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Conjugation of Antisense Oligonucleotides to PEGylated Carbon Nanotubes Enables Efficient Knockdown of PTPN22 in T Lymphocytes Lucia G. Delogu,† Andrea Magrini,‡,§ Antonio Bergamaschi,| Nicola Rosato,‡,⊥ Marcia I. Dawson,# Nunzio Bottini,*,† and Massimo Bottini*,‡,⊥,# Department of Biochemistry and Molecular Biology and Institute for Genetic Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, California 90033, NAST Centre for Nanoscience & Nanotechnology & Innovative Instrumentation, Department of Environmental, Occupational, and Social Medicine, and Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy, Institute of Occupational Medicine, Universita` Cattolica del Sacro Cuore, Largo Agostino Gemelli 8, 00168 Rome, Italy, and Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, California 92037. Received December 12, 2008; Revised Manuscript Received February 4, 2009
PEGylated-carbon nanotubes (PNTs) were evaluated as nanocarriers of antisense oligonucleotides into T-cells using protein tyrosine phosphatase N22 (PTPN22) as a model target gene. PTPN22 is an important predisposing gene and drug target in type 1 diabetes and several other human autoimmune diseases. Here, we generated the first anti-PTPN22 20-mer antisense oligonucleotides (ASOs) and tethered them to PNTs through a cleavable disulfide bond. Spectroscopic and atomic force microscopy analyses were used to determine the loading of ASO onto PNTs, whereas the cleavable nature of the disulfide bond connecting the oligonucleotide to the nanocarrier was confirmed by incubation with dithiothreitol followed by agarose gel electrophoresis. PNT-conjugated ASOs achieved efficient (>50%) knockdown of PTPN22 expression in T-lymphocytes in culture at the mRNA and protein level, as measured by quantitative real-time PCR and Western blotting, respectively. Considering the high biocompatibility and low in vivo toxicity of PNTs, we expect that our approach will be easily translated to achieve in vivo knockdown of PTPN22 and other T lymphocyte targets, thus enabling novel ASO-mediated immunotherapies for type 1 diabetes and other autoimmune diseases.
Gene silencing can be achieved using antisense oligomers, ribozymes, DNAzymes, and RNA interference (1). Antisense technology permits knockdown in expression of a specific gene for both research and medicinal applications. Typically, a 20mer antisense oligonucleotide (ASO) is designed to complement the mRNA of a particular target for cleavage by RNaseH endonuclease or by inhibiting translation (2). ASOs have been chemically modified to improve their affinity for target mRNA, potency, nuclease resistance, and biostability and to elicit RNaseH-mediated cleavage of target mRNA (3, 4). The socalled first generation of ASOs is based on phosphorothioate deoxynucleotides. These sequences have a net negative charge, elicit RNase H-mediated message degradation, and are stable to nuclease degradation (5). Systemic injection of ASOs enables the therapeutic knockdown of disease-related genes in important tissues, including liver and bone (6). However, their use for immunotherapy has been hampered by inefficient transport * Address correspondence to Nunzio Bottini, M.D., Ph.D., nunzio@ usc.edu, Institute for Genetic Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USA, Phone (323) 442-2634; or Massimo Bottini, Ph.D.,
[email protected], Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA, Phone (858) 646-3100 x3063. † Keck School of Medicine of the University of Southern California. ‡ NAST Centre for Nanoscience & Nanotechnology & Innovative Instrumentation, University of Rome Tor Vergata. § Department of Environmental, Occupational, and Social Medicine, University of Rome Tor Vergata. | Universita` Cattolica del Sacro Cuore. ⊥ Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata. # Burnham Institute for Medical Research.
across the plasma membranes of immune cells. For example, the negatively charged phosphorothioates have very low bioavailability in B- and T-lymphocytes (7). Chemically engineered nanotechnology-derived carriers (nanocarriers) have recently improved drug delivery into difficult cell targets (8). These nanocarriers have been shown to be able to deliver cargo (nucleic acids, peptides, small molecules, etc.) into such specific cell types as B- and T-lymphocytes after injection in mice without causing significant toxicity. Carbon nanotubes (NTs), due to their extremely high aspect ratio (length/diameter), represent a potential scaffold for constructing multivalent delivery systems capable of binding many membrane receptors (9, 10). In particular, NTs functionalized with poly(ethylene glycol) (PEG) chains (PEGylated NTs or PNTs) have been reported to deliver smallinterfering RNA into cultured cells (including T-lymphocytes) (11) and target integrin-positive tumors in mice (12). Moreover, PNTs exhibit long circulation times in the blood, low uptake by the reticuloendothelial system (RES) (13), and lack of toxicity in vivo (14). Both in vitro and in vivo, PNTs were taken into the cytoplasm of T-lymphocytes (15). These reports led us to investigate whether PNTs could be used as nanocarriers to deliver ASOs into immune cells. Here, we used PNT-conjugated ASOs to achieve knockdown of protein tyrosine phosphatase N22 (PTPN22) in T cells. PTPN22 is an important drug target for autoimmune diseases (16-18). A mutation in its gene is a well-recognized predisposing factor for type 1 diabetes (19-22), rheumatoid arthritis (23, 24), and several other autoimmune diseases (25). PTPN22 is found in white blood cells and has a particularly critical function in T-lymphocytes, in which it negatively regulates growth and activation (26-29). The autoimmune-predisposing C1858T mutation (R620W) (29, 30) in the PTPN22 gene produces a
10.1021/bc800540j CCC: $40.75 2009 American Chemical Society Published on Web 02/25/2009
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Scheme 1. Fabrication of Antisense Oligonucleotide- And Carboxyfluorescein-Functionalized PEGylated Carbon Nanotubesa
a Pristine carbon nanotubes (NTs) were sonicated in the presence of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly(ethylene glycol))2000] (PL-aPEG), fractionated by stepwise centrifugation, and purified away from small molecules by ultrafiltration. The amino-functionalized PEGylated NTs (aPNTs) obtained were capped with 5-carboxyfluorescein by reaction with 5-carboxyfluorescein N-hydroxysuccinimide ester to give fluorescent PEGylated NTs (fPNTs, (1)). Alternatively, aPNTs were capped with ASO8 phosphorothioate by stepwise activation with sulfosuccinimidyl 6-(3′-[2-pyridyldithio]propionamido)hexanoate (sulfo-LC-SPDP) (to give pyridyldithio-functionalized PEGylated NTs, PNTPDP (2)) followed by displacement with 5′-mercapto-terminated ASO8 to give the PTPN22 antisense oligonucleotide tethered to PEGylated NTs through a disulfide bond (PNT-SS-ASO, (3)). This procedure was repeated to produce DNA tethered to PNTs through a disulfide bond as a model conjugate (PNT-SS-DNA).
gain-of-function-W620 variant that is a more potent inhibitor of T-lymphocyte activation. Reduced T-lymphocyte growth and activation are important predisposing factors for autoimmune disease (31, 32). Thus, systemic inhibition of PTPN22 expression is considered to be beneficial for prevention or early treatment of autoimmunity (18). Our goal is to develop nucleotide-based PTPN22 inhibitors and optimize their delivery into T-lymphocytes for treatment of type 1 diabetes and other autoimmune diseases. PNTs were obtained by hydrophobic adsorption of phospholipids terminated with amino-functionalized PEG chains onto NT sidewalls by using a procedure previously reported by us (15) and others (11). Pristine (nonfunctionalized) NTs were ultrasonicated with phospholipids terminated with approximately 12-nm-long amino-functionalized PEG chains {1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)2000]} (PL-aPEG, Scheme 1), fractionated by stepwise ultracentrifugation to isolate short hydrophilic amino-terminated PEGylated NTs and purified by ultrafiltration to remove free phospholipids (aPNTs). The collected nanocarriers were found to disperse under physiologic conditions and did not display any visible flocculation upon standing for several months at room temperature in PBS pH 7.4. Atomic force microscopy (AFM) images of aPNTs showed the presence of individual particles and small bundles (Figure 1A) having a length of 164 ( 46 nm. We calculated that aPNTs had an approximate molecular weight of 450 kDa and were decorated with 85 ( 14 PEG chains per nanoparticle (see Supporting Information). The exposed amino groups of the aPNTs were acylated with 5-carboxyfluorescein N-hydroxysuccinimide ester to introduce a fluorescent label (Scheme 1). The fluorescent PNTs (fPNTs) were visible by conventional light optical and confocal microscopy (data not shown). The Jurkat human acute lymphoblastoma leukemia T-cell line expressing the SV40 T antigen (TAg) (33)
was kept at logarithmic growth in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES pH 7.3, 2.5 mg/mL D-glucose, 100 units/mL of penicillin, and 100 µg/mL streptomycin. The internalization of fPNTs by Jurkat TAg cells was investigated by flow cytometry using a Beckman FC500 instrument. In the presence of fPNTs, 100% cells showed internalization of the nanocarriers already at 30 min and the internal fluorescence of the cells kept increasing during the first 18 h (Figure 1B, cell fluorescence was read in the presence of Trypan blue in order to quench fluorescence of noninternalized fluorescein). Next, we loaded the aPNTs with oligonucleotides using a tether with a cleavable disulfide bond that enables the release of the oligonucleotide cargo once the nanocarrier is transported into intracellular compartments. We acylated the aPNT primary amino groups with sulfosuccinimidyl 6-[3′-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP) to give pyridyldithiofunctionalized PNTs (PNT-PDP) and, subsequently, used a SHterminated 21-mer single-stranded oligonucleotide to displace the pyridyl group and produce the oligonucleotide-tethered PNTs (PNT-SS-DNA) as a model conjugate (Scheme 1). The PNTSS-DNA were purified by filtration, and the cleavable nature of the disulfide bond connecting the oligonucleotide to the nanocarrier was confirmed by incubation with dithiothreitol (DTT) followed by agarose gel electrophoresis (Figure 1C). The electrophoretic mobility of PNT-SS-DNA (lane 2), which was greater than that of PNT-PDP (lane 1), was decreased after incubation with DTT (lane 3), which cleaved the oligonucleotide off the PNTs by reducing the disulfide bond. Next, we generated and characterized anti-PTPN22 ASOs. PTPN22 ASO sequences have not been reported. We used the Sfold software (http://sfold.wadsworth.org, Supporting Information) to design a set of 20-mer ASOs against human PTPN22 (Accession: NM_017774; GI: 48928053). After passing a
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Figure 1. Characterization of PNT-based nanoassemblies. (A) Atomic force microscope (AFM) image of aPNTs (bar length 500 nm). (B) Flow cytometry plot of Jurkat TAg cell fluorescence after incubation with 6 nM fPNT for 30 min (blue line), 1 h (red line), 6 h (green line), or 18 h (black line) compared to nontreated control cells after the same times (dotted lines). Cell fluorescence was measured in the presence of 0.4% (w/v) trypan blue in order to quench fluorescence from noninternalized fluorescein. The histogram shows ungated data from approximately 5000 events; cell fluorescence is reported in arbitrary units (a.u.). (C) Reversible conjugation of a 21-mer single-stranded SH-terminated DNA oligomer to activated aPNTs through a disulfide bond. PNT-PDP (lane 1), PNT-SS-DNA (lane 2), and PNT-SS-DNA incubated with 0.1 M DTT for 30 min (lane 3) were analyzed by 1% agarose gel electrophoresis and visualized under visible light.
Figure 2. Characterization of a new anti-PTPN22 ASO. (A) Dose-dependent knockdown of PTPN22 using ASO. JTAg cells were electroporated with 5 µM (lanes 1, 2), 10 µM (lanes 3, 4), or 20 µM (lanes 5, 6) scrambled ASO (lanes 1, 3, 5) or ASO8 (lanes 2, 4, 6). Cells were lysed 24 h after transfection. Upper panel shows anti-PTPN22 blot, and lower panel shows control anti-ERK2 blot of total lysates. (B) PTPN22-specific knockdown by ASO8. Jurkat TAg cells were electroporated with a plasmid encoding PTP-PEST and with 10 µM scrambled ASO (lane 1) or ASO8 (lane 2) and lysed 24 h later. Cell lysates were subjected to Western blotting using antibodies to PTPN22, PTP-PEST, and Erk2. (C) ASO8 possesses endonuclease-inducing activity. Jurkat TAg cells were electroporated with 10 µM scrambled ASO or ASO8 and lysed 24 h later for isolation of total RNA. PTPN22 mRNA levels were measured in triplicate by quantitative real-time PCR. The histogram shows residual PTPN22 mRNA (%) in cells treated with ASO8 relative to those treated with scrambled ASO (100%). The statistical significance of differences between the two samples was calculated on the raw data by two-tailed Student’s t test. (D) ASO8 induces increased T cell receptor (TCR) signaling and rescues the inhibitory effects of PTPN22 overexpression on a TCR reporter activation. Jurkat TAg cells were electroporated with a NFAT/AP1-firefly luciferase reporter plasmid and a pGL3 Renilla control plasmid alone (columns 1, 2, 3, 6, 7, 8) or combined with 10 µM scrambled ASO (columns 2, 4, 7, 9) or ASO8 (columns 3, 5, 8, 10) and with a plasmid for hemagglutinin (HA)-tagged PTPN22 (columns 4, 5, 9, 10). Samples represented in columns 1 and 6 represent negative controls and contained no ASO. At 24 h after transfection, half of the samples were stimulated using 1.5 mg/mL of OKT3 for 6 h (columns 6-10) to induce reporter activation, while the remainder (columns 1-5) were not stimulated. The histogram shows reporter activation (firefly/renilla luciferase ratios) as measured by the dual-luciferase assay (see Supporting Information for details). Statistical significance of differences was calculated by one-way ANOVA with Tukey post-test.
counter-screen to disallow nonspecific cross-hybridizers, the three top-scoring antisense sequences (ASO7, ASO8, and ASO9) were synthesized as phosphorothioates. A 20-mer scrambled ASO (34) was used as a control in the experiments. Jurkat TAg were transfected with the ASO by electroporation at 240 V with a single 25-ms pulse (29), and the expression of PTPN22 protein was assessed by Western blotting (WB) using an anti-PTPN22 antibody (R&D Systems, Minneapolis, MN) compared to that of ERK2, which was detected using anti-ERK2 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) and used as a loading control. Comparison of PTPN22 protein levels in the ASO7, ASO8, ASO9, and scrambled ASO-electroporated Jurkat TAg cells after 24 and 48 h revealed that ASO8 had most effectively knocked down expression by 50% or more (as assessed by densitometric scanning of Western blotting) when compared with the scrambled ASO (Figure S1 in Supporting Information) and showed the
highest potency at 24 h. In ASO8-electroporated T-cells PTPN22, knockdown was dose-dependent and became evident at 5 µM concentration (Figure 2A). The specificity of knockdown by ASO8 was next evaluated by controlling for knockdown of the closely related PTP proline, glutamate, serine, and threonine-rich sequence (PTP-PEST) (35). Jurkat TAg cells were electroporated with 10 µM of ASO8 or scrambled ASO and with a plasmid encoding PTP-PEST. Western blots of T-cell lysates using an anti-PTPN22 antibody compared to anti-PTPPEST and anti-ERK2 antibodies showed that ASO8 only caused knockdown of PTPN22 expression without affecting the levels of PTP-PEST (Figure 2B). RT-PCR analysis of T-cells electroporated with 10 µM ASO8 or scrambled ASO showed that PTPN22 mRNA levels were reduced in cells treated with ASO8 compared to cells treated with scrambled ASO, suggesting that ASO8 possesses RNase-activating effects (Figure 2C). We
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Figure 3. PNT-SS-ASO mediated knock-down of PTPN22 expression in Jurkat TAg cells. (A) Dose-dependent knockdown of PTPN22 mRNA using ASO8-conjugated PEGylated nanotubes (PNT-SS-ASO). Jurkat TAg cells were incubated with approximately 3.5 nM (column 1), 12 nM (column 3), or 35 nM (column 5) aPNT (black column) or approximately 3.5 nM (approximately 0.3 µM ASO8, column 2); 12 nM (approximately 1 µM ASO8, column 4), or 35 nM (approximately 3 µM ASO8, column 6) PNT-SS-ASO (lined columns). Total mRNA was extracted after 24 h. PTPN22 mRNA levels were measured in triplicate by quantitative real time PCR. Y-axis shows residual PTPN22 mRNA (%) in cells after treatment with PNT-SS-ASO relative to cells treated with aPNTs (100%). Statistical significance of differences was calculated on the raw data by two-tailed Student’s t test. (B) Knockdown of PTPN22 protein expression using PNT-SS-ASO. Jurkat TAg cells were incubated with 12 nM aPNT alone (lane 1) or 12 nM PNT-SSASO (approximately 1 µM ASO8 or 85 ASO8/PNT, lane 2) and then lysed 24 h later. Anti-PTPN22 antibody staining of total cell lysates is shown in upper panel compared to anti-ERK2 antibody staining in lower panel.
completed the characterization of ASO8 with a functional evaluation of its effect on T cell receptor (TCR) signaling using an NFAT/AP1-luciferase reporter assay in Jurkat TAg cells. This assay monitors the TCR-induced activation of the distal IL-2 promoter following its recruitment of activated NFAT and AP1 transcription factors (see Supporting Information). As shown in Figure 2D, ASO8-mediated knockdown of PTPN22 expression led to increased TCR signaling. ASO8 was also able to rescue the TCR-inhibiting effect of PTPN22 overexpression in Jurkat TAg, as indicated by reduced inhibition of TCR-induced reporter activation in cells overexpressing PTPN22 and treated with ASO8 compared to cells treated with scrambled ASO. Finally, we conjugated anti-PTPN22 ASO to PNTs and assessed the ability of the PNT-ASO conjugate to enable knockdown of PTPN22 expression in Jurkat TAg cells. Activated PNT-PDP was treated with 5′-SH-terminated-ASO8 (purchased as 5′-C6-S-S(disulfide)-modified ASO from IDT, modification cat. no. 5ThioMC6-D) to produce PNTs conjugated with ASO8 through a cleavable disulfide bond (PNT-SS-ASO). The cleavable nature of the disulfide bond was confirmed by incubation with DTT followed by agarose gel electrophoresis (data not shown). Knockdown of PTPN22 message and protein expression in Jurkat TAg cells incubated for 24 h with PNTSS-ASO or the same amount of aPNTs was assessed using RTPCR and Western blotting, respectively. Incubation of cells with PNT-SS-ASO enabled a dose-dependent knockdown of PTPN22 mRNA (Figure 3A) and a significant reduction of PTPN22 protein levels (Figure 3B). Interestingly, a significant reduction of PTPN22 expression was also achieved in these experiments by treating cells with approximately 1 µM PNT-conjugated ASO, which is below the threshold ASO concentration needed to achieve detectable knockdown when ASO8 was introduced by electroporation. The increased potency of the PNT-SS-ASO could be due to the SH termination of the ASO, or it might indicate that silencing of PTPN22 expression through PNT
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delivery is more efficient than that achieved by the electroporation method. In conclusion, we generated and characterized the first antiPTPN22 antisense oligomer and achieved its PNT-mediated delivery into T-cells. Incubation of T cells with PNT-SS-ASO enabled knockdown of PTPN22 expression at the mRNA and protein levels. Considering the high biocompatibility and low in vivo toxicity of PNTs (14), we expect that, after appropriate functionalization for targeting of selected immune cell subpopulations, PNT-ASO conjugates could enable ASO-mediated in vivo immunotherapy. Previously, we achieved selective delivery of PNTs to T-cells in vivo by functionalization with anti-CD3 and anti-CD28 antibodies (15). Future objectives are to prepare antibody-conjugated PNT-ASOs to achieve specific T-cell specific knockdown of PTPN22 and later translate this approach to in vivo knockdown of PTPN22 for therapy of type 1 diabetes and other autoimmune diseases.
ACKNOWLEDGMENT This work was supported by a Baxter Foundation Grant (N.B.), PRIN Grant No. 2006069554 from the Italian Ministry of University and Scientific Research (A.M.) and a Master & Back Fellowship from the Sardinian government (L.G.D.). The authors are grateful to Sanjay K. Pandey for the gift of the scrambled ASO and for critically revising the manuscript. Supporting Information Available: Materials and methods, characterization of PEGylated carbon nanotubes and knockdown of PTPN22 by ASOs. This material is available free of charge via the Internet at http://pubs.acs.org.
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