Membrane Fusion-Mediated Loading of Therapeutic siRNA into Exosome for Tissue-Specific Application.

Tissue-specific delivery of oligonucleotide therapeutics beyond the liver remains a key challenge in nucleic acid drug development. To address this issue, exploiting exosomes as a novel carrier has emerged as a promising approach for efficient nucleic acid drug delivery. However, current exosome-based delivery systems still face multiple hurdles in their clinical applications. Herein, this work presents a strategy for constructing a hybrid exosome vehicle (HEV) through a DNA zipper-mediated membrane fusion approach for tissue-specific siRNA delivery. As a proof-of-concept, this work successfully fuses a liposome encapsulating anti-NFKBIZ siRNAs with corneal epithelium cell (CEC)-derived exosomes to form a HEV construct for the treatment of dry eye disease (DED). With homing characteristics inherited from exosomes, the siRNA-bearing HEV can target its parent cells and efficiently deliver the siRNA payloads to the cornea. Subsequently, the NFKBIZ gene silencing significantly reduces pro-inflammatory cytokine secretions from the ocular surface, reshapes its inflammatory microenvironment, and ultimately achieves an excellent therapeutic outcome in a DED mouse model. As a versatile platform, this hybrid exosome with targeting capability and designed therapeutic siRNAs may hold great potential in various disease treatments.

Factors affecting long-term changes of meibomian gland in MGD patients.

PURPOSE: To explore the long-term course of patients with meibomian gland dysfunction (MGD), and to analyse potential factors affecting the recovery of meibomian gland (MG) dropout. METHODS: Seventy-nine MGD patients (79 eyes) aged 36.03±15.78 years old who underwent more than one year of follow-up were enrolled in this retrospective study. Corneal fluorescein staining (CFS), tear meniscus height (TMH), noninvasive breakup time (NIBUT), and noncontact meibography at baseline and last visit were collected and analysed. Then an automatic MG analyzer was used to measure the morphological and functional parameters of MGs, including their area ratio (AR), tortuosity index (TI), and signal index (SI). The patients whose AR increased by more than 5% were defined as MG improvement, and AR decreased by more than 5% was MG worsening. RESULTS: A total of 79 patients (79 eyes) were assessed with at least 1-year of follow-up. More than 1/3 of MGD patients (27 eyes, 34.2%) underwent MG improvement, and 30.4% of MGs became worsened. Age (P=0.002), gender (P<0.001), IPL treatment (P=0.013), the change of CFS (P=0.0015), and the recovery of SI (P=0.035) showed significant differences among different recovery groups. Age(P<0.001), female sex (P=0.003), ΔCFS (P<0.001), AR at baseline (P<0.001) were negative correlation with AR recovery, and the change of SI (P=0.003) and IPL treatment (P=0.003) had a positive correlation with it. Among them, age (P=0.038), the change of CFS (P=0.004), and AR at baseline (P=0.007) were confirmed as negatively correlated factors predicting the long-term change of the MG. CONCLUSION: Although the MGD treatment has continued for more than 1 year, only 34.2% of MGD patients were observed to undergo MG improvement. Younger patients and patients with better CFS recovery seem to have more opportunities to improve their MGs.

In vivo CRISPR gene editing in patients with herpetic stromal keratitis.

In vivo CRISPR gene therapy holds large clinical potential, but the safety and efficacy remain largely unknown. Here, we injected a single dose of herpes simplex virus 1 (HSV-1)-targeting CRISPR formulation in the cornea of three patients with severe refractory herpetic stromal keratitis (HSK) during corneal transplantation. Our study is an investigator-initiated, open-label, single-arm, non-randomized interventional trial at a single center (NCT04560790). We found neither detectable CRISPR-induced off-target cleavages by GUIDE-seq nor systemic adverse events for 18 months on average in all three patients. The HSV-1 remained undetectable during the study. Our preliminary clinical results suggest that in vivo gene editing targeting the HSV-1 genome holds acceptable safety as a potential therapy for HSK.

Alpha 7-nicotinic cholinoceptor regulation of pericyte-containing retinal capillaries.

BACKGROUND AND PURPOSE: Local blood flow regulation relies on the coordination between neurons and pericyte-containing capillaries. Pericyte relaxation and contraction are influenced by vasoactive substances and regulated by neurotransmitters. α7 nicotinic acetylcholine receptors (α7-nAChRs), involved in the regulation of vascular function and inhibitory γ-aminobutyric acid (GABA) systems, have neuroprotective effects against CNS diseases. Although α7-nAChRs are found throughout the retina, their contribution to the retinal capillary tone remains unknown. Here, we investigated the neurovascular coupling mechanism underlying α7-nAChR-mediated retinal capillary tone regulation. EXPERIMENTAL APPROACH: Changes in capillary diameter and pericyte transverse diameter during drug perfusion were observed using differential interference contrast (DIC) microscopy, to help elucidate signalling pathways underlying α7-nAChR-mediated regulation of capillary blood flow at the whole retinal level. Patch clamp technique was used to investigate α7-nAChR-mediated regulation of the GABA synaptic circuit. Immunofluorescence was used to explore the expression of α7-nAChRs and GABA receptors. KEY RESULTS: Activating α7-nAChRs on the endothelial cell membrane caused perinuclear accumulation of endothelial nitric oxide synthase (eNOS), resulting in dilated retinal capillaries and pericytes via the nitric oxide synthase (NOS)/nitric oxide (NO)/guanosine 3',5'- monophosphate (cGMP) signalling pathway. Neuronal α7-nAChR activation directly relaxed retinal capillaries and pericytes via a neurovascular coupling mechanism. α7-nAChR also increased the vesicular release of GABA, possibly promoting the release of NO by binding to GABAA receptors in retinal ganglion cells (RGCs) and relaxing blood vessels via eNOS-NO, with GABA binding to GABAB receptors on retinal capillary endothelial cells. CONCLUSION AND IMPLICATIONS: α7-nAChR activation causes vasorelaxation of retinal capillaries.

Rapid and quantitative detection of tear MMP-9 for dry eye patients using a novel silicon nanowire-based biosensor.

Dry eye disease (DED) is the most common chronic eye disease characterized by ocular surface inflammation that affects hundreds of millions of people worldwide. The diagnosis and monitoring of DED require fast and reliable tools in the clinical setting. Matrix metalloproteinase 9 (MMP-9) has been proven to be a reliable indicator of DED owing to its close relationship with inflammation. A novel biosensor based on silicon nanowire-based field-effect transistor (SiNW FET) devices was fabricated for the quantitative measurement of MMP-9 in human tears. A modified controllable process was applied to improve the uniformity of the SiNWs in size and stabilize their performance with optical calibration at low salt concentrations for clinical application. With this protocol, correlation analysis proved the high agreement between the biosensor and enzyme-linked immunosorbent assay (correlation coefficient of 0.92 for DED patients and 0.90 for healthy controls). A diagnostic sensitivity of 86.96% and specificity of 90% were achieved in human tear samples from DED patients and healthy subjects in real-world clinical settings. Furthermore, the tear MMP-9 concentrations monitored using the device correlated with the therapeutic response of the patients with DED. Our enhanced SiNW biosensor device demonstrated its potential as an alternative tool for real-time diagnosis and monitoring for prognostic prediction toward point-of-care testing for DED.

Safety and Feasibility of Low Fluence Intense Pulsed Light for Treating Pediatric Patients with Moderate-to-Severe Blepharitis.

To explore the safety and feasibility of low fluence intense pulsed light (IPL) for treating pediatric patients with moderate-to-severe blepharitis and to analyze potential factors associated with the recovery of meibomian glands (MG) dropout, a retrospective, noncomparative study, including 17 blepharitis patients (33 eyes) aged between 5 and 16 years old was conducted. All of the participants were given 4 continuous sessions of low-fluence (9−12 J/cm2) IPL at 3−4 week intervals. Corneal fluorescein staining (CFS), tear breakup time (BUT), inferior tear meniscus height, Demodex presence, and MG morphology were examined before and after the treatment. Results indicated that CFS, BUT and MG morphology (central/total gland area ratio and gland signal index) had significantly improved (p < 0.05). Symptoms and signs such as severe corneal neovascularization, limbal pannus and conjunctival congestion also subsided. Among age, gender, presence of Demodex and interval before diagnosis, age initiating the formal treatment was confirmed as a negatively correlated factor of the recovery of MG dropout (p = 0.032, B = −1.755). No notable adverse events were reported. In conclusion, low fluence IPL seems to be a safe and effective alternative for moderate-to-severe pediatric blepharitis, and MG dropout is prone to recover in younger patients.

Morphological and Functional Changes of Meibomian Glands in Pediatric and Adult Patients with Allergic Conjunctivitis.

Allergic conjunctivitis (AC) is one of the most common ocular disorders in clinical practice and is associated with meibomian gland dysfunction. This study aimed to explore the morphological and functional changes of meibomian glands (MGs) in pediatric and adult patients with AC and to analyze their potential predictors. In our prospective, observational cohort study, a total of 59 patients with AC were enrolled, with 30 patients aged ≤16 years in the pediatric group and 29 patients in the adult group. All patients underwent examinations at baseline and last visit when the complete resolution of conjunctival papillae was identified. An automatic MG analyzer was used to measure the morphological and functional parameters of MGs, including their area ratio (GA), tortuosity index (TI), and signal index (SI). Two groups were comparable at baseline in terms of characteristics and MG parameters (p > 0.05). The morphological (length, square, and GA) and functional MG parameters (SI) of AC patients significantly improved in the pediatric group after treatment (all p < 0.05), but not in the adult group. The change in the GA correlated with age, sex, GA, TI, and SI at baseline (all p < 0.05). Age (p = 0.001) and GA (p < 0.001) at baseline were predictors of an improvement in the GA of MGs. The findings showed that the structure and function of MGs in pediatric patients with AC seem to improve after the conjunctival papillae disappear, but not in adult patients.

WAY-100635 Alleviates Corneal Lesions Through 5-HT1A Receptor-ROS-Autophagy Axis in Dry Eye.

Purpose: To explore whether 5-HT1A receptors are involved in the dry eye disease (DED) mouse model and reveal its underlying mechanism. Methods: A C57BL/6J mouse DED model was established via the administration of 0.2% benzalkonium chloride twice a day for 14 days. Corneal fluorescein sodium staining score and Schirmer I test were checked before, and on days 7, 14, and 21 after treatment. The experiment was randomly divided into control, DED, 5-HT1A receptor agonist with or without N-acetylcysteine (NAC) and 5-HT1A receptor antagonist with or without NAC groups. The mRNA expression of inflammatory cytokines was measured by reverse transcription-quantitative polymerase chain reaction. Cellular reactive oxygen species (ROS) were detected by 2', 7'-dichlorodihydrofluorescein diacetate assays. Western blot analysis was used to measure the expression levels of autophagic proteins microtubule-associated protein 1 light chain 3 (LC3B-I/II) and autophagy-related gene 5 (ATG5). Results: 5-HT1A receptor agonist (8-OH-DPAT) increased corneal fluorescein sodium staining spots and 5-HT1A receptor antagonist (WAY-100635) decreased them. Treatment with 8-OH-DPAT was associated with the gene expression of more inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 10 (CXCL10) compared with treatment with WAY-100635. An increased expression of LC3B-I/II and ATG5 was observed in corneal epithelial cells in the mouse model of DED. 8-OH-DPAT significantly enhanced the expression of LC3B-I/II and ATG5 by disrupting ROS levels. WAY-100635 alleviates autophagy by inhibiting ROS production. Conclusion: Excessive ROS release through 8-OH-DPAT induction can lead to impaired autophagy and increased inflammatory response in DED. WAY-100635 reduces corneal epithelial defects and inflammation in DED, as well as alleviates autophagy by inhibiting ROS production. The activation of the 5-HT1A receptor-ROS-autophagy axis is critically involved in DED development.

Long Non-coding RNAs Gabarapl2 and Chrnb2 Positively Regulate Inflammatory Signaling in a Mouse Model of Dry Eye.

Purpose: To elucidate the expression profile and the potential role of long non-coding ribonucleic acids (RNAs) (lncRNAs) in a dry eye disease (DED) model. Methods: A DED model was established in C57BL/6J mice with 0.2% benzalkonium chloride (BAC) twice a day for 14 days. The differentially expressed lncRNAs were detected by RNA-seq technology (Gene Expression Omnibus, GEO GSE186450) and the aberrantly expressed lncRNAs were further verified by RT-qPCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to predicate the related candidate genes and potential pathological pathways. Cells from a human corneal epithelial cell line (HCECs) were cultured under hyperosmolarity. The regulation of inflammatory factors by silencing potential targeted lncRNAs was verified in vitro in HCECs. Results: In our study, a significant increase in corneal fluorescence staining and a reduction in tear production were observed in DED mice at all follow-ups compared with the controls, and the differences were increasing over time. In total, 2,649 upregulated and 704 downregulated lncRNAs were identified in DED mice. We selected six aberrantly expressed and most abundant lncRNAs and performed RT-qPCR using the samples for RNA-seq. Chrnb2, Gabarapl2, and Usp31 were thereby confirmed as the most significantly altered lncRNAs. Pathway analysis revealed that the neuroactive ligand-receptor interaction signaling pathway was the most enriched, followed by the calcium signaling pathway and cytokine-cytokine receptor interaction. Following treatment of Gabarapl2 siRNA and Chrnb2 siRNA, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 were significantly downregulated in the HCECs. Conclusion: Our study suggests that Chrnb2 and Gabarapl2 may be involved in the inflammation response by regulating TNF-α, IL-1ß, and IL-6 in DED. These candidate lncRNAs may be both potential biomarkers and therapeutic targets for DED.

Nanomedicines for the treatment of glaucoma: Current status and future perspectives.

Glaucoma is the global leading cause of irreversible blindness. It is a chronic progressive disorder and, therefore, often requires long-term management with drugs on patients' discretion. However, there is a shortage of antiglaucoma drugs in the current market due to their low bioavailability. This is because there are multiple biological barriers of the human eyes, thereby leading to increased demands for frequent dosage regimen per day of these drugs, which could result in concomitant side effects and eventually reduced patient compliance. Recently, nanomedicines have become optimized alternatives to conventional ophthalmic formulations due to advantages of improved barrier permeability, sustained drug release, tissue targeting, and lowered systemic absorption of instilled medications. These merits provide the active ingredients in these nanomedicines an effective manner to reach the ideal concentrations at sites of damaged nerves, offering a promising platform for neuroprotective treatment of these conditions. In this study, nanomedicines and nanomedicine-based novel strategies for pharmacotherapy of glaucoma were reviewed, including liposomes, niosomes, nanoparticles, and dendrimers. This article intends to offer a comprehensive review of frontier progresses as well as hotspots and issues that appeared in the field of nanomedicines, which may enable a practical flourish in the future. STATEMENT OF SIGNIFICANCE: Recent novel pharmaceutical strategies toward glaucoma, a chronic blinding ocular disease that currently requires frequent daily dosage regimen, based on nanomedicines and nanomaterials have been comprehensively reviewed in this manuscript. The collection of field hotspots and issues in the late years should offer a quick grasp of the general concept and up-to-date threads upon the refinement of existing treatment patterns for glaucoma. Meanwhile, the Conclusion and Future Perspective section given at the end of the text brings out the possible shortages and opinions in terms of ideal research direction, which hopefully could facilitate a future practical flourish in the area.

Comparison of the Meibomian Gland Openings by Optical Coherence Tomography in Obstructive Meibomian Gland Dysfunction and Normal Patients.

PURPOSE: The aim of this study was to use swept-source anterior segment optical coherence tomography (OCT) to explore imaging the meibomian gland openings and to identify their in vivo characteristics in patients with obstructive meibomian gland dysfunction (MGD) and healthy participants. METHODS: We enrolled 49 patients with MGD and 54 health controls in this case-control study. Each participant underwent slit-lamp examination, meibography, and OCT scanning. Sixteen patients with MGD underwent a repeat OCT examination after eyelid massage. The outcome measures included determinations of meibomian gland openings (orifices and terminal ducts) from OCT images and comparisons of the meibomian openings between patients with MGD and normal controls before and after meibomian gland massage. RESULTS: Using the same OCT scanning model, the number of visible orifices of the meibomian glands was similar between eyes with MGD and normal eyes (9.2 ± 2.3 vs. 9.7 ± 2.4). The mean diameter of the terminal ducts in patients with MGD was larger (120.22 ± 27.92 µm vs. 100.96 ± 20.30 µm) than in the normal controls, and had a larger coefficient of variation. Significant differences were observed in the mean diameter of the terminal ducts of patients with MGD before and after meibum gland massage (133.73 ± 27.81 µm vs. 102.26 ± 24.30 µm, p < 0.001). CONCLUSIONS: Patients with MGD have more diversified orifices and larger meibomian gland terminal duct diameters than normal subjects. In addition, meibomian gland terminal duct diameters seem to decrease in patients with MGD after meibum gland massage.