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Non-hybridisation saturable mechanisms play a role in the uptake of 68Ga-labelled LNA-DNA mixmer antisense oligonucleotides in rats
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
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2009 (English)In: Oligonucleotides, ISSN 1545-4576 (Print), Vol. 19, no 3, 223-231 p.Article in journal (Refereed) Published
Abstract [en]

Oligonucleotides (ODN) are key molecules for the aim of preventing   translation of a gene product or monitoring gene expression in tissues.   However, multiple methodological and biological hurdles need to be   solved before in vivo application in humans will be possible. For   positron emission tomography (PET) investigations, a 20-mer DNA-locked   nucleic acid (LNA) mixmer ODN specifi c for rat chromogranin-A mRNA was   labeled with Ga-68 and its uptake was examined in vivo in rats with and   without blocking of scavenger receptors by polyribo-nucleotides. In   addition, uptake studies of Ga-68-LNA were performed with respect to   time and concentration in human and rat cell lines. The human cell   lines did not express the target mRNA. Both polyinosinic acid (poly-I)   and polyadenylic acid (poly-A) reduced the uptake in rat tissues and in   human cell lines. Poly-I was found to be more effective in the liver   whereas poly-A was more effective in the kidney. In addition, the   blockade by poly-I was statistically significant in the pancreas,   adrenal gland, bone marrow, intestine, testis, urinary bladder, muscle,   parotid gland, and heart, whereas poly-A also caused significant   reduction in pancreas, adrenal gland, and bone marrow but not as much   as in kidney. Cell culture study showed a 2-phase dose-dependent uptake   characteristic with a saturable and a passive diffusion-like phase;   however, these 2 phases were not so well expressed in the rat cell   line. The results suggest that scavenger receptors or other saturable   processes unrelated to hybridization may be involved in the tissue   uptake of Ga-68-LNA and in the clearance of antisense ODN through the   liver, kidney, spleen, and bone marrow. The fact that these processes   may be sequence-dependent suggests that proof of in vivo hybridization   through imaging may not be obtained by only comparing sense and   antisense sequences and proving dose-dependency.

Place, publisher, year, edition, pages
2009. Vol. 19, no 3, 223-231 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-96301DOI: 10.1089/oli.2009.0192ISI: 000270785700002OAI: oai:DiVA.org:uu-96301DiVA: diva2:170831
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2014-04-15Bibliographically approved
In thesis
1. Radiolabelled Oligonucleotides for Evaluation of in vivo Hybridisation Utilising PET Methodology
Open this publication in new window or tab >>Radiolabelled Oligonucleotides for Evaluation of in vivo Hybridisation Utilising PET Methodology
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression in vivo, i.e. to accomplish in vivo hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples.

This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced in vivo imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents.

The present study aimed to investigate 76Br- and 68Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-O-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of in vivo hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 90 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 279
Molecular medicine, Gene expression, Antisense oligonucleotides, Positron emission tomography, In vivo hybridisation, In vivo biodistribution, Chromogranin-A, 68Ga, RT-PCR, Scavenger receptors, Molekylärmedicin
urn:nbn:se:uu:diva-8262 (URN)978-91-554-6990-0 (ISBN)
Public defence
2007-11-09, Skoogsalen, University Hospital, Ingång 78/79, Uppsala, 09:15
Available from: 2007-10-18 Created: 2007-10-18Bibliographically approved

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Monazzam, Azita
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Department of Medical SciencesDepartment of Oncology, Radiology and Clinical ImmunologyDepartment of Biochemistry and Organic ChemistryDepartment of Pharmaceutical Biosciences
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