Study On The Hypotensive Effect Of Viral Vector-mediated C3 Gene Expression In Rats And Mice

Objective:To study the IOP lowering effect of viral vector mediated exoenzyme C3transferase(C3)gene therapy on intraocular pressure in rats and mice,and to provide experimental basis for its safety and effectiveness.Methods:Thirty male C57BL/6 mice were purchased.Using self-complementary adeno-associated viral vector type 2(scAAV2)as a vector,C3 and enhanced green fluorescent protein can be expressed,respectively.The viral vectors expressing the EGFP gene(scAAV2-C3 and scAAV2-EGFP)were injected into the anterior chamber of 30 C57BL/6 mice with normal IOP,of which 15 mice were injected with scAAV2-C3(single eye dose:5 x 10¹¹ vg,1?l),and 15 mice with normal IOP were injected with scAAV2-EGFP(single eye dose:1 x 10¹² vg,0.5?l)as a blank control.IOP on day 7 was recorded and enhanced green fluorescent protein expression in the atrial angle was observed using a Micron IV retinal imager.Twelve male SD rats were purchased,using lentivirus(LV)as a vector and expressing C3 and enhanced green fluorescent protein,respectively.Viruses of the EGFP gene(LV-C3-EGFP and LV-EGFP)were injected into the anterior chamber of 16 rats with normal IOP,LV-C3-EGFP was injected into the left eye(single eye dose:8 x 10⁸ TU,5?l),LV-EGFP was injected into the right eye(single eye dose:5 x 10⁸ TU,8?l),and the left and right eyes were compared.IOP was recorded on day 0,day 7,day 21,and day 35,respectively.During the experiment,slit-lamp biomicroscopy was used to observe whether there was inflammation in the anterior chamber of rats.Micron IV retinal imager was used to observe the expression of enhanced green fluorescent protein in the atrial angle.Results:1.Mice injected with scAAV2-C3 and scAAV2-EGFP in the anterior chamber and rats injected with LV-C3-EGFP and LV-EGFP in the anterior chamber did not cause significant anterior chamber inflammatory reactions throughout the experiment.2.LV and scAAV2 can be transfected into the anterior chamber angle of the eye with the target gene for stable expression for a period of time,and the fluorescence of the anterior chamber angle of the rat with LV vector appeared on the 7th day,the brightest on the21st day,and lasted at least until the 35th day.By measuring IOP of experimental mice,it was found that sc AAV2-C3 group was significantly lower than scAAV2-EGFP group and blank control group.4.The IOP measurement of experimental rats showed that the LV-C3-EGFP group was significantly lower than the LV-EGFP group.Conclusion:The above results showed that anterior chamber injection of scAAV2-EGFP and scAAV2-C3,anterior chamber injection of LV-C3-EGFP and LV-EGFP did not trigger significant anterior chamber and intraocular inflammation,and had little effect on their growth activities.ScAAV2 and LV-mediated gene therapy of exoenzyme C3 transferase can reduce IOP in rats and mice,and viral vector-mediated exoenzyme C3 transferase and enhanced green fluorescent protein can be stably expressed in the anterior segment for a certain period of time.Using viral vector to transfect C3 gene into trabecular meshwork tissue to reduce IOP after expression,as a gene therapy method for glaucoma has certain potential.