Correction Of Aberrant Splicing Research Articles

Correction of the Splicing Defect Caused by a Recurrent Variant in ABCA4 (c.769-784C>T) That Underlies Stargardt Disease

Stargardt disease is an inherited retinal disease caused by biallelic mutations in the ABCA4 gene, many of which affect ABCA4 splicing. In this study, nine antisense oligonucleotides (AONs) were designed to correct pseudoexon (PE) inclusion caused by a recurrent deep-intronic variant in ABCA4 (c.769-784C>T). First, the ability of AONs to skip the PE from the final ABCA4 mRNA transcript was assessed in two cellular models carrying the c.769-784C>T variant: a midigene assay using HEK293T cells and patient-derived fibroblasts. Based on the splicing-correcting ability of each individual AON, the three most efficacious AONs targeting independent regions of the PE were selected for a final assessment in photoreceptor precursor cells (PPCs). The final analysis in the PPC model confirmed high efficacy of AON2, -5, and -7 in promoting PE exclusion. Among the three AONs, AON2 is chosen as the lead candidate for further optimization, hereby showcasing the high potential of AONs to correct aberrant splicing events driven by deep-intronic variants.

A Chemical Biology Perspective to Therapeutic Regulation of RNA Splicing in Spinal Muscular Atrophy (SMA).

Manipulation of RNA splicing machinery has emerged as a drug modality. Here, we illustrate the potential of this novel paradigm to correct aberrant splicing events focused on the recent therapeutic advances in spinal muscular atrophy (SMA). SMA is an incurable neuromuscular disorder and at present the primary genetic cause of early infant death. This Review summarizes the exciting journey from the first reported SMA cases to the currently approved splicing-switching treatments, i.e., antisense oligonucleotides and small-molecule modifiers. We emphasize both chemical structures and molecular bases for recognition. We briefly discuss the advantages and disadvantages of these treatments and include the remaining challenges and future directions. Finally, we also predict that these success stories will contribute to further therapies for human diseases by RNA-splicing control.

Splicing Modulation as a Promising Therapeutic Strategy for Lysosomal Storage Disorders: The Mucopolysaccharidoses Example

Over recent decades, the many functions of RNA have become more evident. This molecule has been recognized not only as a carrier of genetic information, but also as a specific and essential regulator of gene expression. Different RNA species have been identified and novel and exciting roles have been unveiled. Quite remarkably, this explosion of novel RNA classes has increased the possibility for new therapeutic strategies that tap into RNA biology. Most of these drugs use nucleic acid analogues and take advantage of complementary base pairing to either mimic or antagonize the function of RNAs. Among the most successful RNA-based drugs are those that act at the pre-mRNA level to modulate or correct aberrant splicing patterns, which are caused by specific pathogenic variants. This approach is particularly tempting for monogenic disorders with associated splicing defects, especially when they are highly frequent among affected patients worldwide or within a specific population. With more than 600 mutations that cause disease affecting the pre-mRNA splicing process, we consider lysosomal storage diseases (LSDs) to be perfect candidates for this type of approach. Here, we introduce the overall rationale and general mechanisms of splicing modulation approaches and highlight the currently marketed formulations, which have been developed for non-lysosomal genetic disorders. We also extensively reviewed the existing preclinical studies on the potential of this sort of therapeutic strategy to recover aberrant splicing and increase enzyme activity in our diseases of interest: the LSDs. Special attention was paid to a particular subgroup of LSDs: the mucopolysaccharidoses (MPSs). By doing this, we hoped to unveil the unique therapeutic potential of the use of this sort of approach for LSDs as a whole.

Cytoprotective activities of kinetin purine isosteres

Kinetin (N6-furfuryladenine), a plant growth substance of the cytokinin family, has been shown to modulate aging and various age-related conditions in animal models. Here we report the synthesis of kinetin isosteres with the purine ring replaced by other bicyclic heterocycles, and the biological evaluation of their activity in several in vitro models related to neurodegenerative diseases. Our findings indicate that kinetin isosteres protect Friedreich́s ataxia patient-derived fibroblasts against glutathione depletion, protect neuron-like SH-SY5Y cells from glutamate-induced oxidative damage, and correct aberrant splicing of the ELP1 gene in fibroblasts derived from a familial dysautonomia patient. Although the mechanism of action of kinetin derivatives remains unclear, our data suggest that the cytoprotective activity of some purine isosteres is mediated by their ability to reduce oxidative stress. Further, the studies of permeation across artificial membrane and model gut and blood-brain barriers indicate that the compounds are orally available and can reach central nervous system. Overall, our data demonstrate that isosteric replacement of the kinetin purine scaffold is a fruitful strategy for improving known biological activities of kinetin and discovering novel therapeutic opportunities.

Investigation of New Morpholino Oligomers to Increase Survival Motor Neuron Protein Levels in Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal-recessive childhood motor neuron disease and the main genetic cause of infant mortality. SMA is caused by deletions or mutations in the survival motor neuron 1 (SMN1) gene, which results in SMN protein deficiency. Only one approved drug has recently become available and allows for the correction of aberrant splicing of the paralogous SMN2 gene by antisense oligonucleotides (ASOs), leading to production of full-length SMN protein. We have already demonstrated that a sequence of an ASO variant, Morpholino (MO), is particularly suitable because of its safety and efficacy profile and is both able to increase SMN levels and rescue the murine SMA phenotype. Here, we optimized this strategy by testing the efficacy of four new MO sequences targeting SMN2. Two out of the four new MO sequences showed better efficacy in terms of SMN protein production both in SMA induced pluripotent stem cells (iPSCs) and SMAΔ7 mice. Further, the effect was enhanced when different MO sequences were administered in combination. Our data provide an important insight for MO-based treatment for SMA. Optimization of the target sequence and validation of a treatment based on a combination of different MO sequences could support further pre-clinical studies and the progression toward future clinical trials.

Antisense Oligonucleotide-Based Splice Correction of a Deep-Intronic Mutation in CHM Underlying Choroideremia.

Choroideremia is a progressive genetic eye disorder caused by mutations in the CHM gene that encodes the Rab escort protein-1 (REP-1). One of the many CHM mutations described so far is a deep-intronic variant, c.315-4587T>A, that creates a novel splice acceptor site resulting in the insertion of a 98-bp pseudoexon in the CHM transcript. Antisense oligonucleotides (AONs) are a potential therapeutic tool for correcting splice defects, as they have the properties to bind to the pre-mRNA and redirect the splicing process. Previously, we used AONs to correct aberrant splicing events caused by a recurrent intronic mutation in CEP290 underlying Leber congenital amaurosis. Here, we expand the use of these therapeutic molecules for the c.315-4587T>A deep-intronic mutation in CHM by demonstrating splice correction in patient-derived lymphoblast cells.

The Use of Tricyclo-DNA Oligomers for the Treatment of Genetic Disorders.

Antisense Oligonucleotides (ASOs) represent very attractive therapeutic compounds for the treatment of numerous diseases. The antisense field has remarkably progressed over the last few years with the approval of the first antisense drugs and with promising developments of more potent and nuclease resistant chemistries. Despite these recent clinical successes and advances in chemistry and design, effective delivery of ASOs to their target tissues remains a major issue. This review will describe the latest advances obtained with the tricyclo-DNA (tcDNA) chemistry which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration. We will examine the variety of therapeutic approaches using both fully modified tcDNA-ASOs and gapmers, including splice switching applications, correction of aberrant splicing, steric blocking strategies and targeted gene knock-down mediated by RNase H recruitment. We will then discuss the merits and potential liabilities of the tcDNA chemistry in the context of ASO drug development.

Abstract 1232: Development of novel therapeutic splice-switching oligonucleotides targeting oncogenic RNA isoforms driving race-related aggressive prostate cancer

Abstract Background: Age-adjusted incidence and mortality rates for prostate cancer (PCa) among African American (AA) men are 1.6- and 2.4-fold greater, respectively, than among white men. More aggressive characteristics of AA PCa account for a significant component of the PCa disparity, in addition to social determinants of health. Previous work from our laboratory and others have identified deregulation of the androgen receptor (AR) and epidermal growth factor receptor (EGFR) pathways in AA versus white PCa. Thus, there is an urgent need to develop a novel therapeutic strategy capable of inhibiting oncogenic AR and EGFR RNA isoforms enriched for in AA PCa. Methods: We have designed and synthesized novel chemically modified splice switching oligonucleotides (SSOs) to: 1) correct aberrant splicing leading to production of AR-V7 (AR-V7 SSO), an AR variant that lacks the ligand binding domain, is constitutively active and associates with castration-resistant PCa, poorer clinical outcomes and resistance to androgen ablation/AR inhibition therapies, 2) drive production of AR45 (AR45 SSO), an AR variant that has a unique, non-functional transactivation domain and acts in a dominant-negative manner and 3) drive production of inhibitory or dominant-negative EGFR isoforms (EGFR-TM or EGFR-TK SSOs), lacking the transmembrane or tyrosine kinase domain, respectively. Results: Transfection of a panel of PCa cell lines derived from AA and white patients with AR-V7 SSO decreases AR-V7 RNA and protein in a dose-dependent manner. Preliminary data suggests this biochemical response correlates with a biologically significant phenotype, as AR-V7 SSO also decreases proliferation, proliferation in the presence of enzalutamide and colony forming ability of PCa cells. Following transfection of PCa cell lines with AR45 SSO, an increase in AR45 RNA is seen and simultaneous decreases in wild type AR RNA and AR-V7 RNA are seen. In addition, modulation of AR signaling is detected, with decreases in RNAs corresponding to AR-induced target genes and increases in RNAs corresponding to AR-repressed target genes. Transfection of PCa cell lines with EGFR-TM or EGFR-TK SSOs increase RNA corresponding to these isoforms in a dose-dependent manner. Furthermore, a decrease in phosphorylated EGFR protein is detected. Further studies to examine the effects of these SSOs on transactivation activity, signaling and PCa cell biology and the therapeutic efficacy of these SSOs in AA and white PCa patient-derived explants are underway. Conclusions: These studies suggest that SSOs can be developed to modulate AR and EGFR signaling. Such SSOs could represent a novel therapeutic strategy with the potential to reduce PCa disparities for AA men and improve outcomes for men of all races with aggressive disease driven by these mechanisms. Citation Format: Jennifer A. Freedman, Timothy Robinson, Bonnie LaCroix, Brendon Patierno, Daniel George, Bruce Sullenger, Steven Patierno. Development of novel therapeutic splice-switching oligonucleotides targeting oncogenic RNA isoforms driving race-related aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1232. doi:10.1158/1538-7445.AM2017-1232

Abstract B18: Development of novel therapeutic splice-switching oligonucleotides against aggressive prostate cancer in men of African descent

Abstract African American (AA) men exhibit a nearly 2-fold higher incidence and 3-fold higher mortality rate from prostate cancer compared to Caucasian American (CA) men and disparities in tumor aggressiveness remain after controlling for social determinants of health. Previous work from our laboratory and others has revealed differences in gene expression that contribute to prostate cancer health disparities among AAs. Oncogenic signaling pathways exhibiting up-regulation more often in AA prostate cancer include androgen receptor (AR) and epidermal growth factor receptor (EGFR). Thus, novel therapeutic strategies capable of driving production of inhibitory AR and EGFR isoforms and capable of limiting aberrant constitutively active AR isoforms are urgently needed. Such strategies will increase our understanding of these molecular mechanisms underlying prostate cancer in AA men. In addition, such strategies have the potential to ultimately result in novel specific approaches for treatment that will help reduce prostate cancer disparities for AAs and will improve treatment of advanced stage disease in all men with aggressive disease driven by these mechanisms. Splice-switching oligonucleotides (SSOs) represent a novel therapeutic strategy to combat prostate cancer. Unlike the strategy of using RNA interference to inhibit the expression of a gene, SSOs simultaneously limit the production of pathogenic proteins and induce the expression of protein variants with therapeutic value. SSOs modulate pre-mRNA splicing by binding to target pre-mRNAs and blocking access of the splicing machinery to a particular splice site, and can be used to produce novel splice variants or correct aberrant splicing. Herein we have designed and synthesized novel SSOs targeting AR and EGFR pre-mRNAs to produce inhibitory splice variants and correct aberrant splicing as follows. We have synthesized a SSO to correct the aberrant splicing that leads to production of the constitutively active AR-V7 variant leading to restoration of ligand dependency. Transfection of prostate cancer cells with this chemically modified SSO, which targets the splice site of the cryptic exon included in AR-V7, decreases AR-V7 RNA. In addition, we have synthesized a SSO to drive production of a naturally occurring dominant-negative AR variant (AR45) leading to interference with wild type AR transactivation activity. Transfection of prostate cancer cells with this chemically modified SSO, which targets the splice site of the first exon of wild type AR and leads to inclusion of an alternative first exon encoding the unique N-terminal extension characterizing AR45, increases AR45 RNA. Furthermore, we have synthesized SSOs targeting exons in the transmembrane domain and the tyrosine kinase domain of EGFR to drive production of a soluble, naturally expressed inhibitory EGFR isoform and a dominant-negative EGFR isoform, respectively. Transfection of prostate cancer cells with these chemically modified SSOs increase RNA corresponding to these isoforms. Studies are underway to examine the effects of these SSOs on AR and EGFR signaling, respectively, and the effects of these SSOs on prostate tumor cell biology. These studies suggest that SSOs can be developed to modulate AR and EGFR signaling. Such SSOs have the potential to further our understanding of the contribution of the targeted molecular mechanisms to AA prostate cancer and have the potential to yield novel therapeutic modalities to combat prostate cancer in AA men as well as men of all races with aggressive disease driven by these mechanisms. Citation Format: Jennifer A. Freedman, Timothy J. Robinson, Bonnie LaCroix, Brendon M. Patierno, Daniel J. George, Bruce A. Sullenger, Steven R. Patierno. Development of novel therapeutic splice-switching oligonucleotides against aggressive prostate cancer in men of African descent. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B18.

Cardiac glycosides correct aberrant splicing of IKBKAP‐encoded mRNA in familial dysautonomia derived cells by suppressing expression of SRSF3

The ability to modulate the production of the wild-type transcript in cells bearing the splice-altering familial dysautonomia (FD) causing mutation in the IKBKAP gene prompted a study of the impact of a panel of pharmaceuticals on the splicing of this transcript, which revealed the ability of the cardiac glycoside digoxin to increase the production of the wild-type, exon-20-containing, IKBKAP-encoded transcript and the full-length IκB-kinase-complex-associated protein in FD-derived cells. Characterization of the cis elements and trans factors involved in the digoxin-mediated effect on splicing reveals that this response is dependent on an SRSF3 binding site(s) located in the intron 5' of the alternatively spliced exon and that digoxin mediates its effect by suppressing the level of the SRSF3 protein. Characterization of the digoxin-mediated effect on the RNA splicing process was facilitated by the identification of several RNA splicing events in which digoxin treatment mediates the enhanced inclusion of exonic sequence. Moreover, we demonstrate the ability of digoxin to impact the splicing process in neuronal cells, a cell type profoundly impacted by FD. This study represents the first demonstration that digoxin possesses splice-altering capabilities that are capable of reversing the impact of the FD-causing mutation. These findings support the clinical evaluation of the impact of digoxin on the FD patient population.

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.

Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice

Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or beta-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals.

Correction of Aberrant Pre-mRNA Splicing by Antisense Oligonucleotides in β-Thalassemia Egyptian Patients With IVSI-110 Mutation

The splicing mutation in intron 1 of beta-globin gene (IVS1-110) is the most common mutation in Egyptian thalassemics that causes aberrant splicing of pre-mRNA and deficient beta-globin chain synthesis. Antisense oligonucleotides (ASONs) are compounds that redirect pre-mRNA splicing and modify gene expression. Our aim was ex vivo correction of the aberrant splicing of beta-globin110 pre-mRNA by ASON against the 3' aberrant splice site. Peripheral blood mononuclear cells of 10 thalassemic patients with IVS1-110 mutation were duplicated and 1 was treated with 20 micromoL/mL morpholino ASON targeted against the 3' aberrant splice site. The level of total hemoglobin (Hb), fetal Hb, and mRNA were estimated in the duplicate samples. Five cases (50%) showed correction with ASON treatment, of which 2 cases showed the appearance of corrected mRNA band with absence of the aberrant band and 3 cases showed an increased ratio of the corrected to the aberrant mRNA band from 2:1 to 3:1, and 4:1. The total Hb showed significant increase in the 5 corrected cases. In conclusion, ASON can restore correct splicing of beta-globin pre-mRNA leading to correct gene product in cultured erythropoietic cells. These results suggest the applicability of ASON for the treatment of thalassemia.

Switching on transgene expression by correcting aberrant splicing using multi‐targeting steric‐blocking oligonucleotides

Mutations leading to aberrant splicing are found as a cause of numerous pathologies. Splice-switching oligonucleotides (SSOs), which modify aberrant expression patterns of alternatively spliced mRNAs, are a novel means of potentially controlling such diseases. We used an experimental model in which a mutated beta-globin intron, carrying an aberrant splice site at nucleotide 705, interrupts the coding region of the luciferase reporter gene inserted in HeLa pLuc/705 cells. We have optimized delivery of splice correcting, steric-blocking 2'-O-methyl SSOs targeting the 705 mutated region (2'-O-Me SSO(705)) with DLS (DLS: delivery liposomal system) lipoplexes. Optimal luciferase activity for DLS/2'-O-Me SSO(705) was achieved at 100 nM and was detectable at concentrations as low as 10 nM in serum-containing culture medium, confirming the potential of DLS lipoplex-mediated nuclear SSO delivery as observed in cellular uptake studies. We confirmed by cytofluorometry and epifluorescence microscopy the high potential of the DLS lipoplex for cellular and nuclear oligonucleotide uptake. The DLS lipoplex was then used to directly compare the intracellular efficacy of various SSO chemistries and sequences in correction of aberrant splicing. 2'-O-Methoxyethyl-oligodeoxyribonucleoside phosphorothioates had a greater activity than 2'-O-methyl phosphodiester or 2'-O-methyl-phosphorothioate oligoribonucleotides. Targeting the splicing enhancer 623 region upstream was as efficient as targeting the 705 splice site, and, remarkably, simultaneous targeting of both sites was more efficient than treatment of the cells either with 2'-O-Me SSO(705) or 2'-O-Me SSO(623) alone. We demonstrated that SSOs can switch on luciferase activity in HeLa cells previously transfected with the pLuc/705 plasmid via the same DLS vector and provides a novel approach to modulate the expression of a transgene.

Induction of splice correction by cell‐penetrating peptide nucleic acids

Directing splicing using oligonucleotides constitutes a promising therapeutic tool for a variety of diseases such as beta-thalassemia, cystic fibrosis, and certain cancers. The rationale is to block aberrant splice sites, thus directing the splicing of the pre-mRNA towards the desired protein product. One of the difficulties in this setup is the poor bioavailability of oligonucleotides, as the most frequently used transfection agents are unsuitable for in vivo use. Here we present splice-correcting peptide nucleic acids (PNAs), tethered to a variety of cell-penetrating peptides (CPPs), evaluating their mechanism of uptake and ability to correct aberrant splicing. HeLa cells stably expressing luciferase containing an aberrant splice site were used. A previously described PNA sequence, capable of correcting the aberrant splicing, was conjugated to the CPPs, Tat, penetratin and transportan, via a disulfide bridge. The ability of the CPP-PNA conjugates to correct splicing was measured, and membrane disturbance and cell viability were evaluated using LDH leakage and WST-1 assays. Lysosomotropic agents, inhibition of endocytosis at 4 degrees C and confocal microscopy were used to investigate the importance of endocytosis in the uptake of the cell-penetrating PNAs. All the three CPPs were able to promote PNA translocation across the plasma membrane and induce splice correction. Transportan (TP) was the most potent vector and significantly restored splicing in a concentration-dependent manner. Interestingly, TP also rendered a concentration-dependent splice correction in serum, in contrast to Tat and penetratin. Addition of the lysosomotrophic agent chloroquine increases the splice correction efficacy of the CPP-PNA conjugates up to 4-fold, which together with experiments at 4 degrees C and the visual information from confocal microscopy, indicate that the mechanism of uptake responsible for internalization of CPP-PNA conjugates is mainly endocytic. Finally, co-localization studies with dextran further indicate that conjugates, at least in the case of TP, internalize via endocytosis and in particular macropinocytosis. These data demonstrate that CPPs can be used for the delivery of splice-correcting PNAs, with potential to be used as a therapeutic approach for regulating splicing in a variety of diseases. Transportan presents itself as the overall most suitable vector in this study, generating the most efficient conjugates for splice correction.

Evaluation of Transfection Protocols for Unmodified and Modified Peptide Nucleic Acid (PNA) Oligomers

We have compared the efficacy of different transfection protocols reported for peptide nucleic acid (PNA) oligomers. A precise evaluation of uptake efficacy was achieved by using a positive readout assay based on the ability of a PNA oligomer to correct aberrant splicing of a recombinant luciferase gene. The study comprised transfection of PNA conjugated to acridine, adamantyl, decanoic acid, and porphyrine (acr-PNA, ada-PNA, deca-PNA, and por-RNA, respectively) and unmodified PNA partially hybridized to a DNA oligomer (PNA/DNA cotransfection). Furthermore, the effect of conjugation to a nuclear localization signal (NLS) was evaluated as part of the PNA/DNA cotransfection protocol. Transfection of the tested PNAs was systematically optimized. PNA/DNA cotransfection was found to produce the highest luciferase activity, but only after careful selection of the DNA oligonucleotide. Both a cationic lipid, Lipofectamine, and a nonliposomal cationic polymer, polyethylenimine (PEI, ExGen 500), were efficient transfection reagents for the PNA/DNA complex. However, Lipofectamine, in contrast to PEI, showed severe side effects, such as cytotoxicity. acr-PNA, ada-PNA, and por-PNA were transfectable with efficacies between 5 and 10 times lower than that seen with PNA/DNA cotransfection. Conjugation of PNA to NLS had no effect on PNA/DNA cotransfection efficacy. An important lesson from the study was the finding that because of uncontrollable biologic variations, even optimal transfection conditions differed to a certain extend from experiment to experiment in an unpredictable way.

Selective Inhibition of Topoisomerase I and Various Steps of Spliceosome Assembly by Diospyrin Derivatives

Pre-mRNA splicing is an essential step of the expression of most metazoan protein-coding genes, which is often regulated in a cell type-specific or developmental manner. We have demonstrated previously that human DNA topoisomerase I, an extensively studied target for anticancer drugs, also has an intrinsic protein kinase activity that specifically phosphorylates proteins involved in splice site selection. Therefore, DNA topoisomerase I was recently shown to play a critical role in alternative splicing. Here, we have exploited these novel properties of DNA topoisomerase I to develop entirely novel diospyrin derivatives targeting its protein kinase activity and thereby modulating pre-mRNA splicing. Although some derivatives indeed inhibit kinase activity of topoisomerase I, they did not block reactions of topoisomerase I on DNA. However, these drugs interfere with camptothecin-dependent topoisomerase I-mediated DNA cleavage, implying that diospyrin derivatives mediate a conformational change of topoisomerase I. It is note-worthy that in vitro splicing reactions revealed that diospyrin derivatives alter various steps of splicing. Some diospyrin derivatives inhibit either the first or the second catalytic step of splicing but not spliceosome assembly, whereas diospyrin itself prevents the formation of full spliceosome. Our data revealed for the first time that diospyrin derivatives are able to stall the dynamic assembly of the spliceosome and open the exciting possibility of using these derivatives to correct aberrant splicing in human genetic diseases.

(2′‐O‐Methyl‐RNA)‐3′‐PNA Chimeras: A New Class of Mixed Backbone Oligonucleotide Analogues with High Binding Affinity to RNA

The automated on-line synthesis of DNA-3′-PNA chimeras 1–4 and (2′-O-methyl-RNA)-3′-PNA chimeras 5–8 is described, in which the 3′-terminal part of the oligonucleotide is linked to the N-terminal part of the PNA via N-(ω-hydroxyalkyl)-N-[(thymin-1-yl)acetyl]glycine units (alkyl=Et, Ph, Bu, and pentyl). By means of UV thermal denaturation, the binding affinities of all chimeras were directly compared by determining their Tm values in the duplex with complementary DNA and RNA. All investigated DNA-3′-PNA chimeras and (2′-O-methyl-RNA)-3′-PNA chimeras form more-stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. Interestingly, a N-(3-hydroxypropyl)glycine linker resulted in the highest binding affinity for DNA-3′-PNA chimeras, whereas the (2′-O-methyl-RNA)-3′-PNA chimeras showed optimal binding with the homologous N-(4-hydroxybutyl)glycine linker. The duplexes of (2′-O-methyl-RNA)-3′-PNA chimeras and RNA were significantly more stable than those containing the corresponding DNA-3′-PNA chimeras. Surprisingly, we found that the charged (2′-O-methyl-RNA)-3′-PNA chimera with a N-(4-hydroxybutyl)glycine-based unit at the junction to the PNA part shows the same binding affinity to RNA as uncharged PNA. Potential applications of (2′-O-methyl-RNA)-3′-PNA chimeras include their use as antisense agents acting by a RNase-independent mechanism of action, a prerequisite for antisense-oligonucleotide-mediated correction of aberrant splicing of pre-mRNA.

Antisense properties of tricyclo-DNA.

Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2'-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10-17 nt in length, show enhanced selectivity and enhanced thermal stability by approximately 1 degrees C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37 degree C. Moreover, tc-DNA-RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human beta-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in beta-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3'-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2'-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2'-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentrations.

Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping.

Alternative splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both flanking introns (IVS8 and IVS9). Several studies have identified in the IVS8 intron 3' splice site a regulatory element that is composed of a polymorphic (TG)m(T)n repeated sequence. At present, no cellular factors have been identified that recognize this element. We have identified TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding specifically to the (TG)m sequence. Transient TDP-43 overexpression in Hep3B cells results in an increase in exon 9 skipping. This effect is more pronounced with concomitant overexpression of SR proteins. Antisense inhibition of endogenous TDP-43 expression results in increased inclusion of exon 9, providing a new therapeutic target to correct aberrant splicing of exon 9 in CF patients. The clinical and biological relevance of this finding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(DeltaF508)/TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.