The antioxidant effect of tetrahedral framework nucleic acid-based delivery of small activating RNA targeting DJ-1 on retinal oxidative stress injury.

Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are the world's leading causes of blindness. The retinal pigment epithelium (RPE) and vascular endothelial cell exposed to oxidative stress is the major cause of AMD and DR. DJ-1, an important endogenous antioxidant, its overexpression is considered as a promising antioxidant treatment for AMD and DR. Here, we modified the tetrahedral frame nucleic acids (tFNAs) with DJ-1 saRNAs as a delivery system, and synthesized a novel nanocomplex (tFNAs-DJ-1 saRNAs). In vitro studies show that tFNAs-DJ-1 saRNAs can efficiently transfer DJ-1 saRNAs to human umbilical vein endothelial cells (HUVECs) and ARPE-19s, and significantly increased their cellular DJ-1 level. Reactive oxygen species expression in H2O2-treated HUVECs and ARPE-19s were decreased, cell viability was enhanced and cell apoptosis were inhibited when tFNAs-DJ-1 saRNAs were delivered. Moreover, tFNAs-DJ-1 saRNAs preserved mitochondrial structure and function under oxidative stress conditions. In the aspect of molecular mechanism, tFNAs-DJ-1 saRNAs activated Erk and Nrf2 pathway, which might contribute to its protective effects against oxidative stress damage. To conclude, this study shows the successfully establishment of a simple but effective delivery system of DJ-1 saRNAs associated with antioxidant effects in AMD and DR, which may be a promising agent for future treatment in oxidative stress-related retinal disorders.

Tetrahedral framework nucleic acids-based delivery of MicroRNA-22 inhibits pathological neovascularization and vaso-obliteration by regulating the Wnt pathway.

The objective of this study was to investigate the effects and molecular mechanisms of tetrahedral framework nucleic acids-microRNA22 (tFNAs-miR22) on inhibiting pathological retinal neovascularization (RNV) and restoring physiological retinal vessels. A novel DNA nanocomplex (tFNAs-miR22) was synthesised by modifying microRNA-22 (miR22) through attachment onto tetrahedral frame nucleic acids (tFNAs), which possess diverse biological functions. Cell proliferation, wound healing, and tube formation were employed for in vitro assays to investigate the angiogenic function of cells. Oxygen-induced retinopathy (OIR) model was utilised to examine the effects of reducing pathological neovascularization (RNV) and inhibiting vascular occlusion in vivo. In vitro, tFNAs-miR22 demonstrated the ability to penetrate endothelial cells and effectively suppress cell proliferation, tube formation, and migration in a hypoxic environment. In vivo, tFNAs-miR22 exhibited promising results in reducing RNV and promoting the restoration of normal retinal blood vessels in OIR model through modulation of the Wnt pathway. This study provided a theoretical basis for the further understanding of RNV, and highlighted the innovative and potential of tFNAs-miR22 as a therapeutic option for ischemic retinal diseases.

Cm-siRPred: Predicting chemically modified siRNA efficiency based on multi-view learning strategy.

The rational modification of siRNA molecules is crucial for ensuring their drug-like properties. Machine learning-based prediction of chemically modified siRNA (cm-siRNA) efficiency can significantly optimize the design process of siRNA chemical modifications, saving time and cost in siRNA drug development. However, existing in-silico methods suffer from limitations such as small datasets, inadequate data representation capabilities, and lack of interpretability. Therefore, in this study, we developed the Cm-siRPred algorithm based on a multi-view learning strategy. The algorithm employs a multi-view strategy to represent the double-strand sequences, chemical modifications, and physicochemical properties of cm-siRNA. It incorporates a cross-attention model to globally correlate different representation vectors and a two-layer CNN module to learn local correlation features. The algorithm demonstrates exceptional performance in cross-validation experiments, independent dataset, and case studies on approved siRNA drugs, and showcasing its robustness and generalization ability. In addition, we developed a user-friendly webserver that enables efficient prediction of cm-siRNA efficiency and assists in the design of siRNA drug chemical modifications. In summary, Cm-siRPred is a practical tool that offers valuable technical support for siRNA chemical modification and drug efficiency research, while effectively assisting in the development of novel small nucleic acid drugs. Cm-siRPred is freely available at https://cellknowledge.com.cn/sirnapredictor/.

Tetrahedral framework nucleic acids inhibit pathological neovascularization and vaso-obliteration in ischaemic retinopathy via PI3K/AKT/mTOR signalling pathway.

This study aimed to explore the effect and the molecular mechanism of tetrahedral framework nucleic acids (tFNAs), a novel self-assembled nanomaterial with excellent biocompatibility and superior endocytosis ability, in inhibition of pathological retinal neovascularization (RNV) and more importantly, in amelioration of vaso-obliteration (VO) in ischaemic retinopathy. tFNAs were synthesized from four single-stranded DNAs (ssDNAs). Cell proliferation, wound healing and tube formation assays were performed to explore cellular angiogenic functions in vitro. The effects of tFNAs on reducing angiogenesis and inhibiting VO were explored by oxygen-induced retinopathy (OIR) model in vivo. In vitro, tFNAs were capable to enter endothelial cells (ECs), inhibit cell proliferation, tube formation and migration under hypoxic conditions. In vivo, tFNAs successfully reduce RNV and inhibit VO in OIR model via the PI3K/AKT/mTOR/S6K pathway, while vascular endothelial growth factor fusion protein, Aflibercept, could reduce RNV but not inhibit VO. This study provides a theoretical basis for the further understanding of RNV and suggests that tFNAs might be a novel promising candidate for the treatment of blind-causing RNV.

Advances in regenerative medicine applications of tetrahedral framework nucleic acid-based nanomaterials: an expert consensus recommendation.

With the emergence of DNA nanotechnology in the 1980s, self-assembled DNA nanostructures have attracted considerable attention worldwide due to their inherent biocompatibility, unsurpassed programmability, and versatile functions. Especially promising nanostructures are tetrahedral framework nucleic acids (tFNAs), first proposed by Turberfield with the use of a one-step annealing approach. Benefiting from their various merits, such as simple synthesis, high reproducibility, structural stability, cellular internalization, tissue permeability, and editable functionality, tFNAs have been widely applied in the biomedical field as three-dimensional DNA nanomaterials. Surprisingly, tFNAs exhibit positive effects on cellular biological behaviors and tissue regeneration, which may be used to treat inflammatory and degenerative diseases. According to their intended application and carrying capacity, tFNAs could carry functional nucleic acids or therapeutic molecules through extended sequences, sticky-end hybridization, intercalation, and encapsulation based on the Watson and Crick principle. Additionally, dynamic tFNAs also have potential applications in controlled and targeted therapies. This review summarized the latest progress in pure/modified/dynamic tFNAs and demonstrated their regenerative medicine applications. These applications include promoting the regeneration of the bone, cartilage, nerve, skin, vasculature, or muscle and treating diseases such as bone defects, neurological disorders, joint-related inflammatory diseases, periodontitis, and immune diseases.

HODD: A Manually Curated Database of Human Ophthalmic Diseases with Symptom Characteristics and Genetic Variants Towards Facilitating Quick and Definite Diagnosis.

Ophthalmic diseases are disorders that affect the eyes. Hundreds of causal genes and biological pathways have been reported to be closely correlated with ophthalmic diseases. However, these information are scattered across various resources, which has hindered a thorough and deep understanding of ophthalmic diseases. In the present work, we proposed the Human Ophthalmic Diseases Database (HODD), which currently deposits 730 ophthalmic diseases and 653 related genes and is available at http://bio-bigdata.cn/HODD/ . The disease-related information and genes related to ophthalmic diseases were collected from the several well-known databases. To comprehensively understand the ophthalmic diseases, the basic information was provided for each disease, including disease description, related genes, gene location, ocular and extraocular effect of the disease, protein-protein interaction and disease-associated pathways. All these data were reorganized and made accessible through multiple entrances. We hope that HODD will facilitate studies on ophthalmic diseases. The workflow for the construction of the HODD (Human Ophthalmic Diseases Database, http://bio-bigdata.cn/HODD/ ) database.

Isolation, Structural Elucidation, and α-Glucosidase Inhibitory Activities of Triterpenoid Lactones and Their Relevant Biogenetic Constituents from Ganoderma resinaceum.

Ganoderma resinaceum has been used as an ethnomedicine for lowering blood sugar. To clarify the bioactive chemical constituents contributing to lower blood sugar, chemical investigation on the fruiting bodies of Ganoderma resinaceum was conducted by chromatographic techniques, and led to the isolation of 14 compounds. Their structures were elucidated as triterpenoid lactones (1⁻4 and 8) and ganoderma acids (5⁻7 and 9⁻14) based on the analysis of extensive spectroscopy (mass spectrometry (MS), nuclear magnetic resonance (NMR), infrared (IR), and ultraviolet (UV)) and comparison with literature data. Compounds 3, 5, 6, and 9⁻14 were evaluated for α-glucosidase inhibitory activity. Compounds 1⁻7 are new compounds. Compounds 1⁻4 and 8 were characteristic of an oxaspirolactone moiety, consisting of a five-membered ether ring, a five-membered lactone ring, and a characteristic C-23 spiro carbon. It is rare for natural products that such an oxaspirolactone moiety occurred in the lanostane-type triterpenoids. Compounds 5⁻7 and 9⁻14 may be important intermediates of the biosynthetic pathways of 1⁻4 and 8. Compounds 1 and 2 showed more potent inhibitory activity against α-glucosidase compared with the positive control drug acarbose with IC50 value of 0.75 ± 0.018 mM and 1.64 ± 0.022 mM, respectively.

A novel peptide specifically binding to VEGF receptor suppresses angiogenesis in vitro and in vivo.

Vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, plays an essential role in both physiological and pathological angiogenesis through binding to VEGF receptors (VEGFRs). Here we report a novel peptide designated HRHTKQRHTALH (peptide HRH), which was isolated from the Ph.D. -12 phage display library using VEGFR-Fc fusion protein as the bait. This peptide was found to dose-dependently inhibit the proliferation of human umbilical vein endothelial cells stimulated by VEGF. The anti-angiogenesis effect of the HRH peptide was further confirmed in vivo using the chick chorioallantoic membrane assay, which was also dose-dependent. Besides, peptide HRH was proved to inhibit corneal neovascularization in an alkali-burnt rat corneal model and a suture-induced rat corneal model. Taken together, these findings suggest that the HRH peptide can inhibit angiogenesis both in vitro and in vivo. Consequently, the HRHTKQRHTALH peptide might be a promising lead peptide for the development of potential angiogenic inhibitors.

Quantification of MicroRNAs in human aqueous humor by miRFLP assay.

MiRNAs are expressed in a tissue-specific fashion in the eyes and changes in miRNAs levels in aqueous humor (AH) may reflect the function of the eye and eye disease. Due to the low concentration of total miRNA in human aqueous humor, high volume of sample is required for RNA extraction prior to routine quantification such as RT-qPCR. However, limited volume of AH could be collected through surgery because of the characteristic of the eye. In addition, inefficiency of RNA-extraction kits could affect target miRNA quantification dramatically. AH-direct miRFLP assay was developed for quantification of target miRNAs in human aqueous humor samples. For the first time, accurate miRNA quantification in human AH was achieved with microliter scale sample loading. Higher copy numbers of target miRNAs were obtained in direct detection than in RNA-extraction solution. It indicates that AH-direct miRFLP assay was able to quantify target miRNAs more accurately with no requirement for RNA-extraction to avoid sampling variability.

Analysis and quality control of carbohydrates in therapeutic proteins with fluorescence HPLC.

Conbercept is an Fc fusion protein with very complicated carbohydrate profiles which must be carefully monitored through manufacturing process. Here, we introduce an optimized fluorescence derivatization high-performance liquid chromatographic method for glycan mapping in conbercept. Compared with conventional glycan analysis method, this method has much better resolution and higher reproducibility making it excellent for product quality control.

Observation of total VEGF level in hyperglycemic mouse eyes after intravitreal injection of the novel anti-VEGF drug conbercept.

PURPOSE: Conbercept (KH902), a novel recombinant, soluble vascular endothelial growth factor (VEGF) receptor-IgG fusion protein, has been developed as a new drug for ocular neovascularization and macular edema. The present study aims to clarify the changes in conbercept levels, VEGF, and intraocular pressure (IOP) after the intravitreal injection of conbercept into diabetic mouse eyes. METHODS: Five-week-old C57BL/6 mice were injected with streptozotocin to induce diabetes. Total VEGF and conbercept levels in the eyes were detected using an ELISA kit at -2 h, 1 h, 1 d, 4 d, 8 d, 16 d, 28 d, and 34 d after intravitreal injection of conbercept into diabetic and control mice. IOP was measured with a noninvasive TonoLab tonometer 7 d after intravitreal conbercept injection. RESULTS: The concentration of conbercept in the treated eyes increased immediately after injection and remained at high levels for 4 d (29.77±27.19 ng/ml, 20.28±28.85 ng/ml, and 42.43±36.51 ng/ml for days 1, 2, and 4, respectively). The concentration of conbercept in the untreated fellow eyes increased from day 2 to day 4 after injection with a level of about 1% of that in the injected eyes. Conbercept concentrations in both the treated and fellow eyes decreased from day 7 after intravitreal injection. The concentration of VEGF in the treated eyes increased significantly 1 h after injection when compared with the baseline measured 2 h before injection in both the diabetic and control mice (645.91±86.47 pg/ml versus 296.10±76.11 pg/ml and 860.50±201.47 pg/ml versus 377.69±70.72 pg/ml, respectively). VEGF concentration reached its peak 24 h after injection and then decreased thereafter. At day 7 after intravitreal injection, the difference in IOP between mice that received conbercept and mice that received PBS injections was not significant (p>0.05). CONCLUSIONS: Conbercept and total VEGF levels in the mouse eyes were elevated after intravitreal injection of conbercept. Increased VEGF levels likely reflect VEGF sequestered by conbercept. These data could be helpful in understanding the metabolism of anti-VEGF drugs in the eye and for determining the protocol of multiple intravitreal injections of conbercept in patients.

Safety and efficacy of conbercept in neovascular age-related macular degeneration: results from a 12-month randomized phase 2 study: AURORA study.

PURPOSE: To assess the safety and efficacy of multiple injections of 0.5 and 2.0 mg conbercept using variable dosing regimens in patients with neovascular age-related macular degeneration (AMD). DESIGN: Randomized, double-masked, multicenter, controlled-dose, and interval-ranging phase 2 clinical trial divided into a 3-month loading phase followed by a maintenance phase. PARTICIPANTS: Patients with choroidal neovascularization secondary to AMD with lesion sizes of 12 disc areas or less and a best-corrected visual acuity (BCVA) letter score of between 73 and 24 were enrolled. METHODS: Patients were randomized 1:1 to receive either 0.5 or 2.0 mg intravitreal conbercept for 3 consecutive monthly does. After the third dose, each group was reassigned randomly again to monthly (Q1M group) or as-needed (pro re nata [PRN] group) treatment without changing the drug assignment. MAIN OUTCOME MEASURES: The primary end point was the mean change in BCVA from baseline to month 3, with secondary end points being the mean change in BCVA, mean change in central retinal thickness (CRT), and safety at month 12. RESULTS: We enrolled 122 patients. At the primary end point at month 3, mean improvements in BCVA from baseline in the 0.5- and 2.0-mg groups were 8.97 and 10.43 letters, respectively. At month 12, mean improvements in BCVA from baseline were 14.31, 9.31, 12.42, and 15.43 letters for the 0.5-mg PRN, 0.5-mg Q1M, 2.0-mg PRN, and 2.0-mg Q1M regimens, respectively. At month 12, mean reductions in CRT in the 4 regimens were 119.8, 129.7, 152.1, and 170.8 µm, respectively. There were no significant differences for the pairwise comparisons between all study groups. The difference in the number of injections between the 2 PRN groups was not statistically significant. Treatment with conbercept generally was safe and well tolerated. CONCLUSIONS: The significant gains in BCVA at 3 months were the same or better at 12 months in all conbercept dosing groups of neovascular AMD patients. During the 12 months, repeated intravitreal injections of conbercept were well tolerated in these patients. Future clinical trials are required to confirm its long-term efficacy and safety.

Novel VEGF decoy receptor fusion protein conbercept targeting multiple VEGF isoforms provide remarkable anti-angiogenesis effect in vivo.

VEGF family factors are known to be the principal stimulators of abnormal angiogenesis, which play a fundamental role in tumor and various ocular diseases. Inhibition of VEGF is widely applied in antiangiogenic therapy. Conbercept is a novel decoy receptor protein constructed by fusing VEGF receptor 1 and VEGF receptor 2 extracellular domains with the Fc region of human immunoglobulin. In this study, we systematically evaluated the binding affinity of conbercept with VEGF isoforms and PlGF by using anti-VEGF antibody (Avastin) as reference. BIACORE and ELISA assay results indicated that conbercept could bind different VEGF-A isoforms with higher affinity than reference. Furthermore, conbercept could also bind VEGF-B and PlGF, whereas Avastin showed no binding. Oxygen-induced retinopathy model showed that conbercept could inhibit the formation of neovasularizations. In tumor-bearing nude mice, conbercept could also suppress tumor growth very effectively in vivo. Overall, our study have demonstrated that conbercept could bind with high affinity to multiple VEGF isoforms and consequently provide remarkable anti-angiogenic effect, suggesting the possibility to treat angiogenesis-related diseases such as cancer and wet AMD etc.

Structural characterization of a recombinant fusion protein by instrumental analysis and molecular modeling.

Conbercept is a genetically engineered homodimeric protein for the treatment of wet age-related macular degeneration (wet AMD) that functions by blocking VEGF-family proteins. Its huge, highly variable architecture makes characterization and development of a functional assay difficult. In this study, the primary structure, number of disulfide linkages and glycosylation state of conbercept were characterized by high-performance liquid chromatography, mass spectrometry, and capillary electrophoresis. Molecular modeling was then applied to obtain the spatial structural model of the conbercept-VEGF-A complex, and to study its inter-atomic interactions and dynamic behavior. This work was incorporated into a platform useful for studying the structure of conbercept and its ligand binding functions.

A phase 1 study of KH902, a vascular endothelial growth factor receptor decoy, for exudative age-related macular degeneration.

PURPOSE: To determine the safety, tolerability, and bioactivity of KH902, a fully human fusion protein containing key domains from vascular endothelial growth factor receptors 1 and 2 with human immunoglobulin Fc. DESIGN: Prospective, single-center, open-label, dose-escalating, interventional case series. PARTICIPANTS: Twenty-eight patients with choroidal neovascularization (CNV) resulting from exudative age-related macular degeneration (AMD) with lesion size of 12 disc areas or less and best-corrected visual acuity (VA) of 55 letters or worse. METHODS: A single intravitreal injection of KH902 at 1 of 6 escalating doses if no dose-limiting toxicity (DLT) occurred through postinjection day 14 of the previous dose level. Follow-up examinations were performed on postinjection days 1, 3, 5, 7, 14, 28, and 42. The primary end point was at 42 days, and patients were monitored for an additional 6 weeks (12 weeks total). MAIN OUTCOME MEASURES: The primary safety measures were changes from baseline in VA, intraocular pressure (IOP), intraocular inflammation, and production of anti-KH902 antibody. Dose-limiting toxicity was defined as intraocular inflammation, elevated IOP, significantly reduced vision, or retinal hemorrhage within 42 days after injection. Bioactivity measures included mean change from baseline in VA, central retinal thickness, and total macular volume on optical coherence tomography and CNV changes on fluorescein angiography. RESULTS: All patients completed the study with no DLT and no serious or drug-related adverse events. Ocular adverse events were mild to moderate in severity, including transient IOP elevation and injection-site subconjunctival hemorrhage after KH902 injections. No serum anti-KH902 antibodies were detected. On day 42 after injection, the mean change in VA from baseline was +19.6 letters with no subjects losing 1 letter or more and 57% of patients gaining 15 letters or more from baseline. The mean change in center point thickness from baseline was -77.2 µm and the mean decrease in CNV area was 12.6%. CONCLUSIONS: No safety concerns were detected after a single, intravitreal injection of KH902 up to 3.0 mg in this phase 1 study. Bioactivity of KH902 was suggested with improvements in VA, reduction in central retinal thickness, and a decrease in CVN area in patients with CNV resulting from exudative AMD, indicating that further study is warranted.