Antisense Oligonucleotides with Different Backbones

Abstract

A novel, positive read-out assay that quantifies only sequence-specific nuclear activity of antisense oligonucleotides was used to evaluate morpholino and 2-Omethyl sugar-phosphate oligonucleotides. The assay is based on modification of the splicing pathway of human ␤-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.Antisense oligonucleotides and RNAs show great promise as sequence-specific agents able to down-regulate the expression of targeted genes. In this capacity, extensively reviewed in a recent volume and review articles (1-4), they have advanced not only to clinical trials (5-8) but also to clinical practice (9); they have also proven to be useful as research tools (1). However, the application of antisense technology both in research and in clinical studies presents a number of outstanding issues.In principle, the most important feature of antisense oligonucleotides is their ability to block mRNA function by sequence-specific hybridization to the RNA. Surprisingly, the oligonucleotides may also exert their effects by binding directly to a number of proteins in a sequence-dependent but not sequence-specific manner, resulting in unpredictable and nonspecific effects (10 -14). This non-antisense mechanism of binding was shown to be particularly pronounced in commonly used oligodeoxynucleoside phosphorothioates (15); other mechanisms also contribute to sequence-independent oligonucleotide effects (16).Our ability to select appropriate target sequences within the RNA is still limited (17). Cell-free selection of susceptible sites is not always helpful (18, 19) because in vivo cellular RNAs are always complexed with proteins (20), which may block the sites and/or change the secondary and tertiary structure of the target RNA. Therefore, oligonucleotide targeting is frequently carried out by trial and error, requiring the synthesis of large numbers of compounds (21,22).Equally unsatisfactory is our understanding of the intracellular site of action of oligonucleotides. Most of the antisense oligonucleotides are designed against sequences within mRNA which presumably represents a...

Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients

“…2, days 13-15), the mononuclear cells from a thalassemic patient carrying the IVS2-654͞␤ E mutations were treated with morpholino oligomers (24,25) complementary to aberrant splice sites activated in IVS2-654 ␤-globin pre-mRNA. Morpholino analogs of oligonucleotides were selected because they are resistant to nucleases, do not allow degradation of RNA in the RNA-oligonucleotide hybrids by RNase H, and bind to targeted splice sites forming duplexes of high Tm (22).…”

“…The morpholino analogs of oligonucleotides have numerous desirable characteristics as modifiers of splice site selection and in model systems performed better than the oligonucleotides with other backbones (22). However, their free uptake into the erythropoietic cells was limited and even syringe loading of the cells, which entails mechanical disturbance of the cell membrane (see Materials and Methods), required high concentrations of the compounds.…”

“…Aberrant splicing of several of the ␤-globin splicing mutants has been shown in cell free extracts (18), in transfected HeLa based cell lines (19)(20)(21)(22), and in a transgenic mouse model (23).…”

Receptor-mediated endocytosis of phosphodiester oligonucleotides in the HepG2 cell line: evidence for non-conventional intracellular trafficking

“…Capping at the 5′-end, and particularly the 3′-end, protects ON against exonucleases (7,14). Backbone modifications yield additional ON protection, at the risk of compromising selectivity of interactions (13), but novel ON structures have been developed that combine improved stability and specificity (15). The second problem is accessibility.…”

Restoration of Correct Splicing of Thalassemic β-Globin Pre-mRNA by Modified U1 snRNAs

“…Restoration of gene activity was accomplished by targeting the splice sites created or activated by several mutations in thalassemia (23)(24)(25)(26), cystic fibrosis (27), and in a mouse mdx model of Duchenne muscular dystrophy (28). Thus, modification of splicing by treatment with antisense oligonucleotides provides a potential alternative to gene therapy protocols involving the replacement of defective genes.…”