Design of shRNA and miRNA for Delivery to the CNS

Abstract

Neurologic diseases tend to target various areas of the central nervous system (CNS) and can therefore result in paralysis, dementia, and death. Neurodegenerative diseases distinguish themselves from other diseases by affecting nerve cells, which unlike many other cells in our body cannot regenerate when severely injured. The discovery of RNA interference (RNAi) has enabled scientist to design new therapeutic approaches based on specific gene silencing rather than the canonical gene therapy through gene augmentation. Two types of molecules can be used for viral vector-mediated gene silencing: short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs) that have the ability to enter the RNAi pathway. Although both shRNAs and miRNAs can be used to silence genes, they enter the RNAi pathway at different points. Unlike shRNAs, miRNAs require an additional cleavage step inside the nucleus before being exported to the cytoplasm. These molecules can then be incorporated into the RNA-induced silencing complex (RISC) which utilizes sequence complementarity to recognize target mRNAs and activate either translational repression, in the case of partial complementarity, or induce mRNA cleavage in the case of complete complementarity. Elevated amounts of shRNAs, which are commonly driven by strong polymerase III promoters, can cause saturation of the endogenous RNAi machinery due to competition between endogenous and artificial molecules. Switching to a DNA polymerase II promoter is an alternative to reduce shRNA production, thereby reducing toxicity. Even though the molecules are designed to target specific mRNAs there may be off-target effects due to nonspecific binding that must be accounted for during the design process. In this chapter we discuss the design and in vitro screening of shRNAs and artificial miRNAs.

Safe and Efficient Silencing with a Pol II, but Not a Pol lII, Promoter Expressing an Artificial miRNA Targeting Human Huntingtin

“…These sequences were selected on the basis of known targeting rules 16 . We cloned two copies of the artificial miRNA in tandem into a backbone on the basis of the endogenous miRNA 155 and placed the entire artificial miRNA into the 3′-UTR of EGFP (Figure 1B, top), which was expressed under the control of a chicken beta-actin promoter.…”

“…Artificial miRNAs were designed and cloned according to the protocol detailed by Toro Cabrera and Mueller 16 . AAVs were packaged by the University of Massachusetts vector core, according to published protocols 30 …”

Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

“…Our results are inconsistent with the conclusion in a previous study, where virus shRNA delivery was used as gene knockdown approach (Hwang et al, 2014 ). However, because different experimental approaches are subject to different limitations, such as potential alternation in other gene expression in gene knockout mice and potential off-target effect in shRNA gene knockdown method (Toro Cabrera and Mueller, 2016 ), caution should be exercised in viewing the discrepancy resulting from different experimental approaches, and a more definite experimental approach would be highly valuable to resolve this inconsistency in the future.…”

Silencing Alpha Synuclein in Mature Nigral Neurons Results in Rapid Neuroinflammation and Subsequent Toxicity

“…α-syn (GAAGGACCAGATGGGCAAG), myocardin (TGCAACTGCAGAAGCAGAA), or scrambled (CACAAGATGAAGAGCACCA) siRNAs were designed using standard algorithms (Toro Cabrera and Mueller, 2016 ), and sequences were confirmed against available genomic data in order to ensure α-syn specificity. shRNAs were expressed by the H1 promoter (Benskey et al, 2014 ).…”