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The therapeutic application of siRNA shows promise as an alternative approach to small molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the difficulty in delivering these large anionic molecules in vivo. In this study, we have investigated whether siRNA-mediated knockdown of p38 MAP kinase mRNA in mouse lung is influenced by conjugation to the non-viral delivery vector cholesterol and the cell penetrating peptides (CPP) TAT(48-60) and penetratin. Initial studies in the mouse fibroblast L929 cell line, showed that siRNA conjugated to cholesterol, TAT(48-60) and penetratin but not siRNA alone achieved a limited reduction of p38 MAP kinase mRNA expression. Intratracheal administration of siRNA resulted in localisation within macrophages and scattered epithelial cells and produced a 30-45% knockdown of p38 MAP kinase mRNA at 6hrs. As with increasing doses of siRNA, conjugation to cholesterol improved upon the duration but not the magnitude of mRNA knockdown whilst penetratin and TAT(48-60) had no effect. Importantly, administration of the penetratin or TAT(48-60) peptides alone caused significant reduction in p38 MAP kinase mRNA expression whilst the penetratin-siRNA conjugate activated the innate immune response. Overall, these studies suggest that conjugation to cholesterol may extend but not increase siRNA mediated p38 MAP kinase mRNA knockdown in the lung. Furthermore, the use of CPP may be limited due to as yet uncharacterized effects upon gene expression and a potential for immune activation.
The discovery of RNA interference (RNAi) and the application of short interference RNA (siRNA) for the knockdown of protein expression through mRNA degradation has revolutionised the area of functional genomics (1, 2). Thus, siRNA macromolecules composed of double stranded RNA 21-23 nucleotides in length are now commonly used in cell based studies to investigate gene function. Furthermore, a number of academic groups and biotechnology companies are investigating the utility of siRNA as a therapeutic approach in the treatment of diseases such as macular degeneration, hepatitis C infection and cancer (3-6).
Compared with small molecule inhibitors, the relatively large size and anionic charge of siRNA means that one of the key barriers to their in vivo use is the availability of effective approaches for delivery both to specific tissues and across the plasma membrane. Since intravenous siRNA administration is subject to first-pass metabolism, unless a liver condition is being targeted (i.e. hepatitis C infection), therapeutic application is limited to tissues where siRNA localisation for extended periods is possible, e.g. following intra-ocular injection for the treatment of macular degeneration. In the case of the lung, the structure of the tissue permits direct access to large numbers of cells within the branching airways and alveoli, an estimated surface area of 400 m2 in man. Importantly, the facility of topical delivery via either intranasal, intratracheal or aerosol administration suggests the lung as an ideal target organ for siRNA-based therapeutics. Indeed, studies have shown siRNA-mediated attenuation of cytokine (7) and heme oxygenase-1 (8) induced expression during acute lung injury. Similarly, intranasal and intratracheal siRNA administration have been shown to prevent para-influenza and respiratory syncytial virus (PIV and RSV respectively) infection in mice (9-12) and severe acute respiratory syndrome (SARS) infection in rhesus monkeys (13).
In the majority of these studies, cationic lipids (e.g. Lipofectin) and polymers (e.g. PEI and dendrimers) have been employed to package and deliver the siRNA to the lung. However, generic application of these strategies is limited by drawbacks such as toxicity and siRNA-mediated induction of immune responses through Toll like receptors (TLR) (14-16). In an attempt to circumvent these problems, we examined the utility of chemical conjugation of siRNA to the non-viral delivery vector cholesterol and the cell penetrating peptides (CPP) penetratin and TAT(48-60) to improve siRNA mediated mRNA knockdown in the mouse lung. Specifically, we attempted to target the constitutively expressed p38 mitogen activated protein (MAP) kinase (also known as MAPK14). Activation of this protein is known to be important in the release of multiple pro-inflammatory mediators including tumour necrosis factor (TNF)-α and interleukin (IL)-1. Indeed, many pharmaceutical companies are developing small molecule inhibitors that target p38 MAP kinase to be used in the treatment of a host of inflammatory diseases including rheumatoid arthritis, Crohn's disease, chronic obstructive pulmonary disease and psoriasis (17, 18).
Chemical conjugation of siRNA to cholesterol has been shown to facilitate intracellular siRNA uptake in vitro (19) and in vivo (20). In the latter case, intravenous administration of cholesterol conjugated siRNA was shown to silence endogenous apolipoprotein B (ApoB) gene expression in the liver and jejunum resulting in decreased plasma levels of apoB protein and total cholesterol (20). Interestingly, a recent study has shown that the delivery of siRNA targeted to ApoB can be further increased ~20-fold compared to cholesterol conjugation by formulation in stabilised nucleic acid lipid particles (SNALP) (21). Additional evidence of the utility of cholesterol for oligonucleotide delivery is provided in a recent report showing cholesterol-mediated delivery of 2′-O-methyl and phosphorothioate-modified single stranded RNA for the knockdown of microRNA expression in the liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenal glands following intravenous injection (22).
The CPP TAT(48-60) and penetratin are short cationic peptides derived from the HIV-1 TAT trans-activator protein (23, 24) and the insect Antennaedia homeoprotein (25) that have been extensively used for the in vitro and in vivo delivery of biologically active peptides and proteins (26, 27). Significantly, CPP have been shown to mediate uptake of a range of biological and non-biological cargos, which has led to the suggestion that they may represent a universal, non-toxic approach for the delivery of oligonucleotides (28-31). Interestingly, although no studies have examined the utility of CPP for the in vivo delivery of siRNA, this possibility is supported by reports showing penetratin mediated delivery of siRNA and antisense into isolated neurons (32-34) and TAT(48-60)-siRNA conjugate mediated knockdown of eGFP and CDK9 expression in HeLa cells (35).
In this report, we demonstrate that intratracheal administration of siRNA achieved a limited attenuation of mRNA expression in the lung for an endogenous, constitutively expressed target, p38 MAP kinase. However, although conjugation to cholesterol, TAT(48-60) or penetratin facilitated knockdown of p38 MAP kinase mRNA in a mouse cell line, only cholesterol-siRNA conjugates were found to influence target gene mRNA levels in vivo. Importantly, since both CPP inhibited p38 MAP kinase mRNA expression in vivo, but not in vitro, we document the existence of as yet uncharacterised CPP bioactivity. Moreover, the detection of innate immune response induction by penetratin-siRNA but not TAT(48-60)-siRNA conjugates suggests differential cellular uptake mechanisms between the two CPP.
Initial screening studies were performed with pre-annealed and PAGE purified siRNA obtained from Dharmacon, Inc. (Lafayette, USA) using the following sequences: A - sense: 5′-GCACACUGAUGAUGAGAUGUU-3′; antisense: 5′-CAUCUCAUCAUCAGUGUGCUU-3′; B - sense: 5′-ACAUUCGGCUGACAUAAUUUU-3′; antisense: 5′-AAUUAUGUCAGCCGAAUGUUU-3′; C - sense: 5′-GGGAGGUGCCCGAACGAUAUU-3′; antisense: 5′-UAUCGUUCGGGCACCUCCCUU-3′; Mismatch 1 (MM1) – sense 5′-CCGAGGUGGCGGAACGAUAUU : antisense : 5′-UAUCGUUCCGCCACCUCGGUU-3′ ; Mismatch 2 (MM2) – sense : 5′-GCGAGCUGCGCGAAGGAUAUU-3′ ; antisense : 5′-UAUCCUUCGCGCAGCUCGCUU-3′.
The synthesis of disulphide linked constructs was performed by Dr Brian Sproat at Integrated DNA Technologies (Leuven, Belgium). Npys activated C-terminal Cys containing TAT(48-60 – grkkrrqrrrppqc) and penetratin (rqikiwfqnrrmkwkkc) peptides with N-terminal acetyl and C-terminal amide functions were obtained from American Peptide Company Inc (Sunnyvale, USA) and conjugated to the 5′-end of the sense strand of sequence C (HS-(CH2)6-OP(O2−)-GGGAGGUGCCCGAACGAUAUidT) via disulfide exchange under denaturing conditions in the presence of urea. The two peptide-RNA conjugates were purified by preparative anion-exchange HPLC under denaturing conditions, desalted and supplied as lyophilized sodium salts. The purity of the final products was determined by analytical anion-exchange HPLC and electrospray mass spectroscopy with deconvolution. For analytical HPLC, constructs were eluted through MonoQ 5/5 columns (Amersham Biosciences, Piscataway, NJ, USA) with a linear gradient from 10-50% buffer B in buffer A+B during 30min at a flow rate of 1 ml/min, the effluent monitored by UV spectroscopy at 260 and 280nm. For HS-(CH2)6-OP(O2−)-GGGAGGUGCCCGAACGAUAUidT, analytical HPLC buffer A consisted of 10mM LiClO4, 20mM Tris-HCl, 50μM EDTA and 8M urea, pH 7.4 and buffer B was as buffer A with a [LiClO4] of 600mM. For the penetratin and TAT(48-60) peptide-RNA conjugates, LiClO4 was substituted with NaCl at 10mM and 1M for buffers A and B respectively. For Chol-(CH2)6-S-S-(CH2)6-O-P(O2−)-O-GGGAGGUGCCCGAACGAUAUidT, a linear gradient of 10-100% buffer B in A+B over 40min elution at 1 ml/min was used, with buffers prepared in 20% v/v acetonatrile, pH 7.4, in the absence of urea, and 10mM or 1 M NaCl (buffers A and B respectively). The cholesterol conjugated sense strand RNA was synthesized directly on solid phase using the commercially available cholesterol phosphoramidite coupled to a C6-SS-C6 linker at the 5′-end of the RNA. Mismatch studies in animals were performed with MM2 - 5′- H2N-CO-CH2-S-(CH2)6-OP(O2)-GCGAGCUGCGCGAAGGAUAUidT-3′ ; antisense : 5′-UAUCCUUCGCGCAGCUCGCUidT-3′.
For annealing, 240nmol of lyophilized siRNA-sense strand or sense strand conjugates were resuspended at 240μM concentrations in sterile Dublecco's phosphate buffered saline (PBS; Sigma, Poole, UK) and then mixed by pipetting with an equal volume of antisense strand (5′-UAUCGUUCGGGCACCUCCCUidT-3′) resuspended in PBS at equimolar concentrations, to give a final siRNA concentration of 1nmol/μl. Annealing was performed by heating to 95 °C for 5 min on a peltier element thermal cycler block followed by slow cooling over a period of 1hr.
Annealing products were examined upon a precast 20% polyacrylamide Tris borate ethylenedinitrilotetraacetic acid (EDTA) gel (Invitrogen, Carlsbad, USA), RNA visualised by SybrGold staining as recommended by the manufacturer (Invitrogen) and documented using a computer-controlled UVP GelDoc-It Imagining System (Fisher) fitted with an ethidium bromide filter. Image analysis was performed using the UVP Labworks™ Image Acquisition and Analysis Software version 4.6.00.0 (Media Cybernetics, Inc., Atlanta, GA, USA). LPS content in dosing preparations was determined using the PyroGene® Recombinant Factor C Endotoxin Detection System according to the manufacturer's instructions (Cambrex Bio Science Wokingham Ltd, Wokingham, UK).
To assess siRNA and siRNA conjugate susceptibility to RNases in a simulated lung microenvironment, stability studies were carried out in murine bronchoalveolar lavage (BAL) fluid. This was collected from three terminally anaesthetized male BALB/c mice (20-25g) following exposure of the lungs by three repeat lavages of 0.3ml of RPMI media (Invitrogen). All collected media was pooled and stored at −20°C. To assess siRNA stability in BAL fluid, annealed siRNA and siRNA constructs were incubated in excess volumes of 95% v/v murine BAL fluid at 37°C. 20 ul volumes were removed at set timepoints, gel loading buffer II (Invitrogen) was quickly added to a final 1x concentration and the mixture was snap frozen in liquid nitrogen. Samples were thawed on ice, and 10 pmol quantities were analysed by polyacrylamide gel electrophoresis (PAGE).
L929 cells were grown to 70% confluence in DMEM (Sigma) supplemented with 10% foetal calf serum, 1% penicillin/strepavidin/glutamine and 5 mM non-essential amino acids (Invitrogen). On the day of transfection, cells were trypsinised and resuspended at 2 × 105 in media. siRNA, siRNA-lipofectamine 2000 (Invitrogen) complexes prepared to the manufacturer's instructions or siRNA-conjugates were diluted to 2x the final indicated concentration in OptiMEM (Invitrogen) and mixed with an equal volume of cell suspension to give a final serum concentration of 5% FCS. The cells were then plated into a 96-well plate in triplicate in a volume of 100 μl (1 × 104 cells/well), incubated for 24hrs at 37 °C before either RNA extraction or measurement of cell viability using the MTT assay (Sigma) was carried out.
All in vivo procedures were carried out under local ethics approval and in strict accordance to the 1986 Animals (Scientific Procedures) Act. Groups of 6-18 male BALB/c mice (20-25g) were sedated with 4% Halothane in O2 and given the indicated dose of siRNA, mismatch, siRNA conjugates, TAT(48-60), penetratin, polyinosinic: polycytidylic acid (poly(I:C); Sigma) or LPS from Escherichia coli 0111:B4 (Sigma) in 20μl volumes by intratracheal administration. Animals were sacrificed at the indicated timepoints using an overdose of pentobarbitone (200mg/kg intraperitoneally).
Cell culture samples were extracted using the Qiagen RNeasy mini kits according to the manufacturer's instructions (Qiagen, Crawley, UK). Animal tissues were gently removed immediately after confirmation of death and stored in either RNAlater according to the manufacturers' instructions (Sigma). Total RNA was extracted by rotor/stator homogenisation in Tri Reagent (Sigma) using an Ultraturrax T18 homogenizer, followed by isopropanol precipitation of the aqueous phase and reconstitution in RNase-free water (Promega, Southampton, UK). Yield and purity were determined by spectrophotometric measurement of the absorbance at 260nm and 280nm.
For the determination of p38 MAP kinase mRNA expression, mouse-specific Taqman primers and probes were obtained from the ‘Assay on Demand’ service provided by Applied Biosystems (assay no. Mm00442497_m1, Applied Biosystems, Applera Corp., Warrington, UK). Real-time PCR was performed using the one-step Quanti-Tect RT-PCR Kit (Qiagen) in a MicroAmp 96-well Reaction Plate (Applied Biosystems). Each well contained 25 ng total RNA, 300 nM of forward and reverse transcription primers and 125 nM Taqman probe in a reaction volume of 25 μl. All sample and non-template control reactions were performed in the ABI Prism 7700 Sequence Detection System (Applied Biosystems) in triplicate. Comparative slopes of the relationship between log [RNA] and Ct (gradient = −3.3) for both the gene of interest and the endogenous control 18S (Applied Biosystems) indicated that the comparative Ct method (ΔΔCt) could be used for the relative quantification of gene expression. Data was analysed on the Prism version 4.03 statistic analysis package (GraphPad Software, Inc., San Diego, CA, USA) using non-parametric analysis of variance followed by Tukey post-tests where appropriate.
Lungs were gently removed immediately after confirmation of death and snap frozen in liquid nitrogen, then stored at −80°C. Frozen lung was homogenised in ice-cold RIPA buffer (25 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 5 mM dithithreitol, 0.5 mM phenylmethylsulphonyl fluoride, 2 mM sodium orthovanadate, 1.25 mM sodium fluoride, 1 mM sodium pyrophosphate; Sigma) at a ratio of 1 ml per 100 mg tissue, using an Ultraturrax T18 homogenizer (Fisher Scientific). The homogenates were then spun in a refrigerated (4°C) tabletop microcentrifuge at 21,000 × g for 20 mins. The supernatants were removed and stored at −20°C. Total protein content was determined by Bradford Assay (Bio-Rad, Hertfordshire, UK) according to the manufacturer's instructions. Tissue tumour necrosis factor (TNF)-α, interleukin (IL)-12 p40 (DuoSet ELISA kits; R&D Systems Europe, Abingdon, UK) and interferon (IFN)-α (PBL laboratories, Piscataway, NJ, USA) were measured by sandwich ELISA and normalised for total protein content.
5′ end Cy3 labeled antisense strand (Dharmacon) was annealed to the appropriate sense strand and the 10nmol of the resulting Cy3 labelled siRNA, penetratin-siRNA, TAT-siRNA, cholesterol-siRNA or equimolar amounts of Cy3 alone were given by intratracheal administration. Whole lungs were collected 3 hours later and fixed in 4% formaldehyde-supplemented phosphate buffered saline (Sigma). After fixation the samples were rinsed in 70% EtOH, dehydrated, and embedded in paraffin. Paraffin sections (3 μm, Leica microtome, Leica Microsystems, Milton Keynes, UK) were rehydrated, rinsed in Tris-Buffered Salie pH 7.6 (Sigma), and stained with the DNA-binding fluorochrome Hoechst 33342 (Sigma). After mounting in Aqua Perm mountant (484975 Life Sci. International) Cy3 (red channel) and Hoechst 33342 (green channel) were by visualized on an epifluorosence microscope (Olympus BX10, Olympus Sveridge AB, Solna, Sweden) equipped with a high resolution Olympus DP-50 digital camera (Olympus Sveridge AB) linked to a computerized image analysis system (Viewfinder Lite, v1, Pixera Co and Image-Pro Plus v 4.5, Media Cybernetics). Cy3-siRNA positive cell phenotypes in the lung parenchyma alveolar macrophages were distinguished from structural alveolar cells by immunohistochemical visualization of the macrophage marker F4/80 (36). Briefly, after rehydration the slides were subjected to antigen retrieval through mild proteinase K digestion (Sigma). After blocking of non-specific binding sites, sections were incubated with a monoclonal rat anti-murine F40/80 (dilution 1:50, clone CI:A3-1, Serotec Ltd, Oxford, UK) for 1hr. After repeated rinsing the immunoreactivity was detected by an Alexa 448 conjugated goat anti rat antibody (green channel).
Initial studies were focused upon the identification of an optimum siRNA sequence for p38 MAP kinase mRNA knockdown. To this end, the concentration dependent knockdown achieved by three siRNAs was determined in the L929 mouse fibroblast cell line at 24 hrs (Figure S1) following reverse transfection with Lipofectamine 2000. Thus, an efficacious sequence (C) was selected that gave a maximal knockdown of 87% and an EC50 of 139pM when transfected into the cells with lipofectamine (Figure 1). As a negative control, we identified a 4 mismatch siRNA sequence that gave no significant knockdown except at the highest concentration of 100nM, where we observed a p38 MAP kinase mRNA reduction of ~20% (Figure 1). To facilitate the conjugation of TAT(48-60), penetratin or cholesterol upon siRNA, the 5′ end of the siRNA sense strand was modified with a C6-thiol linker, providing a thiol group for disulphide conjugation with a cysteine residue on the CPP (Analytical HPLC and electrospray mass spectrograms available in Figure S2). C6-thiol linker modified sense strands and CPP-sense strand conjugates were then annealed to the antisense strand to yield siRNA duplexes. To evaluate the impact of the conjugation of the C6-thiol linker group on the biological action of the siRNA, C6-thiol modified siRNA duplexes were transfected using lipofectamine 2000 in cultured L929 fibroblasts. Addition of the C6-thiol linker to the siRNA had no significant effect upon the magnitude of knockdown but caused a shift in the EC50 to 798pM (Figure 1). Visual examination of CPP-siRNA constructs showed no precipitation, whilst separation on 20% polyacrylamide gels demonstrated the formation of discrete annealed products (Figure S2). In contrast, annealing of the cholesterol-siRNA gave a higher molecular weight product, suggesting the formation of higher order structures.
The biological action of siRNAs conjugated to TAT(48-60), penetratin or cholesterol was then examined in vitro following a 24hr incubation in the absence of any transfection reagent (i.e. Lipofectamine 2000). Figure 2 shows that equimolar concentrations of siRNA incubated in the absence of lipofectamine 2000, TAT(48-60) peptide or penetratin peptide had no effect upon p38 MAP kinase mRNA levels. In contrast, the TAT(48-60)-, penetratin- and cholesterol-siRNA conjugates produced a small but significant knockdown of 36% ± 6%, 20% ± 3% and 28% ± 7%, respectively at 10μM (Figure 2). Examination of all the constructs showed no significant cellular toxicity at 24hrs (Figure S4).
Examination of the relative levels of p38 MAP kinase expression in mouse tissues indicated constitutive expression in the lung (Figure S5). To ascertain the capacity of siRNA to down-regulate p38 MAP kinase mRNA levels, increasing doses of the C6-thiol linker modified siRNA were administered intratracheally and the effect upon p38 MAP kinase mRNA expression was determined over a 24hr period. From Figure 3, it can be seen that all doses of the linker-modified siRNA (50nmol, 10nmol or 1nmol siRNA) produced a significant (~ 30-45%) mRNA knockdown at 6hrs. However, this effect appeared transient and only animals dosed with 50nmol showed significantly attenuated gene expression at 12hrs (30 ± 5%) and 24hrs (20 ± 6%). Administration of 10nmol mismatch control produced no significant down-regulation of p38 MAP kinase expression over the 24hrs period (Figure 4).
We next investigated whether the duration and magnitude of p38 MAP kinase mRNA knockdown could be influenced by conjugation of the siRNA to TAT(48-60), penetratin or cholesterol (Figure 4). TAT(48-60)-siRNA showed 20-30% knockdown at all time points although this was only significant at 12hrs. However, though this knockdown was comparable to that achieved by equimolar doses of unconjugated siRNA, administration of the TAT(48-60) peptide alone produced a comparable and statistically significant knockdown at all time points examined, suggesting the CPP as a modulator of p38 MAP kinase expression. Similarly, although the penetratin-siRNA conjugate appeared to show increased knockdown (47 ± 9%) at 6hrs, the peptide alone also caused significant reduction (30 ± 7%) of p38 MAP kinase mRNA levels. In contrast, cholesterol conjugated siRNA increased the duration but not the magnitude of the response: significant reduction of p38 MAP kinase mRNA expression was seen both at 6hrs (28 ± 9%) and 12hrs (42 ± 13%), at levels comparable to those achieved by the 50 nmol dose of unconjugated siRNA. Because of the highly lipophilic nature of cholesterol and the resultant low solubility in water, we were unable to determine if this was a direct result of the administration of cholesterol to the lung.
To determine the distribution of the siRNA and siRNA-conjugates in the lung, the 5′ end of the antisense strand was labelled with the fluorochrome Cy3. At 3hrs post administration, histological analysis showed intense staining of macrophages within the parenchyma and sub-epithelial tissue of bronchi and bronchioles which suggested that these might represent the principal target cell population (Figure 5). In addition, we observed intense and scattered staining of epithelial cells within the bronchi and bronchioles which appeared to be an all-or-nothing response. Only rarely were positively stained alveolar epithelial cells detected. siRNA conjugation to penetratin or cholesterol did not influence the cellular labelling pattern (results not shown), however conjugation to TAT(48-60) appeared to cause a small increase in the staining intensity and number of Cy3 positive cells, with some increase in nuclear staining (Figure 5). In contrast, dosing with equimolar amounts of Cy3 dissolved in PBS produced diffuse staining around the large bronchi and blood vessels (data not shown).
To determine whether our observations might have been influenced by siRNA and siRNA-conjugate stability, we examined the degradation profile following exposure to mouse BAL fluid. Although BAL fluid incubation resulted in the appearance of a single double-stranded cleavage product, full length siRNA could still be detected at >7hrs (Figure 6). Identical stability and cleavage patterns were seen with the TAT(48-60)-siRNA, penetratin-siRNA and cholesterol-siRNA, implying that conjugation of such delivery moieties has no significant effect upon siRNA stability, nor that the higher-order structures observed in annealed chol-siRNA gels were capable of improving siRNA stability (Figure S3). Importantly, as the shift in gel mobility appears to be entirely due to siRNA cleavage, both the TAT(48-60) and penetratin peptides and the conjugation chemistry are stable in BAL fluid over this time range. Examination of the breakdown product using MALDI-TOF mass spectral analysis showed that the AUidT was cleaved from the 3′ end of the sense strand whilst the U residue was cleaved from the 5′ end of the antisense strand, most probably by the action of an RNase A-like activity, to produce a blunt ended siRNA (37).
Previous reports have suggested that siRNAs are capable of stimulating the innate immune response through binding to Toll like receptors (TLR)-3, -7 and -8 within the endosomes (38). To eliminate the possibility that the observed reductions in p38 MAP kinase mRNA expression might have resulted from the activation of this response, we measured the levels of the immune markers IFN-α, TNF-α and IL-12 p40 in whole lung. As expected, the positive controls lipopolysaccharide (LPS) and poly(I:C), known activators of the immune response through of TLR4 and TLR3 respectively, induced a robust increase in IFNα production at 6hrs whilst LPS increased TNFα at 1hr (Figure 7). Interestingly, with the exception of penetratin-siRNA, none of the experimental agents induced an immune response following intratracheal administration; in the case of penetratin-siRNA we observed a significant increase in the expression of TNFα and IL-12p40, as well as elevated release of IFNα (Figure 7). As a means of controlling for contaminating LPS in the dosing preparations, potentially influencing p38 MAP kinase expression or inducing immune activation, measurement of LPS content was included in our siRNA quality control process. Crucially, LPS levels were <0.01 EU among all preparations, significantly below the concentration believed to activate TLR-4 mediated responses (14).
In this paper, we have investigated the utility of the cell penetrating peptides TAT(48-60) and penetratin and the lipidic carrier cholesterol in improving siRNA-mediated knockdown of endogenous mRNA expression in the mouse lung. The chosen target was p38 MAP kinase, a key component of inflammatory signal transduction pathways whose inhibition can ameliorate inflammatory responses (17, 18), constitutively expressed in a variety of lung-associated cell types including epithelial, endothelial, macrophage, eosinophil, fibroblast and smooth muscle cell types. The quantities of siRNA employed in our investigation, which were approximately 37.5mg/kg (50nmol), 7.5mg/kg (10nmol) and 0.75mg/kg (1nmol), are comparable to those reported as efficacious in lung. Thus, a 2mg/kg siRNA dose was employed to attenuate heme oxygenase 1 (HO-1) expression (8) and a 5mg/kg dose was found to reduce RSV and PIV titer in the lung (12). Following intratracheal administration, sequence-specific siRNA-mediated p38 MAP kinase mRNA knockdown was observed as early as 6hrs, implying rapid uptake and action of naked siRNA upon the target gene mRNA levels. However, the short duration of knockdown observed with 1nmol and 10nmol doses, quickly reversed within 12hrs, suggested that naked siRNA might be rapidly degraded and that its action might be extended through chemical stabilization. Indeed, BAL fluid stability studies showed that even though full length siRNA could still be recovered at 7 hrs, cleavage did indeed occur, detectable as early as 15 min.
An alternative hypothesis might be the existence of feedback mechanisms that maintain the mRNA level of the constitutively expressed p38 MAP kinase through increased transcription. Failure to detect this in vitro could be ascribable to the cell line used, however, our in vitro studies allowed the identification of a potent and selective siRNA sequence. Insofar as the utility of siRNA in the regulation of gene expression in the lung has been pursued, to our knowledge we are the first to report an attempt to manipulate an endogenous, constitutively expressed gene. Indeed, little is known about the biochemistry of p38 MAP kinase mRNA level homeostasis as research has focused on phosphorylation of the protein, a key part of the inflammatory signal transduction process (17,18). In this respect, we have observed no significant change in p38 MAP kinase mRNA expression in the mouse lung 4 hrs after a 30 min challenge with 0.1 mg/ml aerosolised LPS (0.88±0.29-fold p38 MAP kinase relative mRNA levels to saline challenged controls; Moschos., unpublished data). Therefore, either MAPK14 mRNA levels are either not affected by cellular stress signals such LPS-induced inflammation, or are closely monitored and strictly maintained at specific levels. Our findings of an apparent limit on the maximal mRNA knockdown achievable in vivo lends further weight to this hypothesis; we were unable to overcome this threshold, since increasing the siRNA dose to 50nmol improved the duration but not the magnitude of the response. Notwithstanding homeostasis, additional contributors to the limited knockdown could be uneven lung distribution, localisation in the immediate epithelial cell layer or poor cellular uptake. We therefore examined the distribution of Cy3 labelled siRNA by histology and showed uptake into macrophages and scattered airway epithelial cells. However, the total tissue mRNA downregulation by about 30-45% was comparable to that seen in the liver following intravenous administration of cholesterol linked and stabilised siRNA (20).
To improve upon the duration and magnitude of p38 MAP kinase mRNA knockdown, we examined the effect of chemical conjugation of siRNA to a number of non-viral molecular delivery vectors, including cholesterol and the cell penetrating peptides TAT(48-60) and penetratin. Since a free hydroxyl group at the 5′ -end of the antisense strand is thought to be crucial to the mechanism of RNA interference (39, 40), these delivery moieties were conjugated to the 5′ end of the sense strand via a C6-thiol linker. To dissociate the sense strand from the delivery vector in the cytosol, we included a disulphide bond within our conjugation chemistry, believed to be cleaved intracellularly. Examination of the biological action of this modified siRNA in vitro showed that conjugation of the C6-thiol linker increased the EC50 but did not affect the maximal level of mRNA knockdown (~85%). This magnitude of efficacy in cell culture is similar to that reported for other siRNAs that have been successfully used in vivo (41), thus ratifying our choice of conjugation chemistry.
Unlike the mouse studies, examination of the p38 MAP kinase knockdown in cells showed no effect of naked siRNA at concentrations up to 10μM. In contrast, the cholesterol, penetratin and TAT(48-60) constructs produced a small (20 - 40%) but significant mRNA knockdown without inducing toxicity. Importantly, this was determined to be siRNA-specific as no effect was observed by equimolar quantities of the two unconjugated CPP. However, the uptake mechanism appears highly inefficient since these concentrations were > 100,000x higher than those effective with Lipofectamine 2000. Surprisingly, previous cell based studies using siRNA conjugated to cholesterol-, penetratin or TAT(48-60) (35) gave values reportedly in the sub-micromolar range. The reason for these differences is presently unknown although they could be related to the gene target, cell type and/or the type of synthetic construct. The majority of investigators have, like ourselves, attached the delivery vector to the 5′ -end of the sense strand via a disulphide bond (19, 32-34), although Chiu et al (35) attached TAT(48-60) to the 3′-end of the antisense strand and in the report by Soutschek et al. (20) the cholesterol moiety was conjugated to the 3′ -end of the sense strand via a pyrrolidone linker. Crucially, unlike ourselves, many investigators do not report post-conjugation HPLC purification in the presence of denaturing agents to remove excess, unconjugated cationic CPP from CPP-siRNA conjugates. As with electrostatic based delivery systems such as cationic lipids and polymers, omission of this purification step is likely to result in the formation of complexes between excess CPP and CPP-siRNA conjugates that might increase the efficacy of uptake (42).
Despite the low potency of our conjugates in culture, we proceeded to in vivo investigations since we were administrating solutions of 500μM (20μl of 10nmol), concentrations 50x greater than those effective in vitro. Significantly, these studies failed to show an effect of conjugation to the cell penetrating peptides TAT(48-60) or penetratin upon either lung distribution, magnitude or duration of p38 MAP kinase mRNA knockdown. Strikingly, penetratin peptide alone induced a significant downregulation in p38 MAP kinase mRNA expression at 6hrs and, in the case of TAT(48-60), this was also observed at 12 and 24hrs. This data therefore document a biological action of these peptides upon gene expression, or associated regulatory processes. The mechanism underlying this response is not currently known, nor is the extent or consequence of the gene modulation enacted by CPP in vivo. The lack of adverse reactions or behavior at 24hrs following peptide administration implies that this does not involve lung toxicity in the form of necrosis or apoptosis. Indeed, in associated studies in our laboratory using the lung epithelial cell line A549 exposed to TAT(48-60) and penetratin, we have been unable to detect toxicity at concentrations up to 30μM (43). Alternatively, down-regulation of p38 MAP kinase mRNA expression might occur following the interaction of CPP with extracellular or intracellular targets. Indeed, extracellular CPP-protein interactions are likely as Fotin-Mleczek et al. (44) have shown that both TAT(48-60) and penetratin induce clathrin-dependent uptake of the TNFα and EGF receptors without their activation, though the exact mechanism is unknown. Also, given the fact that CPP are derived from the DNA binding domains of transcription factors and are highly positively charged, it might be envisaged that once taken up into cells at high concentrations, these peptides directly interfere with gene transcription. Interestingly, the high-affinity binding of CPP to both plasma membranes and genomic DNA has previously been shown to result in erroneous conclusions about the mechanism of CPP uptake (45, 46). Interpretation of our findings as an indication of global gene expression perturbation by CPP is restricted by the lack of effect of CPP on the expression of MAPK14 in vitro even at concentrations as high as 10 μM. In terms of a direct effect of CPPs in the regulation of MAPK14 gene expression, lack of specific effects on the MAPK14 locus by means of CPP-DNA interactions can be postulated only under the assumption that no significant sequence differences exist between the genome of the BALB/c mice used in our in vivo experiments and that of the L929 cell line (derived from C3H/An). Instead, a more likely scenario would favor CPPs targeting of the signal transduction pathways or transcriptional regulators of MAPK14 gene expression. Alternatively, the viral origin of some CPP coupled to the internalization of cytokine receptors might be expected to activate the anti-viral innate response, resulting in inhibition of transcription. However, such a mechanism would appear unlikely as associated pro-inflammatory marker release in vivo was not detected.
Investigation of the immunological response in vivo showed that penetratin-siRNA but not TAT(48-60) or cholesterol conjugate administration induced an innate immune response, possibly through activation of TLR-3, -7 and/or TLR8 (38). Liposomal delivery of siRNA containing immunostimulatory sequences has been shown to be necessary for activation of these toll-like receptors, underscoring the importance of the uptake mechanism in TLR mediated immune activation by siRNA. Significantly, none of the currently known immunostimulatory siRNA motifs are found in the siRNA sequence we administered in vivo, nor did siRNA, TAT or penetratin alone induce an innate immune response. Thus, our data suggest that either penetratin mediated siRNA delivery is accomplished in a different manner from that of the TAT and cholesterol-conjugates, or that the individual conjugates are subject to different biochemical fates once in the cytosol.
In contrast to the CPP, although cholesterol conjugation failed to increase the magnitude of p38 MAP kinase knockdown, it extended the duration of mRNA knockdown. However, although gel electrophoresis indicated the formation of higher molecular weight complexes in cholesterol siRNA conjugates, this did not improve upon siRNA stability in BAL fluid, nor was it found to improve upon lung distribution according to our histological investigations.
Overall, these studies have demonstrated siRNA mediated knockdown of the endogenous, constitutively expressed gene, p38 MAP kinase, in the mouse lung. Although cholesterol increased the duration of the knockdown, the fact that the magnitude of the responses was unaffected following conjugation to cholesterol, TAT(48-60) or penetratin suggests that either these CPP are unable to increase cellular delivery and distribution or alternatively that, under these circumstances, maximal responses can be achieved with naked siRNA alone. However, since both TAT and penetratin alone induced changes in gene expression we believe that, despite the widespread success of CPP for the delivery of biologically active peptides and proteins in vivo (26, 27), these results highlight a need to continue to elucidate the mechanisms of CPP bioactivity in order to determine their application in the delivery of biopharmaceuticals. This contention is further supported by our findings that penetratin-siRNA but not TAT(48-60)-siRNA conjugates caused activation of the innate immune response, which implies that these have different intracellular fates.
Sterghios Moschos is supported by BBSRC (BB/C508234/1), Simon W. Jones and John J Turner are supported by a grants from the EU framework 5 program, Mark M. Perry and Mark A Lindsay are supported by the Wellcome Trust (076111). Jonas Erjefelt is supported by the Swedish Heart and Lung Foundation. We would like to thank Prof. Maria Belvisi and Dr Mark A Birrell, Department of Respiratory Pharmacology, National Heart and Lung Institute, Imperial College for their advice on using mice models.
CPP and cholesterol-siRNA conjugates in mouse lung
Supporting Information Available: Figure S1, dose-dependent percentile p38 MAP kinase mRNA levels in vitro following lipofection with three mouse p38 MAP kinase siRNA or two mismatch controls; Figure S2, analytical HPLC and electrospray mass spectrograms of peptide- and cholesteryl-RNA constructs; Figure S3, analytical gels of CPP and cholesterol conjugate annealing products; Figure S4, cell viability following incubation with siRNA, CPP or siRNA conjugates; Figure S5, p38 MAP kinase expression across a mouse tissue panel. This information is available free of charge via the Internet at http://pubs.acs.org