Biologists use a variety of approaches to disable a gene in order to gain an understanding about its function. Methods to disable genes include gene-editing techniques (TALEN or CRISPR), knockout animal models and RNA interference (RNAi). Since its discovery in the late 1990s, RNAi has been proven to robustly and selectively suppress expression of target genes, which is why it has rapidly become a major tool for in vitro analysis of protein function.
In addition to being a research tool, RNAi has been explored as a therapy for a host of diseases that are caused by gene overactivity. Biopharmaceutical companies working on treatments for cancer, AIDS, neurodegenerative diseases and liver diseases have been racing to develop successful RNAi-based drugs. In 2018, the FDA approved Alnylam’s Onpattro® (patisiran), a drug indicated for hereditary transthyretin amyloidosis that works by targeting the TTR gene, making it the first drug that uses an RNAi strategy to be FDA-approved.
How Does RNAi Work?
RNAi is a naturally occurring process of gene silencing in the cells. During RNAi, long double-stranded RNA (dsRNA) is cut into small fragments, about 21 nucleotides long, by an enzyme called "Dicer." These small RNA fragments, referred to as small interfering RNAs (siRNA), can direct enzymes (RNA-induced silencing complex, or RISC) to degrade sequence-matched mRNA molecules and thus decrease their activity by preventing translation via post-transcriptional gene silencing. Synthetic siRNA can be introduced into cells exogenously – via transfection, electroporation or viral-mediated approaches – and trigger the subsequent silencing processes. Technology for making synthetic siRNA has evolved rapidly, and algorithms continue to improve silencing specificity and potency while also reducing off-target effects and toxicity. Functionally-validated synthetic siRNAs that match most genes of interest are now commercially available.
Primary Hepatocytes and the HEPATOPAC System
Primary hepatocytes are the gold standard for in vitro liver-related research because they more closely resemble the in vivo liver than commonly used hepatoma cell lines1. The functionality of primary hepatocytes can allow for more accurate elucidation of hepatocellular processes, drug metabolism, toxicity and efficacy, all of which are crucial for developing novel drugs. BioIVT’s HEPATOPAC® system, a micro-patterned co-culture of primary hepatocytes and non-parenchymal stromal cells, has proven to maintain in vivo-like liver function for up to four weeks (Figure 1 and HEPATOPAC kits ). Because of its longevity and physiologically-relevant function, the HEPATOPAC system has demonstrated unparalleled utility for in vitro evaluation of hepatic toxicity, metabolism and disease models.2-4.
siRNA Delivery and Gene Silencing in HEPATOPAC Cultures
Using siRNA to knockdown hepatic gene expression in in vitro liver models can be highly useful in various research areas of drug development and discovery. While siRNA is readily available, its utility is limited to cell types that are amenable to transfection of synthetic oligonucleotides. In general, it can be challenging to transfect primary hepatocytes. An additional complexity comes when working with HEPATOPAC co-cultures due to stromal cells surrounding the hepatocyte islands. The stromal cells, which are fibroblasts, are one of the most easily transfected cell types for DNA/RNA transfection. Attempting to deliver siRNA in HEPATOPAC co-cultures using traditional lipid-based transfection reagents may result in most siRNA introduced into fibroblasts and none or little into the hepatocytes.
Hepatocyte-targeted siRNA Delivery in HEPATOPAC cultures
At BioIVT, we are particularly interested in transfection agents that can specifically target hepatocytes and deliver siRNA
(as opposed to DNA). We compared a panel of general and hepatocyte-targeting transfection reagents and found that Promofectin-Hepatocyte (PromoCell) could deliver siRNA specifically and efficiently5 into the hepatocytes in the HEPATOPAC cultures (Figure 2). Very little siRNA went into the stromal cells, even though these cells comprise up to 90% of the culture.
After we determined that the siRNA had been transfected into the hepatocytes, we assessed whether the siRNA was silencing the target gene. We transfected commercially available siRNA (ON-TARGETplus SMARTpool, Dharmacon) targeting CYP3A4 using Promofectin-Hepatocyte. CYP3A4 activity was measured at 24, 48, 72 and 96 hours after siRNA transfection, and a time-dependent knockdown was observed from wells transfected with CYP3A4-targeting siRNA, but not from those transfected with control siRNA. At 96 hours post transfection, CYP3A4 activity was reduced to 50% by siRNA, compared to control (Figure 3). IL-6 treatment, which reduces CYP3A4 expression, was used as a positive control for down-regulation of CYP3A4 activity.
These results showed that PromoFectin-Hepatocyte reagent specifically introduced siRNA into the hepatocytes in HEPATOPAC co-cultures, and the delivered siRNA caused functional knockdown of the target gene CYP3A4. The duration of the gene silencing effect after transfection is currently being examined.
Ubiquitous siRNA Delivery in HEPATOPAC
In the evaluation of transfection reagents, we noticed that some transfection reagents did not effectively target specific cell types but did deliver siRNA to the “difficult-to-transfect” hepatocytes, in addition to fibroblasts. For example, we used Lipofectamine RNAiMAX (ThermoFisher) and fluorescent control siRNA to transfect the cultures, and we detected the fluorescent signal in both hepatocytes and stromal cells (Figure 4). When CYP3A4-targeting siRNA was transfected in HEPATOPAC using RNAiMAX, a significant knockdown of CYP3A4 activity was detected at 24, 48, 72 and 96 hours after transfection (Figure 3). In contrast, other transfection reagents that don’t target certain cell types (e,g, METAFECTENE® SI⁺ from Biontex) only introduced siRNA into fibroblasts, but not hepatocytes (data not shown).
This suggests that RNAiMAX provides an alternative option when it is preferable to knockdown a target gene simultaneously in the hepatocytes and in stromal cells in HEPATOPAC co-cultures.
HEPATOPAC System is Amenable to Gene Modulation
Having the capability to manipulate gene expression in HEPATOPAC cultures further extends the utility of this system. It would be immensely beneficial to achieve greater knockdown (> 50%) of target genes in HEPATOPAC co-cultures either by making refinements to the siRNA delivery procedures described here or by identifying other reagents or methodology for optimal siRNA transfection and gene silencing. BioIVT’s scientists are working to optimize this application to enable more relevant in vitro studies that can take advantage of the HEPATOPAC model.
- Sison-Young RL, Park BK, et al. Comparative Proteomic Characterization of 4 Human Liver-Derived Single Cell Culture Models Reveals Significant Variation in the Capacity for Drug Disposition, Bioactivation, and Detoxication. Toxicol Sci. (2015) 147:412-24.
- Khetani SR, Will Y, et al. Use of micropatterned cocultures to detect compounds that cause drug-induced liver injury in humans. Toxicol Sci. (2013) 132:107-17.
- Kratochwil NA, Fowler S, et al. Simultaneous Assessment of Clearance, Metabolism, Induction, and Drug-Drug Interaction Potential Using a Long-Term In Vitro Liver Model for a Novel Hepatitis B Virus Inhibitor. J Pharmacol Exp Ther. (2018) 365:237-248.
- Ware BR, Khetani S, et al. Exploring Chronic Drug Effects on Microengineered Human Liver Cultures Using Global Gene Expression Profiling. Toxicol Sci. (2017) 157:387-398.
- PromoCell claims that PromoFectin-Hepatocyte reagent has been designed for transfection of cells expressing galactose-specific membrane lectins and has been very successfully used to transfect primary hepatocytes as well as hepatocyte cell lines (e.g. BNL CL.2) and human hepatocarcinoma cells (e.g. HepG2) with up to 50% transfection efficiency and excellent cell viability.