From the last three decades, several antisense drugs have entered into clinical trials and market for the treatment of a broad variety of diseases, and numerous oligonucleotides are under clinical. Antisense therapy is a form of treatment that uses antisense oligonucleotides asos to target messenger rna mrna. Antisense oligonucleotides can alter gene expression through. A unique mechanism of toxicity for asos is hybridization. Antisense oligonucleotides in solution or encapsulated in. The mutation in the intron causes aberrant splicing of luciferase premrna, preventing translation of luciferase. A possible candidate is the u1 small nuclear rnaprotein complex, which hybridizes to the 5. Other splicing defect mutations also occur in cftr 9, representing additional potential targets for the antisense approach. Modification of splicing the surprisingly small number of human genes, which has recently been estimated to be approximately 30,000, suggests. The basics of antisense a sense strand is a 5 to 3 mrna molecule or dna molecule. Antisense technology is the process in which the antisense strand hydrogen bonds with the targeted sense. We show here that high levels of expression of bclxl promoted apoptosis of cells treated with an antisense oligonucleotide 5bclx as t. These experiments made use of a published anti bclx l sequence and control mismatch sequence 44. This was demonstrated with the double mismatch oligonucleotides which showed largely reduced antisense efficacy, and with a nonsense 18mer tcdna which showed no antisense effect at all.
Aminoglycoside enhances the delivery of antisense morpholino. Jul 08, 2004 a recently developed antisense splicing assay was used to determine the relative activities of 2. Rna interference and antisense oligonucleotides downregulate gene expression by inducing enzymedependent degradation of targeted mrna. Interestingly, delivery of both oligonucleotides simultaneously had an additive effect, a result not seen with. Here, we describe the synthesis, binding properties, and biologic properties of peptideoligonucleotide conjugates comprised of the tat and ant cellpenetrating peptides with 2. It also suggests a specificity of the oligonucleotide effects since noncancerous cells with low levels of bclxl should resist the treatment. Lack of antisense inhibition in this case is suggestive, particularly if the antisense oligomer is still effective when the wildtype target is forcibly overexpressed. Modification of premrna splicing by antisense oligonucleotides.
Antisense rna asrna, also referred to as antisense transcript, natural antisense transcript nat or antisense oligonucleotide, is a single stranded rna that is complementary to a protein coding messenger rna mrna with which it hybridizes, and thereby blocks its translation into protein. Some, through various mechanisms, inhibit aso activity, for example, by competing with rnase h1 for binding to the rnaaso heteroduplex 14. Department of pharmacology and lineberger comprehensive cancer center. Additive antisense effects of different pnas on the in vitro. The biological effect of an antisense oligonucleotide. Antisense properties of tricyclodna nucleic acids research. Cellular response to an antisensemediated shift of bclx pre. However, treatment of the cells with a 2omethyloligoribonucleotide targeted to. A laboratory manual, cold spring harbor laboratory.
Another example of a secondgeneration oligonucleotide is the n33 p5 pn, which result from the replacement of. The effect of 3tubulinspecific antisense oligonucleotide. Gabriela schmajuk, halina sierakowska, and ryszard kole. Such oligonucleotides have successfully been used in the past to shift premrna splicing of cftr, il5r, cmyc, tau, smn2, bclx 8. An antisense oligonucleotide 20 mer targeted to the parasite ptubulin gene and encap sulated in cationic liposomes.
In this study, we investigated a few aminoglycosides ags for their potential to improve the. Control of alternative splicing by antisense oligonucleotides. The antisense oligonucleotide isis 388626 inhibits the synthesis of the renal antisense sodiumglucose cotransporter 2 sglt2 receptor, which accounts for 90% of the reabsorption of glomerular filtrated glucose zanardi et al. Jci the experimental use of antisense oligonucleotides. Pros and cons of antisense therapy antisense therapy. Feb 12, 2019 also, the new chemistries and better antisense oligonucleotide designs further improve the unwanted side effects, safety, and tolerability. The antisense effect of a synthetic oligonucleotide sequence was first demonstrated in the late 1970s by zamecnik and stephenson 1. Effects of an antisense oligonucleotide inhibitor of. The potential of antisense oligonucleotide therapies for. Oligos as short as 11 bases can have potent antisense effects 26. Overexpression of bclxl, an antiapoptotic member of the bcl2 family, negatively correlates with the sensitivity of various cancers to chemotherapeutic agents. Also here nuclear antisense effects increased with increasing dose and length of the oligonucleotides. Phase 12 trial of antisense oligonucleotide tofersen for. Additive antisense effects of different pnas on the in vitro translation of the pmlrar.
Cellular response to an antisensemediated shift of bclx. Correction of aberrant splicing of the cystic fibrosis. Sglt2 inhibition affects renal glucose reabsorption, resulting in glucosuria and lowered serum. Antisense oligonucleotides that are stable against nucleolytic degradation and that do not induce rnase h catalyzed hydrolysis of complementary mrna were shown to be powerful modulators of premrna splicing in vitro and in vivo 1,2. Efficient and persistent splice switching by systemically delivered. Bclx as on inducing apoptosis mercatante and kole, unpublished data. However, therapeutic applications of aos remain limited, particularly because of the lack of efficient cellular delivery methods imperative for achieving efficacy. Surprisingly, the oligonucleotides may also exert their effects by binding directly to a number of proteins in a sequencedependent but not sequencespecific. The third generation is more heterogeneous because it includes a large number of modifications aiming to improve binding.
Nuclear antisense effects in cyclophilin a premrna splicing. Antisense oligonucleotides with different backbones journal of. Antisense properties of tricyclodna oxford academic journals. Enhanced antisense effects resulting from an improved streptolysin o protocol for oligodeoxynucleotide delivery into human leukaemia. Upon adequate delivery of an appropriate spliceswitching antisense oligonucleotide sso to the nucleus, the intron is spliced out and luciferase is expressed, thus providing a very sensitive positive readout of oligonucleotide delivery and subsequent biological effect sazani and kole, 2003. Sequence specificity of the in vivo effects of antisense oligomers was ascertained after i. Methods study design we conducted a phase i, doubleblind, placebocontrolled, adaptive, doseresponse study to evaluate the effects of isiscrp rx on the acutephase response to endotoxin challenge in healthy volunteers. Phosphoramidate oligonucleotides as potent antisense. In the assay, rnase hinactive oligonucleotides are used to block aberrant splicing and restore correct splicing of an enhanced green fluorescence protein. Nielsen3 and carlo gambacortipasserini1, 1division of experimental oncology d, istituto nazionale tumori, via venezian 1, 203 milan, italy, 2mario negri. During the last 2 decades, many reports have been published using this backbone to generate antisense effects both in tissue culture and in vivo. Targeting splicing by antisense oligonucleotides allows rna modifications that. Omoe, or morpholino oligomers containing three mismatches to the target sequence oligomers 79, table 1.
In 90% of people with erythropoietic protoporphyria epp, the disease results from the inheritance of a common hypomorphic fech allele, encoding ferrochelatase, in trans to a private deleterious fech mutation. Antisense efficacy of pna is influenced by the number of attached lysine residues to further examine the effects of the backbones on the antisense properties of the oligomers, we compared antisense pnas modified with one, two and four positively charged lys residues at the cterminus pna1, 2 and 4. Effects of base modifications on antisense properties of 2. Exonskipping antisense oligonucleotides to correct missplicing in. To test for antisense effects of tcdna in vivo, we used a tissue culture model for alternative splicing established by kole and coworkers 23, 24. Moe phosphorothioate oligonucleotides containing base modifications. Rna modulation, repair and remodeling by splice switching. Jul 30, 1999 a mismatch in on705wt targeted to ivs2705 5.
Furdon pj, dominski z, kole r 1989 rnase h cleavage of rna hybridized. Antisense effects kole pdf aberrant splicing can result in deleterious consequences for the organism. The attainment of effective intracellular delivery remains an important issue for pharmacologic applications of antisense oligonucleotides. Effects of lntrahypothalamic administration of antisense dna for. Conceptual simplicity, the possibility of rational design, relatively inexpensive cost, and developments in the sequencing of human genome have led to the use of short fragments of nucleic acid, commonly called oligonucleotides, either as therapeutic agents or as tools to study gene function. Such modifications are of potential interest if the target rna must not be degraded. Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs by peter sazani, shinhong kang, martin a.
Jan 20, 2012 here, we discuss three rnabased therapeutic technologies exploiting various oligonucleotides that bind to rna by base pairing in a sequencespecific manner yet have different mechanisms of action and effects. Furthermore, we found that all antisense effects observed were sequencedependent, and thus true antisense effects. Upon adequate delivery of an appropriate spliceswitching antisense oligonucleotide sso to the nucleus, the intron is spliced out and luciferase is expressed, thus providing a very sensitive positive readout of oligonucleotide delivery and subsequent biological effect sazani and kole. Antisense oligonucleotides oligonucleotidebased antisense techniques represent the most common and, to date, the most successful approach to achieving suppression or elimination of a genetic message. Research in ryszard kole s laboratory is supported in part via a sponsored research agreement between the university of north carolina and ercole biotech inc. Therefore, this study aimed to study the effects of the dnazymes. Critically, they are also capable of activating rnase h activity. Antisensebased therapy for the treatment of spinal. Intrathecal administration of tofersen is being studied for the treatment of amyotrophic lateral sclerosis als due to sod1 mutations methods. Feb 08, 20 to make sure that the effects of retro1 on antisense actions are not confined to a single sequence, we examined a second target, namely the apototic regulator bclx l. Tofersen is an antisense oligonucleotide that mediates the degradation of superoxide dismutase 1 sod1 messenger rna to reduce sod1 protein synthesis.
Mar 31, 2020 the antisense effect of this type of aso is probably due to steric blockade of translation. Antisense transcription as a tool to tune gene expression. Kole r, sazani p 2001 antisense effects in the cell nucleus. The small molecule retro1 enhances the pharmacological. To test for antisense effects in vivo, we used hela. Control of alternative splicing by antisense oligonucleotides as a. Molecular mechanisms of antisense oligonucleotides. Properties in the nature of antisense effects by sumati sundaram a dissertation submitted to the graduate schoolnew brunswick rutgers, the state university of new jersey in partial fulfillment of the requirements for the degree of doctor of philosophy graduate program in chemical and biochemical engineering written under the direction of dr. We also show that they are nontoxic and that the sequencespecific antisense effects of a singledose regimen of lna sso persists for more than 3 weeks. Jci therapeutic potential of antisense oligonucleotides. Nuclear antisense effects of neutral, anionic and cationic. However, phosphorothioate oligonucleotides are highly soluble and have excellent antisense activity. Apr 01, 2017 we have characterized the mechanisms accounting for the effects on antisense activity of a number of these proteins. Jul 08, 2019 antisense effects kole pdf aberrant splicing can result in deleterious consequences for the organism.
Control of alternative splicing by antisense oligonucleotides as a potential chemotherapy. However, to utilize antisense oligonucleotides as molecular chemotherapeutic agents, the global effects of these molecules need to be examined. Molecular mechanisms of antisense oligonucleotides nucleic. Pdf nuclear antisense effects of neutral, anionic and. Pmos are charge neutral and exert their antisense effect by. Renal effects of antisensemediated inhibition of sglt2. In one series of experiments, the cationic lipid reagent lipofectin invitrogen life technologies was shown to enhance the entry of an 18mer phosphorothioate oligonucleotide into huvec cells by 20fold and to effect a fold increase in biological antisense activity. Upregulation of luciferase gene expression with antisense. While frequent, the roles of antisense transcription in regulation are poorly understood. Oct 06, 2019 antisense effects kole pdf october 6, 2019 admin aberrant splicing can result in deleterious consequences for the organism. Modification of alternative splicing of premrna by antisense. The effects of antisense oligonucleotides should be highly specific because only the latter cells contain the target sequence. Oct 21, 2019 antisense effects kole pdf posted on october 21, 2019 by admin aberrant splicing can result in deleterious consequences for the organism.
Asos are capable of altering mrna expression through a variety of mechanisms, including ribonuclease h mediated decay of the premrna, direct steric blockage, and exon content modulation through splicing site binding on premrna. Such marked changes are not commensurate with the change in the t m of the duplexes but may be due to competition with splicing factors that bind to the 5. Antisense oligonucleotides are designed to specifically hybridize to a target messenger rna mrna and interfere with the synthesis of the encoded protein. Targeting splicing by antisense oligonucleotides allows rna modifications that are not possible with. The biological effect of an antisense oligonucleotide depends. Jci therapeutic potential of antisense oligonucleotides as. Antisense oligonucleotides with different backbones. Pdf systemically delivered antisense oligomers upregulate. Apr 18, 2018 antisense oligonucleotide aso therapeutics are single. Jul 18, 2002 the use of antisense oligonucleotides to modify aberrant expression patterns of alternatively spliced mrnas is a novel means of potentially controlling such diseases. Hela tetoff cells were transfected transiently as well as stably with a recombinant plasmid pluc705 carrying the luciferase gene interrupted by a mutated human. Antisense pna accumulates in escherichia coli and mediates a. Effects of a trinucleotide ethyl phosphotriester, gmpetgmpetu, on mammalian cells in.
The complementary strands or mirror strand to the sense is called an antisense. Effect of antisense oligonucleotides against cholesteryl ester transfer protein on the. Antisense oligonucleotide ao therapy has been the specific treatment for duchenne muscular dystrophy, with ongoing clinical trials. Antisense therapeutics in chronic myeloid leukaemia. We conducted a phase 12 ascendingdose trial evaluating tofersen in adults with als. Nuclear antisense effects in cyclophilin a premrna. Intrathecal administration of tofersen is being studied for the treatment of amyotrophic lateral sclerosis als due to sod1 mutations. The restoration of correct splicing by targeting the cryptic 3. The antisense activity of oligomers with 2omethyl 2ome phosphorothioate, 2omethoxyethyl 2omoe phosphorothioate, morpholino and peptide nucleic acid pna backbones was investigated using a splicing assay in which the. Maier, changfu wei, jennifer dillman, james summerton, muthiah manoharan and ryszard kole. Feb 06, 2018 antisense oligonucleotides are an emerging therapeutic option to treat diseases with known genetic origin. The permeation of cells with streptolysin o is another method that has been used for the cellular delivery of morpholino oligonucleotides 42. Conjugates of antisense oligonucleotides with the tat and. In the age of personalised medicines, antisense oligonucleotides can sometimes be designed to target and bypass or overcome a patients genetic mutation, in particular those lesions that compromise normal premrna processing.
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