AAV-Mediated CAG-Targeting Selectively Reduces Polyglutamine-Expanded Protein and Attenuates Disease Phenotypes in a Spinocerebellar Ataxia Mouse Model.

Polyglutamine (polyQ)-encoding CAG repeat expansions represent a common disease-causing mutation responsible for several dominant spinocerebellar ataxias (SCAs). PolyQ-expanded SCA proteins are toxic for cerebellar neurons, with Purkinje cells (PCs) being the most vulnerable. RNA interference (RNAi) reagents targeting transcripts with expanded CAG reduce the level of various mutant SCA proteins in an allele-selective manner in vitro and represent promising universal tools for treating multiple CAG/polyQ SCAs. However, it remains unclear whether the therapeutic targeting of CAG expansion can be achieved in vivo and if it can ameliorate cerebellar functions. Here, using a mouse model of SCA7 expressing a mutant Atxn7 allele with 140 CAGs, we examined the efficacy of short hairpin RNAs (shRNAs) targeting CAG repeats expressed from PHP.eB adeno-associated virus vectors (AAVs), which were introduced into the brain via intravascular injection. We demonstrated that shRNAs carrying various mismatches with the CAG target sequence reduced the level of polyQ-expanded ATXN7 in the cerebellum, albeit with varying degrees of allele selectivity and safety profile. An shRNA named A4 potently reduced the level of polyQ-expanded ATXN7, with no effect on normal ATXN7 levels and no adverse side effects. Furthermore, A4 shRNA treatment improved a range of motor and behavioral parameters 23 weeks after AAV injection and attenuated the disease burden of PCs by preventing the downregulation of several PC-type-specific genes. Our results show the feasibility of the selective targeting of CAG expansion in the cerebellum using a blood-brain barrier-permeable vector to attenuate the disease phenotype in an SCA mouse model. Our study represents a significant advancement in developing CAG-targeting strategies as a potential therapy for SCA7 and possibly other CAG/polyQ SCAs.

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.

Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models.

BACKGROUND: The majority of genes in the human genome is present in two copies but the expression levels of both alleles is not equal. Allelic imbalance is an aspect of gene expression relevant not only in the context of genetic variation, but also to understand the pathophysiology of genes implicated in genetic disorders, in particular, dominant genetic diseases where patients possess one normal and one mutant allele. Polyglutamine (polyQ) diseases are caused by the expansion of CAG trinucleotide tracts within specific genes. Spinocerebellar ataxia type 3 (SCA3) and Huntington's disease (HD) patients harbor one normal and one mutant allele that differ in the length of CAG tracts. However, assessing the expression level of individual alleles is challenging due to the presence of abundant CAG repeats in the human transcriptome, which make difficult the design of allele-specific methods, as well as of therapeutic strategies to selectively engage CAG sequences in mutant transcripts. RESULTS: To precisely quantify expression in an allele-specific manner, we used SNP variants that are linked to either normal or CAG expanded alleles of the ataxin-3 (ATXN3) and huntingtin (HTT) genes in selected patient-derived cell lines. We applied a SNP-based quantitative droplet digital PCR (ddPCR) protocol for precise determination of the levels of transcripts in cellular and mouse models. For HD, we showed that the process of cell differentiation can affect the ratio between endogenous alleles of HTT mRNA. Additionally, we reported changes in the absolute number of the ATXN3 and HTT transcripts per cell during neuronal differentiation. We also implemented our assay to reliably monitor, in an allele-specific manner, the silencing efficiency of mRNA-targeting therapeutic approaches for HD. Finally, using the humanized Hu128/21 HD mouse model, we showed that the ratio of normal and mutant HTT transgene expression in brain slightly changes with the age of mice. CONCLUSIONS: Using allele-specific ddPCR assays, we observed differences in allele expression levels in the context of SCA3 and HD. Our allele-selective approach is a reliable and quantitative method to analyze low abundant transcripts and is performed with high accuracy and reproducibility. Therefore, the use of this approach can significantly improve understanding of allele-related mechanisms, e.g., related with mRNA processing that may be affected in polyQ diseases.

Atrophin-1 Function and Dysfunction in Dentatorubral-Pallidoluysian Atrophy.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a rare, incurable genetic disease that belongs to the group of polyglutamine (polyQ) diseases. DRPLA is the most common in the Japanese population; however, its global prevalence is also increasing due to better clinical recognition. It is characterized by cerebellar ataxia, myoclonus, epilepsy, dementia, and chorea. DRPLA is caused by dynamic mutation of CAG repeat expansion in ATN1 gene encoding the atrophin-1 protein. In the cascade of molecular disturbances, the pathological form of atrophin-1 is the initial factor, which has not been precisely characterized so far. Reports indicate that DRPLA is associated with disrupted protein-protein interactions (in which an expanded polyQ tract plays a crucial role), as well as gene expression deregulation. There is a great need to design efficient therapy that would address the underlying neurodegenerative process and thus prevent or alleviate DRPLA symptoms. An in-depth understanding of the normal atrophin-1 function and mutant atrophin-1 dysfunction is crucial for this purpose. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Implications of Poly(A) Tail Processing in Repeat Expansion Diseases.

Repeat expansion diseases are a group of more than 40 disorders that affect mainly the nervous and/or muscular system and include myotonic dystrophies, Huntington's disease, and fragile X syndrome. The mutation-driven expanded repeat tract occurs in specific genes and is composed of tri- to dodeca-nucleotide-long units. Mutant mRNA is a pathogenic factor or important contributor to the disease and has great potential as a therapeutic target. Although repeat expansion diseases are quite well known, there are limited studies concerning polyadenylation events for implicated transcripts that could have profound effects on transcript stability, localization, and translation efficiency. In this review, we briefly present polyadenylation and alternative polyadenylation (APA) mechanisms and discuss their role in the pathogenesis of selected diseases. We also discuss several methods for poly(A) tail measurement (both transcript-specific and transcriptome-wide analyses) and APA site identification-the further development and use of which may contribute to a better understanding of the correlation between APA events and repeat expansion diseases. Finally, we point out some future perspectives on the research into repeat expansion diseases, as well as APA studies.

Regulatory Potential of Competing Endogenous RNAs in Myotonic Dystrophies.

Non-coding RNAs (ncRNAs) have been reported to be implicated in cell fate determination and various human diseases. All ncRNA molecules are emerging as key regulators of diverse cellular processes; however, little is known about the regulatory interaction among these various classes of RNAs. It has been proposed that the large-scale regulatory network across the whole transcriptome is mediated by competing endogenous RNA (ceRNA) activity attributed to both protein-coding and ncRNAs. ceRNAs are considered to be natural sponges of miRNAs that can influence the expression and availability of multiple miRNAs and, consequently, the global mRNA and protein levels. In this review, we summarize the current understanding of the role of ncRNAs in two neuromuscular diseases, myotonic dystrophy type 1 and 2 (DM1 and DM2), and the involvement of expanded CUG and CCUG repeat-containing transcripts in miRNA-mediated RNA crosstalk. More specifically, we discuss the possibility that long repeat tracts present in mutant transcripts can be potent miRNA sponges and may affect ceRNA crosstalk in these diseases. Moreover, we highlight practical information related to innovative disease modelling and studying RNA regulatory networks in cells. Extending knowledge of gene regulation by ncRNAs, and of complex regulatory ceRNA networks in DM1 and DM2, will help to address many questions pertinent to pathogenesis and treatment of these disorders; it may also help to better understand general rules of gene expression and to discover new rules of gene control.

Genetic tests based on the RT-PCR reaction in the diagnostics of SARS-CoV-2 infection.

INTRODUCTION: Since the SARS-CoV-2 emergence in 2019/2020, at least 158 million infections with this pathogen have been recorded, of which 3.29 million infected people have died. Due to the non-specific symptoms of SARS-CoV-2 infection, laboratory tests based on RT-PCR (reverse transcription and polymerase chain reaction) are mainly used in the diagnosis of COVID-19 disease. AIM: The aim of this study is to compare the molecular tests available on the Polish market for the diagnosis of SARS-CoV2 infection. RESULTS: Based on the data provided by the manufacturers and the performed laboratory analyses, we have shown that the available diagnostic kits differ mainly in the sensitivity and duration of the reaction. CONCLUSION: Due to the ongoing COVID-19 pandemic, the indicated parameters are key to effective control of the spread of SARS-CoV2, and therefore should be mainly taken into account when choosing and purchasing by diagnostic centres.

What, When and How to Measure-Peripheral Biomarkers in Therapy of Huntington's Disease.

Among the main challenges in further advancing therapeutic strategies for Huntington's disease (HD) is the development of biomarkers which must be applied to assess the efficiency of the treatment. HD is a dreadful neurodegenerative disorder which has its source of pathogenesis in the central nervous system (CNS) but is reflected by symptoms in the periphery. Visible symptoms include motor deficits and slight changes in peripheral tissues, which can be used as hallmarks for prognosis of the course of HD, e.g., the onset of the disease symptoms. Knowing how the pathology develops in the context of whole organisms is crucial for the development of therapy which would be the most beneficial for patients, as well as for proposing appropriate biomarkers to monitor disease progression and/or efficiency of treatment. We focus here on molecular peripheral biomarkers which could be used as a measurable outcome of potential therapy. We present and discuss a list of wet biomarkers which have been proposed in recent years to measure pre- and postsymptomatic HD. Interestingly, investigation of peripheral biomarkers in HD can unravel new aspects of the disease pathogenesis. This especially refers to inflammatory proteins or specific immune cells which attract scientific attention in neurodegenerative disorders.

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.

Strategies for mutant gene expression silencing in Huntington's disease therapy / Strategie wyciszania ekspresji zmutowanego genu w terapii choroby Huntingtona.

Huntington's disease (HD) is a genetic disease caused by expanded CAG repeat tract in exon 1 of the HTT gene that codes for huntingtin. Since the first symptoms of the disease the average life expectancy is 15-20 years, when the symptoms resulting from neurodegeneration are progressing. Therefore, there is a great demand for an effective HD treatment method. Various therapeutic strategies are being developed based on mechanisms of gene expression silencing, including DNA editing techniques. Here, we present the most important currently tested approaches, with particular emphasis on strategies based on the use of antisense oligonucleotides (ASO), RNA interference (RNAi) technology and CRISPR-Cas9. Currently ongoing clinical trials as well as different pharmacological agents are discussed.

RAN Translation of the Expanded CAG Repeats in the SCA3 Disease Context.

Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the ATXN3 gene encoding the ataxin-3 protein. Despite extensive research the exact pathogenic mechanisms of SCA3 are still not understood in depth. In the present study, to gain insight into the toxicity induced by the expanded CAG repeats in SCA3, we comprehensively investigated repeat-associated non-ATG (RAN) translation in various cellular models expressing translated or non-canonically translated ATXN3 sequences with an increasing number of CAG repeats. We demonstrate that two SCA3 RAN proteins, polyglutamine (polyQ) and polyalanine (polyA), are found only in the case of CAG repeats of pathogenic length. Despite having distinct cellular localization, RAN polyQ and RAN polyA proteins are very often coexpressed in the same cell, impairing nuclear integrity and inducing apoptosis. We provide for the first time mechanistic insights into SCA3 RAN translation indicating that ATXN3 sequences surrounding the repeat region have an impact on SCA3 RAN translation initiation and efficiency. We revealed that RAN translation of polyQ proteins starts at non-cognate codons upstream of the CAG repeats, whereas RAN polyA proteins are likely translated within repeats. Furthermore, integrated stress response activation enhances SCA3 RAN translation. Our findings suggest that the ATXN3 sequence context plays an important role in triggering SCA3 RAN translation and that SCA3 RAN proteins may cause cellular toxicity.

Generation of human iPS cell line IBCHi002-A from spinocerebellar ataxia type 3/Machado-Joseph disease patient's fibroblasts.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is autosomal-dominant neurodegenerative disease caused by an expansion of polyglutamine-encoding CAG repeats in the ATXN3 gene. Here we established IBCHi002-A induced pluripotent stem cells (iPSCs) line generated from SCA3 patient fibroblasts by using non-integrative Sendai-virus delivery system of four reprogramming factors. This cellular model provides a valid platform for study SCA3 pathogenesis and potential therapies for this so far incurable disease.

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.

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.

A potential role of extended simple sequence repeats in competing endogenous RNA crosstalk.

MicroRNA (miRNA)-mediated crosstalk between coding and non-coding RNAs of various types is known as the competing endogenous RNA (ceRNA) concept. Here, we propose that there is a specific variant of the ceRNA language that takes advantage of simple sequence repeat (SSR) wording. We applied bioinformatics tools to identify human transcripts that may be regarded as repeat-associated ceRNAs (raceRNAs). Multiple protein-coding transcripts, transcribed pseudogenes, long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) showing this potential were identified, and numerous miRNAs were predicted to bind to SSRs. We propose that simple repeats expanded in various hereditary neurological diseases may act as sponges for miRNAs containing complementary repeats that would affect raceRNA crosstalk. Based on the representation of specific SSRs in transcripts, expression data for SSR-binding miRNAs and expression profiling data from patients, we determined that raceRNA crosstalk is most likely to be perturbed in the case of myotonic dystrophy type 1 (DM1) and type 2 (DM2).

Reduction of Huntington's Disease RNA Foci by CAG Repeat-Targeting Reagents.

In several human polyglutamine diseases caused by expansions of CAG repeats in the coding sequence of single genes, mutant transcripts are detained in nuclear RNA foci. In polyglutamine disorders, unlike other repeat-associated diseases, both RNA and proteins exert pathogenic effects; therefore, decreases of both RNA and protein toxicity need to be addressed in proposed treatments. A variety of oligonucleotide-based therapeutic approaches have been developed for polyglutamine diseases, but concomitant assays for RNA foci reduction are lacking. Here, we show that various types of oligonucleotide-based reagents affect RNA foci number in Huntington's disease cells. We analyzed the effects of reagents targeting either CAG repeat tracts or specific HTT sequences in fibroblasts derived from patients. We tested reagents that either acted as translation blockers or triggered mRNA degradation via the RNA interference pathway or RNase H activation. We also analyzed the effect of chemical modifications of CAG repeat-targeting siRNAs on their efficiency in the foci decline. Our results suggest that the decrease of RNA foci number may be considered as a readout of treatment outcomes for oligonucleotide reagents.

Mutant CAG Repeats Effectively Targeted by RNA Interference in SCA7 Cells.

Spinocerebellar ataxia type 7 (SCA7) is a human neurodegenerative polyglutamine (polyQ) disease caused by a CAG repeat expansion in the open reading frame of the ATXN7 gene. The allele-selective silencing of mutant transcripts using a repeat-targeting strategy has previously been used for several polyQ diseases. Herein, we demonstrate that the selective targeting of a repeat tract in a mutant ATXN7 transcript by RNA interference is a feasible approach and results in an efficient decrease of mutant ataxin-7 protein in patient-derived cells. Oligonucleotides (ONs) containing specific base substitutions cause the downregulation of the ATXN7 mutant allele together with the upregulation of its normal allele. The A2 ON shows high allele selectivity at a broad range of concentrations and also restores UCHL1 expression, which is downregulated in SCA7.

Silencing of genes responsible for polyQ diseases using chemically modified single-stranded siRNAs.

Polyglutamine (polyQ) diseases comprise a group of nine genetic disorders that are caused by the expansion of the CAG triplet repeat, which encodes glutamine, in unrelated single genes. Various oligonucleotide (ON)-based therapeutic approaches have been considered for polyQ diseases. The very attractive CAG repeat-targeting strategy offers selective silencing of the mutant allele by directly targeting the mutation site. CAG repeat-targeting miRNA-like siRNAs have been shown to specifically inhibit the mutant gene expression, and their characteristic feature is the formation of mismatches in their interactions with the target site. Here, we designed novel single-stranded siRNAs that contain base substitutions and chemical modifications, in order to develop improved therapeutic tools with universal properties for several polyQ diseases. We tested these ONs in cellular models of Huntington's disease (HD), spinocerebellar ataxia type 3 (SCA3) and dentatorubral-pallidoluysian atrophy (DRPLA). Selected siRNAs caused the efficient and selective downregulation of the mutant huntingtin, ataxin-3 and atrophin-1 levels in cultured human fibroblasts. We also prove the efficiency of novel ONs, with chemical modification pattern mainly containing 2'-fluoro (2'F), in HD mouse striatal cells.

Oligonucleotide-based strategies to combat polyglutamine diseases.

Considerable advances have been recently made in understanding the molecular aspects of pathogenesis and in developing therapeutic approaches for polyglutamine (polyQ) diseases. Studies on pathogenic mechanisms have extended our knowledge of mutant protein toxicity, confirmed the toxicity of mutant transcript and identified other toxic RNA and protein entities. One very promising therapeutic strategy is targeting the causative gene expression with oligonucleotide (ON) based tools. This straightforward approach aimed at halting the early steps in the cascade of pathogenic events has been widely tested for Huntington's disease and spinocerebellar ataxia type 3. In this review, we gather information on the use of antisense oligonucleotides and RNA interference triggers for the experimental treatment of polyQ diseases in cellular and animal models. We present studies testing non-allele-selective and allele-selective gene silencing strategies. The latter include targeting SNP variants associated with mutations or targeting the pathologically expanded CAG repeat directly. We compare gene silencing effectors of various types in a number of aspects, including their design, efficiency in cell culture experiments and pre-clinical testing. We discuss advantages, current limitations and perspectives of various ON-based strategies used to treat polyQ diseases.