A method for detecting and preventing negative RNA interference in preparation of lentiviral vectors for siRNA delivery.

The lentiviral vector is a useful tool for delivery of hairpin siRNA (shRNA) into mammalian cells. However, the efficiency of this system for carrying double-stranded siRNA (dsRNA) has not been explored. In this study we cloned the two forms of siRNA-coding sequence, a palindromic DNA with a spacer loop for shRNA and a double-stranded DNA with opposing Pol III promoters for dsRNA, into lentiviral DNA vectors, and compared their viral vector production yields. Our results indicate that sharply lower titer vector was obtained for dsRNA while much higher titer vector was produced for shRNA, posing a fundamental concern whether siRNA-carrying viral RNA itself is an inherent target of RNAi. Further experimental analyses using packaging cells that either allow or do not allow siRNA transcription indicate that the shRNA-carrying viral RNA is resistant to RNAi but the viral RNA carrier for dsRNA is not, offering a linker of RNAi bias-target secondary structure that causes shRNA vector to evade RNAi degradation. More importantly, the poor yield of dsRNA vector production was restored when a novel packaging cell line was used that blocks the antisense strand from dsRNA duplexes. This method has important implications for the RNAi field, especially for those who are using lentiviral dsRNA and dsRNA libraries for various biological discovery and therapeutic interventions.

A 96-well surrogate survival assay coupled with a special short interfering RNA vector for assessing cancer gene targets with enhanced signal/noise ratio and its utility in HTS for cancer therapeutic targets.

Transient transfection of short interfering RNAs to inactivate cancer therapeutic genes in cancer cells is an important method to induce therapeutic phenotypes (cell apoptosis, growth arrest, etc.) for cancer target validation. These phenotypes can be initially assessed by cell survival via colorimetric/fluorescence readings, e.g., alamarBlue (Trek Diagnostic Systems, Cleveland, OH) and WST-1. However, intrinsic problems exist for transient transfection-varying toxicity, inconsistent transfection efficiency, as well as other cell-specific determinants-which contribute to a low signal:noise ratio of the assays, rendering of the assay ineffective particularly when applied in high-throughput screening (HTS) multiplexed for different cells. This report describes a method using reporter as a "normalized surrogate" for the conventional survival readout in a 96-well format. In this approach, only the transfected surviving cells produce reporter activities, and many variables associated with transient transfection are excluded. A constitutively expressed reporter gene (luciferase or LacZ) expression cassette is co-transfected into cells along with a specially designed RNA interference (RNAi) vector (or a transgene for that matter). The reporter activity in either liquid cultures or in soft agar cultures in 96-well formats is then quantitated in situ. The RNAi vector construction is simplified so that it can be adapted to a 96-well format. Our data demonstrated that the relative reporter readings for survival are independent of both transfection efficiency and cellular toxicity. The signal:noise ratio is markedly increased, particularly for cells with low transfection efficiency. The assay is versatile and robust and can be applied in multiplexed HTS for cancer target identification and validation.