A successful oncolytic virus is critical to therapeutic success, requiring the identification of an effective viral platform and immunomodulatory molecules that potently promote host immune responses and reprogram the tumor microenvironment (TME). In the present study, we developed V01, an oncolytic virus derived from the Western Reserve (WR) strain of vaccinia virus, featuring double deletions of the vaccinia growth factor (VGF) and thymidine kinase (TK) genes, along with the expression of interleukin-21 (IL-21). Locally administered intratumoral (i.t.) V01 elicited systemic anti-tumor effects against both the primary injected tumor and distant non-injected tumors in immunocompetent tumor-bearing mice. Abscopal effects of V01 were observed in homologous tumors but not in heterologous tumors. Combination treatment of V01 with immunotherapeutic agents-PD-1 antibody or natural killer (NK) cell therapy-exhibited synergistic tumor regression. Together, these results suggest that V01 holds significant promise as a novel immunotherapeutic or immunoadjuvant for solid tumor treatment, warranting further clinical investigation.

Systematic evolution of ligands by exponential enrichment (SELEX)-based aptamer discovery can be constrained by target availability, selection biases, and optimization requirements. We introduce Xelari, a modular pipeline for de novo DNA and RNA aptamer design from protein structures without target-specific SELEX data or retraining. Xelari combines algorithmic modules with surrogate models trained on physical or structural references. InNA reproduced nucleotide-amino-acid interaction energies with RMSE of 0.19 kcal/mol across 483,735 reference pairs. AliNA achieved a mean F1 of 0.757 on 339 PDB-derived RNA evaluation sequences. Edgar correlated with experimental folding energies but did not reproduce their absolute scale, supporting its use as a ranking heuristic. Under the reported calibration, ValentiNA showed MAE of 0.931 kcal/mol and Spearman ρ of 0.795 across 32 retrospective records associated with 27 structural templates; this analysis does not establish family-independent validation. Prospectively, three DNA aptamers designed against Treponema pallidum protein structures showed cell-associated binding in a treponeme assay (apparent KD, 0.91-2.57 μM). A fourth DNA aptamer served as a capture reagent in a serum-based hybrid aptamer-antibody assay for Tau441. These applications support feasibility without establishing intended-target engagement in the cellular assay or a general success rate, positioning structure-based design as a complement to experimental selection.

Intracellular immune checkpoints regulate activation thresholds in cytotoxic immune cells. Casitas B-lineage lymphoma proto-oncogene B (CBL-B) is an E3 ubiquitin ligase that negatively regulates antigen receptor signaling and T cell activation. APN401 is an autologous peripheral blood mononuclear cell (PBMC) therapy incorporating transient small interfering RNA (siRNA)-mediated CBL-B silencing. Two phase 1 studies evaluated escalating doses of APN401 in patients with advanced treatment-refractory solid tumors. APN401 was manufactured from leukapheresis-derived PBMCs using the EPiC point-of-care platform. Seventeen patients received APN401 across three dose levels. APN401 demonstrated a favorable safety profile, with no dose-limiting toxicities, cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, or grade ≥3 treatment-related adverse events. Manufacturing was reproducible across sites. Although no objective responses were observed, disease stabilization occurred in 5 of 10 Response Evaluation Criteria in Solid Tumors (RECIST)-evaluable patients (50.0%), corresponding to 29.4% of all 17 treated patients. Exploratory analyses identified treatment-associated systemic immune changes, including enrichment of circulating CD8[+] T cells, induction of cytotoxic mediators, and expansion of dominant T cell receptor β (TCRβ) clonotypes. These changes were most pronounced in patients with stable disease but remain exploratory and hypothesis-generating. These findings support further evaluation of APN401 in larger, controlled studies.

Neonatal mouse hearts exhibit a transient regenerative capacity that enables recovery from injury and certain pathological stresses; however, their response to pathological hypertrophic stimuli remains poorly defined. Here, we show that neonatal hearts within the regenerative window are protected from isoproterenol-induced pathological hypertrophy and maintain cardiac function, whereas hearts beyond this window develop hypertrophy and functional impairment. Resistance to hypertrophic stress during the regenerative phase was associated with robust cardiomyocyte proliferation, which was largely absent in the non-regenerative phase. Transcriptomic profiling identified the long non-coding RNA Lockd as the most upregulated transcript during the regenerative window, in an E2F1-dependent manner. Cardiomyocyte-specific AAV9-mediated overexpression of Lockd in non-regenerative neonatal hearts attenuated isoproterenol-induced hypertrophy, preserved cardiac function, and restored cardiomyocyte proliferative capacity. Mechanistically, Lockd directly interacted with and stabilized α-enolase (ENO1), leading to enhanced β-catenin stability, nuclear accumulation, and induction of the pro-proliferative target gene Myc. Consistently, Eno1 knockdown impaired β-catenin signaling, Myc expression, and cardiomyocyte proliferation. These findings identify Lockd as a key mediator of regenerative resistance to pathological hypertrophic stress in the neonatal heart.

Efficient and cell-type-specific delivery remains a major barrier to CRISPR-based genome editing in vascular endothelial cells. Here, we systematically evaluate lipid nanoparticle (LNP)-mediated mRNA and CRISPR delivery in human umbilical vein endothelial cells (HUVECs). While a conventional SM-102-based LNP formulation enables highly efficient mRNA delivery to both HEK293T cells and HUVECs, CRISPR-SpCas9 gene editing is observed exclusively in HEK293T cells, revealing strong cell-dependent limitations. We identify reduced SpCas9 mRNA translation as a key bottleneck in endothelial cells and demonstrate that rational modification of LNP lipid composition dramatically enhances SpCas9 protein expression and restores robust genome editing in HUVECs. Using the optimized LNP formulation, we achieve efficient editing of VEGFA and HIF1A in both 2D cultures and physiologically relevant 3D microfluidic endothelial systems mimicking flow conditions in vivo. Together, our results establish lipid composition as a critical determinant of endothelial CRISPR editing efficiency and provide a scalable, non-viral strategy for genome engineering in the vascular endothelium.

Our objective is to develop a universal gene editing therapeutic for individuals with cystic fibrosis (CF). Due to the large number of CFTR variants, we sought to develop a correction methodology utilizing targeted integration of CFTR 2-27 sequence into CFTR intron 1 (TI-1). Following electroporation of CF primary airway basal stem cells with intron 1-targeted Cas9/gRNA, we delivered CFTR 2-27 donor, flanked by a splice acceptor and polyadenylation signal, via an integrase defective lentiviral vector. Molecular characterization revealed highly efficient integration of the CFTR 2-27 sequence into the native CFTR intron 1 with most insertion events reflecting the desired homology-directed repair. Fully differentiated airway epithelium, derived from TI-1 treated basal cells without enrichment for corrected cells, exhibited functional restoration of CFTR channel activity with significant improvement of mucus hydration and transport. ATAC-seq revealed that the native CFTR chromatin architecture was unaffected by the CFTR 2-27 sequence insertion, and importantly, scRNA-seq demonstrated that the cell-type-specific expression of the introduced transgene replicated that of the endogenous CFTR. In this report, we demonstrate an almost completely mutation-agnostic therapeutic approach that is capable of functional CFTR restoration. These results may also be relevant to CFTR 2-27 integration via other editing and delivery methodologies.

Chronic hepatitis B (CHB) remains difficult to cure because nucleos(t)ide analogs suppress hepatitis B virus (HBV) replication but do not eliminate covalently closed circular DNA (cccDNA) or integrated HBV DNA, which sustain viral RNA and hepatitis B surface antigen (HBsAg) production. RNA-targeted therapeutics, including small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), and CRISPR-Cas13 platforms, offer sequence-directed approaches to suppress HBV transcripts and reduce antigen burden. Clinical evaluation of emerging HBV-directed therapies, including JNJ-3989, bepirovirsen, imdusiran, and related candidates, has demonstrated meaningful declines in circulating HBsAg levels. The recent phase III B-Well data further support the role of ASO-mediated antigen reduction as a potential therapeutic approach to increase the likelihood of functional cure within finite treatment regimens for selected patients. However, therapeutically induced HBsAg reduction alone rarely guarantees durable off-treatment control. This review summarizes HBV-directed RNA and oligonucleotide therapeutics from a translational pharmacology perspective, emphasizing transcript-silencing mechanisms, delivery constraints, pharmacodynamic endpoints, safety considerations, biomarker-guided patient selection, and integration with nucleos(t)ide analogs and immunomodulatory interventions. We propose that RNA-mediated antigen reduction may support finite therapy in biomarker-selected patients and may also serve as an antigen-conditioning component within staged functional cure strategies.

X-linked myotubular myopathy (XLMTM) is caused by mutations in the MTM1 gene that encodes the myotubularin protein, essential for normal muscle development and function. XLMTM manifests as profound muscle weakness and hypotonia at birth, leading to severe respiratory insufficiency, and in many cases, early death. Resamirigene bilparvovec (rAAV8-Des-hMTM1) is a muscle-directed, adeno-associated virus (AAV)-based gene therapy in clinical development for the treatment of XLMTM. In the first-in-human clinical trial (ASPIRO; NCT03199469), rAAV8-Des-hMTM1 was associated with reduced ventilator dependence and unprecedented improvements in motor function in boys with XLMTM. However, four of the 24 participants who received rAAV8-Des-hMTM1 died after developing cholestatic liver failure. Safety results from large-animal studies of rAAV8-Des-MTM1 are presented herein and discussed retrospectively in the context of the adverse events observed in the clinical trial. Healthy infant cynomolgus monkeys and juvenile XLMTM dogs received relatively high doses of rAAV8-Des-hMTM1, or its canine version (rAAV8-Des-cMTM1), respectively. In both species, vector genomes were highest in liver, while myotubularin expression was highest in cardiac or skeletal muscle. rAAV8-Des-MTM1 was generally well-tolerated; importantly, there was no evidence of hyperbilirubinemia or other liver-associated toxicity following rAAV8-Des-MTM1 administration to cynomolgus monkeys or XLMTM dogs.

Dravet Syndrome (DS) is a catastrophic developmental and epileptic encephalopathy (DEE) primarily caused by SCN1A haploinsufficiency. It is characterized by refractory hyperthermia-induced seizures, neurodevelopmental delay, and an elevated risk of sudden unexpected death in epilepsy (SUDEP) compared to other childhood epilepsies. While the small molecule fenfluramine (FFA) provides profound clinical benefit, its potential for systemic side effects and a poorly defined mechanism of action (MOA) underscores the need for more targeted interventions. Using a Scn1a[+/-] rat model of DS, we demonstrate that chronic FFA treatment reduces the frequency of hyperthermia-induced seizures and attenuates pathological EEG network hypersynchrony. Furthermore, FFA administration ameliorates hyperactivity and depressive-like comorbidities, an effect associated with a marked reduction in hippocampal neuroinflammation and microglial overactivation. Transcriptomic analysis implicated serotonergic signaling in the therapeutic response to FFA, and subsequent anatomical and functional analyses revealed enhanced activity of dorsal raphe nucleus (DRN) serotonergic neurons following FFA treatment. Cell-type-specific AAV-mediated modulation of DRN serotonergic neurons was sufficient to reproduce the key antiseizure and electrophysiological effects of FFA. Together, our findings identify the DRN serotonergic circuit as a therapeutically relevant circuit and provide proof-of-concept for mechanism-driven, circuit-specific neuromodulation strategies for Dravet syndrome.

Although in vitro methods for aptamer screening are well established, quantitative systematic in vivo screening of multiple aptamers remains challenging due to time and cost constraints associated with rodent models. To address this bottleneck, we developed a strategy for rapid screening of tumor-targeting aptamers using light-sheet fluorescent imaging after systemic administration of aptamers in a zebrafish larvae neuroblastoma model. Correlated imaging and qPCR-based aptamer detection identified an optimal screening window of 30-90 min post-injection, enabling rapid in vivo evaluation of five aptamers and one negative control oligonucleotide. We compared this strategy with in vitro screening of the same aptamers against a panel of neuroblastoma cell lines and found that zebrafish larvae screening ranked lead aptamers differently from in vitro assays, suggesting that in vivo screening may identify more physiological tumor-preferential aptamers for therapeutic development than in vitro screening alone. To demonstrate the therapeutic potential, the lead aptamer was conjugated to fusogenic nanovesicles and showed reproducible neuroblastoma-preferential accumulation 1-2 h post-injection. This strategy enables rapid in vivo evaluation of aptamers selected in vitro to identify candidates with tumor-targeting properties. Importantly, this in vivo approach can also assess off-target binding to healthy tissues without the time and cost associated with rodent models.

DNA-based non-viral gene therapy offers a promising platform for titratable gain-of-function treatments, enabling multi-dosing and sustained efficacy. However, its clinical translation has been constrained by unintended immune activation. We systematically investigated the pathogenesis of DNA drug-induced toxicity in mouse models, with emphasis on the contribution of innate immune responses. Intravenous administration of lipid nanoparticle (LNP)-DNA induces systemic toxicity, characterized by body weight loss, hepatic injury, and cytokine storm. Mechanistically, cyclic GMP-AMP synthase (cGAS) acts as the primary sensor for LNP-delivered DNA, activating two major downstream pathways: type Ⅰ interferon signaling and inflammatory responses. The interferon response contributes partially to overall toxicity and suppresses transgene expression. The suppressive effect on DNA drug stability is mediated by interferon-induced exonuclease three-prime repair exonuclease 1 (TREX1), which actively degrades DNA therapeutics and reduces their bioavailability. In parallel, inflammatory activation constitutes another major source of adverse effects, with TNF-α as a key mediator of liver injury. Importantly, we demonstrated that prophylactic immune modulation with dexamethasone or Janus kinase (JAK) inhibitor alleviates LNP-DNA-induced toxicity. Multi-target agent dexamethasone exhibited a significantly stronger inhibitory effect on adverse effects. Our findings uncover central innate immune mechanisms governing the safety and stability of LNP-DNA formulations and underscore the necessity of immunomodulatory strategies to unlock the potential of DNA-based gene therapy.