Nme2 Cas9-mediated therapeutic editing in inhibiting angiogenesis after wet age-related macular degeneration onset.

BACKGROUND: Age-related macular degeneration (AMD), particularly wet AMD characterised by choroidal neovascularization (CNV), is a leading cause of vision loss in the elderly. The hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) pathway contributes to CNV pathogenesis. Previous gene editing research indicated that disrupting these genes in retinal pigment epithelial cells could have a preventive effect on CNV progression. However, no studies have yet been conducted using gene editing to disrupt VEGF signalling after CNV induction for therapeutic validation, which is critical to the clinical application of wet AMD gene editing therapies. METHOD: Here, we employed the single-adeno-associated virus-mediated Nme2 Cas9 to disrupt key molecules in VEGF signalling, Hif1α, Vegfa and Vegfr2 after inducing CNV and estimated their therapeutic effects. RESULTS: We found that Nme2 Cas9 made efficient editing in target genes up to 71.8% post 11 days in vivo. And only Nme2 Cas9-Vegfa treatment during the early stage of CNV development reduced the CNV lesion area by 49.5%, compared to the negative control, while Nme2 Cas9-Hif1α or Nme2 Cas9-Vegfr2 treatment did not show therapeutic effect. Besides, no off-target effects were observed in Nme2 Cas9-mediated gene editing in vivo. CONCLUSIONS: This study provides proof-of-concept possibility of employing Nme2 Cas9 for potential anti-angiogenesis therapy in wet AMD.

Screening an Effective Dual-Adeno-Associated Virus Split-Cytosine Base Editor System for C-to-T Conversion In Vivo.

The cytosine base editor (CBE) has shown promise as a gene editing tool for gene therapy, as it can convert cytidine to thymidine. Adeno-associated virus (AAV) has been widely used for in vivo gene therapy, but its limited 4.7 kb packing capacity presents challenges in delivering CBE by a single AAV. To address this, one feasible solution is to split CBE into two sections for dual-AAV delivery. In this study, we utilized BE3 as an example and constructed 22 potential split-BE3 pairs with the combination of 11 splitting sites and two split-inteins (Npu and Rma). These split-BE3 pairs were initially screened in the green fluorescent protein (GFP) reporter system, with six split-BE3 pairs selected for further evaluation. The subsequent screening of split-BE3 pairs was performed at two endogenous sites in 293T and HeLa cells, revealing that the split-BE3-Rma674, split-BE3-Rma713, and split-BE3-Rma1005 displayed effective C-to-T conversion after transfection. The effectiveness of dual-AAV split-BE3 was further validated in culture cells and adult mouse eyes. Of note, the split-BE3-Rma674 demonstrated the most efficient C-to-T editing after AAV infection, with a maximal editing efficiency of 23.29% ± 10.98% in the mouse retinal pigment epithelium cells in vivo. Overall, our study presents a novel split-BE3 system with effective C-to-T conversion, which could be applied to CBE-based in vivo gene therapy.

Dual-AAV delivering split prime editor system for in vivo genome editing.

Prime editor (PE), a new genome editing tool, can generate all 12 possible base-to-base conversions, insertion, and deletion of short fragment DNA. PE has the potential to correct the majority of known human genetic disease-related mutations. Adeno-associated viruses (AAVs), the safe vector widely used in clinics, are not capable of delivering PE (∼6.3 kb) in a single vector because of the limited loading capacity (∼4.8 kb). To accommodate the loading capacity of AAVs, we constructed four split-PE (split-PE994, split-PE1005, split-PE1024, and split-PE1032) using Rma intein (Rhodothermus marinus). With the use of a GFP-mutated reporter system, PE reconstituting activities were screened, and two efficient split-PEs (split-PE1005 and split-PE1024) were identified. We then demonstrated that split-PEs delivered by dual-AAV1, especially split-PE1024, could mediate base transversion and insertion at four endogenous sites in human cells. To test the performance of split-PE in vivo, split-PE1024 was then delivered into the adult mouse retina by dual-AAV8. We demonstrated successful editing of Dnmt1 in adult mouse retina. Our study provides a new method to deliver PE to adult tissue, paving the way for in vivo gene-editing therapy using PE.

YY1-Induced Transcriptional Activation of FAM111B Contributes to the Malignancy of Breast Cancer.

BACKGROUND: Family with sequence similarity 111 member B (FAM111B) is an oncoprotein associated with multiple malignancies. We investigated the potential mechanisms of FAM111B in breast cancer. PATIENTS AND METHODS: We tested the expression of FAM111B in breast cancer tissues and the survival rate of breast cancer patients with high or low level of FAM111B through TCGA data. The expression of FAM111B in breast cancer tissues and adjacent tissues was detected using western blotting. Then we used siRNA to construct a low expression model of FAM111B in SKBR3 and MDA-MB-468. EdU, CCK-8, wound healing, and transwell assays were performed to monitor the proliferation, migration, and invasion of breast cancer cells. Western blotting was used to detect the expression of EMT-related indicators. Chromatin Immunoprecipitation (ChIP) and qPCR were used to evaluate the regulatory effect of Yin Yang 1 (YY1) on FAM111B. RESULTS: The expression of FAM111B in breast cancer tissues was higher than that in normal tissues. Patients who had high FAM111B expression had a worse prognosis. Knockdown of FAM111B inhibited the proliferation, migration, and invasion of breast cancer cells. Knockdown of FAM111B resulted in increased expression of EMT-related protein E-cadherin and decreased expression of N-cadherin and Vimentin. ChIP-qPCR analysis demonstrated that YY1 could bind to the promoter of FAM111B gene and strengthen its transcription activity. CONCLUSION: YY1-induced transcriptional activation of FAM111B accelerated the progression of breast cancer.

Application of Bioactive Hydrogels for Functional Treatment of Intrauterine Adhesion.

Intrauterine adhesion (IUA) is a common endometrial disease and one of the main causes of infertility in women of childbearing age. Current treatment strategies, such as hysteroscopic adhesion resection, hysteroscopic transcervical resection of adhesion (TCRA), the use of local hormone drugs, and anti-adhesion scaffold implantation, do not provide a satisfactory pregnancy outcome for moderate-severe IUA, which presents a great challenge in reproductive medicine. With the development of material engineering, various bioactive and functional hydrogels have been developed using natural and synthetic biomaterials. These hydrogels are not only used as barely physical barriers but are also designed as vectors of hormone drugs, growth factors, and stem cells. These characteristics give bioactive hydrogels potentially important roles in the prevention and treatment of IUA. However, there is still no systematic review or consensus on the current advances and future research direction in this field. Herein, we review recent advances in bioactive hydrogels as physical anti-adhesion barriers, in situ drug delivery systems, and 3D cell delivery and culture systems for seeded cells in IUA treatment. In addition, current limitations and future perspectives are presented for further research guidance, which may provide a comprehensive understanding of the application of bioactive hydrogels in intrauterine adhesion treatment.

Therapeutic Agents for the Treatment of Temporomandibular Joint Disorders: Progress and Perspective.

Temporomandibular joint disorders (TMD) are a common health condition caused by the structural or functional disorders of masticatory muscles and the temporomandibular joint (TMJ). Abnormal mandibular movement in TMD patients may cause pain, chronic inflammation, and other discomfort, which could be relieved by a variety of drugs through various delivery systems. In this study, we summarized commonly used therapeutic agents in the management of TMD as well as novel bioactive molecules in preclinical stage and clinical trials. The emerging therapy strategies such as novel intra-TMJ delivery systems and implants based on tissue engineering are also discussed. This comprehensive review will strengthen our understanding of pharmacological approaches for TMD therapy.

The Neuroprotective Effect of Astaxanthin on Pilocarpine-Induced Status Epilepticus in Rats.

Cognitive dysfunction is one of the serious complications induced by status epilepticus (SE), which has a significant negative impact on patients' quality of life. Previous studies demonstrated that the pathophysiological changes after SE such as oxidative stress, inflammatory reaction contribute to neuronal damage. A recent study indicated that preventive astaxanthin (AST) alleviated epilepsy-induced oxidative stress and neuronal apoptosis in the brain. In the present study, rats were treated with vehicle or AST 1 h after SE onset and were injected once every other day for 2 weeks (total of seven times). The results showed that the cognitive function in SE rats was significantly impaired, and AST treatment improved cognitive function in the Morris water maze (MWM). Magnetic resonance imaging (MRI), hematoxylin-eosin (HE) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining showed obvious damage in the hippocampus of SE rats, and AST alleviated the damage. Subsequently, we evaluated the effect of AST on relative pathophysiology to elucidate the possible mechanisms. To evaluate the oxidative stress, the expression of malondialdehyde (MDA) and superoxide dismutase (SOD) in plasma were detected using commercially available kits. NADPH oxidase-4 (Nox-4), p22phox, NF-E2-related factor 2 (Nrf-2), heme oxygenase 1 (Ho-1) and sod1 in the parahippocampal cortex and hippocampus were detected using western blot and real-time polymerase chain reaction (RT-PCR). The levels of MDA in plasma and Nox-4 and p22phox in the brain increased in SE rats, and the levels of SOD in plasma and Nrf-2, Ho-1 and sod1 in the brain decreased. Treatment with AST alleviated these changes. We also detected the levels of inflammatory mediators like cyclooxygenase-2 (cox-2), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and NF-κB phosphorylation p65 (p-p65)/p65 in the brain. The inflammatory reaction was significantly activated in the brain of SE rats, and AST alleviated neuroinflammation. We detected the levels of p-Akt, Akt, B-cell lymphoma-2 (Bcl-2), Bax, cleaved caspase-3, and caspase-3 in the parahippocampal cortex and hippocampus using western blot. The levels of p-Akt/Akt and Bcl-2 decreased in SE rats, Bax and cleaved caspase-3/caspase-3 increased, while AST alleviated these changes. The present study indicated that AST exerted an reobvious neuroprotective effect in pilocarpine-induced SE rats.