Missense Oligonucleotides Research Articles

Antisense oligodeoxynucleotides selectively suppress expression of the mutant alpha 2(I) collagen allele in type IV osteogenesis imperfecta fibroblasts. A molecular approach to therapeutics of dominant negative disorders.

We are investigating the use of antisense oligodeoxynucleotides to selectively suppress expression of the mutant type I collagen allele in osteogenesis imperfecta (OI). In this report, we target a human collagen mutation in its natural cellular context. We used cultured fibroblasts from a case of type IV OI, in which the mutant alpha 2(I) allele produces mRNA with exon 16 deleted due to a point mutation in the splice donor site. Lipid-mediated transfection was used to deliver antisense, sense and missense phosphorothioates targeted to both the abnormal mRNA exon 15/17 junction and the nuclear level point mutation. Significant suppression of the mutant protein chain and mRNA was achieved with antisense oligonucleotide to both mRNA and nuclear levels. Mutant protein was suppressed to 44-47% and mutant alpha 2(I) mRNA to 37-43% of their levels in control cells, indicating decreased mRNA as the basis for suppression. Selectivity of mutant allele suppression was better with an mRNA target: suppression was sequence specific and normal mRNA was expressed at 79% of its level in untreated cells. With a nuclear target, significant suppression of mutant mRNA occurred not only with antisense and sense, but also with missense oligonucleotide, which suppressed mutant mRNA to 60% of its level in untreated cells. We also investigated the time course of suppression of protein and mRNA in response to a 4 h transfection of antisense oligonucleotide. From 24-72 h after transfection, mutant protein was suppressed to approximately 50% of its untreated level and suppression of mutant message was significantly greater than that of normal message. The suppression achieved in these studies is insufficient for clinical intervention, but our results provide support for further development of antisense therapy as an approach to the treatment of dominant negative disorders.

Antisense oligonucleotides to CRABP I and II alter the expression of TGF-beta 3, RAR-beta, and tenascin in primary cultures of embryonic palate cells.

The cellular retinoic acid-binding proteins (CRABPs) are thought to modulate the responsiveness of cells to retinoic acid (RA). We have previously shown that primary cultures of murine embryonic palate mesenchymal (MEPM) cells express both CRABP-I and CRABP-II genes and that this expression is regulated by RA and transforming growth factor beta (TGF-beta). These cells also express high levels of TGF-beta 3, which is also regulated by RA and TGF-beta. We have used an antisense strategy to investigate the role of the CRABPs in retinoid-induced gene expression. Subconfluent cultures of MEPM cells were treated for several days with phosphorothioate modified 18-mer oligonucleotides antisense to CRABP-I or CRABP-II and then with all-trans-retinoic acid at a concentration of 3.3 microM or 0.33 microM for 5 or 22 h. Total RNA was then extracted and the expression of TGF-beta 3, retinoic acid receptor beta (RAR-beta), and tenascin was assessed by northern blot analysis. Antisense oligonucleotides to CRABP-I partially inhibited the RA-induced TGF-beta 3, RAR-beta, and tenascin mRNA expression. The corresponding mis-sense oligonucleotides were without effect. Antisense oligonucleotides to CRABP-II also partially inhibited RA-induced expression of these genes. As with the CRABP-I antisense, mis-sense oligonucleotides to CRABP-II had no effect. These data suggest that both CRABPs modulate the responsiveness of MEPM cells to retinoic acid. Inhibition of endogenous CRABP expression renders MEPM cells less responsive to RA with respect to induction of TGF-beta 3, RAR-beta, and tenascin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of the glutamate receptor δ2 subunit in the long-term depression of glutamate responsiveness in cultured rat Purkinje cells

An antisense oligonucleotide against the glutamate receptor δ2 subunit mRNA, which is selectively expressed only in Purkinje neurons, suppressed the induction of long-term depression (LTD) of glutamate responsiveness in the rat cerebellar culture. LTD of glutamate response is induced by pairing glutamate application and depolarization of a Purkinje cell. Treatment of the culture with the antisense oligonucleotide exerted no appreciable effect on basic physiological and morphological properties of Purkinje cells, except for LTD induction and reduction of δ2 immunoreactivity which was intense in distal dendrites. Sense and missense oligonucleotides, which were used as controls, did not block LTD induction. These results suggest that the glutamate receptor δ2 subunit is involved in the cerebellar LTD.

C-myc antisense oligonucleotides inhibit the colony-forming capacity of Colo 320 colonic carcinoma cells.

Colo 320 cells are colonic carcinoma cells known to express abundant c-myc mRNA. Based on the response of several hematopoietic cell lines to chemical inducers of differentiation, we reasoned that such agents might have similar inductive activity in Colo 320 cells. Accordingly, we exposed Colo 320 cells to 5 mM sodium butyrate (NaBT) for 7 d. C-myc expression decreased threefold and self-replicative potential decreased (defined as a greater than 60% decrease in colony-forming capacity in soft agar that did not contain inducer). In an effort to demonstrate a direct cause and effect between myc expression and the colony-forming capacity of Colo 320 cells, we exposed these cells to a 15-base antisense c-myc oligonucleotide (complementary to the translation initiation region of exon II). Cells were also exposed to equimolar (20 microM) amounts of sense and missense oligonucleotides. Subsequently, cells were incubated at 10, 20, 30, and 40 microM antisense DNA for 16 h, then washed and plated in oligonucleotide-free agar medium. We demonstrated that: (a) the oligomers were stable in the extracellular medium and in the cell cytoplasm; (b) the uptake of the oligonucleotides was 0.7%; (c) sense and missense oligonucleotides had no effect on colony-forming capacity; and (d) the antisense c-myc oligonucleotide resulted in a 40-75% concentration-dependent decrease in colony-forming capacity. The specific inhibition of colony-forming capacity by antisense DNA suggests that the role of myc expression in Colo 320 cells is similar to its role in hematopoiesis, and that the failure to inhibit myc expression maintains colony-forming capacity. This system provides a new strategy for inducing differentiation and may provide further insight into the genetic factors that govern the process of colonic carcinogenesis.

C-kit Expression by CD34+ Bone Marrow Progenitors and Inhibition of Response to Recombinant Human Interleukin-3 Following Exposure to c-kit Antisense Oligonucleotides

The c-kit proto-oncogene encodes a receptor having tyrosine-specific kinase activity and has been mapped to chromosome 4 in the human and chromosome 5 in the mouse, at the dominant white spotting locus (W). Mutations at the W locus affect various aspects of murine hematopoiesis. The c-kit proto-oncogene has been shown to be expressed by leukemic myeloblasts, but not by normal unseparated human bone marrow cells. The role of this oncogene in differentiation and proliferation of human hematopoietic progenitors is presently undefined. To determine c-kit expression by normal hematopoietic progenitors, CD34+ cells were isolated from disease-free human bone marrow, and RNA-based polymerase chain reaction (PCR) techniques were used to assess expression. By this method, we have demonstrated c-kit expression by CD34+ bone marrow progenitors. To address the functional requirement for c-kit expression in normal human hematopoiesis, CD34+ cells were incubated in the presence of sense, antisense, or missense oligonucleotides to c-kit, and subsequently cultured in the presence of either recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or recombinant human interleukin-3 (rhIL-3). Exposure of CD34+ cells to c-kit antisense oligonucleotides significantly inhibited colony-forming ability of cells cultured in the presence of rhIL-3, but had no effect on colony formation of cells cultured in rhGM-CSF. Together, these data suggest a possible role for c-kit in hematopoietic proliferation and differentiation that may be linked to some, but not all, stimulatory factors.

C-kit expression by CD34+ bone marrow progenitors and inhibition of response to recombinant human interleukin-3 following exposure to c-kit antisense oligonucleotides

The c-kit proto-oncogene encodes a receptor having tyrosine-specific kinase activity and has been mapped to chromosome 4 in the human and chromosome 5 in the mouse, at the dominant white spotting locus (W). Mutations at the W locus affect various aspects of murine hematopoiesis. The c-kit proto-oncogene has been shown to be expressed by leukemic myeloblasts, but not by normal unseparated human bone marrow cells. The role of this oncogene in differentiation and proliferation of human hematopoietic progenitors is presently undefined. To determine c-kit expression by normal hematopoietic progenitors, CD34+ cells were isolated from disease-free human bone marrow, and RNA-based polymerase chain reaction (PCR) techniques were used to assess expression. By this method, we have demonstrated c-kit expression by CD34+ bone marrow progenitors. To address the functional requirement for c-kit expression in normal human hematopoiesis, CD34+ cells were incubated in the presence of sense, antisense, or missense oligonucleotides to c-kit, and subsequently cultured in the presence of either recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or recombinant human interleukin-3 (rhIL-3). Exposure of CD34+ cells to c-kit antisense oligonucleotides significantly inhibited colony-forming ability of cells cultured in the presence of rhIL-3, but had no effect on colony formation of cells cultured in rhGM- CSF. Together, these data suggest a possible role for c-kit in hematopoietic proliferation and differentiation that may be linked to some, but not all, stimulatory factors.