Protein therapeutic binders provide targeted treatment options for autoimmune, cardiovascular, and neurodegenerative diseases, as well as cancer. However, these therapies can cause off-site toxicities when they bind to healthy tissue that also expresses their target. One method to combat this issue is through the addition of a masking moiety that prevents binding until released by a particular stimulus. Both protein and peptide-derived masks can inhibit binding, either via steric hinderance using large domains or by affinity-based direct competition. In diseased tissue, protease expression levels and pH are often altered, which can be leveraged for site-specific removal or deactivation of a masking moiety. Therefore, emergent strategies have been used to design conditionally activated, disease-targeted, protein-based therapeutics. This review will focus on the design, effectiveness, and mechanism of masks for cytokines, antibodies, and their derivatives, and further discuss strategies for future therapeutic avenues.
Corneal chemical injury, including mustard vesicant injury, remains a clinical challenge with limited therapeutic options. To enable effective mechanistic and therapeutic studies, we developed a mouse model of nitrogen mustard (NM)-induced corneal injury using a 3D-molded, cap-shaped filter paper conforming to the corneal surface to generate reproducible injuries. Multimodal in vivo imaging and histological assessment revealed an injury course characterized by acute epithelial disruption, stromal edema, and inflammation, followed by transient partial recovery and chronic stromal remodeling with persistent endothelial loss. After NM exposure, bromodomain (BD)-containing protein 4 (BRD4) expression and activity were upregulated. Short-term topical BRD4 inhibition with JQ1 reduced acute corneal inflammation (e.g., reducing CD45+ cells by 86%) and oxidative stress, and conferred long-term preservation of corneal clarity, stromal organization, and endothelial integrity. Selective inhibition of BD1 or BD2 recapitulated JQ1’s effects, indicating that blockade of either bromodomain is sufficient. BRD4 inhibition also significantly attenuated NM-induced corneal pathology in a rabbit model, supporting cross-species efficacy and translational relevance. Mechanistically, BRD4 inhibition selectively counteracted key NM-driven pathogenic programs, including inflammation, oxidative stress, and extracellular matrix remodeling. These findings identify BRD4 as a central epigenetic driver of vesicant-induced corneal injury and position BRD4 inhibition as a promising translational therapeutic strategy.
Gentamicin-induced inner-ear injury can cause disabling vestibular dysfunction and hearing loss; however, the regulated death programs driving vestibular and cochlear hair-cell loss remain incompletely defined. In this study, we identify ferroptosis as a shared and central mechanism of gentamicin-induced vestibular and cochlear toxicity in mice. Gentamicin exposure induced robust lipid peroxidation in both cochlear and vestibular tissues and altered the expression of ferroptosis-associated proteins, most notably glutathione peroxidase 4 (GPX4), a key antioxidant enzyme that was markedly downregulated and closely associated with hair-cell degeneration. We further identify ammonium tetrathiomolybdate (TM) as a novel agonist of nuclear factor erythroid 2-related factor 2 (NRF2) and evaluate its therapeutic potential via semicircular canal injection. Mechanistically, TM promoted NRF2 nuclear accumulation and upregulated downstream antioxidant programs, restoring GPX4 expression and suppressing lipid peroxidation and ferroptosis-linked signaling in inner-ear hair cells. Structurally and functionally, TM preserved cochlear and vestibular hair-cell integrity after gentamicin exposure and significantly improved auditory and vestibular performance. Together, these findings establish ferroptosis as a convergent mechanism underlying gentamicin-induced cochleovestibular injury and identify TM as a pathway-directed candidate for mitigating gentamicin-induced cochleovestibular ototoxicity.
Despite the clinical promise of cytokine-induced memory-like (CIML) NK cells, widespread translation is hindered by the high cost and complexity of ex vivo manufacturing. Here, we show that the lung functions as an in vivo programming niche that generates functional CIML NK cells for systemic cancer immunotherapy and immunoprevention. By decoupling immunostimulation from silicosis, we identify an obligate alveolar macrophage (AM)-NK cell axis. Upon engulfing biodegradable amorphous nanosilica (nSiO2), AMs release a transient IL-12/15/18 triad that reprograms circulating NK cells into memory-like effectors. The resulting NK cells meet all three canonical CIML criteria: proliferative expansion, transition to an armed resting state, and enhanced IFN-γ recall upon tumor rechallenge, with specific surface area (SSA) serving as a key physical determinant of programming potency. In mouse models, a brief prophylactic pulmonary nSiO2 regimen (120 μg/dose, 4 doses) establishes durable (≥ 60 days) protective immunity, suppresses melanoma growth, prevents postsurgical recurrence, and synergizes with anti-PD-1 therapy in an NK-dependent manner. Crucially, this therapeutic regimen does not induce pulmonary fibrosis; the amorphous nSiO2 is completely biodegraded and cleared within 90 days, with no detectable systemic toxicity. Our study establishes a cell-free framework for in vivo NK cell programming, informing next-generation cancer immunotherapies.
Cbln1 is a secreted synaptic organizer required for parallel fiber-Purkinje cell (PF-PC) synapse integrity, climbing fiber (CF) refinement, and cerebellar motor learning, but has not previously been implicated in human disease. We identified biallelic CBLN1 missense variants (A63P and Y112C) in two unrelated families with early-onset cerebellar ataxia accompanied by oculomotor abnormalities, cerebellar atrophy, and variable cognitive delay. In heterologous cells, both variants showed reduced steady-state protein abundance, impaired maturation through the early secretory pathway, and little or no detectable secretion, resulting in markedly reduced extracellular CBLN1 availability. Consistently, cerebellar granule cells expressing CBLN1-Y112C failed to induce excitatory synapses onto glutamate receptor δ2 (GluD2)-expressing cells in vitro. A knock-in mouse harboring Y112C lacked synaptic Cbln1 and recapitulated key features of Cbln1 deficiency, including disrupted PF-PC synapse organization, persistent CF multi-innervation, impaired PF-PC transmission and long-term depression, and deficits in motor coordination and oculomotor learning. Notably, systemic delivery of an astrocyte-targeted adeno-associated virus expressing wild-type CBLN1 in adult mutant mice restored synaptic CBLN1 localization, cerebellar synaptic function, plasticity, and behavior. These findings establish CBLN1 deficiency as a cause of hereditary ataxia and identify extracellular CBLN1 replacement as a therapeutic strategy for a reversible cerebellar synaptopathy.
A compact and programmable epigenome editor can be utilized for long-term gene silencing, which becomes a powerful tool for biological research or biomedical applications. Here, we present an enhanced CRISPR-based epigenome editor based on deactivated AsCas12f1 (dAsCas12f1) variants, which incorporate epigenetic modulators for histone methylation and DNA methylation. First, we compared the gene silencing achieved by various dCas12f1 variants fused with Krüppel-associated box (KRAB). The optimized variant dCas12f1-V8.1-KRAB, named AminiCRi, demonstrated high efficacy of gene silencing, which is comparable to dSpCas9-KRAB. To establish a miniature Cas-based system for long-term silencing, we generated dCas12f1-V8.1 fused with KRAB and DNA methyltransferases (Dnmt3A/3L), named AminiCRoff. AminiCRoff enables simultaneous deposition of repressive histone H3K9 trimethylation marks and DNA methylation at target loci. Transient delivery of AminiCRoff allows stable suppression of H2B gene expression to 30% of baseline levels up to 50 days examined, which is more durable than dSpCas9-based CRISPRoff. The compact size of the system is potentially packed into a single Adeno-associated virus (AAV), which is frequently used for gene therapy. Here we developed a highly efficient miniature Cas-based epigenetic editor for long-term gene silencing, which provides a good foundation for epigenetic therapy of genetic or chronic diseases with abnormal gene expression.
Despite the revolutionary impact of genome engineering tools in medicine, the safe and effective intracellular delivery of CRISPR remains a major obstacle for clinical applications. Here, we utilize precision molecular targeting and delivery strategies based on CRISPR/dCas9 systems adapted for epigenetic repression (dCas9-KRAB) to silence oncogenic drivers with high selectivity. As proof-of-principle, we target the EWSR1-FLI1 translocation, which encodes a chimeric and hard-to-drug oncogenic transcription factor driving approximately 85% of the cases of Ewing Sarcoma (EWS)-an aggressive childhood malignancy. We describe the development of a programmable non-viral polymeric system for the delivery of dCas9-KRAB as ribonucleoprotein (RNP) payloads for EWSR1-FLI1 repression. We demonstrate highly efficient intracellular delivery of RNPs loaded in polyamide-amine (PAMAM) polymers functionalized by guanidino groups, resulting in robust silencing of EWSR1-FLI1 both in established cell line xenografts and in EWS-related patient-derived xenografts (PDXs) of EWS. We show that silencing of EWSR1-FLI1 is accompanied by potent anti-tumor effects. Collectively, we characterize an effective non-viral platform for in vivo delivery of dCas9-KRAB/RNPs, which could be adapted for the repression of any oncogene. We further outline dCas9/RNP formulations for future therapeutic applications to treat poor-prognosis cancers driven by hard-to-drug oncogenes.
Damage to cochlear spiral ganglion neurons (SGNs) causes irreversible sensorineural hearing loss (SNHL), yet the underlying degenerative mechanisms remain elusive, impeding targeted therapies. Here, we established an auditory neuropathy model using the ototoxic drug ouabain to induce selective SGN injury. Transcriptomic profiling revealed a ferroptosis-linked gene signature, with functional assays confirming ferroptotic damage in SGNs. Pharmacological inhibition of ferroptosis mitigated SGN loss and hearing impairment. Lipidomics demonstrated dysregulated fatty acid metabolism with an elevated saturated-to-monounsaturated fatty acid ratio, accompanied by downregulation of stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme for monounsaturated fatty acid synthesis. Restoring SCD1 activity through SCD1 overexpression or monounsaturated fatty acid supplementation mitigated ouabain-induced ferroptosis in SGN and preserved auditory function, whereas genetic ablation of SCD1 aggravated degeneration. Bioinformatic screening and chromatin immunoprecipitation-PCR identified microphthalmia-associated transcription factor (MITF) as a direct upstream transcriptional regulator of SCD1. MITF overexpression restored SCD1 expression, suppressed ferroptosis, and improved SGN survival, while disruption of the MITF-SCD1 axis abrogated these protective effects, which were further validated in a cisplatin ototoxicity model. Collectively, these findings reveal a MITF-SCD1 lipid metabolic axis that safeguards SGNs against ferroptosis by sustaining SCD1-dependent lipid desaturation, highlighting a promising therapeutic target for SNHL.
The CRISPR-Cas12f system is an ultracompact genome-editing platform, yet only a few orthologs exhibit robust activity in mammalian cells. Here, we systematically screened 23 Cas12f orthologs and identified two active nucleases, PspCas12f1 and TcCas12f1, capable of genome editing in human cells. sgRNA scaffold optimization enhanced the basal activity of PspCas12f1. To further improve its performance, we combined structure-guided rational design with protein language model-assisted filtering. Candidate mutations predicted by SaProt were further screened based on structural proximity to the DNA-binding interface and electrostatic compatibility. This integrative strategy identified Q100R and E293R, whose combination yielded the optimized variant enPspCas12f1. enPspCas12f1 achieved genome-editing efficiencies comparable to SpCas9 across multiple endogenous loci while maintaining high specificity. Collectively, our results demonstrate that integrating protein language model-assisted filtering with structure-guided rational design provides an effective strategy for engineering PspCas12f1 and may facilitate the optimization of additional compact CRISPR nucleases.
Mounting evidence indicates that interleukin-6 (IL-6) plays an essential role in the development of cancer cachexia. Particularly, recent work showed that IL-6 drives cancer cachexia through neurons in the area postrema of the brainstem. However, there are currently no approved drugs for treating cancer cachexia. Here we developed a splice-switching antisense oligonucleotide (ASO)-based therapy for treating cancer cachexia by reducing IL-6 receptor (IL-6R) expression in the brain. In two mouse models of cancer cachexia, a single dose of ASOs, administered by intracerebroventricular injection after cancer onset, reduces IL-6R levels in the brainstem and ameliorates cachectic symptoms. It also extends survival in one of the models. In parallel, the ASO treatment reduces cancer-associated transcriptomic activation of inflammatory pathways in both the brainstem and skeletal muscle. We also developed ASOs that suppress human IL-6R expression, paving the road for clinical studies. Our study thus provides a new approach for treating cancer cachexia.
Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract disease in infants and older adults; however, vaccine strategies capable of inducing robust respiratory mucosal immunity remain limited. Here, we developed a receptor-guided intranasal circular RNA (circRNA) vaccine platform based on a vasoactive intestinal peptide-nucleocapsid fusion protein (VIP-N), which enables the self-assembled delivery of circRNA encoding RSV antigens. Mechanistically, VIP-N enters cells through VIP receptor-dependent early endocytosis and preferentially enhances circRNA delivery in respiratory epithelial cells and antigen-presenting cells. In mice, intranasal immunization with VIP-N+circRNA vaccines induced serum IgG responses comparable to those elicited by intramuscular administration. VIP-N+circRNA vaccines uniquely promoted IgA responses in bronchoalveolar lavage fluid and increased T follicular helper 1 cells. Moreover, the VIP-N+circRNA vaccine encoding pre-F elicited the strongest neutralizing activity and conferred cross-protection against both RSV A and B subtypes, significantly reducing pulmonary viral burden and lung inflammation. Notably, the immune response was predominantly T helper type 1 (Th1) cell-skewed, without an accompanying increase in Th2 cytokines associated with vaccine-enhanced respiratory disease. Collectively, these findings suggest VIP-mediated circRNA delivery as a promising intranasal strategy to induce comprehensive immunity against RSV, potentially improving protection in vulnerable populations.
