H-ras-targeted genetic therapy remarkably surpassed docetaxel treatment in inhibiting chemically induced hepatic tumors in rats.

AIMS: Hepatocellular carcinoma (HCC) is still a leading cause of cancer-related death worldwide. But its chemotherapeutic options are far from expectation. We here compared H-ras targeted genetic therapy to a commercial docetaxel formulation (DXT) in inhibiting HCC in rats. MAIN METHODS: After the physicochemical characterization of phosphorothioate-antisense oligomer (PS-ASO) against H-ras mutated gene, the PS-ASO-mediated in vitro hemolysis, in vivo hepatic uptake, its pharmacokinetic profile, tissue distribution in some highly perfused organs, its effect in normal rats, antineoplastic efficacy in carcinogen-induced HCC in rats were evaluated and compared against DXT treatment. Mutated H-ras expression by in situ hybridization, hep-par-I, CK-7, CD-15, p53 expression patterns by immunohistochemical methods, scanning electron microscopic evaluation of hepatic architecture, various hepatic marker enzyme levels and caspase-3/9 apoptotic enzyme activities were also carried out in the experimental rats. KEY FINDINGS: PS-ASO showed low in vitro hemolysis (<3 %), and had a sustained PS-ASO blood residence time in vivo compared to DTX, with a time-dependent hepatic uptake. It showed no toxic manifestations in normal rats. PS-ASO distribution was although initially less in the lung than liver and kidney, but at 8 h it accumulated more in lung than kidney. Antineoplastic potential of PS-ASO (treated for 6 weeks) excelled in inhibiting chemically induced tumorigenesis compared to DTX in rats, by inhibiting H-ras gene expression, some immonohistochemical modulations, and inducing caspase-3/9-mediated apoptosis. It prevented HCC-mediated lung metastatic tumor in the experimental rats. SIGNIFICANCE: PS-ASO genetic therapy showed potential to inhibit HCC far more effectively than DXT in rats.

Identification of circRNA-Interacting Proteins by Affinity Pulldown.

Circular RNAs (circRNAs) comprise a vast class of covalently closed transcripts, generated primarily via backsplicing. Most circRNAs arise from full or partial exons, but they can also arise from introns, and from combinations of introns and exons. While high-throughput RNA-sequencing analysis has identified tens of thousands of circRNAs expressed in different tissues and growth conditions, the function of circRNAs has only been described for a handful of them. As most circRNAs appear not to encode peptides, their function is presumed to be linked to their interaction with a range of molecules, particularly other nucleic acids (notably microRNAs) and proteins. A major impediment to identifying circRNA-associated molecules is a lack of suitable methodologies capable of analyzing specifically circRNAs and not their linear RNA counterparts with which they share most of their sequence. Here, we describe a flexible and robust method for identifying the proteins that associate with a given circRNA. The affinity pulldown assay is based on the use of a biotinylated antisense oligomer that recognizes the circRNA-specific junction sequence. Following pulldown using streptavidin beads, the proteins are eluted from the circRNP (circribonucleoprotein) complex and identified by mass spectroscopy; validation by Western blot analysis and other methods would then confirm the identity of the circRNA-associated proteins. We present a detailed step-by-step protocol, tips to optimize the analysis, troubleshooting suggestions, and assistance in interpreting the results. In sum, this protocol enables the discovery of proteins present in circRNPs, a critical effort toward elucidating circRNA function.

Zn-based physiometacomposite nanoparticles: distribution, tolerance, imaging, and antiviral and anticancer activity.

The aim of this study was to investigate the distribution, tolerance, and anticancer and antiviral activity of Zn-based physiometacomposites (PMCs). Manganese, iron, nickel and cobalt-doped ZnO, ZnS or ZnSe were synthesized. Cell uptake, distribution into 3D culture and mice, and biochemical and chemotherapeutic activity were studied by fluorescence/bioluminescence, confocal microscopy, flow cytometry, viability, antitumor and virus titer assays. Luminescence and inductively coupled plasma mass spectrometry analysis showed that nanoparticle distribution was liver >spleen >kidney >lung >brain, without tissue or blood pathology. Photophysical characterization as ex vivo tissue probes and LL37 peptide, antisense oligomer or aptamer delivery targeting RAS/Ras binding domain (RBD) was investigated. Treatment at 25 µg/ml for 48 h showed ≥98-99% cell viability, 3D organoid uptake, 3-log inhibition of ß-Galactosidase and porcine reproductive respiratory virus infection. Data support the preclinical development of PMCs for imaging and delivery targeting cancer and infectious disease.

Translational development of splice-modifying antisense oligomers.

INTRODUCTION: Antisense nucleic acid analogues can interact with pre-mRNA motifs and influence exon or splice site selection and thereby alter gene expression. Design of antisense molecules to target specific motifs can result in either exon exclusion or exon inclusion during splicing. Novel drugs exploiting the antisense concept are targeting rare, life-limiting diseases; however, the potential exists to treat a wide range of conditions by antisense-mediated splice intervention. Areas covered: In this review, the authors discuss the clinical translation of novel molecular therapeutics to address the fatal neuromuscular disorders Duchenne muscular dystrophy and spinal muscular atrophy. The review also highlights difficulties posed by issues pertaining to restricted participant numbers, variable phenotype and disease progression, and the identification and validation of study endpoints. Expert opinion: Translation of novel therapeutics for Duchenne muscular dystrophy and spinal muscular atrophy has been greatly advanced by multidisciplinary research, academic-industry partnerships and in particular, the engagement and support of the patient community. Sponsors, supporters and regulators are cooperating to deliver new drugs and identify and define meaningful outcome measures. Non-conventional and adaptive trial design could be particularly suited to clinical evaluation of novel therapeutics and strategies to treat serious, rare diseases that may be problematic to study using more conventional clinical trial structures.