmed by Western blotting. A modified version of this protocol involving precise excision of the CAG repeats and insertion of a new DNA sequence by homology directed repair may also be used for the generation of new isogenic cellular models of HD.

Generation of human iPS cell line IBCHi001-A from dentatorubral-pallidoluysian atrophy patient's fibroblasts.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an incurable autosomal dominant disease caused by an expansion of a CAG repeats in ATN1 gene encoding atrophin 1 protein. Here we report the generation of IBCHi001-A, an induced pluripotent stem cell (iPSC) line derived from DRPLA patient fibroblasts using non-integrative reprogramming technology with OCT4, SOX2, cMYC and KLF4 reprogramming factors. The pluripotency of iPSC was confirmed by immunocytochemistry and PCR for pluripotency markers and by the ability to form three germ layers in vitro. The established iPSC line offers a useful resource to study the pathogenesis of DPRLA.

qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites.

Genome editing technology based on engineered nucleases has been increasingly applied for targeted modification of genes in a variety of cell types and organisms. However, the methods currently used for evaluating the editing efficiency still suffer from many limitations, including preferential detection of some mutation types, sensitivity to polymorphisms that hamper mismatch detection, lack of multiplex capability, or sensitivity to assay conditions. Here, we describe qEva-CRISPR, a new quantitative method that overcomes these limitations and allows simultaneous (multiplex) analysis of CRISPR/Cas9-induced modifications in a target and the corresponding off-targets or in several different targets. We demonstrate all of the advantages of the qEva-CRISPR method using a number of sgRNAs targeting the TP53, VEGFA, CCR5, EMX1 and HTT genes in different cell lines and under different experimental conditions. Unlike other methods, qEva-CRISPR detects all types of mutations, including point mutations and large deletions, and its sensitivity does not depend on the mutation type. Moreover, this approach allows for successful analysis of targets located in 'difficult' genomic regions. In conclusion, qEva-CRISPR may become a method of choice for unbiased sgRNA screening to evaluate experimental conditions that affect genome editing or to distinguish homology-directed repair from non-homologous end joining.

Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases.

Huntington's disease (HD) is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of CAG repeats in the first exon of the huntingtin gene (HTT). The accumulation of polyglutamine-rich huntingtin proteins affects various cellular functions and causes selective degeneration of neurons in the striatum. Therapeutic strategies used to date to silence the expression of mutant HTT include antisense oligonucleotides, RNA interference-based approaches and, recently, genome editing with the CRISPR/Cas9 system. Here, we demonstrate that the CAG repeat tract can be precisely excised from the HTT gene with the use of the paired Cas9 nickase strategy. As a model, we used HD patient-derived fibroblasts with varied numbers of CAG repeats. The repeat excision inactivated the HTT gene and abrogated huntingtin synthesis in a CAG repeat length-independent manner. Because Cas9 nickases are known to be safe and specific, our approach provides an attractive treatment tool for HD that can be extended to other polyQ disorders.

Stress-induced changes in miRNA biogenesis and functioning.

MicroRNAs (miRNAs) are small, noncoding RNAs that play key roles in the regulation of cellular homeostasis in eukaryotic organisms. There is emerging evidence that some of these processes are influenced by various forms of cellular stresses, including DNA damage, pathogen invasion or chronic stress associated with diseases. Many reports over the last decade demonstrate examples of stress-induced miRNA deregulation at the level of transcription, processing, subcellular localization and functioning. Moreover, core miRNA biogenesis proteins and their interactions with partners can be selectively regulated in response to stress signaling. However, little is known about the role of isomiRs and the interactions of miRNA with non-canonical targets in the context of the stress response. In this review, we summarize the current knowledge on miRNA functions under various stresses, including chronic stress and miRNA deregulation in the pathogenesis of age-associated neurodegenerative disorders.

Novel microemulsion-based gels for topical delivery of indomethacin: Formulation, physicochemical properties and in vitro drug release studies.

HYPOTHESIS: Microemulsion-based semisolid systems may be considered as an interesting alternative to the traditional dosage forms applied in topical drug delivery. Mechanical properties of topical products are important both in terms of application and dosage form effectiveness. In this study we designed and evaluated novel microemulsion-based gels with indomethacin and analyzed the factors affecting their mechanical characteristics and drug release. EXPERIMENTS: The impact of the microemulsion composition on the extent of isotropic region was investigated with the use of pseudoternary phase diagrams. Selected microemulsions were analyzed in terms of electrical conductivity and surface tension in order to determine the microemulsion type. Microemulsions were transformed into polymer-based gels and subjected to rheological and textural studies. Finally, the indomethacin release from the analyzed gels was studied and compared to commercially available product. FINDINGS: The extent of isotropic domain in pseudoternary phase diagrams seems to be dependent on the polarity of the oil phase. The surface tension and conductivity monitored as a function of water content in microemulsion systems revealed possible structural transformations from w/o through bicontinuous systems into o/w. The mechanical properties of semisolid microemulsion-based systems depended on the composition of surface active agents and the drug presence. The drug release profiles observed in the case of the investigated gels differed from those recorded for the commercially available product which was most probably caused by the different structure of both systems.

siRNA release from pri-miRNA scaffolds is controlled by the sequence and structure of RNA.

shmiRs are pri-miRNA-based RNA interference triggers from which exogenous siRNAs are expressed in cells to silence target genes. These reagents are very promising tools in RNAi in vivo applications due to their good activity profile and lower toxicity than observed for other vector-based reagents such as shRNAs. In this study, using high-resolution northern blotting and small RNA sequencing, we investigated the precision with which RNases Drosha and Dicer process shmiRs. The fidelity of siRNA release from the commonly used pri-miRNA shuttles was found to depend on both the siRNA insert and the pri-miR scaffold. Then, we searched for specific factors that may affect the precision of siRNA release and found that both the structural features of shmiR hairpins and the nucleotide sequence at Drosha and Dicer processing sites contribute to cleavage site selection and cleavage precision. An analysis of multiple shRNA intermediates generated from several reagents revealed the complexity of shmiR processing by Drosha and demonstrated that Dicer selects substrates for further processing. Aside from providing new basic knowledge regarding the specificity of nucleases involved in miRNA biogenesis, our results facilitate the rational design of more efficient genetic reagents for RNAi technology.

Sequence-non-specific effects generated by various types of RNA interference triggers.

RNA interference triggers such as short interfering RNA (siRNA) or genetically encoded short hairpin RNA (shRNA) and artificial miRNA (sh-miR) are widely used to silence the expression of specific genes. In addition to silencing selected targets, RNAi reagents may induce various side effects, including immune responses. To determine the molecular markers of immune response activation when using RNAi reagents, we analyzed the results of experiments gathered in the RNAimmuno (v 2.0) and GEO Profiles databases. To better characterize and compare cellular responses to various RNAi reagents in one experimental system, we designed a reagent series in corresponding siRNA, D-siRNA, shRNA and sh-miR forms. To exclude sequence-specific effects the reagents targeted 3 different transcripts (Luc, ATXN3 and HTT). We demonstrate that RNAi reagents induce a broad variety of sequence-non-specific effects, including the deregulation of cellular miRNA levels. Typical siRNAs are weak stimulators of interferon response but may saturate the miRNA biogenesis pathway, leading to the downregulation of highly expressed miRNAs, whereas plasmid-based reagents induce known markers of immune response and may alter miRNA levels and their isomiR composition.

The role of the immune system in triplet repeat expansion diseases.

Trinucleotide repeat expansion disorders (TREDs) are a group of dominantly inherited neurological diseases caused by the expansion of unstable repeats in specific regions of the associated genes. Expansion of CAG repeat tracts in translated regions of the respective genes results in polyglutamine- (polyQ-) rich proteins that form intracellular aggregates that affect numerous cellular activities. Recent evidence suggests the involvement of an RNA toxicity component in polyQ expansion disorders, thus increasing the complexity of the pathogenic processes. Neurodegeneration, accompanied by reactive gliosis and astrocytosis is the common feature of most TREDs, which may suggest involvement of inflammation in pathogenesis. Indeed, a number of immune response markers have been observed in the blood and CNS of patients and mouse models, and the activation of these markers was even observed in the premanifest stage of the disease. Although inflammation is not an initiating factor of TREDs, growing evidence indicates that inflammatory responses involving astrocytes, microglia, and the peripheral immune system may contribute to disease progression. Herein, we review the involvement of the immune system in the pathogenesis of triplet repeat expansion diseases, with particular emphasis on polyglutamine disorders. We also present various therapeutic approaches targeting the dysregulated inflammation pathways in these diseases.

RNA imaging in living cells - methods and applications.

Numerous types of transcripts perform multiple functions in cells, and these functions are mainly facilitated by the interactions of the RNA with various proteins and other RNAs. Insight into the dynamics of RNA biosynthesis, processing and cellular activities is highly desirable because this knowledge will deepen our understanding of cell physiology and help explain the mechanisms of RNA-mediated pathologies. In this review, we discuss the live RNA imaging systems that have been developed to date. We highlight information on the design of these systems, briefly discuss their advantages and limitations and provide examples of their numerous applications in various organisms and cell types. We present a detailed examination of one application of RNA imaging systems: this application aims to explain the role of mutant transcripts in human disease pathogenesis caused by triplet repeat expansions. Thus, this review introduces live RNA imaging systems and provides a glimpse into their various applications.

RAN translation and frameshifting as translational challenges at simple repeats of human neurodegenerative disorders.

Repeat-associated disorders caused by expansions of short sequences have been classified as coding and noncoding and are thought to be caused by protein gain-of-function and RNA gain-of-function mechanisms, respectively. The boundary between such classifications has recently been blurred by the discovery of repeat-associated non-AUG (RAN) translation reported in spinocerebellar ataxia type 8, myotonic dystrophy type 1, fragile X tremor/ataxia syndrome and C9ORF72 amyotrophic lateral sclerosis and frontotemporal dementia. This noncanonical translation requires no AUG start codon and can initiate in multiple frames of CAG, CGG and GGGGCC repeats of the sense and antisense strands of disease-relevant transcripts. RNA structures formed by the repeats have been suggested as possible triggers; however, the precise mechanism of the translation initiation remains elusive. Templates containing expansions of microsatellites have also been shown to challenge translation elongation, as frameshifting has been recognized across CAG repeats in spinocerebellar ataxia type 3 and Huntington's disease. Determining the critical requirements for RAN translation and frameshifting is essential to decipher the mechanisms that govern these processes. The contribution of unusual translation products to pathogenesis needs to be better understood. In this review, we present current knowledge regarding RAN translation and frameshifting and discuss the proposed mechanisms of translational challenges imposed by simple repeat expansions.