Inherited retinal degenerations (IRD) are a group of diseases with high genetic heterogeneity and differences in inheritance patterns, age of onset and severity of visual dysfunction. It is one of the leading causes of blindness. In recent years, gene therapy becomes a popular research area in the treatment of genetic diseases due to the rapid development of gene diagnosis technology. Several clinical trials worldwide have proved the safety and effectiveness of gene therapies in IRD. Clinical application of adeno-associated virus -mediated gene therapies for Leber congenital amaurosis and choroideremia clinical trials indicate that patients' retinal functions were improved at different levels after treatment. There are a number of other IRD clinical trials ongoing currently, which bring new possibilities to treat IRD. This article reviews the pathogenesis of IRD, gene vectors and clinical trials in IRD.
Objective To observe the effects of immunologic cytokines or anti-angiogenesis gene transfer mediated by electroporation for choroidal melanoma cells.Methods The human embryo kidney cells and malignant choroidal melanoma cells were transfected with plasmids pNGVL-mIL2, pNGVL-mIL12, pCI-sFLK-1, pCR3.1-antiVEGF121,pCI-ExTek. Then the expression of mIL2, mIL12, sFLK-1, VEGF and ExTek were detected by enzymelinked immunosorbentassay (ELISA) and Western blot. Nude mice models of malignant choroidal melanoma were established and they were divided into four groups randomly. Each group was treated with 30 mu;l of 0.9% NaCl, 30 mu;g pNGVL, 30 mu;g antiVEGF121+sFLK-1+ExTek and 30 mu;g mIL2+mIL12 respectively by electroporation. Seven，14, 21, 28, 35 and 42 days after treatment, the tumor volumes were measured to calculate the tumor inhibition rate. Results ELISA and Western blot showed that mIL2，mIL12，sFLK-1 and ExTek were expressed after electroporation，VEGF expression was decreased remarkably. After treatment，the tumors of mIL2+mIL12 group were greatly inhibited with a tumor inhibition rate of 97.33%，while the tumors of antiVEGF121+sFLK-1+ExTek and pNGVL group were partially inhibited with tumor inhibition rates of 53.33% and 36.33% respectively.Conclusions Immunologic cytokines transfer mediated by electroporation can inhibit the growth of melanoma，but anti-angiogenesis only have a mild effects.
Vascular endothelial growth factor (VEGF) is a multifunctional factor that promotes blood vessel formation and increases vascular permeability. Its abnormal elevation plays a key role in common retinal diseases such as wet age-related macular degeneration and diabetic macular edema. Anti-VEGF therapy can inhibit angiogenesis, reduce vascular leakage and edema, thereby delaying disease progression and stabilizing or improving vision. Currently, the clinical application of anti-VEGF drugs has achieved satisfactory therapeutic effects, but there are also issues such as high injection frequency, heavy economy burden, potential systemic side effects, and non-responsiveness. To address these issues, current research and development mainly aim on biosimilars, multi-target drugs, drug delivery systems, oral anti-VEGF drugs, and gene therapy. Some drugs have shown great potential and are expected to turn over a new leaf for anti-VEGF treatment in ophthalmology.
Objective To introduce the studies on gene therapy for peripheral arterial disease(PAD) using plasmid DNA encoding human hepatocyte growth factor(HGF) gene. Methods Recent articles including preclinical and clinical studies were reviewed. Results Intramuscular injection of human HGF plasmid DNA into rat, rabbit, dog and diabetic hindlimb ischemic models, resulted in a significant increase in capillary density, blood flow and blood pressure. but no influence on tumor growth in mice. A clinical trial wasperformed in ischemic limbs of 6 critical limb ischemic patients, the result showed that no side effect caused by gene transfer was detected in all 6 patients.The pain scale and long diameter of ischemic ulcers were reduced. Conclusion Intramuscular injection of naked HGF plasmid DNA could be a safe and potential treatment for PAD.
OBJECTIVE: To study the effect of overexpression of truncated type II TGF-beta receptor on transforming growth factor-beta 1(TGF-beta 1) autoproduction in normal dermal fibroblasts. METHODS: In vitro cultured dermal fibroblasts were treated with recombinant human TGF-beta 1(rhTGF-beta 1) (5 ng/ml) or recombinant adenovirus containing truncated type II TGF-beta receptor gene (50 pfu/cell). Their effects on regulating gene expression of TGF-beta 1 were observed with Northern blotting. RESULTS: rhTGF-beta 1 up-regulated the gene expression of TGF-beta 1 and type I procollagen. Overexpression of truncated receptor II down-regulated the gene expression of TGF-beta 1. CONCLUSION: Overexpression of the truncated TGF-beta receptor II decreases TGF-beta 1 autoproduction via blocking TGF-beta receptor signal. The results may provided a new strategy for scar gene therapy.
ObjectiveTo observe the safety of 2-port non-vitrectomized subretinal injection (SRI) for the treatment of Bietti crystalline dystrophy (BCD). MethodsA exploratory clinical study. From February to May 2023, 6 BCD patients with 6 eyes who were confirmed by examination in Xiamen Eye Center of Xiamen University and were treated with SRI adeno-associated virus vector transgenic drugs were included in the study. Among them, 2 males had 2 eyes and 4 females had 4 eyes. Age were 34-60 years old. The study eye underwent adeno associated virus gene therapy via 2-port non-vitrectomized SRI. Two scleral ports were created using 25G vitrectomy trocar to place the light pipe and injection cannula. Anterior chamber paracentesis was performed to lower intraocular pressure. Under the silicone oil infusion mode of the vitrectomy machine, a 38G injection cannula penetrated the retina to reach the subretinal space. The injection speed was controlled by the foot pedal of the vitrectomy machine, and the drug was slowly injected into the subretinal space to create a subretinal bleb. if intra-ocular pressure assessed by finger palpation was high at the end of injection, drainage of the aqueous humor can be made by compressing the cornea incision until the intraocular pressure was normal. Patients were followed for 9-12 months and be examined using the same equipment and methods as before. ResultsRetinal pigment epithelium and choroidal atrophy were observed in all 6 eyes of 6 patients were graded as stage Ⅲ by the fundus examination revealing atrophy of retinal pigmented epithelium and choroid, with or without yellow-white crystals and/or complex lipid. The range were operation time 9-14 minutes. No vitreous prolapse, retinal hemorrhage, or retinal tear was observed during surgery. After 24 hours, optical coherence tomogrophy examination showed absorption of subretinal fluid and retinal reattachment. None of the six patients showed corneal keratic precipitates, anterior chamber cells, vitreous cells, inflammation, high intraocular pressure, or retinal tear within the 9-month follow-up. ConclusionSubretinal injection without vitrectomy using two ports is a safe and feasible alternative for adult gene therapy, and it shortens the surgical time.
Objective To use an improved technique to construct the recombinant adeno-associated virus 2 (rAAV2) mediated gene which can transfer human tissue inhibitor of metalloproteinase-1 (TIMP1). Methods Human TIMP1 gene was amplified from pDNR-LIB plasmid by PCR and cloned into the rAAV2 vector pSNAV to recombinant pSNAV-TIMP1, then was transferred into BHK-21 cells by means of lipofectamine. Using G418 selection, a mixed cell named BHK-21/rAAV2-TIMP1 was isolated, which was capable to express TIMP1. The cell was subsequently infected with recombinant herpes simplex virus 1 (rHSV1-rc/△UL2) that was able to package the rAAV2-TIMP1. After purification, rAAV2-TIMP1 was obtained. Results The rAAV2 carrying human TIMP1 gene was constructed successfully. The viral titer of the rAAV2-TIMP1 was 1×1012 v.g./ml. Conclusion rAAV2-TIMP1 was constructed successfully, which would provide experimental basis for carrying the TIMP1 into hepatocellular carcinoma effectively and inhibiting the invasiveness and migratory capacity of hepatocellular carcinoma in vitro and in vivo models.
ObjectiveTo study the effect of Triton X-100 promoting liposome-mediated bone morphogenetic protein 2 (BMP-2) gene transfection of rat bone marrow mesenchymal stem cells (BMSCs).
MethodsBMSCs were separated and cultured from the femur and tibia of healthy Wistar rats (8-week-old, male). The 3rd passage BMSCs identified by detecting the surface antigen were used to transfect. The optimum concentration of Triton X-100 for liposome mediated gene transfection was determined with ELISA meter by the way of MTT. In optimum concentration of Triton X-100, liposome mediated BMP-2 gene was transfected to BMSCs. The experiment was divided into 3 groups according to types of trasfection agents:BMSCs were transfected with Triton X-100+liposome+BMP-2 (experimental group), with liposome+ BMP-2 (conventional transfection group), and untransfected BMSCs served as blank control group. After 48 hours of transfecting, the green fluorescent protein (GFP) in cells was detected through inverted fluorescence microscope. After 72 hours of transfection, real-time fluorescence quantitative PCR was applied to measure the mRNA expression of BMP-2.
Results0.01% Triton X-100 was determined to be the optimum concentration for not only making the BMSCs maintain vitality, but also achieving a certain effect on BMSCs. After trasfecting for 48 hours, GFP was observed through inverted fluorescence microscope in the experimental group and conventional transfection group, but was not observed in the blank control group. After trasfecting for 72 hours, the relative BMP-2 mRNA expression level was 5.94±0.12 in the experimental group, and was 4.99±0.08 in the conventional transfection group, showing significant difference (t=360.28, P=0.02). The transfection efficiency was increased by 19% in the experimental group.
Conclusion0.010%Triton X-100 can promote the liposome mediated BMP-2 gene transfection of rat BMSCs, and can improve the transfection efficiency.
OBJECTIVE This paper aims to explore the new method of continuous delivery of epidermal growth factor to wounds by transfected fibroblasts to promote wound repair. METHODS It was constructed a novel chimeric expression plasmid in which the biologically active portion of the human epidermal growth factor (EGF) gene was fused in-frame to the human granulocyte colony-stimulating factor signal sequence. RESULTS Clonally selected human fibroblasts transfected with this construct could secrete biologically active EGF. After the transplantation of irradiated gene-transfected fibroblasts suspended in fibrin glue to murine full-thickness wounds, EGF could be demonstrated for at least seven days in the wounds, slowly decreasing from initially 470 ng/L to 140 ng/L in 7 days. No EGF was found in the wound at 14 days. CONCLUSION A single application of irradiated EGF gene transfected fibroblasts to wounds can continuously deliver the transgene in vivo and can be used to administer drugs to the wound bed during the crucial first seven days of wound-healing.
Retinoblastoma (RB) is the most common intraocular malignant tumor in children. With advanced clinical technologies there are more and more methods available to treat retinoblastoma, and make it is possible to delivery individualized protocol combined traditional treatments with modern regimen to patients now. In order to improve the survival rate and the life quality of RB patients in China, it is very important to make a suitable system of standardized therapy based on results from developed countries and health policies of our own country.
