Molecular Therapy - Nucleic Acids

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder caused by loss of dystrophin. Upregulation of utrophin (UTRN), a dystrophin paralogue, is a promising therapeutic avenue. Here, we present a CRISPR-Cas9-mediated strategy to increase utrophin expression by disrupting microRNA (miR) binding sites (BS). Using a Cas9/gRNA ribonucleoprotein (RNP) complex we disrupted several miR BS in DMD myoblasts and selected the Let-7c BS has crucial for UTRN repression. Interestingly, Cas9/gRNA indels were as efficient as the complete removal of Let-7c BS in upregulating UTRN expression, without any major off-targets. In three-dimensional human DMD cultures, Cas9/gRNA-mediated editing resulted in significant utrophin upregulation and functional improvements of calcium dysregulation and muscle contraction. Finally, Let-7c BS disruption in mdx animals by systemic rAAVs mediated delivery of Cas9 and gRNA resulted in utrophin upregulation and amelioration of the muscle histopathological phenotype. These findings provide the foundations for a universal (mutation-independent) gene editing therapeutic strategy for DMD. One Sentence SummaryCRISPR-Cas9 has the potential to upregulate utrophin to treat all DMD patients.

MicroRNAs (miRNAs) are short RNA molecules that bind to target mRNAs, resulting in translational repression and gene silencing. Overexpression of microRNA-21 (miR-21) is associated with various human diseases, including autosomal dominant polycystic kidney disease (ADPKD) and pulmonary fibrosis. In this study, a previously described heterobifunctional molecule, TGP-21-RiboTAC, that degrades the miR-21 precursor (pre-miR-21) in triple negative breast cancer cells was investigated in polycystic kidney cell lines and a lung fibroblast cell line. In the former, TGP-21-RiboTAC degraded pre-miR-21 and de-repressed of miR-21s downstream target, Programmed Cell Death 4 (PDCD4) and Peroxisome Proliferator-Activated Receptor alpha (PPAR), known drivers of ADPKD. The heterobifunctional molecule also inhibited cyst growth and rescued the metabolic alterations that occur in ADPKD. In the lung fibroblast cell line, MRC-5, TGP-21-RiboTAC also reduced pre- and mature miR-21 levels, rescued Transforming Growth Factor {beta} (TGF-{beta})-induced repression of SMAD Family Member 7 (SMAD7) and inhibited cell invasion. Collectively, these studies demonstrate the potential of targeted RNA degradation as therapeutic agents that retard the development of organ fibrosis.

Facioscapulohumeral muscular dystrophy (FSHD) is a potentially devastating muscle disease caused by de-repression of the toxic DUX4 gene in skeletal muscle. FSHD patients may benefit from DUX4 inhibition therapies, and although several experimental strategies to reduce DUX4 levels in skeletal muscle are being developed, no approved disease modifying therapies currently exist. We developed a CRISPR-Cas13b system that cleaves DUX4 mRNA and reduces DUX4 protein level, protects cells from DUX4-mediated death, and reduces FSHD-associated biomarkers in vitro. In vivo delivery of the CRISPR-Cas13b system with adeno-associated viral vectors reduced acute damage caused by high DUX4 levels in a mouse model of severe FSHD. However, protection was not sustained over time, with decreases in Cas13b and guide RNA levels between 8 weeks and 6 months after injection. In addition, wild-type mice injected with AAV6.Cas13b showed muscle inflammation with infiltrates containing Cas13b-responsive CD8+ cytotoxic T cells. Our RNA-seq data confirmed that several immune response pathways were significantly increased in human FSHD myoblasts transfected with Cas13b. Overall, our findings suggest that CRISPR-Cas13b is highly effective for DUX4 silencing but successful implementation of CRISPR/Cas13-based gene therapies may require strategies to mitigate immune responses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus 2019 (COVID-19). No treatment is available. Micro-RNAs (miRNAs) in mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are potential novel therapeutic agents because of their ability to regulate gene expression by inhibiting mRNA. Thus, they may degrade the RNA genome of SARS-CoV-2. EVs can transfer miRNAs to recipient cells and regulate conditions within them. MSC-EVs harbor major therapeutic miRNAs that play important roles in the biological functions of virus-infected host cells. Here, we examined their potential impact on viral and immune responses. MSC-EVs contained 18 miRNAs predicted to interact directly with the 3 UTR of SARS-CoV-2. These EVs suppressed SARS-CoV-2 replication in Vero E6 cells. In addition, five major miRNAs suppressed virus activity in a luciferase reporter assay by binding the 3 UTR. MSC-EVs showed strong regenerative effects and potent anti-inflammatory activity which may prevent lethal cytokine storms. We confirmed that EVs regulated inflammatory responses by several cell types, including human brain cells that express the viral receptor ACE2, suggesting that the brain may be targeted by SARS-CoV-2. miRNAs in MSC-EVs have several advantages as therapeutic agents against SARS-CoV-2: 1) they bind specifically to the viral 3 UTR, and are thus unlikely to have side effects; 2) because the 3 UTR is highly conserved and rarely mutates, MSC-EV miRNAs could be used against novel variants arising during viral replication; and 3) unique cargoes carried by MSC-EVs can have diverse effects, such as regenerating damaged tissue and regulating immunity.

Adeno-associated virus (AAV) vectors are one of the leading platforms for gene delivery for the treatment of human genetic diseases, but the antiviral cellular mechanisms that interfere with optimal transgene expression are incompletely understood. Here, we performed two genome-scale CRISPR screens to identify cellular factors that restrict transgene expression from recombinant AAV vectors. Our screens revealed several components linked to DNA damage response, chromatin remodeling and transcriptional regulation. Inactivation of the Fanconi Anemia gene FANCA, the Human Silencing Hub (HUSH) associated methyltransferase SETDB1 and the gyrase, Hsp90, histidine kinase and MutL (GHKL)-type ATPase MORC3 led to increased transgene expression. Moreover, SETDB1 and MORC3 knockout improved transgene levels of several AAV serotypes as well as other viral vectors, such as lentivirus and adenovirus. Finally, we demonstrated that inhibition of FANCA, SETDB1 or MORC3 also enhanced transgene expression in human primary cells, suggesting that these could be physiologically relevant pathways that restrict AAV transgene levels in therapeutic settings. IMPORTANCERecombinant AAV (rAAV) vectors have been successfully developed for the treatment of genetic diseases. The therapeutic strategy often involves the replacement of a defective gene by expression of a functional copy from the rAAV vector genome. However, cells possess antiviral mechanisms that recognize and silence foreign DNA elements thereby limiting transgene expression and its therapeutic effect. Here, we utilize a functional genomics approach to uncover a comprehensive set of cellular restriction factors that inhibit rAAV-based transgene expression. Genetic inactivation of selected restriction factors increased rAAV transgene expression. Hence, modulation of identified restriction factors has the potential to enhance AAV gene replacement therapies.

MEG3, a long non-coding RNA (lncRNA), has been shown to play a critical role in regulating apoptosis. Its downregulation inhibits apoptosis in cancer cells, whereas its upregulation has been associated with cell death in both cardiovascular disease and, more recently, Alzheimers Disease. Here we show that MEG3 is upregulated in Myotonic Dystrophy 1 (DM1). Specifically, we show MEG3 upregulation by several-fold in DM1 human muscle cells and in two DM1 mouse models, HSA-LR and LC15. In human DM1 muscle cells we observe nuclear retention of MEG3 and an increase in its transcript diversity. Furthermore, we observe a general trend of nuclear retention in DM1 affecting lncRNAs and microRNAs (miRNAs), in contrast to mRNAs, when compared to healthy cells. This altered nuclear retention may contribute to the pathological effects of non-coding RNA dysregulation in DM1. Importantly, we demonstrate that treatment with antisense conjugates targeting the repeat expansion causing DM1, an approach currently being tested in Clinical Trials, corrects MEG3 levels in HSA-LR mice, without additional therapeutic interventions targeting MEG3.

BackgroundVLA-4 is a disease progression biomarker in patients with Duchenne Muscular Dystrophy (DMD) including those treated with corticosteroids. ATL1102 an antisense oligonucleotide (ASO) inhibitor of human CD49d chain of adhesion molecule VLA-4 shows promising phase-2 results in non-ambulant DMD stabilizing upper limb function (PUL2.0), grip strength, and MRI muscle fat fraction, compared to worsening with corticosteroids. MethodsASO to mouse CD49d (ISIS348574) was used in combination with a known mdx dystrophin morpholino exon-23 skipping restoration drug (PMO), dose-time chosen for low dystrophin restoration. Mdx mice were treated once weekly for 8 weeks with ASO, control mismatch oligonucleotide, saline, or PMO alone for 4 weeks, and PMO in combination with ASO or mismatch. ResultsASO+PMO demonstrated increased specific maximum force and eccentric muscle force retention in the exterior digitorum longus (EDL) muscle after 1-7 muscle eccentric contractions relative to saline. Area-under-curve force remaining after 8-10 contractions was higher for ASO+PMO versus PMO monotherapy and ASO monotherapy, PMO monotherapy versus saline, and the ASO monotherapy versus saline and mismatch. RNA-sequencing of quadriceps muscle evaluated gene expression changes, with false discovery rate (FDR) adjusted p-values up to <0.1. ASO monotherapy at FDR<0.05 showed 2 unique genes, Gm2a in immune neutrophil function and Trdn with Ryr2 muscle calcium channel function, versus mdx saline and none with MM. The lowest FDR PMO gene was another calcium channel Cacna1s, with PMO+MM FDR<0.05. ASO+PMO treatment at FDR<0.05 modulated 55 genes, 53 unique to the combination and 2* also in ASO FDR<0.06. Affected genes were involved in immune response (Btla*, Ppml1, Tnfsm13), lipolysis (Fabp4*, G0s2), fibrosis (Igfbp-7, Calu), muscle cell (Asb15) and muscle stem cell function (Adam10, Mt-Tp, Myom1). ConclusionASO+PMO EDL muscle function protection in mdx mice, and gene expression pathways affected, support the development of ATL1102 in combination therapy with conditionally approved morpholino dystrophin restoration drugs in DMD patients.

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.

Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the ABCA4 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in ABCA4 cause the skipping of exon 39-40 and exon 33-34 respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these ABCA4 splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing ABCA4 variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced ABCA4 c.5461-10T>C variants and up to 78% reduction of the ABCA4 c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for ABCA4, providing an important step to advance the development of gene therapy to treat STGD1.

Valosin-containing protein (VCP) related disease, also known as multisystem proteinopathy 1 (MSP1), is an autosomal dominant disease caused by gain-of-function pathogenic variants of the VCP gene. The disease is associated with inclusion body myopathy, early-onset Pagets disease of bone, frontotemporal dementia, and familial amyotrophic lateral sclerosis. There is currently no treatment for this progressive disease associated with early demise resulting from proximal limb girdle and respiratory muscle weakness. We hypothesize that regulating VCP hyperactivity to normal levels can reduce the disease pathology. In this study, we assessed the effect of antisense oligonucleotides (ASOs) specifically targeting the human VCP gene in the patient (R155H) iPSC-derived skeletal muscle progenitor cells (SMPCs). ASOs were well tolerated up to a concentration of 5 {micro}M, and significantly reduced VCP protein expression in the SMPCs by 48% (95% CI [39-56]). We also treated the transgenic mouse model of VCP disease with the overexpressed humanized VCP gene severe A232E pathogenic variant with weekly subcutaneous ASO injections starting from 6 months of age for 3 months. In the skeletal muscle of transgenic mice, ASOs resulted in 30% (95%CI [27-32]) knockdown of VCP protein compared to control ASO. The ASO-mediated reduction of VCP expression in muscle tissue was associated with improvement in autophagy flux and reduction in TDP-43 expression, hallmarks of VCP disease. In addition, ASO-treated VCP A232E mice showed improvements in functional tests of muscle strength, such as rotarod and inverted screen test compared to mice treated with control ASO. These results suggest that targeting VCP could be beneficial in preventing the progression of the VCP myopathy and hold promise for the treatment of patients with VCP MSP1.

Long non-coding RNAs (lncRNAs) orchestrate various biological processes and regulate the development of cardiovascular diseases. Their potential therapeutic benefit to tackle disease progression has recently been extensively explored. Our study investigates the role of lncRNA Nudix Hydrolase 6 (NUDT6) and its antisense target Fibroblast Growth Factor 2 (FGF2) in two vascular pathologies: abdominal aortic aneurysms (AAA) and carotid artery disease. Using tissue samples from both diseases, we detected a substantial increase of NUDT6, whereas FGF2 was downregulated. Targeting Nudt6 in vivo with antisense oligonucleotides in three murine and one porcine animal models of carotid artery disease and AAA limited disease progression. Restoration of FGF2 upon Nudt6 knockdown improved vessel wall morphology and fibrous cap stability. Overexpression of NUDT6 in vitro impaired smooth muscle cell (SMC) migration, while limiting their proliferation and augmenting apoptosis. By employing RNA pulldown followed by mass spectrometry as well as RNA immunoprecipitation, we identified Cysteine and Glycine Rich Protein 1 (CSRP1) as another direct NUDT6 interaction partner, regulating cell motility and SMC differentiation. Overall, the present study identifies NUDT6 as a well-conserved antisense transcript of FGF2. NUDT6 silencing triggers SMC survival and migration and could serve as a novel RNA-based therapeutic strategy in vascular diseases.

BackgroundDICAR plays a cardioprotection of diabetic models. DICAR-JP, a short sequence of DICAR, may represent DICARs active functional domain. NAC is necessary for the heart tissue development. Addtionally, OGDHL is a metabolism regulator. In this study, we examine a new mechanism of DICAR via NAC/OGDHL on regulating cardiomyocyte metabolism in DCM. We also sought to elucidate the function of DICAR/DICAR-JP in modulating NAC-dependent production of OGDHL nascent peptides and their mitochondrial translocation, and we also aimed to optimize the DICAR-JP sequence to be better therapy on DCM. MethodsSPR was to investigate the binding interaction between DICAR-JP and NAC. The function of DICAR-JP and DICAR-JP45 on OGDHL nascent peptide transfection was detected by in vitro translation approach. Biotin-DICAR-JP was transfected to study the function of DICAR-JP, NAC and OGDHL. Untargeted metabolomics was utilized to characterize the metabolic reprogramming of cardiomyocytes. AAV9-DICAR-JP and DICAR-JP45 were constructed and administered to db/db mice for 1-2 months to assess their cardioprotective effects detected by Echocardiography confirmed mouse cardiac function. ResultsOur findings demonstrated that DICAR-JP be the functional domain that interacts with NAC to regulate OGDHL nascent peptide expression. Disruption of OGDHL expression reversed the metabolic reprogramming observed in diabetic cardiomyocytes, highlighting its crucial role in maintaining cardiac metabolic homeostasis. DICAR-JP facilitated the translocation of OGDHL nascent peptides from the cytoplasm to the mitochondria, leading us to hypothesize that DICAR-JP plays a key role in regulating ribosomal migration from the endoplasmic reticulum to the mitochondria. We refer to this process as the Ribosome Migration. In our studies using AAV9-mediated DICAR-JP and DICAR-JP45 overexpression in heart tissue. DICAR-JP and DICAR-JP45 both exhibited significant cardioprotective effects against diabetic cardiomyopathy (DCM), comparable to those observed with Empagliflozin. ConclusionsIn a conclusion, we propose a new endogenous nucleic acid candidate drug library, and a new molecular framework, the Ribosome Migration, which implicates the DICAR-JP/NAC/OGDHL nascent peptide axis in metabolic reprogramming related to DCM. This theory constructs a new mitochondrion protein from nuclear original protein. Moreover, DICAR-JP45 shows strong potential as a nucleic acid-based therapeutic candidate for treating DCM. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSDICAR is a new protective circular noncoding RNA for diabetic cardiomyopathy. NAC is located in ribosome and regulates new peptide production, which is also a key factor in heart tissue development. OGDHL can regulate different energy metabolism in mitochondria. What New Information Does This Article Contribute?DICAR-JP is the functional domain that interacts with NAC to regulate OGDHL nascent peptide expression. There is an interaction between DICAR-JP and NAC. DICAR-JP sequence is protected by NAC and NAC function is mediated by DICAR-JP. We called this RNA functional domain. OGDHL expression reversed the metabolic reprogramming observed in diabetic cardiomyocytes, highlighting its crucial role in maintaining cardiac metabolic homeostasis. DICAR-JP takes part in tanscription and translocation of OGDHL nascent peptides from the cytoplasm to the mitochondria via plasma ribosome. We call this process the Ribosome Migration Theory. DICAR-JP and DICAR-JP45 both exhibited significant cardioprotective effects against diabetic cardiomyopathy (DCM), comparable to those observed with Dapagliflozin (DAPA). Our study revealed a novel molecular framework, the Ribosome Migration Theory, which implicates the DICAR-JP/NAC/OGDHL nascent peptide axis in metabolic reprogramming related to DCM. Moreover, DICAR-JP45 shows strong potential as a nucleic acid-based therapeutic candidate for treating DCM.

Upstream open reading frames (uORFs) are cis-regulatory motifs that are predicted to occur in the 5' untranslated region (UTR) of the majority of human protein-coding transcripts. uORFs are typically associated with repression of the downstream primary open reading frame (pORF) at either the level of translation, or by promoting mRNA turnover via the nonsense-mediated decay pathway. Interference with uORF activity provides a potential mechanism for targeted upregulation of the expression of specific transcripts. It was recently reported that steric block antisense oligonucleotides (ASOs) can bind to and mask uORF start codons in order to inhibit translation initiation, and thereby disrupt uORF-mediated gene regulation. Given the relative maturity of the oligonucleotide field, such a uORF blocking mechanism might have widespread therapeutic utility. Here, we re-synthesised three of the most potent ASOs targeting the RNASEH1 uORF described in the study by Liang et al. and investigated their potential for RNASEH1 protein upregulation. No upregulation (of endogenous or reporter protein expression) was observed with any of the oligonucleotides tested at doses ranging from 25 nM to 300 nM. Conversely, we observed downregulation of expression in some instances, consistent with well-established mechanisms of blocking ribosome procession. Experiments were performed using multiple transfection protocol setups, with care taken to replicate the conditions of the original study. Transfection efficiency was confirmed using a MALAT1-targeting gapmer ASO as a positive control. We conclude that previously-described RNASEH1 uORF-targeting steric block ASOs are incapable of upregulating pORF protein expression in our hands.

Autosomal recessive thymidine kinase 2 (TK2) mutations causes TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with deoxycytidine and thymidine ameliorates mitochondrial defects and extends lifespan of Tk2 knock-in mouse (TK2-/-); however, efficacy is limited by age- and tissue-dependent expression of the cytosolic enzymes Tk1 and Dck. Thus, therapies aimed at systemic restoration of TK2 activity are needed. Here, we demonstrate that delivery of human TK2 cDNA to Tk2-/- mice using AAV9 efficiently rescued Tk2 activity in all the tissues tested except kidney, delayed disease onset, and increased lifespan. Sequential treatment of Tk2-/- mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2-/- mice dramatically improved mtDNA copy numbers in liver and kidney, animal growth, and lifespan. These data indicate that combined pharmacological and gene therapies may be highly efficacious for human TK2 deficiency.

Delivery of cells or vectors in advanced therapies is probably the major challenge for genetic disorders that affect a large part of the body such as Duchenne Muscular Dystrophy (DMD). Here, we describe a novel approach for systemic cell delivery based upon an implantable bio-scaffold composed of aligned polycaprolactone nanofibers coated with laminin, able to support adhesion and extensive proliferation of mesoderm cells both in vitro and when implanted subcutaneously in a DMD mouse model. The scaffold is rapidly vascularised leading to cell entering the circulation and colonising multiple distal organs, including distant skeletal muscles and heart. Cells survive in colonized muscles and differentiate into muscle fibres that produce well detectable levels of dystrophin and -sarcoglycan. These results are game changing for cell therapy, as they allow colonization of life essential but "difficult to reach" muscles such as diaphragm and heart while avoiding invasive catheterization. Once optimised, this approach will rapidly enter clinical experimentation for DMD, other muscular dystrophies, and possibly other genetic disorders of the mesoderm. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/715524v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@11dfd34org.highwire.dtl.DTLVardef@1da6599org.highwire.dtl.DTLVardef@14427f0org.highwire.dtl.DTLVardef@19a242a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Study design and therapeutic outcome. Muscle biopsies were obtained from Duchenne muscular dystrophy (DMD) patients to isolate human DMD mesangioblasts (DMD-hMabs). Cells were genetically corrected using a lentivirus carrying a snRNA able to induce exon skipping (U7snRNA), generating U7-hMabs (1). U7-hMabs were seeded onto laminin-coated polycaprolactone (Lam-PCL) nanofiber scaffolds and implanted into the back muscle of DMD-NSG mice. This platform enabled systemic distribution of hMabs cells through circulation, resulting in engraftment across multiple muscle groups, including tibialis anterior, triceps, diaphragm and heart. C_FIG

LMNA-associated congenital muscular dystrophy is a currently incurable rare genetic disorder characterized by early-onset muscle weakness, dilated cardiomyopathy and respiratory failure, resulting from mutations in the LMNA gene. In this study, we assessed the potential of a CRISPR-mediated strategy to eliminate the mutant allele Lmna c.745C>T, p.R249W using a mutation specific guide (sg745T). Results from R249W-mutation-carrying cellular models showed specific activity of the Cas9/sg745T complex towards the mutant allele. This property varied depending on the concentration of CRISPR components, with a loss of specificity observed with increased dosage. We tested this strategy in vivo using adeno-associated virus delivery in LmnaR249W mice. Despite being associated with a modest CRISPR activity, this therapeutic approach resulted in a 10% increase in the survival of R249W homozygous mice. Interestingly, a similar CRISPR activity improved the cardiac pathology developed by Lmna+/R249W animals, significantly extending their median survival. These results represent the first therapeutic validation of a CRISPR/Cas9-mediated gene editing strategy for the treatment of LMNA-associated congenital muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal pediatric striated muscle disease caused by loss of dystrophin for which there is no cure. Cardiomyopathy is the leading cause of death amongst individuals with DMD, and effective therapeutics to treat DMD cardiomyopathy are a major unmet clinical need. This work investigated adeno-associated viral (AAV) gene therapy approaches to treat DMD cardiomyopathy by overexpression of the calcium binding proteins S100A1 and apoptosis repressor with caspase recruitment domains (ARC). Using the severe D2.mdx mouse model of DMD, we identified that S100A1 gene therapy improves the diastolic dysfunction associated with DMD cardiomyopathy, whereas ARC gene therapy prolongs survival. The combination of both S100A1 and ARC in a single bicistronic vector improves the long-term cardiac outcome of D2.mdx mice, development of heart failure caused by micro-dystrophin expression, and exhibits safety via intracoronary delivery in a canine model of DMD. Furthermore, S100A1-ARC gene therapy provides functional benefits when expressed in D2.mdx skeletal muscle. Together, these findings indicate that S100A1-ARC gene therapy represents an effective treatment for DMD cardiomyopathy and may be effective in treating other forms of cardiomyopathy and muscle pathologies. SIGNIFICANCE STATEMENTCardiomyopathy is the leading cause of death amongst individuals with Duchenne muscular dystrophy (DMD). Effective therapeutics to treat DMD cardiomyopathy represent a major unmet clinical need. This work identifies the dual gene therapy approach of S100A1 and ARC as an effective treatment that improves long-term cardiac function and life-expectancy in severe mouse model of DMD. Intracoronary delivery of this AAV-based gene therapy also exhibits safety and evidence of efficacy in dystrophic canines. Furthermore, functional benefits in skeletal muscle are also incurred via S100A1-ARC expression in striated muscle. These findings indicate that S100A1-ARC therapy is an effective treatment for DMD cardiomyopathy whose benefits may be applicable for other forms of cardiac and muscle disease.

Adeno associated virus (AAV)-mediated delivery of CRISPR associated nucleases (AAV-CRISPR) is a promising solution to treat genetic diseases such as Duchenne Muscular Dystrophy (DMD) and is now in early clinical trials. However, genotoxicity and immunogenicity concerns have hindered clinical translation. Due to the complex etiology associated with DMD, the post-transduction consequences of double-stranded breaks induced by AAV-CRISPR in disease models are unclear. This barrier is partially conferred by conventional sequencing methods where common outcomes of AAV-CRISPR editing often escape detection. However, recent reports of novel long-read sequencing approaches permit comprehensive variant detection using a broader sequence context. Here, we comprehensively investigated genomic and transcriptomic post-AAV-CRISPR transduction consequences in myoblast cells and a DMD mouse model following intramuscular and intravenous AAV-CRISPR therapy using both long- and short-read sequencing techniques. Structural variant characterization indicates that unintended on-target large insertions and inversions are common editing outcomes. We demonstrate that combining adaptive sampling with nanopore Cas9-targeted sequencing (AS-nCATS) for long-read quantification of AAV integration is synergistic for detecting difficult-to-amplify editing events. This unbiased data suggests that full-length AAV integration is equally as probable as the on-target deletion. Further, we develop a Nanopore Rapid Amplification of cDNA Ends (nRACE-seq) pipeline for long-read detection of unknown 5 or 3 ends of edited transcripts. The nRACE-seq approach effectively detects the presence of AAV-Dmd chimeric transcripts, erroneous splicing events, and off-target AAV integration sites. In summary, our findings offer insights into the adaptation of AAV-CRISPR DSB-mediated therapeutics for monogenic diseases and promote the standardization of CRISPR evaluation. We highlight the importance of coupling polymerase-based and polymerase-free methods in long-read sequencing to assess editing outcomes as the field progresses toward clinical applications.

The cellular and molecular consequences of lack of dystrophin in humans are only partially known, which is crucial for the development of new therapies aiming to slow or stop the progression Duchenne and Becker muscular dystrophies. We analyzed muscle biopsies of DMD patients and controls using single nuclei RNA sequencing (snRNAseq) and correlated the results with clinical data. DMD samples displayed an increase in regenerative fibers, satellite cells and fibro-adipogenic progenitor cells (FAPs) and a decrease in slow fibers and smooth muscle cells. Samples from patients with stable mild weakness were characterized by an increase in regenerative fibers, while those from patients with progressive weakness had fewer muscle fibers and increased FAPs. DMD muscle fibers displayed a strong regenerative signature, while DMD FAPs upregulated genes producing extracellular matrix and molecules involved in several signaling pathways. An analysis of intercellular communication profile identified FAPs as a key regulator of cell signaling in DMD samples. We show significant differences in the gene expression profiled of the different cell populations present in DMD muscle samples compared to controls.

Duchenne muscular dystrophy (DMD) is a severe, progressive genetic disorder primarily affecting boys, characterized by muscle degeneration due to mutations in the DMD gene encoding dystrophin, a crucial protein for muscle fiber integrity. The disease leads to significant muscle weakness and eventually to loss of ambulation. AAV-microdystrophin gene therapy shows promise in preclinical and clinical settings. However, muscle fibrosis, a consequence of chronic inflammation and extracellular matrix (ECM) remodeling, exacerbates disease progression and may hinder therapeutic efficacy. Periostin, a matricellular protein involved in fibrosis, is upregulated in DMD rodent models and correlates with collagen deposition. We previously developed an antisense oligonucleotide strategy to induce exon 17 skipping and so reduce periostin expression and collagen accumulation in the fibrotic D2.mdx mouse model of DMD. Here, we investigated the combined effects of periostin modulation and AAV-microdystrophin (AAV-MD1) treatment. We found that systemic periostin splicing modulation with intramuscular AAV-MD1 administration significantly improved muscle function, assessed by grip strength and treadmill performance, compared to either single treatment. Importantly, periostin exon skipping increases the microdystrophin protein expression. These findings suggest that targeting periostin in conjunction with microdystrophin therapy could represent a valid therapeutic strategy for DMD.