DNAzymes to β1 and β3 mRNA Down-regulate Expression of the Targeted Integrins and Inhibit Endothelial Cell Capillary Tube Formation in Fibrin and Matrigel

Abstract

A novel approach based on DNA-cleaving deoxyribozymes (DNAzymes) was developed to control expression of ␤ 1 and ␤ 3 integrins in endothelial cells. To engineer a specific cleavage site in mRNA, the flanking domains of DNAzymes were derived from oligodeoxynucleotides complementary to sequences corresponding to 1053-1070 and 1243-1267 in ␤ 1 and ␤ 3 mRNA, respectively. Phosphorothioate analogues of these antisense oligodeoxynucleotides, designated ␤1-1053 and ␤3-1243, significantly inhibited expression of ␤ 1 and ␤ 3 integrin subunits in endothelial and K562 cells at the level of mRNA and protein synthesis. They also specifically decreased the cell surface expression of corresponding subunits in endothelial cells and K562 cells, as measured by flow cytometry. In functional tests, ␤1-1053 and ␤3-1243 markedly reduced adhesion of cells to fibronectin and vitronectin, respectively. We designed DNAzymes to ␤ 1 and ␤ 3 mRNAs containing a 15-deoxynucleotide catalytic domain that was flanked by two substrate recognition segments of 8 and 10 deoxynucleotides for ␤ 1 and ␤ 3 DNAzymes, respectively. Both DNAzymes in the presence of Mg 2؉ specifically cleaved their substrates, synthetic ␤ 1 and ␤ 3 mRNA fragments. Although DNAzymes were partially modified with phosphorothioate and with 2-O-methyl groups at both the 5 and 3 ends indicated similar kinetic parameters, they were significantly more potent than the unmodified DNAzymes because of their much higher resistance to nuclease degradation. Similar to the antisense oligonucleotides, DNAzymes abolished microvascular endothelial cell capillary tube formation in fibrin and Matrigel. In conclusion, DNAzymes to ␤ 1 and ␤ 3 mRNAs with 2-O-methyl modifications are potentially useful as gene-inactivating agents and may ultimately provide a therapeutic means to inhibit angiogenesis in vivo.Angiogenesis is a multistep sequence of cellular reactions beginning with degradation of extracellular matrix and then proliferation and migration of endothelial cells followed by lumen formation and maturation (1). The formation of new blood vessels is critical to the development of normal tissues as well as the growth of solid tumors and to a large extent depends on specific molecular interactions between vascular cells and extracellular matrix (2, 3). Aberrant angiogenesis is also a key process for progress of many disorders including atherosclerosis (4), diabetic retinopathy (5) and restenosis (6). Currently, two groups of integrin receptors are known to regulate adhesive interactions during angiogenesis. Integrins in the ␤ 1 subfamily, particularly ␣ 5 ␤ 1 and ␣ 2 ␤ 1 , which are substantially expressed in human angiogenic blood vessels, promote endothelial cell morphogenesis, migration, and tube formation (7,8). The second group is composed of vitronectin receptors ␣ V ␤ 3 and ␣ V ␤ 5 , which are poorly expressed in quiescent vasculature (9, 10) but can be substantially expressed by neoplastic vasculature and cells (11,12). Expression of ␣ V ␤ 3 and ␣ V ␤ 5 can be differentially up-regulate...

DNAzymes to mouse β1 integrin mRNA in vivo: targeting the tumor vasculature and retarding cancer growth

“…31 This particular DNA sequence (hb1DE, 5 0 CAAGGTGAGg 1 g 2 c 3 t 4 a 5 g 6 c 7 t 8 a 9-c 10 a 11 a 12 c 13 g 14 a 15 AATAGAAG 3 0 ) was previously analyzed for its enzymatic activity, specificity, exonuclease resistance and ability to inhibit an expression of human b1 integrins in endothelial cells. 3 It also inhibited adhesive properties of cells as evaluated by analysis of both adhesion and migration to fibronectin, laminin and collagen. To increase the stability of this DNAzyme, modified oligonucleotides with 2 0 -O-methyl-substituted residues were introduced at both the 5 0 and 3 0 sides.…”

“…Matrigel plugs were removed 7 days postimplantation and photographed. Vascularization of plugs was determined by measuring hemoglobin concentration by Drabkin's reagent (Sigma, St Louis, MO) and calculated per 100 mm 3 .…”

“…Figure 3 shows that solid prostate carcinoma growth in athymic nude mice is significantly inhibited by mb1DE when compared with control mb1DE SC . In this experiment, DNAzymes at 1.25 mg per injection, either active or inactive, were administered into solid PC-3 tumors every second day after the tumor volume was assessed to be 150 mm 3 . Mouse body weight was assessed at the beginning and at the end of the experiment.…”

“…2 As they show high substrate flexibility, DNAzymes are frequently developed specifically to recognize the AU nucleotides of the start codon, which are usually located within the region having little secondary structure. Thus, DNAzymes bind the RNA substrate through complementary base-pairing, cleave their target mRNA in a gene-specific manner, 3 and can be used to control even complex biological processes. As examples, DNAzymes that were designed to (a) cleave b1 and b3 integrin mRNA-reduced expression of the targeted integrin subunits in endothelial cells and blocked proliferation, migration and network formation in a fibrin and Matrigel matrix, 3 and (b) cleave 12-lipoxygenase mRNA specifically downregulated the expression of this protein and its metabolites, known to play a crucial role in tumor angiogenesis.…”

“…Thus, DNAzymes bind the RNA substrate through complementary base-pairing, cleave their target mRNA in a gene-specific manner, 3 and can be used to control even complex biological processes. As examples, DNAzymes that were designed to (a) cleave b1 and b3 integrin mRNA-reduced expression of the targeted integrin subunits in endothelial cells and blocked proliferation, migration and network formation in a fibrin and Matrigel matrix, 3 and (b) cleave 12-lipoxygenase mRNA specifically downregulated the expression of this protein and its metabolites, known to play a crucial role in tumor angiogenesis. 4 In addition, (c) DNAzyme made to cleave VEGFR2 mRNA degraded its substrate efficiently and inhibited the proliferation of endothelial cells with a concomitant reduction of vascular endothelial growth factor (VEGF)R2 mRNA, and blocked tumor growth in vivo, 5 and (d) a DNAzyme for cleaving survivin mRNA markedly increased apoptosis and inhibited the growth of human pancreatic carcinoma cells.…”