Biomedical subjects

Ryszard Kole

Publications and source records attributed to Ryszard Kole.

16 recordsLinked to original sources

PubMed2006

Efficient and persistent splice switching by systemically delivered LNA oligonucleotides in mice.

Locked nucleic acid (LNA) oligomers were found to be very effective in their ability to modulate alternative splicing in vivo in transgenic mice that ubiquitously express a modified EGFP pre-mRNA containing an aberrantly spliced beta-globin intron (IVS2-654). Following intraperitoneal injections, the splice-switching oligonucleotide LNA SSO-654 targeted to the aberrant 5' splice site in EGFP-654 pre-mRNA corrected aberrant splicing and increased production of repaired EGFP mRNA mainly in the liver, colon, and small intestine. Little or no effect was detected in heart, lung, or kidney, the organ where most of the oligonucleotide was distributed after four consecutive daily injections. In the liver, LNA SSO-654 had an EC(50) of 3 mg/kg, approximately 17-fold more potent than its 2'-O-methyl congener. Moreover, in the liver, colon, and small intestine oral doses of 50 mg/kg resulted in detectable levels of splice switching. The effects of four daily injections at 25 mg/kg persisted for up to 29 days but did not result in liver toxicity. The results indicate that the LNA backbone confers sequence- and organ-specific functional biodistribution of the oligonucleotides and that these potent compounds have the potential to be safe and long-acting modulators of diseases treatable by splicing manipulation.

Administration, Oral↗

PubMed2006

Analysis of prostate-specific membrane antigen splice variants in LNCap cells.

The prostate-specific membrane antigen (PSMA), a product of the folate hydrolase (FOLH1) gene, is highly expressed as a largely extracellular membrane-anchored protein in malignant prostate tissues and in nonprostatic tumor neovasculature. Treatment of prostate cancer LNCap cells with spliceswitching oligonucleotides (SSOs) modulated splicing of FOLH1 pre-mRNA from the full-length PSMA splice variant to three splice variants: the cytoplasmic PSM', alternatively spliced at exon 1, and the previously unexamined PSMADelta6 and PSMADelta18 variants, which lack exons 6 and 18, respectively. Application of SSOs decreased membrane PSMA levels and increased PSM', PSMADelta6, and PSMADelta18 transcripts. As a result, PSM' protein was translocated to the cytoplasm, and switching to PSMADelta6 and PSMADelta18 downregulated PSMA expression. NAALADase assays showed that PSM' retained enzymatic activity. PSMADelta6 and PSMADelta18 were not active, presumably due to a change in a reading frame that eliminated the NAALDase active site or the dimerization domain or both in these proteins.

Alternative Splicing↗

PubMed2004

Modification of alternative splicing by antisense therapeutics.

Alternative splicing allows the production of several different proteins from a single pre-mRNA, resulting in an increased diversity of proteins derived from a relatively limited number of transcribed genes. Although it is necessary for normal development, alternative splicing and its aberrations are also implicated in disease states from thalassemia and cancer to neurodegenerative disorders. Techniques that trick the splicing machinery to alter the splicing pathways can be of high therapeutic value. Antisense technology, used mostly for RNA downregulation, recently has been adapted to alter the splicing process. The promise of this approach is now being realized as a result of chemical modification of oligonucleotides and improvements in their delivery in vivo.

Alternative Splicing↗

PubMed2004

RNA modulation, repair and remodeling by splice switching oligonucleotides.

Targeting splicing by antisense oligonucleotides allows RNA modifications that are not possible with RNA interference or other antisense techniques that destine the RNA for destruction. By changing the ratio of naturally occurring splice variants the expression of mRNA is modulated. By preventing the use of an aberrant splice site created by a mutation and enforcing re-selection of correct splice sites the RNA is repaired. Antisense induced skipping of the exon that carries a nonsense mutation remodels the mRNA and restores the reading frame of the defective protein. All of the above approaches have clinical applications. Modulation of splice variants is particularly important since close to 60% of all genes code for alternatively spliced pre-mRNA.

Alternative Splicing↗

PubMed2003

HIV-1 preferentially binds receptors copatched with cell-surface elastase.

Human leukocyte elastase (HLE) interacts with HIV-1 glycoprotein (gp)41, suggesting a nonenzymatic receptor function for HLE in the context of HIV-1. HLE is found localized to the cell surface, but not granules in HIV permissive clones, and to granules, but not the cell surface of HIV nonpermissive clones. Inducing cell-surface HLE expression on HLE null, HIV nonpermissive clones permits HIV infectivity. HIV binding and infectivity diminish in proportion to HLE RNA subtraction. HIV binding and infectivity show dose dependence for the natural HLE ligand alpha1 proteinase inhibitor (alpha1antitrypsin, alpha1PI). Chemokines prevent, whereas alpha1PI promotes, copatching of HLE with the canonical HIV receptors. Recent demonstration that decreased viral RNA is significantly correlated with decreased circulating alpha1PI in HIV seropositive individuals is consistent with a model in which HLE and alpha1PI can serve as HIV coreceptor and cofactor, respectively, and potentially participate in the pathophysiology of HIV disease progression.

CD4 Antigens↗

PubMed2003

Superoxide-dependent iron uptake: a new role for anion exchange protein 2.

Lung cells import iron across the plasma membrane as ferrous (Fe2+) ion by incompletely understood mechanisms. We tested the hypothesis that human bronchial epithelial (HBE) cells import non-transferrin-bound iron (NTBI) using superoxide-dependent ferri-reductase activity involving anion exchange protein 2 (AE2) and extracellular bicarbonate (HCO3-). HBE cells that constitutively express AE2 mRNA by reverse transcriptase-polymerase chain reaction and AE2 protein by Western analysis avidly transported NTBI after exposure to either Fe2+ or Fe3+, but reduction of Fe3+ to Fe2+ was first required. The ability of HBE cells to reduce Fe3+ and transport Fe2+ was inhibited by active extracellular superoxide dismutase (SOD). Similarly, HBE cells that overexpress Cu,Zn SOD after adenoviral infection with AdSOD1 showed diminished iron uptake. The role of AE2 in iron uptake was indicated by three lines of evidence: (i) lack of both iron reduction and iron transport in bicarbonate-free buffer at controlled pH, (ii) failure of HBE cells treated with stilbene AE inhibitors to reduce Fe3+ or transport iron, and (iii) inhibition of iron uptake in HBE cells by inhibition of AE2 protein expression with antisense oligonucleotides. We thus disclose a novel ferri-reductase mechanism of NTBI uptake by human lung cells that employs superoxide exchange for HCO3- by AE2 protein in the plasma membrane.

Adenoviridae↗

PubMed2003

Effects of base modifications on antisense properties of 2'-O-methoxyethyl and PNA oligonucleotides.

A recently developed antisense splicing assay was used to determine the relative activities of 2'-O-methoxyethoxy (2'-MOE) phosphorothioate oligonucleotides containing base modifications. In the assay, RNase H-inactive oligonucleotides are used to block aberrant splicing and restore correct splicing of an Enhanced Green Fluorescence Protein (EGFP) reporter pre-mRNA stably expressed in HeLa cells. Thus, the extent of EGFP upregulation is proportional to the antisense activity of the tested molecule. The base modifications included C-5 propynyl analogs of uridine and cytidine and phenoxazine and G-clamp analogs of cytosine. Base-modified 2'-MOE oligonucleotides were delivered to the HeLa EGFP-654 test cells by cationic lipid transfection or scrape-loading or without any delivery method (free uptake). When delivered with a cationic lipid, the G-clamp and phenoxazine oligomers showed increases in activity over the unmodified 2'-MOE parent compound. However, when delivered by scrape-loading or without a delivery method, the unmodified oligomer performed best. The results suggest that base modifications do not enhance the free uptake activity of RNase H inactive 2'-MOE oligomers.

Alternative Splicing↗

PubMed2003

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.

Alternative Splicing↗

PubMed2002

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 beta-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.

Animals↗

PubMed2002

Temperature-dependent splicing of beta-globin pre-mRNA.

A T-->G mutation at nucleotide 705 of human beta-globin intron 2 creates an aberrant 5' splice site and activates a cryptic 3' splice site upstream. In consequence, the pre-mRNA is spliced via aberrant splice sites, despite the presence of the still functional correct sites. Surprisingly, when IVS2-705 HeLa or K562 cells were cultured at temperatures below 30 degrees C, aberrant splicing was inhibited and correct splicing was restored. Similar temperature effects were seen for another beta-globin pre-mRNA, IVS2-745, and in a construct in which a beta-globin intron was inserted into a coding sequence of EGFP. Temperature-induced alternative splicing was affected by the nature of the internal aberrant splice sites flanking the correct sites and by exonic sequences. The results indicate that in the context of thalassemic splicing mutations and possibly in other alternatively spliced pre-mRNAs, temperature is one of the parameters that affect splice site selection.

Globins↗

PubMed2002

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.

Alternative Splicing↗

PubMed2002

High-level expression of hemoglobin A in human thalassemic erythroid progenitor cells following lentiviral vector delivery of an antisense snRNA.

Mutations at nucleotides 654, 705, or 745 in intron 2 of the human beta-globin gene activate aberrant 3' and 5' splice sites within the intron and prevent correct splicing of beta-globin pre-mRNA, resulting in inhibition of beta-globin synthesis and in consequence beta-thalassemia. Transfection of HeLa cells expressing the 3 thalassemic mutants with modified U7 snRNA (U7.623), containing a sequence antisense to a region between the aberrant splice sites, reduced the incorrect splicing of pre-mRNA and led to increased levels of the correctly spliced beta-globin mRNA and protein. A lentiviral vector carrying the U7.623 gene was effective in restoration of correct splicing in the model cell lines for at least 6 months. Importantly, the therapeutic value of this system was demonstrated in hematopoietic stem cells and erythroid progenitor cells from a patient with IVS2-745/IVS2-1 thalassemia. Twelve days after transduction of the patient cells with the U7.623 lentiviral vector, the levels of correctly spliced beta-globin mRNA and hemoglobin A were approximately 25-fold over background. These results should be regarded as a proof of principle for lentiviral vector-based gene therapy for beta-thalassemia.

Erythroid Precursor Cells↗

PubMed2002

Control of alternative splicing by antisense oligonucleotides as a potential chemotherapy: effects on gene expression.

Expression of alternatively spliced mRNA variants at specific stages of development or in specific cells and tissues contributes to the functional diversity of the human genome. Aberrations in alternative splicing were found as a cause or a contributing factor to the development, progression, or maintenance of various diseases including cancer. The use of antisense oligonucleotides to modify aberrant expression patterns of alternatively spliced mRNAs is a novel means of potentially controlling such diseases. However, to utilize antisense oligonucleotides as molecular chemotherapeutic agents, the global effects of these molecules need to be examined. The advent of gene expression array technology has now made it possible to simultaneously examine changes that occur in the expression levels of several thousand genes in response to antisense treatment. This analysis should help in the development of more specific and efficacious antisense oligonucleotides as molecular therapeutics.

Alternative Splicing↗

PubMed2002

Repair of a splicing defect in erythroid cells from patients with beta-thalassemia/HbE disorder.

A HeLa cell line stably expressing the human beta-globin gene carrying thalassemic mutations beta(E)/IVS1-6 served as a thalassemia model for repair of aberrant splicing of beta(E)-globin pre-mRNA with antisense oligonucleotides. Treatment of beta(E)/IVS1-6 HeLa cells with a morpholino oligonucleotide targeted immediately upstream of the aberrant 5' splice site activated by the mutations resulted in an increase in the amount of correctly spliced beta(E)-globin mRNA in a dose-dependent and sequence-specific fashion. The repaired beta(E)-globin mRNA was stable and could be translated into full-length beta(E)-globin polypeptide. Application of the same oligonucleotide to erythroid progenitor cells from two beta-thalassemia/HbE patients resulted in an approximately 70% increase in correct beta(E)-globin mRNA and 36% increase in hemoglobin E. The erythroid progenitor cells represent the actual targets for the clinical application of antisense repair of defective pre-mRNAs.

Alternative Splicing↗

PubMed2002

Short-term and long-term modulation of gene expression by antisense therapeutics.

To achieve effective modulation of gene expression by antisense oligonucleotides, novel oligonucleotide chemistries that do not promote RNase H degradation of target RNA are needed. In addition to short-term oligonucleotide effects, long-term gene regulation can be accomplished by intracellularly expressed antisense RNAs delivered by viral vectors.

Gene Expression Regulation↗

PubMed2002

Restoration of human beta-globin gene expression in murine and human IVS2-654 thalassemic erythroid cells by free uptake of antisense oligonucleotides.

Correct human beta-globin mRNA has been restored in erythroid cells from transgenic mice carrying the human gene with beta-globin IVS2-654 splice mutation and from thalassemia patients with the IVS2-654/beta(E) genotype. This was accomplished in a dose- and time-dependent manner by free uptake of morpholino oligonucleotide antisense to the aberrant splice site at position 652 of intron 2 in beta-globin pre-mRNA. Under optimal conditions of oligonucleotide uptake, the maximal levels of correct human beta-globin mRNA and hemoglobin A in patients' erythroid cells were 77 and 54%, respectively. These levels of correction were equal to, if not higher than, those obtained by syringe loading of the oligonucleotide into the cells. Comparison of splicing correction results with the cellular uptake of fluorescein-labeled oligonucleotide indicated that the levels of mRNA and hemoglobin A correlate well with the nuclear localization of the oligonucleotide and the degree of erythroid differentiation of cultured cells. Similar but not as pronounced results were obtained after the oligonucleotide treatment of bone marrow cells from IVS2-654 mouse. The effectiveness of the free antisense morpholino oligonucleotide in restoration of correct splicing of IVS2-654 pre-mRNA in cultured erythropoietic cells from transgenic mice and thalassemic patients suggests the applicability of this or similar compounds in in vivo experiments and possibly in treatment of thalassemia.

Animals↗