( David Brown (Se...)RNAi as a Tool for Mammalian Gene Analysis: Applications of siRNAs

David Brown (Senior Scientist, Ambion, Inc.)

The realization that RNA interference (RNAi) can be used as a general tool for mammalian gene function studies occurred with the seminal Elbashir et al. (1) publication describing the use of small interfering RNAs (siRNAs) in mammalian cells in 2001. In less than two years, siRNAs have become the cornerstone of many research programs. This rapid uptake has resulted primarily from the ease of use of siRNAs and the strong need for a method to reduce the expression of individual genes in mammalian cells in order to establish a link between gene identity and gene function. Provided below are some of the more prominent applications for RNAi in mammalian systems and examples of these applications from the literature.

Testing Hypotheses of Gene Function
Array analysis and other methods for identifying differentially expressed genes have created an enormous database of genes and associated phenotypes. In many cases, scientists make predictions about gene function based on expression patterns in different samples. Other predictions of mammalian gene function are developed using homology searches with genes whose functions are known in model organisms like Drosophila, C. elegans, and S. cerevisiae. In many cases, testing the accuracy of these predictions can be accomplished using siRNAs.

* Al-Khalili et al (2) treated myotubes with serum and showed that increased glucose uptake correlated with increased cell-surface content of glucose transporter (GLUT1). To confirm that glucose transport depends on GLUT1 expression, cells were treated with GLUT1 siRNA and were shown to have reduced levels of serum-stimulated glucose transport.

* In another report, Chen and Barritt (3) used siRNAs to study the transient receptor potential canonical 1 (TRPC1) gene. The TRPC1 gene was thought to encode a non-selective cation channel activated by depletion of cellular storage and/or an intracellular messenger. When liver cells were treated with the TRPC1 siRNA, they exhibited increased cell volume and decreased inflow of Ca2⁺, Mn²⁺, and ATP in hypotonic solutions supporting the hypothesis.

Target Validation
Much of the excitement surrounding siRNAs has been due to their almost seamless incorporation into the development process for therapeutics. In its simplest form, drug development follows the path of target identification -> target validation -> therapeutic compound development -> compound testing in model systems -> clinical trials. Because they are easy to use and highly specific, siRNAs provide the ultimate tool for validation studies. Reducing the expression of a potential therapeutic target and determining if the desired phenotype results provides confidence that an inhibitor of the same target gene should have therapeutic value.

* Filleur et al (4) showed that the antiangiogenic molecule thrombospondin-1 (TSP-1) could reduce vascularization and delay tumor onset. Over time, tumor cells producing active TSP1 began to form exponentially growing tumors. These tumors were composed of cells secreting unusually high amounts of the angiogenic stimulator, vascular endothelial growth factor (VEGF), which were sufficient to overcome the inhibitory TSP1. Treating tumor cells with a combination of TSP1 and a VEGF-specific siRNA caused a striking reduction in cell proliferation. This result suggested that using a combination of TSP1 and an anti-VEGF compound would slow or eliminate tumor growth.

* Based on the observation that fatty acid synthase (FASE) is over-expressed in human epithelial cells, De Schrijver et al (5) considered the gene to be an interesting target for antineoplastic therapy. The researchers used siRNAs to reduce the expression of FASE in lymph node carcinoma of the prostate (LNCaP) cells. The FASE siRNAs caused several phenotypes in the LNCaP cells, including induction of apoptosis. Interestingly, the FASE siRNAs had no effect on the growth rate or viability of nonmalignant cultured skin fibroblasts. These data point out the potential of cancer drugs that selectively inhibit FASE.

* Cyclin E is overexpressed in a number of tumor cells. To determine the potential value of the gene as a drug target, Li et al (6) used siRNAs to reduce cyclin E expression in hepatocellular carcinoma (HCC) cells. As expected, the cyclin E siRNA promoted apoptosis of HCC cells and blocked cell proliferation. In addition, the cyclin E siRNA inhibited HCC tumor growth in nude mice demonstrating the potential for creating drugs targeting cyclin E.

Pathway Analysis
Another key application for siRNAs is pathway analysis. Reducing the expression of a single gene has implications on the expression and activities of genes that are in the same pathway(s). For instance, reducing the levels of a transcription factor such as p53 will reduce the expression of any gene that relies on the p53 transcription factor for activity. Furthermore, the expression of genes that are regulated by gene products that are controlled by p53 should likewise be impacted. Treating cells with an siRNA targeting a given gene and then monitoring the expression of other genes using a microarray will make it possible to identify genes that are associated with the target gene. Furthermore, a specific pathway can be dissected by treating cells sequentially with siRNAs targeting the various genes in the pathway and assaying which genes are affected. This will make it possible to assign a position in the pathway for each gene.

* Ramos-Nino et al (7) exposed RPM cells to crocidolite asbestos and monitored gene expression using arrays. Genes were categorized based on their response. The genes that were highly and quickly up-regulated included the proto-oncogene, fra-1. siRNA-induced reduction in fra-1 expression caused an increase in the expression of both cd44 and c-met, connecting fra-1 with genes governing cell motility and invasion in mesothelioma.

Gene Redundancy
In many cases, eliminating the expression of a single gene in higher eukaryotes can be tolerated even if that gene product functions in a critical pathway. This is because many critical cell functions are accomplished by more than one gene product. When one gene product is eliminated, the redundant gene product compensates to allow the cell or animal to survive. Identifying redundant genes could be achieved by co-transfecting siRNAs and assaying for a given phenotype. For example, a gene that is identified as being important in cell cycle regulation might fail to elicit a cell-cycle defective phenotype. Co-transfecting this siRNA with other siRNAs targeting other cell cycle genes and assaying for a cell cycle phenotype could identify genes that might serve at the same point in the cell cycle. Evaluating each of the candidate genes alone to ensure that they only cause the cell cycle defect when reduced in combination with the target gene would help pinpoint the most likely redundant gene.

* Glucose levels are thought to be regulated by the family of Akt serine/threonine kinases. When Katome et al (8) reduced the expression of Akt2 with an siRNA, they noted a slight change in cellular glucose regulation. However, when they targeted the two isoforms of the Akt gene (Akt1 and Akt2), they noted a significant change in glucose regulation. Experiments with isoform-specific siRNA ultimately showed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and glucose uptake in two different cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis.

Functional Screening
Libraries of siRNAs targeting broad collections of genes will enable screening experiments to tie genes to cellular function. To date, libraries with more than a couple of hundred siRNAs have been limited to a few large research organizations. Recognizing the benefits of siRNA libraries, Ambion is preparing a collection of more than 1800 siRNAs targeting the known human kinases. There have been no published reports on the application of siRNA libraries in screening experiments, but screens in Drosophila and C. elegans using dsRNA libraries exemplify the opportunities.

RNAi libraries targeting more than 10,000 genes have been used in C. elegans to identify genes that regulate fat (9), life expectancy (10), and mutation control (11). A similar RNAi library for Drosophila has been used to identify the genes responsible for regulating the phosphorylation of Down-Syndrome cell-adhesion molecule (12). In each of the screening applications, the keys to the experiments have been robust phenotypic assays and high quality RNAi libraries.

siRNAs as Therapeutics: The Next Frontier
While many researchers are exploiting siRNAs in their drug development processes, some scientists are evaluating siRNAs as therapeutic agents (reviewed in 13). If realized, siRNAs could make it possible to target virtually any gene for therapeutic intervention. Researchers have already shown that the RNAi pathway is active in mice and that siRNAs are tolerated and effective in several different tissues (14). Synthetic siRNAs and siRNA expression vectors (both plasmid and viral) have been injected systemically and into defined tissues and elicited target-specific responses. A number of publications have shown that siRNAs can inhibit the replication HIV (15,16) and Hepatitis B (17). Additionally, an siRNA targeting a prion-prone protein was capable of inhibiting prion formation in cells, creating an alternative therapeutic approach to prion diseases (18).

As the RNAi field continues to develop, moving into animal models, therapeutics, and drug discovery and targeting, Ambion will continue to develop innovative products that harness the power of RNAi for applications to basic, applied and therapeutic research efforts.

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