Strong RNA Splicing Enhancers Identified by a Modified Method of Cycled Selection Interact with SR Protein

A modified method of cycled selection was used to characterize splicing enhancers for exon inclusion from a pool of beta-globin-based three exon/two intron pre-mRNAs with a variable number of random nucleotides incorporated in the internal exon. The pre-mRNAs generated by this method contained random sequences ranging from 0 to 18 nucleotides in length. This method was used to isolate particular splicing enhancer motifs from a previously enriched pool of extremely diverse enhancers. After four cycles of selection for mRNA containing the internal exon, a distinct enhancer motif (GACGAC...CAGCAG) was highly enriched. This motif served as strong splicing enhancers in a heterogeneous exon. We have shown here that the selected enhancer motif promotes exon inclusion through specific interaction with SRp30. We have also shown that although present in many of our selected splicing enhancers conforming to this motif, a typical purine-rich enhancer sequence is dispensable for either enhancer activity or binding with SRp30

Modification of HER2 pre-mRNA alternative splicing and its effects on breast cancer cells

The oncogene HER2 is overexpressed in a variety of human tumors, providing a target for anti-cancer molecular therapies. Here we employed a 2’-O-methoxyethyl (MOE) splice switching oligonucleotide, SSO111, to induce skipping of exon 15 in HER2 pre-mRNA, leading to significant downregulation of full-length HER2 mRNA, and simultaneous upregulation of Δ15HER2 mRNA. SSO111 treatment of SK-BR-3 cells, which highly overexpress HER2, led to inhibition of cell proliferation and induction of apoptosis. The novel Δ15HER2 mRNA encodes a soluble, secreted form of the receptor. Treating SK-BR-3 cells with exogenous Δ15HER2 protein reduced membrane-bound HER2 and decreased HER3 transphosphorylation. Δ15HER2 protein thus has similar activity to an autoinhibitory, natural splice variant of HER2, Herstatin, and to the breast cancer drug Herceptin. Both SSO111 and Δ15HER2 may be potential candidates for the development of novel HER2-targeted cancer therapeutics

Therapeutic Potential of Splice-Switching Oligonucleotides

Alternative splicing enables a single pre-messenger RNA transcript to yield multiple protein isoforms, making it a major contributor to the diversity of the proteome. While this process is essential for normal development, aberrations in alternative splicing are the cause of a multitude of human diseases. Methods for manipulating alternative splicing would thus be of therapeutic value. Chemically modified antisense oligonucleotides that alter alternative splicing by directing splice site selection have been developed to achieve this end. These splice-switching oligonucleotides (SSOs) have been applied to correct aberrant splicing, induce expression of a therapeutic splice variant, or induce expression of a novel therapeutic splice variant in a number of disease-relevant genes. Recently, in vivo efficacy of SSOs has been reported using animal disease models, as well as in results from the first clinical trial

The Importance of Bcl-x L in the Survival of Human RPE Cells

PURPOSE: In normal eyes and in diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cell survival is critically important. Bcl-x(L) has been shown to be among the most highly expressed survival factors in cultured human RPE cells. In the current study the effect of Bcl-x(L) blockade on human RPE cell survival was determined under normal conditions and after induced oxidative stress. METHODS: Cultured human RPE cells from three different donors were transfected with modified, 2'-O-methoxyethoxy Bcl-x(L)-mismatched control antisense oligonucleotides (ASOs), Bcl-x(L)-specific ASOs, and Bcl-x(L) splice switching oligonucleotides (SSOs), which shift the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) into Bcl-x(S), a proapoptotic factor. RNA and protein were harvested at various time points after transfection. Bcl-x(L) and Bcl-x(S) mRNA transcript levels were analyzed using gene-specific primers with reverse transcription-polymerase chain reaction. Bcl-x(L) protein levels were analyzed using Western blot. Cell viability was measured by WST-1 and lactate dehydrogenase (LDH) assays. The mode of cell death was determined with a cell death ELISA and an M30 assay. To study the effects of oxidative stress, the cells were stimulated after transfection with various concentrations of H(2)O(2.) Cell viability was analyzed by WST-1 (Roche, Indianapolis, IN) and LDH assays RESULTS: After Bcl-x(L)-specific ASO and SSO transfections, Bcl-x(L) mRNA and protein levels were significantly reduced. Bcl-x(S) levels were increased after transfection with SSO. By day 8 after plating, the cells transfected with Bcl-x(L)-specific ASO had significantly decreased viability, which was further reduced by day 10. The SSO had an even more potent effect. Cell viability was reduced on day 4 after plating and by day 10, less than 10% of the cells were viable. Apoptotic cell death occurred as early as day 4 after plating. H(2)O(2), used as a model oxidant, further enhanced cell death induced by Bcl-x(L)-specific ASO and SSO. CONCLUSIONS: Bcl-x(L) plays an important role in human RPE cell survival under normal conditions and when cells are exposed to oxidative stress. Treatment strategies that enhance Bcl-x(L) expression and/or prevent conversion of Bcl-x(L) to Bcl-x(S) may be useful in preventing RPE cell death in AMD. Treatments that reduce Bcl-x(L) and enhance Bcl-x(S) may be useful in inhibiting unwanted RPE cell proliferation in PVR

Cellular Response to an Antisense-mediated Shift of Bcl-x Pre-mRNA Splicing and Antineoplastic Agents

Overexpression of Bcl-xL, an anti-apoptotic member of the Bcl-2 family, negatively correlates with the sensitivity of various cancers to chemotherapeutic agents. We show here that high levels of expression of Bcl-xL promoted apoptosis of cells treated with an antisense oligonucleotide (5'Bcl-x AS) that shifts the splicing pattern of Bcl-x pre-mRNA from the anti-apoptotic variant, Bcl-xL, to the pro-apoptotic variant, Bcl-xS. This surprising finding illustrates the advantage of antisense-induced modulation of alternative splicing versus down-regulation of targeted genes. It also suggests a specificity of the oligonucleotide effects since non-cancerous cells with low levels of Bcl-xL should resist the treatment. 5'Bcl-x AS sensitized cells to several antineoplastic agents and radiation and was effective in promoting apoptosis of MCF-7/ADR cells, a breast cancer cell line resistant to doxorubicin via overexpression of the mdr1 gene. Efficacy of 5'Bcl-x AS combined with chemotherapeutic agents in the PC3 prostate cancer cell line may be translated to clinical prostate cancer since recurrent prostate cancer tissue samples expressed higher levels of Bcl-xL than benign prostate tissue. Treatment with 5'Bcl-x AS may enhance the efficacy of standard anti-cancer regimens and should be explored, especially in recurrent prostate cancer

Antibodies to hnRNP core proteins inhibit in vitro splicing of human β-globin pre-mRNA

In vitro splicing of human beta-globin pre-mRNA can be fully inhibited by treatment of the splicing extract with polyclonal antibodies against hnRNP core proteins prior to the addition of pre-mRNA. Inhibition of the first step in the splicing pathway, cleavage at the 5' splice site and lariat formation, requires more antibodies than inhibition of the second step, cleavage at the 3' splice site and exon ligation. The anti-hnRNP antibodies can also inhibit the splicing reaction after the formation of the active nucleoprotein splicing complex which is known to occur during the initial lag period. Thus, hnRNP core proteins appear to be present in the complex that performs pre-mRNA splicing

Correction of Aberrant Splicing of the Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR ) Gene by Antisense Oligonucleotides

The CFTR splicing mutation 3849 + 10 kb C --> T creates a novel donor site 10 kilobases (kb) into intron 19 of the gene and is one of the more common splicing mutations that causes cystic fibrosis (CF). It has an elevated prevalence among patients with atypically mild disease and normal sweat electrolytes and is especially prominent in Ashkenazi Jews. This class of splicing mutations, reported in several genes, involves novel splice sites activated deep within introns while leaving wild-type splice elements intact. CFTR cDNA constructs that modeled the 3849 + 10 kb C --> T mutation were expressed in 3T3 mouse fibroblasts and in CFT1 human tracheal and C127 mouse mammary epithelial cells. In all three cell types, aberrant splicing of CFTR pre-mRNA was comparable to that reported in vivo in CF patients. Treatment of the cells with 2'-O-methyl phosphorothioate oligoribonucleotides antisense toward the aberrant donor and acceptor splice sites or to the retained exon-like sequence, disfavored aberrant splicing and enhanced normal processing of CFTR pre-mRNA. This antisense-mediated correction of splicing was dose- and sequence-dependent and was accompanied by increased production of CFTR protein that was appropriately glycosylated. Antisense-mediated correction of splicing in a mutation-specific context represents a potential gene therapy modality with applicability to many inherited disorders

Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine-rich sequence in the 3'-untranslated region of rat tyrosine hydroxylase mRNA

Reduced oxygen tension (hypoxia) induces a 3-fold increase in stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. To investigate the possibility that RNA-protein interactions are involved in mediating this increase in stability, RNA gel shift assays were performed using different fragments of labeled TH mRNA and the S-100 fraction of PC12 cytoplasmic protein extracts. We identified a sequence within the 3'-untranslated region of TH mRNA that binds cytoplasmic protein

Modification of Alternative Splicing of Bcl-x Pre-mRNA in Prostate and Breast Cancer Cells: ANALYSIS OF APOPTOSIS AND CELL DEATH

There is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5'-splice site of bcl-x(L), an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) to Bcl-x(S), a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-x(L), Bcl-x(S), and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells

Size-Uniform 200 nm Particles: Fabrication and Application to Magnetofection

We report on the fabrication of arrays of mono- and multimetallic particles via metal evaporation onto lithographically patterned posts, as well as the magnetic force calibration and successful magnetofection of iron particles grown via this method. This work represents the first instance in which metal evaporation onto post structures was used for the formation of released, shape-defined metal particles. Also, our work represents the first use of lithographically defined particles as agents of magnetofection. Using these techniques it is possible to create particles with complex shapes and lateral dimensions as small as 40 nm. Our demonstrated compositionally flexible particles are highly size-uniform due to their photolithographically defined growth substrates, with particle dimensions along two axes fixed at 200 nm; the third axis dimension can be varied from 20 nm to 300 nm during the deposition procedure. Atomic percent of metals incorporated into the particle volume is highly tunable and particles have been synthesized with as many as four different metals. We performed magnetic force calibrations on a single particle size for iron particles using an axially magnetized NeFeB permanent magnet and comparisons are made with commercially available magnetic beads. In order to evalutate their usefulness as magnetofection agents, an antisense oligonucleotide (ODN) designed to correct the aberrant splicing of enhanced green fluorescent protein mRNA, was successfully transfected into a modified HeLa cell line. Magnetically enhanced gene delivery was accomplished in vitro using antisense ODN-laden iron particles followed by application of a field gradient. Magnetically enhanced transfection resulted in a 76% and 139% increase in fluorescence intensity when compared to Lipofectamine and antisense ODN-loaded particles delivered without magnetic treatment, respectively. To our knowledge, these experiments constitute the first use of lithographically defined particles as successful agents for magnetically enhanced transfection of an antisense oligonucleotide