Molecular Therapy Nucleic Acids

Haploinsufficiency in SYNGAP1 causes a severe neurodevelopmental syndrome. SYNGAP1 protein is mainly detected in neuronal synapses. However, SYNGAP1 RNA is more widely expressed and strongly regulated via alternative splicing: alternative 3' splice site (A3SS) inclusion leads to non-productive transcripts that are degraded through nonsense-mediated decay. Recently, splice-switching oligonucleotides (SSOs) that redirect SYNGAP1 splicing to increase SYNGAP1 protein levels were developed. However, we hypothesized that during neuronal maturation, non-productive splicing may decrease to enhance functional transcripts in mature neurons. This would reduce the abundance of the SSO target transcript, limiting the potential for SSO treatment to increase neuronal SYNGAP1 expression. Using neural differentiation of human induced pluripotent stem cells, we show that the A3SS transcript is abundant in neural progenitors, astrocytes, microglia and immature neurons, with minimal presence in mature neurons. These data imply that SSOs targeting A3SS might lack therapeutic efficacy to rescue the neuronal phenotypes associated with SYNGAP1 haploinsufficiency.

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.

Antisense oligonucleotides (ASOs) are a versatile therapeutic modality for inborn genetic diseases. ASOs can induce skipping of ''dispensable'' exons containing disease-causing variants to rescue protein amount and function, but this approach has been studied for only a small number of genes. We developed a high-throughput in silico tool for assessing exon dispensability and designing exon-skipping ASO sequences. Parameters were optimized using known dispensable and in-frame indispensable exons. Across 72,644 exons of 5,057 disease genes, we identified thousands of new targets for exon-skipping ASOs (3.4% of exons with most stringent filters, 24.5% with less stringent filters) that collectively include 0.97%-15.6% of disease-causing variants in large-scale databases. To facilitate recognition of DNA variants potentially amenable to exon skipping as a therapeutic strategy, we established the HAWK-EYE database as a repository of exon dispensability predictions and corresponding in silico-optimized ASO sequences, available as an open-access web application (https://hawk-eye.research.sickkids.ca/). To illustrate translational utility, we experimentally validated a subset of the in silico-optimized ASO sequences that were generated for all Dispensable exons in the HAWK-EYE database, and showed that skipping a Dispensable exon in SOX5 preserves protein function using in vivo and in vitro assays. This scalable platform approach to exon-skipping ASOs will accelerate identification and testing of amenable genetic variants.

Background & Aims: Adeno-associated virus (AAV) vectors are attractive delivery vehicles for therapeutic gene delivery, and a notable feature of most AAVs is their natural tropism for the liver, which leads to significant hepatic uptake following systemic administration. In previous work, we identified 266G as a conserved motif on a variable region on the capsid of many commonly used AAV variants that controls liver uptake in both mice and non-human primates. This single amino acid could be functionally leveraged to engineer AAVs to either de-target from or enhance tropism to the liver. Here, we explored whether these observations extended to the human context. Methods: Two human hepatocyte models were tested: Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice with humanized livers and a bioengineered human microliver platform in vitro. A barcoded AAV capsid library including standard control serotypes were used to assess the role of the 266G motif on gene transfer and transgene expression in both liver systems. Results: In vivo, 266G containing AAVs indeed targeted human hepatocytes superiorly, with some noted dependency on the degree of human-hepatocyte replacement in the chimeric mouse model. Initial studies in the micropatterned primary human hepatocyte co-culture model however demonstrated enrichment of heparin-binding AAVs, and not 266G variants. Notably, incorporation of polyethylene glycol (PEG) into the system modified the AAV transduction potential of those capsids including the liver-targeting motif, recapitulating the hepatocyte transduction pattern observed in vivo. Importantly, when PEG was used, the two human models, both at the DNA and RNA level, did correlate significantly. Conclusions: Our results showed the potential of a combinatorial AAV library for model validation and revealed the human microliver platform-PEG as a reliable system for the development of AAV therapeutics.

A challenge of "once-and-done" adeno associated virus (AAV)-based gene therapy is the inability to modulate the level of therapeutic protein expression post-administration. Herein, we demonstrate the utility of an adenosine deaminase acting on RNA (ADAR) - mediated gene switch to control AAV-delivered gene expression. Using a premature termination codon (PTC) in the human Factor IX (hFIX) transgene, we established an ON switch, where expression of hFIX is contingent on rescuing the PTC mutation via RNA editing. In vitro and in vivo studies demonstrated silencing of the hFIX transgene by the PTC mutation and induction of protein expression by administration of an ADAR-recruiting trigger RNA. Mice transduced with a hepatotropic AAV capsid encoding an ApoE-hAAT hFIX-PTC transgene expression cassette showed a dose-dependent response between the levels of LNP-delivered trigger RNA and the amount of plasma hFIX expression achieved. We observed predictable and reproducible levels of hFIX expression upon multiple rounds of RNA editing and demonstrated that this system can achieve clinically relevant levels of hFIX. This work suggests that ADAR-mediated RNA editing may be a valuable tool for tunable expression of therapeutic transgenes in applied gene therapies.

Oligonucleotide based therapeutics continue to rise as a significant class of medicines for the treatment of human disease. However, achieving delivery to non-hepatic tissues remains a challenge in the field. While substantial advances have been realized, largely through the use of antibody or protein based targeting agents to tissues including muscle and the CNS, these large protein agents present complications in synthesis and carry the potential for immune responses. In an effort to identify a simpler, smaller and robust means of delivery, we have generated and evaluated aptamers targeting the human transferrin receptor (hTfR) for the delivery of both ASO and siRNA cargoes to muscle. Using a fully backbone modified anti-TfR aptamer, 36 nt in length, that binds hTfR and does not compete for binding with the natural ligand, transferrin, we evaluated the ability to deliver ASOs to skeletal muscle following systemic delivery. Using optimized linker chemistry, aptamer-ASO conjugates led to >50% target gene knockdown in muscle tissue for up to 42 days following a single dose at 3 mg/kg ASO ([~]11 mg/kg total drug) in mice. Taken as a whole, these results offer significant promise for the use of aptamers in the development of future therapeutics.

Duchenne muscular dystrophy is a lethal neuromuscular disorder caused by the absence of dystrophin, for which no curative treatment is available. RNA-based approaches have shown promising results; however, their evaluation is hindered by the lack of robust and rapid delivery methods for differentiated human muscle cells, which represent the most physiologically relevant in vitro models for assessing therapeutic strategies. Here, we establish a versatile lipid nanoparticle platform enabling efficient delivery of diverse RNA therapeutics across a range of human muscle models, including myotubes, induced pluripotent stem cell-derived myotubes, myoblasts, cardiomyocytes, and 3D engineered skeletal muscle tissues. Remarkably, a single commercially available lipid nanoparticle formulation supports delivery of cargos spanning more than 300-fold in size, from short antisense oligonucleotides ([~]20 nt) to complex CRISPR-based editors (up to [~]6.7 kb), including Cas9 nucleases, adenine base editors, and CRISPRa systems. This enables efficient gene correction and transcriptional modulation, resulting in dystrophin restoration or compensatory utrophin upregulation in relevant Duchenne muscular dystrophy models. Together, our results establish a single lipid nanoparticle formulation as a versatile platform for RNA delivery in human muscle systems and provide a practical framework for the rapid preclinical assessment of emerging therapies for Duchenne muscular dystrophy and other neuromuscular disorders. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/741396v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@a18d71org.highwire.dtl.DTLVardef@4d0faforg.highwire.dtl.DTLVardef@140b078org.highwire.dtl.DTLVardef@1a235_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Leclerc, D. (2026) https://BioRender.com/90h0fme

Messenger RNA (mRNA) is canonically composed of ribonucleotides, with sporadic incorporation of deoxyribonucleotides into natural RNA transcripts being traditionally regarded as a rare, deleterious error arising from transcriptional infidelity. Here, we challenge this paradigm by demonstrating controlled partial substitution of ribonucleotides with deoxyribonucleotides during in vitro transcription (IVT) generates intact, stable and fully translationally competent IVT-mRNA. Unexpectedly, chimeric DNA-RNA backbone modification exhibits markedly enhanced IVT-mRNA translation several fold across multiple cell types and in vivo via diverse dosing routes relative to their ribonucleotide-based counterparts. 25% substitution of cytidine triphosphate with deoxycytidine triphosphate achieved best-performing translational output, surpassing the current gold-standard N1-methylpseudouridine (m1{Psi})-modified IVT-mRNA in a B16-OVA tumor vaccination model. These findings identify nucleotide class composition as a previously unrecognized parameter governing IVT-mRNA function and establish hybrid ribonucleotide-deoxyribonucleotide backbone engineering as a versatile strategy to expand the chemical space for next-generation mRNA therapeutics.

Antisense oligonucleotide (ASO) activity is jointly influenced by nucleotide sequence, chemical modification, target-RNA context, dose, delivery protocol, and cellular environment. Most existing computational screening methods model only a subset of these factors, limiting their ability to predict experimentally measured activity across heterogeneous screening conditions and previously unseen biological contexts. We introduce ASOCompass, a context-and chemistry-aware framework for ASO activity prediction and candidate ranking. ASOCompass integrates contextualized ASO and target-RNA sequence representations with position-specific molecular representations of chemical modifications. It further incorporates dose and delivery information together with prototype-adapted transcriptomic representations of target genes and cell lines. To encourage chemically and biophysically informative representations, the model is jointly trained on auxiliary molecular-property and sequence-derived thermodynamic prediction tasks. We evaluate ASOCompass on ASO Atlas, a large patent-derived dataset of RNase H-mediated gapmer ASOs, under held-out drug, target-gene, cell line, and joint gene-cell line settings. ASOCompass achieves an overall Spearman correlation of 0.5970, improving over the strongest ASO-specific baseline by 0.0421, and consistently performs best across all four distribution shifts. When adapted to unseen SOD1 and KLKB1 targets, ASOCompass also provides more accurate candidate ranking across different annotation budgets, reaching correlations of 0.830 and 0.696 with 1,024 target-specific labels. Additional analyses suggest that molecular-property supervision improves modification-specific ranking, while the auxiliary thermodynamic task produces representations more closely aligned with measured inhibition. These results demonstrate the potential of jointly modeling sequence, chemistry, and experimental-biological context for transferable ASO screening.

Trauma to the spinal cord disrupts the blood-spinal cord barrier and triggers a secondary injury cascade characterized by inflammation and progressive neuronal and glial cell death. Therapeutic cytokines and growth factors have shown promise as a treatment in preclinical studies, though their clinical translation is limited by short protein half-lives and the need for invasive intraspinal administration. Lipid nanoparticle-mediated delivery of mRNA offers an alternative strategy that enables transient protein production. Here, we investigated whether intravenously administered mRNA-lipid nanoparticles could leverage the injury-induced disruption of the blood-spinal cord barrier to access the injured spinal cord for local transgene expression. After spinal cord injury in a rat, lipid nanoparticles loaded with reporter mRNA were administered intravenously, and transgene expression was quantified in the spinal cord and peripheral organs. Intravenous delivery within a 6-hours post-injury resulted in local transgene expression in the injured spinal cord, demonstrating that mRNA-lipid nanoparticles cross the disrupted blood-spinal cord barrier. Transgene expression was observed in astrocytes, oligodendrocytes, microglia, and neurons, detected within 3 hours and remained elevated for up to 5 days post-injury. These findings demonstrate that systemic mRNA-lipid nanoparticles delivery exploit transient blood-spinal cord barrier disruption to achieve local gene expression in the injured spinal cord.

Osteoarthritis is the most common joint disease for which disease-modifying therapies remain unavailable. Intra-articular gene delivery of interleukin-1 receptor antagonist (IL-1Ra) using high-capacity adenovirus (HCAd) has shown therapeutic promise; however, the duration of therapeutic benefit and the feasibility of repeat dosing under anti-adenoviral immunity remain unresolved. Using the murine anterior cruciate ligament transection model of osteoarthritis, we show that a single intraarticular injection of HCAd5-NF{kappa}B-IL-1Ra provides structural preservation and functional improvement in early-stage osteoarthritis but fails to sustain cartilage protection as disease progresses. However, HCAd5 transduction following repeated treatment is limited due to pre-existing immunity against this serotype. Notably, exchanging serotypes for repeated treatments effectively restores vector transduction and transgene expression in both healthy and osteoarthritic joints. Leveraging this strategy, we demonstrate that sequential intra-articular HCAd-NF{kappa}B-IL-1Ra administration does not further improve pain or motor function compared to the initial treatment, but preserves cartilage as assessed by histopathology and phase-contrast CT, irrespective of serotype. These findings establish HCAd serotype switching as a feasible approach to overcome immune barriers to repeat intra-articular gene therapy. Importantly, sequential HCAd-NF{kappa}B-IL-1Ra administration enhances therapeutic durability in post-traumatic osteoarthritis, providing a translational framework for repeat intra-articular gene delivery strategies aimed at long-term disease modification in osteoarthritis.

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.

The blood-brain barrier (BBB) is a highly selective cell layer that restricts the diffusion of diverse chemical entities into the central nervous system (CNS) from systemic circulation. Macromolecular therapeutics including oligonucleotides, peptides, and monoclonal antibodies exhibit only minimal brain distribution after systemic dosing due to exclusion by the BBB. Receptor-mediated transcytosis (RMT) has evolved to transport vital cargo across the BBB through a specialized vesicular transport pathway. Transferrin receptor 1 (TfR1) shuttles transferrin, its natural ligand, across the BBB, as well as TfR1-binding IgG antibodies and conjugates. Here, we describe a novel monovalent TfR1-binding VHH-Fc for the delivery of oligonucleotide cargo, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) across the BBB in rodents and non-human primates (NHPs), supporting the translational potential of the VHH-antisense RMT platform for the treatment of neurological disorders. We explore the role of binding affinity, conjugation site, drug-antibody ratio (DAR), and conjugation chemistry, and determine that binding affinity, DAR and conjugation site are major determinants of RMT capacity and brain activity of siRNAs delivered across the BBB. Graphical Abstract / Highlights O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/744307v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@d1d648org.highwire.dtl.DTLVardef@4b22d3org.highwire.dtl.DTLVardef@db8b6borg.highwire.dtl.DTLVardef@19e5ac3_HPS_FORMAT_FIGEXP M_FIG C_FIG - Anti-TfR1 (-TfR1) VHH ligands formatted as heterodimeric, 2-chain monovalent VHH-Fc were engineered for conjugation to siRNA and ASO. - Systematic in vivo evaluation of VHH clones spanning a range of TfR1 binding affinities revealed a relationship between TfR1 binding affinity and the CNS activity of intravenously dosed VHH-Fc-siRNA conjugates. - By optimizing TfR1 binding affinity, conjugation site, and conjugation chemistry, we identified VHH-Fc-siRNA molecules that efficiently cross the BBB via receptor-mediated transcytosis and reduce target mRNA across CNS tissues, including deeper brain regions, after intravenous (IV) or subcutaneous (SC) dosing in mice and non-human primates (NHPs).

BackgroundLMNA-related congenital muscular dystrophy (L-CMD) is a rare, life-threatening genetic disorder caused by point mutations in the LMNA gene, for which no effective treatment currently exists. It is characterized by early-onset muscle weakness, dropped-head syndrome, hypotonia, cardiac complications, and restrictive lung disease, frequently leading to premature death. The LMNA c.745C>T (p.R249W) mutation is the most prevalent amongst L-CMD patients. Given its monogenic nature, L-CMD represents a compelling candidate for gene therapy approaches. ResultsIn this study, we investigated the therapeutic potential of adenine base editing (ABE) to correct the pathogenic LMNA c.745C>T (p.R249W) mutation in human myoblasts. We evaluated multiple ABE variants and single-guide RNAs (sgRNAs), identifying optimal combinations that achieved efficient and specific correction of the mutant allele. However, we found that editing can also introduce an adjacent bystander mutation, c.743T>C (p.L248P). To determine the functional consequences of base editing, we established clonal cell lines reverted to wild type or harboring the p.L248P variant. Whereas wild-type edited cells showed a clear correction for all the studied parameters that were abnormal in R249W myoblasts, we found that L248P cells show nuclear abnormalities resembling those of R249W mutant cells, and their cellular function is partially compromised. These results demonstrate that ABE can effectively target the LMNA c.745C>T mutation but also reveal the significant impact of bystander edits on cellular physiology. ConclusionsOur findings provide proof-of-concept for the application of base editing as a therapeutic strategy for L-CMD, while underscoring the necessity of precise editing technologies to ensure both efficacy and safety in future clinical translation.

Targeting GC-rich gene loci is a major challenge owing to their high duplex stability, repetitive sequence composition, and propensity to adopt alternative DNA structures. Triplex-forming oligonucleotides (TFOs) provide a programmable strategy towards the recognition of GC-rich DNA, but their application is restricted by the limited recognition capabilities of natural nucleobases in a cellular setting. Here, we overcome this barrier using parallel-binding TFOs containing the synthetic nucleobase 6-amino-5-nitropyridin-2-one (Z), which enables pH-independent recognition of G-C base pairs. Using two structurally distinct regulatory elements within the MYC promoter, we show that Z-modified TFOs form stable, sequence-selective triplexes that repress promoter activity by 50-80% in both episomal reporter assays and at endogenous gene loci. Notably, the greatest repression was observed at a GC-rich quadruplex-forming element that functions as a structural hub for transcription factor recruitment. To our knowledge, this represents the first demonstration that a simple nucleobase modification alone is sufficient to enable parallel-binding TFOs to repress expression of an endogenous gene, establishing a general strategy for targeting GC-rich regulatory elements through programmable DNA recognition. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/741700v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d23b1corg.highwire.dtl.DTLVardef@126de76org.highwire.dtl.DTLVardef@d75631org.highwire.dtl.DTLVardef@15bd46d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Facioscapulohumeral muscular dystrophy (FSHD) is a rare neuromuscular disease caused by aberrant re-expression of the embryonic transcription factor DUX4 in skeletal muscle, which activates a toxic transcriptional program that drives progressive muscle wasting. No approved disease-modifying therapies currently exist. Prior work in mammalian and zebrafish models has shown that a truncated form of DUX4 retaining only its DNA-binding domain (DBD) lacks transactivation capacity and can suppress DUX4-FL-driven pathology; separately, dCas9/KRAB-based epigenetic repressors have demonstrated efficacy in silencing DUX4 expression, though CRISPR-based strategies face challenges from the repetitive nature of the D4Z4 locus, the immunogenicity associated with bacterial Cas proteins, and the payload limitations of gene delivery vehicles. Building on these findings, we corroborate that the DUX4 DBD, comprising both homeodomains, acts as a non-toxic competitive inhibitor of full-length DUX4 (DUX4-FL) at its genomic target sites, and extend this strategy by fusing the DBD to a human KRAB(ZNF10) domain, converting DUX4 from a transcriptional activator into a fully humanized epigenetic silencer of its own targets. Using a fluorescent DUX4-responsive reporter, we show that DBD alone produces dose-dependent repression of DUX4-FL transcriptional activity in HEK293T cells (200-fold at the highest inducible dose tested), while a constitutively expressed DBD-KRAB fusion produces significantly greater repression than DBD alone (949-fold versus 17-fold at a 25x molar ratio), with a similar trend observed in C2C12 myoblasts (47-fold versus 3.3-fold knockdown). To contextualize these findings and explore dosing considerations, we developed three complementary computational models - a transcription factor competitive binding model, a myotube diffusion model, and an ordinary differential equation (ODE) compartmental model - that illustrate how DBD concentration, intracellular diffusion, and population-level cell state transitions may relate to therapeutic efficacy. Together, these results corroborate and extend existing approaches into a single, fully humanized construct that may help circumvent the immunogenicity and delivery limitations of Cas-based systems.

In an era of increasing drug resistance, exploring alternative molecules is crucial for the efficient management and treatment of viral diseases. Nucleic acid aptamers have emerged as highly promising candidates due to their exceptional target specificity, low immunogenicity, and versatile mechanisms for viral blocking. This manuscript describes AptViralDB, a manually curated database providing comprehensive information on experimentally validated antiviral aptamers. It contains 1,768 entries of antiviral aptamers against 40 viral species and 104 molecular targets, compiled from literature and existing databases. Each entry provides detailed annotations, including sequence, aptamer type, target, chemical modifications, binding affinity, antiviral activity, stability, and cytotoxicity. We also provide predicted secondary structures and their corresponding minimum free energy (MFE) values. Additionally, a knowledge graph created using ArcadeDB/openCypher enables users to seamlessly explore connections among aptamers, viruses, molecular targets, and biological activities. Finally, the platform offers advanced search and browsing tools, BLAST-based sequence similarity searches, GC-content analysis, downloadable datasets, and REST API access to support computational applications. (https://webs.iiitd.edu.in/raghava/aptviraldb/).

The blood-brain barrier (BBB) is a highly selective, semi-permeable border of endothelial cells that prevents solutes and therapeutic agents in systemic circulation from passively crossing into the central nervous system (CNS) parenchyma. The Transferrin receptor 1 (TfR1) endocytosis pathway for iron homeostasis is one of the most well-characterized strategies for therapeutic delivery across the BBB. The work presented here showcases the discovery of novel anti-TfR1 NANOBODY(R) shuttles. The identified anti-TfR1 NANOBODY(R) molecules display cross-reactivity and pH-dependent binding to human, cynomolgus (cyno), and mouse TfR1. Structural data further explain and support the underlying mechanism of this pH-dependent binding. These anti-TfR1 NANOBODY(R) molecules were successfully conjugated to both short-interfering RNA (siRNA) and antisense oligonucleotide (ASO) tool payloads. anti-TfR1 NANOBODY(R)-siRNA conjugates can induce up to 60% knockdown of the target mRNA transcript in skeletal muscle up to two weeks post a single IV dose in mice and up to 35-40% at four weeks post dose. Furthermore, extending the half-life of the anti-TfR1 NANOBODY(R)-ASO shuttles enhances heart, sciatic nerve, and brain exposure and enables up to 30-60% target knockdown in different CNS cell types. Altogether, these results highlight important features for the development of anti-TfR1 shuttles for the purpose of downregulating target mRNA transcripts in muscle and CNS for a variety of neurologic and neuromuscular indications.

Chimeric antigen receptor (CAR)-T cell therapies have transformed the treatment of various tumor types by redirecting and activating T cells against tumor cells. However, CAR-T cell manufacturing approaches remain challenging and limit their widespread use in clinical settings. In vivo CAR-T therapy bypasses ex vivo cell manufacturing and patient preconditioning limitations; however, it faces a significant safety concern as CAR proteins on viral packaging cells are incorporated into budding virions, leading to off-target transduction of tumor cells. Here, we address this risk by developing the CAR-Less ER-Anchor Vector (CLEAN-V) system. By exploiting endoplasmic reticulum (ER) retention, CLEAN-V prevents the CAR protein from trafficking to the cell surface during viral packaging, thereby blocking its incorporation into the viral envelope. CLEAN-V particles exhibit near-complete loss of CAR-mediated tumor cell transduction. Furthermore, CLEAN-V integrates seamlessly into existing third-generation LVV workflows in four- or five-plasmid formats and generates CAR-T cells with preserved phenotypic and functional integrity. These results establish CLEAN-V as a robust platform for developing safe, targeted lentiviral vectors for in vivo CAR-T therapy.

Periodontal disease and COVID-19 are linked by convergent immunoinflammatory pathways, yet the molecular basis of their interaction remains poorly defined. Here, we present a comprehensive salivary microRNA profile from individuals with prior SARS-CoV-2 infection, sampled approximately 3-6 months after diagnosis and meeting criteria for long COVID, providing new insight into the post-viral oral microenvironment. Salivary miRNA sequencing revealed widespread repression in patients with PD, consistent with persistent immune dysregulation. Relative to COVID-19-negative/PD-negative controls, thirty-two miRNAs were differentially expressed in COVID-19-positive/PD-positive individuals, all significantly downregulated. A similar signature was observed in a post-vaccination cohort for the selected dysregulated miRNAs. Integrative pathway analyses identified these miRNAs as regulators of core inflammatory circuits, including Ras, MAPK, and NF{kappa}B signaling, converging on IL-1{beta}- and TNF-centered networks relevant to both PD and COVID-19. Mechanistically, restoration of three downregulated miRNAs, miR- miR-30e-3p 106-3p-3p, and miR-652-3p attenuated NF{kappa}B activation and cytokine release in TLR-stimulated human oral keratinocytes, while their functional suppression using inhibitors potentiates inflammation. These miRNAs were also predicted to target SARS-CoV-2 spike and nucleocapsid transcripts, an interaction validated by dual-luciferase reporter assays. Their overexpression further reduced spike and nucleocapsid expression in Beta- and Omicron-infected epithelial cells, as measured by flow cytometry and RT-qPCR confirming host miRNAs as potent endogenous SARS-CoV-2 restriction factor. Together, these findings identify salivary host miRNAs as mechanistic regulators of oral inflammatory tone and viral persistence, establishing a molecular link between periodontal inflammation and post-COVID oral pathology.