C. A. Stein

Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

For the past 15–20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called ‘gymnosis’) that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

miR-21 mediates hematopoietic suppression in MDS by activating TGF-β signaling

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. We observed that SMAD7, a negative regulator of transforming growth factor-beta (TGF-β) receptor-I kinase, is markedly reduced in MDS and leads to ineffective hematopoiesis by overactivation of TGF-β signaling. To determine the cause of SMAD7 reduction in MDS, we analyzed the 3UTR of the gene and determined that it contains a highly conserved putative binding site for microRNA-21. We observed significantly elevated levels of miR-21 in MDS marrow samples when compared with age-matched controls. miR-21 was shown to directly bind to the 3UTR of SMAD7 and reduce its expression in hematopoietic cells. Next, we tested the role of miR-21 in regulating TGF-β signaling in a TGF-β-overexpressing transgenic mouse model that develops progressive anemia and dysplasia and thus serves as a model of human bone marrow failure. Treatment with a chemically modified miR-21 inhibitor led to significant increases in hematocrit and led to an increase in SMAD7 expression in vivo. Inhibition of miR-21 also led to an increase in erythroid colony formation from primary MDS bone marrow progenitors, demonstrating its ability in stimulating hematopoiesis in vitro. Taken together, these studies demonstrate the role of miR-21 in regulating overactivated TGF-β signaling in MDS.

Antisense RNA Down-Regulation of bcl-2 Expression in DU145 Prostate Cancer Cells Does Not Diminish the Cytostatic Effects of G3139 (Oblimersen)

Purpose: Inhibition of the function of the bcl-2 protein has been postulated to sensitize cells to cytotoxic chemotherapy, and thus provides an attractive target for investigative therapies. G3139, a phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the bcl-2 mRNA, is currently being evaluated in several Phase II and Phase III clinical trials. However, the mechanism of action of this molecule appears to depend on a combination of antisense plus nonantisense events. Indeed, the very idea that bcl-2 is a critical target is, at least in part, an extrapolation from experiments in which intracellular bcl-2 protein concentrations have been dramatically increased, yielding chemoresistant cells. Experimental Design: In this work, we down-regulated the expression of bcl-2 protein by 80–90% by two different antisense RNA strategies (antisense RNA and small interfering RNA) in DU145 prostate cancer cells. Results: Even after down-regulation of bcl-2 protein expression by either one of these strategies, the cellular phenotype induced by subsequent G3139 treatment (inhibition of cellular growth and the generation of reactive oxygen species) was essentially identical to that induced in mock-infected or wild-type DU145 cells in which bcl-2 protein expression had not been down-regulated previously. Conclusions: These results strongly suggest that bcl-2 expression in DU145 cells is not strongly associated with the prolife phenotype and that the mechanism by which G3139 produces its cytostatic effects in this cell line is bcl-2 independent.

Silencing of gene expression by gymnotic delivery of antisense oligonucleotides.

Antisense oligodeoxyribonucleotides have been used for decades to achieve sequence-specific silencing of gene expression. However, all early generation oligonucleotides (e.g., those with no other modifications than the phosphorothioate backbone) are inactive in vitro unless administered using a delivery vehicle. These delivery vehicles are usually lipidic but can also be polyamines or some other particulate reagent. We have found that by employing locked nucleic acid (LNA) phosphorothioate gap-mer nucleic acids of 16 mer or less in length, and by carefully controlling the plating conditions of the target cells and duration of the experiment, sequence-specific gene silencing can be achieved at low micromolar concentrations in vitro in the absence of any delivery vehicle. This process of naked oligonucleotide delivery to achieve gene silencing in vivo, which we have termed gymnosis, has been observed in many both adherent and nonadherent cell lines against several different targets genes.

Gene profiling study of G3139- and Bcl-2-targeting siRNAs identifies a unique G3139 molecular signature

G3139 is a phosphorothioate oligodeoxyribonucleotide that is targeted to the initiation codon region of the Bcl-2 mRNA, which downregulates Bcl-2 protein and mRNA expression via an antisense mechanism. In previous work, we have demonstrated that the phenotype observed in several prostate and melanoma cell lines after treatment with G3139 appears to be Bcl-2 independent. In contrast, downregulation of Bcl-2 expression by a small interfering RNA (siRNA) produced little or no phenotype change. In the present work, we performed an Agilent oligonucleotide microarray assay on mRNA isolated from PC3 prostate cancer cells that were treated with two different oligonucleotide gene-silencing reagents. G3139 and a Bcl-2-targeting siRNA both downregulate Bcl-2 expression, but via different mechanisms. A side-by-side comparative analysis showed that the expression profile generated by these molecules differs substantially. The study revealed upregulation of the expression of stress-inducible genes in PC3 cells at 1 and 3 days after a 5-h transfection with G3139 complexed with Lipofectamine 2000. In contrast, only a very diminished stress response was seen 1 and 3 days after a 24-h transfection of siRNA/Lipofectamine 2000 complexes. These results highlight the profound differences in off-target effects in cells treated with the phosphorothioate oligonucleotide G3139 and with an siRNA targeted to the same gene, and provide further evidence that the mechanism of action of G3139 is not related to Bcl-2 silencing.

Changes in Gene Expression Induced by Phosphorothioate Oligodeoxynucleotides (Including G3139) in PC3 Prostate Carcinoma Cells Are Recapitulated at Least in Part by Treatment with Interferon-β and -γ

Purpose: G3139 is an antisense bcl-2 phosphorothioate oligodeoxyribonucleotide that is currently being evaluated in Phase III clinical trials in several human cancers. The aim of the present work was to further identify the apparent non-bcl-2-dependent mechanism of this action of this compound in PC3 prostate cancer cells. Experimental Design: We performed Affymetrix U95A oligonucleotide microarray studies on mRNA isolated from cells treated with G3139 and related oligonucleotides. Results: Hierarchical clustering revealed the presence of a set of genes of which the expression was elevated on both 1 and 3 days after oligonucleotide treatment. Significantly, the persistence of expression of the up-regulation of these genes, many of which are members of the IFN cascade, was greater for G3139 than for any other oligomer evaluated. Furthermore, many of the genes with the greatest up-regulation of expression are also those of which the expression is up-regulated after treatment of cells with IFNs. Treatment of PC3 cells with either IFN-β or -γ recapitulated some of the aspects of the molecular and phenotypic changes observed after treatment with a G3139/Lipofectin complex. These include down-regulation of bcl-2 protein expression itself, down-regulation of protein kinase C α protein expression (but not that of other protein kinase C isoforms), alteration in p21/Waf1/Cip1 protein expression, up-regulation of MHC-I cell surface expression, and profound suppression of cell growth in the absence of a notable increase in cellular apoptosis. However, G3139 (when complexed with Lipofectin) did not induce the up-regulation of expression of either type I or type II IFNs, nor could IFNs be found in conditioned media from treated cells. Conclusions: Oligonucleotide microarray experiments demonstrated that G3139 could induce elements of the IFN cascade in PC3 cells in vitro. In addition, the cellular phenotype obtained after treatment with exogenous IFN could, at least in part, recapitulate that obtained after G3139 treatment. Nevertheless, the oligonucleotide microarray experiments we performed also demonstrated that there are extremely large qualitative and quantitative differences between the two treatments.

Bcl-2 Protein in 518A2 Melanoma Cells In vivo and In vitro

Purpose: Bcl-2 is an apoptotic protein that is highly expressed in advanced melanoma. Several strategies have been employed to target the expression of this protein, including G3139, an 18-mer phosphorothioate oligodeoxyribonucleotide targeted to the initiation region of the Bcl-2 mRNA. This compound has recently completed phase III global clinical evaluation, but the function of Bcl-2 as a target in melanoma has not been completely clarified. To help resolve this question, we have permanently and stably down-regulated Bcl-2 protein and mRNA expression in 518A2 cells by two different technologies and evaluated the resulting clones both in vitro and in vivo. Experimental Design: 518A2 melanoma cells were transfected with plasmids engineered to produce either a single-stranded antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA or a short hairpin RNA also targeted to the Bcl-2 mRNA. In vitro growth, the apoptotic response to G3139, and the G3139-induced release of cytochrome c from isolated mitochondria were evaluated. Cells were then xenografted into severe combined immunodeficient mice and tumor growth was measured. Results:In vitro, down-regulation of Bcl-2 expression by either method produced no change either in the rate of growth or in sensitivity to standard cytotoxic chemotherapeutic agents. Likewise, the induction of apoptosis by G3139 was entirely Bcl-2 independent. In addition, the G3139-induced release from isolated mitochondria was also relatively independent of Bcl-2 expression. However, when xenografted into severe combined immunodeficient mice, cells with silenced Bcl-2, using either technology, either failed to grow at all or grew to tumors of low volume and then completely regressed. In contrast, control cells with “normal” levels of Bcl-2 protein expression expanded to be large, necrotic tumors. Conclusions: The presence of Bcl-2 protein profoundly affects the ability of 518A2 melanoma cells to grow as human tumor xenografts in severe combined immunodeficient mice. The in vivo role of Bcl-2 in melanoma cells thus differs significantly from its in vitro role, and these experiments further suggest that Bcl-2 may be an important therapeutic target even in tumors that do not contain the t14:18 translocation.

Therapeutic oligonucleotides: the road not taken.

Antisense oligonucleotide therapeutics have been in development for almost 25 years without a single U.S. Food and Drug Administration–approved product in cancer. The reasons for this absence stem, in part, from a deep lack of understanding about how to deliver these molecules to cancer cells in vivo. Clin Cancer Res; 17(20); 6369–72. ©2011 AACR.

G3139, an anti-Bcl-2 antisense oligomer that binds heparin-binding growth factors and collagen I, alters in vitro endothelial cell growth and tubular morphogenesis.

Purpose: We examined the effects of G3139 on the interaction of heparin-binding proteins [e.g., fibroblast growth factor 2 (FGF2) and collagen I] with endothelial cells. G3139 is an 18-mer phosphorothioate oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA. A randomized, prospective global phase III trial in advanced melanoma (GM301) has evaluated G3139 in combination with dacarbazine. However, the mechanism of action of G3139 is incompletely understood because it is unlikely that Bcl-2 silencing is the sole mechanism for chemosensitization in melanoma cells. Experimental Design: The ability of G3139 to interact with and protect heparin-binding proteins was quantitated. The effects of G3139 on the binding of FGF2 to high-affinity cell surface receptors and the induction of cellular mitogenesis and tubular morphogenesis in HMEC-1 and human umbilical vascular endothelial cells were determined. Results: G3139 binds with picomolar affinity to collagen I. By replacing heparin, the drug can potentiate the binding of FGF2 to FGFR1 IIIc, and it protects FGF from oxidation and proteolysis. G3139 can increase endothelial cell mitogenesis and tubular morphogenesis of HMEC-1 cells in three-dimensional collagen gels, increases the mitogenesis of human umbilical vascular endothelial cells similarly, and induces vessel sprouts in the rat aortic ring model. Conclusions: G3139 dramatically affects the behavior of endothelial cells. There may be a correlation between this observation and the treatment interaction with lactate dehydrogenase observed clinically.

“Extreme” Renal Preservation: Neoadjuvant Chemotherapy and Percutaneous Resection for Upper-Tract Urothelial Carcinoma in a Patient with Solitary Kidney—A Case Report

BACKGROUND AND PURPOSEnRenal preservation in selected patients with upper-tract urothelial cancer (UTUC) has been well described, offering an alternative to radical nephroureterectomy. We present our experiences in performing percutaneous treatments after neoadjuvant chemotherapy in one such patient with a large, complex, high-grade UTUC in a solitary kidney.nnnCASE REPORTnA 55-year-old woman with a solitary kidney presented with a 5.2 cm enhancing mass with calcifications involving the left renal pelvis and lower pole. Cystoscopy and retrograde pyelography demonstrated normal bladder mucosa. Ureteroscopy revealed a large, papillary tumor occupying the renal pelvis. Ureteroscopic treatment was deemed impossible because of the lesions volume. We proceeded with percutaneous resection after downsizing the tumor after a course of neoadjuvant chemotherapy. Using a 25F resectoscope via a percutaneous tract, resection was performed to fully excise the tumor, and the patient received two postoperative chemotherapy courses. A recurrence developed within an isolated calix 8 months postoperatively, which was also managed percutaneously.nnnCONCLUSIONnA multimodal approach in a highly motivated patient could represent a reasonable strategy for patients in whom such a therapy is desired.