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.

Site Selective Antibody-Oligonucleotide Conjugation via Microbial Transglutaminase.

Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types.

Synthesis of Azide-Modified Chondroitin Sulfate Precursors: Substrates for "Click"- Conjugation with Fluorescent Labels and Oligonucleotides.

Azidopropyl-modified precursors of chondroitin sulfate (CS) tetrasaccharides have been synthesized, which, after facile conversion to final CS structures, may be conjugated with alkyne-modified target compounds by a one-pot "click"-ligation. RP HPLC was used for the monitoring of the key reaction steps (protecting group manipulation and sulfation) and purification of the CS precursors (as partially protected form, bearing the O-Lev, O-benzoyl, and N-trichloroacetyl groups and methyl esters). Subsequent treatments with aqueous NaOH, concentrated ammonia, and acetic anhydride (i.e., global deprotection and acetylation of the galactosamine units) converted the precursors to final CS structures. The azidopropyl group was exposed to a strain-promoted azide-alkyne cycloaddition (SPAAC) with a dibenzylcyclooctyne-modified carboxyrhodamine dye to give labeled CSs. Conjugation with a 5'-cyclooctyne-modified oligonucleotide was additionally carried out to show the applicability of the precursors for the synthesis of biomolecular hybrids.

Solid-Supported Porphyrins Useful for the Synthesis of Conjugates with Oligomeric Biomolecules.

meso-Tris(pyridin-4-yl)(4-carboxyphenyl)porphyrin and 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (Photochlor, HPPH) were amide-coupled to 1R,2S,3R,4R-2,3-dihydroxy-4-(hydromethyl)-1-aminocyclopentane and immobilized via an ester linkage to long chain alkyl amine-derivatized controlled pore glass (LCAA-CPG). The applicability of these supports (5 and 6) for the synthesis of porphyrin conjugates with oligomeric biomolecules was demonstrated using an automated phosphoramidite coupling chemistry. Cleavage from the support with concentrated ammonia gave the products, viz., porphyrin conjugates of oligonucleotides (7-9) and dendritic glycoclusters (10-13) and a cyclooctyne derivative (14) in 23-58% yield. In addition, the synthesized cyclooctyne derivative of meso-tris(pyridin-4-yl)(4-carboxyphenyl)porphyrin (14) was conjugated with an azidopropyl-modified hyaluronic acid (19). The hyaluronic acid-porphyrin conjugate (15) was radiolabeled with (64)Cu and its (15[(64)Cu]) receptor binding affinity to CD44-expressing tumor cells was evaluated.

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.

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 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.

Design, Synthesis, and Biochemical Analysis of a Molecule Designed to Enhance Endosomal Escape.

RNA therapeutics, including siRNAs, ASOs, and PMOs, have great potential to treat human disease. However, RNA therapeutics are too large, too charged, and/or too hydrophilic to cross the cellular membrane and are instead taken up into cells by endocytosis. Unfortunately, the vast majority of RNA therapeutics remain trapped inside endosomes (≥ 99%), which is the sole reason preventing their use to treat cancer, COVID, and other diseases. In contrast, enveloped viruses, such as influenza, also have an endosomal escape problem, but have evolved a highly efficient endosomal escape mechanism using trimeric hemagglutinin (HA) fusogenic protein. HA contains an outer hydrophilic domain (HA1) that masks an inner hydrophobic fusogenic/endosomal escape domain (HA2). Once inside endosomes, HA1 is shed to expose HA2 that, due to hydrophobicity, buries itself into the endosomal lipid bilayer, driving escape into the cytoplasm in a non-toxic fashion. To begin to address the RNA therapeutics rate-limiting endosomal escape problem, we report here a first step in the design and synthesis of a universal endosomal escape domain (uEED) that biomimics the enveloped virus escape mechanism. uEED contains an outer hydrophilic mask covalently attached to an inner hydrophobic escape domain. In plasma, uEED is inert and highly metabolically stable; however, when placed in endo/lysosomal conditions, uEED is activated by enzymatic removal of the hydrophilic mask, followed by self-immolation of the linker resulting in exposure of the hydrophobic indole ring domain in the absence of any hydrophilic tags. Thus, uEED is a synthetic biomimetic of the highly efficient viral endosomal escape mechanism.

Delivery of RNA Therapeutics: The Great Endosomal Escape!

RNA therapeutics, including siRNAs, antisense oligonucleotides, and other oligonucleotides, have great potential to selectively treat a multitude of human diseases, from cancer to COVID to Parkinson's disease. RNA therapeutic activity is mechanistically driven by Watson-Crick base pairing to the target gene RNA without the requirement of prior knowledge of the protein structure, function, or cellular location. However, before widespread use of RNA therapeutics becomes a reality, we must overcome a billion years of evolutionary defenses designed to keep invading RNAs from entering cells. Unlike small-molecule therapeutics that are designed to passively diffuse across the cell membrane, macromolecular RNA therapeutics are too large, too charged, and/or too hydrophilic to passively diffuse across the cellular membrane and are instead taken up into cells by endocytosis. However, similar to the cell membrane, endosomes comprise a lipid bilayer that entraps 99% or more of RNA therapeutics, even in semipermissive tissues such as the liver, central nervous system, and muscle. Consequently, before RNA therapeutics can achieve their ultimate clinical potential to treat widespread human disease, the rate-limiting delivery problem of endosomal escape must be solved in a clinically acceptable manner.

Synthesis, Characterization, and Cytotoxicity Evaluation of Polyethylene Glycol-Coated Iron Oxide Nanoparticles for Radiotherapy Application.

BACKGROUND: Treatment methods for cancer that are widely being utilized affect both normal and cancerous cells. We report synthesis polyethylene glycol (PEG)-coated Fe3O4 nanoparticles (NPs) and its characteristic properties and appraise its potential as a promising radiation sensitizer candidate in radiotherapy that improves cancer treatment and reduces side effects of radiation. MATERIALS AND METHODS: PEG-coated Fe3O4 NPs were synthesized by chemical coprecipitation method and characterized by studying their size, structure, functional group, stability, magnetization, and cytotoxicity using different techniques. X-ray powder diffraction, Fourier transform infrared spectroscopy, and thermogravimetric analysis results show that Fe3O4 NPs have been functionalized with PEG molecules during the course of synthesis. RESULTS: Synthesized NPs have good stability based on zeta-potential study. Dynamic light-scattering results reveal that PEG-coated Fe3O4 has a greater hydrodynamic size than bare Fe3O4. Transmission electron microscopy (TEM) micrograph exhibited that NPs are roughly spherical with size in range of 10-20 nm. Saturation magnetization value of PEG-coated and bare Fe3O4 also confirms coating and shows superparamagnetic behavior. Cytotoxicity evaluation study indicated that PEG-coated Fe3O4 is biocompatible on L929 and toxic on Michigan Cancer Foundation-7 (MCF-7) (breast cancer cells). CONCLUSION: These characterized properties of PEG-coated Fe3O4 NPs show that it could be used as a potential radiosensitizer candidate in radiotherapy to significantly improve cancer treatment and minimize painful side effects of radiation.