Molecular Therapy Nucleic Acids

The need for safe, allogeneic cell therapies for cancer is driving a growing interest in CAR-NK-based therapies, which, unlike CAR-T cell therapies, offer the potential for off-the-shelf administration. Lentiviruses pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) are commonly used for genetic modification of cell therapy products. Their use in NK cells, however, is limited by low transduction efficiency. This study explores the complexities of NK cell transduction using lentiviral vectors pseudotyped with VSV-G. We demonstrate that efficient transduction depends on multiple factors such as NK cell activation, construct design, lentivirus pseudotype selection, and the use of transduction enhancers. By optimizing these elements, we achieved effective transduction, facilitating the use of VSV-G-pseudotyped LVs for therapeutic NK cell production. Our optimized workflow comprises NK cell activation with interleukins, followed by transduction with a NK cell-specific CAR construct using VSV-G-pseudotyped LVs in the presence of BX795 and Retronectin, resulting in excellent transduction efficiency without compromising NK cell phenotype or growth. This allows for the use of a widely used gene transfer vector with an excellent safety record for producing therapeutic NK cell products.

Rotavirus is a major cause of severe diarrheal disease in children under the age of five, with reduced vaccine effectiveness in low-resource settings causing substantial morbidity and mortality. In the absence of approved antiviral therapeutics, treatment is largely supportive, urging the need for targeted and precision-based interventions. VP4 protein plays an essential role in viral attachment, entry, and infectivity, making it a suitable target for targeted therapy. In this context, RNA interference is a specific method for inhibiting viral gene expression with its efficacy depending on sequence conservation, target accessibility, and compatibility with the RISC-loading machinery. In the present study, an integrative in silico approach was employed to design and evaluate siRNAs targeting conserved regions of the VP4 gene across six geographically diverse countries. Candidate siRNAs were screened using established design rules and regression-based scoring with off-target filtering. Three optimized siRNAs were further assessed through structural modeling, molecular docking, and molecular dynamics simulations to examine interactions with human Dicer, TRBP, and Argonaute-2. Comparative dynamic analyses identified one siRNA with enhanced structural compatibility, reduced conformational fluctuations, and stable interactions with RISC-loading proteins. These findings provide a rational computational basis for VP4-targeted siRNA development, facilitating experimental validation.

Duchenne muscular dystrophy (DMD) is the most common, lethal X-linked neuromuscular disorder of childhood and is caused by mutations in the Dmd gene that disrupt dystrophin expression. Although adeno-associated virus-mediated gene therapies hold tremendous promise for DMD treatment, their clinical applications have been limited by dose-dependent vector and genome-level toxicities. Here, we developed and tested a single-vector adenine base editing strategy as a potentially safer genome editing approach to recode the pathogenic nonsense mutation into a benign missense mutation in mdx4cvDMD mouse model. Delivered using a muscle-tropic adeno-associated virus (MyoAAV) at a clinically-feasible dose (4E13 VG/kg), this strategy enabled detectable molecular recoding of the mdx4cv mutation in mice ranging in age from 3 days to 6 months. Yet, the overall efficiency and therapeutic impact of in vivo base editing with this system was highest in mice treated at the juvenile stage, with animals administered MyoAAV vectors at 3 weeks of age showing robust recovery of dystrophin expression and significant improvement in muscle contractile properties only one month later. Notably, introduction of adenine base editors either earlier in development, in neonatal mice, or later, in adulthood, yielded substantially lower editing efficiencies, particularly in muscle satellite cells whose editing is essential to ensure durable rescue of dystrophin expression in growing and regenerating muscle. Taken together, these results demonstrate the therapeutic potential of single-vector adenine base editing for DMD and underscore the importance of recipient age and disease stage in achieving optimal treatment outcomes for this and other genetic muscle disorders.

Differentiation-based therapies represent a promising strategy for the treatment of neuroblastoma; however, single-agent approaches frequently yield incomplete and transient responses due to the robustness of underlying gene regulatory networks. MicroRNAs (miRNAs) are endogenous regulators of gene expression that modulate entire gene programs rather than individual molecular targets, making them attractive candidates for network-level therapeutic intervention. While individual miRNAs have been investigated as therapeutic agents, the potential for synergistic interactions between miRNAs remains largely unexplored. Here, we developed a scalable high-content phenotypic screening platform to identify synergistic miRNA combinations that promote neuronal differentiation and growth arrest in neuroblastoma cells. Using SK-N-BE(2)-C cells and automated quantification of neurite outgrowth and confluence, we screened pairwise combinations of differentiation-associated miRNAs at submaximal doses. Candidate synergistic interactions were identified using the Highest Single Agent framework and subsequently validated by dose-response interaction modeling. We identified a robust synergistic interaction between miR-124-3p and miR-363-3p that exceeded zero-interaction potency expectations by approximately 20.9% and increased maximal differentiation-associated phenotypic response by 73% relative to single-miRNA treatments. Target gene and pathway enrichment analyses revealed that miR-124-3p and miR-363-3p regulate largely distinct but functionally complementary target gene sets. These complementary targets converged on neuronal differentiation and cell cycle control pathways, providing a mechanistic basis for their cooperative activity. Together, these findings establish miRNA combinations as programmable network regulators capable of inducing complex cellular phenotypes with greater efficacy than single agents. This work provides a conceptual and experimental framework for the rational discovery of synergistic miRNA therapeutics and suggests new avenues for differentiation-based treatment strategies in neuroblastoma and other diseases driven by dysregulated regulatory networks.

Recombinant adeno-associated virus (rAAV) vectors are pivotal for gene therapy; however, the encapsidation of residual DNA, particularly plasmid backbone sequences, pose significant safety risks. Recent studies have identified the p5 promoter, which contains a Rep-binding element and a terminal resolution site (TRS), as a cryptic origin of replication that facilitates packaging of upstream sequences. In this study, we investigated the effect of p5 TRS modifications on impurity DNA levels in a single-plasmid All-in-One (AiO) AAV production system. Wild-type p5 (p5wt) promoted significant packaging of upstream plasmid backbone DNA, especially when the backbone was positioned between p5wt and the inverted terminal repeat. Introducing mutations or deletions in the p5 TRS significantly reduced encapsidation of plasmid-derived sequences, including kanamycin resistance genes, and improved the ratio of full to partial particles, as seen with the p5{Delta}loop variant. Furthermore, the p5{Delta}loop-AiO system showed higher rAAV yields than both conventional triple-transfection methods and previously reported p5-spacer variants. Thus, our findings suggest a robust vector design strategy for minimizing DNA impurities, thereby enhancing the safety and efficacy of AAV-based gene therapy.

Recombinant Adeno-associated viruses (AAVs) are widely used for gene delivery in the central nervous system and have become central tools in both gene therapy and basic neuroscience research. However, although AAV serotypes have been extensively characterized in rodent models, their performance in human neurons, particularly those derived from induced pluripotent stem cells (iPSCs), remains poorly characterized. While human iPSC-derived neurons are increasingly used for disease modeling and drug screening, their susceptibility to viral transduction varies and remains difficult to predict. In this study, we systematically evaluated the transduction efficiency and toxicity profiles of 18 wild-type and engineered AAV serotypes across three distinct types of iPSC-derived neurons, relevant to disease modeling and drug discovery: cortical projection neurons, NGN2- induced forebrain-like neurons, and dopaminergic neurons and four doses (1E3, 1E4, 1E5 and 2E5 genome copies per cell). Using automated high-throughput confocal imaging and quantification of reporter gene expression, we identified several serotypes with robust and efficient transduction across all neuronal subtypes. Among these, three serotypes AAV6, AAV6.2 and AAV2.7m8 showed consistently high performance. To assess safety, we quantified cell number and neurite morphology, finding that while high transduction and gene expression correlate with toxicity, sensitivity varied across neuronal subtypes, with NGN2 neurons being most vulnerable and dopaminergic neurons most resilient. Finally, we validated our findings in a more complex 3D model by testing one of the best-performing serotypes, AAV2.7m8, in both whole and dissociated human cerebellar organoids. Together, our results establish a benchmark dataset for AAV performance in human iPSC- derived neurons and provide practical guidance for AAV based gene delivery in human in vitro neural models. This resource will be valuable for both basic research and preclinical applications aiming to manipulate gene expression in human neurons and understanding AAV tropism in disease-relevant cell types.

Usher syndrome, the leading cause of hereditary deaf-blindness affecting approximately 1 in 15,000 individuals worldwide, is currently still untreatable. Antisense oligonucleotide-based exon skipping has shown significant therapeutic promise for USH2A-associated retinal dysfunction. Selection of (combinations of) exons suitable for therapeutic exon skipping within the fibronectin type 3 (FN3) domain-encoding region of USH2A currently requires that skipped exons exactly align with complete protein domains. However, only few exon combinations meet this criterion, which significantly restricts the therapeutic potential of this strategy. Our study addresses this limitation by incorporating AlphaFold2 structural modelling into the exon skipping target selection pipeline. Following this adjusted framework, we can predict exon skipping combinations that allow remaining domain fragments to form structurally viable hybrid domains. As a proof-of-concept, we examined and confirmed the functionality of usherin{Delta}exon54-58 that contains a hybrid FN3 domain, using zebrafish as a model. This highligts the potential of the newly developed paradigm for identifying exon skipping targets with potential therapeutic relevance. Our results emphasize the value of structural modeling in identifying new therapeutic exon skipping targets, aiming to improve precision, efficiency, applicability, and cost-effectiveness in the development of genetic therapies for hereditary diseases such as Usher syndrome.

With the increasing interest in RNA-based therapies, there is a pressing need to incorporate new chemistries into more complex RNA molecules. These modifications can protect RNA from degradation, improve its pharmacokinetics, and enhance its targeting properties. Here we describe the enzymatic synthesis of chemically modified RNA derivatives using a mutant T7 RNA polymerase to incorporate 23 different base modifications alongside stabilizing ribose modifications, such as 2'-fluoro and 2'-deoxy groups. To investigate the impact on transcription efficiency and fidelity, we employed a pool of 38 template sequences and analyzed the transcripts by next-generation sequencing of the cDNA. Results demonstrated that all modifications were successfully incorporated into RNA, with transcription efficiency influenced by three main factors: type of modification, base modified, and the sequence context. Misincorporation levels during transcription and reverse transcription into cDNA were generally low (<1%) but included noticeable exceptions for some nucleobase-modification combinations. As a robust proof-of-concept we demonstrated the selection of Histidine-U modified aptamer, relying on multiple rounds of transcription and amplification, binding Influenza hemagglutinin protein with low nanomolar KD. We anticipate that this work will significantly contribute to the design and production of chemically modified RNAs with novel functionalities, advancing applications in biomedicine and synthetic biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/720138v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@184d010org.highwire.dtl.DTLVardef@77fa67org.highwire.dtl.DTLVardef@d89e2eorg.highwire.dtl.DTLVardef@178ebc7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Diffuse midline gliomas (DMGs) are a deadly class of pediatric high-grade brain cancers. Approximately 80% of pontine DMGs feature a dominant, somatic, heterozygous point mutation in the non-canonical histone H3.3-coding gene H3-3A. This dominant-negative mutation replaces lysine 27 with methionine (K27M) and prevents global K27 di- and tri-methylation of all wild-type histone H3 proteins. We aimed to target the H3.3K27M onco-histone pre-mRNA with splice-switching antisense oligonucleotides (ASOs) designed to promote skipping of H3-3A exon 2, as this constitutive exon comprises both the K27M mutation and the natural in-frame start codon of the gene. The lead ASO identified in a systematic screen specifically induced H3-3A exon 2 skipping, did not affect expression or splicing of the paralog gene H3-3B--which also encodes histone H3.3--and restored global H3K27me3 marks in patient-derived DMG cells grown as neurospheres. In a patient-derived orthotopic xenograft tumor mouse model, the lead ASO reduced proliferation and extended survival. Our results show the potential of exon-skipping ASOs targeting H3-3A exon 2 as a therapeutic option for H3.3K27M-altered DMG. More generally, they exemplify the strategy of using ASOs to induce skipping of a constitutive exon to effectively achieve gene downregulation.

Gene therapies have demonstrated transformative potential for a range of genetic disorders, including immunodeficiencies, hematopoietic conditions, and neuromuscular diseases. However, the application of these approaches to cystic fibrosis (CF) and other airway diseases remains constrained by the challenge of efficient gene delivery to target epithelial cells. Adeno-associated virus (AAV) vectors are widely used for in vivo gene delivery due to their favorable safety profile and capacity for long-term transgene expression in non-dividing cells. Nonetheless, current AAV capsids require high doses to achieve therapeutic efficacy in the airways, raising safety concerns. Here we report the development of novel AAV capsid variants with markedly enhanced transduction efficiency of airway epithelial cells. Using unbiased peptide-modified AAV libraries and round-over-round screening in well-differentiated primary cultures of human airway epithelia (HAE), we identified 20 novel capsids that efficiently transduced cells at doses 10- to 100-fold lower than those required by existing vectors (termed AAV-AE). These variants demonstrated high transgene expression in HAE, primary human basal cells, tracheal explants from nonhuman primates, and murine airways in vivo. These optimized AAV capsids represent a significant advancement in pulmonary gene therapy, offering a versatile platform for the delivery of gene addition and editing reagents to treat CF and other respiratory diseases.

Exacerbations of respiratory viral infections significantly contribute to morbidity and healthcare burden. Among these viruses, Human Rhinoviruses (HRVs) are the most frequent causative agents of upper respiratory tract infections. To date, over 150 HRV serotypes have been identified, classified into three species: HRV-A, HRV-B, and HRV-C. No antiviral therapies are currently available against this viral family, largely due to the high serotype diversity and limited cross-protection. The major group of HRVs relies on the Intercellular Adhesion Molecule-1 (ICAM-1) receptor to infect airway epithelial cells, making ICAM-1 an attractive target for broad-spectrum therapeutic interventions. Here, we report the development of nucleic acid-based aptamers designed to disrupt ICAM-1-HRV binding and thereby prevent viral infection. Aptamers are single-stranded DNA molecules that fold into precise three-dimensional structures, enabling highly specific protein recognition. Using a Systematic Evolution of Ligands by EXponential Enrichment (SELEX) approach guided by a minimal peptide mimicking the ICAM-1 viral binding interface, a library of >1024 random single-stranded DNA sequences was screened. Through iterative rounds of selection, we identified eight candidate 77-nt DNA aptamers, which were subsequently evaluated for their potential using in silico and in vitro assays, as well as functional assays in human epithelial cells. From this strategy, two lead aptamers were selected that effectively inhibited HRV-A16 replication in a concentration-dependent manner, as measured by viral titers (TCID assay) and viral RNA quantification by RT-PCR. These findings demonstrate the potential of ICAM-1-targeting aptamers as antiviral agents capable of preventing HRV entry. By targeting a host receptor and creating a protective barrier at the cell surface, this approach may offer a broadly applicable strategy against multiple HRV serotypes, paving the way for the development of novel antiviral interventions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/717810v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@50e61dorg.highwire.dtl.DTLVardef@1338142org.highwire.dtl.DTLVardef@6b45e8org.highwire.dtl.DTLVardef@bac228_HPS_FORMAT_FIGEXP M_FIG C_FIG

Alzheimers disease (AD), the leading cause of dementia, affects over 33 million people worldwide, with pathogenesis tied to amyloid-{beta} (A{beta}) accumulation. Although anti-A{beta} monoclonal antibodies have shown clinical benefits, they often cause side effects including amyloid-related imaging abnormalities and brain microhemorrhage, especially in APOE E4 allele carriers. Here we used PHP.eB serotype adeno-associated virus (AAV), a vector with enhanced central nervous system (CNS) tropism, to deliver an A{beta} antibody expression vector (AAV-LEC) into the CNS of APP/PS1 and 5xFAD mice intravenously. The AAV-LEC-mediated expression of anti-A{beta} antibodies in the CNS significantly reduced the number and size of A{beta} plaques at various stages in both APP/PS1 and 5xFAD mice, alongside improved spatial learning and memory. It also reversed abnormal glial activation with reduced disease-associated microglia and astrocytes, and restored oligodendrocyte differentiation and myelin formation. No brain microhemorrhage or liver damage was detected following the AAV-antibody treatment. Thus, this AAV-mediated strategy offers a promising, convenient and safe AD therapeutic approach in the future.

Recombinant adeno-associated virus (rAAV) vectors are a leading platform for gene delivery in basic and clinical research, yet large-scale manufacturing remains constrained by residual nucleic-acid impurities that compromise safety. In this study, we profiled the DNA species packaged within rAAV capsids and identified plasmid backbone sequences and host cell genomic DNA (hcDNA) as predominant contaminants. To mitigate this critical quality attribute, we implemented upstream strategies designed to fragment or excise backbone DNA, including TelN/TelROL excision, I-SceI meganuclease digestion, CRISPR/Cas9 cleavage, and Cre/LoxP recombination. Quantitatively, TelN/TelROL and I-SceI reduced encapsidated plasmid backbone DNA to approximately 20-30% and 20-40% of baseline levels, respectively, while CRISPR/Cas9 lowered it to about 10-20%. Notably, the Cre/LoxP system eliminated detectable plasmid backbone DNA without compromising vector-genome titers, indicating preserved genomic integrity. Additionlly, supplementating cell culture with a caspase inhibitor significantly reduced hcDNA contamination in rAAV particles to 1-5% of the baseline level. Collectively, these interventions provide practical bioprocess frameworks that markedly enhance rAAV purity via targeted DNA minimization and prevention of hcDNA fragmentation, thereby strengthening the safety profile of rAAV therapeutics in alignment with current Good Manufacturing Practice (cGMP) expectations.

Instability of the inverted terminal repeats (ITRs) in AAV transfer plasmids has long hindered consistent and efficient production of therapeutic AAV vectors. The palindromic, GC-rich ITR sequence readily forms secondary structures, making them highly mutable in transfer plasmids. Indeed, a recent survey observed mutated ITRs in [~]40% of AAV transfer plasmids from labs around the world. Conventional strategies to mitigate this issue - such as using specialized E. coli strains, suboptimal culture conditions, or modified ITR sequences - have limited effect and often compromise plasmid and AAV yield. Here, by combinatorial optimization of the plasmid backbone structure and ITR flanking sequences, we established MuteFree, an AAV transfer plasmid system that eliminated ITR mutations for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV). Specifically, MuteFree reduced ITR mutation rates from a range of 32-100% in various transfer plasmids tested to 0% after serial passage of host E. coli for >160 population doublings. Moreover, in three GMP-grade AAV plasmid manufacturing projects initially cancelled due to severe and incurable ITR mutations, replacing conventional backbone with MuteFree completely solved the problem, reducing mutation occurrence to zero under standard GMP manufacturing conditions. Notably, MuteFree supports the packaging of potent AAV virus. The MuteFree system thus presents a robust solution to ITR instability, enabling high-fidelity and high-yield AAV production of AAV-based gene therapy vectors that is fully compatible with existing GMP manufacturing workflows.

PurposeTo characterize the cellular tropism and temporal dynamics of adeno-associated virus 2 (AAV2)-retro-mediated gene delivery in the adult mouse retina following intravitreal injection. MethodsAdult C57BL/6J mice received single or sequential intravitreal injections of AAV2-retro carrying the mGreenLantern (mGL) reporter gene. Retinas were collected at 1-, 3-, and 14-days post-injection (dpi) and processed for immunofluorescence analysis. Transduced cell types were identified using cell-type markers, including cone arrestin, RBPMS, and AP-2. The number and distribution of mGL-positive cells were quantified on whole retinas or retinal cross-sections to assess transduction efficiency, specificity, and spatial coverage. ResultsReporter expression was detected in the outer retina at 1 dpi and increased markedly at 3 and 14 dpi. AAV2-retro demonstrated strong tropism for photoreceptors and retinal pigment epithelium (RPE), with robust labeling of both rods and cones. In contrast to the robust outer retinal expression, transduction in the inner nuclear layers was limited to a few retinal ganglion and amacrine cells, reflecting strong cell-type specificity. Reporter expression was distributed widely across the retina, exceeding the localized pattern typically observed following subretinal delivery with conventional AAV2 vectors. Sequential injections further increased reporter expression and spatial coverage compared with single injections. ConclusionsAAV2-retro enables efficient, outer retina-specific gene delivery following intravitreal administration. This approach overcomes the limitations of traditional intravitreal gene transfer and provides a minimally invasive alternative to subretinal injection. AAV2-retro- mediated transduction may facilitate preclinical studies of retinal degeneration and support the development of gene therapies aimed at preserving photoreceptors and RPE function.

BackgroundThe optic nerve serves as a vital conduit for visual signaling, and its degeneration in optic neuropathy results in irreversible vision loss. It is also a widely used model for studying central nervous system (CNS) injury and repair. Although adeno-associated virus (AAV) and lentivirus are extensively applied in CNS research, their transduction efficiency and cell-type specificity within the optic nerve remain poorly characterized. This study aimed to identify the most effective viral vector, serotype, and promoter for direct gene delivery to the adult rat optic nerve. MethodsSprague-Dawley rats (7-10 weeks) received intra-optic nerve injections of lentiviral or AAV vectors encoding GFP under different promoters (CAG, CMV, or GFAP). Two to three weeks post-injection, optic nerves were collected for immunohistochemistry with markers of oligodendrocytes (Olig2), astrocytes (GFAP, Sox9), and microglia (IBA1). Transduction efficiency and cell-type specificity were assessed using confocal microscopy. ResultsAAV2, AAV5, and lentivirus showed minimal transduction, with only sparse GFP-positive cells observed near injection sites. In contrast, AAV-PHP.eB carrying the CAG promoter yielded robust and widespread GFP expression near the injection site. Quantitative analysis revealed that approximately 90% of transduced cells were Olig2-positive oligodendrocytes, indicating strong tropism for this glial population. ConclusionAAV-PHP.eB driven by the CAG promoter enables efficient gene delivery to the optic nerve, with a predominant tropism for oligodendrocytes. This targeted intra-optic nerve injection approach offers a reliable platform for manipulating oligodendrocytes and investigating mechanisms of CNS development, injury, and repair relevant to both optic neuropathies and other CNS diseases.

Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) lowers low-density lipoprotein cholesterol, a major risk factor for cardiovascular disease. Although several gene therapy strategies targeting Pcsk9 have been developed, direct comparisons across modalities are limited. To address this, we systematically evaluated cytosine base editing, nuclease-based CRISPR-Cas9, and epigenetic gene editing for Pcsk9 suppression. We first engineered a cytosine base editor to introduce a premature stop codon, then optimized and characterized an epigenetic editor, and finally delivered all modalities as mRNA formulated in Arcturus lipid nanoparticles (LUNAR(R)) into wild-type mice, benchmarking them against conventional CRISPR-Cas9 and GalNAc-siRNA. Remarkably, epigenetic editing achieved the most efficient and sustained repression of PCSK9, maintaining low protein levels throughout the entire 30-day study period. By comparison, cytosine base editing reduced PCSK9 with minimal double-stranded DNA breaks and off-target effects, but editing precision requires further improvement, while GalNAc-siRNA produced only transient suppression, limiting its suitability for a one-time therapeutic approach. Collectively, these findings highlight the superior durability and efficacy of epigenetic gene editing and provide proof-of-concept for its combination with LUNAR(R) delivery as a promising strategy for long-lasting hepatic-targeted therapy.

BackgroundSmall non-coding RNAs (sncRNAs) play central roles in post-transcriptional gene regulation. In addition to canonical microRNAs (miRNAs), fragments derived from vault RNAs (vtRNAs), called small vault RNAs (svtRNAs), have been reported in human cells. However, the absence of a standardized annotation framework has hindered their systematic detection, quantification, and comparison across small RNA sequencing (small RNA-seq) studies. MethodsWe developed an expression-based annotation strategy to identify svtRNAs from human small RNA-seq datasets. Using FlaiMapper followed by structure and expression-based filtering, we generated two annotation sets: a stringent "miRNA-like" set enriched in Argonaute-associated datasets, and (ii) a broader "Total" set derived from total small RNA-seq libraries under relaxed structural constraints. We explored the expression of the annotated svtRNAs across the different datasets analyzed: multiple normal and tumor-derived human cell lines, including Argonaute immunoprecipitation datasets. ResultsWe identified a repertoire of svtRNAs that are detected across independent datasets and, in several cases, reach abundance levels comparable to canonical miRNAs. Several highly abundant svtRNAs correspond to molecules with experimental validation from prior studies, supporting the robustness of our annotation strategy. Importantly, the same "dominant" (in terms of gene expression) svtRNAs emerged independently from Argonaute-associated and total small RNA datasets, supporting the idea of enzymatically consistent, reproducible svtRNA processing. We further identified svtRNAs derived from distinct vtRNA precursors that could share identical seed sequences, suggesting the possibility of svtRNA families with potential miRNA-like regulatory properties. We provide a standardized annotation that enables reproducible svtRNA quantification. ConclusionsOur study establishes a comprehensive expression-based annotation resource for human svtRNAs. By enabling their systematic detection and reproducible quantification, we show that svtRNAs appear to represent an abundant component of the human small RNA landscape.

PurposeRetinal ganglion cells (RGCs) are essential for visual signal transmission, yet they are vulnerable to injury and degeneration. Gene modulation in RGCs using adeno-associated virus (AAV) offers a promising avenue for neuroprotection and regeneration, but promoters lack sufficient RGC specificity, limiting precision needed for preclinical studies. This study aims to identify novel promoter-enhancer combinations (PECs) to achieve gene expression preferentially in RGCs. MethodsWe evaluated existing transcriptomic data to identify Neuritin 1(Nrn1) as a gene with highly restricted RGC expression in the retina. Synthetic PECs derived from human and mouse Nrn1 loci were incorporated into AAV2 vectors driving expression of a nuclear-targeted reporter GreenLantern. AAVs were delivered via intravitreal injection into C57BL6/J mice, and transduction efficiency and RGC specificity were evaluated in both young and aged retinas and those subjected to intraorbital optic nerve crush (ONC), using immunohistochemistry and quantitative analysis of RBPMS+ cells. ResultsWe found that AAV2 with a human Nrn1 PEC drives gene expression in RGCs. Quantitative analysis revealed that over 83% of transduced cells were RBPMS-positive, indicating robust RGC selectivity and significantly outperforming ubiquitous promoters. Notably, the Nrn1 PEC retained strong and selective transgene expression in RGCs in aged mice and following ONC, demonstrating its resilience under aged and injury conditions. ConclusionThe Nrn1 PEC enables efficient and injury-resilient gene expression in RGCs, addressing a key limitation in cell-specific targeting. This AAV-incorporated PEC offers a robust platform for evaluating neuroprotective interventions and accelerates translational development of gene therapies for glaucoma and other optic neuropathies.

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