Ryszard Kole

Systemically delivered antisense oligomers upregulate gene expression in mouse tissues

Systemically injected 2′-O-methoxyethyl (2′-O-MOE)-phosphorothioate and PNA-4K oligomers (peptide nucleic acid with four lysines linked at the C terminus) exhibited sequence-specific antisense activity in a number of mouse organs. Morpholino oligomers were less effective, whereas PNA oligomers with only one lysine (PNA-1K) were completely inactive. The latter result indicates that the four-lysine tail is essential for the antisense activity of PNA oligomers in vivo. These results were obtained in a transgenic mouse model designed as a positive readout test for activity, delivery, and distribution of antisense oligomers. In this model, the expressed gene (EGFP-654) encoding enhanced green fluorescence protein (EGFP) is interrupted by an aberrantly spliced mutated intron of the human β-globin gene. Aberrant splicing of this intron prevented expression of EGFP-654 in all tissues, whereas in tissues and organs that took up a splice site-targeted antisense oligomer, correct splicing was restored and EGFP-654 expression upregulated. The sequence-specific ability of PNA-4K and the 2′-O-MOE oligomers to upregulate EGFP-654 provides strong evidence that systemically delivered, chemically modified oligonucleotides affect gene expression by sequence-specific true antisense activity, validating their application as potential therapeutics.

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.

Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing

An estimated 60% of all human genes undergo alternative splicing, a highly regulated process that produces splice variants with different functions. Such variants have been linked to a variety of cancers, and genetic diseases such as thalassemia and cystic fibrosis. This Perspective describes a promising approach to RNA repair based on the use of antisense oligonucleotides to modulate alternative splicing and engender the production of therapeutic gene products.

Selection of splice sites in pre-mRNAs with short internal exons.

Model pre-mRNAs containing two introns and three exons, derived from the human beta-globin gene, were used to study the effects of internal exon length on splice site selection. Splicing was assayed in vitro in HeLa nuclear extracts and in vivo during transient expression in transfected HeLa cells. For substrates with internal exons 87, 104, and 171 nucleotides in length, in vitro splicing proceeded via a regular splicing pathway, in which all three exons were included in the spliced product. Primary transcripts with internal exons containing 23, 29, and 33 nucleotides were spliced by an alternative pathway, in which the first exon was joined directly to the third one. The internal exon was missing from the spliced product and together with two flanking introns was included in a large lariat structure. The same patterns of splicing were retained when transcripts containing 171-, 33-, and 29-nucleotide-long internal exons were spliced in vivo. A transcript containing a 51-nucleotide-long exon was spliced in vitro via both pathways but in vivo generated only a correctly spliced product. Skipping of short internal exons was reversed both in vitro and in vivo when purines in the upstream polypyrimidine tract were replaced by pyrimidines. The changes in the polypyrimidine tract achieved by these substitutions led in vitro to complete (transcripts containing 28 pyrimidines in a row) or partial (transcripts containing 15 pyrimidines in a row) restoration of a regular splicing pathway. Splicing in vivo of these transcripts led exclusively to the spliced product containing all three exons. These results suggest that a balance between the length of the uninterrupted polypyrimidine tract and the length of the exon is an important determinant of the relative strength of the splice sites, ensuring correct splicing patterns of multiintron pre-mRNAs.

Cellular Response to an Antisense-mediated Shift of Bcl-x Pre-mRNA Splicing and Antineoplastic Agents

Overexpression of Bcl-xL, an anti-apoptotic member of the Bcl-2 family, negatively correlates with the sensitivity of various cancers to chemotherapeutic agents. We show here that high levels of expression of Bcl-xL promoted apoptosis of cells treated with an antisense oligonucleotide (5′Bcl-x AS) that shifts the splicing pattern of Bcl-x pre-mRNA from the anti-apoptotic variant, Bcl-xL, to the pro-apoptotic variant, Bcl-xS. This surprising finding illustrates the advantage of antisense-induced modulation of alternative splicing versus down-regulation of targeted genes. It also suggests a specificity of the oligonucleotide effects since non-cancerous cells with low levels of Bcl-xL should resist the treatment. 5′Bcl-x AS sensitized cells to several antineoplastic agents and radiation and was effective in promoting apoptosis of MCF-7/ADR cells, a breast cancer cell line resistant to doxorubicin via overexpression of the mdr1 gene. Efficacy of 5′Bcl-x AS combined with chemotherapeutic agents in the PC3 prostate cancer cell line may be translated to clinical prostate cancer since recurrent prostate cancer tissue samples expressed higher levels of Bcl-xL than benign prostate tissue. Treatment with 5′Bcl-x AS may enhance the efficacy of standard anti-cancer regimens and should be explored, especially in recurrent prostate cancer.

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.

Antisense pharmacodynamics: critical issues in the transport and delivery of antisense oligonucleotides.

This review critically examines current understanding of the kinetics and biodistribution of antisense oligonucleotides, both at the cellular level and at the level of the intact organism. The pharmacodynamic relationships between biodistribution and the ultimate biological effects of antisense agents are considered. The problems and advantages inherent in the use of delivery systems are discussed in the light of further enhancing in vivo pharmacological actions of oligonucleotides.

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.

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 β-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.

Anti-tumor activity of splice-switching oligonucleotides

Alternative splicing has emerged as an important target for molecular therapies. Splice-switching oligonucleotides (SSOs) modulate alternative splicing by hybridizing to pre-mRNA sequences involved in splicing and blocking access to the transcript by splicing factors. Recently, the efficacy of SSOs has been established in various animal disease models; however, the application of SSOs against cancer targets has been hindered by poor in vivo delivery of antisense therapeutics to tumor cells. The apoptotic regulator Bcl-x is alternatively spliced to express anti-apoptotic Bcl-x(L) and pro-apoptotic Bcl-x(S). Bcl-x(L) is upregulated in many cancers and is associated with chemoresistance, distinguishing it as an important target for cancer therapy. We previously showed that redirection of Bcl-x pre-mRNA splicing from Bcl-x(L) to -x(S) induced apoptosis in breast and prostate cancer cells. In this study, the effect of SSO-induced Bcl-x splice-switching on metastatic melanoma was assessed in cell culture and B16F10 tumor xenografts. SSOs were delivered in vivo using lipid nanoparticles. Administration of nanoparticle with Bcl-x SSO resulted in modification of Bcl-x pre-mRNA splicing in lung metastases and reduced tumor load, while nanoparticle alone or formulated with a control SSO had no effect. Our findings demonstrate in vivo anti-tumor activity of SSOs that modulate Bcl-x pre-mRNA splicing.