Split AAV8 Mediated ABCA4 Expression for Gene Therapy of Mouse Stargardt Disease (STGD1).

Adeno-associated virus (AAV)-based gene therapy has been shown to be safe and effective in numerous animal models and clinical trials for various ophthalmic diseases. Stargardt disease (STGD1; MIM #248200) is the most common autosomal recessive macular dystrophy disease, and the most common form is caused by mutations in the ABCA4 gene, a gene with 6.8 kb coding sequence. Split intein approaches increase the capacity of dual AAV gene therapy, but at the cost of reduced protein expression, which may be insufficient to achieve a therapeutic effect. In this study, we designed various dual split intein ABCA4 vectors and showed that the efficiency of expression of full-length ABCA4 protein is dependent on combinations of types and split sites of the intein system. The most efficient vectors were identified through in vitro screening, and a novel dual AAV8-ABCA4 vector was constructed and subsequently proven to express full-length ABCA4 protein at a high level, reducing bisretinoid formation and correcting the visual function of ABCA4-knockout mice. Furthermore, we evaluated therapeutic effects of different dosages by subretinal injection in mice model. Both therapeutic effects and safety were guaranteed under the treatment of 1.00 × 109 GC/eye. These results support the optimized dual AAV8-ABCA4 approach in future clinical translation for treatment of Stargardt disease.

Delivery of nVEGFi using AAV8 for the treatment of neovascular age-related macular degeneration.

Inhibition of vascular endothelial growth factor (VEGF) is the standard therapy for neovascular age-related macular degeneration (nAMD). However, anti-VEGF agents used in the clinic require repeated injections, causing adverse effects. Gene therapy could provide sustained anti-VEGF levels after a single injection, thereby drastically decreasing the treatment burden and improving visual outcomes. In this study, we developed a novel VEGF Trap, nVEGFi, containing domains 1 and 2 of VEGFR1 and domain 3 of VEGFR2 fused to the Fc portion of human IgG. The nVEGFi had a higher expression level than aflibercept under the same expression cassettes of adeno-associated virus (AAV)8 in vitro and in vivo. nVEGFi was found to be noninferior to aflibercept in binding and blocking VEGF in vitro. AAV8-mediated expression of nVEGFi was maintained for at least 12 weeks by subretinal delivery in C57BL/6J mice. In a mouse laser-induced choroidal neovascularization (CNV) model, 4 × 108 genome copies of AAV8-nVEGFi exhibited a significantly increased reduction in the CNV area compared with AAV8-aflibercept (78.1% vs. 63.9%, p < 0.05), while causing no structural or functional changes to the retina. In conclusion, this preclinical study showed that subretinal injection of AAV8-nVEGFi was long lasting, well tolerated, and effective for nAMD treatment, supporting future translation to the clinic.

YinQiSanHuang Jiedu decoction for the treatment of hepatitis B-related compensated liver cirrhosis: study protocol for a multi-center randomized controlled trial.

INTRODUCTION: Hepatitis B-related compensated liver cirrhosis is related to a higher risk of hepatocellular carcinoma, and antiviral therapy is the preferred method. As the pathological mechanisms of liver fibrosis are complex, drugs developed for a single target are difficult to be effective in clinical practice, so there are no chemical drugs or biological drugs with clear efficacy available for clinical application at present. Traditional Chinese medicine is a kind of medical science that has been gradually formed during thousands of years and continuously enriched by the people of all ethnic groups in China. Traditional Chinese medicine shows curative effects in the treatment of liver diseases, especially in the field of liver fibrosis prevention and treatment. This study aims to test the integrative medicine (Chinese medicine plus antiviral therapy) effective on lowing hepatocellular carcinoma risk among patients with hepatitis-related compensated liver cirrhosis. METHODS AND ANALYSIS: This is a multi-center randomized controlled trial, and a total of 5 hospitals and 802 patients will be involved in. All the subjects are randomly allocated to the YinQiSanHuang Jiedu decoction (YQSHD) group (n = 401) or the placebo group (n = 401). The YQSHD group receives YQSHD granule with entecavir (ETV), and the placebo group receives YQSHD placebo with ETV. The treatment period will last for 52 weeks, and the follow-up period for 52 ± 2 weeks. The primary outcome measure is the annual incidence of HCC. Outcomes will be assessed at baseline and after treatment. The objective of this trial is "the integrative of YQSHD with ETV reduce the annual incidence of HCC to 1%." ETHICS AND DISSEMINATION: The protocol has been approved by the Medical Ethics Committee of Guang'anmen Hospital, China (No.2019-006-KY), and the other centers in the trial will not begin recruiting until the local ethical approval has been obtained. Trial final results will be disseminated via publication. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR1900021532 . Registered on February 26, 2019.

In vivo PCSK9 gene editing using an all-in-one self-cleavage AAV-CRISPR system.

Adeno-associated virus (AAV)-mediated delivery of the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9) has shown promising results in preclinical models. However, the long-term expression of Cas9 mediated by AAV in the post-mitotic cells raises concerns with specificity and immunogenicity. Thus, it would be advantageous to limit the duration of Cas9 expression following delivery. In this study, we have engineered an all-in-one self-cleavage AAV-CRISPR-Cas9 system to restrict the expression of Cas9 nuclease, which consists of a Cas9 nuclease from Staphylococcus aureus (SaCas9), a chimeric single guide RNA (sgRNA) molecule targeting PCSK9, and flanking sites targeted by this sgRNA. The self-cleavage system generated a negative feedback loop where Cas9 cut both the target genomic locus and the AAV vector, thus self-limiting the expression of Cas9. We demonstrated that this system could reduce ∼60% expression of SaCas9 protein and had a 20-fold reduction in off-target activity at 24 weeks post-vector administration in vivo. Moreover, the on-target editing efficacy was not compromised and resulted in a stable reduction in circulating PCSK9 and serum cholesterol. The inclusion of this self-cleavage system in gene-editing approaches could increase the safety profile of AAV-delivered genome-editing nucleases and thereby promote its clinical transformation.

BATF2 prevents glioblastoma multiforme progression by inhibiting recruitment of myeloid-derived suppressor cells.

The basic leucine zipper ATF-like transcription factor 2 (BATF2) has been implicated in inflammatory responses and anti-tumour effects. Little, however, is known regarding its extracellular role in maintaining a non-supportive cancer microenvironment. Here, we show that BATF2 inhibits glioma growth and myeloid-derived suppressor cells (MDSCs) recruitment. Interestingly, extracellular vesicles (EVs) from BATF2-overexpressing glioma cell lines (BATF2-EVs) inhibited MDSCs chemotaxis in vitro. Moreover, BATF2 inhibited intracellular SDF-1α and contributes to decreased SDF-1α in EVs. In addition, BATF2 downregulation-induced MDSCs recruitment were reversed by blocking SDF-1α/CXCR4 signalling upon AMD3100 treatment. Specifically, detection of EVs in 24 pairs of gliomas and healthy donors at different stages revealed that the abundance of BATF2-positive EVs in plasma (BATF2+ plEVs) can distinguish stage III-IV glioma from stage I-II glioma and healthy donors. Taken together, our study identified novel regulatory functions of BATF2 in regulating MDSCs recruitment, providing a prognostic value in terms of the number of BATF2+ plEVs in glioma stage.

Hypomethylation of GDNF family receptor alpha 1 promotes epithelial-mesenchymal transition and predicts metastasis of colorectal cancer.

Tumor metastasis is the major cause of poor prognosis and mortality in colorectal cancer (CRC). However, early diagnosis of highly metastatic CRC is currently difficult. In the present study, we screened for a novel biomarker, GDNF family receptor alpha 1 (GFRA1) based on the expression and methylation data in CRC patients from The Cancer Genome Altlas (TCGA), followed by further analysis of the correlation between the GFRA1 expression, methylation, and prognosis of patients. Our results show DNA hypomethylation-mediated upregulation of GFRA1 in invasive CRC, and it was found to be correlated with poor prognosis of CRC patients. Furthermore, GFRA1 methylation-modified sequences were found to have potential as methylation diagnostic markers of highly metastatic CRC. The targeted demethylation of GFRA1 by dCas9-TET1CD and gRNA promoted CRC metastasis in vivo and in vitro. Mechanistically, demethylation of GFRA1 induces epithelial-mesenchymal transition (EMT) by promoting AKT phosphorylation and increasing c-Jun expression in CRC cells. Collectively, our findings indicate that GFRA1 hypomethylation can promote CRC invasion via inducing EMT, and thus, GFRA1 methylation can be used as a biomarker for the early diagnosis of highly metastasis CRC.

CRISPR-Cas9-Mediated In Vivo Gene Integration at the Albumin Locus Recovers Hemostasis in Neonatal and Adult Hemophilia B Mice.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 loaded by vectors could induce high rates of specific site genome editing and correct disease-causing mutations. However, most monogenic genetic diseases such as hemophilia are caused by different mutations dispersed in one gene, instead of an accordant mutation. Vectors developed for correcting specific mutations may not be suited to different mutations at other positions. Site-specific gene addition provides an ideal solution for long-term, stable gene therapy. We have demonstrated SaCas9-mediated homology-directed factor IX (FIX) in situ targeting for sustained treatment of hemophilia B. In this study, we tested a more efficient dual adeno-associated virus (AAV) strategy with lower vector dose for liver-directed genome editing that enables CRISPR-Cas9-mediated site-specific integration of therapeutic transgene within the albumin gene, and we aimed to develop a more universal gene-targeting approach. We successfully achieved coagulation function in newborn and adult hemophilia B mice by a single injection of dual AAV vectors. FIX levels in treated mice persisted even after a two-thirds partial hepatectomy, indicating stable gene integration. Our results suggest that this CRISPR-Cas9-mediated site-specific gene integration in hepatocytes could transform into a common clinical therapeutic method for hemophilia B and other genetic diseases.

Nanomicelle protects the immune activation effects of Paclitaxel and sensitizes tumors to anti-PD-1 Immunotherapy.

Paclitaxel (PTX) has shown pleiotropic immunologic effects on the tumor microenvironment, and nanomicelle has emerged as a promising strategy for PTX delivery. However, the detailed mechanisms remain to be fully elucidated. Meanwhile, immunogenic cell death (ICD) is an effective approach to activate the immune system. This study investigated the ICD effect of PTX and how nanomicelle affected the immune-activation ability of PTX. Methods: The ICD effects of PTX were identified via the expression of ICD markers and cell vaccine experiment. Tumor size and overall survival in multiple animal models with treatment were monitored to evaluate the antitumor effects. The mechanisms of PTX-induced ICD and antitumor immunity were determined by detecting gene expression related to ER stress and analyzing immune cell profile in tumor after treatment. Results: We revealed the immune-regulation mechanism of PTX nanomicelle by inducing ICD, which can promote antigen presentation by dendritic cells (DCs) and activate antitumor immunity. Notably, nanomicelle encapsulation protected the ICD effects and immune activation, which were hampered by immune system impairment caused by chemotherapy. Compared with traditional formulations, a low dose of nanomicelle-encapsulated PTX (nano-PTX) treatment induced immune-dependent tumor control, which increased the infiltration and function of both T cells and DCs within tumors. However, this antitumor immunity was hampered by highly expressed PD-1 on tumor-infiltrating CD8+ T cells and upregulated PD-L1 on both immune cells and tumor cells after nano-PTX treatment. Combination therapy with a low dose of nano-PTX and PD-1 antibodies elicited CD8+ T cell-dependent antitumor immunity and remarkably improved the therapeutic efficacy. Conclusions: Our results provide systemic insights into the immune-regulation ability of PTX to induce ICD, which acts as an inducer of endogenous vaccines through ICD effects, and also provides an experimental basis for clinical combination therapy with nano-PTX and PD-1 antibodies.

Targeted demethylation of the SARI promotor impairs colon tumour growth.

SARI (suppressor of activator protein 1, regulated by IFN) functions as a tumour suppressor and is inactivated in various cancers. However, the mechanism underlying SARI inactivation in cancer remains elusive. In this study, we detected a high level of DNA methylation of the SARI promoter and an inverse correlation between SARI promoter methylation and expression in malignant tissues from patients with colon cancer. Furthermore, we found that the SARI promoter methylation status is a prognostic indicator for patients with colon cancer. A dCas9-multiGCN4/scFv-TET1CD-sgRNA-based SARI-targeted demethylation system (dCas9-multiGCN4/scFv-TET1CD-sgSARI) was constructed to precisely and specifically demethylate regions of SARI; this system resulted in the substantial activation of SARI expression. Further in vitro and in vivo data confirmed that dCas9-multiGCN4/scFv-TET1CD-sgSARI exerts anti-tumour effects by regulating tumour proliferation, apoptosis, and angiogenesis. Collectively, specific demethylation of the SARI promoter and restoration of endogenous SARI expression by dCas9-multiGCN4/scFv-TET1CD-SARI have therapeutic applications for colon cancer and perhaps for other cancers.

Progress in the treatment of infantile hemangioma.

Infantile hemangioma (IH) is a common benign tumor, which mostly resolves spontaneously; however, children with high-risk IH need treatment. Currently, the recognized first-line treatment regimen for IH is oral propranolol, but research on the pathogenesis of IH has led to the identification of new therapeutic targets, which have shown good curative effects, providing more options for disease treatment. This article summarizes the applications of different medications, dosages, and routes of administration for the treatment of IH. In addition to drug therapy, this article also reviews current therapeutic options for IH such as laser therapy, surgical treatment, and observation. To provide the best treatment, therapeutic regimens for IH should be selected based on the child's age, the size and location of the lesion, the presence of complications, the implementation conditions, and the potential outcomes of the treatment.

Modulating the Tumor Microenvironment via Oncolytic Viruses and CSF-1R Inhibition Synergistically Enhances Anti-PD-1 Immunotherapy.

Immunotherapy based on the immune checkpoint blockade has emerged as the most promising approach for cancer therapy. However, the proportion of colorectal cancer patients who benefit from immunotherapy is small due to the immunosuppressive tumor microenvironment. Hence, combination immunotherapy is an ideal strategy to overcome this limitation. In this study, we developed a novel combination of CSF-1R (colony-stimulating factor 1 receptor) inhibitor (PLX3397), oncolytic viruses, and anti-PD-1 antibody. Our results demonstrated that the triple treatment synergistically conferred significant tumor control and prolonged the survival of mouse models of colon cancer. Approximately 43% and 82% of mice bearing the CT26 and MC38 tumor, respectively, survived long term following the triple treatment. This combination therapy reprogrammed the immunosuppressive tumor microenvironment toward a CD8+ T cell-biased anti-tumor immunity by increasing T cell infiltration in the tumor and augmenting anti-tumor CD8+ T cell function. Our results provide a robust strategy for clinical combination therapy.

Follistatin-like protein 5 inhibits hepatocellular carcinoma progression by inducing caspase-dependent apoptosis and regulating Bcl-2 family proteins.

Hepatocellular carcinoma (HCC) is one of the most common and deadly malignant tumors in the world, especially in China. Follistatin-like protein 5 (FSTL5) is a member of the FSTL family, which is involved in cell proliferation, migration, differentiation, and embryo development. We aimed to investigate the function and underlying mechanism of FSTL5 in HCC. FSTL5 expression was determined by immunohistochemistry staining in a liver cancer tissue microarray (TMA) and the correlation between FSTL5 and the prognosis of HCC patients was analysed. Further proliferation assay, colony formation assay, flow cytometry, and xenograft tumor model were performed to investigate the bioeffects of FSTL5 in HCC in vitro and in vivo. We found that FSTL5 expression was downregulated in HCC tissues and positively correlated with the prognosis of patients with HCC at tumor node metastasis stage I/II. Overexpression of FSTL5 efficiently impaired HCC growth both in vivo and in vitro with an exogenous manner. Mechanistic investigation demonstrated that FSTL5 promoted HCC cell apoptosis in a caspase-dependent manner and regulated Bcl-2 family proteins. These results indicate that FSTL5 may be a potential novel target for HCC treatment, and a biomarker for tumor prognosis.

DDA1, a novel oncogene, promotes lung cancer progression through regulation of cell cycle.

Lung cancer is globally widespread and associated with high morbidity and mortality. DDA1 (DET1 and DDB1 associated 1) was first discovered and registered in the GenBank database by our colleagues. DDA1, an evolutionarily conserved gene, might have significant functions. Recent reports have demonstrated that DDA1 is linked to the ubiquitin-proteasome pathway and facilitates the degradation of target proteins. However, the function of DDA1 in lung cancer was previously unknown. This study aimed to investigate whether DDA1 contributes to tumorigenesis and progression of lung cancer. We found that the expression of DDA1 in normal lung cells and tissue was significantly lower than that in lung cancer and was associated with poor prognosis. DDA1 overexpression promoted proliferation of lung tumour cells and facilitated cell cycle progression in vitro and subcutaneous xenograft tumour progression in vivo. Mechanistically, this was associated with the regulation of S phase and cyclins including cyclin D1/D3/E1. These results indicate that DDA1 promotes lung cancer progression, potentially through promoting cyclins and cell cycle progression. Therefore, DDA1 may be a potential novel target for lung cancer treatment, and a biomarker for tumour prognosis.

SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin.

SARI, also called as BATF2, belongs to the BATF family and has been implicated in cancer cell growth inhibition. However, the role and mechanism of SARI in tumour angiogenesis are elusive. Here we demonstrate that SARI deficiency facilitates AOM/DSS-induced colonic tumorigenesis in mice. We show that SARI is a novel inhibitor of colon tumour growth and angiogenesis in mice. Antibody array and HUVEC-related assays indicate that VEGF has an essential role in SARI-controlled inhibition of angiogenesis. Furthermore, Co-IP/PAGE/mass spectrometry indicates that SARI directly targets ceruloplasmin (Cp), and induces protease degradation of Cp, thereby inhibiting the activity of the HIF-1α/VEGF axis. Tissue microarray results indicate that SARI expression inversely correlates with poor clinical outcomes in colon cancer patients. Collectively, our results indicate that SARI is a potential target for therapy by inhibiting angiogenesis through the reduction of VEGF expression and is a prognostic indicator for patients with colon cancer.

Increased density of macrophage migration inhibitory factor (MIF) in tuberculosis granuloma.

Granulomas, the pathologic hallmarks of tuberculosis, are composed of tightly numerous immune cells that respond to a variety of persistent stimuli during pathogen-host interaction. The granuloma is essential for host containment of mycobacterial infection, however, the mechanism of host and pathogen determinants to recruit immune cells at the site of inflammation and the formation of granulomas remains elusive until now. Macrophage migration inhibitory factor (MIF), a cytokine produced by many cell types, modulates cellular and humoral immune responses and promote lymphocytes migration to the site of infection. In this study, we evaluate the expression of MIF in tuberculous granulomas by three different models of diseases: mouse, human tissues and zebrafish. The overall results demonstrated that the expression of MIF positive signals markedly increased in the tissues which have been infected with mycobacterium, whereas a few presence of MIF in the PBS-treated animals (means the control group). In the mycobacterial-infected animals, the MIF positives distributed extensively within the granuloma especially in the multinucleated giant cells. Thus, three independent lines of evidence support the hypothesis that MIF may be an important player in aggregate immune cells to the granuloma microenvironments in these animal models of tuberculosis.