Molecular Therapy

Clinical applications of gene editors including CRISPR-Cas9 are limited by the cell-type specificity and tissue penetrance of current in vivo delivery options. Cell-mediated gene editor delivery could overcome these obstacles. To date, systems utilizing immortalized cell lines have demonstrated gene editor delivery in vitro. Because T cells traffic throughout the organism and engage in intercellular communication with individual target cells, we wondered whether they could be engineered to deliver gene editing machinery. Here, we demonstrate that primary human T cells can produce retroviral-based enveloped delivery vehicles (EDVs) packaging functional Cas9 ribonucleoproteins. When paired with fusogenic VSV-G, T cell-generated Cas9-EDVs edited a diverse array of target cells. We developed a miniaturized Moloney murine leukemia virus-based system that proved superior to existing architectures in this context and optimized both lentiviral and site-specific knock-in approaches for T cell engineering. These Enveloped Particle Information Courier- (EPIC-) T cells offer key advantages as delivery systems.

Mitochondrial DNA (mtDNA) base editors (mtBEs) are promising tools for studying and correcting pathogenic mtDNA mutations. However, the application of mtBEs is often hindered by substantial mtDNA-wide off-target editing that accumulates with prolonged mtBE expression resulting from plasmid DNA or viral delivery. To overcome this limitation, we developed mtBE-VLPs, a virus-like-particle (VLP) platform for transiently delivering mtBEs in vitro and in vivo with minimal off-target effects. While canonical protein-packaging VLPs yielded undetectable mtDNA base editing, engineered mRNA-packaging VLPs enabled efficient but transient mtBE cargo delivery. By iteratively engineering mRNA-packaging VLP compositions, we improved mtBE-VLP-mediated base editing efficiencies from less than 1% to greater than 50% across multiple target mtDNA loci in bulk populations of treated cultured cells. To further improve the packaging and delivery of certain mtBEs, we developed a strategy to selectively reduce mtBE cargo expression in VLP producer cells but not transduced cells, which increased VLP-mediated base editing efficiencies for certain mtBEs by 7.1-fold. Compared to plasmid DNA delivery in vitro, mtBE-VLPs exhibited equivalent on-target editing but 23- to 41-fold lower mtDNA-wide off-target editing, highlighting the improved editing specificity offered by transient mtBE delivery. mtBE-VLPs efficiently installed a pathogenic mtDNA mutation in mouse embryonic fibroblasts (88% efficiency) and corrected a pathogenic mtDNA mutation in a human patient-derived cell line (41% efficiency). Additionally, a single subretinal injection of mtBE-VLPs yielded 6-20% (average 12%) mtDNA base editing in the mouse retinal pigment epithelium with minimal mtDNA-wide off-target editing. These findings establish mRNA-packaging mtBE-VLPs as promising vehicles for efficient and precise mitochondrial base editing via transient mtBE delivery.

Antisense oligonucleotides (ASOs) inducing exon-skipping during pre-mRNA splicing offer promising treatment for diseases such as Duchenne muscular dystrophy. Recently, dual-targeting ASOs that simultaneously bind two splicing regulatory sequences have been shown to improve exon-skipping efficiency. Current ASOs are designed to interact with target RNAs solely based on Watson-Crick base pairing. Here we report the development of dual-targeting three-dimensional bridge (3D-bridge) ASOs in which tertiary interactions are engineered to increase target-binding affinity and the number of target residues occupied. We designed a 3D-bridge ASO for skipping exon 44 of dystrophin mRNA and biochemically validated its enhanced binding affinity for the target RNA over a similar ASO with the tertiary interactions disrupted. We subsequently optimized the 3D-bridge ASO by varying its ends and ribose chemical modifications using patient-derived induced directly reprogrammed myotubes and identified an LNA/DNA mixmer as the most potent. We further demonstrate enhanced exon-skipping efficacy and potency of the 3D-bridge ASO compared to previously reported ASOs in myotubes differentiated from patient-derived primary myoblasts and MyoD-reprogrammed fibroblasts harboring mutations amenable to reframing through exon 44 skipping. In conclusion, the 3D-bridge ASO technology improves the current dual-targeting design, demonstrating the utility and power of structure-based ASO design methods.

Adeno-associated virus (AAV) mediated gene therapies continue to show promise for the treatment of genetic diseases. However, delivery and safety still represent significant prohibitive barriers restricting translation of these delivery modalities to humans. We describe a novel AAV9 variant, AAV9fyhr or FYHR, identified from screening a controlled randomization point mutation AAV9 library in mice and nonhuman primates followed by directed evolution of in vivo CNS-derived libraries screened in human iPSC differentiated cortical neurons. Capsid variants of interest were identified by coupling long read sequencing and computational structure modeling. Structure-function analysis of our lead candidate reveal that two targeted amino acid changes are sufficient to result in beneficial pleiotropic effects across viral stability, cell surface attachment, and gene delivery. We developed a novel multiplexed barcoding reporter system, enabling a time and cost-effective option to gather spatially resolved single cell capsid validation data in mice and nonhuman primates. When administered intrathecally, AAV9fyhr exhibits 2-to-6-fold higher gene delivery across several tissues in the CNS while minimally altering the tropism to peripheral tissues in comparison to AAV9. These findings demonstrate the potential of whole capsid engineering for the rational development of translational next generation AAV capsids.

Safe, efficient gene delivery throughout the CNS remains a central obstacle to treating genetic diseases of the brain and spinal cord. We describe BI-TfR1 CapX, a human transferrin receptor (TfR1)-binding AAV capsid that, after a low intravenous dose in adult humanized TFRC mice, transduced more than 80% of cortical and spinal neurons and ~60% of cortical astrocytes, with reduced distribution and expression in several peripheral tissues relative to AAV9. Unlike other engineered BBB-crossing capsids, CapX bound the CNS vasculature within minutes and a large fraction of its genomes reached parenchymal nuclei within 24 hours. Delivering a Prnp-targeted epigenetic silencer (Prnp-CHARM), CapX reduced brain Prnp mRNA by over 95% requiring 18-fold fewer vector genomes per brain cell than AAV-PHP.eB for 50% lowering. A CapX delivered dual-vector cytosine base editor installed a protein-truncating PRNP stop codon lowering brain prion protein by 80%. These properties make CapX an attractive vehicle for human CNS gene therapy.

Mislocalization and aggregation of the RNA-binding proteins (RBPs) FUS and TDP-43 are pathological hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), contributing to combined loss- and gain-of-function toxicity. Here, we present a protein engineering strategy that appends optimized anti-aggregation motifs to FUS and TDP-43, thereby enhancing their nuclear localization and solubility, and enabling functional replacement of disease-associated proteins. We show that these engineered chimeric RBPs reduce levels of aggregation-prone endogenous proteins through autoregulatory mechanisms. In addition, chimeric FUS and TDP-43 recapitulate key nuclear functions, including DNA repair and mRNA processing. Notably, chimeric FUS restores neurite outgrowth in diseased iPSC-derived neurons, while chimeric TDP-43 suppresses cryptic exon inclusion in patient-derived fibroblasts. Collectively, these results demonstrate that engineered chimeric RBPs can rebalance RBP homeostasis by simultaneously addressing protein aggregation and mislocalization, thus mitigating RBP-associated loss- and gain-of-function toxicity, highlighting a generalizable strategy for therapeutic protein replacement in neurodegenerative disease.

Adeno-associated virus (AAV) vectors are widely used for gene delivery to the central nervous system, but natural capsid tropism is broad and cell-type restriction is typically imposed transcriptionally using promoters and enhancers that consume packaging capacity and often drive weak expression. Here, we engineer neuronal targeting directly into the AAV-DJ capsid by ablating its endogenous heparan sulfate proteoglycan (HSPG) affinity and genetically displaying a nanobody against the Group 1 metabotropic glutamate receptor mGluR5 within the VP1 subunit at a permissive capsid loop (T456), generating AAV-m5. Western blot confirmed incorporation of the nanobody-VP1 fusion into assembled capsids. In primary hippocampal neuron cultures, ablating HSPG binding abolished infectivity and nanobody display rescued transduction while restricting GFP expression almost exclusively to mGluR5-positive neurons. Heparin competition assays showed that, unlike wild-type AAV-DJ, AAV-m5 transduction was unaffected by exogenous heparin, confirming that entry occurs independently of HSPG binding. Packaged with a strong constitutive promoter (CAG), AAV-m5 achieved neuron-restricted expression comparable to or exceeding that of wild-type AAV-DJ driven by a neuron-specific promoter (hSyn) and produced negligible expression under an astrocyte-specific promoter (GFAP) despite promoter activity in glia, demonstrating that capsid-level targeting can substitute, or complement, transcriptional restriction. Following stereotactic injection into the mouse hippocampus, AAV-m5 achieved an eight-fold higher proportion of transduced neurons than wild-type AAV-DJ at equivalent titers. Delivery to Grm5-null hippocampus reduced both the intensity and the spatial extent of transduction, confirming that the broad hippocampal transduction achieved by AAV-m5 is mGluR5-dependent. Together, these results establish nanobody-functionalized AAV-DJ as a modular, single-component platform for precision CNS gene delivery that circumvents the packaging and expression trade-offs of promoter-based cell-type restriction, with potential for retargeting to additional CNS cell types and disease-relevant receptors.

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.

Spinocerebellar ataxia type 3 (SCA3) is most common subtype of autosomal dominant spinocerebellar ataxia worldwide and the most prevalent CAG expansion disorder in the east Asian population, but disease-modifying therapies remain unavailable. SCA3 is caused by the accumulation of polyglutamine- (polyQ-) expanded mutant ataxin-3 (mAtxn3), while the wild-type ataxin-3 (wtAtxn3) plays an important cellular role. Thus, allele-selective clearance of mAtxn3 without affecting wtAtxn3 is highly desired for SCA3 treatment. To achieve this goal, we tested previously reported compounds that have been shown to selectively degrade the Huntingtons disease causing mutant HTT protein by tethering the expanded polyQ to the autophagosome protein LC3. Here, we demonstrated that two of these compounds, AN2 and GW5074, selectively reduced both soluble and aggregated mAtxn3 with expanded polyQ (84Q), but not wtAtxn3, in both cellular and mouse models of SCA3. Mechanistically, both compounds lowered mAtxn3 by enhancing its autophagic degradation without obvious activation of global autophagy. At the molecular level, both compounds increased mAtxn3-LC3 interaction and colocalization to enhance the allele-selectively autophagic degradation of mAtxn3. Phenotypically, both compounds rescued cell viability and ATP content of SCA3 cellular models. In SCA3 patient pluripotent stem cells (iPSCs) derived cortical neurons, both compounds allele-selectively reduced mAtxn3 and partially rescued neurite shortening. Importantly, in a well-established SCA3 transgenic mouse model (MJD84.2), intracerebroventricular, intraperitoneal, and oral administration allele-selectively reduced mAtxn3 in multiple brain regions and rescued motor function deficits. Oral dosing achieved nanomolar cerebellar exposure and showed no overt liver, kidney, or glial toxicity over 28 days. Analyses of 1303 Chinese patients with SCA3 and ATXN3 constructs of different polyglutamine lengths further supported the compounds polyQ length-dependent activity with relative sparing of normal-length ataxin-3. Together, these findings provide preclinical proof of concept that utilizing orally available small molecule compounds to allele-selectively enhance mAtxn3 autophagic degradation for SCA3 treatment. One-sentence summarySmall-molecule autophagosome-tethering compounds selectively lower mutant ataxin-3 through an LC3- and autophagy-dependent mechanism and improve disease-related phenotypes in preclinical models of SCA3.

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.

Recombinant vectors derived from adeno-associated viruses (rAAVs) are a mainstay of human gene therapy. rAAVs are produced from plasmids containing transgene cassettes flanked by inverted terminal repeats (ITRs), which form structured DNA elements that stabilize the ends of the single-stranded viral genome and are the only viral sequences required in cis for genome packaging. For decades, it has been recognized that propagation of ITR-containing plasmids can result in deletions and other mutations, prompting the use of specialized bacterial strains, modified growth conditions, and truncated or altered ITRs. Despite these practices, ITR instability remains a persistent source of plasmid heterogeneity. To identify determinants of ITR stability, we evaluated ITR integrity in one of the original cloned AAV2 genome isolates, a reconstructed AAV2 plasmid, and a synthetic rAAV vector containing full-length native AAV2 ITRs. We established a quantitative bioinformatic workflow for analyzing ITR-containing plasmids and virus preparations from raw Oxford Nanopore sequencing data. These experiments showed that ITRs were highly stable during short-term culture, whereas prolonged culture revealed strong positional effects, with preferential loss or mutation of the ITR nearest the plasmid origin of replication. Consistent with this model, a survey of 7,041 sequence-verifiable AAV plasmids from the Addgene repository identified a widely disseminated 11-bp ITR deletion in 4,773 plasmids; among analyzable two-ITR plasmids, this deletion was located in the origin-proximal ITR in 95.3% of cases. Guided by these findings, we constructed a novel rAAV entry vector with stable full-length native AAV2 ITRs that enabled efficient packaging of a 4,750-bp all-in-one CRISPR-Cas9 cassette. Finally, we developed a cell-based strategy to compare the effects of ITR mutations on rAAV genome integration, providing preliminary evidence that ITR sequence variation can influence integration outcomes. Together, these findings show that ITR instability is a preventable, position-dependent property of plasmid architecture and identify ITR integrity as an important variable in rAAV vector design and quality control.

Immunotoxins (ITs) are potent targeted cancer therapeutics that couple tumor-selective receptor binding with efficient intracellular delivery and cytotoxic enzymatic activity of bacterial toxins. While ITs have shown impressive efficacy in hematologic malignancies, dose-limiting toxicity remains a major barrier to their broader clinical application, particularly in solid tumors. The exceptional potency of immunotoxin payloads contributes to off-tumor toxicity because even limited uptake by receptor-low cells can be sufficient to cause cell death. Here, we systematically investigated how receptor-binding domain (RBD) affinity and avidity interact with catalytic payload activity to determine IT selectivity. Using CD123-, HER3-, and HER2-targeted ITs, we found that increasing receptor-binding affinity or avidity altered potency but provided limited control over the selectivity between receptor-high and receptor-low cells. In contrast, attenuating catalytic activity produced profound gains in selectivity when payload potency was appropriately matched to receptor engagement. The optimal level of attenuation varied with receptor context and toxin scaffold, demonstrating that maximal selectivity emerges from balancing cellular delivery with catalytic activity rather than maximizing either property alone. In vivo, payload attenuation increased the maximum tolerated dose of a HER2-targeted IT by 400-fold and enabled robust antitumor activity well below its toxicity limit, whereas the WT counterpart showed no detectable efficacy at tolerated doses. Together, these findings uncover and establish payload activity as a tunable determinant of immunotoxin selectivity and provide a framework for matching receptor engagement with catalytic potency to develop safer and more broadly applicable targeted toxins.

The immunosuppressive tumor microenvironment (TME) poses formidable challenges for cancer immunotherapies. Among the complex TME landscape, the cytokine TGF-{beta}1 is an attractive soluble target for reinvigoration of anti-tumor immunity due to its pivotal function in immune cell suppression, extracellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT) promotion, while immune checkpoint receptors exemplified by PD-1 and CD24 are promising surface targets. Though adaptive immune checkpoint inhibition is well established in clinical practice, TGF-{beta} blockade or innate immune checkpoint blockade is yet to be harnessed for tumor treatment. Here, leveraging CD24 overexpression on multiple tumor tissues and its unique recycling kinetics, we designed an innate Immune Checkpoint TArgeting Chimera (ICTAC) G01, a bifunctional CD24 antibody-TGF-{beta}1 receptor fusion protein that disrupts TGF-{beta}1 signaling by TGF-{beta}1 degradation instead of simple blockade and simultaneously inhibits the "don't eat me" CD24 signaling. We demonstrated that G01 effectively suppressed TGF-{beta}1-Smad signaling, reversed the EMT, and attenuated fibrotic collagen deposition. Additionally, it antagonized CD24 signaling to unleash robust antibody-dependent effector activity, further reprogramming the immunologically cold TME toward inflammatory phenotype. By selectively targeting malignant cells while sparing CD24-expressing B cells, G01 demonstrated a favorable safety profile. Notably, in patient-derived tumor fragments (PDTFs), G01 also inflamed the TME, underscoring its translational potential. Together, these results establish ICTAC as an effective strategy for tumor-specific receptor-mediated extracellular protein depletion, which supports its application for reversing TGF-{beta}1; and innate immune checkpoint-mediated tumor immunosuppression by concurrent targeting of both surface and soluble tumor-promoting factors.

NOS1AP is a genetically validated driver of tau pathology in Alzheimer's disease (AD), yet the NOS1AP-tau interaction itself has remained pharmacologically untargeted. We report CAPTOR-1, a first-in-class small molecule that disrupts this interaction by binding a pocket in the N-terminal region of NOS1AP (residues 1-126). A drug-resistant NOS1AP Y16A/F42A variant that retained tau binding but exhibited a 78-fold reduction in CAPTOR-1 affinity provides genetic evidence supporting an on-target mechanism. In human iPSC-derived neurons, CAPTOR-1 dose-dependently reduces endogenous NOS1AP-tau association, tau seeding activity, and pathogenic tau phosphorylation, phenocopying genetic NOS1AP knockdown; these effects extend to neurons from patients with sporadic AD. Orally dosed CAPTOR-1 crosses the blood-brain barrier and engages NOS1AP in brain dose-dependently. Chronic oral dosing rescues pathology, synaptic function, and memory in a tauopathy model (PS19) and an amyloidosis model (APP/PS1), sparing wild-type littermates in both. This work establishes pharmacological disruption of the NOS1AP-tau interaction as a viable, disease-modifying strategy for AD and positions CAPTOR-1 as a biomarker-ready chemical probe (pTau217, neurofilament light) for translational studies.

MAPT (Tau) dysregulation is implicated in several neurodegenerative diseases, but its contribution to amyotrophic lateral sclerosis (ALS) is poorly understood. Here we show that mRNA isoforms encoding 4-repeat (4R) Tau are upregulated and cytoplasmically enriched in iPSC-derived motor neurons (MNs) from VCP-mutant and sporadic ALS, without a corresponding change in Tau protein. Using splice-switching antisense oligonucleotides and isoform-specific siRNAs, we find that enhanced 4R expression reduces MN viability, whereas its selective knockdown improves survival, with kinetics more consistent with an RNA-intrinsic effect than altered protein synthesis. Exon 10-containing MAPT RNA shows increased predicted secondary structure, self-association and altered Tau biocondensation in vitro. In post-mortem ALS cervical spinal cord, increased relative exon 10 usage is associated with a higher-risk clinical phenotype and shorter disease duration These findings identify an isoform-specific contribution of MAPT to MN vulnerability in ALS and nominate 4R MAPT RNA as a therapeutic target.

Retinitis pigmentosa (RP) is genetically heterogeneous, yet initiating mutations converge on primary rod degeneration followed by cone dysfunction and loss. Activated microglia are implicated in this secondary phase, but whether selective immunometabolic reprogramming of microglia preserves retinal function remains unknown. In human iPSC-derived microglia and retinal CD11b cells, PGC1 induction suppressed LPS-driven interferon signaling and cytokine release, yet Seahorse analysis and isotopologue tracing showed that energy metabolism remained altered. We therefore fused PGC1 to a multivalent p65-HSF1 module, yielding ~2.5-fold higher nuclear receptor output than native PGC1 and coordinated engagement of oxidative and anti-inflammatory programs in mouse retinal CD11b cells. Microglia-restricted expression in humanized RHOP347L mice preserved photopic responses through six months, increased retinal thickness and promoted ramified subretinal IBA1 morphology. Microglial immunometabolism thus represents a potentially genotype-independent neuroprotective target in RP, and engineered PGC1 co-activators provide a means to sustain oxidative metabolism under inflammatory pressure.

COVID-19 highlighted the need for antivirals that can be rapidly deployed against both current and future coronavirus threats. CMX990 is a clinical stage coronavirus main protease (Mpro) inhibitor bearing a unique trifluoromethoxymethyl ketone warhead. Here, we broadly evaluate its pan-coronavirus target engagement, antiviral breadth, and pharmacologic differentiation. Across a reporter panel of 24 Mpro enzymes spanning alpha-, beta-, delta-, and gamma-coronaviruses, CMX990 retained robust potent activity, including against nirmatrelvir-resistant SARS-CoV-2 variants. In direct comparison, nirmatrelvir and ensitrelvir showed diminished potency against non-SARS-CoV-2 coronaviruses. Live-virus assays corroborated this breadth, with potent, selective activity against SARS-CoV-2, SARS-CoV-1, MERS-CoV, and additional human coronaviruses. Structural studies revealed a conserved covalent binding mode centered on the catalytic Cys of coronavirus Mpro. In serial-passage studies, resistance emerged only after approximately twice as many serial passages under CMX990 selection as under nirmatrelvir selection, without selecting for the canonical nirmatrelvir-resistance substitution E166V. Extracellular washout experiments revealed superior persistence of antiviral activity relative to nirmatrelvir, consistent with durable target engagement. In a murine SARS-CoV-2 infection model, once-daily CMX990 protected against weight loss and reduced lung viral titers to near baseline levels. Collectively, these findings identify CMX990 as a structurally and functionally validated pan-coronavirus protease inhibitor supporting its potential as a pandemic-ready antiviral candidate.

Background: Recurrent ocular herpetic disease is a leading infectious cause of corneal blindness and is associated with psychological distress, anxiety, and social withdrawal. No ocular herpes vaccine or immunotherapy currently exists. Hypothesis: A prime/pull/keep (PPK) T-cell immunotherapy, built on HSV-1 epitopes preferentially recognized by CD8+/CD4+ T cells from naturally protected asymptomatic (ASYMP) individuals, would reduce viral reactivation, shedding, recurrent corneal disease, and associated neurobehavioral deficits. Methods: Immunodominant HLA-restricted CD8+/CD4+ epitopes from HSV-1 gD/gB, tegument proteins VP11/12/VP13/14, and helicase UL9 were delivered via topical ocular AAV8 vector (prime) to latently infected HLA-A*0201/DRB1 mice, combined with chemokine CXCL11 (pull) and IL-2/IL-15 cytokines to sustain TRM cells (keep), then UVB-exposed to trigger reactivation and recurrent corneal disease. Results: PPK immunotherapy conferred robust, near-complete protection against recurrent HSV-1 disease and improved survival after UVB-induced reactivation, unlike its individual components. This protection was associated with site-specific accumulation of HSV-1-specific CD8+/CD4+ TRM cells in the cornea and trigeminal ganglia (TG), but not in the spleen, with peak retention from combined IL-2/IL-15, alongside durable central/effector memory populations. PPK-induced T cells showed enhanced polyfunctionality (cytokine production, cytotoxicity, proliferation), reduced exhaustion markers, and a TCF-1+ self-renewing phenotype. PPK also elicited robust, Th1-biased HSV-1-specific IgG responses complementing mucosal immunity, and restored spatial memory, reduced depression-like behavior, and reversed social withdrawal in mice with recurrent ocular herpes. Conclusion: PPK immunotherapy induces potent, durable antiviral TRM cells at primary sites of HSV-1 latency and reactivation, protecting against recurrent ocular herpetic disease and alleviating associated neurobehavioral deficits, supporting its translational potential in humans.

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.

Nemaline myopathy is a severe inherited skeletal muscle disorder characterized by profound muscle weakness, impaired mobility, and early mortality in severe cases. Despite the devastating clinical burden, no disease-modifying therapies are currently available. Nemaline myopathy-causing genes either encode sarcomeric structural proteins or regulators of sarcomeric protein stability, resulting in primary defects in sarcomere structure and function. Recessive mutations in KLHL40 cause one of the most severe forms of nemaline myopathy, frequently resulting in perinatal lethality. Given its loss-of-function mechanism, KLHL40-associated nemaline myopathy is an attractive candidate for gene replacement therapy. We therefore evaluated the therapeutic potential of systemic AAV9-mediated KLHL40 gene replacement in a mouse model of severe nemaline myopathy. A single systemic administration of an AAV9 vector encoding human KLHL40 during early disease produced durable rescue of survival, skeletal muscle pathology, motor performance, and contractile function in KLHL40-deficient mice. Therapeutic benefit was accompanied by restoration of sarcomere organization and normalization of myofiber size without detectable long-term histopathological abnormalities in major organs. Collectively, our findings provide a strong preclinical foundation for developing AAV9-mediated KLHL40 gene replacement as a treatment for severe KLHL40-associated nemaline myopathy.