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- Lendvai, Gabor, et al.
(author)
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Biodistribution of Ga-68-labeled LNA-DNA mixmer antisense oligonucleotides for rat Chromogranin-A
- 2008
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In: Oligonucleotides. - : Mary Ann Liebert Inc. - 1545-4576 .- 1557-8526. ; 18:1, s. 33-49
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Journal article (peer-reviewed)abstract
- In vivo monitoring of gene expression may be accomplished using a most advanced imaging technology such as positron emission tomography (PET). However, a range of methodological and biological hurdles needs exploration. In the present study, 20-mer DNA-LNA (locked nucleic acid) mixmer oligonucleotides specific for rat Chromogranin-A (Chg-A) mRNA were labeled with Ga-68 and their biodistribution were investigated in rats; namely, two Antisense (LNA1, LNA2-differing only in the positioning of LNA modification), Mismatched, and Sense sequences. In addition, in vivo and in vitro metabolite analysis of LNA1 and LNA2 was compared, and hybridization in solution was performed to verify the hybridization ability after labeling. Furthermore, semiquantitative polymerase chain reaction was carried out to find organs expressing Chg-A mRNA in the rat. The biodistribution patterns altered according to the sequence and the positioning of LNA modification. The pattern of Mismatched-differing only in two nucleotides from the two Antisenses-was similar to that of Sense, whereas the pattern of LNA1 and LNA2 showed differences. Uptake in the adrenal gland was twofold higher with LNA2 compared to the other three oligonucleotides. Intact LNA2 could be observed in the 60-minute sample in vivo, whereas in vitro, the intact compound of both Antisenses could also be detected after 2 hours. Hybridization in solution revealed that the two Antisenses retained their hybridization abilities after Ga-68-labeling. With decreasing magnitude, Chg-A mRNA was expressed in the adrenal gland, intestine, testis, and pancreas. This study further supported LNA-DNA mixmer to be a favorable modification for antisense targeting approach with respect to hybridization and longer plasma residence; however, the organ uptake was dominated by processes irrelevant to specific hybridization.
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- Lendvai, Gabor, et al.
(author)
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Non-hybridisation saturable mechanisms play a role in the uptake of 68Ga-labelled LNA-DNA mixmer antisense oligonucleotides in rats
- 2009
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In: Oligonucleotides. - : Mary Ann Liebert Inc. - 1545-4576 .- 1557-8526. ; 19:3, s. 223-231
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Journal article (peer-reviewed)abstract
- 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.
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- Lendvai, Gábor, 1969-
(author)
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Radiolabelled Oligonucleotides for Evaluation of in vivo Hybridisation Utilising PET Methodology
- 2007
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Doctoral thesis (other academic/artistic)abstract
- 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.
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- Lendvai, Gabor, et al.
(author)
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Radiolabelled Oligonucleotides for Imaging of Gene Expression with PET
- 2009
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In: Current Medicinal Chemistry. - : Bentham Science Publishers Ltd.. - 0929-8673 .- 1875-533X. ; 16:33, s. 4445-4461
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Research review (peer-reviewed)abstract
- Our understanding of altered patterns of gene expression being responsible for many diseases has been growing thanks to modern molecular biological methods. Today, these changes can only be identified when tissue samples are available. Therefore, a noninvasive method allowing us to monitor gene expression in vivo would be valuable, not only as a research tool, but also for patient stratification before treatment and for treatment follow-up. Antisense oligonucleotides (ODN) have been considered to be suitable molecules to trace active genes in vivo, as well as to treat diseases by hybridising to its complementary messenger RNA (mRNA) sequence in the cells thereby preventing the synthesis of the peptide. However, the use of ODNs in the organisms are endangered by many hurdles such as physical barriers to pass and enzyme attack to be avoided. Positron emission tomography (PET) provides a most advanced in vivo imaging technology that allows the exploration of 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. This review provides a general summary about the antisense concept and displays the present status of the antisense imaging field with the major achievements and remaining challenges on the long journey towards accomplishing in vivo monitoring of gene expression using PET.
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