Background

Oligonucleotide therapeutics represent a new class of drug that can target any genetically defined disorder, by silencing the expression of mutant proteins. Specifically, siRNAs are double stranded oligonucleotides that are loaded into the RNA induced silencing complex (RISC) and can silence mRNA before it is translated. However, unmodified siRNAs are unstable and cannot enter cells without the help of cationic lipid formulation, which can be toxic to primary cells such as neurons. In this protocol, we use self-delivering, hydrophobically modified siRNAs (hsiRNAs) for mRNA silencing. Recent progress in the chemistry of oligonucleotides has enabled the design of these stabilized hsiRNAs, which promote cellular internalization, efficient entry into RISC, and potent knockdown of target genes (Byrne et al., 2013; Alterman et al., 2015; Ly et al., 2017). These compounds contain 2’-O-methyl and 2’-fluoro modifications on all sugars and phosphorothioate backbone modifications; the oligonucleotides are often conjugated to a hydrophobic moiety, such as cholesterol, to support membrane binding and cellular internalization without toxicity. This new class of compounds offers researchers a straightforward method for silencing various genes in the context of biologically relevant, and hard to transfect, primary cortical neurons (Alterman et al., 2015).

Today, in the early stage of drug discovery, most high-throughput tests are performed in cell-based assays for lead oligonucleotide identification and validation. Cell-based assays improve and accelerate drug screening, providing more relevant in vivo biological information than biochemical assays and reducing the need for animal testing. High-throughput assays performed in primary neurons have emerged as a powerful tool to discover new therapies for the treatment of neurodegenerative disorders, such as Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS) or Alzheimer’s disease (AD) (Sharma et al., 2012). Cell based assays using primary neurons provide a more natural (relevant) environment for studying neurodegenerative disorders than clonal neuronal lines. Transfecting oligonucleotide-based therapeutics into primary neurons generally relies on approaches such as electroporation (Mertz et al., 2002; Gresch et al., 2004; Zhang et al., 2016), viral transduction (Naldini et al., 1996; Hughes et al., 2002; Janas et al., 2006) or lipid-mediated transfection (Ohki et al., 2001; Dalby et al., 2004; Zhang et al., 2016). However, these methods can be laborious, show low efficiency, and induce cellular toxicity. Thus, the self-delivering properties of hsiRNAs represent an effective method to identify new leads in vitro in complex cellular models, such as primary neurons. We have recently demonstrated that hsiRNAs efficiently bind neuronal-cell membranes within seconds after treatment, enter cells, and induce potent gene silencing, both in vitro, in primary neurons, and in vivo, in mouse brain, all without the use of transfection reagents (Alterman et al., 2015; Ly et al., 2017).

Our laboratory has established a rapid high-throughput platform to identify and validate hsiRNA leads in primary neurons in vitro in 96-well format. To quantify the amount of target mRNA upon hsiRNA treatment, we use the QuantiGene 2.0 branched DNA (bDNA) assay, a high-throughput, 96-well plate-based mRNA quantification assay. This technique is designed to directly quantify the target mRNA from sample lysate without the need to purify the RNA, thus minimizing sample manipulation (Kern et al., 1996; Coles et al., 2015). This assay enables accurate and precise detection of even low abundance mRNAs, minimizing experimental variability and error (Collins et al., 1997; Canales et al., 2006). The combination of both self-delivering hsiRNAs and high-throughput quantification of mRNA accelerates the identification of efficacious oligonucleotides in primary neurons.

Here, we describe the workflow of this high throughput assay for performing large-scale screening and dose response validation of hsiRNAs in primary cortical neurons. We detail our methods for primary cortical neuron preparation in 96-well plate format (Figure 2), neuronal treatment with hsiRNA, assay plate management, mRNA quantification, and data analysis. This platform can be used for the screening of oligonucleotide therapeutics in primary neurons for the potential treatment of neurodegenerative diseases.