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.

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.

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.

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.

PAMAM dendrimers as delivery agents for antisense oligonucleotides.

PURPOSE: To investigate the potential use of PAMAM dendrimers for the delivery of antisense oligonucleotides into cells under conditions that mimic the in vivo environment. METHODS: We used HeLa cells stably transfected with plasmid pLuc/705 which has a luciferase gene interrupted by a human beta-globin intron mutated at nucleotide 705, thus causing incorrect splicing. An antisense oligonucleotide overlapping the 705 splice site, when delivered effectively, corrects splicing and allows luciferase expression. The ability of dendrimers to deliver oligonucleotides to HeLa Luc/705 cells was evaluated in the absence or presence of serum. RESULTS: PAMAM dendrimers formed stable complexes with oligonucleotides that had modest cytotoxicity and showed substantial delivery activity. The dose of the oligonucleotide, the charge ratio of oligonucleotide to dendrimer, and the size (generation) of the dendrimers were all critical variables for the antisense effect. The physical properties of dendrimer/oligonucleotide complexes were further investigated using sedimentation and gel electrophoresis methods. Effective oligonucleotide/generation 5 dendrimer complexes were macromolecular rather than particulate in nature, and were not sedimented at 100,000 RPM. Compared to other types of delivery agents, PAMAM dendrimers were more effective in delivering oligonucleotides into the nucleus of cells in the presence of serum proteins. CONCLUSIONS: Our results suggest that PAMAM dendrimers form nonparticulate delivery complexes that function in the presence of serum proteins and thus may be suited for in vivo therapeutic applications.