NAR Molecular Medicine

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex multisystem disorder. Its hallmark feature, post-exertional malaise (PEM), is a delayed and prolonged worsening of symptoms following even minor exertion. As PEM commonly includes muscle pain, weakness, exercise intolerance and impaired recovery, skeletal muscle dysfunction may contribute to its pathophysiology, but the underlying molecular processes remain poorly understood. We generated induced pluripotent stem cell-derived skeletal muscle myotubes from individuals with ME/CFS (n = 6) and healthy controls (n = 7) to investigate disease-associated transcriptomic alterations and identify potential therapeutic candidates. Myogenic progenitor cells from both groups differentiated into elongated, multinucleated myotubes expressing titin and myosin heavy chain, while qPCR profiling of myogenic differentiation markers further supported successful differentiation. Gene-level analysis identified three differentially expressed genes at an FDR-adjusted p < 0.05: ERVH48-1, CDX4 and SLC2A14. Isoform-switching analysis identified 16 genes with significant differential isoform usage, including CACNA1H, MIEF2, SMAD2 and CHD4. Gene set enrichment analysis identified broader pathway-level alterations involving RNA processing, protein synthesis, proteostasis, mitochondrial quality control, metabolism and muscle-related processes. Library of Integrated Network-based Cellular Signatures Phase 2 (LINCS2) analysis further prioritised 14 compounds associated with mitochondrial metabolism, glucose and lipid regulation, PPAR signalling, calcium and excitatory signalling, neuroimmune activity and cellular stress responses. Overall, ME/CFS iPSC-derived myotubes showed modest gene-level differences alongside broader isoform- and pathway-level alterations, supporting their use for investigating skeletal muscle biology and prioritising candidates for functional validation.

Myotonic dystrophy type 1 (DM1) is caused by a CTG expansion in the 3' untranslated region of the DMPK gene that results in the expression of expanded CUG RNA which trap splicing factors resulting in widespread alternative splicing dysregulation. A panel of these splicing events, or splicing index (SI), is currently used to measure splicing dysregulation in DM1 patients. Despite significant interest in splicing, little is known about its progression over time in DM1. The objective of this study was to evaluate the changes in SI over 3 years in a well-phenotyped cohort of DM1 participants and identify factors contributing to these changes. RNA was extracted from vastus lateralis (VL) biopsies of 20 DM1 participants taken 3 years apart and the SI score was measured using targeted RNA sequencing. Baseline SI scores correlated with percent predicted muscle strength of the ankle dorsiflexors, knee extensors and hand grip. On average, SI scores worsened over 3 years, but the progression varied markedly among participants. Adult/juvenile phenotypes and lower baseline SI scores were among the factors associated with higher progression of SI scores over time. These findings emphasize the importance of integrating clinical phenotype and baseline molecular severity when interpreting longitudinal SI change, particularly in the context of clinical trials.

Pitt-Hopkins syndrome (PTHS) is a severe neurodevelopmental disorder caused by haploinsufficient expression of transcription factor 4 (TCF4). Increasing expression from the remaining functional TCF4 allele could provide a mutation-independent strategy for patients with diverse pathogenic variants. We integrated genomic datasets with strand-specific RNA analysis to identify transcribed regions of the TCF4 promoter suitable for targeting by activating double-stranded RNAs (dsRNAs). Screening of overlapping promoter-targeted dsRNAs identified a hotspot for increased TCF4 expression. Activation was retained by a lead dsRNA containing extensive 2'-fluoro, 2'-O-methyl, and phosphorothioate modifications used in therapeutic oligonucleotides. Anti-AGO2 immunoprecipitation showed association with RNA transcribed from the targeted promoter region. dsRNA-mediated activation was associated with increased RNA polymerase II occupancy. These findings establish promoter-targeted dsRNAs as chemically tractable starting points for increasing TCF4 expression and support their investigation as a mutation-independent strategy for haploinsufficient disease.

Emerging and re-emerging zoonotic RNA viruses, including filoviruses, henipaviruses and Rift Valley Fever virus (RVFV), pose a significant threat to human health. Due to their epidemic potential and high pathogenicity, these viruses are categorized as WHO priority pathogens, yet we still lack effective medical countermeasures (MCMs) against most of these dangerous pathogens, supporting that host-directed antivirals will be advantageous for broadly acting MCMs. Here we assessed the antiviral efficacy of SLR14, an immunomodulatory stem-loop RNA that activates RIG-I, for its activity against a panel of highly pathogenic viruses. SLR14 treatment provided broad-spectrum protection, markedly reducing infection with Ebola virus (EBOV), Marburg virus, and Sudan virus in human primary monocyte-derived macrophages, and Nipah virus replication in iPSC-derived endothelial cells, whether administered prophylactically or therapeutically. In vivo, a single dose of SLR14 protected mice against lethal RVFV infection when administered prophylactically, and, notably, retained significant efficacy when administered up to two days post-infection. Therapeutic SLR14 administration also improved survival rates of EBOV-infected mice. Although EBOV infection and SLR14 treatment activated overlapping innate immune pathways in human macrophages, viral infection induced a nonprotective response, whereas SLR14 elicited a well-regulated, markedly stronger and protective interferon-stimulated and inflammatory gene signature while avoiding prolonged IFN gene upregulation. Our data identify SLR14 as a potent host-directed therapeutic with broad antiviral efficacy against some of the most lethal viral pathogens.

As we have entered the endemic phase of COVID-19, there is a need to assess the long-term effects of SARS-CoV-2 infection on the trajectory of neurodegenerative disorders. Herein, we investigate whether the SARS-CoV-2 RNA-binding nucleocapsid protein (NCP; N-protein) may influence molecular processes relevant to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We detected N-protein immunoreactivity in ALS spinal cord motor neurons, including cytoplasmic N-protein-positive puncta that colocalized with cytoplasmic TAR DNA-binding protein 43 (TDP-43). In vitro, optogenetically induced N-protein biomolecular condensates (BMCs) showed reversible, liquid-like behavior and recovered after photobleaching. Using this model, we found that these condensates colocalized with TDP-43 and the RNA-binding proteins G3BP1/2, PABPC1 and TIA1. Condensate formation was associated with TDP-43 cytoplasmic mislocalization, altered TARDBP expression, and altered exon 3 splicing of POLDIP3. The N-terminal portion of N-protein influenced BMC formation, whereas truncated C-terminal proteoforms did not form detectable BMCs or show detectable TDP-43 colocalization in this assay. Extending these observations to a human cell model, we show that optogenetically induced N-protein BMCs colocalize with TDP-43 in human iPSC-derived motor neurons. Collectively, these findings support further investigation into potential molecular links between SARS-CoV-2 exposure and the trajectory of neurodegenerative disease.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by repetitive behavior and impaired social interactions. Recent reports from human postmortem brain samples of ASD show transcriptome-wide dysregulations, including changes in differential gene expression and alternative splicing. An important mechanism regulating transcriptome function is the editing of double-stranded RNA by adenosine deaminases acting on RNA (ADARs), which play regulatory roles in neurodevelopment and innate immunity. We explore the ADAR editing changes associated with the use of Selective Serotonin Reuptake Inhibitors (SSRIs) in Arid1b haploinsufficient mouse models. Our results show dynamic changes in site-specific editing rates in autistic mice. We further demonstrate the transcriptome-wide effects of differential ADAR editing in terms of changes in miRNA-mRNA interactions. Moreover, our findings highlight a site-specific change in editing rates for evolutionarily conserved sites in mammals and ASD associated risk genes, such as Gria2. Our analysis of significant biological processes shows the association of differentially edited genes with processes vital for neurotransmission. Overall, our findings imply that the use of SSRIs can induce distinct ADAR editing signatures, potentially contributing to ASD pathology.

We report a neonate with IOPD who had high pre-existing anti-AAV9 antibody titers, which precluded eligibility for AAV9-mediated gene therapy (GC301). Preconditioning with a single dose of the IgG-degrading enzyme GC801 (0.2 mg/kg) safely reduced serum IgG from 6.25 g/L to a nadir of 0.72 g/L within 5 days. This pharmacological window facilitated successful GC301 vector transduction, driving a robust increase in GAA activity to 19.38 mmol/L/16h by day 84. Crucially, no adverse events beyond the underlying IOPD were observed. Our findings support the potential utility of GC801 as an agent to overcome pre-existing humoral barriers in neonatal gene therapy candidates.

Adeno-associated viral (AAV) vectors possess immense potential for gene therapy applications. However, microglia can mount an immune response against AAV vectors. Yet, microglia are therapeutic targets since they play a driving role in the progression of neurodegenerative diseases. Thus, it is critical to understand the cellular processes driving the microglia-mediated immune response against AAV vectors. To this end, we modelled AAV6 induced inflammation upon retinal gene therapy in minipigs. Upon AAV6 administration, the IBA1+ cells demonstrated a morphological transition to the amoeboid form, indicating microglial reactivity. We also observed the invasion of peripheral immune cells in the retina. To elucidate the underlying molecular mechanisms, we transduced human iPSC derived microglia-like cells (iMGLs) in vitro with AAV6 and AAV8 respectively. Only AAV6 transduced iMGLs exhibited morphological features and a transcriptomic signature of microglial reactivity. The magnitude of intracellular AAV6 viral genomes was significantly higher as compared to the AAV8 condition. The elevated levels of intracellular AAV6 genomes induced a DNA damage response in AAV6-transduced iMGLs, which is a key feature of neuroinflammation-linked microglial reactivity. Thus, our work highlights the impact of intracellular viral genome accumulation on microglial reactivity and its implications for retinal gene therapy associated inflammation.

Previously exercise trained skeletal muscle is more growth-responsive to retraining. Using a murine training-detraining-retraining approach and multi-omics, we find that Pecam1 gene expression is lower, but capillarization is appreciably higher in previously trained (preconditioned) relative to naive trained control muscle. Greater capillarity could be permissive for accelerated hypertrophic adaptation. Exercise preconditioned myonuclei feature differential promoter CpG regulation in genes related to Wnt signaling. These epigenetic alterations with retraining align with our prior observations of methylation changes within the same pathway after a longer period of chronic training, suggesting a more rapid response due to preconditioning. Methylome-transcriptome integration and single myonucleus RNA-sequencing expose the polyamine metabolism enzyme Smox as a target that relates to heightened hypertrophic adaptability with retraining. Smox induction is sufficient to cause hypertrophy in aligned myotubes cultured on a stiffness-tuned substrate along with a growth-supportive transcriptional program. Integration of our multi-omics data suggests that Smox regulates repression of Ddit4/Redd1 (an inhibitor of mTORC1 signaling) after retraining. Smox may govern a favorable muscle fiber growth environment in previously trained muscle by sensitizing anabolic potential through polyamine metabolism. A lower adaptive threshold mediated by Smox could contribute to myometaplasticity, or a change to how subsequent muscle adaptations are made.

Multiple sclerosis (MS) is approximately three times more common in females than in males, but sex-linked epigenetic mechanisms remain incompletely understood. We tested whether X-chromosome DNA methylation differs in females with MS and whether associated sites preferentially map to genes normally subject to X-chromosome inactivation (XCI). An X-chromosome-focused epigenome-wide association study was performed in whole blood from three datasets comprising 496 females with MS and 509 female controls, with independent evaluation in isolated CD4+ T cells (80 cases, 76 controls). At FDR < 0.01, 3 182 X-chromosome CpGs were hypomethylated in MS (mean {Delta}{beta} = -0.035). Hypomethylated CpGs were strongly enriched in non-escape genes normally subject to XCI relative to escape genes (OR 6.77; p = 1.55x10-57) and mapped to immune and chromatin-related processes. Exploratory cis-mQTL analysis identified strong local genetic effects at a small subset of loci, including WAS, IRAK1 and TLR7. In CD4+ T cells, 2 080 of the 3,182 whole-blood hypomethylated CpGs (65.4%) were also significantly hypomethylated. These findings identify a reproducible X-chromosome hypomethylation signature in female MS and support direct investigation of inactive-X regulation, XCI escape and X-linked immune gene dosage.

Reactivation of telomerase confers immortality to approximately 90% of human tumors by enabling continuous elongation of the DNA at chromosome ends, or telomeres. The telomerase catalytic subunit TERT adds TTAGGG repeats using a portion of the telomerase RNA component hTR as a template. Because telomerase is inactive in most normal somatic cells, it remains an attractive therapeutic target; however, no telomerase inhibitor has yet demonstrated robust clinical efficacy with acceptable safety. Here we evaluate thiomorpholino oligonucleotides (TMOs) as a new class of antisense oligonucleotides targeting the template region of hTR. TMOs incorporate morpholino rings and phosphorothioate linkages, which enhance nuclease resistance, RNA binding and nuclear uptake. Two anti-hTR TMOs inhibited telomerase activity in vitro with an IC50 below 1 nM, whereas two control TMOs were at least 100-fold less active. HeLa cells treated with anti-hTR TMOs showed progressive telomere shortening, detectable after one week of treatment. Growth inhibition was observed after substantial telomere erosion, and both telomere length and proliferation recovered upon withdrawal of TMOs. These findings establish TMOs as a promising new chemistry for telomerase-targeted therapeutics.

The long-range RNA-RNA interaction between the termini of a flavivirus genome is controlled by multiple local RNA secondary and tertiary structures. However, a complete picture of this essential genome cyclization process is still elusive. Herein, by using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), SHAPE and mutational profiling, in vitro RNA binding assays, and reverse genetics approaches, we demonstrated that the hairpin 2 in the capsid-coding region (cHP2) is required for efficient replication of yellow fever virus and functionally interacts with the 3'-small hairpin (sHP) during genome cyclization. Interestingly, through a combination of multidimensional chemical probing and functional characterization assays, it was discovered that the sHP forms previously unknown tertiary interactions with an immediately upstream GACG region embedded within the 3'-cyclization sequence. This crucial tertiary RNA motif accounts for a potential negative regulatory mechanism of genome cyclization and is universally conserved among the mosquito-borne-related flaviviruses, which constitute a major phylogenetic branch within the subgenus Euflavivirus, genus Orthoflavivirus. Our study highlights the intricacy of RNA-based regulation in flaviviruses.

Purpose. Neuroblastoma is the most common malignancy of infancy, and cancer cells strongly depend on iron metabolism for proliferation. Whether sub-cytotoxic iron load reprograms mitochondrial and redox metabolism, and how this relates to the malignant behavior of neuroblastoma cells, is poorly defined. We hypothesized that exposure of human neuroblastoma cells to a non-toxic iron overload perturbs mitochondrial and redox metabolism, thereby affecting cancer cell properties. Methods. SH-SY5Y neuroblastoma cells were exposed to 100 M iron sulfate for 24 h. The resulting changes were monitored using complementary methods, including cell viability, cytotoxicity, proliferation and migration assays, intracellular iron quantification by ICP-MS, confocal imaging of mitochondrial morphology and membrane potential ({Delta}{Psi}m), high-resolution respirometry with flux control ratios, probe-based oxidant and H2O2 measurements, and label-free quantitative proteomics with Ingenuity Pathway Analysis. Results. Non-toxic iron loading raised intracellular iron about four-fold, promoted cell proliferation and reduced migration. Iron induced mitochondrial fragmentation and {Delta}{Psi}m depolarization while preserving mitochondrial content and OXPHOS coupling efficiency. Among respiratory states, only proton-leak respiration increased, paralleled by a higher leak/ETS coupling ratio. Total cellular oxidant levels and H2O2 output were strongly reduced. The proteome showed downregulation of ribosomal proteins and translation/RNA-processing components and upregulation of histones and ferritin. Conclusion. Mild iron exposure reprograms mitochondrial and redox metabolism in neuroblastoma cells. The observed selective increase in proton leak lowers {Delta}{Psi}m to a range that biophysically limits oxidant production while OXPHOS coupling is preserved. Interfering with this adaptive, proton leak-dependent and preventive antioxidant program may offer a way to alter neuroblastoma progression.

Defective interfering particles (DIPs) represent promising antivirals against influenza A virus (IAV), yet strategies to further enhance their antiviral activity remain largely unexplored. Here, we engineered a next-generation OP7 DIP by truncating the viral interferon (IFN) antagonist non-structural protein 1 (NS1) to enhance IFN-mediated antiviral activity. A previously validated mathematical multiscale model predicted that loss of NS1 function would enhance antiviral activity, providing a mechanistic rationale for this design. We engineered OP7-trNS1 DIP, established a cell culture-based production process yielding infectious virus-free material, and evaluated its antiviral activity in human lung epithelial cells in vitro. OP7-trNS1 inhibited IAV replication by approx. two orders of magnitude more effectively than the parental OP7 DIP while inducing a stronger IFN response, consistent with the model predictions. Together, these findings demonstrate that reducing viral IFN antagonism is a viable strategy for rationally enhancing DIP antiviral activity and establish OP7-trNS1 as a promising candidate for further preclinical development.

Transfer RNA (tRNA) is highly modified and difficult to profile using reverse-transcription-based sequencing. Here, we established a direct RNA sequencing (tRNA-DRS) workflow to simultaneously characterize tRNA abundance and modification signals in mouse liver. The workflow included small-RNA enrichment, deacylation, splint-adapter ligation, barcoding, reverse transcription, and nanopore sequencing on PromethION with modification-aware basecalling. From 1,678,666 raw reads, 1,421,279 (84.67%) reads were retained after 20-200 nt length filtering, and 17,094 uniquely mapped reads (MAPQ [&ge;] 10) were obtained. These reads covered 244 tRNA genes and represented all 23 tRNA isotypes. tRNA abundance was strongly skewed, with Asp, Gly, Ser, and Lys accounting for 56.6% of uniquely mapped reads. Read-body coverage indicated that most uniquely mapped reads spanned substantial portions of tRNA molecules (mean 71.1%, median 75.9%; 83.3% reads covering [&ge;]50% of the reference). Modification analysis identified 41,013 candidate sites, of which 711 passed stringent filtering (modification fraction [&ge;]50%, coverage [&ge;]5), spanning eight modification types and dominated by pseudouridine and m5C. Filtered sites showed position-specific clustering and isotype-dependent patterns. Together, these results support tRNA-DRS as a practical single-assay framework for simultaneous tRNA expression and epitranscriptomic profiling in mammalian tissue.

Duchenne muscular dystrophy (DMD) is an X-linked disorder mainly affecting young boys characterized by repeated cycles of myofiber injury/regeneration and chronic muscle inflammation. Although glucocorticoids can slow disease progression, their potential long-term adverse effects highlight the need for new steroid-sparing therapies for DMD. Annexin A1 (ANXA1) is a glucocorticoid-induced pro-resolving protein that signals primarily through formyl peptide receptor 2 (FPR2). We investigated whether the ANXA1 mimetic peptide Ac2-26 could reduce inflammation, stimulate tissue regeneration, and improve skeletal muscle function in muscular dystrophy. 3-week-old dystrophin-deficient male mdx mice were randomized to receive daily intraperitoneal injections of vehicle control, deflazacort (1 mg/kg/day), or Ac2-26 (1 mg/kg/day) for a duration of 5-weeks. Body composition, skeletal muscle mass, contractile function, and tissue histopathology/immune-cell infiltration were assessed at 8-weeks of age. Direct effects on myogenesis were also examined in vitro using primary D2-mdx myoblasts. ANXA1 was expressed predominantly by myeloid cells rather than resident dystrophic muscle cells. Both deflazacort and Ac2-26 markedly reduced intramuscular inflammation and increased myofiber size. However, Ac2-26 produced broader anabolic and functional benefits, increasing body-weight gain, lean body mass, hindlimb muscle weights, and absolute contractile force. Ac2-26 treatment also directly promoted myogenesis of dystrophic myoblasts in vitro, while pharmacological FPR2 blockade impaired myotube development. Overall, Ac2-26 mimicked the anti-inflammatory effects of deflazacort while providing superior benefits for body weight and composition, skeletal muscle growth, myofiber regeneration, and absolute muscle strength. These findings support therapeutic targeting of the ANXA1-FPR2 axis as a potential safe and effective steroid-sparing immunomodulatory strategy for DMD.

Antisense oligonucleotides (ASOs) represent a promising therapeutic modality for RNA-based disease mechanisms, but identifying high-efficacy candidates from large sequence spaces remains a major bottleneck in drug development. Here, we present ASOFormer, a novel Transformer-based neural network that predicts the inhibition efficiency of RNase H-mediated ASOs from sequence, predicted secondary structure, and chemical modification features. ASOFormer was trained on a knockdown efficacy dataset spanning over 170,000 ASO-target pairs compiled largely from published patents. Ablation analysis confirmed that the addition of chemical modification features to the primary sequence drove the largest performance gains, with predicted secondary structure providing complementary benefit when combined with modification information. Further analysis highlighted the importance of the wings of gapmer ASOs and other features. As independent validation, ASOFormer was applied to ASOs targeting seven genes that were not in the training dataset gene list. For four out of the seven genes, the ground truth ASO inhibition efficiency was obtained from a public dataset, whereas the others were measured in-house via qPCR. Compared to two published state-of-the-art methods, ASOFormer achieved the best overall accuracy and prioritized the strongest inhibitors. Crucially, only ASOFormer consistently exceeded random expectation for top-candidate recovery across all seven test genes.

Pyrogenic black carbon, such as biochar and wildfire char, possesses a reversible electron storage capacity (ESC), allowing it to serve as an electron donor or acceptor for microorganisms. However, not all the ESC is bioavailable, and the proportion available varies by culture. We hypothesized microbes capable of producing soluble redox mediators could access the ESC in biochar interior and thus utilize a higher percentage of the ESC than organisms without a mediator. To test this hypothesis, we grew wild-type Shewanella oneidensis (MR-1) and {Delta}bfe MR-1, a mutant incapable of exporting flavins, on air-oxidized wood biochar. Cell counts and redox titration show that both strains of MR-1 grew to similar cell densities and accessed comparable amounts of biochar ESC (~19%). The addition of exogenous flavin mononucleotide (FMN) yielded no measurable increase in final cell density, due to the strong adsorption of FMN to biochar. SEM images show attachment of MR-1 and {Delta}bfe to biochar surfaces and the presence of nanowires. This study demonstrates MR-1 can utilize biochar ESC for growth, through direct physical contact without the involvement of flavins. Understanding how microbes metabolize biochar provides insight into the impacts and biogeochemical roles of pyrogenic carbon.

Naive T cells are poised for activation and contain a pool of translationally repressed ribosomal protein (RP) mRNA prepared to induce ribosome biogenesis to support protein synthesis, cell growth and proliferation. RP mRNA are the prototypical members of a class of transcripts initiating at cytosine followed by a CU rich element called terminal oligo pyrimidine (TOP) RNAs. TOP RNAs are regulated by an RNA binding protein LARP1, which promotes transcript stabilisation and translational repression. We investigated LARP1 function in T cell activation by generating cross-linking immunoprecipitation (CLIP) datasets detailing the LARP1-RNA interactions in naive and activated CD4+ T cells and identifying novel TOP RNAs. TOP RNAs identified by this analysis were functionally diverse. RP mRNAs were typified by high stability, and translational repression in naive T cells followed by MTORC1-dependent translation increases following T cell activation. However, other TOP RNAs varied in these aspects of their regulation. Notably, TOP RNAs with longer 3'UTRs had a relaxed dependency on LARP1 for stability and a reduced dependency on MTORC1 for their translation. Transcription start site heterogeneity also impacted TOP RNA regulation by generating a mixture of transcript isoforms with different TOP motif lengths. Longer terminal oligo pyrimidine stretches were associated with a greater dependency on MTORC1 for translation. Differential regulation of TOP RNAs may allow tuneable translational responses to MTORC1 and indicates potential roles for LARP1 beyond translation regulation and stability.

Therapeutic siRNAs are a major clinical modality for hepatic gene silencing, but efficient extrahepatic delivery and multi-gene modulation remain significant barriers. Nucleic acid nanotechnology offers programmable assembly, but conventional structures suffer from enzymatic instability in vivo . Here we report fully chemically modified RNA nanostructures (FMRNs): a vehicle-free platform entirely composed of stabilized therapeutic nucleic acids that integrate structural precision with metabolic durability. Following systemic administration, FMRNs exhibit enhanced pharmacokinetics and extrahepatic accumulation, with in vivo behavior influenced greatly by hydrodynamic size rather than molecular weight. Using triangular and square geometries, we demonstrate programmable control over siRNA stoichiometry to achieve sustained, simultaneous silencing of up to four genes from a single entity. Nanostructure assembly alone drives productive delivery, especially to heart and muscle, matching lipophilic conjugation. Furthermore, cerebrospinal fluid administration reveals that larger FMRNs improve central nervous system distribution and retention revealing physicochemical parameters that impact brain exposure. FMRNs establish a modular and effective platform combining improved pharmacokinetics, extrahepatic delivery, and multi-gene silencing for complex diseases.