B RNA Interference Screening and Oncology

Whereas cDNA screens are useful for identifying genes that cause cell transformation and possibly cancer, RNA interference (RNAi) inhibition screens may be used to identify which genes can be inhibited to reverse the disease phenotype. Since RNAi inhibition of gene expression is in some respects analogous to inhibition of the same gene product by a small molecule antagonist, the utility of RNAi for drug target identification and validation has caught the interest of the drug discovery community. The phenomenon of RNAi, first discovered in the nematode Caenorhabditis elegans, has become a favored method for studying "loss-of-function" phenotypes in a high throughput and unbiased manner (Fire et al., 1998). The direct use of RNAi in mammalian tissue culture cells has now become a feasible approach for functional genomics studies. The utility of this approach stems largely from efficient design and delivery of siRNA to mammalian cells, and the availability of the human genome sequence to design targeting-oligos for whole genome screens in mammalian cells.

The availability of the human genome sequence enables design of specific inhibitory RNA sequences against all of the predicted human genes, and advances in screening methodologies and automation allow one to interrogate the effects of knocking down every gene, one-by-one, in parallel in cellular assays just as described for cDNAs. In mammalian cells, gene silencing has been achieved by transient transfection of synthetic short double-stranded (ds) siRNA [in vitro synthesized siRNA, and plasmid-based short hairpin RNAs (shRNAs) (Aza-Blanc et al., 2003; Berns et al., 2004; Brummelkamp et al., 2002; Hsieh et al., 2004; Paddison et al., 2004; Yang et al., 2002; Zheng et al., 2004]. The short hairpins are processed by ribonuclease III activity of the Dicer enzyme to generate effective 21-22 nt siRNA (Bernstein et al., 2001). Besides Moloney-based retroviral delivery of shRNAs, other viral delivery systems including adenoviral RNAi vectors or lentiviral vectors have additional benefits (Arts et al., 2003; Berns et al., 2004; Paddison et al., 2004; Rubinson et al., 2003). Lentiviruses, in contrast to retroviruses, have the capacity to integrate into the host genome of nondividing cells and enable stable expression of the delivered shRNAs (Naldini et al., 1996a,b). Adenoviral vectors are also used to achieve long-term expression and high gene transfer efficiency. Both types of RNAi reagents, including chemically synthesized siRNAs and vector-encoded shRNAs, have their own unique limitations that impact the success of the screen. For instance, it has been shown that some siRNAs, and more often shRNAs are prone to induce a nonspecific interferon-mediated response, which can complicate readouts (Bridge et al., 2003; Sledz et al., 2003). In addition, excessive concentrations of RNAi reagents can lead to significant off-target effects (Persengiev et al., 2004). The ability to control the concentration of exogenous siRNA in contrast to the noncontrollable effective dose of shRNA transcribed inside the cell minimizes this risk of nonspecific effects.

Toward oncology drug targets, initial proof of concept for an arrayed synthetic siRNA library and the feasibility of large-scale RNAi screens in mammalian cells was first demonstrated by Aza-Blanc et al. (2003). siRNA

oligos targeting 510 human kinases were tested for their ability to modulate the induction of apoptosis by TNF-related apoptosis inducing-ligand (TRAIL, a "biologic" drug in clinical development for oncology applications) in a cell viability assay. The goal of the screen was to identify genes which when selectively knocked down could enhance sensitivity of the cells to TRAIL-mediated apoptosis, or alternatively block apoptosis. Both known and previously uncharacterized genes, including downstream of bid (DOBI) and MIRSA, were identified. In addition, a functional linkage between MYC, WNT, JNK, and BMK1/ERK5 genes to the TRAIL-mediated response pathways was revealed. A genome-wide library comprising ^49,000 synthetic siRNAs spotted in an arrayed format (two siRNAs per gene) has also been designed (Huesken et al., 2005). An artificial intelligence algorithm (BIOPREDsi, www.biopredsi.org) that computationally predicts 21-nt siRNA sequences that have an optimal knockdown effect for a given gene was used. BIOPREDsi implements a neuronal network, based on the Stuttgart Neural Net Simulator (http://www-ra.informatik. uni-tuebingen.de/SNNS/). This collection was used to interrogate the response to hypoxia by induction/suppression of HIF-1a levels measured by the use of a luciferase reporter gene containing the hypoxia-response element. HIF-1a and aryl hydrocarbon receptor nuclear translocator (ARNT, HIF-1/3), which heterodimerizes with HIF-1a on hypoxic shock, scored among the top hits, thus validating this approach. These examples demonstrate the powerful nature of siRNA design when coupled with genome-wide screening. Genes required for cell division in HeLa cells were screened by using a high-throughput cell viability assay followed by a high content video-microscopy assay to quantify the frequency of cells in mitosis for arrest phenotypes. Thirty-seven genes were identified, including several splicing factors whose silencing generated mitotic spindle defects. Another example of a vector library directed against the family of deubiquitinating enzymes revealed CYLD, the familial cylindromatosis tumor suppressor gene, as a suppressor of NF-kB, thus establishing a direct link between a tumor suppressor gene and NF-kB signaling (Brummelkamp et al., 2003). It was also shown that inhibition of CYLD enhanced protection from apoptosis and can be reversed by aspirin derivatives, such as salicylate, which are established NF-kB inhibitory molecules. This result led to the suggestion of a therapeutic strategy for treating cylindromatosis with existing drugs and provides an insight as to how genetic screening approaches can reveal potential drug targets and intervention strategies.

As a result of the power of siRNA-based genetic screens, several groups have combined their efforts to expand this resource. The RNAi consortium (TRC), a collaborative effort among six research institutions and five international life sciences organizations, released a lentiviral shRNA library consisting of ^35,000 shRNA constructs targeting 5300 human and 2200

mouse genes. The first-generation library named MISSION™ TRC-Hs 1.0 (Sigma-Aldrich) or Expression Arrest™ TRC (Open Biosystems) is based on the self-inactivating lentiviral vector pLKO.1, wherein expression of the hairpin sequence is driven by the U6 promoter (Stewart et al., 2003). The effectiveness of this newly released lentiviral library has not been reported, however, the results of several aforementioned screens indicate the potential utility of this resource.

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook

Post a comment