Delivery of a hammerhead ribozyme specifically down-regulates the production of fibrillin-1 by cultured dermal fibroblasts
Delivery of a hammerhead ribozyme specifically down-regulates the production of fibrillin-1 by cultured dermal fibroblastsMichael W. Kilpatrick1,*, Leonidas A. Phylactou1, Maurice Godfrey2, Catherine H. Wu3, George Y. Wu3 and Petros Tsipouras1
1Department of Pediatrics and 3Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030, USA and 2Department of Pediatrics and Munroe Center for Human Genetics, University of Nebraska Medical Center, Omaha, NE, USA
Received July 1, 1996;Revised and Accepted September 6, 1996
The hammerhead ribozyme is a small catalytic RNA molecule. Potential hammerhead ribozymes that possess a catalytic domain and flanking sequence complementary to a target mRNA can cleave in trans at a putative cleavage site within the target molecule. We have investigated the potential of hammerhead ribozymes to down-regulate the product of the fibrillin-1 gene (FBN1). Fibrillin is a 347 kDa glycoprotein that is a major constituent of the elastin-associated microfibrils. Mutations in the FBN1 gene are responsible for Marfan syndrome (MFS), a common systemic disorder of the connective tissue. Many FBN1 mutations responsible for MFS appear to act in a dominant-negative fashion, raising the possibility that reduction of the amount of product from the mutant FBN1 allele might be a valid therapeutic approach for MFS. A trans-acting hammerhead ribozyme (FBN1-RZ1) targeted to the 5' end of the human FBN1 mRNA has been designed and synthesized, and shown to cleave its target efficiently in vitro. FBN1-RZ1 cleavage is magnesium dependent and efficient at both 37 and 50oC. Delivery of the FBN1-RZ1 ribozyme into cultured dermal fibroblasts, by receptor-mediated endocytosis of a ribozyme-transferrin-polylysine complex, specifically reduces both cellular FBN1 mRNA and the deposition of fibrillin in the extracellular matrix. These results suggest that the use of hammerhead ribozymes is a valid approach to the study of fibrillin gene expression and possibly to the development of a therapeutic approach to MFS.
Ribozymes are catalytic RNA molecules (1 ). The hammerhead ribozyme is a small RNA molecule whose catalytic activity resides in a core of <40 ribonucleotides, arranged in a typical three-stem structure (2 ). Potential hammerhead ribozymes that possess a catalytic domain and flanking sequence complementary to a target mRNA can cleave in trans at a three base target sequence NUY (where N is any base and Y is any base except G) within the target molecule (2 ,3 ). This ability has led to the synthesis of hammerhead ribozymes designed to cleave a variety of target RNA molecules (4 -7 ). However, few examples exist of the use of hammerhead ribozymes to reduce the amount of an endogenous gene product. Thus, the ability of hammerhead ribozymes to cleave a target mRNA in vivo and, therefore, down-regulate the expression of a particular gene product needs to be investigated for individual systems (8 ). We have determined the potential of hammerhead ribozymes to down-regulate the product of the fibrillin-1 gene (FBN1). Fibrillin-1 is a large structural glycoprotein that is a major constituent of the elastin-associated microfibrils (9 ). The protein has a molecular weight of 347 kDa and is coded for by an mRNA of 9663 bases (9 ,10 ). Mutations in the gene coding for fibrillin on human chromosome 15 (FBN1) have been shown to be responsible for Marfan syndrome (MFS) (11 -27 ).
To determine whether the FBN1-RZ1 ribozyme (Fig. 1 ) is capable of cleaving its target sequence, an FBN1 mRNA fragment was treated with ribozyme FBN1-RZ1 in vitro. A radiolabelled 86 base FBN1 mRNA fragment was prepared and mixed with an equimolar amount of ribozyme at 50oC as described in Materials and Methods. The results of this treatment are shown in Figure 2 (i). Panel B shows treatment with FBN1-RZ1 and panel A shows treatment with control ribozyme. The control ribozyme is identical to FBN1-RZ1 in the sequence of its catalytic domain and stem-loop II, but differs in the sequence of stems I and III, having no complementarity to the 86 base FBN1 mRNA fragment. Treatment with the control ribozyme leaves the FBN1 mRNA fragment intact, whereas treatment with FBN1-RZ1 produces the 56 and 30 base subfragments expected if cleavage is occurring at the GUC target sequence in the FBN1 mRNA fragment.
It has been shown previously that ribozyme activity requires the presence of magnesium (1 ). To determine whether the observed FBN1-RZ1 cleavage of FBN1 mRNA was magnesium dependent, the 86 base FBN1 mRNA fragment was treated with FBN1-RZ1 ribozyme in the presence of 0-40 mM MgCl2. To assess thermal stability of ribozyme activity, cleavage studies were performed at 37 as well as 50oC. Figure 3 shows the result of treating the 86 base FBN1 mRNA fragment with an equimolar amount of FBN1-RZ1 ribozymefor 30 min at 37 or 50oC. Ribozyme treatment in the absence of magnesium (lanes 0) shows no cleavage of the 86 base fragment, whereas treatment in the presence of 10-40 mM magnesium chloride leads to the production of the expected 56 and 30 base subfragments at both 37 and 50oC.
Having demonstrated that the FBN1-RZ1 ribozyme is capable of cleaving its target sequence efficiently in vitro at 37oC, we next wanted to determine the effect of the ribozyme on cultured fibroblasts. To introduce the FBN1-RZ1 ribozyme into cultured dermal fibroblasts, we utilized receptor-mediated endocytosis via the transferrin receptor. Receptor-mediated endocytosis has been used previously to introduce and express nucleic acid successfully in specific eukaryotic cells and tissues (36 -38 ). Total cellular RNA was isolated from cultured fibroblasts 48 h after transfection with FBN1-RZ1 or a control RNA of sequence complementary to that of FBN1-RZ1 and subjected to RT-PCR using FBN1 and [beta]-actin primer sets. Figure 4 shows that transfection with FBN1-RZ1 (lane RZ1) specifically reduces FBN1 mRNA levels relative to those of control [beta]-actin mRNA. In contrast, transfection of fibroblasts with a control RNA of sequence complementary to that of FBN1-RZ1 (lane T2L) or treatment with transferrin-polylysine conjugate alone (lane CA) does not reduce FBN1 mRNA levels.
The effect of delivery of FBN1-RZ1 at the protein level was determined by immunohistochemical analysis of cultured dermal fibroblasts using an anti-fibrillin primary antibody, as described in Materials and Methods. Cells were transfected with FBN1-RZ1-transferrin-polylysine complex or transferrin-polylysine conjugate alone as a control. Figure 5 A shows the results of immunochemical analysis of transfected cells for the presence of fibrillin. The fibrillin can be seen as a meshwork surrounding the cells, with the cell nuclei apparent as brightly stained oval structures (RC). Whereas transfection with transferrin-polylysine conjugate alone (CA) does not appear to diminish the amount of fibrillin produced by cultured fibroblasts, transfection with FBN1-RZ1-transferrin-polylysine complex (RC) dramatically reduces the amount of fibrillin produced. The brightly staining cell nuclei demonstrate the presence of healthy fibroblasts in segment RC. To determine whether the FBN1-RZ1-mediated down-regulation of fibrillin production was specific for fibrillin, the effect of delivery of FBN1-RZ1 on the production of an unrelated matrix protein, fibronectin, by cultured fibroblasts was also examined. Figure 5 B shows the result of immunostaining of transfected cells for the presence of fibronectin. In contrast to its effect on fibrillin production, delivery of FBN1-RZ1 (Fig. 5 B, RC), does not reduce the amount of fibronectin produced by cultured fibroblasts. To determine the reproducibility of the effect of FBN1-RZ1 on the deposition of fibrillin to the extracellular matrix of cultured fibroblasts, this experiment was repeated four times. The specific reduction in fibrillin deposition observed was essentially identical each time.
In summary, we have shown that an antisense hammerhead ribozyme can be used to down-regulate the production of fibrillin by cultured dermal fibroblasts. The down-regulation of fibrillin is specific, in that the production of fibronectin by the fibroblasts is not down-regulated by the presence of the FBN1-RZ1 ribozyme. The ribozyme has been shown to cleave its target mRNA in vitro and to reduce FBN1 mRNA levels specifically in cultured fibroblasts. These results suggest that the use of hammerhead ribozymes is a valid approach to study the expression of endogenous genes. In particular, this approach may provide a powerful method for the study of fibrillin and the analysis of the role played by this and other microfibrillar molecules in the complex process of the assembly of the extracellular microfibrils, about which little is currently known. For example, the design of ribozymes targeted to specific regions of the FBN1 mRNA, such as the 3'-untranslated region (UTR), might provide insights into the complexities of fibrillin synthesis and secretion. Similarly, targeting of ribozymes to other components of the microfibrils, such as the microfibril-associated glycoprotein (MAGP) (28 ), may help elucidate the role of different molecules in microfibrillar assembly.
MFS is a systemic disorder of the connective tissue which manifests itself in the musculoskeletal, cardiovascular and ocular systems (29 -31 ). The disease is one of the most common connective tissue disorders with an estimated prevalence of 1 in 10 000, is inherited in an autosomal dominant manner, imparts significant morbidity and mortality and, if untreated, considerably reduces life expectancy (30 ,32 ). Many FBN1 mutations responsible for MFS appear to act in a dominant-negative fashion (12 ,15 ,33 ), and expression of a mutant human fibrillin allele upon a normal human or murine genetic background has been shown to produce a Marfan cellular phenotype (34 ). The ability successfully to down-regulate cellular FBN1 mRNA and the extracellular deposition of fibrillin suggests that the hammerhead ribozyme may be useful in the development of a therapeutic approach to MFS. For example, it might be possible to utilize this approach to down-regulate specifically the mutant FBN1 allele in fibroblasts derived from individuals with MFS, or to combine the ablation of endogenous FBN1 expression in MFS individuals with the delivery and expression of normal fibrillin. The sensitivity of hammerhead ribozymes to mismatches between ribozyme and target sequence (35 ) supports the feasibility of designing ribozymes to target mutant FBN1 alleles specifically. This approach may also be applicable to other disorders where there is evidence of a mutant gene exerting a dominant-negative effect and where it might, therefore, be valid to develop a therapy based on the ablation of a mutant gene product.
The ribozyme FBN1-RZ1 was designed as a hammerhead ribozyme targeted to the 5' end of the FBN1 mRNA (10 ). FBN1-RZ1 consists of 12 bases complementary to bases 49-60 of the FBN1 mRNA, which will form helix I of the ribozyme, followed by the sequence necessary to form the main body of the hammerhead ribozyme, comprising two regions of conserved sequences connected by helix II (2 ). Then follow 13 bases complementary to bases 62-74 of the FBN1 mRNA which will form helix III of the ribozyme (Fig. 1 ). A control ribozyme, identical to FBN1-RZ1 in the sequence of its catalytic domain and stem-loop II, but differing in the sequence of stems I and III such that it had no complementarity to the FBN1 mRNA sequence targeted by FBN1-RZ1, was also designed. DNA template for in vitro synthesis of FBN1-RZ1 was produced by ligation of a 58 bp DNA fragment containing the FBN1-RZ1 sequence (Fig. 1 ) flanked by BamHI and XbaI restriction sites into the BamHI and XbaI sites of the vector pBSIISK+ (Stratagene). Linearization of the resulting construct (pFBN1-RZ1) with BamHI produced the DNA template for synthesis of FBN1-RZ1 in vitro by transcription off the T3 promoter. Radiolabelled 86 base FBN1 mRNA fragment was produced by in vitro T7 RNA polymerase transcription of a 109 bp DNA fragment containing the FBN1 cDNA sequence adjacent to the T7 promoter.
For large-scale in vitro transcription, linearized DNA template (1 [mu]g) was incubated with 50 U of RNasin ribonuclease inhibitor, 40 mM Tris-HCl pH 7.5, 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol (DTT), 7.5 mM ATP, 7.5 mM CTP, 7.5 mM GTP, 7.5 mM UTP and 50-100 U of T3 RNA polymerase in a total volume of 20 [mu]l, at 37oC for 1-16 h. DNA template was removed by incubation with 2 U of RNase-free DNase I at 37oC for 30 min, and the RNA recovered by ethanol precipitation. For synthesis of radiolabelled transcripts, 0.1-1 [mu]g of DNA template was incubated with 25 U of RNase inhibitor, 40 mM Tris-HCl pH 7.5, 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 1 mM ATP, 1 mM CTP, 1 mM GTP, 50 [mu]Ci of [[alpha]-32P]UTP (400-800 Ci/mmol) and 10 U of T7 polymerase, in a total volume of 20 [mu]l, at 37oC for 30 min-1 h. DNA template was removed by incubation with 2 U of RNase-free DNase I at 37oC for 30 min, and the RNA recovered by ethanol precipitation.
To assay for cleavage of FBN1 mRNA by FBN1-RZ1, an 86 base radiolabelled FBN1 mRNA fragment was prepared by in vitro transcription. Ribozyme FBN1-RZ1 was prepared from plasmid pFBN1-RZ1 by transcription off the T3 promoter. The FBN1 mRNA fragment (5-50 ng) was treated with an equimolar amount of FBN1-RZ1 at 37 or 50oC in 50 mM Tris-HCl pH 7.5, 0-40 mM MgCl2, in a total volume of 50 ml, for 0-3.5 h. RNA fragments were separated by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography.
Treatment of total fibroblast RNA with FBN1-RZ1 was by incubation of 2 [mu]g of total RNA (isolated from ~500 000 fibroblasts) with 2 [mu]g of FBN1-RZ1, in a total volume of 50 [mu]l, at 37oC for 1 h. FBN1 mRNA and control actin mRNA were then quantitated by RT-PCR.
Normal skin fibroblasts were cultured in monolayer in modified Dulbecco's medium (DMEM) supplemented with 10% fetal bovine serum. For transfection, cells were incubated in 100 mm culture dishes until nearly confluent. Transfection was by receptor-mediated endocytosis of transferrin-polylysine conjugates as described previously (36 ). Conjugate was prepared by coupling transferrin to poly-L-lysine by periodate oxidation of the carbohydrate of transferrin followed by coupling to polylysine and reductive amination as described (37 ), or was obtained from Sigma (Cat#T0288). FBN1-RZ1-transferrin-polylysine complex was formed by incubation of 2-5 [mu]g of FBN1-RZ1, 4-10 [mu]g of transferrin-polylysine conjugate and 150 mM NaCl in a total volume of 26-65 [mu]l, at room temperature, for 30 min. The medium was removed from the culture dish and the 65 [mu]l complex added. DMEM (3 ml) was then added and the cells incubated for 4 h. The medium was then replaced with DMEM plus 10% fetal calf serum. Duplicate plates of fibroblasts were then assayed for FBN1 mRNA or fibrillin-1 protein as described below.
Detection of FBN1 mRNA in total cellular RNA was by reverse transcription followed by amplification of FBN1 and, as a control, [beta]-actin-specific cDNA fragments. Total RNA from 150 000- 500 000 fibroblasts was reverse transcribed by incubation, with 100 ng each of FBN1 and [beta]-actin-specific downstream primers, at 95oC for 5 min then at 52oC for 10 min. Reverse transcription buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 5 mM MgCl2), 0.5 mM dNTPs and 100 U of M-MLV reverse transcriptase (GIBCO-BRL) were then added, the reaction volume adjusted to 25 [mu]l and incubation continued at 37oC for 1 h. For amplification of cDNA, 1/10 000th of the reverse transcription reaction was amplified in 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, 0.1 mg/ml gelatin, pH 8.3, 50 ng each of FBN1 and [beta]-actin-specific primers and 1 U of Taq polymerase in a total volume of 10 [mu]l. PCR samples were denatured for 5 min at 95oC and subjected to amplification for 20 cycles (95oC: 1.5 min, 57oC: 1.5 min, 72oC: 1 min). Quantitative amplification conditions were established using the same templates and primers. Some control PCR reactions were supplemented with RNase A (1 mg/ml) or FBN1-RZ1 (2 ng), prior to amplification. Amplified fragments were resolved on polyacrylamide gels and detected using a phosphorimager (Molecular Dynamics).
Detection of fibrillin-1 following transfection was essentially as has been described previously (39 ). Briefly, the fibroblasts were treated with trypsin to remove them from the culture dishes and were transferred to chamber slides (four 1.8 cm2 chambers per slide), at a density of 2.5*105 cells per chamber. Following 48 h incubation at 37oC, the medium and chambers were removed from the slides and the cells fixed with ice-cold acetone. To visualize the fibrillin, the cells were stained with anti-fibrillin-1 primary antibody (9 ) at room temperature for 1-2 h. The slides were then washed with phosphate-buffered saline (PBS) and a secondary antibody (anti-mouse IgG conjugated to phycoerythrin) (Biomeda) added. Following incubation at 37oC for 30 min, the slides were again washed with PBS and, if required, incubated with the nuclear dye propidium iodide to visualize the cells. The slides were then washed and mounted and viewed under fluorescence microscopy. Fibronectin was detected in a similar manner using a monoclonal anti-human fibronectin primary antibody (Sigma, cat#F7387).
We would like to thank Dr Stephen Helfand for advice on the fluorescence microscopy and Drs Triantafyllos Tafas and Gordon Carmichael for helpful discussions. This work was supported by grants from the March of Dimes Birth Defects Foundation 6-0279 (P.T.), American Heart Association 92015820 (P.T.), Coles Family Foundation (P.T.), NIH-HL48126 (M.G.), March of Dimes Clinical Research Grant FY94-0012 (M.G.), American Heart Association NE Affiliate 9307786S (M.G.), NIH-DK92182 (G.Y.W.), and TargeTech Inc./Immune Response Corporation (C.H.W.). G.Y.W. and C.H.W. hold equity in the Immune Response Corporation.
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