Search

Spinal muscular atrophy is a neuromuscular disorder caused by mutations in both copies of the survival motor neuron gene 1 (SMN1), which lead to reduction in the production of the SMN protein. Currently, there are several therapies that have been approved for SMA, with many more undergoing active research. While various biomarkers have been proposed for assessing the effectiveness of SMA treatment, a universally accepted one still has not been identified. This study aimed to describe a fast and reliable method using the number of gems in cell nuclei as a potential tool for assessment of splicing correction of oligonucleotide efficacy in SMA cells. To gain insight into whether the number of gems in cell nuclei varies based on their SMN genotype and whether the increase in gem number is associated with therapeutic response, we utilized fibroblast cell cultures obtained from a patient with SMA type II and from a healthy individual. We discovered a remarkable difference in the number of gems found in the nuclei of these cells, specifically when counting gems per 100 nuclei. The SMA fibroblasts treated with antisense oligonucleotide showed beneficial effects in correcting the abnormal splicing of SMN2 exon 7. It was observed that there was a significant increase in the number of gems in the treated cells compared to the intact SMA cells. The results obtained significantly correlate with an increase of full-length SMN transcript sharing. Based on our findings, we propose using the quantity of gems as a reliable biomarker for SMA drug development. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a devastating neurodegenerative disease caused by mutations in the SMN1 gene. Existing therapies demonstrate positive results on SMA patients but still might be ameliorated in efficacy and price. In the presented study we designed antisense oligonucleotides (AONs), targeting intronic splicing silencer sites, some were modified with 2′-O-methyl, others with LNA. The AONs have been extensively tested in different concentrations, both individually and combined, in order to effectively target the ISS-N1 and A+100G splicing silencer regions in intron 7 of the SMN2 gene. By treating SMA-cultured fibroblasts with certain AONs, we discovered a remarkable increase in the levels of full-length SMN transcripts and the number of nuclear gems. This increase was observed to be dose-dependent and reached levels comparable to those found in healthy cells. When added to cells together, most of the tested molecules showed a remarkable synergistic effect in correcting splicing. Through our research, we have discovered that the impact of oligonucleotides is greatly influenced by their length, sequence, and pattern of modification. [ABSTRACT FROM AUTHOR]

Splicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression. The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. Targeting aberrant RNA(s) may thus provide an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. To that purpose, the use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s—ExSpeU1s) has been applied as a potentially therapeutic strategy to correct splicing mutations, particularly those affecting the 5′ splice-site (5′ss). Here we review and summarize a vast panoply of studies that used either modified U1 snRNAs or ExSpeU1s to mediate gene therapeutic correction of splicing defects underlying a considerable number of genetic diseases. We also focus on the pre-clinical validation of these therapeutic approaches both in vitro and in vivo, and summarize the main obstacles that need to be overcome to allow for their successful translation to clinic practice in the future. [ABSTRACT FROM AUTHOR]

Stargardt disease type 1 (STGD1) is the most common hereditary form of maculopathy and remains untreatable. STGD1 is caused by biallelic variants in the ABCA4 gene, which encodes the ATP-binding cassette (type 4) protein (ABCA4) that clears toxic byproducts of the visual cycle. The c.5461-10T>C p.[Thr1821Aspfs∗6,Thr1821Valfs∗13] variant is the most common severe disease-associated variant, and leads to exon skipping and out-of-frame ABCA4 transcripts that prevent translation of functional ABCA4 protein. Homozygous individuals typically display early onset STGD1 and are legally blind by early adulthood. Here, we applied antisense oligonucleotides (AONs) to promote exon inclusion and restore wild-type RNA splicing of ABCA4 c.5461-10T>C. The effect of AONs was first investigated in vitro using an ABCA4 midigene model. Subsequently, the best performing AONs were administered to homozygous c.5461-10T>C 3D human retinal organoids. Isoform-specific digital polymerase chain reaction revealed a significant increase in correctly spliced transcripts after treatment with the lead AON, QR-1011, up to 53% correct transcripts at a 3 μM dose. Furthermore, western blot and immunohistochemistry analyses identified restoration of ABCA4 protein after treatment. Collectively, we identified QR-1011 as a potent splice-correcting AON and a possible therapeutic intervention for patients harboring the severe ABCA4 c.5461-10T>C variant.

Spinal muscular atrophy is a neuromuscular disorder caused by mutations in both copies of the survival motor neuron gene 1 (SMN1), which lead to reduction in the production of the SMN protein. Currently, there are several therapies that have been approved for SMA, with many more undergoing active research. While various biomarkers have been proposed for assessing the effectiveness of SMA treatment, a universally accepted one still has not been identified. This study aimed to describe a fast and reliable method using the number of gems in cell nuclei as a potential tool for assessment of splicing correction of oligonucleotide efficacy in SMA cells. To gain insight into whether the number of gems in cell nuclei varies based on their SMN genotype and whether the increase in gem number is associated with therapeutic response, we utilized fibroblast cell cultures obtained from a patient with SMA type II and from a healthy individual. We discovered a remarkable difference in the number of gems found in the nuclei of these cells, specifically when counting gems per 100 nuclei. The SMA fibroblasts treated with antisense oligonucleotide showed beneficial effects in correcting the abnormal splicing of SMN2 exon 7. It was observed that there was a significant increase in the number of gems in the treated cells compared to the intact SMA cells. The results obtained significantly correlate with an increase of full-length SMN transcript sharing. Based on our findings, we propose using the quantity of gems as a reliable biomarker for SMA drug development.

Spinal muscular atrophy (SMA) is a devastating neurodegenerative disease caused by mutations in the SMN1 gene. Existing therapies demonstrate positive results on SMA patients but still might be ameliorated in efficacy and price. In the presented study we designed antisense oligonucleotides (AONs), targeting intronic splicing silencer sites, some were modified with 2′-O-methyl, others with LNA. The AONs have been extensively tested in different concentrations, both individually and combined, in order to effectively target the ISS-N1 and A+100G splicing silencer regions in intron 7 of the SMN2 gene. By treating SMA-cultured fibroblasts with certain AONs, we discovered a remarkable increase in the levels of full-length SMN transcripts and the number of nuclear gems. This increase was observed to be dose-dependent and reached levels comparable to those found in healthy cells. When added to cells together, most of the tested molecules showed a remarkable synergistic effect in correcting splicing. Through our research, we have discovered that the impact of oligonucleotides is greatly influenced by their length, sequence, and pattern of modification.

The investigation of gene regulation therapeutics for the treatment of skin-related diseases is rarely explored in part due to inefficient systemic delivery. In this study, a bottlebrush polymer-antisense oligonucleotide (ASO) conjugate, termed pacDNA, designed to target IL-17 receptor A (IL-17RA), which is involved in psoriasis pathogenesis is presented. Systemic administration of pacDNA led to its accumulation in epidermis, dermis, and hypodermis of mouse skin, reduced IL-17RA gene expression in skin, and significantly reversed the development of imiquimod (IMQ)-induced psoriasis in a mouse model. These findings highlight the potential of the pacDNA as a promising nanoconstruct for systemic oligonucleotide delivery to the skin and for treating psoriasis and other skin-related disorders through systemic administration., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

Abstract Bcl-x pre-mRNA splicing serves as a typical example to study the impact of alternative splicing in the modulation of cell death. Dysregulation of Bcl-x apoptotic isoforms caused by precarious equilibrium splicing is implicated in genesis and development of multiple human diseases, especially cancers. Exploring the mechanism of Bcl-x splicing and regulation has provided insight into the development of drugs that could contribute to sensitivity of cancer cells to death. On this basis, we review the multiple splicing patterns and structural characteristics of Bcl-x. Additionally, we outline the cis-regulatory elements, trans-acting factors as well as epigenetic modifications involved in the splicing regulation of Bcl-x. Furthermore, this review highlights aberrant splicing of Bcl-x involved in apoptosis evade, autophagy, metastasis, and therapy resistance of various cancer cells. Last, emphasis is given to the clinical role of targeting Bcl-x splicing correction in human cancer based on the splice-switching oligonucleotides, small molecular modulators and BH3 mimetics. Thus, it is highlighting significance of aberrant splicing isoforms of Bcl-x as targets for cancer therapy.

Bcl-x pre-mRNA splicing serves as a typical example to study the impact of alternative splicing in the modulation of cell death. Dysregulation of Bcl-x apoptotic isoforms caused by precarious equilibrium splicing is implicated in genesis and development of multiple human diseases, especially cancers. Exploring the mechanism of Bcl-x splicing and regulation has provided insight into the development of drugs that could contribute to sensitivity of cancer cells to death. On this basis, we review the multiple splicing patterns and structural characteristics of Bcl-x. Additionally, we outline the cis-regulatory elements, trans-acting factors as well as epigenetic modifications involved in the splicing regulation of Bcl-x. Furthermore, this review highlights aberrant splicing of Bcl-x involved in apoptosis evade, autophagy, metastasis, and therapy resistance of various cancer cells. Last, emphasis is given to the clinical role of targeting Bcl-x splicing correction in human cancer based on the splice-switching oligonucleotides, small molecular modulators and BH3 mimetics. Thus, it is highlighting significance of aberrant splicing isoforms of Bcl-x as targets for cancer therapy. [ABSTRACT FROM AUTHOR]

Antisense oligonucleotides hold therapeutic promise for various lung disorders, but their efficacy is limited by suboptimal delivery. To address this challenge, we explored the use of inhaled bottlebrush polymer-DNA conjugates, named pacDNA, as a delivery strategy. Inhaled pacDNA exhibits superior mucus penetration, achieving a uniform and sustained lung distribution in mice. Targeting the 5' splice site of an aberrant enhanced green fluorescence protein ( EGFP ) pre-mRNA in EGFP-654 mice, inhaled pacDNA more efficiently corrects splicing than a B-peptide conjugate and restores EGFP expression in the lung. Additionally, in an orthotopic NCI-H358 non-small-cell lung tumor mouse model, inhaled pacDNA targeting wild-type KRAS mRNA effectively suppresses KRAS expression and inhibits lung tumor growth, requiring a substantially lower dosage compared to intravenously injected pacDNA. These findings demonstrate the potential of bottlebrush polymer-DNA conjugates as a promising agent for enhanced oligonucleotide therapy in the lung and advancing the treatment landscape for lung disorders.

One of most important factors for messenger RNA (mRNA) transcription is the spliceosomal component U1 small nuclear RNA (snRNA), which recognizes 5′ splicing donor sites at specific regions in pre‐mRNA. Mutations in these sites disrupt U1 snRNA binding and cause abnormal splicing. In this study, we investigated mutations at splice sites in SLC26A4 (HGNC 8818), one of the major causative genes of hearing loss, which may result in the synthesis of abnormal pendrin, the channel protein encoded by the gene. Seventeen SLC26A4 variants with mutations in the U1 snRNA binding sites were assessed by minigene splicing assays, and 11 were found to result in abnormal splicing. Interestingly, eight of the 11 pathogenic mutations were intronic, suggesting the importance of conserved sequences at the intronic splice site. The application of modified U1 snRNA effectively rescued the abnormal splicing for most of these mutations. Although three were cryptic mutations, they were rescued by cotransfection of modified U1 snRNA and modified antisense oligonucleotides. Our results demonstrate the important role of snRNA in SLC26A4 mutations, suggesting the therapeutic potential of modified U1 snRNA and antisense oligonucleotides for neutralizing the pathogenic effect of the splice‐site mutations that may result in hearing loss. [ABSTRACT FROM AUTHOR]

Leber congenital amaurosis (LCA) is a rare inherited retinal disorder affecting approximately 1:50,000 people worldwide. So far, mutations in 25 genes have been associated with LCA, with CEP290 (encoding the Centrosomal protein of 290 kDa) being the most frequently mutated gene. The most recurrent LCA-causing CEP290 mutation, c.2991+1655A>G, causes the insertion of a pseudoexon into a variable proportion of CEP290 transcripts. We previously demonstrated that antisense oligonucleotides (AONs) have a high therapeutic potential for patients homozygously harbouring this mutation, although to date, it is unclear whether rescuing one single allele is enough to restore CEP290 function. Here, we assessed the AON efficacy at RNA, protein and cellular levels in samples that are compound heterozygous for this mutation, together with a protein-truncating mutation in CEP290. We demonstrate that AONs can efficiently restore splicing and increase protein levels. However, due to a high variability in ciliation among the patient-derived cell lines, the efficacy of the AONs was more difficult to assess at the cellular level. This observation points towards the importance of the severity of the second allele and possibly other genetic variants present in each individual. Overall, AONs seem to be a promising tool to treat CEP290-associated LCA, not only in homozygous but also in compound heterozygous carriers of the c.2991+1655A>G variant. [ABSTRACT FROM AUTHOR]

While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect.

Inherited retinal diseases are an extremely diverse group of genetically and phenotypically heterogeneous conditions characterized by variable maturation of retinal development, impairment of photoreceptor cell function and gradual loss of photoreceptor cells and vision. Significant progress has been made over the last two decades in identifying the many genes implicated in inherited retinal diseases and developing novel therapies to address the underlying genetic defects. Approximately one-quarter of exonic mutations related to human inherited diseases are likely to induce aberrant splicing products, providing opportunities for the development of novel therapeutics that target splicing processes. The feasibility of antisense oligomer mediated splice intervention to treat inherited diseases has been demonstrated in vitro, in vivo and in clinical trials. In this review, we will discuss therapeutic approaches to treat inherited retinal disease, including strategies to correct splicing and modify exon selection at the level of pre-mRNA. The challenges of clinical translation of this class of emerging therapeutics will also be discussed.

Stargardt disease type 1 (STGD1) is the most common hereditary form of maculopathy and remains untreatable. STGD1 is caused by biallelic variants in the ABCA4 gene, which encodes the ATP-binding cassette (type 4) protein (ABCA4) that clears toxic byproducts of the visual cycle. The c.5461-10T>C p.[Thr1821Aspfs∗6,Thr1821Valfs∗13] variant is the most common severe disease-associated variant, and leads to exon skipping and out-of-frame ABCA4 transcripts that prevent translation of functional ABCA4 protein. Homozygous individuals typically display early onset STGD1 and are legally blind by early adulthood. Here, we applied antisense oligonucleotides (AONs) to promote exon inclusion and restore wild-type RNA splicing of ABCA4 c.5461-10T>C. The effect of AONs was first investigated in vitro using an ABCA4 midigene model. Subsequently, the best performing AONs were administered to homozygous c.5461-10T>C 3D human retinal organoids. Isoform-specific digital polymerase chain reaction revealed a significant increase in correctly spliced transcripts after treatment with the lead AON, QR-1011, up to 53% correct transcripts at a 3 μM dose. Furthermore, western blot and immunohistochemistry analyses identified restoration of ABCA4 protein after treatment. Collectively, we identified QR-1011 as a potent splice-correcting AON and a possible therapeutic intervention for patients harboring the severe ABCA4 c.5461-10T>C variant., Competing Interests: M.K., P.d.B., K.D., T.H., W.B., G.P., and J.S. were employed by ProQR Therapeutics during this project. K.D. is inventor on the international patent application (WO2018189376) that has been filed by ProQR Therapeutics describing methods and means for AON therapy for Stargardt disease type 1. The rest of the co-authors declare that they have no competing interests., (© 2023 The Authors.)

Abstract: Expression of alternatively spliced mRNA variants at specific stages of development or in specific cells and tissues contributes to the functional diversity of the human genome. Aberrations in alternative splicing were found as a cause or a contributing factor to the development, progression, or maintenance of numerous diseases. The use of antisense oligonucleotides (ON) to modify aberrant expression patterns of alternatively spliced mRNAs is a novel means of potentially controlling such diseases. Oligonucleotides can be designed to repair genetic mutations, to modify genomic sequences in order to compensate for gene deletions, or to modify RNA processing in order to improve the effects of the underlying gene mutation. Steric block ON approach have proven to be effective in experimental model for various diseases. Here, we describe our experience in investigating two strategies for ON delivery: ON conjugation with basic peptides and lipid-based particulate system (lipoplex). Basic peptides or Cell Penetrating Peptides (CPP) such as the TAT-derived peptide appear to circumvent many problems associated with ON and drug delivery. This strategy may represent the next paradigm in our ability to modulate cell function and offers a unique avenue for the treatment of disease. Lipoplexes result from the intimate interaction of ON with cationic lipids leading to ON carrying particles able to be taken up by cells and to release ON in the cytoplasm. We have used as an experimental model the correction of a splicing alteration of the mutated β-globin intron causing thalassemia. Data on cell penetration and efficacy of correction of specific steric block ON delivered either by basic peptides or lipoplex are described. A comparison of the properties of both delivery systems is made respective to the use of this new class of therapeutic molecules. [Copyright &y& Elsevier]

Abstract: Splicing correction by steric-blocking oligonucleotides (ON) might lead to important clinical applications but requires efficient delivery to cell nuclei. The conjugation of short oligolysine tails has been used to deliver a correcting peptide nucleic acid (PNA) sequence in a positive readout assay in which ON hybridization to the cryptic splice site is strictly required for the expression of a luciferase reporter gene. We have investigated the mechanism of cellular uptake and the efficiency of a (Lys)8-PNA-Lys construction in this model system. Cell uptake is temperature-dependent and leads to sequestration of the conjugate in cytoplasmic vesicles in keeping with an endocytic mechanism of internalization. Accordingly a significant and sequence-specific splicing correction is achieved only in the presence of endosome-disrupting agents as chloroquine or 0.5 M sucrose. These endosome-disrupting agents do not affect the activity of free PNA, and do not increase (Lys)8-PNA-Lys uptake. [Copyright &y& Elsevier]

Inherited retinal diseases are an extremely diverse group of genetically and phenotypically heterogeneous conditions characterized by variable maturation of retinal development, impairment of photoreceptor cell function and gradual loss of photoreceptor cells and vision. Significant progress has been made over the last two decades in identifying the many genes implicated in inherited retinal diseases and developing novel therapies to address the underlying genetic defects. Approximately one-quarter of exonic mutations related to human inherited diseases are likely to induce aberrant splicing products, providing opportunities for the development of novel therapeutics that target splicing processes. The feasibility of antisense oligomer mediated splice intervention to treat inherited diseases has been demonstrated in vitro, in vivo and in clinical trials. In this review, we will discuss therapeutic approaches to treat inherited retinal disease, including strategies to correct splicing and modify exon selection at the level of pre-mRNA. The challenges of clinical translation of this class of emerging therapeutics will also be discussed., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Oligonucleotide therapeutics hold great promise for the treatment of various diseases and the antisense field is constantly gaining interest due to the development of more potent and nuclease resistant chemistries. Despite a rather low success rate with only three antisense drugs being clinically approved, the frontiers of AON therapeutic applications have increased over the past three decades and continue to expand thanks to a steady increase in understanding the mechanisms of action of these molecules, progress in chemical modification and delivery.In this review, we will examine the recent advances obtained with the tricyclo-DNA chemistry which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration. We will review their specific properties and their therapeutic applications mainly for neuromuscular disorders, including exon-skipping for Duchenne muscular dystrophy and exon-inclusion for spinal muscular atrophy, but also aberrant splicing correction for Pompe disease. Finally, we will discuss their advantages and potential limitations, with a focus on the need for careful toxicological screen early in the process of AON drug development.

Antisense oligonucleotides (ASOs) can be used to alter the splicing of a gene and either restore production of a required protein or eliminate a toxic product. In this issue of Genes & Development, Hua and colleagues (pp. 1634-1644) show that ASOs directed against an intron splice silencer (ISS) in the survival motor neuron 2 (SMN2) gene alter the amount of full-length SMN transcript in the nervous system, restoring SMN to levels that could correct spinal muscular atrophy (SMA). [ABSTRACT FROM AUTHOR]

survival of motor neuron 2, centromeric (SMN2) exon 7 inclusion to express more full-length SMN protein in motor neurons is a promising approach to treat spinal muscular atrophy (SMA), a genetic neurodegenerative disease. Previously, we identified a potent 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified antisense oligonucleotide (ASO) that blocks an SMN2 intronic splicing silencer element and efficiently promotes exon 7 inclusion in transgenic mouse peripheral tissues after systemic administration. Here we address its efficacy in the spinal cord--a prerequisite for disease treatment--and its ability to rescue a mild SMA mouse model that develops tail and ear necrosis, resembling the distal tissue necrosis reported in some SMA infants. Using a micro-osmotic pump, we directly infused the ASO into a lateral cerebral ventricle in adult mice expressing a human SMN2 transgene; the ASO gave a robust and long-lasting increase in SMN2 exon 7 inclusion measured at both the mRNA and protein levels in spinal cord motor neurons. A single embryonic or neonatal intracerebroventricular ASO injection strikingly rescued the tail and ear necrosis in SMA mice. We conclude that this MOE ASO is a promising drug candidate for SMA therapy, and, more generally, that ASOs can be used to efficiently redirect alternative splicing of target genes in the CNS. [ABSTRACT FROM AUTHOR]

Splicing is an important and highly regulated step in gene expression. The ability to modulate it can offer a therapeutic option for many genetic disorders. Antisense-mediated splicing-correction approaches have recently been successfully exploited for some genetic diseases, and are currently demonstrating safety and efficacy in different clinical trials. Their application for the treatment of retinal dystrophies could potentially solve a vast panel of cases, as illustrated by the abundance of mutations that could be targeted and the versatility of the technique. In this review, we will give an insight of the different therapeutic strategies, focusing on the current status of their application for retinal dystrophies., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Alternative splicing is a key molecular mechanism for increasing the functional diversity of the eukaryotic proteomes. A large body of experimental data implicates aberrant splicing in various human diseases, including cancer. Both mutations in cis-acting splicing elements and alterations in the expression and/or activity of splicing regulatory factors drastically affect the splicing profile of many cancer-associated genes. In addition, the splicing profile of several cancer-associated genes is altered in particular types of cancer arguing for a direct role of specific splicing isoforms in tumor progression. Deciphering the mechanisms underlying aberrant splicing in cancer may prove crucial to understand how splicing machinery is controlled and integrated with other cellular processes, in particular transcription and signaling pathways. Moreover, the characterization of splicing deregulation in cancer will lead to a better comprehension of malignant transformation. Cancer-associated alternative splicing variants may be new tools for the diagnosis and classification of cancers and could be the targets for innovative therapeutical interventions based on highly selective splicing correction approaches.