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treatment part 1

Submitted by ziweiwang on Thu, 12/12/2019 - 21:23

Despite the imperfect animal model and lack of specific treatment for RP, there have been certain methods that have been tested for retinitis pigmentosa, including gene therapy, small molecules that stabilize the protein and microchips that take place of retinas. However, microchips that are currently in development would only be viable for those who have lost their sight completely. Those who still retain their eyesight does not have many treatment options. One treatment option for those people who still retain some eyesight is gene therapy. Because the eye is immunologically isolated from the rest of the body and easily accessible, some complications from gene therapy is not a factor in the eye. In the research paper, Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery, the paper aimed at validating a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant while preserving the WT allele in vitro and translate the approach in vivo by delivering the CRISPR/Cas9 component in RHO mutant retina.  In the first experiment that was done, the researchers created various Cas 9 containing vectors to test with one disrupted the P23H mutation most efficiently. The cleavage efficiency was measured using T7El assay, TIDE, and Sanger sequencing. The result indicated that sgRNA-mMUT discriminated with the highest specificity of the mutant RHO sequence, and no off-target indel was detected. The result indicates that sgRNA-mMUT is the best candidate and should be used in further studies regarding the gene therapy of the retina. In the second experiment, to test whether the treatment would work in vivo, the vectors were inserted into the retina of the mice by in vivo electroporation and the product of the genes was assessed for the behavior of the RHO protein and mutation rate in vivo. The final result of the experiment is that Cas9-VQR/sgRNA-mMUT system enabled an efficient and selective targeting of the P23H mutant capable of a high degree of inactivation in the RHO allele in vivo.