Noninvasive and Quantitative Monitoring of the Distributions and Kinetics of MicroRNA-Targeting Molecules in Vivo by Positron Emission Tomography.

MicroRNAs (miRNAs) are endogenous, small, noncoding ribonucleic acids (RNAs) that bind to the 3' untranslated regions of messenger RNAs (mRNAs) and induce translational repression or mRNA degradation. Although numerous studies have reported that miRNAs are of potential use for disease diagnostics and gene therapy, little is known about their fates in vivo. This study elucidated the whole-body distributions and kinetics of intravenously administered miRNA-targeting molecules in vivo by positron emission tomography (PET) imaging. A 22-mer sequence targeting miR-15b was conjugated with three different chelators and labeled with gallium-68 (68Ga). These tracers were compared with a scrambled 22-mer sequence; 22-mer with two single base substitutions; anti-miR-34 22-mer; hexathymidylate (T6), a 6-mer sequence; and an unconjugated chelator. miR-15b was chosen as a target because it is important for bone remodeling. All three 68Ga-labeled anti-miR-15b molecules had similar biodistributions and kinetics, and they all accumulated in the bones, kidneys, and liver. The bone accumulation of these tracers was the highest in the epiphyses of long tubular bones, maxilla, and mandible. By contrast, the scrambled 22-mer sequence, the 6-mer, and the unconjugated chelator did not accumulate in bones. PET imaging successfully elucidated the distributions and kinetics of 68Ga-labeled chelated miRNA-targeting molecules in vivo. This approach is potentially useful to evaluate new miRNA-based drugs.

In Vivo Bone-Targeting of Bis(phosphonate)-Conjugated Double Helical RNA Monitored by Positron Emission Tomography.

A bis(phosphonate) conjugate of 2'-O-methyl oligoribonucleotide (microRNA-21) was synthesized and used as a bone-targeting carrier in the systemic delivery of a (68)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-chelated 2'-O-methyl oligoribonucleotide (anti-microRNA-21). The whole-body biodistribution of the double helical RNA was monitored by positron emission tomography (PET), which verified the expected bis(phosphonate)-induced bone accumulation in healthy rats.

Synthesis and In Vivo PET Imaging of Hyaluronan Conjugates of Oligonucleotides.

Synthesis for (68)Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-chelated oligonucleotide hyaluronan (HA) tetra- and hexasaccharide conjugates is described. A solid-supported technique is used to introduce NOTA-chelator into the 3'-terminus of oligonucleotides and a copper-free strain promoted azide alkyne cycloaddition (SPAAC) to HA/oligonucleotide conjugation. Protecting group manipulation, required for the HA-moieties, is carried out after the SPAAC-conjugation. Positron emission tomography (PET) is used (1) in the whole-body distribution kinetic studies of the conjugates in healthy rats and (2) to show the potential of hyaluronan-induced targeting of oligonucleotides into the infarcted area of rats with myocardial infarction.

68Ga-DOTA-Siglec-9--a new imaging tool to detect synovitis.

INTRODUCTION: Vascular adhesion protein-1 (VAP-1) is an adhesion molecule, which upon inflammation is rapidly translocated from intracellular sources to the endothelial cell surface. We have recently discovered that sialic acid- binding immunoglobulin-like lectin 9 (Siglec-9) is a leukocyte ligand of VAP-1 and that 68Ga-labeled Siglec-9 motif peptide facilitates in vivo imaging of inflammation. This study evaluated the feasibility of 68Ga-DOTA-Siglec-9 positron emission tomography (PET) for the assessment of synovitis. METHODS: Rabbits with synovial inflammation were injected with 18F-FDG or 68Ga-DOTA-Siglec-9 and studied by gamma counting and autoradiography. Certain rabbits were also examined with magnetic resonance imaging (MRI). After PET imaging, rabbits were intravenously administered with anti-VAP-1 antibody to evaluate luminal expression of VAP-1 by immunohistochemistry. Finally, binding of Siglec-9 peptide and VAP-1 positive vessels were evaluated by double staining of rheumatoid arthritis synovium. RESULTS: Intra-articular injection of hemagglutinin induced mild synovial inflammation in rabbit knee with luminal expression of VAP-1. Synovitis was clearly visualized by 68Ga-DOTA-Siglec-9 PET in addition to 18F-FDG-PET and MRI. Compared with the 18F-FDG, the ex vivo inflamed-to-control synovium ratio of 68Ga-DOTA-Siglec-9 was similar (1.7 ± 0.4 vs. 1.5 ± 0.2, P = 0.32). Double staining revealed that Siglec-9 peptide binds to VAP-1 positive vessels in human rheumatoid synovium. CONCLUSION: Ga-DOTA-Siglec-9 PET tracer detected VAP-1 positive vasculature in the mild synovitis of rabbits comparable with 18F-FDG, suggesting its potential for in vivo imaging of synovial inflammation in patients with rheumatic diseases.

Synthesis of multi-galactose-conjugated 2'-O-methyl oligoribonucleotides and their in vivo imaging with positron emission tomography.

(68)Ga labelled 2'-O-methyl oligoribonucleotides (anti-miR-15b) bearing one, three or seven d-galactopyranoside residues have been prepared and their distribution in healthy rats has been studied by positron emission tomography (PET). To obtain the heptavalent conjugate, an appropriately protected 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) precursor bearing a 4-[4-(4,4'-dimethoxytrityloxy)butoxy]phenyl side arm was first immobilized via a base labile linker to the support and the oligonucleotide was assembled on the detritylated hydroxyl function of this handle. A phosphoramidite building block bearing two phthaloyl protected aminooxy groups and one protected hydroxyl function was introduced into the 5'-terminus. One acetylated galactopyranoside was coupled as a phosphoramidite to the hydroxyl function, the phthaloyl protections were removed on-support and two trivalent galactopyranoside clusters were attached as aldehydes by on-support oximation. A two-step cleavage with aqueous alkali and ammonia released the conjugate in a fully deprotected form, allowing radiolabelling with (68)Ga in solution. The mono- and tri-galactose conjugates were obtained in a closely related manner. In vivo imaging in rats with PET showed remarkable galactose-dependent liver targeting of the conjugates.

Accumulation of genomic alterations in 2p16, 9q33.1 and 19p13 in lung tumours of asbestos-exposed patients.

We have previously demonstrated an association between genomic alterations in 19p13, 2p16, and 9q33.1 and asbestos exposure in patients' lung tumours. This study detected allelic imbalance (AI) in these regions in asbestos-exposed lung cancer (LC) patients' histologically normal pulmonary epithelium. We extended the analyses of tumour tissue to cover a large LC patient cohort and studied DNA copy number alteration (CNA) and AI in 19p13, 2p16, and 9q33.1 for the first time in combination. We found both CNA and AI in ≥2/3 of the regions to be significantly and dose-dependently (P < 0.001) associated with pulmonary asbestos fibre count. Twenty percent of the exposed patients' LC showed CNA in ≥2/3 of the regions, whereas none of the non-exposed patients' LC showed CNA in more than one region. AI was evident in 89% of the exposed and in only 26% of the non-exposed patients' LC. The genomic alterations in 19p13, 2p16, and 9q33.1 in compilation identified asbestos-exposed patients' lung tumours better than each of the regions alone. These alterations form the basis for the development of a combinatorial molecular assay that could be used to identify asbestos-related LC.

Solid-supported NOTA and DOTA chelators useful for the synthesis of 3'-radiometalated oligonucleotides.

Esterified precursors of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA; 18) and 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA; 17,19) ligands bearing a dimethoxytritylated hydroxyl side arm were prepared and immobilized via an ester linkage to long chain alkyl amine derivatized controlled pore glass (LCAA-CPG). Oligonucleotide chains were then assembled on the hydroxyl function and conjugates were released and deprotected by a two-step cleavage with aqueous alkali and ammonia. The 3'-DOTA and 3'-NOTA conjugated oligonucleotides were converted to (68)Ga chelates by a brief treatment with [(68)Ga]Cl(3) at elevated temperature. Applicability of the conjugates for in vivo imaging with positron emission tomography (PET) was verified.