Modulation of alternative splicing by antisense oligonucleotides.
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Alternative Splicing↗
Biomedical subjects
R Kole
Publications and source records attributed to R Kole.
Antisense-mediated redirection of mRNA splicing.
Antisense technology has been used to study basic biological processes, and to block these processes when they deleteriously lead to human disease. A separate, equally important application of antisense technology is to upregulate the gene expression lost in the diseased state by shifting alternative splicing of pre-messenger RNA. This strategy has commonly relied upon the use of antisense oligonucleotides; however, another approach is to use a plasmid construct to generate antisense RNA inside the cell. Antisense therapeutics based on expression vectors and viral vectors offers a gene therapy approach, whereas those based on oligonucleotides offers a more drug like approach.
Alternative Splicing↗
Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs.
The antisense activity of oligomers with 2'-O-methyl (2'-O-Me) phosphorothioate, 2'-O-methoxyethyl (2'-O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing assay in which the modified oligonucleotides blocked aberrant and restored correct splicing of modified enhanced green fluorescent protein (EGFP) precursor to mRNA (pre-mRNA), generating properly translated EGFP. In this approach, antisense activity of each oligomer was directly proportional to up-regulation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers confirmed validity of the functional assay. The results showed that the free uptake and the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher than that of negatively charged 2'-O-Me and 2'-O-MOE congeners. The effects of the PNA oligomers were observed to be dependent on the number of L-lysine (Lys) residues at the C-terminus. The experiments suggest that the PNA containing Lys was taken up by a mechanism similar to that of cell-penetrating homeodomain proteins and that the Lys tail enhanced intracellular accumulation of PNA oligomer without affecting its ability to reach and hybridize to the target sequence.
Anions↗
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.
Amino Acid Motifs↗
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.
Alternative Splicing↗
Spontaneous and MNNG-induced reversion of an EGFP construct in HeLa cells: an assay for observing mutations in living cells by fluorescent microscopy.
A HeLa cell line stably expressing the enhanced green fluorescence protein (EGFP) gene, interrupted by the HBB IVS2-654 intron, was studied without treatment and after treatment with a single standard dose of 15 microM of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done in order to prove that such a construct can revert by a variety of mechanisms and that it produces a visible phenotype, i.e., green fluorescence. The system permits visual detection of living mutant cells among a background of non-mutant cells and does not require a selective medium. The results show that the construct reverts by large deletions (-62, -100, and -162 bp), small insertions (+4 bp), small rearrangements (19 bp duplication), base substitutions at purines (G652, G653, A655, G579), and a pyrimidine (T654) between nucleotide positions 579 and 837. Splice-site mutations were recovered, and some of the mechanisms underlying these mutations are discussed. Because of the ease of detection of revertant cells under fluorescent light and the wide variety of mutations that can be recovered, further development of this system could make it a useful new mammalian cell mutagenicity assay.
Base Sequence↗
Modification of alternative splicing by antisense oligonucleotides as a potential chemotherapy for cancer and other diseases.
It has been estimated that greater than 35% of all human genes undergo alternative splicing. The process of alternative splicing is highly regulated and disruption of a splicing pattern can produce splice variants that have different functions. Certain splice variants that are associated with induction of cell death, regulation of cellular proliferation and differentiation, cell signaling, and angiogenesis are present in a variety of cancers. Several of these cancer-related alternatively spliced genes will be discussed in this review. In addition, alternative splicing is associated with several genetic disorders such as beta-thalassemia, cystic fibrosis, and muscular dystrophy. Control of pre-mRNA splicing patterns with antisense oligonucleotides presents an attractive way to potentially treat and manage a variety of diseases. This review will discuss potential gene targets for antisense oligonucleotide induced modification of alternative splicing patterns. Furthermore, the chemistries and delivery strategies of antisense oligonucleotides will be discussed.
Alternative Splicing↗
Antisense effects in the cell nucleus: modification of splicing.
The surprisingly small number of human genes, which has recently been estimated to be approximately 30,000, suggests that RNA processing, and in particular alternative RNA splicing, is in large part responsible for the diversity of gene products in human and mammalian cells. The ability to manipulate alternative splicing using antisense oligonucleotides, as demonstrated in several studies during the past year, makes it an important and attractive approach to altering gene expression. A review of these studies leads to the conclusion that antisense oligonucleotides, whether designed to affect the cytoplasmic mRNA or nuclear pre-mRNA, function predominantly in the nucleus and not the cytoplasm.
Alternative Splicing↗
Restoration of correct splicing of thalassemic beta-globin pre-mRNA by modified U1 snRNAs.
The T-->G mutation at nucleotide 705 in the second intron of the beta-globin gene creates an aberrant 5' splice site and activates a 3' cryptic splice site upstream from the mutation. As a result, the IVS2-705 pre-mRNA is spliced via the aberrant splice sites leading to a deficiency of beta-globin mRNA and protein and to the genetic blood disorder thalassemia. We have shown previously that in cell culture models of thalassemia, aberrant splicing of beta-thalassemic IVS2-705 pre-mRNA was permanently corrected by a modified murine U7 snRNA that incorporated sequences antisense to the splice sites activated by the mutation. To explore the possibility of using other snRNAs as vectors for antisense sequences, U1 snRNA was modified in a similar manner. Replacement of the U1 9-nucleotide 5' splice site recognition sequence with nucleotides complementary to the aberrant 5' splice site failed to correct splicing of IVS2-705 pre-mRNA. In contrast, U1 snRNA targeted to the cryptic 3' splice site was effective. A hybrid with a modified U7 snRNA gene under the control of the U1 promoter and terminator sequences resulted in the highest levels of correction (up to 70%) in transiently and stably transfected target cells.
Animals↗
Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients.
Mononuclear cells from peripheral blood of thalassemic patients were treated with morpholino oligonucleotides antisense to aberrant splice sites in mutant beta-globin precursor mRNAs (pre-mRNAs). The oligonucleotides restored correct splicing and translation of beta-globin mRNA, increasing the hemoglobin (Hb) A synthesis in erythroid cells from patients with IVS2-654/beta(E), IVS2-745/IVS2-745, and IVS2-745/IVS2-1 genotypes. The maximal Hb A level for repaired IVS2-745 mutation was approximately 30% of normal; Hb A was still detectable 9 days after a single treatment with oligonucleotide. Thus, expression of defective beta-globin genes was repaired and significant level of Hb A was restored in a cell population that would be targeted in clinical applications of this approach.
Cell Nucleus↗
Antisense oligonucleotides and RNAs as modulators of pre-mRNA splicing.
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Alternative Splicing↗
Modification of alternative splicing pathways as a potential approach to chemotherapy.
Many cancer-associated genes are alternatively spliced; their expression leads to the production of multiple splice variants. Although the functions of most of these variants are not well-defined, some have antagonistic activities related to regulated cell death mechanisms. In a number of cancers and cancer cell lines, the ratio of the splice variants is frequently shifted so that the anti-apoptotic splice variant predominates. This observation suggests that modification of splicing, which restores the proper ratio of alternatively spliced gene products, may reverse the malignant phenotype of the cells and offer a gene-specific form of anticancer chemotherapy. Our laboratory has extensively investigated the use of antisense oligonucleotides for shifting the splicing patterns of several genes. Potential application of this method for treatment of cancers, as well as of certain genetic disorders, is discussed.
Alternative Splicing↗
Antisense oligonucleotides as modulators of pre-mRNA splicing.
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Electrophoresis, Polyacrylamide Gel↗
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.
3T3 Cells↗
Novel cationic amphiphiles as delivery agents for antisense oligonucleotides.
There has been great interest recently in therapeutic use of nucleic acids including genes, ribozymes and antisense oligonucleotides. Despite recent improvements in delivering antisense oligonucleotides to cells in culture, nucleic acid-based therapy is still often limited by the poor penetration of the nucleic acid into the cytoplasm and nucleus of cells. In this report we describe nucleic acid delivery to cells using a series of novel cationic amphiphiles containing cholic acid moieties linked via alkylamino side chains. We term these agents 'molecular umbrellas' since the cationic alkylamino chains provide a 'handle' for binding of nucleic acids, while the cholic acid moieties are likely to interact with the lipid bilayer allowing the highly charged nucleic acid backbone to traverse across the cell membrane. Optimal gene and oligonucleotide delivery to cells was afforded by a derivative (amphiphile 5) containing four cholic acid moieties. With this amphiphile used as a constituent in cationic liposomes, a 4-5 log increase in reporter gene delivery was measured. This amphiphile used alone provided a 250-fold enhancement of oligo-nucleotide association with cells as observed by flow cytometry. A substantial fraction of cells exposed to complexes of amphiphile 5 and fluorescent oligo-nucleotide showed nuclear accumulation of the fluorophore. Enhanced pharmacological effectiveness of antisense oligonucleotides complexed with amphiphile 5 was observed using an antisense splicing correction assay that activates a Luciferase reporter. Intracellular delivery, nuclear localization and pharmacological effectiveness of oligonucleotides using amphiphile 5 were similar to those afforded by commercial cytofectins. However, in contrast to most commercial cytofectins, the umbrella amphiphile showed substantial delivery activity even in the presence of high concentrations of serum.
3T3 Cells↗
Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake.
A novel, positive read-out assay that quantifies only sequence-specific nuclear activity of antisense oligonucleotides was used to evaluate morpholino and 2'-O-methyl sugar-phosphate oligonucleotides. The assay is based on modification of the splicing pathway of human beta-globin pre-mRNA. In addition, scrape-loading of cells with oligonucleotides allows the separate assessment of intracellular antisense activity of the oligonucleotides and their ability to penetrate the cell membrane barrier. The results show that, with scrape-loading, the morpholino oligonucleotides were approximately 3-fold more effective in their intrinsic antisense activity than alternating phosphodiester/phosphorothioate 2'-O-methyl-oligoribonucleotides and 6-9- and almost 200-fold more effective than the exclusively phosphorothioate and phosphodiester derivatives, respectively. The morpholino oligonucleotides were over 20-fold more effective than the phosphorothioate 2'-O-methyl-oligoribonucleotides in free uptake from the culture media. The antisense activity of the morpholino oligonucleotides was detectable not only in monolayer HeLa cells but also in suspension K562 cells. Time course experiments suggest that both the free uptake and efflux of morpholino oligonucleotides are slow.
Alternative Splicing↗
Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides.
The mdx mouse, which carries a nonsense mutation in exon 23 of the dystrophin gene, has been used as an animal model of Duchenne muscular dystrophy to evaluate cell or gene replacement therapies. Despite the mdx mutation, which should preclude the synthesis of a functional dystrophin protein, rare, naturally occurring dystrophin-positive fibres have been observed in mdx muscle tissue. These dystrophin-positive fibres are thought to have arisen from an exon-skipping mechanism, either somatic mutations or alternative splicing. Increasing the frequency of these fibres may offer another therapeutic approach to reduce the severity of Duchenne muscular dystrophy. Antisense oligonucleotides have been shown to block aberrant splicing in the human beta-globin gene. We wished to use a similar approach to re-direct normal processing of the dystrophin pre-mRNA and induce specific exon skipping. Antisense 2'-O-methyl-oligoribonucleotides, directed to the 3' and 5' splice sites of introns 22 and 23, respectively in the mdx pre-mRNA, were used to transfect myoblast cultures. The 5' antisense oligonucleotide appeared to efficiently displace factors normally involved in the removal of intron 23 so that exon 23 was also removed during the splicing of the dystrophin pre-mRNA. Approximately 50% of the dystrophin gene mRNAs were missing this exon 6 h after transfection of primary mdx myotubes, with all transcripts showing skipping of exon 23 after 24 h. Deletion of exon 23 does not disrupt the reading frame and should allow the synthesis of a shorter but presumably functional Becker-like dystrophin. Molecular intervention at dystrophin pre-mRNA splicing has the potential to reduce the severity of a Duchenne mutation to the milder Becker phenotype.
Alternative Splicing↗
Sensitivity of splice sites to antisense oligonucleotides in vivo.
A series of HeLa cell lines which stably express beta-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 5' splice sites and activate a common 3' cryptic splice site upstream leading to aberrantly spliced beta-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 5' splice sites affected sensitivity of both the 5' and 3' splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 5' splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA.
Base Sequence↗