Knockdown of wild-type mouse rhodopsin using an AAV vectoredribozyme as part of an RNA replacement approach

[摘要] Purpose: To develop a hammerhead ribozymes (Rz) that might beexploited in a "digest and replace" gene therapy strategy for autosomaldominant retinitis pigmentosa (ADRP) caused by mutations in the gene forrhodopsin (RHO).Methods: A ribozyme (Rz397) was designed to hybridize with anaccessible region in rhodopsin mRNA. It was tested in vitro to determinethe kinetics of cleavage of a target oligonucleotide. Followingtransfection of cultured cells, reduction of rhodopsin mRNA in responseto Rz397 was measured RT-PCR. The gene for the ribozyme (Rz397) wasinserted in an adeno-associated virus (AAV2) vector and packaged in AAV2capsids. The virus was injected subretinally in the eyes of C57BL/6J(RHO+/+) and rhodopsin knockout hemizygous (RHO+/-) mice atpostnatal days 6 (P6) and 30 (P30). Mice were analyzed by full-fieldelectroretinography (ERG). The reduction of opsin protein was measuredby western blot analysis and visualized by immunocytochemistry.Reduction of rhodopsin mRNA was assessed using in situ hybridization.Morphometric microscopy of fluorescent antibody-antigen complexes andautoradiography of retinas were used to quantify levels of rhodopsinprotein and mRNA, respectively.Results: Transient co-transfection of HEK 293 cells with a wild-typerhodopsin cDNA and Rz397 resulted in an approximately 60% reduction ofRHO mRNA one day after transfection. RHO+/--mice injected withAAV2-Rz397 at P6 showed a 50% reduction in b-wave amplitudes in injectedeyes relative to saline injected contralateral eyes. However, injectionof RHO+/--animals at one month and of RHO+/+-animals ateither age had no impact on ERG. Nevertheless, we detected an 80%reduction of opsin protein in ribozyme-injected eyes of hemizygous mice(by western blot) and a 50% reduction in opsin content in RHO+/+mice (by morphometry). These reductions were confirmed by in situhybridization.Conclusions: AAV2-Rz397 led to significant (greater than or equal to50%) reduction of rhodopsin mRNA and protein in mice. It affected ERGamplitudes only when injected in hemizygous RHO knockout pups. This RNAinhibitor may prove useful in treating animal models of ADRP as part ofan RNA replacement approach.

[学科分类] 生物化学/生物物理