NAR Molecular Medicine

Spinal Muscular Atrophy (SMA) stands as a devastating ailment arising from the dearth of functional SMN (Survival Motor Neuron) protein due to genetic anomalies within the SMN1 gene. This condition is marked by the consequential attrition of motor neurons, precipitating a progressive decline in muscular strength and culminating in the disruption of neuromuscular junctions. Existing therapeutic approaches encompassing Zolgensma, Nursinersen, and Evrysdi employ innovative genetic therapeutic strategies involving transgene delivery, Antisense Oligonucleotide (ASO) technology, and modulation of pre-mRNA processing to enhance functional SMN protein expression. However, the ASO therapeutics remain suboptimal in establishing a sustained panacea for SMA, as they inadequately maintain consistent levels of functional SMN protein expression. In this study, we present a discerning inquiry into focusing on XNA-DNA-ASO products that exhibit enhanced safety and stability compared to conventional DNA/RNA-ASO sequences. Through precise targeting of the ISSN-1 region within SMN2 genes intron 7, our approach seeks to amplify SMN protein expression. Employing Xeno Nucleic Acid (XNA) bases, known for their augmented hydrophobicity and stability, our strategy surmounts previous limitations associated with chemical modifications, showcasing heightened endonuclease resistance. Comparative analyses with conventional DNA/RNA-ASO products substantiate the superiority of XNA-DNA-ASO sequences, underscoring elevated SMN protein expression and reduced toxicity. In a comprehensive evaluation, our gene therapy paradigm is scrutinized within a type 1 SMA fibroblast cell line. Utilizing diverse analytical methodologies, encompassing Annexin V-PI analysis for cytotoxicity, MTT assays for mitochondrial activity, and flow cytometry for SMN protein expression profile, we gauge therapeutic impact and potential toxicity. In conclusion, our investigation not only spotlights the promise of XNA-DNA-ASO sequences but also holds implications for refining SMA treatment strategies, converging on minimized dosages, lowered toxicity, and heightened therapeutic efficacy, thus shaping the landscape of gene therapy for SMA.

BackgroundHuntingtons disease (HD) is a fatal neurodegenerative disorder caused by a mutation in the huntingtin gene (HTT), characterized by an expanded CAG trinucleotide repeat. At the time of writing no cure or disease-modifying treatments exist. Currently, the most explored investigational therapeutic strategy targets HTT gene expression, either lowering both alleles (total lowering) or selectively lowering the mutant allele. These approaches require reliable pharmacodynamic biomarkers to measure an allele-selective knockdown. However, allele specific quantification of wild-type and mutant HTT RNA or protein remains a challenge. ResultsHere we optimized a droplet digital PCR (ddPCR) assay to distinguish between mutant and wild-type HTT (mHTT and wtHTT respectively) mRNA expression based on differential amplification of HTT mRNA molecules with different CAG repeat lengths under limited dNTP conditions. This assay, combined with our novel automated analysis pipeline reliably detects allele-specific expression in HD patient cell lines. We simulated various mHTT to wtHTT mRNA ratios by mixing RNA from respective homozygous cell lines to demonstrate the assays accuracy under varying allele ratios. We also validated the assays utility in 13 cell lines from HD patients and their family members. Additionally, we optimized a one-step RT-ddPCR method, offering a streamlined alternative to a two-step ddPCR method. We further confirmed the assays clinical relevance by demonstrating allele-selective siRNA mediated HTT lowering in HD patient fibroblast cell lines. ConclusionsOur optimized ddPCR assay, with its pipeline for automated data analysis, enables the precise quantification of allele-selective HTT mRNA knockdown. The method does not require prior knowledge of patients SNP genotypes, previously a prerequisite for assays aiming to determine the mHTT transcript expression in patient samples. Our HTT ddPCR assay is universally applicable regardless of patient genotype. The ability to accurately monitor allele-specific HTT mRNA expression levels holds great promise for developing effective treatments for Huntingtons disease.

TAR DNA/RNA-binding protein 43 kDa (TDP-43) proteinopathy is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis, in which cytoplasmic aggregates containing TDP-43 and its C-terminal fragments, such as TDP25, are observed in degenerative neuronal cells. However, few reports have focused on small molecules that can reduce their aggregation and cytotoxicity. Here, we show that short RNA repeats of GGGGCC and AAAAUU are aggregation-suppressors of TDP-43 and TDP25. TDP25 interacts with these RNAs, as well as TDP-43, despite the lack of major RNA-recognition motifs, using fluorescence cross-correlation spectroscopy. Expression of these RNAs significantly decreases the cells harboring cytoplasmic aggregates of TDP-43 and TDP25 and ameliorates rounded and shrinking cells by mislocalized TDP-43; furthermore, the cellular transcriptome is not altered. Consequently, these RNAs can maintain proteostasis by preventing aggregation of TDP-43 and TDP25.

Recent advances in "omics" technologies allow for the identification of an increasing number of individuals with diseases caused by nano-rare mutations. These difficult-to-diagnose individuals are uniquely disadvantaged and pose significant challenges to healthcare systems and society. Despite having diseases caused by actionable single gene mutations, in many cases, there is no commercial path for treatments for such small patient populations. We have defined nano-rare mutations as, mutations with a known worldwide prevalence <30. Since antisense oligonucleotide (ASO) technology has proven to be suited to address the needs of a portion of these patients, the n-Lorem Foundation is establishing an industrialized approach that couples detailed genotypic and phenotypic data to the immediate potential for ASO therapy. In this manuscript we have leveraged our experience in assessing the causality of nano-rare genetic variants and associated proximal molecular pathological events to attempt a correlation between detailed genetic data with patient specific phenotypic observations in 173 nano-rare individuals from diverse age groups evaluated for experimental ASO therapy. We found that the time required to achieve a molecular diagnosis varies from 1 month to 36 years, with the mean and median times from symptom onset to diagnosis estimated to be 4.32 years and 2 years, respectively. Amongst submitted cases there is a significant bias toward neurological diseases, with diverse genes and functional families involved and a marked preponderance of mutations in ion channel genes. The variability in phenotypic expression associated with nano-rare variants in genes such as GNAO1, H3F3A, GBE1, UBTF, or PACS1 clearly supports previous observations that phenotypes associated with same variants in the same gene can vary. We also observe that different, but functionally equivalent variants can result in both similar (e.g., TARDBP) and different phenotypes (e.g., GNAO1). Despite the relatively small size of the patient population investigated, this first compilation of its kind allows a variety of insights into the genotype and phenotype relationships in nano-rare conditions. Moreover, we show that our unique patient population presents a remarkable opportunity to apply "modern omics" approaches to begin to understand the various homeostatic, compensatory, and secondary effects of these genetic variants on the networks that result in expression of their unique phenotypes. To provide a more detailed description of the processes involved to provide a personalized antisense medicine, we have included nonclinical and clinical data on three exemplary patients who display disease in three different organs, the CNS, the eye and the kidney and are treated with ASOs of different designs. In contrast to traditional drug development, each patient presents unique genomic, ASO design, clinical treatment and management and evaluation challenges.

TDP-43, an essential nucleic acid binding protein and splicing regulator, is broadly disrupted in neurodegeneration. TDP-43 nuclear localization and function depend on the abundance of its nuclear RNA targets and its recruitment into large ribonucleoprotein complexes, which restricts TDP-43 nuclear efflux. To further investigate the interplay between TDP-43 and nascent RNAs, we aimed to employ 5-ethynyluridine (5EU), a widely used uridine analog for click chemistry labeling of newly transcribed RNAs. Surprisingly, 5EU induced the nuclear accumulation of TDP-43 and other RNA-binding proteins and attenuated TDP-43 mislocalization caused by disruption of the nuclear transport apparatus. RNA FISH demonstrated 5EU-induced nuclear accumulation of polyadenylated and GU-repeat-rich RNAs, suggesting increased retention of both processed and intronic RNAs. TDP-43 eCLIP confirmed that 5EU preserved TDP-43 binding at predominantly GU-rich intronic sites. RNAseq revealed significant 5EU-induced changes in alternative splicing, accompanied by an overall reduction in splicing diversity, without any major changes in RNA stability or TDP-43 splicing regulatory function. These data suggest that 5EU may impede RNA splicing efficiency and subsequent nuclear RNA processing and export. Our findings have important implications for studies utilizing 5EU and offer unexpected confirmation that the accumulation of endogenous nuclear RNAs promotes TDP-43 nuclear localization.

Personalized antisense oligonucleotides (ASOs) have achieved positive results in the treatment of rare genetic disease. As clinical sequencing technologies continue to advance, the ability to identify rare disease patients harboring pathogenic genetic variants amenable to this therapeutic strategy will likely improve. Here, we describe a scalable platform for generating patient-derived cellular models and demonstrate that these personalized models can be used for preclinical evaluation of patient-specific ASOs. We establish robust protocols for delivery of ASOs to patient-derived organoid models and confirm reversal of disease-associated phenotypes in cardiac organoids derived from a Duchenne muscular dystrophy (DMD) patient harboring a structural deletion in the dystrophin gene amenable to treatment with existing ASO therapeutics. Furthermore, we design novel patient-specific ASOs for two additional DMD patients (siblings) harboring a deep intronic variant in the dystrophin gene that gives rise to a novel splice acceptor site, incorporation of a cryptic exon, and premature transcript termination. We show that treatment of patient-derived cardiac organoids with patient-specific ASOs results in restoration of DMD expression and reversal of disease-associated phenotypes. The approach outlined here provides the foundation for an expedited path towards the design and preclinical evaluation of personalized ASO therapeutics for a broad range of rare diseases.

Mutations in the SOD1 gene are among the most significant genetic contributors to amyotrophic lateral sclerosis (ALS), with different variants linked to varying disease severity. To investigate the molecular mechanisms driving this variability, we conducted RNA sequencing on spinal motor neurons (MNs) differentiated from isogenic human induced pluripotent stem cell (iPSC) lines engineered via CRISPR/Cas9. These lines carried two representative SOD1 heterogenous mutations, D91A and G94A, and were analyzed at Days 10 and 20 of neuronal maturation stage to capture the temporal changes of gene expression. We aim to explore how these mutations affect MN function, identify distinct molecular pathways that may explain the variable severity of ALS, and investigate the role of translation and metabolic dysregulation in disease progression.

More than 50 repeat expansion disorders have been identified, with long-read sequencing marking a new milestone in the diagnosis of these disorders. Despite these major achievements, the comprehensive characterization of short tandem repeats in a pathological context remains challenging, primarily due to their inherent characteristics such as motif complexity, high GC content, and variable length. In this study, our aim was to thoroughly characterize repeat expansions in two neuromuscular diseases: myotonic dystrophy type 1 (DM1) and oculopharyngodistal myopathy (OPDM) using CRISPR/Cas9- targeted long-read sequencing (Oxford Nanopore Technologies, ONT). We conducted precise analyses of the DM1 and OPDM loci, determining repeat size, repeat length distribution, expansion architecture and DNA methylation, using three different basecalling strategies (MinKnow software, Dorado and Bonito). We demonstrated the importance of the basecalling strategy in repeat expansion characterization. We proposed guidelines to perform CRISPR-Cas9 targeted long-read sequencing (no longer supported by ONT), from library preparation to bioinformatical analyses. Finally, we showed, for the first time, somatic mosaicism, hypermethylation of LRP12 loci in OPDM symptomatic patients and changes in the repeat tract structure of these patients. We propose a strategy based on CRISPR/Cas9-enrichment long-read sequencing for repeat expansion diseases, which could be readily applicable in research but also in diagnostic settings.

RNA-targeting CRISPR-Cas systems enable modulation of gene expression without permanent genome modification, making them useful for sensitive cell types such as neurons. While CRISPR-Cas technologies have been most extensively applied and validated in primary hippocampal and cortical neurons, their use in sensory neurons remains largely unexplored. Sensory neurons are an established cellular model for studying axon growth and regeneration, pain mechanisms, sensory transduction, and neuron-environment interactions. Here, we evaluated the performance of compact RNA-targeting CRISPR-Cas effectors Cas7-11S, hfCas13X, and hfCas13d in primary rat sensory neurons in culture. Using an endogenous mRNA as the target, we compared knockdown efficiency and assessed the effects of CRISPR-Cas expression on neuronal health. The systems showed distinct differences in performance, with Cas7-11S inducing toxicity, hfCas13X showing minimal knockdown, and hfCas13d providing robust gene silencing with minimal adverse effects on neuronal health. These findings identify hfCas13d as an effective and well-tolerated RNA-targeting CRISPR-Cas tool for sensory neurons and provide important insight into its suitability for neuroscience research and potential therapeutic applications.

Multiple sclerosis (MS) is a fatal neurodegenerative disease that progresses by eroding the myelin sheath and exposing the neuron, leading to neuronal degradation and death. While MS remains without an effective treatment or cure, studies have identified genes that are dysregulated in MS patients and predicted to be involved with disease progression. These genes are primarily involved in controlling DNA methylation: a process required for regulating gene expression, which is critical for cellular health. Having identified potential genetic risk factors, research is focused on how to manipulate the expression of these genes by offsetting DNA methylation errors in patients through the targeting of DNA and RNA secondary structure formation. Serine hydroxy methyltransferase 1 (SHMT1), a key player in DNA methylation, was determined to be upregulated in MS patients. Here, we identified and characterized a hybrid 3+1 G-quadruplex (GQ) and i-motif (iM) structures in the SHMT1 DNA 5 untranslated region and a parallel GQ in the corresponding mRNA. Additionally, we found that the GQ/iM structures suppress the mRNA levels and protein expression of a reporter gene. Together, these data suggest that GQ/iM structures are necessary for SHMT1 regulation, which could serve as a target for therapeutic intervention for MS patients.

Mutations in the mitochondrial DNA (mtDNA) are associated with severe human diseases, lacking efficient therapies. Direct correction of mtDNA mutations may offer a cure for such diseases. We propose a novel strategy based on double-stranded DNA (dsDNA) oligonucleotide delivery into mitochondria and intrinsic microhomology-mediated end joining (MMEJ) for mtDNA editing. This strategy enables introduction of multiple predefined nucleotide changes in mtDNA. For this, the presence of MMEJ activity in the human mitochondrial lysates was confirmed. 49 bp DNA oligonucleotide duplexes, fused to an RNA hairpin previously identified as a mitochondrial import signal, were delivered into the mitochondria of cultured human cells. Delivery of these donor dsDNA molecules, homological to an ND4 site of mtDNA and bearing designed nucleotide changes, led to a low but statistically significant introduction of the designed nucleotide changes into mtDNA. Donor dsDNA delivery combined with the CRISPR/mito-AsCas12a system also resulted in a statistically significant number of an expected concomitant change of five nucleotides distributed across a 16-nucleotide ND4 site of the mitochondrial genome. The proposed strategy may become an efficient mtDNA editing tool suitable for the correction of near-homoplasmic mutations such as Lebers Hereditary Optic Neuropathy (LHON)-associated mutations in the ND4 gene of mtDNA.

-Synucleinopathies are devastating neurodegenerative diseases characterized by pathological accumulation of a neuronal protein, -synuclein (Syn). Lowering soluble Syn levels is a promising therapeutic strategy to limit aggregation and neurotoxicity, but directly targeting this protein is hindered by its intrinsically disordered structure and other factors, such as its conformational heterogeneity and intracellular drug delivery barriers. Consequently, increasing attention has been directed toward targeting the SNCA transcript, which encodes Syn. Here, we developed phosphorodiamidate morpholino oligonucleotide (PMO)-based RNA-degrading chimeras (RDCs) that selectively bind the 5' untranslated region of SNCA mRNA and recruit RNase L for targeted RNA degradation. Through the systematic evaluation of 10 RDCs, we identified and optimized 4-D1, which effectively reduced SNCA mRNA and Syn protein expression in HEK293T cells in an RNase L-dependent manner. 4-D1 lowered SNCA transcript and Syn protein levels in both primary cortical neurons from humanized SNCA mice and in human induced pluripotent stem cell-derived cortical neurons. This reduction prevented prion-like seeding induced by patient-derived Syn fibrils and protected neurons from fibril-induced cytotoxicity. Finally, in vivo studies confirmed the efficacy of 4-D1 in reducing Syn mRNA expression in humanized SNCA mice. These findings indicate that PMO-based RDCs may represent a promising therapeutic modality for -synucleinopathies. Significance StatementAbnormal aggregation of the neuronal protein -synuclein is central to Parkinsons disease and related disorders, yet therapeutic candidates that directly target this protein have yet to demonstrate efficacy. in clinical trials. We developed a new strategy that lowers -synuclein production at the RNA level using phosphorodiamidate morpholino oligonucleotide (PMO)-based RNA-degrading chimeras (RDCs). These molecules recruit a natural RNA-degrading enzyme to selectively destroy the RNA transcript encoding -synuclein. Our lead RDC reduced -synuclein levels in cultured cells, humanized mouse and human neurons, blocked -synuclein pathological aggregation, and protected neurons from toxicity. This study establishes RDCs as a promising therapeutic platform for Parkinsons disease and other neurodegenerative diseases driven by -synuclein.

MotivationTraditional methods for detecting large-scale mitochondrial DNA (mtDNA) deletions (LSMDs) in cells present challenges, i.e. a priori information, high DNA inputs, poor sensitivity and are not always quantitative. Mitigation can be achieved through high throughput DNA sequencing using e.g. Illumina and Oxford Nanopore Technologies (ONT), in combination with LSMD breakpoint identification and quantification using bioinformatic tools. Splice-aware RNA alignment tools increase the sensitivity for detecting LSMD breakpoints compared with DNA aligners. Long-read sequencing (LRS) also offers potential advantages over short read sequencing, e.g. greater read lengths and capturing variants on single reads. No existing pipelines capture the benefits of both a splice-aware alignment tool and LRS. ResultsWe developed "NanoDel", a LRS pipeline, to sensitively and accurately detect cellular LSMDs. Using artificial datasets, "NanoDel" was more sensitive and accurate than other pipelines. In samples diagnosed with mitochondrial disease, it identified both known and previously uncharacterised (including mixtures) of LSMDs, without a priori information. LSMD breakpoints were found in mt-co1, mt-cyb, mt-nd6 and mt-nd5 genes. Analysis of selected LSMDs revealed proximity to repeat and putative G-quadruplex motifs, and occurrence in a range of healthy and pathological tissues, indicating potential for a shared vulnerability landscape in mtDNA, shaped by sequence motifs and structural constraints. "NanoDel" combined with one-amplicon, not two-amplicon, LR-PCR offers a robust strategy with clinical application for detecting LSMDs across a variety of cell/tissue samples, and its application across a broader range of samples, will yield new mechanistic insights into LSMD formation, and further our understanding of mtDNA instability.

Nucleotide repeat expansions, such as the GGGGCC repeats in C9orf72, associated with C9-ALS, are linked to neurodegenerative diseases. These repeat sequences undergo a non-canonical translation known as repeat-associated non-AUG (RAN) translation. Unlike canonical translation, RAN translation initiates from non-AUG codons and occurs in all reading frames. To identify potential regulators of RAN translation, we employed a bottom-up approach using a human factor-based reconstituted cell-free translation system to recapitulate RAN translation. This approach revealed that omission of either eIF1A or eIF5B enhanced the translation in all reading frames of C9orf72-mediated RAN translation (C9-RAN), suggesting that eIF1A and eIF5B act as repressors of RAN translation. eIF1A and eIF5B are known to contribute to the fidelity of translation initiation. In HEK293T cells, double knockdown of eIF1A and eIF5B further promoted C9-RAN compared to single knockdowns, indicating that these factors regulate C9-RAN through distinct initiation steps. Furthermore, under eIF1A knockdown conditions, the enhancement of RAN translation via the integrated stress response (ISR) was not observed in HEK293T cells, indicating that eIF1A is involved in the ISR-mediated non-AUG translation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/654993v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@160e2adorg.highwire.dtl.DTLVardef@1c1cc8forg.highwire.dtl.DTLVardef@5d01d0org.highwire.dtl.DTLVardef@220507_HPS_FORMAT_FIGEXP M_FIG C_FIG

Post-transcriptional modifications expand the information encoded by an mRNA. These dynamic and reversible modifications are specifically recognized by reader RNA-binding proteins (RBPs), which mediate the regulation of gene expression, RNA processing, localization, stability, and translation. Given their crucial functions, any disruptions in the normal activity of these readers can have significant implications for cellular health. Consequently, the dysregulation of these RBPs has been associated with neurodegenerative disorders, cancers, and viral infections. Therefore, there has been growing interest in targeting reader RBPs as a potential therapeutic strategy since developing molecules that restore proper RNA processing and function may offer a promising avenue for treating diseases. In this work, we coupled our previously established live-cell RNA-protein interaction (RPI) assay, RNA interaction with Protein-mediated Complementation Assay (RiPCA), with CRISPR technology to build a new platform, CRISPR RiPCA. As a model for development, we utilized the interaction of eukaryotic translation initiation factor 4E (eIF4E), a reader RBP that binds to the m7GpppX cap present at the 5' terminus of coding mRNAs, with an m7G capped RNA substrate. Using eIF4E CRISPR RiPCA, we demonstrate our technologys potential for measuring on-target activity of inhibitors of the eIF4E RPI of relevance to cancer drug discovery.

Human DExD/H-box RNA helicases are ubiquitous molecular motors that unwind and rearrange RNA secondary structures in an ATP-dependent manner. These enzymes play essential roles in nearly all aspects of RNA metabolism. While their biological functions are well-characterized, the kinetic mechanisms remain relatively understudied in vitro. In this study, we describe the development and optimization of a bioluminescence-based assay to kinetically characterize three human RNA helicases: MDA5, LGP2, and DDX1. The assays were conducted using annealed 24-mer RNA (blunt-ended double-stranded RNA) or double-stranded RNA (ds-RNA) with a 25- nt 3' overhang. These findings establish a robust and high-throughput in vitro assay suitable for a 384-well format, enabling the discovery and characterization of inhibitors targeting MDA5, LGP2, and DDX1. This work provides a valuable resource for advancing our understanding of these helicases and their therapeutic potential in Alzheimers disease.

Chemical modifications in mRNAs, such as pseudouridine (psi), can control gene expression. Yet, we know little about how they are regulated, especially in neurons. We applied nanopore direct RNA sequencing to investigate psi dynamics in SH-SY5Y cells in response to two perturbations that model a natural and unnatural cellular state: retinoic-acid-mediated differentiation (healthy) and exposure to the neurotoxicant, lead (unhealthy). We discovered that the expression of some psi writers change significantly in response to physiological conditions. We also found that globally, lead-treated cells have more psi sites but lower relative occupancy than untreated cells and differentiated cells. Interestingly, examples of highly plastic sites were accompanied by constant expression for psi writers, suggesting trans-regulation. Many positions were static throughout all three cellular states, suggestive of a "housekeeping" function. This study enables investigations into mechanisms that control psi modifications in neurons and its possible protective effects in response to cellular stress.

Spliceosome and ciliary dysfunctions can lead to remarkably similar clinical syndromes. Studying ten individuals with retinal dystrophy, neurological involvement, and skeletal abnormalities, suggestive of both spliceosomopathies and ciliopathies, we involved GPATCH11, a lesser-known GPATCH-domain-containing regulators of RNA metabolism. To elucidate GPATCH11 function, we employed fibroblasts from unaffected individuals and patients carrying a recurring mutation specifically removing the main part of the GPATCH-domain while preserving other domains. Additionally, we generated a mouse model replicating the patients genetic defect, exhibiting behavioural abnormalities and retinal dystrophy. Our findings revealed GPATCH11 unique subcellular localization, marked as foci staining pattern and a diffuse presence in the nucleoplasm, alongside its centrosomal localization, indicating roles in RNA and cilia metabolism. We show dysregulation of U4 snRNA in patient cells and dysregulation in both gene expression and spliceosome activity within the mutant mouse retina, impacting key processes such as photoreceptor light responses, RNA regulation, and primary cilia-associated metabolism. These results highlight GPATCH11 roles in RNA metabolism, spliceosome regulation, and potential ciliary involvement. They underscore its significance in maintaining proper gene expression, contributing to retinal, neurological, and skeletal functions. Our research also demonstrates how studying rare genetic disorders can reveal broader gene functions, providing insights into GPATCH11 multifaceted roles.

Myofibrillar myopathy 6 (MFM6) is a rare childhood-onset myopathy characterized by myofibrillar disintegration, muscle weakness, and cardiomyopathy. The genetic cause of MFM6 is p.Pro209Leu mutation (rs121918312-T) in the BAG3 gene, which generates the disease outcomes in a dominant fashion. Since the consequences of the BAG3 mutation are strong and rapidly progressing, most MFM6 patients are due to de novo mutation. There are no effective treatments for MFM6 despite its well-known genetic cause. Given p.Pro209Leu mutation is dominant, regenerative medicine approaches employing orthologous stem cells in which mutant BAG3 is inactivated offer a promising avenue. Here, we developed personalized allele-specific CRISPR-Cas9 strategies capitalizing on PAM-altering SNP and PAM-proximal SNP. In order to identify the disease chromosome carrying the de novo mutation in our two affected individuals, haplotype phasing through cloning-sequencing was performed. Based on the sequence differences between mutant and normal BAG3, we developed personalized allele-specific CRISPR-Cas9 strategies to selectively inactivate the mutant allele 1) by preventing the transcription of the mutant BAG3 and 2) by inducing nonsense-mediated decay (NMD) of mutant BAG3 mRNA. Subsequent experimental validation in patient-derived induced pluripotent stem cell (iPSC) lines showed complete allele specificities of our CRISPR-Cas9 strategies and molecular consequences attributable to inactivated mutant BAG3. In addition, mutant allele-specific CRISPR-Cas9 targeting did not alter the characteristics of iPSC or the capacity to differentiate into cardiomyocytes. Together, our data demonstrate the feasibility and potential of personalized allele-specific CRISPR-Cas9 approaches to selectively inactivate the mutant BAG3 to generate cell resources for regenerative medicine approaches for MFM6.

RNA oxidation has been implicated in neurodegeneration, but the underlying mechanism for such effects is unclear. Recently, we demonstrated extensive RNA oxidation within the neurons in multiple sclerosis (MS) brain. In this report we identified selectively oxidized mRNAs in neuronal cells that pertained to neuropathological pathways. N-acetyl aspartate transferase 8 like (NAT8L) mRNA is one such transcript, whose translated product enzymatically synthesizes N-acetyl aspartic acid (NAA), a neuronal metabolite important for myelin synthesis. We reasoned that impediment of translation of an oxidized NAT8L mRNA will result in reduction in its cognate protein, thus lowering NAA level. This assertion is directly supported by our studies on a model cellular system, an MS animal model and postmortem human MS brain. Reduced NAA level in the brain hampers myelin integrity making neuronal axons more susceptible to damage, which contributes in MS neurodegeneration. Overall, this work provides a framework for mechanistic understanding of the link between RNA oxidation and neurodegenerative diseases.