text: The development of novel pharmaceutical therapeutics relies on the identification and validation of key regulators of disease processes (“drug targets”). New technologies, reagents and methods that contribute to risk-reduction with respect to predicting therapeutic efficacy in human patients is highly valued. This process of therapeutic target identification and validation ideally utilizes functional therapeutic mimetic technologies in models of human disease. In particular, approaches that emulate therapeutic modalities, where a specific gene-product is inactivated and demonstrated to ameliorate a disease state, are needed.
  RNA interference (RNAi) is a conserved cellular mechanism for regulating gene expression in all cells. RNAi-based technology has rapidly become a significant functional genomics tool and drug target validation approach for the pharmaceutical and biotechnology industries. RNAi technology facilitates the directed inactivation (“silencing”) of virtually any gene and thus the opportunity to associate specific gene-function with specific disease mechanisms.
  Contemporary approaches to constructing RNAi trigger molecules (i.e., an siRNA molecule) build on empirically determined bioinformatic algorithms. Significant progress has been made in understanding the molecular mechanisms underlying in vivo RNAi, and furthermore, artificial RNAi-inducing molecules have been successfully constructed. However, significant challenges remain in the development of this technology, particularly in the context of pharmaceutical target validation.
  Various methods are known in the literature for siRNA vector and library construction. For example, Sen et al., “Restriction enzyme-generated siRNA (REGS) vectors and libraries,” Nature Genetics 36(2): 183-189 (2004) describes a “REGS” method for producing siRNA molecules. However, the REGS method has various drawbacks. The REGS method described in that publication produces shRNA molecules that are shorter than the optimal length for inducing RNAi, and which therefore function inefficiently. Furthermore, the yield at each step of the procedure is low so that prior to cloning the product must be amplified. The method of amplification described (i.e., rolling circle amplification by phi29 polymerase) is prone to bias (the preferential amplification of one product at the expense of amplification of other products), thereby resulting in a library that contains only a fraction of the potential diversity.
  There is a need in the art for improved methods for generating and screening for biologically active molecules that induce RNAi, for example, shRNA or siRNA molecules. There is a need in the art for improved methods for validating candidate drug targets and predicting in vivo responses to inhibitor compounds. Further, there is a need to develop methods that aid in the validation of candidate drug targets, where the validation of drug targets can be done in a dose-dependent manner. The present invention provides compositions and methods that meet this need, overcome the limitations of the REGS method, and provide other benefits that will become apparent upon reading the present disclosure.