糖苷酶 z-bo 2020-11-19 503

　　Introduction: Hereditary retinal degeneration is the main cause of blindness. In most forms, genetic mutations affect cells in the outer retina, namely photoreceptors and retinal pigment epithelium (RPE)-making these cells the prime target of emerging gene therapy. Prove the safety and effectiveness of transgene delivery to RPE via subretinal injection via adeno-associated virus (AAV) vector. In advanced retinal degeneration, the retina will become thin and fragile, making the subretinal delivery of AAV vectors challenging and prone to complications. Another method is intravitreal injection, the technique is not complicated, and the risk of complications is low. The treatment from the vitreous body to the transduction level of the retina presents a challenge and has become the focus of some recent preclinical gene therapy research. AAV-based vectors are currently being developed, which can transduce the outer retina in an animal model after intravitreal injection through reasonable mutagenesis or directed evolution in vivo. Reasonable mutagenesis treatment of viral capsids (surface exposed tyrosine, threonine and lysine residues) to reduce the intracellular ubiquitination and proteosome degradation of the carrier, resulting in increased retinal transduction. Directed evolution selects AAV variants from a combinatorial library, which have ideal cell tropism in vivo. Therefore, through multiple evolutionary cycles, it enriches AAV variants with specific cell orientations or can reach the outer retina from the vitreous body through altered receptor binding properties. These new AAV variants have been shown to produce more effective functional rescue of the disease phenotype in animal models of retinal degeneration.

　　"Another strategy to increase retinal transduction after intravitreal delivery is to solve the physical barrier to retinal barrier penetration. The naturally occurring AAV serotypes produce limited intraretinal transduction and do not efficiently transduce the outer retina through intravitreal transmission because of the vitreous body, inner limiting membrane (ILM), retinal extracellular matrix (ECM) and cell surface proteoglycans Form a substantial barrier to fix the binding site of AAV particles. Using glycosidases, enzymatic cleavage of these barriers increases the depth and efficiency of carrier penetration, resulting in more effective retinal transduction. Digestion of ILM with the non-specific protease Pronase E also enhanced retinal transduction, suggesting that ILM forms an important barrier to carrier penetration. We describe an optimized method in which mice are co-injected with glycosidase to deliver unchanged AAV2 in the vitreous to increase retinal transduction. We conducted a quantitative and qualitative analysis of the transduction efficiency of AAV2 with a variety of glycosidases (including chondroitin ABC lyase, hyaluronic acid lyase, heparinase III and their combinations), and found that heparinase III and hyaluronic acid The combination of lyases produced the greatest improvement in retinal penetration on the AAV2 vector, and the total transduction level was highest in the intact wild-type retina.

　　Method: Adult C57BL/6J (wild type) and C3H/HEJ (RD1) mice were used in this study.

　　Via AAV gene delivery: The AAV2 vector used in this study was obtained from the vector laboratory. A specific cell on the bipolar cell promoter (GRM627), fused with a 200 bp GRM6 gene enhancer sequence, which encodes the bipolar cell-specific metabotropic glutamate receptor, MGLUR6 and SV40 eukaryotic promoter The sub, called AAV2-GRM6GFP, is surrounded by the inverted terminal repeat (ITR) DOMAI. Viral vectors were injected into mice anesthetized with isoflurane at 8 weeks of age. Before the injection, dilate the pupils with tropikamide and phenylephrine. A customized ultra-fine needle was installed on a 5μL glass syringe and passed through the flat part at 45° into the vitreous cavity without retinal perforation. Under the operating microscope, look directly at the tip of the needle for injection, taking care to avoid lens contact and blood vessels. AAV2-CAG-GFP is injected at low or high dose. AAV2-GRM6-GFP is only injected at high doses. Low-dose vector injection 1μl, 2×1011 GC/ml, high-dose vector injection 3μL, 1×1013 GC/ml. Each eye injected with enzyme received 0.5 μl of PBS. 0.125 units of chondroitin ABC lyase was isolated from Proteus acidophilus, heparinase III composed of Flavobacterium heparin or Streptomyces hyaluronic acid lyase alone or in different combinations. The enzyme solution was prepared fresh on the day of injection by dissolving the enzyme in sterile PBS. The carrier and enzyme are mixed in a syringe immediately before injection and administered in a single combined injection.

　　Histology: After taking the cornea and lens under a light microscope, fix the eye with 4% paraformaldehyde (PFA) in PBS. The tissue was then washed in PBS and then incubated in PBS containing 30% sucrose at 4°C. The fixed eyes were rinsed in PBS, and the entire retina was carefully dissected under an optical microscope. Then use fluorescent mounting medium containing DAPI to flatten the retina to stain the nucleus. , The fixed eyes are stored frozen in the optimal cutting temperature medium and frozen at -80°C until further processing. Make each part contain the complete nasal temporal cross-section of the retina. Before analysis, the cut pieces were taken out of the freezer, allowed to air dry at room temperature for 1 hour, and the nuclei were stained with fluorescent mounting medium installed with DAPI.

　　Biological imaging: Use multiple targets to image under an upright fluorescence microscope, and use the CaleSnAPES ES camera to take images and process them with Meta VAVE software. Then use Image J software to analyze the image.

　　Quantitative analysis of vector transduction: For the quantification of GFP+ cells in retinal slices, one slice (6-8 segments) per eye in each treatment group (n=4) was used for analysis, and all slices were taken at 10×. Count GFP+ cells. The transduced GFP+ cells were identified based on the laminar flow position and morphology of the GFP+ cells. Count and record GFP + cells according to the retinal layer, including GCL, INL and ONL.

　　ERG: Evaluation of retinal function in wild-type mice at 1, 6, and 12 months after intravitreal injection. Mice adapt to dark overnight (>12 hours), and prepare ERG records under dark red light. A mixture of ketamine (75 mg/ml, 10%) and xylazine (13.6 mg/ml, 20%) was injected intraperitoneally to induce anesthesia. The pupils are dilated with local mydriasis, and then hydroxypropyl methylcellulose solution is dropped on the contact lens electrode. The electrode is connected to a personal computer through a signal conditioner, which differentially amplifies (×3000) and filters the signal, and a digitizer.

　　Results: Glycosidase increases the in vivo transduction efficiency of AAV2 in the vitreous: In order to describe the expression of reporter gene (GFP) mediated by low-dose AAV2 vector, we alone or in combination with glycosidase, count 2×108 genomes (GC)/eye The AAV2-CGA-GFP vector was injected into the vitreous of adult wild-type mice. As expected, with AAV2-CGA-GFP alone, there was weak gene expression, and this was restricted to the inner retina. On the contrary, when AAV2-CGA-GFP is injected with glycosidase chondroitin ABC lyase, hyaluronic acid lyase or heparinase III, the expression of GFP in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) Significant increase, confirming our previous findings. We tested various combinations of enzymes, including chondroitin ABC lyase + heparinase III, chondroitin ABC lyase + hyaluronic acid lyase, and heparinase III + hyaluronic acid lyase, and found that they were further enhanced GFP expression. The strongest transduction achieves the combination of heparinase III and hyaluronic acid lyase, which produces robust GFP expression throughout GCL and INL, and some areas also have moderate extraretinal transduction. Quantitative evaluation of the transduction efficiency of AAV2-CGA-GFP showed that adding glycosidase hyaluronic acid lyase or heparinase III per millimeter of retinal slices significantly increased the number of GFP + cell bodies. Among them, heparinase III produces more retinal transduction than chondroitin ABC lyase alone. When using a combination of glycosidases, they also showed a significant improvement in transduction efficiency compared to eyes that were not treated with enzymes. In particular, heparinase III and hyaluronic acid lyase resulted in the highest counts of GFP+ cells. Compared with the unenhanced AAV2-CaGGFP-mediated transduction, it is increased by 17 times. In GCL and INL cells, this increase is significant, but not in the outer nuclear layer (ONL) cell body.

　　Application of high-dose AAV2 vector combined with heparinase III and hyaluronic acid lyase in wild-type mice: In order to determine the degree of transduction that can be achieved with this technology, we will combine the most effective combination (heparinase III + transparent Acid lyase) and higher dose carrier application. Wild-type mice were injected with heparinase III and hyaluronic acid lyase, while adding a high dose of AAV2-CGA-GFP (3×1010 GC/eye). No green fluorescent signal was observed in the retina injected with PBS alone. Compared with the high-dose carrier injection alone, increasing the carrier dose in the presence of glycosidase resulted in extensive retinal transduction of cells in GCL and INL. Extra-retinal conduction (ONL) was observed at a low level. This sheet-like expression may be due to the non-uniform diffusion of the enzyme and carrier through the vitreous, leading to more effective retinal transduction near the injection site.

　　AAV2 vector combined with glycosidase leads to robust expression of reporter gene in RD1 retina: We studied the transduction of Adv2 in RD1 retina, a model of advanced retinal degeneration. We used heparinase III and hyaluronic acid lyase to inject AAV2-CGA-GFP vector (3×1010 GC/EVE) into the vitreous of adult RD1 mice. The retina was harvested 6 weeks after the injection, and strong reporter gene expression was observed in GCL, optic nerve cells and INL cells compared with AAV2-CGA-GFP alone. The expression in GCL is consistent, while the INL transgene expression, although pan-retinal, shows more different density and depth. We injected AAV2 with a bipolar-specific promoter combined with glycosidase to drive GFP expression and found that, as expected, the expression of cells in INL was restricted. The GRM6/SV40 enhancer promoter sequence has been shown in multiple studies and is only expressed in bipolar cells.

　　Conclusion: We have developed an AAV-mediated treatment that improves retinal transduction through intravitreal injection, which has excellent long-term safety in rodents. In this way, rod-shaped opsin was delivered to RGC and bipolar cells, successfully saving the late model of retinal degeneration. After intravitreal injection, it is possible to effectively deliver AAVs outside the retina, and it can be further improved by combining engineered carriers with these glycosidases.