RNAi Mediated Silencing of LRRK2G2019S in Parkinson's Disease.

摘要

This proposal will utilize RNA interference technology to diminish LRRK2 kinase activity in both cell culture and animal models of G2019S-mediated neurotoxicity to establish a novel therapy for PD. To achieve this objective we proposed the following Specific Aims: 1. Inhibition of wild-type LRRK2 and G2019S expression using small interfering RNAs (siRNA), 2. In vitro inhibition of wild-type LRRK2 and G2019S expression using shRNA technology and 3. In vivo inhibition of wild-type LRRK2 and G2019S expression using shRNA technology. During this award period we completed Technical Objective 1: Inhibition of wild- type LRRK2 and G2019S expression using small interfering RNAs in a cell line (MN9D) with LRRK2 or G2019S overexpression to attenuate the expression of wild- type and mutant LRRK2, cell death and neurite extension. We have completed this TO. In addition we made progress on Technical Objective 2: In vitro inhibition of wild-type LRRK2 and G2019S expression using shRNA technology. We have designed and expressed shRNAs that target LRRK2 or G2019S, cloned them into viral vector expression vectors and initiated the efficacy testing in vitro. Lastly, we initiated work on Technical Objective 3: In vivo inhibition of wild- type LRRK2 and G2019S expression using shRNA technology. We have constructed and expressed lentivirus with expression of WT and G2019S LRRK2 and rAAV expressing shRNAs.