title: Versatile Viral Vector Strategies for Postscreening Target Validation and RNAi ON-Target Control.
abstract: Our approach aims to optimize postscreening target validation strategies using viral vector-driven RNA interference (RNAi) cell models. The RNAiONE validation platform is an array of plasmid-based expression vectors that each drives tandem expression of the gene of interest (GOI) with one small hairpin RNA (shRNA) from a set of computed candidate sequences. The best-performing shRNA (>85% silencing efficiency) is then integrated in an inducible, all-in-one lentiviral vector to transduce pharmacologically relevant cell types that endogenously express the GOI. VariCHECK is used subsequently to combine the inducible knockdown with an equally inducible rescue of the GOI for ON-target phenotype verification. The complete RNAiONE-VariCHECK system relies on three key elements to ensure high predictability: (1) maximized silencing efficiencies by a focused shRNA validation process, (2) homogeneity of the RNAi cell pools by application of sophisticated viral vector technologies, and (3) exploiting the advantages of inducible expression systems. By using a reversible expression system, our strategy adds critical information to hot candidates from RNAi screens and avoids potential side effects that may be caused by other, irreversible genomic manipulation methods such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas). This approach will add credibility to top-hit screening candidates and protect researchers from costly misinterpretations early in the preclinical drug development process.
authors: SIRION Biotech GmbH, Martinsried, Germany christel@sirion-biotech.de.
authors: Carl J
authors: CJ
authors: Christel
authors: SIRION Biotech GmbH, Martinsried, Germany.
authors: Patricia
authors: P
authors: Schmied
authors: SIRION Biotech GmbH, Martinsried, Germany.
authors: Verena
authors: V
authors: Jagusch
authors: SIRION Biotech GmbH, Martinsried, Germany.
authors: Silke
authors: S
authors: Schrödel
authors: SIRION Biotech GmbH, Martinsried, Germany.
authors: Christian
authors: C
authors: Thirion
authors: SIRION Biotech GmbH, Martinsried, Germany.
authors: Kathrin
authors: K
authors: Schmitt
authors: SIRION Biotech GmbH, Martinsried, Germany.
authors: Michael
authors: M
authors: Salomon
mesh_terms: D002460:Cell Line; D005434:Flow Cytometry; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D010641:Phenotype; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering
keywords: RNAi; inducible gene expression; knockdown; lentivirus