Molecular Therapy - Nucleic Acids

Mutations in MYO7A result in the most severe subtype of Usher syndrome, the leading genetic cause of deafblindness. The large size of MYO7A requires dual adeno-associated virus (AAV) vectors for gene transfer or alternative methods to treat retinal defects. Here, we evaluated two treatment approaches: i) Supplementation of the human MYO7A gene via dual mRNA trans-splicing AAVs, and ii) CRISPR/Cas-mediated activation of the related murine Myo7b gene. Upon MYO7A supplementation, the transgenic MYO7A transcript and protein were expressed and correctly localized in retinal pigment epithelial (RPE) and photoreceptors of mice, pigs, and human retinal organoids. In RPE-and photoreceptor-specific Myo7a knockout mice, we could restore MYO7A expression and localization of melanosomes in RPE cells to wild-type levels. Myo7b activation led to partial restoration of melanosome localization, and the localization of MYO7B protein was largely comparable to MYO7A. These findings indicate that both approaches are in principle suitable for the therapy of Usher syndrome.

Efficient systemic delivery to the lung remains a major barrier for adeno-associated virus (AAV)-mediated pulmonary gene therapy, particularly when pre-existing immunity limits the use of conventional capsids. Here, we evaluated Bovine AAV, a phylogenetically divergent capsid, as candidate vector for lung-directed gene transfer. In adult C57BL/6J mice, intravenous delivery of Bovine AAV resulted in robust and preferential lung transduction comparable to AAV4, with predominant targeting of alveolar type I pneumocytes and pulmonary endothelial cells. In primary human lung-resident cells, Bovine AAV was particularly effective in microvascular endothelial cells, a target poorly transduced by AAV4 in vitro. Bovine AAV demonstrated scalable production with yield, purification performance, capsid quality, and genome integrity comparable to AAV9. In sera from healthy adults from the United States and Switzerland, Bovine AAV showed intermediate neutralization frequencies, lower than AAV2 and AAV4 but higher than AAV5 and AAV9. Of relevance, Bovine AAV maintained in vivo transduction efficiency in mice previously immunized with a pool of human and non-human primate-derived AAV capsids, including AAV4. Together, these results position Bovine AAV as a promising lung-tropic and immune-distinct vector for pulmonary gene therapy, with particular relevance for applications requiring systemic delivery in the presence of pre-existing immunity to conventional serotypes.

Ex vivo genome editing of human hematopoietic stem and progenitor cells (HSPCs) requires targeted integration strategies that support large therapeutic DNA payloads while preserving stem cell fitness. Although CRISPR/Cas9-mediated homology-directed repair using AAV donors is effective, it is constrained by limited cargo capacity and adverse effects on long-term HSPCs function. Integrase-defective lentiviral vectors (IDLVs) offer an alternative donor platform, yet their precise and controlled genomic integration remains inefficient. Here, we describe TILV (Targeted Integration of Lentiviral Vector), a CRISPR-assisted knock-in strategy that exploits Cas9-mediated linearization of episomal IDLV DNA to expose a single homology arm and engage homology-mediated end-joining repair pathways. TILV enables precise, directional and seamless integration of transgenes in multiple loci, enabling constitutive or physiological expression. Using single-cell clonal analyses and targeted long-read sequencing, we define the molecular features of TILV-mediated integration and demonstrate preferential use of CRISPR-linearized episomal substrates. TILV supports accurate insertion of large therapeutic transgenes, without compromising HSPC viability or multilineage potential. We further show that transient modulation of DNA repair pathway, in combination with extended homology arms, enhances integration efficiency and junctional precision. Importantly, optimized TILV enables targeted integration in phenotypically defined long-term HSPCs, highlighting its potential for scalable and durable gene therapy.

Pulmonary hypertension (BPD-PH) associated with bronchopulmonary dysplasia (BPD) in preterm infants associates with high morbidity and mortality within the first two years of life. In a previous unbiased study, we identified a panel miRNAs in tracheal aspirates (TA) that were differentially expressed in extremely low gestational age newborns (ELGANs) with BPD-PH compared to those with BPD but no PH. To explore the predictive potential of these miRNAs, we studied TA exosomes from 7 days old ELGANs and analysed a curated panel of 16 miRNAs through logistic regression and calculated the predictive AUROC to diagnose BPD-PH at 36 weeks PMA. AUROC of TA miRNAs was 0.76 with sensitivity and specificity of 53% and 93%, respectively. Adding sex and gestational age to the variables improved the AUROC to 0.78 with sensitivity and specificity of 61 and 87% respectively. Due to challenges of obtaining TA in non-invasively ventilated infants, we collected saliva samples from ELGANs at 7 days of age and compared the log expression of these 16 miRNAs in both biofluids and found significant correlation in their expression (pearson r=0.92, p<0.001). We calculated the predictive AUROC of the same miRNAs to diagnose BPD-PH at 36 weeks PMA. AUROC of these miRNAs in saliva was = 0.85 with sensitivity and specificity of 82% and 72%, respectively; addition of biological sex and gestational age improved AUROC to 0.86 with sensitivity and specificity of 79% and 76% respectively. Leave-one-sample-out sensitivity analysis demonstrated stable training performance with reduced performance in testing samples, supporting the need for validation in larger independent cohorts. In conclusion, early salivary miRNAs have great potential for risk stratification of ELGANs to develop BPD-PH, while also providing the opportunity to identify target molecules and mechanisms that modulate molecular function.

While adeno-associated virus (AAV) vectors have shown therapeutic benefit in clinical applications, noted challenges include low transduction efficiencies, poor cellular targeting, and vector related adverse events. Recently, it was demonstrated that a rationally designed synthetic inverted terminal repeat (SynITR) altered the AAV vector-induced DNA damage response and abrogated apoptosis in human embryonic stem cells. To explore the utility of AAV-SynITR for diverse gene therapy applications, vector production, transduction, and the cellular response were evaluated in various contexts. Regarding production, SynITR preparations exhibited comparable titers to wtITR in a serotype/transgene-independent manner. Despite slightly decreased transduction efficiency in various cell lines, intravenous administration of AAV8 vectors showed SynITR enhanced transduction in a tissue-specific manner in liver (>7-fold) and kidney and pancreas (>2-fold) at equivalent vector copy numbers; however, no differences were observed in muscle/heart/spleen tissues. Interestingly, persistent {gamma}H2AX, a marker of aging/chronic inflammation, was abundant in the liver and spleen following wtITR (but not SynITR) transduction. In human corneas, SynITR enhanced transduction up to 16-fold over wtITRs. These data demonstrate that SynITRs elicit tissue-specific transduction enhancement and alter the cellular stress response. Importantly, the SynITRs offer an alternative context to elucidate wtITR biology for targeted, enhanced, and potentially safer human gene therapy.

This study enrolled 152 hypertensive patients with an ARR > 3.7 to assess the relationship between the traditional hypokalemia cutoff (3.5 mmol/L) and primary aldosteronism (PA) screening, and to establish a new cutoff. Under the traditional cutoff, only 35.7% of PA patients presented with hypokalemia. ROC curve analysis identified a new cutoff of 4.22 mmol/L, which increased sensitivity from 35.7% to 77.5%, with a specificity of 91.1% and an AUC of 0.897. The findings indicate that the traditional cutoff is insufficiently sensitive, while the new cutoff markedly improves screening sensitivity and facilitates early detection of PA.

To facilitate the translation of NF1 exon 17 skipping as a mutation-specific therapy for Neurofibromatosis type 1 into in vivo testing, we have continued to develop more efficient antisense oligonucleotides (ASOs), humanized mouse models, and explored multiple delivery platforms including an adeno-associated virus (AAV)-U7-SnRNA vector approach. We evaluated both biodistribution and exon skipping efficacy of a U7-SnRNA targeting NF1 exon 17 with an SFFV-driven cassette containing T2A-linked Luciferase (Luc) and eGFP packaged in AAV-9, AAV-F and AAV-B1 capsids. We show that AAV-F is superior to AAV-9 and AAV-B1 for mouse brain delivery based on DNA transduction, GFP expression, and luciferase activity, but AAV-B1 delivers 2-4 fold more to sciatic nerve (SCN). In terms of exon skipping, AAV-F appears to induce the most skipping in liver and optic nerve (ON), while AAV-B1 mediates highest skipping in the liver, SCN, and ON. The identification of AAV serotypes that allow efficient transduction and delivery of transgenes to the mouse CNS and PNS is impactful for preclinical research in murine models of other diseases. Furthermore, this is both the first report of NF1 exon skipping efficacy in vivo and the first successful application of an U7-SnRNA for the restoration of functional neurofibromin for NF1.

Background & Aims: Liver transplantation improves outcomes in hepatocellular carcinoma (HCC), yet treatment options for patients with tumor recurrence remain limited to tyrosine kinase inhibitors. Glypican-3 (GPC3)-targeted CAR T cells offer a tumor-directed immune-based therapeutic strategy, but their efficacy may be limited by post-transplant immunosuppression. We developed a CAR T cell platform combining CRISPR/Cas9-mediated FKBP1A disruption to confer resistance to FKBP12-dependent immunosuppressive agents, including tacrolimus, everolimus, and sirolimus, with TRAC knockout to eliminate endogenous T cell receptor expression and reduce alloreactivity. Methods: Human T cells were edited using Cas9 ribonucleoprotein complexes targeting FKBP1A and TRAC, expanded, and transduced with an anti-GPC3 CAR construct. Cytokine production and cytotoxicity were assessed in vitro. Antitumor activity under tacrolimus treatment was evaluated in a Hep G2 xenograft model, and xenoreactivity was assessed in a graft-versus-host disease model. FKBP1A/TRAC double-knockout T cells were enriched using mTOR inhibitor selection combined with CD3-based MACS depletion. PBMCs from liver transplant recipients were used to evaluate feasibility for clinical translation during the early post-transplant period. Results: Tacrolimus suppressed wild-type CAR T cell function but not FKBP1A/TRAC double-knockout CAR T cells, which retained cytokine production, cytotoxicity, and in vivo antitumor activity. Cyclosporine A remained suppressive, enabling its potential use as a pharmacologic control strategy. TRAC disruption reduced xenoreactivity. CD3-based MACS depletion and mTOR inhibition achieved functional double-knockout efficiencies greater than 98%, without compromising cell viability. Functional FKBP1A/TRAC knockout CAR T cells were generated from patient PBMC samples 30 days post-transplant. Conclusions: Dual-edited GPC3 CAR T cells resist tacrolimus-based immunosuppression while limiting alloreactivity, supporting their use for recurrent HCC after liver transplantation. Sequential, high-viability selection in a modular cellular engineering framework enables adaptation to alternative tumor targets and next-generation CAR T cell designs.

Leptomeningeal disease (LD), involving the metastasis of cells to the leptomeningeal membranes in the central nervous system (CNS), can be a deadly complication of several different types of cancer originating in the periphery or CNS, including breast cancer (BC) and pediatric medulloblastoma (MB). Targeted therapy represents a promising new approach to improve overall survival for LD patients. To this date, angiopep-2 (Ang2) and transactivating transcriptional activator (TAT), two well-known peptides for their brain delivery capability, have been reported to transport therapeutic cargos into the CNS for treatment of disease. Current administration strategies, however, still rely on oral delivery or intravenous injection (IV), where the substances need to travel through complex biological barriers to reach the subarachnoid space (SAS), which is the primary location of LD. Our research group has focused on the intrathecal (IT) route of administration as an alternative approach that can potentially enable high exposure of drug to CSF exposed tissues. However, there is a major field gap in understanding how targeting peptides can access (or not access) LD as a function of their route of administration. Therefore, our work was focused on comparing the targeting capability of Ang2 vs TAT by IT vs IV routes of administration. We first generated two xenograft models of LD by directly infusing breast cancer cells (MDA-MB231) or medulloblastoma cells (HDMB03) into the SAS via intracisternal magna injection (ICM) to form BC-LD and MB-LD models, respectively. These tumor models were characterized for overall survival, tumor growth patterns, and presence of hydrocephalus. Second, we further administered fluorescently labeled Ang2 or TAT peptides either IV or ICM into tumor bearing mice. Neuraxial fluorescence images were examined to evaluate the targeting ability of these two peptides based on colocalization between peptide signal and tumor tissues ex vivo. We discovered that the median survival of both models was negatively related to the number of the cells infused. While HDMB03 cells tended to metastasize preferentially to the brain region, MDA-MB231 cells tended to metastasize preferentially to the spinal cord. Both models present hydrocephalus as one of the common clinical symptoms in LD patients. Compared to the healthy control, MB-LD yielded a 7.3-fold increase and BC-LD a 26.5-fold increase in ventricular volume. Furthermore, targeting achieved by TAT was significantly higher than targeting achieved by Ang2 in thoracic spine for the MB-LD model. For BC-LD model, TAT signal was found to be significantly higher than Ang2 signal in the olfactory bulbs, brain stem, thoracic spine, and lumbar spine regions. While both peptides showed a strong signal at 2 hours post ICM injection, signal was not detectable 24 hours after administration, reflecting washout or degradation. Significantly, these data provide evidence that ICM will be a preferable route of administration over IV for the purpose of maximally targeting LD.

Background: Endothelial progenitor cells (EPCs) contribute to endothelial repair and neovascularization, and EPC dysfunction is closely associated with oxidative stress-related vascular injury. Forkhead box O3a (FoxO3a) regulates cellular stress responses, whereas miR-34a has been implicated in endothelial dysfunction, senescence, and apoptosis. However, the relationship between FoxO3a and miR-34a-3p in oxidatively injured EPCs remains incompletely defined. Objective: This study investigated the role of FoxO3a in H2O2-induced EPC dysfunction and examined whether miR-34a-3p directly interacts with the FoxO3a 3' untranslated region (3'UTR). Methods: Human umbilical cord blood-derived EPCs were identified by DiI-ac-LDL uptake, FITC-UEA-1 binding, and the expression of EPC-related markers. Oxidative stress was induced by H2O2. Cell viability, apoptosis, and angiogenic capacity were evaluated using CCK-8 assay, Annexin V/7-AAD flow cytometry, and Matrigel tube formation assay, respectively. FoxO3a expression was modulated using adenoviral overexpression or knockdown vectors, and miR-34a was modulated using mimics or antagomir. FoxO3a and miR-34a expression levels were detected by Western blot and qPCR. A dual-luciferase reporter assay was used to verify the interaction between hsa-miR-34a-3p and the FoxO3a 3'UTR. Results: H2O2 reduced EPC viability, increased apoptosis, and impaired tube formation in a concentration-dependent manner. H2O2 increased FoxO3a protein abundance and miR-34a expression, whereas FoxO3a mRNA did not change markedly. FoxO3a overexpression aggravated, whereas FoxO3a knockdown partially alleviated, H2O2-induced EPC dysfunction. Similarly, miR-34a mimics further suppressed EPC viability and tube formation, while miR-34a antagomir exerted a protective effect. Dual-luciferase reporter analysis showed that hsa-miR-34a-3p significantly reduced the activity of the wild-type FoxO3a 3'UTR reporter, while mutation of the predicted binding site abolished this suppression. Conclusion: FoxO3a and miR-34a participate in oxidative stress-induced EPC dysfunction. The dual-luciferase data demonstrate that hsa-miR-34a-3p directly targets the FoxO3a 3'UTR, suggesting the presence of miR-34a-3p-mediated post-transcriptional feedback within the FoxO3a-related stress-response network in EPCs.

Oculocutaneous albinism type 1 (OCA1) is a pigmentation disorder caused by biallelic tyrosinase (TYR) mutations, an essential enzyme for melanin synthesis. TYR inactivity results in loss of hair, skin, and eye pigment, which is detrimental for ocular function. Hypopigmentation of iris, retinal pigment epithelium (RPE), and choroid results in severe photosensitivity and low visual acuity. There are currently no FDA-approved pigment restoring therapies for OCA1, making therapeutic development an unmet clinical need. To address this gap, we have advanced an adeno-associated viral (AAV)-mediated Tyr replacement approach for OCA1 ocular pigment restoration. We evaluated the optimal viral delivery strategy and vector cell-type specificity for iris, RPE, and choroid pigmentation in an OCA1 mouse model, testing intraocular and systemic viral delivery methods in conjunction with viral constructs of varying RPE-specificity. Early, systemic delivery of an RPE-directed AAV-Tyr construct, AAV9.2yf-VMD2-Tyr, achieved widespread ocular pigment rescue with minimal off-target expression in non-ocular tissues. Animals treated with AAV9.2yf-VMD2-Tyr demonstrated reduced photophobic behavior compared to untreated controls, indicating that ocular pigmentation restores a debilitating functional consequence of OCA1. Our findings establish a foundation for clinical translation of an AAV-TYR therapy aimed at improving light sensitivity, glare, and low vision through pigment restoration in patients with OCA1.

Peripheral nerve injuries often result in prolonged skeletal muscle denervation, leading to progressive atrophy, fibrosis, neuromuscular instability, and loss of regenerative capacity before axons can reinnervate distal targets. Here, we developed a non-viral strategy using tissue nanotransfection (TNT) to deliver the neurogenic transcription factor cocktail Ascl1, Brn2, and Myt1l (ABM) directly to denervated skeletal muscle. In vitro, ABM-transfected myoblasts sustained expression of the reprogramming factors, acquired neuron-like morphologies, upregulated neuronal markers including Tuj1, Map2, and Syp, and exhibited electrophysiological properties consistent with membrane excitability. RNA sequencing confirmed broad activation of neurogenic transcriptional programs, with enrichment of pathways associated with neuronal fate commitment, neuron differentiation, axon guidance, synaptogenesis, and developmental signaling. In a mouse model of sciatic nerve transection, TNT enabled localized ABM expression in denervated gastrocnemius muscle. ABM-TNT treatment accelerated resolution of denervation-associated fibrillation potentials and showed trends toward improved twitch and tetanic torque, compound muscle action potential amplitudes, and muscle mass preservation. Transcriptomic profiling of treated muscles 5 weeks after injury revealed distinct gene expression programs enriched for muscle regeneration, neuromuscular organization, trophic support, extracellular matrix remodeling, angiogenesis, myogenesis, and metabolic adaptation. Network analyses further identified activation of neurogenic regulators, neurotrophic signaling, and vascular-support pathways. These findings establish TNT-mediated ABM delivery as a non-viral platform for inducing neurogenic and myoprotective programs in denervated muscle, suggesting a potential strategy to preserve muscle viability during the prolonged interval required for peripheral nerve regeneration.

Sedentary behavior promotes chronic low-grade inflammation in adipose tissue, contributing to metabolic dysfunction and insulin resistance. High-intensity interval training (HIIT) is a time-efficient exercise strategy with potent anti-inflammatory and metabolic benefits; however, its effects on adipose tissue inflammatory signaling and microRNA (miRNA) regulation remain incompletely understood. This study investigated the effects of eight weeks of HIIT on inflammatory and epigenetic markers in interscapular white adipose tissue (iWAT) of male Wistar rats. Fourteen rats were randomly assigned to either a sedentary (SED; n = 7) or HIIT (n = 7) group. The HIIT protocol consisted of treadmill running five days per week for eight weeks. Body weight and iWAT mass were assessed, and molecular adaptations were evaluated at multiple regulatory levels using RT-qPCR for mRNA targets (NLRP3, TNF-, PPAR-{gamma}, and IL-10) and miRNAs (miR-21 and miR-30d-5p), while protein levels of NLRP3 and PPAR-{gamma} were assessed using Western blotting. Compared with the SED group, HIIT significantly reduced body weight (p < 0.001) and iWAT mass (p = 0.002). Furthermore, HIIT downregulated the expression of pro-inflammatory mediators, including NLRP3 (gene: p = 0.001; protein: p < 0.001) and TNF- (p = 0.025), while upregulating anti-inflammatory regulators PPAR-{gamma} (gene: p = 0.026; protein: p = 0.020) and IL-10 (p = 0.010). In parallel, inflammation-associated miRNAs, including miR-21 (p = 0.004) and miR-30d-5p (p = 0.002), were markedly downregulated. These coordinated transcriptional, post-transcriptional, and translational adaptations suggest that HIIT attenuates adipose tissue inflammation and promotes a favorable immunometabolic phenotype through integrated molecular and epigenetic mechanisms.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that disrupt the reading frame and abolish expression of functional dystrophin protein. Antisense oligonucleotides (ASO) can restore production of partially functional dystrophins by inducing exon skipping to restore the reading frame of dystrophin transcripts. While exon skipping is an FDA approved therapeutic strategy, there are currently no approved therapies for patients amenable to exon 44 skipping (8% of DMD patients). Here, we carried out a discovery campaign to identify phosphorothioate (PS) ASOs that efficiently induce exon 44 skipping and to define key sequence and chemistry features associated with activity. A tiling and micro-tiling approach with 18mer fully PS and 2-O-methoxyethyl (2MOE) modified ASOs in patient-derived myotubes identified five exonic target regions that promote skipping. ASO activity was strongly correlated across skeletal muscle and iPSC-derived cardiomyocytes, indicating similar exon 44 splicing regulation across cell types. Optimization studies showed that for 2MOE PS ASOs, 16-20mers were generally most active, while longer ASOs often had reduced activity due in part to impaired productive uptake into cells. Swapping out 2MOE modifications at both terminal positions for locked nucleic acids (LNAs) rarely improved activity and could also reduce it. Finally, promising candidates were tested in a humanized mouse model with an exon 44 skippable deletion, where one ASO outperformed others, inducing dose-dependent exon 44 skipping and dystrophin restoration in mouse models. These findings define practical design rules for exon 44-targeted ASOs and provide a foundation for therapeutic development.

Efficient delivery of molecular therapies to the central nervous system (CNS) remains a major barrier to treating neurogenetic disorders such as Niemann Pick type C (NPC) disease. Focused ultrasound-mediated blood-brain barrier opening (FUS-BBBO) has emerged as a non-invasive strategy to enhance delivery of systemically administered therapeutics. In this study, we evaluated whether FUS-BBBO could enable delivery of lipid nanoparticle (LNP)-packaged modified mRNA (modRNA) to the cerebellum in an NPC mouse model. A pilot study in wild-type mice demonstrated successful FUS-mediated BBB opening, delivery of LNP-packaged GFP mRNA, and subsequent protein expression in the cerebellum. We then performed a controlled study in NPC mice comparing delivery of LNP-GFP and LNP-NPC modRNA using intravenous administration with and without FUS-BBBO. BBB opening was confirmed by contrast-enhanced MRI in FUS-treated animals. Quantitative PCR revealed the presence of GFP mRNA in the cerebellum following FUS-BBBO, whereas NPC mRNA was minimal or undetectable across groups. However, no GFP or NPC1 protein expression was detected in the cerebellum by western blot in any experimental group. Consistent with this, no therapeutic effect on Purkinje cell survival was observed. These results demonstrate that while FUS-BBBO reliably induces BBB opening and can facilitate limited delivery of LNP-packaged mRNA to the brain, this did not translate into detectable protein expression or therapeutic benefit in the NPC model under the conditions tested. This discrepancy between successful delivery in wild-type mice and lack of efficacy in diseased animals points to potential important biological and/or formulation-dependent barriers that must be addressed to enable effective CNS delivery of LNP-based mRNA therapies.

Keloids are fibroproliferative skin disorders arising following dermal injury that extend beyond the original wound margins. Their pathogenesis remains poorly understood, and current treatments are associated with high recurrence rates. Identifying transcriptomic biomarkers that distinguish keloids from other skin and scar phenotypes may provide insight into disease mechanisms and facilitate the development of targeted therapeutic approaches. However, previous transcriptomic studies have often been limited by small sample sizes, pairwise comparisons between tissue classes, heterogeneous data-integration strategies, and a reliance on conventional differential gene expression (DGE) analysis. Here, we employed a multi-stage machine learning (ML) workflow for robust keloid biomarker discovery using transcriptomic datasets derived from both bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq). We assembled and harmonized, to the best of our knowledge, the largest curated cross-study keloid transcriptomic cohort currently available, comprising 81 samples from 13 independent studies spanning four clinically relevant tissue classes: normal skin, normotrophic scar, hypertrophic scar, and keloid scar. Through study-aware cross-validation, feature selection, partition-stability analysis, and bootstrap validation across multiple ML classifiers, we identified a panel of eight highly consistent biomarkers capable of distinguishing keloid from non-keloid samples. These biomarkers were associated with dysregulation of extracellular matrix homeostasis, fibrosis-resolution pathways, vascular remodelling, and metabolic reprogramming. Comparison with conventional DGE analysis demonstrated substantial agreement while also highlighting important differences between the two approaches. In particular, FASN was consistently identified by the ML workflow as an upregulated discriminatory biomarker despite exhibiting weak, non-significant differential expression in the DGE analysis. Cell-type-specific analysis further supported this finding, revealing significant FASN upregulation in fibroblast and vascular endothelial populations. These results demonstrate that ML and DGE capture complementary aspects of transcriptomic variation. This study provides a robust strategy for cross-study transcriptomic biomarker discovery and identifies candidate genes and pathways for future mechanistic and therapeutic investigation in keloids. 1 Author SummaryKeloids are abnormal scars that continue to grow beyond the original wound and can be difficult to treat because they frequently recur after therapy. Although many studies have investigated the biology of keloids, the molecular mechanisms that distinguish them from other scar types remain incompletely understood. Identifying biomarkers involved in keloid formation may help inform improved treatment strategies. Previous transcriptomic studies have often been limited by small sample sizes and inconsistent analytical approaches. In this study, we combined gene-expression data from multiple independent studies to create, to the best of our knowledge, the largest cross-study transcriptomic collection available for keloid analysis. We then applied several machine learning approaches to identify genes that consistently distinguished keloids from other skin and scar phenotypes. The identified biomarkers were associated with extracellular matrix remodeling, fibrosis, vascular function, and cellular metabolism. One gene involved in fatty-acid synthesis, FASN, was repeatedly identified by the machine learning analyses despite being overlooked by conventional gene-expression methods. Additional single-cell analyses confirmed elevated FASN expression in specific cell populations within keloid tissue. More broadly, this work provides a strategy for discovering robust biomarkers from heterogeneous biological datasets and identifies molecular targets for future studies of keloid disease.

Various methods have detected miRNA-target interactions via immunoprecipitation of UV-crosslinked Argonaute ribonucleoprotein complexes, followed by intermolecular ligation of bound miRNAs to target strands, forming chimeric RNAs. To date, these methods have relied on conventional viral reverse transcriptases (RTs) to generate cDNAs for sequencing. However, crosslinked RNAs often retain adducts after purification, which can make them poor templates for viral RTs. Here, we adapted OTTR (Ordered Two-Template Relay) techniques to generate cDNAs from Ago2-bound RNAs. OTTR makes use of a modified retroelement-encoded RT, which is strongly processive even on templates with modifications or adducts. We show that this "OTTR-CLASH" method increases the frequency of generating chimeric RNAs compared to previous methods. We also developed an improved bioinformatic pipeline for analysis of these data, and we use this to catalog miRNA-target interactions not previously described in the literature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/738487v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@13bc276org.highwire.dtl.DTLVardef@5beb41org.highwire.dtl.DTLVardef@b204e5org.highwire.dtl.DTLVardef@15f747d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Clinical evidence demonstrates that ex vivo gene therapy and genome engineering of hematopoietic stem and progenitor cells (HSPCs) could represent one-time cures. However, while genome editing itself has become increasingly efficient and precise, the toxic conditioning required for hematopoietic stem cell transplantation remains a major barrier to broad clinical implementation of these otherwise curative therapies. In particular, the use of busulfan for myeloablative conditioning constitutes a major safety concern. While preclinical studies established CD117 as a promising target for antigen-specific therapy, clinical translation faced setbacks balancing efficacy and safety. To overcome current limitations, we generated a new CD117-blocking monoclonal antibody (CIM058) and demonstrate its potency to block wild-type HSPCs. To enable long-term blockade of host HSPCs even after transplantation, we used prime editing to engineer CIM058-resistant human CD34+ HSPCs. When combined, CIM058 and the epitope engineered CD34+ HSPCs ameliorated disease phenotype in a {beta}-thalassemia model. Our results suggest that this approach may overcome the reliance on busulfan or other myeloablative conditioning regimens with their associated morbidities, and by enabling toxin-free conditioning and in vivo selection of edited cells, may facilitate clinical implementation of these highly valuable genetic therapies.

Context. Teprotumumab (TEP) is an emerging treatment for thyroid eye disease (TED), but real-world evidence outside the United States remains limited, and detailed changes in orbital components have not been fully clarified. Objective. To evaluate the effectiveness of TEP based on clinical manifestations and magnetic resonance imaging (MRI) findings, and compare it with that of intravenous glucocorticoid (IVGC) therapy. Methods: The TEP cohort included all 18 patients who started TEP therapy at Kyoto University Hospital by July 31, 2025. A historical IVGC cohort included 20 patients matched to the TEP cohort. Results: During 24 weeks of TEP therapy, proptosis measured using a Hertel exophthalmometer improved from 22 (20-22) to 19 (16-21) mm (p = 0.025), and clinical activity score decreased from 4 (3-5) to 1 (0-1) point (p < 0.001). Among 15 patients with diplopia, a reduction of at least 1 point in Gorman score was observed in 9 patients (60.0%). Thyroid-stimulating antibody titers decreased from 1,180% (349-4,710) to 282% (132-504) (p = 0.013). MRI-based comparisons with the IVGC cohort showed that TEP reduced both extraocular muscle and orbital fat areas, whereas IVGC reduced extraocular muscle area but conversely increased orbital fat area. Inflamed extraocular muscles identified on MRI were enlarged at baseline and showed marked shrinkage after both therapies. Conclusion: TEP showed robust effectiveness in Japanese real-world patients with TED. MRI-based analyses revealed distinct effects of TEP and IVGC on orbital fat and identified inflamed extraocular muscles as treatment-responsive components.

Immunoglobulins (e.g., IgGs) are critical effectors of the adaptive immune system that when overexpressed or dysregulated can result in autoimmune diseases. Thus, depletion of IgGs can be a promising therapeutic avenue. Here we developed genetically-encoded lysosome targeting chimeras (GELYTACs) that target circulating IgGs for clearance and degradation. The GELYTACs comprised two protein modules derived from insulin-like growth factor 2 (IGF2) and an IgG-binding nanobody, respectively, and mediated clearance of plasma IgG via the lysosomal trafficking receptor IGF2R. To achieve long-lasting IgG depletion, we encoded GELYTACs in an AAV gene therapy vector and established continuous expression in mice. We also developed conditional GELYACs that are activatable with disease-specific proteases or small molecule drugs. This work establishes GELYTACs as a possible therapeutic modality that is deliverable using genetic medicine approaches.