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• W1556322054 abstract "Mesangial cell proliferation and matrix accumulation, driven by platelet-derived growth factor (PDGF), contribute to many progressive renal diseases. In a novel approach to antagonize PDGF, we investigated the effects of a nuclease-resistant high-affinity oligonucleotide aptamer in vitro and in vivo. In cultured mesangial cells, the aptamer markedly suppressed PDGF-BB but not epidermal- or fibroblast-growth-factor-2-induced proliferation. In vivo effects of the aptamer were evaluated in a rat mesangioproliferative glomerulonephritis model. Twice-daily intravenous (i.v.) injections from days 3 to 8 after disease induction of 2.2 mg/kg PDGF-B aptamer, coupled to 40-kd polyethylene glycol (PEG), led to 1) a reduction of glomerular mitoses by 64% on day 6 and by 78% on day 9, 2) a reduction of proliferating mesangial cells by 95% on day 9, 3) markedly reduced glomerular expression of endogenous PDGF B-chain, 4) reduced glomerular monocyte/macrophage influx on day 6 after disease induction, and 5) a marked reduction of glomerular extracellular matrix overproduction (as assessed by analysis of fibronectin and type IV collagen) both on the protein and mRNA level. The administration of equivalent amounts of a PEG-coupled aptamer with a scrambled sequence or PEG alone had no beneficial effect on the natural course of the disease. These data show that specific inhibition of growth factors using custom-designed, high-affinity aptamers is feasible and effective. Mesangial cell proliferation and matrix accumulation, driven by platelet-derived growth factor (PDGF), contribute to many progressive renal diseases. In a novel approach to antagonize PDGF, we investigated the effects of a nuclease-resistant high-affinity oligonucleotide aptamer in vitro and in vivo. In cultured mesangial cells, the aptamer markedly suppressed PDGF-BB but not epidermal- or fibroblast-growth-factor-2-induced proliferation. In vivo effects of the aptamer were evaluated in a rat mesangioproliferative glomerulonephritis model. Twice-daily intravenous (i.v.) injections from days 3 to 8 after disease induction of 2.2 mg/kg PDGF-B aptamer, coupled to 40-kd polyethylene glycol (PEG), led to 1) a reduction of glomerular mitoses by 64% on day 6 and by 78% on day 9, 2) a reduction of proliferating mesangial cells by 95% on day 9, 3) markedly reduced glomerular expression of endogenous PDGF B-chain, 4) reduced glomerular monocyte/macrophage influx on day 6 after disease induction, and 5) a marked reduction of glomerular extracellular matrix overproduction (as assessed by analysis of fibronectin and type IV collagen) both on the protein and mRNA level. The administration of equivalent amounts of a PEG-coupled aptamer with a scrambled sequence or PEG alone had no beneficial effect on the natural course of the disease. These data show that specific inhibition of growth factors using custom-designed, high-affinity aptamers is feasible and effective. Specific inhibition of growth factors and cytokines has become a major goal in experimental and clinical medicine. However, this approach is often hampered by the lack of specific pharmacological antagonists. Available alternative approaches are also limited, as neutralizing antibodies often show a low efficacy in vivo and may be immunogenic, and as in vivo gene therapy for these purposes is still in its infancy. In the present study we have investigated a novel approach to specifically inhibit growth factors in vivo, namely, the use of aptamers produced by the systematic evolution of ligands by exponential enrichment (SELEX) method.1Tuerk C Gold L Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.Science. 1990; 249: 505-510Crossref PubMed Scopus (7692) Google Scholar, 2Ellington A Szostak J In vitro selection of RNA molecules that bind specific ligands.Nature. 1990; 346: 818-822Crossref PubMed Scopus (7279) Google Scholar The SELEX method has recently emerged as a powerful tool for screening large sequence-randomized nucleic acid libraries for unique oligonucleotides (aptamers) that bind to various other molecules with high affinity and specificity. For the purpose of this study, we have targeted platelet-derived growth factor (PDGF), the role of which is particularly well established in cardiovascular and renal disease.3Ross R Cell biology of atherosclerosis.Annu Rev Physiol. 1995; 57: 791-804Crossref PubMed Scopus (885) Google Scholar, 4Floege J Johnson RJ Multiple roles for platelet-derived growth factor in renal disease.Miner Electrolyte Metab. 1995; 21: 271-282PubMed Google Scholar A large variety of progressive renal diseases is characterized by glomerular mesangial cell proliferation and matrix accumulation.5Slomowitz LA Klahr S Schreiner GF Ichikawa I Progression of renal disease.N Engl J Med. 1988; 319: 1547-1548Crossref PubMed Scopus (86) Google Scholar PDGF B-chain appears to have a central role in driving both of these processes given that 1) mesangial cells produce PDGF in vitro and various growth factors induce mesangial cell proliferation via induction of auto- or paracrine PDGF B-chain excretion, 2) PDGF B-chain and its receptor are overexpressed in many glomerular diseases, 3) infusion of PDGF-BB or glomerular transfection with a PDGF B-chain cDNA can induce selective mesangial cell proliferation and matrix accumulation in vivo, and 4) PDGF B-chain or β-receptor knock-out mice fail to develop a mesangium (reviewed in 4Floege J Johnson RJ Multiple roles for platelet-derived growth factor in renal disease.Miner Electrolyte Metab. 1995; 21: 271-282PubMed Google Scholar). So far only one study has examined the effect of inhibition of PDGF B-chain in renal disease; Johnson et al, using a neutralizing polyclonal antibody to PDGF, were able to reduce mesangial cell proliferation and matrix accumulation in a rat model of mesangioproliferative glomerulonephritis.6Johnson RJ Raines EW Floege J Yoshimura A Pritzl P Alpers C Ross R Inhibition of mesangial cell proliferation and matrix expansion in glomerulonephritis in the rat by antibody to platelet-derived growth factor.J Exp Med. 1992; 175: 1413-1416Crossref PubMed Scopus (350) Google Scholar In this model, injection of an anti-mesangial cell antibody (anti-Thy-1.1) results in complement-dependent lysis of the mesangial cells, followed by an overshooting reparative phase that resembles human mesangioproliferative nephritis.7Floege J Eng E Young BA Johnson RJ Factors involved in the regulation of mesangial cell proliferation in vitro and in vivo.Kidney Int Suppl. 1993; 39: S47-S54PubMed Google Scholar Limitations of the study of Johnson et al6Johnson RJ Raines EW Floege J Yoshimura A Pritzl P Alpers C Ross R Inhibition of mesangial cell proliferation and matrix expansion in glomerulonephritis in the rat by antibody to platelet-derived growth factor.J Exp Med. 1992; 175: 1413-1416Crossref PubMed Scopus (350) Google Scholar included the necessity to administer large amounts of heterologous IgG and a limitation of the study duration to 4 days due to concerns that the heterologous IgG might elicit a humoral immune reaction. In the present study we have therefore used the anti-Thy-1.1 nephritis model to evaluate the feasibility and efficacy of inhibiting PDGF B-chain in vivo with high-affinity DNA-based aptamers. All aptamers and their sequence-scrambled controls were synthesized by the solid-phase phosphoramidite method on controlled pore glass using an 8800 Milligen DNA synthesizer and deprotected using ammonium hydroxide at 55°C for 16 hours. 2-Fluoropyrimidine nucleoside phosphoramidites were obtained from JBL Scientific (San Luis Obispo, CA). 2′-O-Methylpurine phosphoramidites were obtained from PerSeptive Biosystems (Boston, MA). Hexaethylene glycol (18-atom) spacer was obtained from Glen Research (Sterling, VA). All other nucleoside phosphoramidites were from PerSeptive Biosystems. To prolong the in vivo half-time of the aptamers in plasma, they were coupled to 40-kd polyethylene glycol (PEG). The covalent coupling of PEG to the aptamer (or to its sequence-scrambled control) was accomplished by treating 40-kd PEG N-hydroxysuccinimide ester (Shearwater Polymers, Huntsville, AL) with a primary amine group introduced at the 5′ end of the aptamer using trifluoroacetyl-protected pentylamine phosphoramidite. All PEG-aptamer conjugates were purified by anion exchange followed by reverse-phase high-pressure liquid chromatography (HPLC). The binding affinities of various aptamers for PDGF-AB or -BB (R&D Systems, Minneapolis, MN) were determined by the nitrocellulose filter binding method8Green LS Jellinek D Jenison R Östman A Heldin CH Janjić N Inhibitory DNA ligands to platelet-derived growth factor B-chain.Biochemistry. 1996; 35: 14413-14424Crossref PubMed Scopus (362) Google Scholar or by the competition electrophoresis mobility shift assay.9Willis MC Collins BD Zhang T Green LS Sebesta D Bell C Kellogg E Gill SC Magillanez A Knauer S Bendele RA Gill PS Janjić N Liposome-anchored vascular endothelial growth factor aptamers.Bioconj Chem. 1998; 9: 573-582Crossref PubMed Scopus (174) Google Scholar Rat PDGF-BB for cross-reactivity binding experiments was derived from Escherichia coli transfected with sCR-Script Amp SK(+) plasmid containing the rat PDGF-BB sequence. Rat PDGF-BB sequence was derived from rat lung poly A+ RNA (Clontech, San Diego, CA) through reverse transcription polymerase chain reaction (RT-PCR) using primers that amplify sequence encoding the mature form of PDGF-BB. Rat PDGF-BB protein expression and purification was performed at R&D Systems. The stabilities of DNA-based aptamers in vitro were examined in rat serum at 37°C. Serum was obtained from a Sprague-Dawley rat and was filtered through a 0.45-μm cellulose acetate filter and buffered with 20 mmol/L sodium phosphate buffer. Test ligands were added to the serum at the final concentration of 500 nmol/L. The final serum concentration was 85% as a result of the addition of buffer and aptamer. From the original 900-μl incubation mixture, 100-μl aliquots were withdrawn at various time points and added to 10 μl of 500 mmol/L EDTA (pH 8.0), mixed and frozen on dry ice, and stored at −20°C until the end of the experiment. The amount of full-length oligonucleotide ligand remaining at each of the time points was quantitated by HPLC analysis. To prepare the samples for HPLC injections, 200 μl of a mixture of 30% formamide, 70% 25 mmol/L Tris buffer (pH 8.0) containing 1% acetonitrile was added to 100 μl of thawed time point samples, mixed for 5 seconds, and spun for 20 minutes at 14,000 rpm in an Eppendorf microcentrifuge. The analysis was performed using an anion exchange chromatography column (NuceoPac, Dionex, PA-100, 4 × 50 mm) applying a LiCl gradient. The amount of full-length oligonucleotide remaining at each time point was determined from the peak areas. The pharmacokinetic properties of the modified PDGF aptamer conjugated to 40-kd PEG were determined in Sprague-Dawley rats. Before animal dosing, the aptamer was diluted with sterile PBS from a stock solution (also in sterile PBS), to final concentrations between 1 and 2 mg/ml (based on oligonucleotide molecular weight and the ultraviolet absorption at 260 nm with an extinction coefficient of 0.037 per mg oligo/ml). A single dose of the aptamer was administered to three Sprague-Dawley rats by i.v. bolus injection through the tail vein. Blood samples (approximately 400 μl) were obtained by venipuncture under isofluorane anesthesia and placed in EDTA-containing tubes. The EDTA blood samples were immediately processed by centrifugation to attain plasma and stored frozen at ≤−20°C. Time points for blood sample collection ranged from 2 to 480 minutes To prepare plasma samples for HPLC analysis, 200 μl of methanol was added to 100 μl of plasma sample, mixed for 30 seconds, and spun in a centrifuge for 10 minutes at 15,000 × g. All of the supernatant was transferred to a new tube and dried under vacuum. Samples were resuspended by addition of 100 μl of 50% formamide and 4% perchloric acid, mixed, and spun as described above. Ninety microliters of supernatant was transferred to a 250-μl limited-volume insert vial for HPLC analysis. Samples were analyzed by anion exchange HPLC (DNAPac PA-100, 4 × 50 mm) with LiCl gradient elution in 30% formamide and monitoring of ultraviolet absorption at 270 nm. Aptamer concentrations were determined based on peak area from a standard curve of the PEG-aptamer conjugate. The HPLC-based analysis described here and the double hybridization analysis described previously9Willis MC Collins BD Zhang T Green LS Sebesta D Bell C Kellogg E Gill SC Magillanez A Knauer S Bendele RA Gill PS Janjić N Liposome-anchored vascular endothelial growth factor aptamers.Bioconj Chem. 1998; 9: 573-582Crossref PubMed Scopus (174) Google Scholar have produced comparable results for a similar aptamer conjugated to 40-kd PEG (specific binding to vascular endothelial growth factor, Stanley C. Gill, unpublished observations), suggesting that the HPLC analysis indicates the levels of undegraded (full-length) aptamer. Nevertheless, as the PEG moiety has a strong influence on the HPLC retention times of the PEG-aptamer conjugate, we have not at this time ruled out the possibility that some of the partially degraded aptamer may migrate with a similar retention time as the full-length aptamer. Human and rat mesangial cells were established in culture, characterized, and maintained as described previously.10Radeke HH Gessner JE Uciechowski P Magert HJ Schmidt RE Resch K Intrinsic human glomerular mesangial cells can express receptors for IgG complexes (hFcγRIII-A) and the associated FcεRI γ-chain.J Immunol. 1994; 153: 1281-1292PubMed Google Scholar To examine the antiproliferative effect of the aptamers on the cultured mesangial cells, cells were seeded in 96-well plates (Nunc, Wiesbaden, Germany) and grown to subconfluency. They were then growth-arrested for 48 hours in MCDB 302 medium (Sigma, Deisenhofen, Germany) (human mesangial cells) or RPMI 1640 with 1% bovine serum albumin (rat mesangial cells). After 48 hours, various stimuli together with PDGF B-chain aptamer or sequence-scrambled aptamer were added: medium alone, human recombinant PDGF-AA, -AB, or -BB (kindly provided by J. Hoppe, University of Würzburg, Germany), human recombinant epidermal growth factor (EGF; Calbiochem, Bad Soden, Germany), or recombinant human fibroblast growth factor-2 (kindly provided by Synergen/Amgen, Boulder, CO). DNA synthesis in the rat mesangial cells was determined by [3H]thymidine incorporation as described11Floege J Topley N Hoppe J Barrett TB Resch K Mitogenic effect of platelet-derived growth factor in human glomerular mesangial cells: modulation and/or suppression by inflammatory cytokines.Clin Exp Immunol. 1991; 86: 334-341Crossref PubMed Scopus (81) Google Scholar after 24 hours of stimulation (of which the last 4 hours were in the presence of [3H]thymidine). In the case of human mesangial cells, after 72 hours of incubation, numbers of viable cells were determined using 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT; Sigma) as described.12Lonnemann G Shapiro L Engler Blum G Muller GA Koch KM Dinarello CA Cytokines in human renal interstitial fibrosis. I. Interleukin-1 is a paracrine growth factor for cultured fibrosis-derived kidney fibroblasts.Kidney Int. 1995; 45: 837-844Crossref Scopus (73) Google Scholar All animal experiments were approved by the local review boards. Anti-Thy-1.1 mesangial proliferative glomerulonephritis was induced in 33 male Wistar rats (Charles River, Sulzfeld, Germany) weighing 150 to 160 g by injection of 1 mg/kg monoclonal anti-Thy-1.1 antibody (clone OX-7; European Collection of Animal Cell Cultures, Salisbury, UK). Rats were treated with aptamers or PEG (see below) from days 3 to 8 after disease induction. Treatment consisted of twice-daily i.v. bolus injections of the substances dissolved in 400 μl of PBS, pH 7.4, for a total of 12 injections. The treatment duration was chosen to treat rats from ∼1 day after the onset to the peak of mesangial cell proliferation, which in the OX-7-induced anti-Thy-1.1 nephritis occurs between days 6 and 9 after disease induction. Four groups of rats were studied: 1) 9 rats that received a total of 4 mg (0.33 mg/injection) of the PDGF-B aptamer (coupled to 15.7 mg 40 kd PEG), 2) 10 rats that received an equivalent amount of PEG-coupled, scrambled aptamer, 3) 8 rats that received an equivalent amount (15.7 mg) of 40-kd PEG alone, and 4) 6 rats that received 400-μl bolus injections of PBS alone. Renal biopsies for histological evaluation were obtained on day 6 by intravital biopsy and postmortem on day 9 after disease induction. For intravital biopsies the left kidney was exposed by a flank incision under general anesthesia. A 3- to 4-mm slice was then cut off the lower pole, and bleeding was stopped immediately by gently applying a collagen sponge, followed by wound closure. As judged from serum creatinines, this biopsy technique does not disturb renal function (unpublished observations). Twenty-four-hour urine collections were performed from days 5 to 6 and 8 to 9 after disease induction. The thymidine analogue 5-bromo-2′-deoxyuridine (BrdU; Sigma; 100 mg/kg body weight) was injected intraperitoneally at 4 hours before sacrifice on day 9. Normal ranges of proteinuria and renal histological parameters (see below) were established in 10 nonmanipulated Wistar rats of similar age. Tissue for light microscopy and immunoperoxidase staining was fixed in methyl Carnoy's solution13Johnson RJ Garcia RL Pritzl P Alpers CE Platelets mediate glomerular cell proliferation in immune complex nephritis induced by anti-mesangial cell antibodies in the rat.Am J Pathol. 1990; 136: 369-374PubMed Google Scholar and embedded in paraffin. Four-micron sections were stained with the periodic acid Schiff (PAS) reagent and counterstained with hematoxylin. In the PAS-stained sections the number of mitoses within 100 glomerular tufts was determined. Four-micron sections of methyl Carnoy's-fixed biopsy tissue were processed by an indirect immunoperoxidase technique as described.13Johnson RJ Garcia RL Pritzl P Alpers CE Platelets mediate glomerular cell proliferation in immune complex nephritis induced by anti-mesangial cell antibodies in the rat.Am J Pathol. 1990; 136: 369-374PubMed Google Scholar Primary antibodies were identical to those described previously14Burg M Ostendorf T Mooney A Koch KM Floege J Treatment of experimental mesangioproliferative glomerulonephritis with non-anticoagulant heparin: therapeutic efficacy and safety.Lab Invest. 1997; 76: 505-516PubMed Google Scholar, 15Yoshimura A Gordon K Alpers CE Floege J Pritzl P Ross R Couser WG Bowen-Pope DF Johnson RJ Demonstration of PDGF B-chain mRNA in glomeruli in mesangial proliferative nephritis by in situ hybridization.Kidney Int. 1991; 40: 470-476Crossref PubMed Scopus (106) Google Scholar and included a murine monoclonal antibody (clone 1A4) to α-smooth muscle actin; a murine monoclonal antibody (clone PGF-007) to PDGF B-chain; a murine monoclonal IgG antibody (clone ED1) to a cytoplasmic antigen present in monocytes, macrophages, and dendritic cells; affinity-purified polyclonal goat anti-human/bovine type IV collagen IgG preabsorbed with rat erythrocytes; an affinity-purified IgG fraction of a polyclonal rabbit anti-rat fibronectin antibody; plus appropriate negative controls as described previously.14Burg M Ostendorf T Mooney A Koch KM Floege J Treatment of experimental mesangioproliferative glomerulonephritis with non-anticoagulant heparin: therapeutic efficacy and safety.Lab Invest. 1997; 76: 505-516PubMed Google Scholar, 15Yoshimura A Gordon K Alpers CE Floege J Pritzl P Ross R Couser WG Bowen-Pope DF Johnson RJ Demonstration of PDGF B-chain mRNA in glomeruli in mesangial proliferative nephritis by in situ hybridization.Kidney Int. 1991; 40: 470-476Crossref PubMed Scopus (106) Google Scholar Evaluation of all slides was performed by an observer who was unaware of the origin of the slides. To obtain mean numbers of infiltrating leukocytes in glomeruli, more than 50 consecutive cross sections of glomeruli were evaluated and mean values per kidney were calculated. For the evaluation of the immunoperoxidase stains for α-smooth muscle actin and PDGF B-chain, each glomerular area was graded semiquantitatively, and the mean score per biopsy was calculated. Each score reflects mainly changes in the extent rather than intensity of staining and depends on the percentage of the glomerular tuft area showing focally enhanced positive staining: I, 0% to 25%; II, 25% to 50%; III, 50% to 75%; IV, >75%. We have recently described that data obtained using this scoring system are highly correlated with those obtained by computerized morphometry.16Kliem V Johnson RJ Alpers CE Yoshimura A Couser WG Koch KM Floege J Mechanisms involved in the pathogenesis of tubulointerstitial fibrosis in 5/6-nephrectomized rats.Kidney Int. 1996; 49: 666-678Crossref PubMed Scopus (257) Google Scholar, 17Hugo C Pichler R Gordon K Schmidt R Amieva M Couser WG Furthmayr H Johnson RJ The cytoskeletal linking proteins, moesin and radixin, are upregulated by platelet-derived growth factor, but not basic fibroblast growth factor in experimental mesangial proliferative glomerulonephritis.J Clin Invest. 1996; 97: 2499-2508Crossref PubMed Scopus (70) Google Scholar Double immunostaining for the identification of the type of proliferating cells was performed as reported previously16Kliem V Johnson RJ Alpers CE Yoshimura A Couser WG Koch KM Floege J Mechanisms involved in the pathogenesis of tubulointerstitial fibrosis in 5/6-nephrectomized rats.Kidney Int. 1996; 49: 666-678Crossref PubMed Scopus (257) Google Scholar, 17Hugo C Pichler R Gordon K Schmidt R Amieva M Couser WG Furthmayr H Johnson RJ The cytoskeletal linking proteins, moesin and radixin, are upregulated by platelet-derived growth factor, but not basic fibroblast growth factor in experimental mesangial proliferative glomerulonephritis.J Clin Invest. 1996; 97: 2499-2508Crossref PubMed Scopus (70) Google Scholar by first staining the sections for proliferating cells with a murine monoclonal antibody (clone BU-1) against bromodeoxyuridine-containing nuclease in Tris-buffered saline (Amersham, Braunschweig, Germany) using an indirect immunoperoxidase procedure. Sections were then incubated with the IgG1 monoclonal antibodies 1A4 against α-smooth muscle actin and ED1 against monocytes/macrophages. Cells were identified as proliferating mesangial cells or monocytes/macrophages if they showed positive nuclear staining for BrdU and if the nucleus was completely surrounded by cytoplasm positive for α-smooth muscle actin or ED1 antigen. Negative controls included omission of either of the primary antibodies, in which case no double staining was noted. In situ hybridization was performed on 4-μm sections of biopsy tissue fixed in buffered 10% formalin using a digoxigenin-labeled antisense RNA probe for type IV collagen18Eitner F Westerhuis R Burg M Weinhold B Gröne HJ Ostendorf T Rüther U Koch KM Rees AJ Floege J Role of interleukin-6 in mediating mesangial cell proliferation and matrix production in vivo.Kidney Int. 1997; 51: 69-78Crossref PubMed Scopus (52) Google Scholar as described.14Burg M Ostendorf T Mooney A Koch KM Floege J Treatment of experimental mesangioproliferative glomerulonephritis with non-anticoagulant heparin: therapeutic efficacy and safety.Lab Invest. 1997; 76: 505-516PubMed Google Scholar Detection of the RNA probe was performed with an alkaline-phosphatase-coupled anti-digoxigenin antibody (Genius nonradioactive nucleic acid detection kit, Boehringer-Mannheim, Mannheim, Germany) with subsequent color development. Controls consisted of hybridization with a sense probe to matched serial sections by hybridization of the antisense probe to tissue sections that had been incubated with RNAse A before hybridization or by deletion of the probe, antibody, or color solution.15Yoshimura A Gordon K Alpers CE Floege J Pritzl P Ross R Couser WG Bowen-Pope DF Johnson RJ Demonstration of PDGF B-chain mRNA in glomeruli in mesangial proliferative nephritis by in situ hybridization.Kidney Int. 1991; 40: 470-476Crossref PubMed Scopus (106) Google Scholar Glomerular mRNA expression was semiquantitatively assessed using the scoring system described above. Urinary protein was measured using the Bio-Rad protein assay (Bio-Rad Laboratories, München, Germany) and bovine serum albumin (Sigma) as a standard. All values are expressed as means ± SD. Statistical significance (defined as P < 0.05) was evaluated using Student t-tests or analysis of variance and Bonferroni t-tests. High-affinity DNA aptamers to the PDGF B-chain were identified previously by the SELEX process.8Green LS Jellinek D Jenison R Östman A Heldin CH Janjić N Inhibitory DNA ligands to platelet-derived growth factor B-chain.Biochemistry. 1996; 35: 14413-14424Crossref PubMed Scopus (362) Google Scholar The consensus secondary structure motif of these aptamers is a three-way helix junction with a conserved single-stranded loop at the point of strand exchange (Figure 1A). To improve nuclease resistance of one of the minimal aptamers (a truncated version of aptamer 36t in 8Green LS Jellinek D Jenison R Östman A Heldin CH Janjić N Inhibitory DNA ligands to platelet-derived growth factor B-chain.Biochemistry. 1996; 35: 14413-14424Crossref PubMed Scopus (362) Google Scholar), we have synthesized and tested a series of 2′-O-methyl-or 2′-fluoro-substituted aptamers to identify positions that tolerate such substitutions without a loss of binding affinity. In addition to identifying a pattern of allowed 2′-O-methyl- and 2′-fluoro substitutions, we found that trinucleotide loops on helices II and III in the aptamer could be replaced with hexaethylene glycol (18-atom) non-nucleotide spacers without compromising high-affinity binding to PDGF-AB or -BB (Figure 1A). This finding is in agreement with the notion that the helix junction domain of the aptamer represents the core of the structural motif required for high-affinity binding.8Green LS Jellinek D Jenison R Östman A Heldin CH Janjić N Inhibitory DNA ligands to platelet-derived growth factor B-chain.Biochemistry. 1996; 35: 14413-14424Crossref PubMed Scopus (362) Google Scholar In practical terms, the replacement of six nucleotides with two spacers is advantageous in that it reduces by four the number of coupling steps required for the synthesis of the aptamer. Another support for the importance of helix junction domain in binding comes from the control aptamer, in which eight nucleotides in the helix junction region were interchanged without formally changing the consensus secondary structure (Figure 1A). The binding affinity of this scrambled aptamer for PDGF-BB (Kd ≈ 1 μmol/L) is 10,000-fold lower compared with the binding affinity of the aptamer used in the experiments described below (Kd ≈ 0.1 nmol/L). We next compared the stabilities of the modified aptamer and its precursor DNA aptamer in rat serum in vitro. The half-time of the modified aptamer in serum was considerably longer (∼8 hours) compared with that of its all-DNA precursor (∼0.6 hours; Figure 1B). The observed increase in nuclease resistance is in agreement with previous studies with 2′-substituted nucleic acids.19Pieken WA Olsen DB Benseler F Aurup H Eckstein F Kinetic characterization of ribonuclease-resistant 2′-modified hammerhead ribozymes.Science. 1991; 253: 314-317Crossref PubMed Scopus (436) Google Scholar, 20Cummins LL Owens SR Risen LM Lesnik EA Freier SM McGee D Guinosso CJ Cook PD Characterization of fully 2′-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity.Nucleic Acids Res. 1995; 23: 2019-2024Crossref PubMed Scopus (301) Google Scholar In addition to the modifications mentioned above, for all experiments reported here, the modified DNA aptamer was conjugated to 40-kd PEG. Importantly, the addition of the PEG moiety to the 5′ end of the aptamer has no effect on the binding affinity of the aptamer for PDGF-BB (Kd ≈ 0.1 nmol/L). The concentration of the modified PDGF aptamer conjugated to 40-kd PEG in rat plasma after i.v. injection (1 mg/kg) is shown in Figure 1C. The clearance of the aptamer-PEG conjugate from plasma is biphasic with approximately 47% of the compound being cleared with a half-life of 32.4 ± 13 minutes and 53% of the compound being cleared with a half-life of 134.5 ± 13 minutes. The concentration of the aptamer in rat plasma after the i.v. injection is 16 μg/ml (1.6 μmol/L) at t = 0 and 0.21 μg/ml (21 nmol/L) at t = 12 hours (extrapolated). Thus, to a first approximation and assuming a linear increase in plasma concentration of the aptamer at the higher injected dose in the experiments described below (2.1 to 2.2 mg/kg i.v. every 12 hours), the aptamer concentration during the treatment period was not lower than 40 nmol/L (ie, 0.4 μg/ml). The sequence of PDGF is highly conserved among species, and human and rat PDGF B-chain sequences are 89% identical.21Herren B Weyer KA Rouge M Lötscher P Pech M Conservation in sequence and affinity of human and rodent PDGF ligands and receptors.Biochim Biophys Acta. 1993; 1173: 294-302Crossref PubMed Scopus (25) Google Scholar, 22Lindner V Giachelli CM Schwartz SM Reidy MA A subpopulation of smooth muscle cells in injured rat arteries expresses platelet-derived growth factor B-chain.Circ Res. 1995; 76: 951-957Crossref PubMed Scopus (102) Google Scholar Nevertheless, in view of the high specificity of aptamers,23Gold L Polisky B Uhlenbeck OC Yarus M Diversity of oligonucleotide functions.Annu Rev Biochem. 1995; 64: 763-797Crossref PubMed Scopus (735) Google Scholar the correct interpretation of th" @default.
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• W1556322054 title "Novel Approach to Specific Growth Factor Inhibition in Vivo" @default.
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