RNA Interference Screen to Identify Pathways That Enhance or Reduce Nonviral Gene Transfer During Lipofection

• Published in: Molecular therapy Vol. 16; no. 9; pp. 1602 - 1608
• Main Authors: Barker, Gregory A, Diamond, Scott L
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2008; Elsevier Limited
• Subjects: Aorta - cytology; Aorta - metabolism; Candidates; Cells, Cultured; Efficiency; Electroporation; Endothelium, Vascular - cytology; Endothelium, Vascular - metabolism; Flow Cytometry; Gene Silencing; Gene therapy; Gene Transfer Techniques; Genetic Vectors - genetics; Genetic Vectors - pharmacology; Genome, Human; Genomes; Humans; Lipids - chemistry; Luciferases - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger - antagonists & inhibitors; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Small Interfering - pharmacology; Signal Transduction; Transfection; United States > US; Pennsylvania
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1038/mt.2008.147

Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from lipofection, with a quantitative PCR–validated knockdown of ∼76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.