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Multiple sclerosis (MS) is an immune-mediated chronic inflammatory and neurodegenerative disease of the central nervous system (CNS) affecting more than 2.5 million patients worldwide. Chronic demyelination in the CNS has an important role in perpetuating axonal loss and increases difficulty in promoting remyelination. Therefore, regenerative, and neuroprotective strategies are essential to overcome this impediment to rescue axonal integrity and function. Neurotrophin 3 (NT-3) has immunomodulatory and anti-inflammatory properties, in addition to its well-recognized function in nervous system development, myelination, neuroprotection, and regeneration. For this study, scAAV1.tMCK.NT-3 was delivered to the gastrocnemius muscle of experimental autoimmune encephalomyelitis (EAE) mice, the chronic relapsing mouse model of MS, at 3 weeks post EAE induction. Measurable NT-3 levels were found in serum at 7-weeks post gene delivery. The treated cohort showed improved clinical scores and performed significantly better in rotarod, and grip strength tests compared to their untreated counterparts. Histopathologic studies showed improved remyelination and axon protection. These data correlated with reduced expression of the pro-inflammatory cytokines in brain and spinal cord, and increased percentage of regulatory T cells in the spleens and lymph nodes. Collectively, these findings demonstrate the translational potential of AAV-delivered NT-3 for chronic progressive MS., Competing Interests: Competing interests: The authors declare no competing interest. Ethical approval: All animal experiments were performed according to the guidelines approved by the Research Institute at Nationwide Children’s Hospital Animal Care and Use Committee, that operates in full accordance with the Animal Welfare Act and the Health Research Extension Act (IACUC approval number: AR21-00140)., (© 2025. The Author(s).)

Surgery of the tracheobronchial tree carries high morbidity, with over half of the complications occurring at the anastomosis. Although fibroblasts are crucial in airway wound healing, the underlying cellular and molecular mechanisms in airway reconstruction remain unknown. We hypothesized that airway reconstruction initiates a surgery-induced stress (SIS) response, altering fibroblast communication within airway tissues. Using single-cell RNA-Seq, we analyzed native and reconstructed airways and identified 5 fibroblast subpopulations, each with distinct spatial distributions across anastomotic, submucosal, perichondrial, and paratracheal areas. During homeostasis, adventitial and airway fibroblasts (Adventitial-Fb and Airway-Fb, respectively) maintained tissue structure and created cellular niches by regulating ECM turnover. Under SIS, perichondrial fibroblasts (PC-Fb) exhibited chondroprogenitor-like gene signatures, and immune-recruiting fibroblasts (IR-Fb) facilitated cell infiltration. Cthrc1-activated fibroblasts (Cthrc1+ Fb), mainly derived from Adventitial-Fb, primarily contributed to fibrotic scar formation and collagen production, mediated by TGF-β. Furthermore, repeated SIS created an imbalance in fibroblast states favoring emergence of CTHRC1+ Fb and leading to impaired fibroblasts-basal cell crosstalk. Collectively, these data identify PC, IR, and Cthrc1+ Fb as a signaling hub, with SIS emerging as a mechanism initiating airway remodeling after reconstruction that, if not controlled, may lead to complications such as stenosis or anastomotic breakdown.

Single-stranded DNA (ssDNA) templates along with Cas9 have been used for knocking-in exogenous sequences in the genome but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 12-19% of internal bases, which we denote as enhanced ssDNA (esDNA), improve knock-in (KI) by 2-3-fold compared to end-modified ssDNA in airway basal stem cells (ABCs), CD34 + hematopoietic cells (CD34 + cells), T-cells and endothelial cells. Over 50% of alleles showed KI in three clinically relevant loci (CFTR, HBB and CCR5) in ABCs using esDNA and up to 70% of alleles showed KI in the HBB locus in CD34 + cells in the presence of a DNA-PKcs inhibitor. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. The esDNA templates did not improve KI in induced pluripotent stem cells (iPSCs). This may be due to the absence of the nuclease TREX1 in iPSCs. Indeed, knocking out TREX1 in other cells improved KI using unmodified ssDNA. esDNA can be used to modify 20-30 bp regions in primary cells for therapeutic applications and biological modeling. The use of this approach for gene length insertions will require new methods to produce long chemically modified ssDNA in scalable quantities., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which the CFTR cDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two-halves of the CFTR cDNA and an enrichment cassette into the CFTR locus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improve CFTR cDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558, which inhibit non-homologous end-joining (NHEJ) and micro-homology mediated end-joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2- to 3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which the CFTR cDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two halves of the CFTR cDNA and an enrichment cassette into the CFTR locus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improve CFTR cDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558 which inhibit non-homologous end joining (NHEJ) and micro-homology mediated end joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2-3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations., Competing Interests: Declaration of Interests None of the authors have any conflict to declare.

Recent clinical studies of single gene replacement therapy for neuromuscular disorders have shown they can slow or stop disease progression, but such therapies have had little impact on reversing muscle disease that was already present. To reverse disease in patients with muscular dystrophy, new muscle mass and strength must be rebuilt at the same time that gene replacement prevents subsequent disease. Here, we show that treatment of FKRP P448L mice with a dual FKRP/FST gene therapy packaged into a single adeno-associated virus (AAV) vector can build muscle strength and mass that exceed levels found in wild-type mice and can induce normal ambulation endurance in a 1-h walk test. Dual FKRP/FST therapy also showed more even increases in muscle mass and amplified muscle expression of both genes relative to either single gene therapy alone. These data suggest that treatment with single AAV-bearing dual FKRP/FST gene therapies can overcome loss of ambulation by improving muscle strength at the same time it prevents subsequent muscle damage. This design platform could be used to create therapies for other forms of muscular dystrophy that may improve patient outcomes., Competing Interests: Declaration of interests P.T.M. has financial conflicts of interest with Sarepta Therapeutics and Genosera Inc. A patent has been filed on this technology., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Neuroinflammation is a miscreant in accelerating progression of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, treatments targeting neuroinflammation alone have led to disappointing results in clinical trials. Both neuronal and non-neuronal cell types have been implicated in the pathogenesis of ALS, and multiple studies have shown correction of each cell type has beneficial effects on disease outcome. Previously, we shown that AAV9-mediated superoxide dismutase 1 (SOD1) suppression in motor neurons and astrocytes significantly improves motor function and extends survival in ALS mouse models. Despite neuron and astrocyte correction, ALS mice still succumb to death with microgliosis observed in endpoint tissue. Therefore, we hypothesized that the optimal therapeutic approach will target and simultaneously correct motor neurons, astrocytes, and microglia. Here, we developed a novel approach to indirectly target microglia with galectin-1 (Gal1) and combined this with our previously established AAV9.SOD1.short hairpin RNA treatment. We show Gal1 conditioning of SOD1 G93A microglia decreases inflammatory markers and rescues motor neuron death in vitro . When paired with SOD1 downregulation, we found a synergistic effect of combination treatment in vivo and show a significant extension of survival of SOD1 G93A mice over SOD1 suppression alone. These results highlight the importance of targeting inflammatory microglia as a critical component in future therapeutic development., Competing Interests: A patent on AAV gene therapy vectors that contain a therapeutic protein and additionally express an anti-inflammatory protein or peptide has been filed by The Research Institute at Nationwide Children’s Hospital with K.C.M. and S.B.L. as inventors., (© 2024 The Authors.)

Leukoencephalopathy with vanishing white matter (VWM) is a progressive incurable white matter disease that most commonly occurs in childhood and presents with ataxia, spasticity, neurological degeneration, seizures, and premature death. A distinctive feature is episodes of rapid neurological deterioration provoked by stressors such as infection, seizures, or trauma. VWM is caused by autosomal recessive mutations in one of five genes that encode the eukaryotic initiation factor 2B complex, which is necessary for protein translation and regulation of the integrated stress response. The majority of mutations are in EIF2B5. Astrocytic dysfunction is central to pathophysiology, thereby constituting a potential therapeutic target. Herein we characterize two VWM murine models and investigate astrocyte-targeted adeno-associated virus serotype 9 (AAV9)-mediated EIF2B5 gene supplementation therapy as a therapeutic option for VWM. Our results demonstrate significant rescue in body weight, motor function, gait normalization, life extension, and finally, evidence that gene supplementation attenuates demyelination. Last, the greatest rescue results from a vector using a modified glial fibrillary acidic protein (GFAP) promoter-AAV9-gfaABC(1)D-EIF2B5-thereby supporting that astrocytic targeting is critical for disease correction. In conclusion, we demonstrate safety and early efficacy through treatment with a translatable astrocyte-targeted gene supplementation therapy for a disease that has no cure., Competing Interests: Declaration of interests J.L.B. is on the board of wFluidx, Inc., owns stock in Orchard Therapeutics, and has consulted for Bluebird bio, Calico Life Sciences, Denali Therapeutics, Enzyvant, and Neurogene. A.M.B. is a beneficiary of a licensing agreement with Axovant Gene Therapies (royalties) and Neurogene (royalties) and has received income from Neurogene (consulting and honorarium). A.M.B., K.M.F., J.L.B., N.K.P., and J.A.H. are inventors on a provisional patent: Materials and Methods for the Treatment of EIF2B5 Mutations and Diseases Resulting Therefrom. Provisional Patent 63/316,241., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Ongoing cardiomyocyte injury is a major mechanism in the progression of heart failure, particularly in dystrophic hearts. Due to the poor regenerative capacity of the adult heart, cardiomyocyte death results in the permanent loss of functional myocardium. Understanding the factors contributing to myocyte injury is essential for the development of effective heart failure therapies. As a model of persistent cardiac injury, we examined mice lacking β-sarcoglycan (β-SG), a key component of the dystrophin glycoprotein complex (DGC). The loss of the sarcoglycan complex markedly compromises sarcolemmal integrity in this β-SG -/- model. Our studies aim to characterize the mechanisms underlying dramatic sex differences in susceptibility to cardiac injury in β-SG -/- mice. Male β-SG -/- hearts display significantly greater myocardial injury and death following isoproterenol-induced cardiac stress than female β-SG -/- hearts. This protection of females was independent of ovarian hormones. Male β-SG -/- hearts displayed increased susceptibility to exogenous oxidative stress and were significantly protected by angiotensin II type 1 receptor (AT 1 R) antagonism. Increasing general antioxidative defenses or increasing the levels of S-nitrosylation both provided protection to the hearts of β-SG -/- male mice. Here we demonstrate that increased susceptibility to oxidative damage leads to an AT 1 R-mediated amplification of workload-induced myocardial injury in male β-SG -/- mice. Improving oxidative defenses, specifically by increasing S-nitrosylation, provided protection to the male β-SG -/- heart from workload-induced injury. These studies describe a unique susceptibility of the male heart to injury and may contribute to the sex differences in other forms of cardiac injury., Competing Interests: Declaration of competing interest The authors have no conflict of interests to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Chronic kidney disease (CKD) is a leading cause of death, and its progression is driven by glomerular podocyte injury and loss, manifesting as proteinuria. Proteinuria includes urinary loss of coagulation zymogens, cofactors, and inhibitors. Importantly, both CKD and proteinuria significantly increase the risk of thromboembolic disease. Prior studies demonstrated that anticoagulants reduced proteinuria in rats and that thrombin injured cultured podocytes. Herein we aimed to directly determine the influence of circulating prothrombin on glomerular pathobiology. We hypothesized that (pro)thrombin drives podocytopathy, podocytopenia, and proteinuria. Glomerular proteinuria was induced with puromycin aminonucleoside (PAN) in Wistar rats. Circulating prothrombin was either knocked down using a rat-specific antisense oligonucleotide or elevated by serial intravenous infusions of prothrombin protein, which are previously established methods to model hypo- (LoPT) and hyper-prothrombinemia (HiPT), respectively. After 10 days (peak proteinuria in this model) plasma prothrombin levels were determined, kidneys were examined for (pro)thrombin co-localization to podocytes, histology, and electron microscopy. Podocytopathy and podocytopenia were determined and proteinuria, and plasma albumin were measured. LoPT significantly reduced prothrombin colocalization to podocytes, podocytopathy, and proteinuria with improved plasma albumin. In contrast, HiPT significantly increased podocytopathy and proteinuria. Podocytopenia was significantly reduced in LoPT vs. HiPT rats. In summary, prothrombin knockdown ameliorated PAN-induced glomerular disease whereas hyper-prothrombinemia exacerbated disease. Thus, (pro)thrombin antagonism may be a viable strategy to simultaneously provide thromboprophylaxis and prevent podocytopathy-mediated CKD progression.

Purpose: For patients with osteosarcoma, disease-related mortality most often results from lung metastasis-a phenomenon shared with many solid tumors. While established metastatic lesions behave aggressively, very few of the tumor cells that reach the lung will survive. By identifying mechanisms that facilitate survival of disseminated tumor cells, we can develop therapeutic strategies that prevent and treat metastasis., Methods: We analyzed single cell RNA-sequencing (scRNAseq) data from murine metastasis-bearing lungs to interrogate changes in both host and tumor cells during colonization. We used these data to elucidate pathways that become activated in cells that survive dissemination and identify candidate host-derived signals that drive activation. We validated these findings through live cell reporter systems, immunocytochemistry, and fluorescent immunohistochemistry. We then validated the functional relevance of key candidates using pharmacologic inhibition in models of metastatic osteosarcoma., Results: Expression patterns suggest that the MAPK pathway is significantly elevated in early and established metastases. MAPK activity correlates with expression of anti-apoptotic genes, especially MCL1. Niche cells produce growth factors that increase ERK phosphorylation and MCL1 expression in tumor cells. Both early and established metastases are vulnerable to MCL1 inhibition, but not MEK inhibition in vivo. Combining MCL1 inhibition with chemotherapy both prevented colonization and eliminated established metastases in murine models of osteosarcoma., Conclusion: Niche-derived growth factors drive MAPK activity and MCL1 expression in osteosarcoma, promoting metastatic colonization. Although later metastases produce less MCL1, they remain dependent on it. MCL1 is a promising target for clinical trials in both human and canine patients., (© 2023. The Author(s).)

Duchenne muscular dystrophy (DMD) is a progressive X-linked disease caused by mutations in the DMD gene that prevent the expression of a functional dystrophin protein. Exon duplications represent 6%-11% of mutations, and duplications of exon 2 (Dup2) are the most common (∼11%) of duplication mutations. An exon-skipping strategy for Dup2 mutations presents a large therapeutic window. Skipping one exon copy results in full-length dystrophin expression, whereas skipping of both copies (Del2) activates an internal ribosomal entry site (IRES) in exon 5, inducing the expression of a highly functional truncated dystrophin isoform. We have previously confirmed the therapeutic efficacy of AAV9.U7snRNA-mediated skipping in the Dup2 mouse model and showed the absence of off-target splicing effects and lack of toxicity in mice and nonhuman primates. Here, we report long-term dystrophin expression data following the treatment of 3-month-old Dup2 mice with the scAAV9.U7.ACCA vector. Significant exon 2 skipping and robust dystrophin expression in the muscles and hearts of treated mice persist at 18 months after treatment, along with the partial rescue of muscle function. These data extend our previous findings and show that scAAV9.U7.ACCA provides long-term protection by restoring the disrupted dystrophin reading frame in the context of exon 2 duplications., Competing Interests: NCH licensed the vector described herein to Audentes Therapeutics, which was subsequently acquired by Astellas Therapeutics. N.W. and K.M.F. have received royalty payments as a result of this license., (© 2023 The Authors.)

Duchenne muscular dystrophy is an X-linked disorder typically caused by out-of-frame mutations in the DMD gene. Most of these are deletions of one or more exons, which can theoretically be corrected through CRISPR-Cas9-mediated knockin. Homology-independent targeted integration is a mechanism for achieving such a knockin without reliance on homology-directed repair pathways, which are inactive in muscle. We designed a system based on insertion into intron 19 of a DNA fragment containing a pre-spliced mega-exon encoding DMD exons 1-19, along with the MHCK7 promoter, and delivered it via a pair of AAV9 vectors in mice carrying a Dmd exon 2 duplication. Maximal efficiency was achieved using a Cas9:donor adeno-associated virus (AAV) ratio of 1:5, with Cas9 under the control of the SPc5-12 promoter. This approach achieved editing of 1.4% of genomes in the heart, leading to 30% correction at the transcript level and restoration of 11% of normal dystrophin levels. Treatment efficacy was lower in skeletal muscles. Sequencing additionally revealed integration of fragmentary and recombined AAV genomes at the target site. These data provide proof of concept for a gene editing system that could restore full-length dystrophin in individuals carrying mutations upstream of intron 19, accounting for approximately 25% of Duchenne muscular dystrophy patients., Competing Interests: A.A.S. and K.M.F. are inventors on a pending patent application filed by Nationwide Children’s Hospital for the gene editing system presented in this work., (© 2023 The Authors.)

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults and affects mainly the skeletal muscle, heart, and brain. DM1 is caused by a CTG repeat expansion in the 3'UTR region of the DMPK gene that sequesters muscleblind-like proteins, blocking their splicing activity and forming nuclear RNA foci . Consequently, many genes have their splicing reversed to a fetal pattern. There is no treatment for DM1, but several approaches have been explored, including antisense oligonucleotides (ASOs) aiming to knock down DMPK expression or bind to the CTGs expansion. ASOs were shown to reduce RNA foci and restore the splicing pattern. However, ASOs have several limitations and although being safe treated DM1 patients did not demonstrate improvement in a human clinical trial. AAV-based gene therapies have the potential to overcome such limitations, providing longer and more stable expression of antisense sequences. In the present study, we designed different antisense sequences targeting exons 5 or 8 of DMPK and the CTG repeat tract aiming to knock down DMPK expression or promote steric hindrance, respectively. The antisense sequences were inserted in U7snRNAs, which were then vectorized in AAV8 particles. Patient-derived myoblasts treated with AAV8. U7snRNAs showed a significant reduction in the number of RNA foci and re-localization of muscle-blind protein. RNA-seq analysis revealed a global splicing correction in different patient-cell lines, without alteration in DMPK expression., Competing Interests: Since the performance of this work, Nationwide Children’s Hospital licensed the vector described herein to Audentes/Astellas Therapeutics. NW and KF have received royalty payments as a result of this license. NW is a Scientific Advisor for Alcyone Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Almeida, Robriquet, Vetter, Huang, Neinast, Hernandez-Rosario, Rajakumar, Arnold, McBride, Flanigan, Weiss and Wein.)

Anc80L65 is a synthetic, ancestral adeno-associated virus that has high tropism toward retinal photoreceptors after subretinal injection in mice and non-human primates. We characterized, for the first time, the post-intravitreal cell-specific transduction profile of Anc80L65 compared with AAV9. Here we use Anc80L65 and AAV9 to intravitreally deliver a copy of the gene encoding GFP into WT C57Bl/6J mice. GFP expression was driven by one of two clinically relevant promoters, chicken β actin (CB) or truncated MECP2 (P546). After qualitative assessment of relative GFP expression, we found Anc80L65 and AAV9 to have similar transduction profiles. Through the development of a novel method for quantifying GFP-positive retinal cells, we found Anc80L65 to have higher tropism in Müller glia and AAV9 to have higher tropism in horizontal cells. In addition, we found P546 to promote GFP expression at a more moderate level compared with the high levels seen under the CB promoter. Finally, for the first time, we characterized Anc80L65 cross-reactivity in human sera; 83% of patients with AAV2 pre-existing antibodies were found to be seropositive for Anc80L65. This study demonstrates the expanded therapeutic applications of Anc80L65 to treat retinal disease and provides the first insights to Anc80L65 pre-existing immunity in humans., Competing Interests: K.M. is receiving royalties from Gene Therapy programs licensed to Amicus Therapeutics and Kiadis. K.M. is a scientific advisor with compensation and stock options and has received research funding from Alcyone Therapeutics., (© 2023 The Authors.)

Background: Tumors are complex tissues containing collections of phenotypically diverse malignant and nonmalignant cells. We know little of the mechanisms that govern heterogeneity of tumor cells nor of the role heterogeneity plays in overcoming stresses, such as adaptation to different microenvironments. Osteosarcoma is an ideal model for studying these mechanisms-it exhibits widespread inter- and intra-tumoral heterogeneity, predictable patterns of metastasis, and a lack of clear targetable driver mutations. Understanding the processes that facilitate adaptation to primary and metastatic microenvironments could inform the development of therapeutic targeting strategies., Results: We investigated single-cell RNA-sequencing profiles of 47,977 cells obtained from cell line and patient-derived xenograft models as cells adapted to growth within primary bone and metastatic lung environments. Tumor cells maintained phenotypic heterogeneity as they responded to the selective pressures imposed during bone and lung colonization. Heterogenous subsets of cells defined by distinct transcriptional profiles were maintained within bone- and lung-colonizing tumors, despite high-level selection. One prominent heterogenous feature involving glucose metabolism was clearly validated using immunofluorescence staining. Finally, using concurrent lineage tracing and single-cell transcriptomics, we found that lung colonization enriches for multiple clones with distinct transcriptional profiles that are preserved across cellular generations., Conclusions: Response to environmental stressors occurs through complex and dynamic phenotypic adaptations. Heterogeneity is maintained, even in conditions that enforce clonal selection. These findings likely reflect the influences of developmental processes promoting diversification of tumor cell subpopulations, which are retained, even in the face of selective pressures., (© 2023. The Author(s).)

Duchenne muscular dystrophy (DMD) is a devastating muscle-wasting disease that arises due to the loss of dystrophin expression, leading to progressive loss of motor and cardiorespiratory function. Four exon-skipping approaches using antisense phosphorodiamidate morpholino oligomers (PMOs) have been approved by the FDA to restore a DMD open reading frame, resulting in expression of a functional but internally deleted dystrophin protein, but in patients with single-exon duplications, exon skipping has the potential to restore full-length dystrophin expression. Cell-penetrating peptide-conjugated PMOs (PPMOs) have demonstrated enhanced cellular uptake and more efficient dystrophin restoration than unconjugated PMOs. In the present study, we demonstrate widespread PPMO-mediated dystrophin restoration in the Dup2 mouse model of exon 2 duplication, representing the most common single-exon duplication among patients with DMD. In this proof-of-concept study, a single intravenous injection of PPMO targeting the exon 2 splice acceptor site induced 45% to 68% exon 2-skipped Dmd transcripts in Dup2 skeletal muscles 15 days post-injection. Muscle dystrophin restoration peaked at 77% to 87% average dystrophin-positive fibers and 41% to 51% of normal signal intensity by immunofluorescence, and 15.7% to 56.8% of normal by western blotting 15 to 30 days after treatment. These findings indicate that PPMO-mediated exon skipping is a promising therapeutic strategy for muscle dystrophin restoration in the context of exon 2 duplications., Competing Interests: K.M.F. has received research support for a clinical trial from Sarepta Therapeutics. K.M.F. and N.W. hold a patent related to therapeutic exon 2 skipping, and along with Nationwide Children’s Hospital receive royalties from Astellas Gene Therapy for that patent, which is not directly related to the data presented here., (© 2022 The Authors.)

In a phase 1/2, open-label dose escalation trial, we delivered rAAVrh74.MCK. GALGT2 (also B4GALNT2 ) bilaterally to the legs of two boys with Duchenne muscular dystrophy using intravascular limb infusion. Subject 1 (age 8.9 years at dosing) received 2.5 × 10 13 vector genome (vg)/kg per leg (5 × 10 13 vg/kg total) and subject 2 (age 6.9 years at dosing) received 5 × 10 13 vg/kg per leg (1 × 10 14 vg/kg total). No serious adverse events were observed. Muscle biopsy evaluated 3 or 4 months post treatment versus baseline showed evidence of GALGT2 gene expression and GALGT2 -induced muscle cell glycosylation. Functionally, subject 1 showed a decline in 6-min walk test (6MWT) distance; an increase in time to run 100 m, and a decline in North Star Ambulatory Assessment (NSAA) score until ambulation was lost at 24 months. Subject 2, treated at a younger age and at a higher dose, demonstrated an improvement over 24 months in NSAA score (from 20 to 23 points), an increase in 6MWT distance (from 405 to 478 m), and only a minimal increase in 100 m time (45.6-48.4 s). These data suggest preliminary safety at a dose of 1 × 10 14 vg/kg and functional stabilization in one patient., Competing Interests: P.T.M. and Nationwide Children’s Hospital report a financial conflict of interest related to this study, as they both receive licensing fees from Sarepta Therapeutics for rAAVrh74.MCK.GALGT2. P.T.M. is also co-founder and President of Genosera Inc., (© 2022 The Authors.)

Duchenne muscular dystrophy (DMD) is typically caused by mutations that disrupt the DMD reading frame, but nonsense mutations in the 5' part of the gene induce utilization of an internal ribosomal entry site (IRES) in exon 5, driving expression of a highly functional N-truncated dystrophin. We have developed an AAV9 vector expressing U7 small nuclear RNAs targeting DMD exon 2 and have tested it in a mouse containing a duplication of exon 2, in which skipping of both exon 2 copies induces IRES-driven expression, and skipping of one copy leads to wild-type dystrophin expression. One-time intravascular injection either at postnatal days 0-1 or at 2 months results in efficient exon skipping and dystrophin expression, and significant protection from functional and pathologic deficits. Immunofluorescence quantification showed 33%-53% average dystrophin intensity and 55%-79% average dystrophin-positive fibers in mice treated in adulthood, with partial amelioration of DMD pathology and correction of DMD-associated alterations in gene expression. In mice treated neonatally, dystrophin immunofluorescence reached 49%-85% of normal intensity and 76%-99% dystrophin-positive fibers, with near-complete correction of dystrophic pathology, and these beneficial effects persisted for at least 6 months. Our results demonstrate the robustness, durability, and safety of exon 2 skipping using scAAV9.U7snRNA.ACCA, supporting its clinical use., Competing Interests: Since the performance of this work, Nationwide Children’s Hospital licensed the vector described herein to Audentes/Astellas Therapeutics. N.W., A.V., and K.M.F. have received royalty payments as a result of this license., (© 2022 The Authors.)

Aims: Dystrophin, the protein product of the DMD gene, plays a critical role in muscle integrity by stabilising the sarcolemma during contraction and relaxation. The DMD gene is vulnerable to a variety of mutations that may cause complete loss, depletion or truncation of the protein, leading to Duchenne and Becker muscular dystrophies. Precise and reproducible dystrophin quantification is essential in characterising DMD mutations and evaluating the outcome of efforts to induce dystrophin through gene therapies. Immunofluorescence microscopy offers high sensitivity to low levels of protein expression along with confirmation of localisation, making it a critical component of quantitative dystrophin expression assays., Methods: We have developed an automated and unbiased approach for precise quantification of dystrophin immunofluorescence in muscle sections. This methodology uses microscope images of whole-tissue sections stained for dystrophin and spectrin to measure dystrophin intensity and the proportion of dystrophin-positive coverage at the sarcolemma of each muscle fibre. To ensure objectivity, the thresholds for dystrophin and spectrin are derived empirically from non-sarcolemmal signal intensity within each tissue section. Furthermore, this approach is readily adaptable for measuring fibre morphology and other tissue markers., Results: Our method demonstrates the sensitivity and reproducibility of this quantification approach across a wide range of dystrophin expression in both dystrophinopathy patient and healthy control samples, with high inter-operator concordance., Conclusion: As efforts to restore dystrophin expression in dystrophic muscle bring new potential therapies into clinical trials, this methodology represents a valuable tool for efficient and precise analysis of dystrophin and other muscle markers that reflect treatment efficacy., (© 2021 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

There has been increasing momentum recently in the biopharmaceutical industry to transition from traditional batch processes to next-generation integrated and continuous biomanufacturing. This transition from batch to continuous is expected to offer several advantages which, taken together, could significantly improve access to biologics drugs for patients. Despite this recent momentum, there has not been a commercial implementation of a continuous bioprocess reported in the literature. In this study, we describe a successful pilot-scale proof-of-concept demonstration of an end-to-end integrated and continuous bioprocess for the production of a monoclonal antibody (mAb). This process incorporated all of the key unit operations found in a typical mAb production process, including the final steps of virus removal filtration, ultrafiltration, diafiltration, and formulation. The end-to-end integrated process was operated for a total of 25 days and produced a total of 4.9 kg (200 g/day or 2 g/L BRX/day) of the drug substance from a 100-L perfusion bioreactor (BRX) with acceptable product quality and minimal operator intervention. This successful proof-of-concept demonstrates that end-to-end integrated continuous bioprocessing is achievable with current technologies and represents an important step toward the realization of a commercial integrated and continuous bioprocessing process., (© 2021 Wiley Periodicals LLC.)

Duchenne muscular dystrophy (DMD) is an X-linked progressive disease characterized by loss of dystrophin protein that typically results from truncating mutations in the DMD gene. Current exon-skipping therapies have sought to treat deletion mutations that abolish an open reading frame (ORF) by skipping an adjacent exon, in order to restore an ORF that allows translation of an internally deleted yet partially functional protein, as is seen with many patients with the milder Becker muscular dystrophy (BMD) phenotype. In contrast to that approach, skipping of one copy of a duplicated exon would be expected to result in a full-length transcript and production of a wild-type protein. We have developed an adeno-associated virus (AAV)-based U7snRNA exon-skipping approach directed toward exon 2, duplications of which represent 10% of all DMD duplication mutations. Deletion of exon 2 results in utilization of an exon 5 internal ribosome entry site (IRES) that allows translation beginning in exon 6 of a highly protective dystrophin protein, providing a wide therapeutic window for treatment. Both intramuscular and systemic administration of this vector in the Dup2 mouse model results in robust dystrophin expression and correction of muscle physiologic defects, allowing dose escalation to establish a putative minimal efficacious dose for a human clinical trial., Competing Interests: Since the performance of this work, Nationwide Children’s Hospital has licensed the vector described herein to Audentes Therapeutics. N.W., A.V.C., and K.M.F. have received royalty payments as a result of this license., (© 2021 The Authors.)