On the in vitro and in vivo Properties of Four Locked Nucleic Acid Nucleotides Incorporated into an Anti-H-Ras Antisense Oligonucleotide

ChemBioChem ◽  
2005 ◽  
Vol 6 (6) ◽  
pp. 1104-1109 ◽  
Author(s):  
Kees Fluiter ◽  
Miriam Frieden ◽  
Jeroen Vreijling ◽  
Christoph Rosenbohm ◽  
Marit B. De Wissel ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Antisense Oligonucleotide ◽  
Locked Nucleic Acid

scholarly journals A Locked Nucleic Acid Antisense Oligonucleotide (LNA) Silences PCSK9 and Enhances LDLR Expression In Vitro and In Vivo

PLoS ONE ◽  
2010 ◽  
Vol 5 (5) ◽  
pp. e10682 ◽  
Author(s):  
Nidhi Gupta ◽  
Niels Fisker ◽  
Marie-Claude Asselin ◽  
Marie Lindholm ◽  
Christoph Rosenbohm ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Antisense Oligonucleotide ◽  
Locked Nucleic Acid

siRNA Versus Antisense Locked Nucleic Acids: Stay Single!.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 614-614
Author(s):  
Phil Kearney ◽  
Majken Westergaard ◽  
Henrik F. Hansen ◽  
Ellen M. Staarup ◽  
Troels Koch ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Therapeutic Option ◽  
Phase 1 ◽  
Locked Nucleic Acid ◽  
Target Validation ◽  
Host Animal ◽  
Clinical Phase ◽  
Potent Inhibition

Abstract Much discussion has centred around the utility and benefits of siRNA in both target validation and as a therapeutic option. This has been driven by significant publications including that of Soutcheck et al (Nature432, 173–177 2004), which demonstrated liver targeting as well as in vivo efficacy when siRNA against ApoB was tethered to a cholesterol moiety. Santaris Pharma has developed a third generation nucleic acid chemistry referred to as locked nucleic acid (LNA) which delivers unmatched affinity and stabiliy benefits, largely overcoming the drawbacks associated with traditional antisense molecules. We therefore sought to compare this chemistry with targets which siRNA has been successfully used in in vivo/in vitro settings. The same motif used in the Soutcheck study was targeted with a LNA molecule, and the free siRNA activity was compared to the cholesterol linked and free LNA molecules in their ability ot down regulate ApoB expression. LNA (SPC3197) inhibited ApoB expression by 90% while at an equimolar concentration siRNA was ineffective in the liver and jejunum. Cholesterol linked siRNA was only effective in the jejunum (c50% reduction in mRNA) Fig1. Only the LNA mediated inhibition of ApoB expression was paralleled by a drop in serum cholesterol in the host animal. In a second model siRNA molecules targeting Hif-1a mRNA (Yu et al Lab Invest84, 553–561 2004) were compared to our lead LNA molecule targeting Hif-1a, SPC2968. Interestingly in in vitro analyses these 2 molecules were equally effective. However in a murine model the increased half life of the LNA molecules translated to a potent inhibition of Hif-1a as measured by QPCR. This effect was noted in jejunum and liver, and persisted for at least 4 days. Hif-1a inhibition mediated by siRNA was not seen in any tissue analysed (Fig 2). Finally a 3rd molecule targeting Bcl-2 has entered clinical Phase 1 trials, and data will be presented documenting its improved affinity and stabitily in relation to competitor molecules such as Genasense. Figure Figure

In Vitro and Vivo Activity Against Multiple Myeloma Cells Of a Novel Locked Nucleic Acid (LNA)-Mir-221 Inhibitor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3166-3166
Author(s):  
Maria Teresa Di Martino ◽  
Annamaria Gullà ◽  
Maria Eugenia Gallo Cantafio ◽  
Emanuela Altomare ◽  
Nicola Amodio ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Nucleic Acid ◽  
Luciferase Activity ◽  
Locked Nucleic Acid ◽  
Phase Cell ◽  
Target Sequence ◽  
Systemic Delivery ◽  
Anti Tumor Activity

Abstract miR-221/222 are two highly homologous microRNAs (miRNAs), encoded in tandem on the chromosome X, whose up-regulation has been found in several malignancies and are thought to promote cell proliferation via down-regulation of p27 and/or p57, two negative regulators of G1 to S phase cell cycle progression. We demonstrated up-regulation of both miRNAs in malignant plasma cells (PCs) from multiple myeloma (MM) patients belonging to distinct TC (translocation/Cyclin) groups, including TC2 and TC4. A rising body of evidence suggests that silencing miRNAs with oncogenic potential could represent a novel approach for human cancer therapy. We previously demonstrated that silencing miR-221/222 exerts significant anti-MM activity and triggers canonical targets in vitro and in vivo. Here, in the aim to progress to clinical translation of our proof-of-principle findings, we investigated the anti-tumor activity and the appropriateness for systemic delivery of a novel and originally designed LNA-miR-221, a 13-mer antisense miR-221 inhibitor, which took advantage of locked nucleic acid (LNA) technology and phosphorothioate backbone chemistry for increasing affinity for miR-221 and nuclease resistance. We found that enforced ectopic expression of LNA-miR-221 in t(4;14) MM cells significantly inhibited growth and survival of MM cells in vitro. In treated cells, we detected knock down of miR-221/222 together and increased levels of both p27Kip1 mRNA and protein. Specific activity of this LNA-miR-221 inhibitor was confirmed by the use of a 3’UTR reporter (luciferase renilla/firefly) constructs containing, miR-221 target site. This construct was co-transfected either with miR-221/222 mimics or LNA-miR-221 inhibitor into MM cells. As predicted, a reduced luciferase activity was detected in miR-221/222 mimics co-transfected cells with each 3’UTR reporter plasmid, while increase luciferase activity was measured in MM cells co-transfected LNA-miR-221 inhibitors indicating an efficient and stable binding to the miRNA target sequence. Importantly, we evaluated the systemic delivery of the LNA-miR-221 inhibitor with saline solution vehicle alone by intraperitoneal or intravenous injection route against MM xenografts in SCID/NOD mice. Significant anti-tumor activity was achieved after 2 weeks of treatment at similar extent by both injection routes. Retrieved tumors from treated animals showed efficient inhibition of miR-221/222, as demonstrated by increased levels of p27Kip1 protein in vivo. H&E staining and immunohistochemical analysis showed wide necrosis areas, reduced Ki67 and a significant increase of p27Kip1 cytoplasmic expression in retrieved tumors from LNA-miR-221 inhibitor-treated mice. No changes in mice behavior or organ toxicity were observed in treated mice. Taken together these findings support the rationale for development of this novel and highly efficient LNA-miR-221 inhibitor as a promising anti-MM drug in subsequent primate toxicology studies. Supported by the Italian Association for Cancer Research (AIRC), PI: PT. “Special Program Molecular Clinical Oncology - 5 per mille” n. 9980, 2010/15. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In vitro and in vivo activity of miR-92a–Locked Nucleic Acid (LNA)–Inhibitor against endometrial cancer

BMC Cancer ◽  
2016 ◽  
Vol 16 (1) ◽  
Author(s):  
Anna Torres ◽  
Joanna Kozak ◽  
Agnieszka Korolczuk ◽  
Paulina Wdowiak ◽  
Ewa Domańska-Glonek ◽  
...  
Keyword(s):  
Endometrial Cancer ◽  
Nucleic Acid ◽  
Locked Nucleic Acid

Pharmacokinetics and Biodistribution of Locked Nucleic Acid (LNA)-Mir-221 Inhibitor: A New Promising Anti-Myeloma Agent

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3025-3025 ◽  
Author(s):  
Maria Eugenia Gallo Cantafio ◽  
Annamaria Gulla ◽  
Nicola Amodio ◽  
Emanuela Leone ◽  
Eugenio Morelli ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Half Life ◽  
Conflicts Of Interest ◽  
Locked Nucleic Acid ◽  
Phase Cell ◽  
Systemic Delivery ◽  
Cynomolgus Monkeys ◽  
Mouse Tissues

Abstract miR-221/222 are highly homologous microRNAs (miRNAs) whose upregulation has been found in several malignancies, including multiple myeloma (MM). Both miRNAs are thought to promote cell proliferation via down-regulation of p27 and/or p57, two negative regulators of G1 to S phase cell cycle progression. We proved that silencing of both miRNAs results in significant anti-MM activity and in downregulation of canonical targets both in vitro and in vivo. In the aim to progress to clinical translation, we designed an original 13mer synthetic inhibitor, specific for systemic delivery, named LNA-i-miR-221, which took advantage from both locked nucleic acid (LNA) technology and phosphorothioate backbone, for increasing the seed sequence binding and nuclease resistance in vivo, respectively. Since no data are presently available, we evaluated the specificity of LNA-i-miR-221 to inhibit endogenous miR-221 and the pharmacokinetic properties (i.e. tissue distribution) in mice and Cynomolgus monkeys. We demonstrated that LNA-i-miR-221 inhibit growth and survival of MM cells bearing high miR-221 levels. Moreover, LNA-i-miR-221-mediated silencing of miR-221 triggered upregulation of p27Kip1 mRNA and protein, evaluated by q-RT-PCR and western blotting, respectively. In vivo, i.p.treatment with 25 mg/kg of LNA-i-miR-221 reduced tumor growth in SCID/NOD mice bearing MM xenografts. Tumors and vital organs (including liver, kidney, bone marrow and heart) were harvested from treated animals and evaluated for pharmacokinetics purposes using in situ hybridization (ISH) assay. After a single i.p. dose of 25 mg/kg, we detected the presence of LNA-i-miR-221 from 2 up to 7 days in tumors and mouse tissues. Interestingly, no toxicity was observed even after long-lasting presence of the inhibitors in vital organs. We also evaluated the LNA-i-miR-221 half-life in mouse plasma using HPLC-MS/MS, which confirmed the rapid uptake of LNA-i-miR-221 in tissues. To evaluate the maximum tolerated doses, in vivo dose escalation treatments was performed using doses from 10 to 100 mg/kg delivered at days 1,4,8,15,22. All treatments were well tolerated. No changes in mice behavior or organ toxicity were observed in treated mice. Finally, a pilot toxicity study was performed in Cynomolgus monkeys. PK results demonstrated that LNA-i-miR-221 has a short serum half-life with a rapid tissue uptake and minimum urinary escretion. In conclusion, our results suggest that LNA-i-miR-221 is a promising anti-MM agent associated with a safety profile in mice and in monkeys, supporting the rationale for development of this novel miR-221 inhibitor in early clinical trials. Disclosures No relevant conflicts of interest to declare.

scholarly journals Erratum to: In vitro and in vivo activity of miR-92a–Locked Nucleic Acid (LNA)–Inhibitor against endometrial cancer

BMC Cancer ◽  
2016 ◽  
Vol 16 (1) ◽  
Author(s):  
Anna Torres ◽  
Joanna Kozak ◽  
Agnieszka Korolczuk ◽  
Paulina Wdowiak ◽  
Ewa Domańska-Glonek ◽  
...  
Keyword(s):  
Endometrial Cancer ◽  
Nucleic Acid ◽  
Locked Nucleic Acid

scholarly journals Exploration of anti EFG1 locked nucleic acid gapmers to control Candida albicans filamentation

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Daniela Araújo ◽  
Dalila Mil-Homens ◽  
Per Trolle Jørgensen ◽  
Arsénio M. Fialho ◽  
Jesper Wengel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nucleic Acid ◽  
Chemical Modification ◽  
Therapeutic Potential ◽  
Locked Nucleic Acid ◽  
Novel Approach ◽  
Candida Infections ◽  
In Vitro Performance

Introduction: Antisense oligonucleotides (ASOs) have been successfully utilized to silence gene expression for the treatment of many genetic human diseases, and particularly the locked nucleic acid (LNA) chemical modification is extensively used with this propose. However, LNA-modified ASOs have never been exploited for controlling virulence genes of Candida. EFG1is an important determinant of virulence that is involved in the switch from yeast to filamentous forms in C. albicans. Thus, our main goal was to explore LNA antisense gapmers for controlling EFG1gene expression and to block C. albicans filamentation. Methods: A set of five LNA-modified gapmers were designed with different chemical modifications (phosphorothioate backbone (PS) and/or palmitoyl-2’-amino-LNA) and ASO length. The in vitro performance of the different ASOs was evaluatedon their ability to control EFG1 gene expression, by qRT-PCR, and to reduce C. albicans’ filamentation, through filaments’ enumeration by microscopy. The in vivo therapeutic potential of ASOs was assessed using a G. mellonella model of infection, through a survival assay. Results: In vitro results showed that all ASOs were able to reduce the levels of EFG1gene expression, consequently reducing the levels of C. albicans filamentation around 50%. Interestingly, in vivo tests showed that the LNA-modified gapmer with PS backbone and palmitoyl-2’-amino-LNA was more effective at preventing G. mellonella infections. Conclusions: Undeniably, this work promotes the development of a novel approach for the treatment of Candida infections based on the delivery of ASOs coupled with LNA chemical modification.

scholarly journals In Vitro and In Vivo Activity of a Novel Locked Nucleic Acid (LNA)-Inhibitor-miR-221 against Multiple Myeloma Cells

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e89659 ◽  
Author(s):  
Maria Teresa Di Martino ◽  
Annamaria Gullà ◽  
Maria Eugenia Gallo Cantafio ◽  
Emanuela Altomare ◽  
Nicola Amodio ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Nucleic Acid ◽  
Locked Nucleic Acid ◽  
Myeloma Cells
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