RNAi has become a corner-stone for directed manipulation of cellular phenotypes, mapping and screening genetic pathways, discovering and validating therapeutic targets, and has significant therapeutic potential1-5
. However, due to their ~14,000 Dalton (Da) size and extensive negative (anionic) charge, simply stated, siRNAs are macromolecules with no ability to enter cells4,5
. In fact, naked siRNAs fail to enter unperturbed cells or induce an RNAi response, even at millimolar concentrations4
. Consequently, significant attention has been focused on devising siRNA cellular delivery approaches, including particle formation by cationic lipids, cholesterol, condensing polymers, antibody-protamine fusions and siRNA encapsulation in liposomes1-5
. However, these siRNA delivery approaches perform best on adherent tumor cells and poorly on primary cells and non-adherent cell types, thereby severely limiting the cell types that can be used for discovery research and large scale RNAi screening. Consequently, there is a significant need for a siRNA delivery approach that: 1) targets the entire cell population of all primary and tumorigenic cell types, 2) is non-cytotoxic, and 3) is independent of siRNA sequence.
In developing a siRNA delivery strategy, we started with the proven macromolecular delivery properties of cationic Peptide Transduction Domain (PTD) delivery vehicle (also termed Cell Penetrating Peptide [CPP]), such as TAT, 8×Arg, and Antp, that have been shown to deliver a wide variety of cargo into primary cells, into most, if not all, tissues in pre-clinical models and are currently being tested in multiple clinical trials6
. Cationic PTDs are rapidly taken up into cells by macropinocytosis, a specialized form of fluid phase uptake that all cells perform7,8
. However, conjugation of cationic PTDs (6-8 positive charges) to anionic siRNAs (~40 negative charges) results in charge neutralization, inactivation of the PTD, aggregation/precipitation, and cytotoxicity with limited siRNA entry into the cells9,10
. To circumvent PTD charge neutralization (inactivation), we generated a TAT PTD fusion protein with a single dsRNA Binding Domain (PTD-DRBD) that binds the siRNA with high avidity (KD
) and thereby masks its negative charge11-13
. DRBDs are small, ~65 residue domains that specifically binds ~12-16 bp of the dsRNA backbone on 90° surface quadrants of the dsRNA helix, resulting in four DBRDs encompassing a single siRNA (4:1 ratio) () (Supplementary Fig. 1
PTD-DRBD mediated siRNA delivery
To determine the ability of PTD-DRBD fusion proteins to deliver siRNAs, we generated a human H1299 lung adenocarcinoma dGFP/dDsRed reporter cell line that allowed for direct determination of the magnitude of a single cell RNAi response and hence, the percentage of cells undergoing a RNAi response. H1299 dGFP/dDsRed reporter cells were treated with PBS (mock), PTD-DRBD plus control control (Con1, Con2) siRNAs or PTD-DRBD plus one of two sequence-independent GFP (GFP1, GFP2) siRNAs and analyzed by flow cytometry for GFP knockdown at 24 h (). PTD-DRBD delivery of GFP specific siRNAs resulted in a significant GFP knockdown with little to no alteration of the internal DsRed control. Similar RNAi responses were induced with 3 additional GFP siRNAs delivered by PTD-DRBD (data not shown). All controls (non-specific control siRNAs, PTD delivery peptide only) failed to induce a RNAi response. PTD-DRBD mediated siRNA delivery also resulted in a significantly stronger RNAi response compared to lipofection delivered siRNAs (). Importantly, we detected little to no alteration of cell viability in PTD-DRBD:siRNAs treated cells, whereas lipofection resulted in varying levels of cytotoxicity (Supplementary Fig. 2
). Single cell flow cytometry analysis of PTD-DRBD:GFP siRNA treated cells showed that the entire cellular population was undergoing a maximal RNAi response at 24 h that was maintained at 48 h (). In contrast, lipofection delivered siRNAs induced a partial penetrant RNAi response with ~20% of cells unresponsive (). Kinetic analysis over 8 days in dividing H1299 cells showed a slow decay of the RNAi response starting 3 days after PTD-DRBD:GFP siRNA treatment that was similar to the lipofection mediated RNAi decay kinetics (). Similar results were obtained in primary human fibroblasts, keratinocytes, macrophage, melanoma and glioma cells containing integrated GFP reporter genes (Supplementary Fig. 2
). To circumvent the RNAi decay curve, we re-treated dividing H1299 cells on days 3 and 6 with PTD-DRBD:GFP siRNAs resulting in maintenance of the extent and magnitude of the GFP RNAi response ().
We next targeted endogenous GAPDH mRNA by PTD-DRBD mediated RNAi. Treatment of H1299 cells with one of two sequence-independent GAPDH siRNAs delivered by PTD-DRBD resulted in a GAPDH RNAi response that was first detected by qRT-PCR at 6 h post-addition and reached a maximal RNAi response by 12 h (). In contrast, all PTD-DRBD negative controls failed to induce a GAPDH RNAi response. Impressively, PTD-DRBD mediated delivery of GAPDH1 siRNA resulted in a near maximal RNAi response by 6 h, significantly (P<0.001) earlier than control lipofection delivery of the same GAPDH siRNAs (), suggesting that PTD-DRBD delivered siRNAs rapidly enter the cytoplasm and are loaded into RISC. To determine if PTD-DRBD mediated siRNA delivery caused any cellular alterations, we examined the transcriptome of treated cells. Whole genome microarrays were probed with total mRNA from PTD-DRBD GAPDH siRNA treated H1299 cells at 12 h and 24 h (). Using a 1.6× fold increase/decrease filter (blue line) of cellular mRNAs, we detected a dramatic reduction in the target GAPDH mRNA along with a limited number of both up and down regulated genes. The up regulated genes were reduced in numbers and to nearly background 1.6× levels at 24 h, while the down regulated genes increased slightly in numbers and maintained a similar magnitude at 24 h (). None of these genes are present in either an innate immune response pathway or congregate into a specific genetic pathway. In contrast, lipofection treated cells showed both a dramatic increase in both the total number of genes altered and the magnitude of the increase (). In addition, the numbers of genes affected increased between 12 h and 24 h, suggesting that lipofection of siRNAs into cells results in a substantial alteration to the transcriptome and may thereby caveat interpretation of experimental outcomes. Moreover, lipofection mediated GAPDH specific knockdown was significantly smaller than PTD-DRBD mediated knockdown. Taken together, these observations demonstrate that PTD-DRBD mediated siRNA delivery efficiently targets the entire cellular population in the absence of cytotoxicity.
Due to inefficient siRNA delivery and associated cytotoxicities, RNAi manipulation of T cells remains problematic. Therefore, we focused on a notoriously difficult cell type to delivery siRNAs into, namely tumorigenic Jurkat T cells. Jurkat T cells containing an integrated GFP reporter gene were treated with GFP siRNA plus either PTD-DRBD or one of two lipofection reagents (Lipofection-2000 and RNAiMAX) at optimal concentrations and analyzed by flow cytometry for GFP knockdown at various time points (). PTD-DRBD delivery of GFP specific siRNAs into Jurkat T cells resulted in a strong GFP RNAi response in the entire population of Jurkat T cells. In comparison, both lipofection reagents induced limited RNAi responses. Moreover, PTD-DRBD delivered GAPDH siRNA into Jurkat T cells resulted in a strong GAPDH RNAi response as measured by qRT-PCR, whereas the two lipofection reagents performed poorly (). We next treated primary murine T cells with PTD-DRBD plus CD4 specific siRNAs and assayed for CD4 cellular levels by flow cytometry (). The entire CD4 cellular population had undergone an RNAi response at 24 h, whereas control siRNAs did not alter CD4 levels. Similarly, PTD-DRBD mediated delivery of CD8 specific siRNAs into primary T cells resulted in a CD8 specific RNAi response with no change in CD4 levels (). Consistent with these observations, we detected PTD-DRBD CD4 and CD8 specific RNAi responses by qRT-PCR at 12 and 24 h (P<0.05) (). Importantly, both flow cytometry and qRT-PCR analyses of internal control CD90 receptor showed little to no alteration in either PTD-DRBD CD4 or CD8 siRNA treated T cells (). In contrast, we were unable to detect any RNAi responses by lipofection of primary T cells (data not shown).
PTD-DRBD siRNA delivery into T cells and HUVECs
Primary human umbilical vein endothelial cells (HUVEC) are an important cell type for large scale RNAi screen; however, lipofection delivery of HUVECs results in both poor siRNA delivery and cytotoxicity. We targeted endogenous GAPDH mRNA by PTD-DRBD mediated RNAi. Treatment of primary HUVECs with one of two sequence-independent GAPDH siRNAs delivered by PTD-DRBD resulted in a GAPDH RNAi response that was first detected by qRT-PCR at 6 h post-addition and reached a maximal RNAi response by 12 h (P<0.01) (). In contrast, all PTD-DRBD negative controls failed to induce a GAPDH RNAi response. Consistent with the observations in H1299 cells above, PTD-DRBD mediated delivery of GAPDH1 siRNA resulted in a maximal RNAi response by 6 h (). Importantly, we detected little to no alteration of HUVEC cell viability in PTD-DRBD:siRNA treated cells compared to mock treated control cells (). In contrast, we were unable to lipofect siRNAs into HUVECs without inducing significant levels of cytotoxicity ().
Human Embryonic Stem Cells (hESCs) have great potential to treat human disease14
; however, manipulation of hESCs into specific cell lineages by RNAi requires protocols that target the entire cell population in a non-cytotoxic manner. We first tested the ability of PTD-DRBD to deliver siRNAs into H9 hESCs stably expressing GFP. Consistent with the observations above, PTD-DRBD mediated delivery of GFP siRNAs induced a marked GFP RNAi response throughout the hESC colony (). We next tested the ability of PTD-DRBD mediated siRNA delivery to affect the fate of hESCs. The Oct4 (POU5F1) transcription factor is required to maintain hESC pluripotency and Oct4 RNAi knockdown results in hESC cell cycle exit and differentiation14
. Treatment of HUES9 hESCs with PTD-DRBD plus Oct4 siRNA resulted in both an Oct4 specific knockdown followed by a reduced growth rate and cell cycle exit indicative of pluripotency loss and initiation of differentiation (). In contrast, both mock and PTD-DRBD plus control siRNA did not alter hESC cellular morphology, growth kinetics or Oct4 expression levels.
PTD-DRBD mediated RNAi responses
Pluripotent hESCs express multiple cell surface markers, including Stage-Specific Embryonic Antigen-4 (SSEA-4)15
. During differentiation into endoderm, hESCs decrease SSEA-4 expression, stop dividing, increase in size and subsequently express the GATA6 differentiation transcription factor16
. PTD-DRBD delivered Oct4 siRNA resulted in loss of Oct4 expression by day 2 with continued SSEA-4 expression (). However, by 10 days post-treatment, Oct4 siRNA treated cells had lost expression of SSEA-4 and induced expression of the GATA6 endoderm specific transcription factor (). In contrast, mock and PTD-DRBD plus control siRNA treated hESCs did not induce differentiation or alter hESC marker expression. Similar results were obtained by simultaneous PTD-DRBD mediated knockdown of Oct4 and Nanog (data not shown). siRNAs have been shown to stimulate activation of Toll-Like Receptors-3, -7, -8 (TLR) to induce innate immune responses17
. However, PTD-DRBD mediated delivery of immunostimulatory siRNAs failed to activate IFN-α or TNF-α responses in primary human peripheral blood mononuclear cells (PBMCs) above background levels (). Taken together, these observations demonstrate the ability of PTD-DRBD to deliver siRNA and rapidly induce RNAi responses in three important and difficult to deliver cell types: T cells, HUVECs, and hESCs.
siRNA-induced RNAi has great potential to treat human disease, including nasal delivery to treat virus infection18
; however, in vivo siRNA delivery remains problematic1-5
. Based on the efficient PTD-DRBD mediated siRNA delivery we observed in primary cell culture systems, we quantified the ability of PTD-DRBD to deliver siRNAs and induce an RNAi response using an intranasal in vivo transgenic Luciferase reporter mouse model19
. Transgenic ROSA26 mice stably expressing tissue restricted luciferase in the nasal and tracheal passage were live animal imaged for Luciferase expression then randomized into groups (). Luciferase mice were then treated intranasally with PBS, PTD-DRBD plus Luc siRNA or control siRNA and monitored daily for 15 days for luciferase expression. Control PBS and PTD-DRBD control siRNA mice showed no change in luciferase expression over the course of the experiment. In contrast, PTD-DRBD delivered Luc siRNA showed extensive reduction of luciferase expression throughout the nasal and trachea at day 1 and gradually recovered luciferase expression by day 15 (). These observations demonstrate the ability of PTD-DRBD mediated siRNA delivery to induce a specific RNAi response to a quantifiable target protein in reporter mouse models in vivo.
siRNA induced RNAi responses are a key experimental procedure for manipulation of cell biology, dissection of genetic pathways, target validation and have great potential for therapeutic intervention. However, due to their macromolecular size (~14,000 Da) and strong anionic charge, siRNAs have limited to no ability to enter cells on their own, even at millimolar concentrations. Indeed, siRNA delivery has become the rate-limiting barrier to efficient cell culture, pre-clinical and clinical usage of siRNA therapeutics1-5
. Consequently, significant effort has been placed on devising efficient siRNA delivery approaches. While current siRNA delivery approaches have merit, they generally fail to target the entire population of cells or a high percentage of cells, especially primary cells, and often result in varying levels of cytotoxicity and alterations in cell biology. In contrast, the PTD-DRBD siRNA delivery approach described here fulfills many of the criteria for an efficient siRNA delivery system into primary cells. PTD-DRBD delivered siRNAs and induced RNAi responses in the entire population of three difficult to deliver primary cell types (T cells, HUVECs, and hESCs) in a rapid and non-cytotoxic fashion. DRBDs bind to dsRNAs (siRNAs) independent of sequence composition and therefore, in theory, PTD-DRBD can deliver any siRNA into cells. Lastly, the intranasal knockdown of luciferase in vivo begins to demonstrate an in vivo potential of PTD-DRBD mediated siRNA delivery; however, significantly more in vivo work needs to be performed to ascertain the full extent of in vivo utility. In summary, PTD-DRBD has broad implications for the scientific community in RNAi basic research, target screening and potential therapeutic applications.