RNAi HTS can be used in diverse biological applications to understand the significance of gene pathways in parallel and to find proteins that are important to the process. RNAi libraries can target large portions of a human or mouse genome; the druggable genome siRNA library corresponds to gene targets that can ultimately be targeted with small-molecule therapies. For finding gene pathways that affect nonviral gene transfer, we developed an RNAi HTS strategy to screen the human druggable genome of siRNAs. We applied the HTS assay to confluent HAECs because nonviral vector transfection efficiencies are typically low, especially in comparison with those of viral vectors. As a model transfection reagent we used Lipofectamine 2000, a commercially available nonviral lipofection reagent, to deliver a reporter pDNA.
After delivery of pooled siRNAs and Renilla pDNA, gene knockdowns that enhanced nonviral gene transfer were identified as potentially rate-limiting (positive hits), and gene knockdowns that reduced nonviral gene transfer were identified as proteins potentially required for nonviral gene transfer (negative hits). After the primary screen of 5,520 druggable gene targets, ~2% of the gene targets were identified as positive hits while ~1.5% of the gene targets were identified as negative hit targets. The pooled siRNAs corresponding to the positive hit targets were subjected to confirmation and validation studies to find which of the individual siRNAs resulted in significant improvements in Lipofectamine 2000 gene transfer. After two rounds of confirmatory screening experiments, 33 genes were confirmed to enhance gene transfer upon silencing with one or more siRNAs, whereas 18 genes were confirmed to enhance gene transfer upon silencing with two or more siRNAs.
In order to confirm that these enhancements in gene transfer could be scaled to a larger format, we carried out the screening experiment in 96-well plates with the same Renilla luciferase pDNA and in 24-well plates with a GFP pDNA. We found that two siRNAs targeting the genes PPP2R3A
, and PRSS23
resulted in an average of 10–65% improvement in luminescence from transfection in 96-well plates. Two of these candidate gene targets, PPP2R3A
, are different isoforms of the protein phosphatase 2A (PP2A) regulatory subunit. PP2A is known to mediate cell-cycle regulation through the M-phase-promoting factor, which regulates the G2/M transition.15
The B regulatory subunit family of PP2A contains multiple subfamilies with multiple isoforms for each protein, each providing similar but unique regulation of PP2A activity. When siRNA 104939, targeting PPP2R2C
, was co-transfected with GFP pDNA, flow cytometric analysis revealed an increase of 10% in the number of cells transfected, relative to the nil-siRNA control. Likewise, when an siRNA targeting an unrelated gene, PRSS23
, was co-transfected with GFP pDNA, flow cytometric analysis revealed an 8% increase in the number of cells transfected. These findings show that the nearly twofold increases in luminescence in 96-well plates can be attributed to a greater number of cells being successfully transfected.
The finding that PPP2R2C targeting can enhance nonviral gene transfer is significant in the light of the known mechanisms of several viruses. Viral proteins such as human immunodeficiency virus-1 Viral Protein R, SV40 small t antigen, and others target subunits within the PP2A complex to aid in viral transduction.9
Human immunodeficiency virus-1 Viral Protein R is known to interact with PP2A, and in fission yeast Viral Protein R was shown to interact specifically with the regulatory B subunits in a manner similar to the findings in this study, locking the cell population in G2/M, a susceptible state for infection.16
SV40 targets the PP2A-A subunit and induces proliferation, which in turn enhances the transduction efficiency of the virus.17
Both of these mechanisms take advantage of the natural mitotic breakdown of the nuclear envelope to shuttle the virus into the nucleus of the target cell. In this study, cell viability data show that silencing of the genes, PPP2R3A
by multiple siRNAs results in cell number increases, thereby suggesting an increase in proliferation. These findings serve as preliminary evidence that the knockdown of PPP2R3A
generates proliferation similar to that produced by several viruses. Despite these findings, further studies of cell-cycle mechanism are warranted in order to fully understand the role that RNAi gene silencing can play in cell-cycle regulation, and the potential applications to lipofection.
Previous work has identified the significance of the nuclear envelope as a transport barrier in gene transfer experiments.18
For example, when a nonclassical nuclear-localization signal was used for assisting lipofection, the transfection was enhanced 100-fold in embryo-derived retinal ganglion cells.19
Reports have also shown that even a high pDNA copy number (~104
) surrounding the nucleus following a typical lipofection, is not a sufficient condition to produce a successful transfection event.20
These previous reports suggest that the nuclear barrier is a significant barrier to successful transfection in confluent or nondividing cells, and that manipulation of the nuclear envelope would produce a successful transfection, such as during cell division. A report by Ludtke et al
. suggested that the increase in transfection efficiency caused by cell division may be limited to twofold in certain cell types.21
Our results are comparable to those of Ludtke et al
., with a 75% increase in raw luminescence (data not shown) and a 10% increase in the number of transfected cells after PPP2R2C
knockdown. However, many genes contribute to cell-cycle regulation and there may be a locus of cell-cycle regulatory gene targets that can ultimately lead to a larger enhancement in nonviral gene transfer, perhaps through similar siRNA or small-molecule inhibition. The limited enhancement observed here could also be a direct result of the use of Lipofectamine 2000 as the gene transfer reagent. Lipofectamine 2000 is known to be relatively efficient (albeit toxic), and other inefficient nonviral reagents may realize larger gains in transfection enhancement after PPP2R2C
The nuclear barrier is just one of many barriers that must be overcome, and appropriate mechanisms need to be incorporated into the vector design. For example, endosome escape is enhanced through the incorporation of neutral helper lipids in nonviral cationic-lipid gene transfer vectors.22
Further research will reveal the best target to enhance gene transfer through cell cycle regulation; the siRNA(s) or small molecule inhibitor(s) targeting the requisite gene(s) or protein(s) could be paired with current non-viral delivery vectors to produce a delivery vehicle with pseudo-viral vector properties.
In this study, we deployed siRNA screening to study nonviral gene transfer in HAECs. This technique can be easily adapted to other cell types and gene transfer techniques, and can point out the appropriate direction for researchers to follow in order to improve efficacy. In our system, we found that several cell- cycle-regulatory genes could be targeted for an enhancement in gene transfer; however screens with different systems may reveal different barriers worth targeting. This work demonstrates not only the utility of RNAi and its applications to nonviral gene transfer mechanisms, but also the potential for coupling siRNA and DNA in delivery vehicles for enhanced gene transfer efficiency.