NANO-01070; No of Pages 12
Nanomedicine: Nanotechnology, Biology, and Medicine xx (2015) xxx – xxx nanomedjournal.com
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Ghulam Hassan Dar, Vijaya Gopal⁎, Madhusudhana Rao⁎
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CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India Received 8 September 2014; accepted 19 January 2015
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Conformation-dependent binding and tumor-targeted delivery of siRNA by a designed TRBP2: Affibody fusion protein
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Abstract
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Efficiency of systemically delivered siRNA in gene silencing is compromised due to lack of target-specific delivery and rapid clearance of siRNA by in vivo elimination pathways. We designed a fusion protein consisting of a dsRNA binding domain of transactivation response RNA binding protein (TRBP2) fused to ErbB2 binding affibody (AF) for target specific delivery of siRNA. Designated as TRAF, the fusion protein is stable and binds efficiently and specifically to siRNA, forming homogenous non-aggregated and nuclease-resistant particles that efficiently and selectively transport siRNA into HER-2 overexpressing cancer cells and tissues. Administration of siRNA by TRAF into cells resulted in significant silencing of chosen genes involved in cell proliferation viz. AURKB and ErbB2. Noticeably, intravenous administration of TRAF:siRNA against these genes resulted in remarkable tumor suppression in the SK-OV-3 xenograft mouse model. Our results establish the potential of engineered proteins for specific and systemic delivery of siRNA for cancer therapy. © 2015 Published by Elsevier Inc.
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Key words: Affibody; Epidermal growth factor receptor 2; Tumor-targeting; siRNA delivery; TRBP2; Fusion proteins
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Background
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RNA interference holds a great potential in cancer therapy due to its high specificity and ability to efficiently silence a large number of different genes involving a number of distinct cellular pathways. 1,2 This is particularly important for a disease as complex as cancer. However, as in other drug targeting therapies, the major challenge for therapeutic use of siRNA is the need for devising safe, stable and effective carriers capable of delivering low doses of siRNA into the cytoplasm. A large number of carriers including organic polymers, lipids and metal nanoparticles have been tested with variable success. 3,4 Successful vehicles designed so far utilized non-covalent complexation of siRNA. 5 While these complexes are sufficient for in vitro cell-based experiments, intravenous administration of these complexes would lead to promiscuous electrostatic interactions with serum proteins, aggregation and nonspecific uptake, leading to low efficiency. 6,7
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We gratefully acknowledge financial support from CSIR (BSC0112). GHD is also grateful to CSIR for a Senior Research Fellowship. ⁎Corresponding authors at: CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500007. A. P. India. E-mail addresses: [email protected], [email protected] (V. Gopal), [email protected] (M. Rao).
Peptides and fusion proteins of varied combinations are emerging as promising vehicles for tissue specific delivery of siRNA. 8 Peptides offer immense sequence variations and could be made target specific by evolving them either by phage display methods or sourcing them from natural sequences. 9,10 Moreover, major advantages of designed modular proteins are the convenience in fine tuning the ratio between payload (siRNA) and carrier, ease in scaling up and their low toxicity than either cationic lipids or polymers. Several targeting peptides attached to either protamine or polyarginine have been successfully used for in vivo delivery of siRNA to cells expressing HIV envelope glycoprotein gp160, 11 primary lymphocytes via integrin ScFvs, 12 and transvascular delivery to brain, 13 However, there have been reports where the presence of positively charged peptides in the fusion proteins could lead to undesired properties such as interaction with serum glycosaminoglycans, 14-16 aggregation at higher concentrations, heterogeneous sizes of peptide-based nanoparticles 17 and difficulty in scaling up production. 18 Double-stranded RNA-binding proteins (dsRBP) offer an excellent alternative over charge-based motifs for conformation– specific binding to siRNA rather than forming ionic complexes of variable sizes. 19 These structurally well-defined domains recognize the A-form of double helix RNA via 2′–OH ribose groups present on minor-major-minor groove of dsRNA. 20 A
http://dx.doi.org/10.1016/j.nano.2015.01.017 1549-9634/© 2015 Published by Elsevier Inc. Please cite this article as: Dar G.H., et al., Conformation-dependent binding and tumor-targeted delivery of siRNA by a designed TRBP2: Affibody fusion protein. Nanomedicine: NBM 2015;xx:1-12, http://dx.doi.org/10.1016/j.nano.2015.01.017
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Methods
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Details of the methods are provided in supporting information
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Cell culture SK-OV-3, MDA-MB-231 and MDA-MB-453 were obtained Medina-Lai Kauwe, University of Southern California Keck School of Medicine, CA. Other cells were obtained from lab stocks. These were maintained in a Dulbecco's Modified Eagle Medium (DMEM): Nutrient Mixture F-12 (DMEM/F-12, Life Technologies) with 10% fetal bovine serum (Life Technologies).
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Flow cytometry and confocal microscopy Cells grown in tissue culture flasks were trypsinized and pelleted. Approximately 50,000 cells were incubated with 40 pmol of FAM-labeled siRNA either alone or complexed with TRAF protein at 5:1 mole ratio. Cells treated with Lipofectamine (Life Technologies) complexed with FAM-labeled siRNA were used as positive control. After 1 h incubation on rotor torque at room temperature, cells were washed with ice-cold PBS containing 1% fetal calf serum, 20 U/ml heparin sulfate and 50 mM ammonium chloride and then analyzed on a FACSCalibur with Cell Quest software (Becton Dickson). For confocal microscopy, cells seeded on coverslips in Petri dishes were transfected with LAMP-1-GFP and RAB-5-GFP plasmid using Lipofectamine. After 48 h, cells were treated with 20 pmol of fluorescent TRAF:siRNA complex (5:1 mole ratio). At the indicated time period, cells were washed with PBS containing 20 U/ml heparin sulfate, stained with Hoechst 33258
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Results
Design, purification and structural properties of TRAF
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Multispectral in vivo fluorescence imaging Female athymic BALB/c-neu mice were used in accordance with the approval from Institute of Animal Ethics Committee (IAEC) (vide IAEC project # 03/11). SK-OV-3 cells (4 million) were resuspended in Matrigel (BD Biosciences) and injected subcutaneously into mice. After the tumors attained 50– 100 mm 3 volumes, TRAF-labeled with Alexa Fluor 633 (TRAF A633) was complexed with siRNA (5:1 mole ratio) and injected by tail vein injection. The mice were imaged using the multispectral in vivo imager (Carestream Molecular Imaging) after indicated time. Snap frozen organs were cryosectioned (4 μm) and stained with Hoechst 33258. Details of methods of tumor suppression by TRAF-mediated siRNA delivery investigated using xenograft tumor model developed in nude mice are provided in the support information.
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(Molecular probes) and fixed with 4% paraformaldehyde (Sigma). The coverslips were mounted in Vectashield mounting medium H-100 (Vector Laboratories, Inc.) and analyzed by confocal laser microscopy (Leica LAS-AF-TCS-SP5). Images were analyzed by LAS AF software.
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dsRBP from IF-activated protein kinase fused in tandem with peptide transduction domain (PTD) was the first successful example for siRNA delivery into primary and transformed cells and was shown to be immunologically and toxicologically safe. 21 However, due to PTD domains and low binding affinity of dsRBD of protein kinase to siRNA, the designed fusion protein delivered siRNA non-specifically in vitro and in vivo. 14 In addition, there are very few studies where fusion proteins have been used to systemically deliver therapeutic-siRNA for suppressing the tumor growth. 22 -24 Human TRBP2 protein, a double-stranded RNA binding protein, plays an important role in RNA interference. 25 TRBP has three dsRBPs and only the first and second domain show high affinity, 220 and 113 nM respectively, to siRNA. 26,27 Based on this, the second domain of TRBP was cloned with well characterized ErbB2-binding affibody for tumor-targeted delivery of siRNA. Erbb2 receptor is a well-known cell-surface marker overexpressed in a majority of tumors. 28 ErbB2 binding affibody (5.8 kDa) molecule, originally evolved from the Z-domain of Staphylococcus aureus protein A, was chosen as the targeting ligand owing to its high affinity to ErbB2 receptors. 27 The recombinant fusion protein (TRAF) was expressed in Escherichia coli and purified to homogeneity by affinity chromatography. In this study, TRAF was used as a siRNA carrier to deliver siRNA specifically into HER-2 expressing cells. To maximize silencing-mediated tumor suppression, we simultaneously delivered siRNA against two genes, HER-2 and AURKB, which are involved in cell proliferation.
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TRAF protein was designed by fusing second domain of TRBP2 (due to its higher affinity) to the DNA sequence of ZHER-2 affibody molecule. To aid proper folding, five glycine codons were inserted between the two domains. During purification, we noticed bacterial RNA associated with TRAF (data not shown) that was removed by adding urea in the lysis buffer. Additionally, 0.1% Triton-X114 was used to remove bacterial endotoxins. 29 The purified protein was characterized by SDS-PAGE and mass spectrometry that revealed a single band of 18.485 kDa matching the predicted size of the protein (Figure 1, A and Supplementary Figure 1). With these purification procedures, we normally obtain yields of ~ 40 mg of purified TRAF protein from one liter of bacterial culture. Far-UV circular dichroism spectra of the TRAF protein showed the presence of a well-defined secondary structure unperturbed at either pH 5.8 or pH 7.8 (Figure 1, B). The percentage of α-helical and α-pleated structures was found to be 56.6% and 18%, respectively. Higher percentage of α-helical structures is also supported by a negative band at 208 nm and 222 nm of far-UV CD data. 30 Thermal stability of TRAF monitored by examining the change in ellipticity at 222 nm revealed a Tm of 62 °C (Supplementary Figure 2). To determine whether binding of TRAF to siRNA resulted in any aggregation of the complexes, we assessed the size of TRAF either alone or in complex with siRNA by dynamic light scattering (DLS). The average diameter of the protein at 2 mg/ml was found to be 2 ± 0.1 nm, while the addition of siRNA (at 5:1 mole ratio of TRAF: siRNA) resulted in particles of mean dimension 6 ± 0.3 nm (Figure 1, C). In addition, the polydispersity index of TRAF and TRAF:siRNA complexes was in the range of 0.1, suggesting homogenous and non-aggregated population of the particles. The sizes of TRAF and TRAF:siRNA complexes were also confirmed by size exclusion chromatography (Supplementary Figure 3). The
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Figure 1. Characterization of recombinant fusion protein TRAF: (A) SDS-PAGE of TRAF. Lane 2 represents purified TRAF. (B) Far-UV CD spectra of TRAF at pH 5.8 and pH 7.8. (C) Hydrodynamic diameter of TRAF and TRAF:siRNA complexes (5:1) measured by dynamic light scatter. (D) Binding of siRNA with TRAF by gel-shift assay. 20 pmol of siRNA was incubated with increasing mole ratios of TRAF. (E and F) ITC-thermograph of TRAF with siRNA (E) and oligo duplex DNA (F). Upper panel represents differential power with time. Lower panel shows integrated energy per injections. (G) RNaseA protection assay of TRAF:siRNA complexes. Details are provided in the support information.
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TRAF delivers siRNA specifically to HER-2 cancer cells
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To ascertain whether TRAF could deliver siRNA specifically into HER-2 positive cancer cells, we initially confirmed the expression levels by western blotting and immunocytochemistry using two HER-2 positive and two HER-2 negative cell lines. SK-OV-3, an ovarian cancer cell line showed the highest expression of HER-2 receptor followed by MDA-MB-453 cells (Figure 2, A and Supplementary Figure 7). However, both MCF-7 and MDA-MB-231 stained negative for HER-2. In MCF-7 cells, we observed HER-2 receptor molecules localized in nucleus of the cells. Similar type of an observation was noticed earlier and it was shown that these receptors also act as a transcription factors to many genes such as cyclin D1, Cox and p53. 32 Due to overexpression of HER-2 receptors, we used SK-OV-3 and MDA-MB-453 cells as HER-2 positive cell lines (designated as HER-2 +) while MCF-7 and MDA-MB-231
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Intracellular trafficking of TRAF:siRNA in SK-OV-3 cells
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To ascertain that siRNA uptake mediated by TRAF is receptor-specific and also to visualize their localization in the cytoplasm at different time points, we labeled TRAF and siRNA with Alexa fluor-633 and Cy3 dyes respectively. Furthermore, to monitor the trafficking of labeled TRAF:siRNA complexes in early and late endosomes, we used SK-OV-3 cells, expressing either GFP-tagged RAB 5 (early endosomal marker) or GFP-tagged lysosomal associated membrane protein 1(LAMP-1), which is present on the surfaces of late endosomes and lysosomes. 33,34 After incubating TRAF A633:siRNA Cy3 complexes for 10 min to allow binding, unbound complexes were removed by PBS washes. No fluorescence was seen in cells incubated with siRNA Cy3 alone. However, within 10 min of incubation, the labeled complexes were seen associated with the cell surface (Figure 3, A). The colocalization of fluorescent probes on the cell surface decreased with time and after 20 min, fluorescence of TRAF A633:siRNA Cy3 complexes were found merging with the fluorescence of GFP-tagged RAB-5 protein, suggesting internalization by endosomal-mediated pathway. Colocalization with RAB-5 indicated that the complexes were in the early-endosomes (Figure 3, B and Supplementary Figure 9). After 1 h of incubation, the colocalization of TRAF A633:siRNA Cy3 complexes with RAB-5 decreased drastically and no colocalization was seen after 5 h. 33,34 Interestingly, within 1 h of incubation, fluorescence of TRAF:siRNA complexes showed strong colocalization with GFP-tagged LAMP-1 protein, indicating the presence of complexes within the late endosomes (Supplementary Figures 10 and 11). Maximal colocalization at 2 h, reflected gradual increase in the transition from early to late endosomes. After 5 h, there was a significant release of the complexes from late endosomes as indicated by a decrease in the colocalization of fluorescent probes. Interestingly, we also observed dissociation of TRAF and siRNA from each other after 5 h of treatment, a time point where they are also released from endosomes. Taken together, the data suggested efficient internalization of the complexes after binding to the HER-2 receptor of the cells followed by the transition from early to late endosomes and eventual release of the complexes into the cytoplasm. Further colocalization studies with transferrin protein, a positive control for clathrin-mediated endocytosis, revealed internalization via clathrin-mediated pathways (Supplementary Figure 12). 35
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To determine the binding of TRAF with siRNA, we initially performed gel-shift assay by preparing complexes at various mole ratios. A faint siRNA band at 1:1 mole ratio and absence of bands at higher mole ratios indicated strong binding of TRAF to siRNA (Figure 1, D). In addition, incubation of the TRAF:siRNA complexes in serum for 6 h at 37 °C did not alter the binding (Supplementary Figure 4). However, incubation of TRAF with oligo DNA of 21 bp length did not show any binding (Supplementary Figure 5). Detailed structural studies of dsRBDs have revealed that the absence of strong ionic interaction, crucial interactions with the 2-hydroxyl groups of dsRNA and preference for binding to A-form (preferred conformation for dsRNA) rather than B-form (preferred conformation of DNA) prevents binding of dsRBD to either dsDNA or RNA-DNA hybrids. 31 To determine the binding affinity we performed isothermal titration calorimetry of purified TRAF and siRNA. Addition of TRAF to siRNA resulted in efficient release of energy that decreased gradually with addition of protein and reached a steady state, reflecting complete binding of TRAF to siRNA (Figure 1, E). When fitted with a model describing single set of identical binding sites, a binding constant of 107 nM was obtained for TRAF, which is similar to that reported (113 nM) for an isolated TRBP2. 27 The stoichiometry factor of ~ 2 suggested that 2 molecules of TRAF bind to one molecule of siRNA. Notably, we did not observe any enthalpy change after addition of TRAF to DNA of similar length, thus, corroborating the specificity of TRAF to siRNA (Figure 1, F). To verify whether TRAF could prevent siRNA degradation, we incubated siRNA alone and TRAF:siRNA complexes with RNase A for 1 h at 37 °C. siRNA complexed with TRAF at higher ratios offered complete protection, which indicates inaccessibility of nucleases to siRNA, while the absence of TRAF led to siRNA degradation (Figure 1G). Similar results were obtained when complexes were treated with human serum (Supplementary Figure 6).
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TRAF binds and protects siRNA from degradation
represented HER-2 negative cells (designated as HER-2 -). After treatment of the cells with TRAF:siRNA FAM complexes, we determined the uptake by flow cytometry. Lipofectamine complexed with siRNA was used as a positive control. As anticipated, siRNA FAM uptake was inefficient in all cell lines due to its large size and negative charge. In contrast, siRNA FAM bound TRAF complexes showed higher uptake in SK-OV-3 cells followed by MDA-MB-453 cells in contrast to MDA-MB-231 and MCF-7 cells, reflecting receptor-specific uptake of siRNA (Figure 2, B). To further verify the specificity, free affibody at different mole ratios was used as a competitor of TRAF:siRNA complex to HER-2 receptors. Uptake of siRNA FAM complexed with TRAF, decreased significantly (N 95%) in presence of affibody while no such effect was observed when siRNA FAM was complexed with Lipofectamine (Supplementary Figure 8).
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major elution peak of TRAF and TRAF:siRNA complexes corresponded to a size of 3.8 nm and 5.7 nm respectively that matched with DLS measurements.
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Figure 2. Uptake of TRAF:siRNA complexes by HER2 positive cells: (A) Immunofluorescence images of SK-OV-3 and MCF-7 cells treated with either HER-2 monoclonal antibody (HER-2 mAb) (left) or isotype control antibody (right). Arrows in MCF-7 panel indicate the presence of HER2 receptor in the nucleus. (B) Representative FACS profiles of cells treated with either 40 pmol siRNA FAM alone or complexed with TRAF as described in the methods. First row: siRNA FAM alone; second row: TRAF:siRNA FAM; third row: Lipofectamine (Lipofec) siRNA FAM complexes.
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Figure 3. Trafficking of TRAF:siRNA complexes in SK-OV-3 cells; (A) confocal fluorescence microscopy images of cells after 10 min of treatment with either siRNACy3 alone (upper panel) or with TRAF A-633 (red):siRNACy3 (blue) complexes. Nuclei were counter-stained with Hoechst (cyan). Pink color in the merged panel represents colocalization of Alexa fluor-633 and Cye-3 dyes. (B) Kinetics of TRAF A-633:siRNACy3 in SK-OV-3 expressing GFP tagged RAB5 (green) protein at indicated time points. Pearson's coefficient of correlation was calculated for determining the percentage of colocalization (see Supplementary Figure 9). Bottom panel represents magnified images corresponding to the areas enclosed in the box. White spots (shown by white arrows) correspond to colocalization of TRAFA633 (red), siRNACy3 (blue) and RAB5 protein (green). Pink color (shown by pink arrows) represents co-localization of TRAFA633 (red) and siRNACy3 (blue).
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The efficiency of gene silencing by siRNA delivered by TRAF was monitored by estimating the mRNA and the protein levels of GAPDH, AURKB and HER-2 genes in SK-OV-3 and MDA-MB-231. SK-OV-3 cells treated with TRAF-bound AURKB siRNA showed a dose-dependent decrease in the expression of AURKB gene, both at mRNA and protein levels (Figure 4, A and B). In addition, we also assessed the knockdown of HER-2 genes by immunofluorescence assays and observed a decrease in the expression of HER-2 receptor levels compared to cells treated with scrambled siRNA (Figure 4, C). However, TRAF-mediated delivery of GAPDH-siRNA to MDA-MB-231 did not result in any gene silencing, which further confirms the role of HER-2 in the uptake of these complexes (Supplementary Figure 13). 36 Similar results were also obtained in MDA-MB-453 cells (Supplementary Figure 14). Moreover, cells treated either with siRNA, TRAF alone or TRAF: siRNA complexes did not reveal any cytotoxic effect as confirmed by MTT assay (Supplementary Figure 15).
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siRNA delivered by TRAF silences endogenous gene expression in HER-2 + cells
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Figure 4. Specific gene silencing in SK-OV-3 cells mediated by siRNA: (A) AURKB mRNA levels, determined by probe-based qRT-PCR, in SK-OV-3 cells after 48 h of treatment as described in methods. The data were normalized with β-actin mRNA levels. AURKB mRNA levels in untreated cells, defined as 1, were used to calculate the relative mRNA expression in treated cells. siRNA complexed with Lipofectamine (Lipofec) was used as a positive control. Scrambled siRNA (ScsiRNA) complexed with TRAF at 50 nM was used as a negative control. Results are shown as mean ± S.E. of three independent experiments (⁎⁎P b 0.05 and ⁎⁎⁎P b 0.005 were considered statistically significant when compared to PBS control, IC50 ~ 13.65 nM). (B) Immunoblotting of AURKB or β-actin (normalization control) expression in SK-OV-3 cells after 72 h of treatment. Bars in (A) correspond to the lanes in (B). (C) Immunofluorescence of SK-OV-3 cells, depicting HER2 expression. The cells were treated with HER-2 siRNA (50 nM) complexed with either TRAF or Lipofectamine. Cells treated with either PBS or scrambled siRNA represent negative control.
Systemic delivery of siRNA:TRAF nanoparticles results in tumor-specific uptake. Based on the promising in vitro results, we examined the pharmacokinetics of the complexes using labeled TRAF A-633:siRNA complexes that were administered intravenously, in a volume of
200 μl, via the tail vein into SK-OV-3 xenograft tumor mice followed by live animal fluorescence imaging. Since TRAF was found to be at optimally active at 5-fold molar excess over siRNA, we chose to fluorescently label TRAF to enable the incorporation of larger amounts of the fluorophore that would facilitate both detection and biodistribution of the systemically-delivered complexes. Besides, the use of Alexa fluor 633, whose fluorescence emission is at a higher wavelength (Emλ = 670 nm) eliminates background noise and reduces photon attenuation in live tissues. 37 Additionally, mice injected with PBS alone did not show any associated background fluorescence. Within 5 h of TRAF A-633:siRNA, we found distinct but diffused fluorescence around the kidneys and tumor, which intensified with time (Figure 5, A). No fluorescence was detected in the liver, spleen and lungs. Accumulation of the complexes in the tumor and kidneys was further confirmed by imaging the organs isolated from the sacrificed mice after 48 h (Figure 5, B). To assess the internalization of the complexes we examined the cryosections from various tissues, isolated from animals after 72 h of treatment. Significant fluorescence was observed only in the tumor and to a small extent in kidney (Supplementary Figure 16). We also assessed the pharmacokinetics of TRAF:siRNA complexes by imaging treated animals at different time points. After 16 h, we observed localization of fluorescence clearly in the tumors and kidney and the signal from the kidneys decreased significantly by 48 h. Fluorescence could be seen in the tumor tissue even at day 6 (Figure 5, C).
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Figure 5. Live-images of animals harboring SK-OV-3 tumor xenografts treated with TRAF A-633:siRNA. (A) Whole body fluorescence of anesthetized animals after 48 h of treatment. 100 μg of labeled TRAF was complexed with siRNA at 5:1 mole ratio in 200 μl of PBS buffer. The complexes were injected intravenously via lateral tail vein. Red spots in the image represent emission of Alexa fluor-633 fluorophores attached to TRAF. (B) Fluorescence images of individual organs of the mice after 48 h of treatment. Red spots in the image indicate presence of fluorescence. K = kidney, S = spleen, L = liver, Ln = Lungs, T = tumor. (C) Pharmacokinetics of TRAF:siRNA complexes after intravenous injection into the animals. Single dose of TRAF:siRNA complexes was given to the animals followed by regular acquisition of fluorescence images. During all experiments, animals were exposed to 620 nm wavelength for 10 s and 670 nm emission filter was used to collect the fluorescence light. For X-ray images, animals were exposed to 5 s for X-ray beam. Inset represents emission of TRAF A-633:siRNA solution under similar conditions.
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To investigate whether siRNA delivered by TRAF could suppress tumor growth by silencing tumor oncogenes, we targeted Aurora B kinase and HER-2 genes. Both the genes have been observed to be overexpressed in a large number of tumors and were implicated in tumorogenesis. 38 -41 It has been observed that reduced expression of AURKB and HER-2 by siRNA-mediated gene silencing, retarded growth of tumors by arresting cells at G1/G0 phase and induced apoptosis. Moreover, combinatorial silencing of AURKB and EGFR in prostate xenograft mouse models by siRNA, has shown synergistic effects as observed by enhanced tumor regression with respect to their individual silencing effects. 42 To determine whether silencing of AURKB and HER-2 genes by siRNA could suppress growth kinetics of SK-OV-3 cells, we performed in vitro cell scratch assay, which mimics cell migration during wound healing in vivo. 43 Migration rates of cells treated with a mixture of AURKB and HER-2 siRNA (50 nM each) (79.6 μm/h) were four times lower when compared to cells treated with scrambled siRNAs (18.3 μm/h), reflecting significant effect on growth kinetics upon knockdown of HER-2 and AURKB genes (Supplementary Figure 17). Based on these results, we used a mixture of AURKB and HER-2 siRNA at 2 mg/kg as described in
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In vivo delivery of siRNA mediated by TRAF suppresses tumor growth
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Figure 6. In vivo gene silencing of HER-2 and AURKB: (A) Graph depicts tumor volume in various treatment groups (n = 6). (B) mRNA expression of AURKB and HER-2 genes in tumor samples of the above mentioned groups. qRT-PCR was run in triplicates and data were analyzed by ΔΔCt method (⁎P ≤ 0.0077, ⁎⁎P ≤ 0.0018 is considered as significant when compared to controls [PBS]). β-actin mRNA level in each sample was used as an internal control. (C) Immunohistochemistry of cryo-sections showing expression level of HER-2 in treated (TRAF complexes with AURKB and HER-2 siRNA) and control (PBS) groups. The nuclei were counter stained with Hoechst (blue) while as HER-2 receptors were detected with antibodies conjugated with FITC (green). B and C represent data of tissues taken from animals after the end of tumor suppression studies (day 15).
methods, by complexing them with TRAF at 5:1 mole ratio in a tumor suppression experiment. Treatment groups that received TRAF: siRNA nanoparticles showed clear and significant suppression in tumor growth with respect to controls, indicating efficient delivery of siRNA mediated by TRAF (Figure 6, A). Further analysis of tumor samples at mRNA and protein levels revealed significant silencing of HER-2 and AURKB gene expression in mice treated with TRAF:siRNA complexes (Figure 6, B and C) However, we did not observe silencing of AURKB and HER-2 genes in all the controls, signifying the role of TRAF in mediating the delivery of therapeutic siRNA into tumor tissues. To assess whether TRAF:siRNA complexes elicit any innate immune response, siRNA, TRAF and TRAF:siRNA complexes were intravenously administered into the balb/c mice. After 12 h to 16 h of post-injection, we determined the serum level of IL-6, TNF-α and IFN-γ cytokines. The serum levels of these cytokines are known to increase considerably due to activation of innate immune response. 44 Administration of siRNA at 2 mg/kg dosage led to significant increase in IL-6, TNF-α and IFN-γ cytokines (Supplementary Figure 18). Interestingly, administration of same amount of siRNA as TRAF:siRNA abrogated immunostimulatory effect of siRNA. The inhibitory effect of TRAF to induce immune response by siRNA could be due to masking of siRNA by TRAF molecules, thereby, preventing binding of siRNA to toll-like receptors.
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Efficient delivery of siRNA is a challenge for realizing its gene silencing potential as these approaches involve protection of siRNA from degradation and cellular uptake followed by timely release inside the cell. Besides, it is important for the delivery vehicle to be stable and free from toxicity. Natural or biosimilar entities have shown promising results in delivering siRNA compared to vehicles that are complex or prepared with synthetic materials. 1,3,5 Moreover, nature has evolved a large repertoire of peptides and proteins that can be engineered to impart target specificity to modulate a variety of functions. In our design, we fused a natural dsRBD protein scaffold of TRBP to an evolved HER-2 antibody mimetic affibody to specifically deliver siRNA into HER-2 positive cancer cells. The overexpressed chimeric protein has shown stability and excellent specificity as observed by target-specific gene silencing in vitro and in vivo. Cellular trafficking studies showed that TRAF has appropriate siRNA releasing properties thus enhancing the bioavailability and targeted gene silencing.
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Design of TRAF
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Cationic polymers and cationic amphiphile structures are considered as gold standard in carrying nucleic acids including siRNA efficiently into the cells in vitro. 23 However, electrostatic interaction between siRNA and cationic polymers leads to the formation of heterogeneous aggregates that are susceptible to adhesion of serum proteins, resulting in rapid extravasation from the blood. 1,3 Additionally, cationic polymers such as polyarginine, protamine sulfate and several cell penetrating peptide sequences are difficult to synthesize or express in heterologous systems. 45 To circumvent these issues, we took advantage of naturally available dsRNA-binding domains whose conformation-dependent binding to dsRNA eliminates the requirement of positively charged peptide for binding to siRNA. In two independent studies pertaining to dsRBD, kinase DRBD and p19 RBD were successfully employed for siRNA delivery. However, these fusion proteins lack tissue specificity and therefore, were limited to only primary cell lines. In addition, subsequent studies found that single dsRBD of protein kinase R was insufficient to stably bind siRNA when fused to targeting peptides other than TAT peptide. 14 Designed TRAF reported here, employs the second domain of TRBP2 that binds siRNA with high affinity (kd 108 nM) and therefore does not require auxiliary domains for binding to siRNA. Stability to variations in pH, temperature or treatment with RNases, serum and heparin further confirm that binding of TRAF to siRNA is very robust. Moreover, fusion of the domain with affibody peptide did not affect its binding affinity as the dissociation constant of TRAF matched with the values reported earlier (113 nM) for the individual domain. 45 FACS-based competition studies between pure affibody and TRAF suggest that the folding of the affibody and receptor-binding properties in TRAF was uncompromised. Importantly, TRAF:siRNA complexes were non-toxic and TRAF reduces the immunostimulatory effect of siRNA in the Balb/c mice. dsRNA is known to trigger innate immune response
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One of the crucial steps in siRNA delivery is its availability to the RISC complex for mediating knockdown of desired genes. Majority of the complexes find their way into the endosomes after taking different entry routes. As endosomes face multiple fates like retrograde or lysosomal route, it is important that siRNA is released from the endosomes into the cytoplasm. 46 Recently it has been observed that only 1-3% of the total siRNA internalized is released into the cytoplasm during early endosomal trafficking events by an unknown mechanism. 47 To increase the efficiency of escape of nanoparticles from endosomes, many strategies including multiple histidines, endosomolytic peptides, cationic peptide or lipids, have been tested with success. It is hypothesized that these peptides enhance endosomal escape of the particles by increasing the osmotic pressure that causes swelling and leakage of the endosomes – a process called proton sponge effect. 46 -49 We believe that the presence of poly-histidine tag in the protein and high uptake of the complexes might have assisted in endosomal escape of the complexes. Colocalization with transferrin suggested clathrinmediated uptake of the complexes. Also labeling early and late endosomes, by expressing GFP tagged RAB-5 and LAMP-1 genes respectively, we were able to monitor the trafficking events of the complexes during transition from early to late endosomes and their eventual release into the cytoplasm. Many cancer intervention methods involve multiple injections of a drug to maintain a constant therapeutic dose. Frequent injections or controlled release strategies have been experimentally tested to maintain the dose. However, non-target distribution and non-specific binding reduce the effective concentrations of the drug at the tumor site. Smaller nanoparticles (b 50 nm) are known to extravasate into the tumor efficiently, while particles of 100 nm sizes may accumulate in target tissue but have poor ability to enter the interstitial spaces. 50 TRAF bound siRNA shows tropism to tumors and also a negative bias toward organs such as liver, spleen and lung. Tumor bias in nanoparticle distribution could be due to specific retention of the nanoparticles in tumors due to HER-2 expression and enhanced permeability and retention (EPR) effect, while poor accumulation in other organs highlights the importance of receptor-specific uptake of nanoparticles. In our earlier study, it was demonstrated that a fusion protein, designed to deliver plasmid DNA, was very effective in regressing ErbB 2 positive MDA-MB-453 derived tumors but ineffective in ErbB 2 minus MDA-MB-231 cells, thus, revealing importance of ErbB2 receptor in tumor targeted delivery of nucleic acids by ErbB2 binding fusion proteins. 51 Ease in synthesis of TRAF and its low toxicity and ability to simultaneously deliver more than one siRNA specifically to tumors, make TRAF a promising delivery vehicle. This is particularly important for treatment of cancer where silencing multiple genes efficiently is crucial. Among various gene targets,
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after binding to toll like receptors such as TL-3, TL-7 and TL-9. Therefore, encapsulating siRNA as in this study could drastically reduce the immune response.
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Appendix A. Supplementary data
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References
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Graphical Abstract
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Conformation-dependent binding and tumor-targeted delivery of siRNA by a designed TRBP2: Affibody fusion protein
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Ghulam Hassan Dar, Vijaya Gopal ⁎, Madhusudhana Rao ⁎
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Recombinant protein with HER-2 targeting and siRNA binding motifs for efficient in vivo siRNA delivery to tumors
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Ghulam Hassan Dar, Vijaya Gopal⁎, Madhusudhana Rao⁎
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CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India Received 8 September 2014; accepted 19 January 2015
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Conformation-dependent binding and tumor-targeted delivery of siRNA by a designed TRBP2: Affibody fusion protein
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Efficiency of systemically delivered siRNA in gene silencing is compromised due to lack of target-specific delivery and rapid clearance of siRNA by in vivo elimination pathways. We designed a fusion protein consisting of a dsRNA binding domain of transactivation response RNA binding protein (TRBP2) fused to ErbB2 binding affibody (AF) for target specific delivery of siRNA. Designated as TRAF, the fusion protein is stable and binds efficiently and specifically to siRNA, forming homogenous non-aggregated and nuclease-resistant particles that efficiently and selectively transport siRNA into HER-2 overexpressing cancer cells and tissues. Administration of siRNA by TRAF into cells resulted in significant silencing of chosen genes involved in cell proliferation viz. AURKB and ErbB2. Noticeably, intravenous administration of TRAF:siRNA against these genes resulted in remarkable tumor suppression in the SK-OV-3 xenograft mouse model. Our results establish the potential of engineered proteins for specific and systemic delivery of siRNA for cancer therapy. © 2015 Published by Elsevier Inc.
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Key words: Affibody; Epidermal growth factor receptor 2; Tumor-targeting; siRNA delivery; TRBP2; Fusion proteins
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RNA interference holds a great potential in cancer therapy due to its high specificity and ability to efficiently silence a large number of different genes involving a number of distinct cellular pathways. 1,2 This is particularly important for a disease as complex as cancer. However, as in other drug targeting therapies, the major challenge for therapeutic use of siRNA is the need for devising safe, stable and effective carriers capable of delivering low doses of siRNA into the cytoplasm. A large number of carriers including organic polymers, lipids and metal nanoparticles have been tested with variable success. 3,4 Successful vehicles designed so far utilized non-covalent complexation of siRNA. 5 While these complexes are sufficient for in vitro cell-based experiments, intravenous administration of these complexes would lead to promiscuous electrostatic interactions with serum proteins, aggregation and nonspecific uptake, leading to low efficiency. 6,7
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We gratefully acknowledge financial support from CSIR (BSC0112). GHD is also grateful to CSIR for a Senior Research Fellowship. ⁎Corresponding authors at: CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500007. A. P. India. E-mail addresses: [email protected], [email protected] (V. Gopal), [email protected] (M. Rao).
Peptides and fusion proteins of varied combinations are emerging as promising vehicles for tissue specific delivery of siRNA. 8 Peptides offer immense sequence variations and could be made target specific by evolving them either by phage display methods or sourcing them from natural sequences. 9,10 Moreover, major advantages of designed modular proteins are the convenience in fine tuning the ratio between payload (siRNA) and carrier, ease in scaling up and their low toxicity than either cationic lipids or polymers. Several targeting peptides attached to either protamine or polyarginine have been successfully used for in vivo delivery of siRNA to cells expressing HIV envelope glycoprotein gp160, 11 primary lymphocytes via integrin ScFvs, 12 and transvascular delivery to brain, 13 However, there have been reports where the presence of positively charged peptides in the fusion proteins could lead to undesired properties such as interaction with serum glycosaminoglycans, 14-16 aggregation at higher concentrations, heterogeneous sizes of peptide-based nanoparticles 17 and difficulty in scaling up production. 18 Double-stranded RNA-binding proteins (dsRBP) offer an excellent alternative over charge-based motifs for conformation– specific binding to siRNA rather than forming ionic complexes of variable sizes. 19 These structurally well-defined domains recognize the A-form of double helix RNA via 2′–OH ribose groups present on minor-major-minor groove of dsRNA. 20 A
http://dx.doi.org/10.1016/j.nano.2015.01.017 1549-9634/© 2015 Published by Elsevier Inc. Please cite this article as: Dar G.H., et al., Conformation-dependent binding and tumor-targeted delivery of siRNA by a designed TRBP2: Affibody fusion protein. Nanomedicine: NBM 2015;xx:1-12, http://dx.doi.org/10.1016/j.nano.2015.01.017
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Methods
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Details of the methods are provided in supporting information
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Cell culture SK-OV-3, MDA-MB-231 and MDA-MB-453 were obtained Medina-Lai Kauwe, University of Southern California Keck School of Medicine, CA. Other cells were obtained from lab stocks. These were maintained in a Dulbecco's Modified Eagle Medium (DMEM): Nutrient Mixture F-12 (DMEM/F-12, Life Technologies) with 10% fetal bovine serum (Life Technologies).
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Flow cytometry and confocal microscopy Cells grown in tissue culture flasks were trypsinized and pelleted. Approximately 50,000 cells were incubated with 40 pmol of FAM-labeled siRNA either alone or complexed with TRAF protein at 5:1 mole ratio. Cells treated with Lipofectamine (Life Technologies) complexed with FAM-labeled siRNA were used as positive control. After 1 h incubation on rotor torque at room temperature, cells were washed with ice-cold PBS containing 1% fetal calf serum, 20 U/ml heparin sulfate and 50 mM ammonium chloride and then analyzed on a FACSCalibur with Cell Quest software (Becton Dickson). For confocal microscopy, cells seeded on coverslips in Petri dishes were transfected with LAMP-1-GFP and RAB-5-GFP plasmid using Lipofectamine. After 48 h, cells were treated with 20 pmol of fluorescent TRAF:siRNA complex (5:1 mole ratio). At the indicated time period, cells were washed with PBS containing 20 U/ml heparin sulfate, stained with Hoechst 33258
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Design, purification and structural properties of TRAF
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Multispectral in vivo fluorescence imaging Female athymic BALB/c-neu mice were used in accordance with the approval from Institute of Animal Ethics Committee (IAEC) (vide IAEC project # 03/11). SK-OV-3 cells (4 million) were resuspended in Matrigel (BD Biosciences) and injected subcutaneously into mice. After the tumors attained 50– 100 mm 3 volumes, TRAF-labeled with Alexa Fluor 633 (TRAF A633) was complexed with siRNA (5:1 mole ratio) and injected by tail vein injection. The mice were imaged using the multispectral in vivo imager (Carestream Molecular Imaging) after indicated time. Snap frozen organs were cryosectioned (4 μm) and stained with Hoechst 33258. Details of methods of tumor suppression by TRAF-mediated siRNA delivery investigated using xenograft tumor model developed in nude mice are provided in the support information.
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(Molecular probes) and fixed with 4% paraformaldehyde (Sigma). The coverslips were mounted in Vectashield mounting medium H-100 (Vector Laboratories, Inc.) and analyzed by confocal laser microscopy (Leica LAS-AF-TCS-SP5). Images were analyzed by LAS AF software.
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dsRBP from IF-activated protein kinase fused in tandem with peptide transduction domain (PTD) was the first successful example for siRNA delivery into primary and transformed cells and was shown to be immunologically and toxicologically safe. 21 However, due to PTD domains and low binding affinity of dsRBD of protein kinase to siRNA, the designed fusion protein delivered siRNA non-specifically in vitro and in vivo. 14 In addition, there are very few studies where fusion proteins have been used to systemically deliver therapeutic-siRNA for suppressing the tumor growth. 22 -24 Human TRBP2 protein, a double-stranded RNA binding protein, plays an important role in RNA interference. 25 TRBP has three dsRBPs and only the first and second domain show high affinity, 220 and 113 nM respectively, to siRNA. 26,27 Based on this, the second domain of TRBP was cloned with well characterized ErbB2-binding affibody for tumor-targeted delivery of siRNA. Erbb2 receptor is a well-known cell-surface marker overexpressed in a majority of tumors. 28 ErbB2 binding affibody (5.8 kDa) molecule, originally evolved from the Z-domain of Staphylococcus aureus protein A, was chosen as the targeting ligand owing to its high affinity to ErbB2 receptors. 27 The recombinant fusion protein (TRAF) was expressed in Escherichia coli and purified to homogeneity by affinity chromatography. In this study, TRAF was used as a siRNA carrier to deliver siRNA specifically into HER-2 expressing cells. To maximize silencing-mediated tumor suppression, we simultaneously delivered siRNA against two genes, HER-2 and AURKB, which are involved in cell proliferation.
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TRAF protein was designed by fusing second domain of TRBP2 (due to its higher affinity) to the DNA sequence of ZHER-2 affibody molecule. To aid proper folding, five glycine codons were inserted between the two domains. During purification, we noticed bacterial RNA associated with TRAF (data not shown) that was removed by adding urea in the lysis buffer. Additionally, 0.1% Triton-X114 was used to remove bacterial endotoxins. 29 The purified protein was characterized by SDS-PAGE and mass spectrometry that revealed a single band of 18.485 kDa matching the predicted size of the protein (Figure 1, A and Supplementary Figure 1). With these purification procedures, we normally obtain yields of ~ 40 mg of purified TRAF protein from one liter of bacterial culture. Far-UV circular dichroism spectra of the TRAF protein showed the presence of a well-defined secondary structure unperturbed at either pH 5.8 or pH 7.8 (Figure 1, B). The percentage of α-helical and α-pleated structures was found to be 56.6% and 18%, respectively. Higher percentage of α-helical structures is also supported by a negative band at 208 nm and 222 nm of far-UV CD data. 30 Thermal stability of TRAF monitored by examining the change in ellipticity at 222 nm revealed a Tm of 62 °C (Supplementary Figure 2). To determine whether binding of TRAF to siRNA resulted in any aggregation of the complexes, we assessed the size of TRAF either alone or in complex with siRNA by dynamic light scattering (DLS). The average diameter of the protein at 2 mg/ml was found to be 2 ± 0.1 nm, while the addition of siRNA (at 5:1 mole ratio of TRAF: siRNA) resulted in particles of mean dimension 6 ± 0.3 nm (Figure 1, C). In addition, the polydispersity index of TRAF and TRAF:siRNA complexes was in the range of 0.1, suggesting homogenous and non-aggregated population of the particles. The sizes of TRAF and TRAF:siRNA complexes were also confirmed by size exclusion chromatography (Supplementary Figure 3). The
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Figure 1. Characterization of recombinant fusion protein TRAF: (A) SDS-PAGE of TRAF. Lane 2 represents purified TRAF. (B) Far-UV CD spectra of TRAF at pH 5.8 and pH 7.8. (C) Hydrodynamic diameter of TRAF and TRAF:siRNA complexes (5:1) measured by dynamic light scatter. (D) Binding of siRNA with TRAF by gel-shift assay. 20 pmol of siRNA was incubated with increasing mole ratios of TRAF. (E and F) ITC-thermograph of TRAF with siRNA (E) and oligo duplex DNA (F). Upper panel represents differential power with time. Lower panel shows integrated energy per injections. (G) RNaseA protection assay of TRAF:siRNA complexes. Details are provided in the support information.
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To ascertain whether TRAF could deliver siRNA specifically into HER-2 positive cancer cells, we initially confirmed the expression levels by western blotting and immunocytochemistry using two HER-2 positive and two HER-2 negative cell lines. SK-OV-3, an ovarian cancer cell line showed the highest expression of HER-2 receptor followed by MDA-MB-453 cells (Figure 2, A and Supplementary Figure 7). However, both MCF-7 and MDA-MB-231 stained negative for HER-2. In MCF-7 cells, we observed HER-2 receptor molecules localized in nucleus of the cells. Similar type of an observation was noticed earlier and it was shown that these receptors also act as a transcription factors to many genes such as cyclin D1, Cox and p53. 32 Due to overexpression of HER-2 receptors, we used SK-OV-3 and MDA-MB-453 cells as HER-2 positive cell lines (designated as HER-2 +) while MCF-7 and MDA-MB-231
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To ascertain that siRNA uptake mediated by TRAF is receptor-specific and also to visualize their localization in the cytoplasm at different time points, we labeled TRAF and siRNA with Alexa fluor-633 and Cy3 dyes respectively. Furthermore, to monitor the trafficking of labeled TRAF:siRNA complexes in early and late endosomes, we used SK-OV-3 cells, expressing either GFP-tagged RAB 5 (early endosomal marker) or GFP-tagged lysosomal associated membrane protein 1(LAMP-1), which is present on the surfaces of late endosomes and lysosomes. 33,34 After incubating TRAF A633:siRNA Cy3 complexes for 10 min to allow binding, unbound complexes were removed by PBS washes. No fluorescence was seen in cells incubated with siRNA Cy3 alone. However, within 10 min of incubation, the labeled complexes were seen associated with the cell surface (Figure 3, A). The colocalization of fluorescent probes on the cell surface decreased with time and after 20 min, fluorescence of TRAF A633:siRNA Cy3 complexes were found merging with the fluorescence of GFP-tagged RAB-5 protein, suggesting internalization by endosomal-mediated pathway. Colocalization with RAB-5 indicated that the complexes were in the early-endosomes (Figure 3, B and Supplementary Figure 9). After 1 h of incubation, the colocalization of TRAF A633:siRNA Cy3 complexes with RAB-5 decreased drastically and no colocalization was seen after 5 h. 33,34 Interestingly, within 1 h of incubation, fluorescence of TRAF:siRNA complexes showed strong colocalization with GFP-tagged LAMP-1 protein, indicating the presence of complexes within the late endosomes (Supplementary Figures 10 and 11). Maximal colocalization at 2 h, reflected gradual increase in the transition from early to late endosomes. After 5 h, there was a significant release of the complexes from late endosomes as indicated by a decrease in the colocalization of fluorescent probes. Interestingly, we also observed dissociation of TRAF and siRNA from each other after 5 h of treatment, a time point where they are also released from endosomes. Taken together, the data suggested efficient internalization of the complexes after binding to the HER-2 receptor of the cells followed by the transition from early to late endosomes and eventual release of the complexes into the cytoplasm. Further colocalization studies with transferrin protein, a positive control for clathrin-mediated endocytosis, revealed internalization via clathrin-mediated pathways (Supplementary Figure 12). 35
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To determine the binding of TRAF with siRNA, we initially performed gel-shift assay by preparing complexes at various mole ratios. A faint siRNA band at 1:1 mole ratio and absence of bands at higher mole ratios indicated strong binding of TRAF to siRNA (Figure 1, D). In addition, incubation of the TRAF:siRNA complexes in serum for 6 h at 37 °C did not alter the binding (Supplementary Figure 4). However, incubation of TRAF with oligo DNA of 21 bp length did not show any binding (Supplementary Figure 5). Detailed structural studies of dsRBDs have revealed that the absence of strong ionic interaction, crucial interactions with the 2-hydroxyl groups of dsRNA and preference for binding to A-form (preferred conformation for dsRNA) rather than B-form (preferred conformation of DNA) prevents binding of dsRBD to either dsDNA or RNA-DNA hybrids. 31 To determine the binding affinity we performed isothermal titration calorimetry of purified TRAF and siRNA. Addition of TRAF to siRNA resulted in efficient release of energy that decreased gradually with addition of protein and reached a steady state, reflecting complete binding of TRAF to siRNA (Figure 1, E). When fitted with a model describing single set of identical binding sites, a binding constant of 107 nM was obtained for TRAF, which is similar to that reported (113 nM) for an isolated TRBP2. 27 The stoichiometry factor of ~ 2 suggested that 2 molecules of TRAF bind to one molecule of siRNA. Notably, we did not observe any enthalpy change after addition of TRAF to DNA of similar length, thus, corroborating the specificity of TRAF to siRNA (Figure 1, F). To verify whether TRAF could prevent siRNA degradation, we incubated siRNA alone and TRAF:siRNA complexes with RNase A for 1 h at 37 °C. siRNA complexed with TRAF at higher ratios offered complete protection, which indicates inaccessibility of nucleases to siRNA, while the absence of TRAF led to siRNA degradation (Figure 1G). Similar results were obtained when complexes were treated with human serum (Supplementary Figure 6).
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represented HER-2 negative cells (designated as HER-2 -). After treatment of the cells with TRAF:siRNA FAM complexes, we determined the uptake by flow cytometry. Lipofectamine complexed with siRNA was used as a positive control. As anticipated, siRNA FAM uptake was inefficient in all cell lines due to its large size and negative charge. In contrast, siRNA FAM bound TRAF complexes showed higher uptake in SK-OV-3 cells followed by MDA-MB-453 cells in contrast to MDA-MB-231 and MCF-7 cells, reflecting receptor-specific uptake of siRNA (Figure 2, B). To further verify the specificity, free affibody at different mole ratios was used as a competitor of TRAF:siRNA complex to HER-2 receptors. Uptake of siRNA FAM complexed with TRAF, decreased significantly (N 95%) in presence of affibody while no such effect was observed when siRNA FAM was complexed with Lipofectamine (Supplementary Figure 8).
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Figure 2. Uptake of TRAF:siRNA complexes by HER2 positive cells: (A) Immunofluorescence images of SK-OV-3 and MCF-7 cells treated with either HER-2 monoclonal antibody (HER-2 mAb) (left) or isotype control antibody (right). Arrows in MCF-7 panel indicate the presence of HER2 receptor in the nucleus. (B) Representative FACS profiles of cells treated with either 40 pmol siRNA FAM alone or complexed with TRAF as described in the methods. First row: siRNA FAM alone; second row: TRAF:siRNA FAM; third row: Lipofectamine (Lipofec) siRNA FAM complexes.
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Figure 3. Trafficking of TRAF:siRNA complexes in SK-OV-3 cells; (A) confocal fluorescence microscopy images of cells after 10 min of treatment with either siRNACy3 alone (upper panel) or with TRAF A-633 (red):siRNACy3 (blue) complexes. Nuclei were counter-stained with Hoechst (cyan). Pink color in the merged panel represents colocalization of Alexa fluor-633 and Cye-3 dyes. (B) Kinetics of TRAF A-633:siRNACy3 in SK-OV-3 expressing GFP tagged RAB5 (green) protein at indicated time points. Pearson's coefficient of correlation was calculated for determining the percentage of colocalization (see Supplementary Figure 9). Bottom panel represents magnified images corresponding to the areas enclosed in the box. White spots (shown by white arrows) correspond to colocalization of TRAFA633 (red), siRNACy3 (blue) and RAB5 protein (green). Pink color (shown by pink arrows) represents co-localization of TRAFA633 (red) and siRNACy3 (blue).
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The efficiency of gene silencing by siRNA delivered by TRAF was monitored by estimating the mRNA and the protein levels of GAPDH, AURKB and HER-2 genes in SK-OV-3 and MDA-MB-231. SK-OV-3 cells treated with TRAF-bound AURKB siRNA showed a dose-dependent decrease in the expression of AURKB gene, both at mRNA and protein levels (Figure 4, A and B). In addition, we also assessed the knockdown of HER-2 genes by immunofluorescence assays and observed a decrease in the expression of HER-2 receptor levels compared to cells treated with scrambled siRNA (Figure 4, C). However, TRAF-mediated delivery of GAPDH-siRNA to MDA-MB-231 did not result in any gene silencing, which further confirms the role of HER-2 in the uptake of these complexes (Supplementary Figure 13). 36 Similar results were also obtained in MDA-MB-453 cells (Supplementary Figure 14). Moreover, cells treated either with siRNA, TRAF alone or TRAF: siRNA complexes did not reveal any cytotoxic effect as confirmed by MTT assay (Supplementary Figure 15).
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Figure 4. Specific gene silencing in SK-OV-3 cells mediated by siRNA: (A) AURKB mRNA levels, determined by probe-based qRT-PCR, in SK-OV-3 cells after 48 h of treatment as described in methods. The data were normalized with β-actin mRNA levels. AURKB mRNA levels in untreated cells, defined as 1, were used to calculate the relative mRNA expression in treated cells. siRNA complexed with Lipofectamine (Lipofec) was used as a positive control. Scrambled siRNA (ScsiRNA) complexed with TRAF at 50 nM was used as a negative control. Results are shown as mean ± S.E. of three independent experiments (⁎⁎P b 0.05 and ⁎⁎⁎P b 0.005 were considered statistically significant when compared to PBS control, IC50 ~ 13.65 nM). (B) Immunoblotting of AURKB or β-actin (normalization control) expression in SK-OV-3 cells after 72 h of treatment. Bars in (A) correspond to the lanes in (B). (C) Immunofluorescence of SK-OV-3 cells, depicting HER2 expression. The cells were treated with HER-2 siRNA (50 nM) complexed with either TRAF or Lipofectamine. Cells treated with either PBS or scrambled siRNA represent negative control.
Systemic delivery of siRNA:TRAF nanoparticles results in tumor-specific uptake. Based on the promising in vitro results, we examined the pharmacokinetics of the complexes using labeled TRAF A-633:siRNA complexes that were administered intravenously, in a volume of
200 μl, via the tail vein into SK-OV-3 xenograft tumor mice followed by live animal fluorescence imaging. Since TRAF was found to be at optimally active at 5-fold molar excess over siRNA, we chose to fluorescently label TRAF to enable the incorporation of larger amounts of the fluorophore that would facilitate both detection and biodistribution of the systemically-delivered complexes. Besides, the use of Alexa fluor 633, whose fluorescence emission is at a higher wavelength (Emλ = 670 nm) eliminates background noise and reduces photon attenuation in live tissues. 37 Additionally, mice injected with PBS alone did not show any associated background fluorescence. Within 5 h of TRAF A-633:siRNA, we found distinct but diffused fluorescence around the kidneys and tumor, which intensified with time (Figure 5, A). No fluorescence was detected in the liver, spleen and lungs. Accumulation of the complexes in the tumor and kidneys was further confirmed by imaging the organs isolated from the sacrificed mice after 48 h (Figure 5, B). To assess the internalization of the complexes we examined the cryosections from various tissues, isolated from animals after 72 h of treatment. Significant fluorescence was observed only in the tumor and to a small extent in kidney (Supplementary Figure 16). We also assessed the pharmacokinetics of TRAF:siRNA complexes by imaging treated animals at different time points. After 16 h, we observed localization of fluorescence clearly in the tumors and kidney and the signal from the kidneys decreased significantly by 48 h. Fluorescence could be seen in the tumor tissue even at day 6 (Figure 5, C).
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Figure 5. Live-images of animals harboring SK-OV-3 tumor xenografts treated with TRAF A-633:siRNA. (A) Whole body fluorescence of anesthetized animals after 48 h of treatment. 100 μg of labeled TRAF was complexed with siRNA at 5:1 mole ratio in 200 μl of PBS buffer. The complexes were injected intravenously via lateral tail vein. Red spots in the image represent emission of Alexa fluor-633 fluorophores attached to TRAF. (B) Fluorescence images of individual organs of the mice after 48 h of treatment. Red spots in the image indicate presence of fluorescence. K = kidney, S = spleen, L = liver, Ln = Lungs, T = tumor. (C) Pharmacokinetics of TRAF:siRNA complexes after intravenous injection into the animals. Single dose of TRAF:siRNA complexes was given to the animals followed by regular acquisition of fluorescence images. During all experiments, animals were exposed to 620 nm wavelength for 10 s and 670 nm emission filter was used to collect the fluorescence light. For X-ray images, animals were exposed to 5 s for X-ray beam. Inset represents emission of TRAF A-633:siRNA solution under similar conditions.
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To investigate whether siRNA delivered by TRAF could suppress tumor growth by silencing tumor oncogenes, we targeted Aurora B kinase and HER-2 genes. Both the genes have been observed to be overexpressed in a large number of tumors and were implicated in tumorogenesis. 38 -41 It has been observed that reduced expression of AURKB and HER-2 by siRNA-mediated gene silencing, retarded growth of tumors by arresting cells at G1/G0 phase and induced apoptosis. Moreover, combinatorial silencing of AURKB and EGFR in prostate xenograft mouse models by siRNA, has shown synergistic effects as observed by enhanced tumor regression with respect to their individual silencing effects. 42 To determine whether silencing of AURKB and HER-2 genes by siRNA could suppress growth kinetics of SK-OV-3 cells, we performed in vitro cell scratch assay, which mimics cell migration during wound healing in vivo. 43 Migration rates of cells treated with a mixture of AURKB and HER-2 siRNA (50 nM each) (79.6 μm/h) were four times lower when compared to cells treated with scrambled siRNAs (18.3 μm/h), reflecting significant effect on growth kinetics upon knockdown of HER-2 and AURKB genes (Supplementary Figure 17). Based on these results, we used a mixture of AURKB and HER-2 siRNA at 2 mg/kg as described in
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Figure 6. In vivo gene silencing of HER-2 and AURKB: (A) Graph depicts tumor volume in various treatment groups (n = 6). (B) mRNA expression of AURKB and HER-2 genes in tumor samples of the above mentioned groups. qRT-PCR was run in triplicates and data were analyzed by ΔΔCt method (⁎P ≤ 0.0077, ⁎⁎P ≤ 0.0018 is considered as significant when compared to controls [PBS]). β-actin mRNA level in each sample was used as an internal control. (C) Immunohistochemistry of cryo-sections showing expression level of HER-2 in treated (TRAF complexes with AURKB and HER-2 siRNA) and control (PBS) groups. The nuclei were counter stained with Hoechst (blue) while as HER-2 receptors were detected with antibodies conjugated with FITC (green). B and C represent data of tissues taken from animals after the end of tumor suppression studies (day 15).
methods, by complexing them with TRAF at 5:1 mole ratio in a tumor suppression experiment. Treatment groups that received TRAF: siRNA nanoparticles showed clear and significant suppression in tumor growth with respect to controls, indicating efficient delivery of siRNA mediated by TRAF (Figure 6, A). Further analysis of tumor samples at mRNA and protein levels revealed significant silencing of HER-2 and AURKB gene expression in mice treated with TRAF:siRNA complexes (Figure 6, B and C) However, we did not observe silencing of AURKB and HER-2 genes in all the controls, signifying the role of TRAF in mediating the delivery of therapeutic siRNA into tumor tissues. To assess whether TRAF:siRNA complexes elicit any innate immune response, siRNA, TRAF and TRAF:siRNA complexes were intravenously administered into the balb/c mice. After 12 h to 16 h of post-injection, we determined the serum level of IL-6, TNF-α and IFN-γ cytokines. The serum levels of these cytokines are known to increase considerably due to activation of innate immune response. 44 Administration of siRNA at 2 mg/kg dosage led to significant increase in IL-6, TNF-α and IFN-γ cytokines (Supplementary Figure 18). Interestingly, administration of same amount of siRNA as TRAF:siRNA abrogated immunostimulatory effect of siRNA. The inhibitory effect of TRAF to induce immune response by siRNA could be due to masking of siRNA by TRAF molecules, thereby, preventing binding of siRNA to toll-like receptors.
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Efficient delivery of siRNA is a challenge for realizing its gene silencing potential as these approaches involve protection of siRNA from degradation and cellular uptake followed by timely release inside the cell. Besides, it is important for the delivery vehicle to be stable and free from toxicity. Natural or biosimilar entities have shown promising results in delivering siRNA compared to vehicles that are complex or prepared with synthetic materials. 1,3,5 Moreover, nature has evolved a large repertoire of peptides and proteins that can be engineered to impart target specificity to modulate a variety of functions. In our design, we fused a natural dsRBD protein scaffold of TRBP to an evolved HER-2 antibody mimetic affibody to specifically deliver siRNA into HER-2 positive cancer cells. The overexpressed chimeric protein has shown stability and excellent specificity as observed by target-specific gene silencing in vitro and in vivo. Cellular trafficking studies showed that TRAF has appropriate siRNA releasing properties thus enhancing the bioavailability and targeted gene silencing.
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Cationic polymers and cationic amphiphile structures are considered as gold standard in carrying nucleic acids including siRNA efficiently into the cells in vitro. 23 However, electrostatic interaction between siRNA and cationic polymers leads to the formation of heterogeneous aggregates that are susceptible to adhesion of serum proteins, resulting in rapid extravasation from the blood. 1,3 Additionally, cationic polymers such as polyarginine, protamine sulfate and several cell penetrating peptide sequences are difficult to synthesize or express in heterologous systems. 45 To circumvent these issues, we took advantage of naturally available dsRNA-binding domains whose conformation-dependent binding to dsRNA eliminates the requirement of positively charged peptide for binding to siRNA. In two independent studies pertaining to dsRBD, kinase DRBD and p19 RBD were successfully employed for siRNA delivery. However, these fusion proteins lack tissue specificity and therefore, were limited to only primary cell lines. In addition, subsequent studies found that single dsRBD of protein kinase R was insufficient to stably bind siRNA when fused to targeting peptides other than TAT peptide. 14 Designed TRAF reported here, employs the second domain of TRBP2 that binds siRNA with high affinity (kd 108 nM) and therefore does not require auxiliary domains for binding to siRNA. Stability to variations in pH, temperature or treatment with RNases, serum and heparin further confirm that binding of TRAF to siRNA is very robust. Moreover, fusion of the domain with affibody peptide did not affect its binding affinity as the dissociation constant of TRAF matched with the values reported earlier (113 nM) for the individual domain. 45 FACS-based competition studies between pure affibody and TRAF suggest that the folding of the affibody and receptor-binding properties in TRAF was uncompromised. Importantly, TRAF:siRNA complexes were non-toxic and TRAF reduces the immunostimulatory effect of siRNA in the Balb/c mice. dsRNA is known to trigger innate immune response
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One of the crucial steps in siRNA delivery is its availability to the RISC complex for mediating knockdown of desired genes. Majority of the complexes find their way into the endosomes after taking different entry routes. As endosomes face multiple fates like retrograde or lysosomal route, it is important that siRNA is released from the endosomes into the cytoplasm. 46 Recently it has been observed that only 1-3% of the total siRNA internalized is released into the cytoplasm during early endosomal trafficking events by an unknown mechanism. 47 To increase the efficiency of escape of nanoparticles from endosomes, many strategies including multiple histidines, endosomolytic peptides, cationic peptide or lipids, have been tested with success. It is hypothesized that these peptides enhance endosomal escape of the particles by increasing the osmotic pressure that causes swelling and leakage of the endosomes – a process called proton sponge effect. 46 -49 We believe that the presence of poly-histidine tag in the protein and high uptake of the complexes might have assisted in endosomal escape of the complexes. Colocalization with transferrin suggested clathrinmediated uptake of the complexes. Also labeling early and late endosomes, by expressing GFP tagged RAB-5 and LAMP-1 genes respectively, we were able to monitor the trafficking events of the complexes during transition from early to late endosomes and their eventual release into the cytoplasm. Many cancer intervention methods involve multiple injections of a drug to maintain a constant therapeutic dose. Frequent injections or controlled release strategies have been experimentally tested to maintain the dose. However, non-target distribution and non-specific binding reduce the effective concentrations of the drug at the tumor site. Smaller nanoparticles (b 50 nm) are known to extravasate into the tumor efficiently, while particles of 100 nm sizes may accumulate in target tissue but have poor ability to enter the interstitial spaces. 50 TRAF bound siRNA shows tropism to tumors and also a negative bias toward organs such as liver, spleen and lung. Tumor bias in nanoparticle distribution could be due to specific retention of the nanoparticles in tumors due to HER-2 expression and enhanced permeability and retention (EPR) effect, while poor accumulation in other organs highlights the importance of receptor-specific uptake of nanoparticles. In our earlier study, it was demonstrated that a fusion protein, designed to deliver plasmid DNA, was very effective in regressing ErbB 2 positive MDA-MB-453 derived tumors but ineffective in ErbB 2 minus MDA-MB-231 cells, thus, revealing importance of ErbB2 receptor in tumor targeted delivery of nucleic acids by ErbB2 binding fusion proteins. 51 Ease in synthesis of TRAF and its low toxicity and ability to simultaneously deliver more than one siRNA specifically to tumors, make TRAF a promising delivery vehicle. This is particularly important for treatment of cancer where silencing multiple genes efficiently is crucial. Among various gene targets,
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after binding to toll like receptors such as TL-3, TL-7 and TL-9. Therefore, encapsulating siRNA as in this study could drastically reduce the immune response.
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Graphical Abstract
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Conformation-dependent binding and tumor-targeted delivery of siRNA by a designed TRBP2: Affibody fusion protein
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Ghulam Hassan Dar, Vijaya Gopal ⁎, Madhusudhana Rao ⁎
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CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
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Recombinant protein with HER-2 targeting and siRNA binding motifs for efficient in vivo siRNA delivery to tumors
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